Lysine: Is it worth more?

Cytotechnology 36: 3–32, 2001.

Lysine:Isitworthmore?

Debatosh Datta

∗

, Akshay Bhinge & Vidya Chandran

School of BioMedical Engineering, Indian Institute of Technology-Bombay, Powai, Mumbai, India

(

∗

Author for correspondence, E-mail: ddatta@cc.iitb.ac.in)

Received 4 April 2001; accepted 30 August 2001

Key words: biological properties, food preservative, immune therapy, lysine

Abstract

Lysine, an essential cationic amino acid, has a positively charged R group. The structure of lysine is given as

-) CH(-COO

−

)-CH

-CH

-N

. While the anabolic role(s) of the molecule has been in focus

for quite a few decades now, its biological properties, e.g. role in cellular proliferation in vitro (both anchorage

dependent and anchorage independent) and in vivo, its ability to induce strong inﬂammatory and immune responses

– both humoral and cell mediated, its role in augmented healing of all types of wounds in animal models as well

as in human subjects (both acute and chronic), as well as its role in inducing extensive angiogenic responses, have

never received reasonable attention so far. In the current brief and indicative review (rather than exhaustive reviews

of each area), we intend to bring these biological properties of the molecule to focus while discussing a few other

interesting aspects – lysine as a food preservative as well as its possible role(s) in immune therapy. While the areas

look extremely divergent, we propose a common denominator in the form of a possible molecular mechanism of

action of the molecule in all these diverse situations.

Lysine as a cell proliferator

Monomeric L-lysine HCl was supportive of cellular

division and growth in vitro in an optimal concentra-

tion range of 7–10 µgml

−1

depending on the cell line

(Datta et al., 1997a, b). We examined the phenomenon

in few anchorage independent (AE9D6, CC9C10,

HUT 78) and anchorage dependent (BHK 21 from

NCCS) cell lines. Peak cellular growth was obtained,

in all cases at 48 hr of culture (which could not be ac-

counted for) and maximal cell concentration depended

on a) initial cell density at seeding and b) cellular

adaptability to culture conditions in terms of number

of passages it went through before lysine stimulation.

Both D- and L-conﬁgurations of oligomeric lysine

(upto 6–7 residues; mol. wt. 1000) supported compar-

able cellular expansion at 48 hr. We examined whether

lysine acts independently as a cellular growth/division

promoting agent or not. It probably acts in conjunc-

tion with the serum derived growth factors. This was

evident from viable cellular expansion data at 48 hr in

serum containing media versus serum-free one. Sup-

plementation of the serum-free medium with insulin,

transferin and selenite (ITS) in contrast (to QBSF

alone), supported the characteristic 48 hr response

in vitro. This evidently shows an indirect facilitat-

ing role(s) of the molecule at cell surface, possibly,

through electrostatic binding of serum derived growth

factor(s) just prior to or as a part of ligand receptor

interaction (Figure 1) (Datta et al., 1997b, 2000). Least

energy conﬁguration model of 6 residue poly-L-lysine

(molecular weight ∼1000) showed characteristically

close molecular orientation (twisting) in a plane of

positively charged – NH

groups. TGF-β(2) (recom-

binant human homodimer molecule), a known agent

promoting wound healing, also showed typical lysine

residue concentrate (4 to 5 in number) at each end. It

is obviously interesting to speculate an active role for

this lysine concentrate in TGF-β and its receptor bind-

ing, in its biological expressions. This corroborates

quite well with the experimental data of poly-lysine

only upto molecular weight 1000 and not beyond, act-

ing as cellular growth promoting agent in vitro (Datta

et al., 1997b, 2000a).

Figure 1. Mechanism of action of lysine.

Wound healing

In this world of cosmesis, a faster and scarless wound

repair is much desired. A want is still felt for a

molecule which can improve the rate and quality of

healing without being prohibitively costly and hav-

ing easy availability and high degree of cell/tissue

compatibility.

A good amount of work has been done in the ﬁeld

of augmented wound healing using growth factors,

which have known mitogenic potential. Epidermal

growth factor (EGF) has been tried in improving

wound strength and accelerating the rate of repair

(Zhou et al., 1997). Delivery of platelet derived growth

factor (PDGF) using cultured dermal ﬁbroblasts trans-

duced retrovirally with PDGF-B gene and adenoviral

mediated gene transfer has been tried to overcome the

ischaemic defect in wound healing (Breitbart et al.,

1999; Liechty et al., 1999). Granulocyte colony stim-

ulating factor (G-CSF) and granulocyte-macrophage

colony stimulating factor (GM-CSF) also has been

claimed to show promise (Jaschke et al., 1999; Grzy-

bowski, 1999). Keratinocyte growth factor-2 (KGF-2)

has been shown to accelerate wound healing with

increase in wound strength and no obvious scarring

(Junienez and Rampy, 1999). Basic ﬁbroblast growth

factor -β (bFGF) carried on a collagen matrix for

inhibiting wound contracture has been tried out in dia-

betic and decubitus ulcers (Ono et al., 1999). TGF-β

delivered through a collagen scaffold has also been

tried in enhancing wound healing (Pandit, 1999).

There is support for addition of protease inhibitors in

conjunction with any treatment using growth factors

(Trengove et al., 1999). Collagen and hyaluronan have

been used by many as carriers for growth factors.

Both per se have been used for tissue augmentation

and wound healing (Ruszczak and Schwartz, 1999;

Baichey et al., 1995; Jia et al., 1998). Platelet release

treatment has also been tried in improving skin heal-

ing in diabetic rats through endogenous growth factor

secretion (Moulin et al., 1998).

Accelerated wound healing has been seen in mice

with a disruption of thrombospondin 2 gene (Kyri-

ahides et al., 1999). Mice lacking Smad 3 have also

shown accelerated healing (Ashcrott et al., 1999b).

Chitosan-heparin membrane is claimed to cause

increased stabilization and concentration of growth

factor in wound area. Immobilized heparin has also

been shown to induce accelerated healing (Kratz et

al., 1998). Water-soluble chitin has also been used

as a wound-healing accelerator (Cho et al., 1999). It

gave high tensile strength and arrangement of colla-

gen ﬁbres similar to normal skin. It has also been

shown that positively charged dextran beads stimulate

wound healing (Tawil et al., 1999). Thymosin beta

4 also has been shown to accelerate wound healing

by increasing re-epithelialisation by stimulating kerat-

inocyte migration, increasing collagen deposition and

causing angiogenesis (Malinda et al., 1999).

A synthetic analogue of prostaglandin E

(PGE

)

has also been evaluated for its effect on wound heal-

ing. It was proposed it might be beneﬁcial during early

stages of inﬂammation rather than during later stages

of remodelling (Talwar et al., 1996). Silver has also

been shown to aid healing in sterile skin, although its

mechanism of action is unknown (Lansdown et al.,

1997). Topical estrogen has also been shown to ac-

celerate cutaneous wound healing especially in aged

humans associated with altered inﬂammatory response

(Ashcroft et al., 1999a). Arnebin-1, a natural product

isolated from Arnebia mobilis, has been shown to ac-

celerate normal and hydrocortisone induced impaired

wound healing (Sidhu et al., 1999a). Curcumin (difer-

uloyl methane), a neutral product from rhizomes of

Curcunia procera, also showed enhanced wound heal-

ing (Sidhu et al., 1999b). The healing potential of

Calotropis procera is also being evaluated (Rasik et

al., 1999).

L-lysine HCl has shown remarkable improvement

in both the rate and quality of wound healing. The

molecule gives qualitatively better and much quicker

wound healing with less scar and deformation in

clean-cut model. Another remarkable feature of the

molecule is its ability to support healing process in

really protracted wounds, e.g. in leprotic ulcer, long

standing diabetic foot ulcer, etc.

On histopathology, lysine treated wounds showed

a remarkable thickening of the dermo-epidermal layer,

suggesting increased cell proliferation from the basal

layer (Keratinocytes). From

C-glycine incorpora-

tion experiments, it was evident that in experimental

wounds, nonspeciﬁc ﬁbrous tissue formation and lay-

ing in and around the immediate vicinity of the

wounds was less than the controls although both the

experimental and the control wounds showed reas-

onable collagen laying in response to the wounds.

Collagen synthesis in free skin of the same animals,

away from the wound sites, was comparable in both

the groups.

Topically lysine treated wound sites (chronic

wound beds), showed a controlled degree of inﬂam-

mation and angiogenesis process whereby, possibly,

required extent of cellular and serum growth factors

entry to the wound beds were ensured, thereby aug-

menting the healing process in situ.

It was postulated that lysine probably acts as a non-

speciﬁc binding molecule between the cell and it is a

growth factor thereby promoting increased cell prolif-

eration (Figure 1). This enhanced cell proliferation is

probably similar to that observed in the scarless foetal

repairs where, because of extremely high rate of cell

proliferation, probably, the wounds normally get more

ﬁlled up with cellular mass rather than by matrix ma-

terial (Datta et al., 2000b, 2001; Datta, 2000; Datta

and Kundu, 1999) with subsequent remodelling.

This contrasts sharply with the current trends in

the development of wound healing products and pro-

cesses: (i) where, mostly, one growth factor (Regranex

from J&J) is delivered to the wound site or (ii) where

one particular cell type (platelet) is delivered to the

wound bed (e.g. from Cytomedix Inc.) or (iii) where

the in vitro process of replacement material devel-

opment is elaborate time consuming and costly (e.g.

Apligraf from Organogenesis Inc.). The unique aspect

of the wound healing property of the molecule (and

its derivatives) lies in the fact that it does not interfere

in the fundamental process of healing and helps the

process peripherally by augmenting angiogenesis.

Angiogenesis

Therapeutic angiogenesis is being attempted in an-

imal model by exogenously added vascular endothelial

growth factor (VEGF) and other angiogenic growth

factors. Therapeutic efﬁcacy of simply injecting na-

ked plasmid DNA or proteins into ischemic tissue

to deliver secreted angiogenic factors holds promise

(Marty and Risau, 1999). Focal angiogen therapy us-

ing intramyocardial delivery of an adenovirus vector

coding for VEGF-121 has been shown to induce fo-

cal angiogenesis sufﬁcient to normalise blood ﬂow to

ischaemic myocardium (Lee et al., 2000). Transfec-

tion of human HGF gene into infarcted myocardium

has been shown to induce a beneﬁcial response to

the decreased endogenous HGF, thereby causing an-

giogenesis (Aoki, 2000). A single intrapericardial

injection of FGF-2 in a porcine model of chronic

myocardial ischaemia has been shown to cause func-

tionally signiﬁcant angiogenesis without any adverse

outcomes (Laham et al., 2000). PR39 and related

compounds are claimed to be potent inducers of an-

giogenesis by selectively inhibiting inducible factor-1

alpha (Li et al., 2000).

Gene therapy to stimulate angiogenesis is sup-

posed to be an effective way of bypassing occluded

arteries (Isner, 1999). Direct in vivo gene transfer us-

ing a replication deﬁcient adenovirus vector has been

carried out in an animal model. It has shown sustained

and localised expression of a potent angiogenic medi-

ator (Magovern, 1996). Autologous transplantation of

bone marrow cells improved damaged heart function

by inducing angiogenesis (Tomita et al., 1999).

Local injection of angiogenic growth factors into

the ischemic site have been attempted by various

routes like catheter based transendocardial injection,

intrapericardial delivery etc. (Kornowski et al., 2000).

Intramyocardial injection of bFGF has been shown to

salvage myocardium in the border zone (Aaregawa,

1999). Transmyocardial laser treatment has been

shown to cause angina relief by destruction of myocar-

dial peripheral nerve endings and improving perfusion

through induction of angiogenesis (Tjomsland, 1999).

On histopathology, topical application of L-lysine

HCl has been been shown to induce profound an-

giogenic response in cutaneous acute and chronic

wounds. Moreover L-lysine HCl supports maximum

cellular growth and expansion in serum containing

media than in a serum-free media suggesting an action

mediated through growth factors (Datta and Kundu,

1999; Datta et al., 2000a, b, 2001; Datta, 2000).

Given that the molecule is basically a C-5 species

with a chiral carbon (with an attached amino group

and linear R-group of 4 carbon chains with terminal

amino group), the amino acid should work as a non-

speciﬁc binding/attachment molecule between any cell

and it is dedicated growth factor. Exactly this has

been observed in all cell types – both in suspen-

sion and adherent cultures as well as in vivo repair

wounds and inducing angiogenesis. Lysine mediated

angiogenesis is postulated to be an end result of the

molecule acting as a cell surface bridge binding the an-

giogenic factors to their receptors (Figure 1). It would

be interesting to study details of angiogenic response

in ischaemic myocardium where normally angiogenic

factors are available at a higher concentration than ad-

equately perfused myocardium (Laham et al., 1999,

2000). Given that the basic molecular mechanism of

angiogenesis is universal and involves binding of the

angiogenic factors to their receptors, the much aug-

mented angiogenesis induced by the molecule may be

the result of ligand-receptor binding process (Figure 1)

(angiogenic factors and their receptors) getting more

stable, widespread and predictable under the inﬂuence

of this ‘new molecular bridge’ compared to the normal

stochastic process.

Food additives

Food additives in a broad sense just means any sub-

stance added to food. It refers to any substance, the

intended use of which results or may reasonably be

expected to result directly or indirectly in its becoming

a component or otherwise affecting the characteristics

of any food. This deﬁnition includes any substance

used in the production, processing, treatment, pack-

aging, transportation or storage of food. If a substance

is added to a food for a speciﬁc purpose in that food,

it is referred to as a direct additive. For example,

the low-calorie sweetener aspartame, which is used in

beverages, puddings, yogurt, chewing gum and other

foods, is considered a direct additive. Some addit-

ives are manufactured from natural sources such as

soybeans and corn, which provide lecithin to main-

tain product consistency, or beets, which provide beet

powder used as food colouring. Other useful additives

are not found in nature and must be man-made. Ar-

tiﬁcial additives can be produced more economically,

with greater purity and more consistent quality than

some of their natural counterparts.

Prebiotic agents are food additives which mainly

consists of oligosaccharides and dietary ﬁbres, mainly

inulin, and are potentially beneﬁal in a number of

ways: 1) potential protective effects against colorectal

cancer and infectious bowel diseases by inhibiting

putrefactive bacteria (Clostridium perfringens)and

pathogen bacteria (Escherichia coli, Salmonella, Lis-

teria and Shigella), respectively; 2) improvement of

glucose and lipid metabolisms; 3) ﬁbre-like properties

by decreasing the renal nitrogen excretion; 4) im-

provement in the bioavailability of essential miner-

als; and 5) low carcinogenic factor (Grizard and

Barthomeuf, 1999).

Colour additives like sulﬁting agents are some-

times used to preserve the colour of foods such as

dried fruits and vegetables, and to inhibit the growth

of microorganisms in fermented foods such as wine.

They are also sometimes used in baked goods, condi-

ments, snack foods and other products.

Erythorbates are food ingredients that are used to

inhibit the change of ﬂavour and colour in food when

exposed to air, such as when a cut apple is exposed to

air. Produced from sugar, erythorbates are similar in

chemical structure to vitamin C. Two forms of eryth-

orbates, erythorbic acid and sodium erythorbate, are

commonly used in hot dogs, luncheon meats, cured

meats, fresh pork, poultry, frozen bananas, dehyd-

rated apples and other foods (Food Additives, 1994,

in cooperation with Food and Drug Administration).

Various kinds of food additives are supplemen-

ted due to some beneﬁcial response either in certain

diseased states or normally to increase growth and

performance. For example: Aminoleban EN contains

branched-chain amino acids and is known to be bene-

ﬁcial for the protein-energy malnutrition in cirrhotic

patients (Meng et al., 1999). Cardiohel (Inrich pro-

duction) which is a biologically active food additive

has been recommended in Ischemic heart disease be-

cause it allows to reduce the dose of hypotensive and

coronarolytic drugs by 35% and cardiac glycosides

by 25%, thus lowering the risk of relevant side ef-

fects (Ivashkin et al., 1999). It has been proposed to

fortify certain grain-based products with Iron EDTA

to increase the bioavailability of iron in human diets

and thereby improve the iron status (Heimbach et al.,

2000). Nutritive factors like vitamin A and retinoids,

vitamin D and selen are used due to important anticar-

cinogenic properties they have. The phytochemicals

promise a lot in anticarcinogenesis to prevent cancer.

Spice extracts act antioxidatively by food supplement-

ation, and turmeric extract has been shown to have

the ability to prevent the deposition of triacylglycerols

in the liver (Sreter, 1999). Food such as blueberries

and spinach which are high in antioxidant capacity

have been examined for their effects to prevent and/or

reverse age-related declines in cerebellar noradrener-

gic receptor function (Bickford et al., 1999). Mixture

of amino acids has been seen to act on the pituitary

to enhance the adrenocorticotropic hormone (ACTH),

lutenizing hormone (LH), and follicle-stimulating hor-

mone (FSH) response to corticotropin-releasing hor-

mone (CRH) + gonadotropic hormone-releasing hor-

mone (GnRH) (di Luigi et al., 1999). Ingestion of

carbohydrate/protein before and after exercise and

dietary supplementation of various nutrients e.g. pro-

tein, glutamine, branched chain amino acid, creatine,

leucine etc. have been purported to promote gains

in fat free mass during resistance training. Creat-

ine and calcium beta-hydroxy beta-methyl-buthylate

(beta-HMB) supplementation during resistance train-

ing have been reported to increase fat-free mass in

athletic and nonathletic populations (Kreider, 1999).

Simplesse, a fat replacement (GRAS status conﬁrmed

by FDA in 1990) made from milk or egg white pro-

tein and olestra, which would partially replace fat have

been evaluated.

Food additives have a great role to play in livestock

production and animal feed industry. Added dietary

pyridoxine has been shown to improve weanling pig

growth performance (Woodworth et al., 2000). Oleam-

ide has been reported to elevate milk oleic acid when

fed to lactating Holstein cows (Jenkins, 2000). Cows

fed diets supplemented with ruminally protected lys-

ine and methionine showed increase in percentage of

milk protein but no effect on production of milk, milk

fat, and milk lactose. Infusion of isoleucine resulted in

a higher milk lactose proportion and tended to produce

more milk and milk lactose (Robinson et al., 1999).

Alpha-ketoglutarate, precursor of glutamine, in com-

bination with ornithine has been suggested to improve

gut morphology and functions and counteract trauma-

induced dysimmunity and exert anabolic/catabolic ac-

tions on protein metabolism. In combination they

increase the synthesis of arginine, proline and polyam-

ines that play key roles in metabolic adaptation to

trauma (Cynober, 1999). Somatotropin has also been

shown to improve body weight gain and feed efﬁ-

ciency (Tripp et al., 1998). Xylitol diet fed partially,

also signiﬁcantly prevented the reductions in body

weight gain and food intake without affecting the

early stage of inﬂammatory responses (Takahashi et

al., 1999). Food additive ascorbic acid has been shown

to have a protective effect against bacterial and viral

diseases due to its effect on heterophil function (An-

dreasen and Frank, 1999). A previous study has shown

that diet supplementation with thioproline, an intracel-

lular sulfhydril antioxidant and free radical scavenger

stimulates lymphocyte functions. It has been shown

to enhance immune function whenever it is depressed

(Correa, 1999). Arginine is said to hold a key po-

sition in the cellular functions and interactions that

occur during inﬂammation and immune responses. L-

arginine improves pain-free and total walking distance

in case of claudication in peripheral arterial disease

due to enhanced activity of endothelium derived nitric

oxide (Maxwell et al., 2000). Dietary supplementation

with L-arginine is said to modify reactivity of endothe-

lium and smooth muscle by at least two mechanisms:

one associated with activation of potassium channels

and the other with receptor-coupled release of nitric

oxide (Ray et al., 1999). Lysine in combination with

arginine is said to be used by bodybuilders for the

combination’s alleged effect of stimulating the re-

lease of growth hormone. According to the European

Federation of Animal Feed Additive Manufacturers

(FEFANA), increased use of amino acids combined

with reduced crude protein levels in feeds is estimated

to have the potential to reduce nitrogen excretion by

up to 20–25%. This is seen as one of the major op-

portunities for reducing nitrogen pollution. Published

work by Brudevold and Southern (1994) showed, in

a series of ﬁve experiments, that diets containing

12% crude protein but supplemented with crystalline

lysine, threonine, glutamic acid, methionine, histid-

ine, isoleucine, tryptophan and valine, were able to

support similar performance in 10–20 kg pigs to con-

ventional control diets containing between 19–22%

crude protein.

Animals in which high rates of lean tissue growth

are achieved through either genetic selection or the

application of growth hormones and repartitioning

agents, lysine requirements are substantially higher

(Goodband, 1990). In a report by Easter (1994) it has

been shown that the level of dietary protein in a stand-

ard maize soybean meal can be reduced by as much as

four percentage units if the diet is adequately fortiﬁed

with lysine, tryptophan and threonine.

Nutritional supplementation with lysine is also re-

commended in case of herpes simplex infection. The

amino acid lysine, in the amount of 1–3 g per day, is

effective in inhibiting the recurrence of herpes simplex

infections in some individuals (Flodin, 1997).

Food preservatives

A long shelf life, antimicrobial properties, no adverse

reactions, harmless metabolites are some of the qual-

ities desired of a food preservative agent. Though

currently many are in use, each is beset with a draw-

back of its own. Hence the search for an ideal and may

be natural preservative continues.

The use of food preservatives, such as benzoic

acid, nitrites, and sulphites, as antimicrobials, and

butylated hydroxyanisole (BHA), butylated hydroxy-

toluene (BHT), ascorbic acid and tocopherols, as

antioxidants, has probably changed food production

patterns and eating habits more than has the use of any

other class of food additive. These food preservative

chemicals confer substantial beneﬁts on man, not only

by the preservation and increased palatability of food,

but also by affording protection against the patholo-

gical effects of reactive oxygen species (ROS) which

are associated with cancer, cardiovascular disease and

aging. Nevertheless, although most preservatives are

now considered to be without potential adverse effects

there have been problems concerning the safety of

some of these chemicals. Benzoic acid and sulphites

probably cause allergies. The formation of nitrosam-

ines from nitrites, which are carcinogenic, and the

possible rodent carcinogenicity of BHA and BHT have

cast a shadow over the safety of these agents (Parke

and Lewis, 1992).

BHT and BHA antioxidants are capable of ag-

gravating symptoms in certain patients with chronic

urticaria (Goodman et al., 1990). The metabol-

ites of BHT, 2,6-di-tert-butyl-p-benzoquinone (BHT-

quinone), 2,6-di-tert-butyl-4-hydroperoxyl-4-methyl-

2, 5-cyclohexadienone (BHT-OOH), and 3,5-di-tert-

butyl-4-hydroxybenzaldehyde (BHT-CHO) are be-

lieved to be carcinnogenic. BHT-OOH participates

in oxidative DNA damage directly, whereas BHT-

quinone causes DNA damage through H

genera-

tion, which leads to internucleosomal DNA fragment-

ation (Oikawa et al., 1996).

Weak organic acid food preservatives are shown

to exert a strong pro-oxidant action on aerobic yeast

cells. In addition these acids are mutagenic toward the

yeast mitochondrial genome, even at levels that are

subinhibitory to growth. This has raised the concern

that the large-scale consumption of these preservatives

in the human diet may generate oxidative stress within

the epithelia of the gastrointestinal tract (Piper, 1999).

Animal and in vitro studies have shown that vit-

amins C and E can effectively inhibit the formation

of carcinogenic nitrosamines. Vitamin C supplement-

ation has been reported to inhibit skin, nerve, and lung

and kidney carcinogenesis. Vitamin E has been shown

to inhibit skin, liver, oral, ear duct, and forestom-

ach carcinogenesis. Several suggested mechanisms of

action include modiﬁcation of the metabolism of poly-

cyclic hydrocarbons, reduction of mutagenic activity

and reaction with genotoxic free radicals (Chen et al.,

1988).

Parabens, methyl, ethyl, propyl, benzyl, and butyl,

are the most common preservatives in use today. They

are the alkyl esters of p-hydroxybenzoic acid and are

used extensively because they are relatively nonirritat-

ing and nontoxic and offer good antimicrobial cover-

age (Pederse et al., 2000). However they have been

shown to evoke an oestrogenic response in vivo,in

sexually immature rainbow trout. Also, isolated cases

of allergic contact dermatitis have also been reported.

But considering their high efﬁcacy and favourble pre-

servative proﬁle they are still the most preferred food

preservatives (Mowad, 2000).

Nisin and pediocin PA-1 are examples of bacteri-

ocins from lactic acid bacteria (LAB) that have found

practical applications as food preservatives. Like other

natural antimicrobial peptides, LAB bacteriocins act

primarily at the cytoplasmic membranes of suscept-

ible microorganisms. The dissipation of proton motive

force is the common mechanism for the lethal activity

of LAB bacteriocin. It has been proposed that nisin

forms poration complexes in the membrane through

a multistep process of binding, insertion, and pore

formation (Montville and Chen, 1998). Combination

of sodium lactate and nisin was particularly effect-

ive in reducing total bacterial counts in this food

product. It also appears that this combination provides

an increased protection against common pathogenic

contaminants of fresh pork sausage, i.e. Staph. aureus

and Salmonella species (Scannell et al., 1997).

Interactions of monolaurin, eugenol (phenolic

compound) and sodium citrate (chelator) on the

growth of common meat spoilage (Lactobacillus

curvatus, Lactobacillus sake, Leuconostoc mesen-

teroides, Brochothrix thermosphacta) and pathogenic

(Escherichia coli O157:H7 and Listeria monocyto-

genes) organisms were investigated. It was shown that

the combinations of these chemicals were required to

have any potent effect. The presence of sodium cit-

rate was necessary to yield potent inhibition of Lb.

curvatus and Lb. sake growth by the monolaurin and

eugenol combinations (Blaszyk and Holley, 1997).

The effects of 5% potassium sorbate (PS) and 3%

lactic acid (LA) applications on total mesophylic aer-

obic bacteria, total psychrotrophic aerobic bacteria,

lactic acid bacteria, staphylococci and coliform bac-

teria, pH values, thiobarbituric acid (TBA) numbers,

and sensorial properties of vacuum-packed chicken

leg and breast meats have been investigated during

storage at 4±1

◦

C. In addition, residual sorbate was

examined. A decrease in bacterial counts of chicken

leg and breast meats was observed in the periods fol-

lowing the treatments of PS and LA; however, towards

the end of the storage period, the effectiveness of PS

was greater than that of LA. Quantities of sorbic acid

found in the samples treated with PS were below the

Acceptable Daily Intake established by the Food and

Agriculture Organization/World Health Organization

(Kolsarici and Candogan, 1995).

Welsh onion ethanol extracts were tested for their

inhibitory activity against the growth and aﬂatoxin

production of Aspergillus ﬂavus and A. parasiticus.It

was seen that the survival of spores of A. ﬂavus and

A. parasiticus depended on both the extract concentra-

tion and the exposure time of the spores to the Welsh

onion extracts. The extracts have been shown to inhibit

mycelial growth of two tested fungi cultured on yeast

extract-sucrose broth. They also inhibited aﬂatoxin

production in the culture at a concentration of 10 mg

−1

or permitted only a small amount of aﬂatoxin

production with extract concentration of 5 mg ml

−1

after 2 weeks of incubation. Welsh onion ethanol

extracts showed more pronounced inhibitory effects

against the two tested aﬂatoxin-producing fungi than

did the same added levels of the preservatives sorbate

and propionate at pH values near 6.5. Thus they ap-

pear to potent biological preservative agents (Fan and

Chen, 1999).

Lysine too has been found to play an interesting

role as a preservative. Production of toxins A and B by

Clostridium difﬁcile is enhanced in a deﬁned medium

with biotin-limited conditions. Asparagine, glutamic

acid and glutamine (10 mM) showed an effect on

growth and toxin production similar to that of bi-

otin. Lysine (10 mM) suppressed growth and inhibited

toxin production. Addition of these toxin-inhibitory

compounds within an incubation period of 2 days in-

hibited the enhanced toxin production, but later addi-

tion showed only slight inhibition of toxin production.

Amino acids contained in the deﬁned medium under

the biotin-limited conditions were actively utilised in

the presence of the three toxin-inhibitory amino acids,

but in the presence of lysine, amino-acid utilisation

was suppressed (Yamakawa et al., 1998). Homocitrate

synthase is the ﬁrst enzyme of the lysine biosynthetic

pathway. It is feedback regulated by L-lysine. The ef-

fect of lysine inhibition was investigated in the strain

Penicillin chrysogenum. Lysine decreases the biosyn-

thesis of penicillin (determined by the incorporation of

[

C] valine into penicillin) by inhibiting and repress-

ing homocitrate synthase, thereby depriving the cell

of alpha-aminoadipic acid, a precursor of penicillin.

Lysine feedback has been shown to inhibit in vivo the

biosynthesis and excretion of homocitrate by a lysine

auxotroph, L2, blocked in the lysine pathway after

homocitrate. The molecular mechanism of lysine feed-

back regulation in Penicillium chrysogenum involves

both inhibition of homocitrate synthase activity and re-

pression of its synthesis. In vitro studies indicated that

L-lysine feedback inhibits and represses homocitrate

synthase both in low- and high-penicillin-producing

strains (Luengo et al., 1980).

Thus it is seen that lysine has remarkable antimi-

crobial properties. Since lysine is an amino acid, the

chances of any immunological response against it are

negligible. These qualities of lysine can be employed

to use it as an effective food peservative.

Adjuvants

Immunological adjuvants are agents that enhance spe-

ciﬁc immune responses to vaccines. The capacity to

identify the nature and form of antigenic epitopes

in proteins allows the speciﬁc design of vaccines to

promote relevant protective immune responses. Such

vaccines, although ideal in terms of speciﬁcity and

purity, may not always achieve the desired levels of

protection through failure to reach relevant cells of

immune system due to simple dilution, elimination

by host enzymes or lack of speciﬁc targeting. Many

vaccines, currently under development and testing, are

composed of synthetic, recombinant, or highly puri-

ﬁed subunit antigens, which are often considered to

be safer than whole-inactivated or live-attenuated vac-

cines. Concomitant with the above there has been de-

velopment of a plethora of adjuvants aimed at enhan-

cing responses to these ‘new’ immunogens (Mrsny,

1998). Parallely, there has been an almost equal rapid

enhancement in understanding the complex nature of

the immune learning response, particularly with re-

spect to antigen processing, the nature and role of

cytokines and the importance of dendritic cells and

T-cell subsets in protection from infections.

Despite substancial progress in the areas of ba-

sic and applied immunology over the past couple of

decades, and drastic modiﬁcations being introduced

to the approach of vaccination, the area of vaccino-

logy is probably facing a formidable and extremely

fundamental requirement of a biocompatible adjuvant.

Although a number of them are available, none really

ﬁts the bill of human use, ideally.

Adjuvants can be grouped according to their phys-

ical characteristics and mode of action. They include

particulate adjuvants, oil and emulsiﬁer-based ad-

juvants, adjuvants providing controlled antigen deliv-

ery, adjuvants based on speciﬁc targeting of antigen,

and gel-type adjuvants (Jennings et al., 1998). They

may act non-speciﬁcally in promoting an immune re-

sponse to an antigen through depot formation, or very

speciﬁcally as in a ‘delivery system’ where an anti-

gen is linked to a cellular protein, or targeted to a

speciﬁc cell receptor. It has also become necessary

that these differing approaches be combined, and an

adjuvant/delivery system designed, to provide slow

release of a targeted antigen (Jennings et al., 1998).

Adjuvants have diverse mechanisms of action and

should be selected for use, based on the route of

administration and the type of immune response (anti-

body, cell-mediated, or mucosal) desired for a partic-

ular vaccine. As mentioned, adjuvant mechanisms of

action include: (i) increasing the biological or immun-

ological half-life of vaccine antigens; (ii) improving

antigen delivery and presentation; and (iii) inducing

the production of immunomodulatory cytokines (2).

Conventionally the efﬁciency of an adjuvant used to be

measured by the capacity to induce enhanced antibody

titres in serum and cell mediated immunity (CMI) to

a given antigen. Recently, the capacity of an adjuvant

is also measured by the quality as well as the mag-

nitude of the induced immune response, where quality

includes isotype and IgG subclass responses, T-helper

cell responses characterized by the cytokine proﬁles

and extent of cytotoxic T cell (CTL) induction. In

the early phase of immunization some adjuvants in-

ﬂuence the antigen administration and uptake by a

so-called depot effect exempliﬁed by aluminium hy-

droxide gel and oil adjuvants, which possibly is not

as desired as alledged. A ‘modern’ depot is exerted

by slow release formulations, continuously releasing

the antigen over a period of time at a steady rate

or by pulses at intervals, although being basically a

‘single injection’ vaccine. Extensive efforts are put

in to formulate efﬁcient delivery combinations target-

ing the antigens from the site of administration to

draining lymph nodes or distant lymphatic tissue or

to mucosal surfaces by parenteral or mucosal admin-

istrations. Nowadays, non-replicating carriers besides

replicating vaccines are formulated to induce mucosal

immune responses encompassing secretory IgA and

CMI (Morein et al., 1996). Efforts to evoke immune

responses on mucosal membranes distant from the site

of administration have resulted mostly in little suc-

cess (Morein et al., 1996). For a long time it was

considered that CTL under the restriction of MHC

Class I only could be evoked by replicating viruses or

intracellular parasites (Morein et al., 1996). However,

novel adjuvant delivery systems readily induce CTL

by delivering the antigen to the APC resulting in intra-

cellular transport to the cytosol for the MHC Class I

presentation system, as well as to the endosomal path-

way for the MHC Class II presentation (Morein et al.,

1996).

Infant immunization is another particularly import-

ant area with multiple challenges for vaccine research

and development. There is, together with a high sus-

ceptibility to infections, a lower efﬁcacy of most vac-

cinations in newborns and young infants, compared to

those administered later in life. Hence adjuvants play

an important role to: (i) rapidly induce strong anti-

body responses of the appropriate isotypes; (ii) elicit

sustained antibody responses extending beyond in-

fancy; (iii) induce efﬁcient Th1 and CTL responses

in spite of the preferential Th2 polarization (in early

life responses); (iv) escape from maternal antibody

mediated inhibition of vaccine responses; (v) show

acceptable reactogenicity in early life; and (vi) allow

incorporation of several vaccine antigens into a single

formulation so as to reduce the number of required

injections (Kovarik and Siegrist, 1998).

Currently, aluminum salts and MF59 are the only

vaccine adjuvants approved for human use. Of the

novel compounds recently evaluated in human trials,

immunostimulatory molecules such as lipopolysac-

charide (LPS) derived monophosphoryl lipid (MPL)

and saponin derivative QS21 appear most promising,

although doubts have been raised as to their safety

in humans (Singh and O’Hagan, 1999). Preclinical

work with particulate adjuvants, such as the MF59

microemulsion and lipid-particle immune-stimulating

complexes (Iscoms), suggest that these molecules are

also potent elicitors of humoral and cellular immune

responses (Singh and O’Hagan, 1999). In addition,

preclinical data on CpG oligonucleotides appear to be

encouraging, particularly with respect to their ability

to selectively manipulate immune responses (Singh

and O’Hagan, 1999).

MF59, an adjuvant approved for human use, typ-

ically elicits higher antibody titers than alum when

used in combination with a variety of recombinant and

natural subunit antigens. The mechanisms responsible

for the adjuvant action of MF59 however are not fully

understood (Dupuis et al., 1999).

MPL has been shown to be a promising adjuvant

due to the following advantages a) MPL retains the

useful immunostimulating activities of the parent LPS

molecule, but with greatly attenuated toxicity; b) pro-

duces diverse effects on the cellular elements of the

immune system, including macrophage activation and

T and B cell interaction, with concomitant cytokine

and lymphokine release; c) proven adjuvant activity,

in both the cellular and humoral effector arms of im-

munity; d) adjuvant activity when used alone, or in

combination with other immunostimulants and deliv-

ery vehicles; and e) safe to humans (Ulrich and Myers,

1995).

Two novel oil adjuvant vaccines, Montanide ISA

25 and 206 are claimed to offer a number of advant-

ages over Al(OH)

, particularly in their ability to raise

better immunity in pigs. Results indicate that vac-

cines, in presence of these adjuvants retain potency

for a longer period and elicit good antibody responses

in both pigs and cattle regardless of injection route

without evidence of toxicity (Barnett et al., 1996).

It has been seen that formulation of a DNA vaccine

encoding hepatitis B surface antigen with calcium-

or aluminum phosphate adjuvants, increases antibody

titers by 10 to 100-fold and decreases the immuno-

genic dose of DNA by 10-fold. Furthermore, boosting

an HBs protein-primed response with the adjuvanted

DNA vaccine resulted in a dramatic increase in the

HBs-speciﬁc IgG2a response reﬂecting a shift to-

wards a TH1 response. It has been suggested that the

mechanism by which aluminum phosphate exerts its

adjuvant effect is not through increased expression of

HBsAg in vivo but by increasing the number and af-

ﬁnity of HBs peptide antigen-speciﬁc IFN-gamma and

IL-2 secreting T cells (Wang et al., 2000).

Microﬂuidized squalene or squalane emulsions act

as efﬁcient adjuvants, eliciting both humoral and cel-

lular immune responses. Squalene or squalane emul-

sions have been administered in human cancer vac-

cines, with mild side effects and evidence of efﬁcacy,

in terms of both immune responses and antitumor

activity (Allison, 1999).

Water-soluble fullerene derivatives have been sug-

gested as prospective adjuvants due to their immunos-

timulating effect (Masalova et al., 1999).

The role of Bacillus Colmette-Guerin (BCG) as

an adjuvant in autologous tumor vaccines has been

examined. BCG results in increased VPLN (vaccine

primed lymph node) cell yield as well as enhanced

type 1 (IFN-gamma release) immune responses of

VPLN cells to autologous tumor without upregulating

type 2 (IL-10 release) response (Li et al., 2000).

Experiments demonstrate that heat killed lysteria

as an adjuvant for immunotherapy mediates im-

mune deviation from a pathological Th2-dominated

response toward a protective immune response in peri-

pheral lymphoid tissues and in the lungs and may be

clinically effective in the treatment of patients with es-

tablished asthma and allergic diseases (Hansen et al.,

2000).

The limited availability of efﬁcient and non-toxic

adjuvants, capable of promoting mucosal responses,

presents with a problem in vaccinology. The poten-

tial usefulness of ﬁbronectin-binding protein I (Sfbl)

of Streptococcus pyogenes as immunological adjuvant

has been assessed using ovalbumin (OVA) as a model

antigen. Phenotypic analysis of proliferating cells

show enrichment in CD4

T cells, producing a pattern

of cytokines (IL-4, IL-5, IL-6 and IL-10) character-

istic of Th2-type cells. In contrast to immunization

with soluble OVA alone, OVA-Sfbl induced the gen-

eration of CD8

OVA-speciﬁc cytotoxic cells. These

results demonstrate that Sfbl represents a promising

mucosal adjuvant able to substantially improve cellu-

lar, humoral and mucosal responses when coupled to

an antigen administered by intranasal route (Medina et

al., 1998).

Norwegian outer membrane vesicle (OMV) vac-

cine against group B meningococcal disease has

proved to be strongly immunogenic when admin-

istered intranasally in mice. All vaccinees developed

marked increases in OMV-speciﬁc IgA antibodies in

nasal secretions. It is thus possible that a nasal OMV

vaccine may induce protection against invasive men-

ingococcal disease, and also that it might be used

as a vehicle for nasal vaccines against other diseases

(Haneberg et al., 1998).

Whole killed meningococci (Nm) and pertussis

bacteria (Bp) has been tested for mucosal immuno-

genicity and as mucosal adjuvants for an inactivated

inﬂuenza virus vaccine given intranasally to unan-

aesthetized mice. With Bp or Nm admixed, serum

IgG and IgA and salivary IgA responses to the inﬂu-

enza virus were substantially augmented (P < 0.005)

(Berstad et al., 2000).

A study has been carried out to demonstrate that

two mutants of Escherichia coli heat-labile toxin (LT),

LTK63, which lacks ADP-ribosylating activity, and

LTR72, which has partial enzyme activity, act as po-

tent mucosal adjuvants for the nasal delivery of an

acellular pertussis (Pa) vaccine (Ryan et al., 1999).

Synthetically prepared N-terminal parts of the lipo-

protein from Enterobacteria carrying three fatty acid

residues or lipopeptide analogs containing one to four

aminoacids bound to S-glycerylcysteine act as potent

immunoadjuvants in vivo in combination with or co-

valently linked to antigens. Thus, bacterial cell wall

components such as lipopolysaccharide, a variety of

membrane proteins, murein, and lipoprotein can act as

immunoadjuvants for bacterial vaccines, thus enhan-

cing protection from bacterial infections. The immun-

oadjuvant properties of the lipopeptides are mediated

by an enhancement of the humoral immune response

(Schlecht et al., 1993).

Though vaccination has been the most cost-

effective way of controlling infectious diseases, the

logistics of delivering at least two to three doses of

conventional vaccines for primary immunization to

achieve protection are difﬁcult and compliance is fre-

quently inadequate, particularly in developing coun-

tries. In recent years biodegradable polymer micro-

spheres have received much attention for the purposes

of controlled release of antigens: (i) to reduce the

number of doses needed for primary immunization

to as few as a single dose and (ii) to better-target an

antigen to microfold cells on mucosal surfaces after

oral administration or to antigen-presenting cells after

parenteral inoculations. A variety of vaccine antigens

have been encapsulated in microspheres usually com-

posed of poly (lactic/glycolic) acid (PLGA). Based

on the size of the microspheres, molecular weight of

polymer and ratio of lactic to glycolic acid in the poly-

mer, the antigen may be targeted to various cells of the

immune system or it may form a depot at the site of in-

jection, allowing slow release of the antigen for exten-

ded periods. Additionally, an adjuvant may be incor-

porated inside microspheres together with the antigen,

further enhancing or modulating the immune response

to the desired type. The major problems in developing

controlled-release vaccines include instability of vac-

cine antigens during micro-encapsulation, storage and

subsequent hydration (Gupta et al., 1998).

Prolonged presence of mIFN gamma at the site

of antigen presentation is crucial for the generation

of systemic immune responses in the B16 melanoma

model. Studies show that liposomal encapsulation of

cytokines proves to be an attractive strategy for parac-

rine cytokine delivery in tumor vaccine development

(Van Slooten et al., 2000).

Archaeosomes act as promising vaccine carriers

capable of facilitating strong primary and memory

responses, both in humoral and cell-mediated im-

munity sectors to entrapped antigens. In contrast,

conventional liposomes induced little cell-mediated

immunity, whereas alum stimulated only an IL-4 re-

sponse. In contrast to alum and Freund’s adjuvant,

archaeosomes composed of Thermoplasma acidophi-

lum lipids, evoked a dramatic memory antibody re-

sponse to the encapsulated protein (at approximately

300 days) after only two initial immunizations (days

0 and 14). This correlated with increased antigen-

speciﬁc cell cycling of CD4

T cells through increase

in synthetic (S) and mitotic (G(2)/M) phase and de-

crease in resting (G(1)) phases of CD4

cell cycle

(Krishnan et al., 2000).

One of the approaches of tumor vaccines is a mix-

ture of irradiated tumor cells with cytokine containing

liposomes. These vaccines are quite easy to prepare

and, in contrast to vaccines consisting of cytokine-

gene transfected-tumor cells, their composition (cell

dosage, cytokine dosage) can be easily varied. Vaccin-

ation efﬁciency depended on (a) the immunogenicity

of the tumor cells (b) vaccination frequency and (c) the

dose of the cytokine encapsulated in the admixed lipo-

some depots. Immunity to the tumors could be induced

only within a narrow cytokine-dose range (‘IL-2-dose

window’) (Krup et al., 1999).

Dendritic cells (DCs) have also been widely con-

sidered to be promising adjuvants for inducing im-

munity to cancer (Thurner et al., 1999). Studies using

mature, monocyte-derived DCs to elicit resistance to

malignant melanoma have been undertaken. In one

of the studies, DCs were pulsed with Mage-3A1 tu-

mor peptide and a recall antigen, tetanus toxoid or

tuberculin. This study proved the principle that DC

‘vaccines’ can frequently expand tumor-speciﬁc CTLs

and elicit regressions even in advanced cancer and, in

addition, provides evidence for an active CD8

CTL-

tumor cell interaction in situ as well as escape by lack

of tumor antigen expression (Thurner et al., 1999).

The co-delivery of inteferon (IFN)-gamma, inter-

leukin (IL)-12, and IL-18 genes along with DNA

vaccine constructs to engineer the immune response

in vivo towards more T-helper Th1-type cellular re-

sponses, has been investigated. Co-immunization of

IFN-gamma and IL-18 in macaques enhanced the level

of antigen-speciﬁc antibody responses. Similarly, co-

delivery of IL-12 and IL-18 also enhanced the level of

antigen-speciﬁc Th proliferative responses (Kim et al.,

1999).

The immunostimulatory sequences can also be

identiﬁed within a pleiotropic cytokine like IL-1

and used in the rational design of novel vaccination

strategies. The human interleukin-1beta (IL-1beta) do-

main in position 163-171, comprising the amino acids

VQGEESNDK, has been synthesized as a nine-amino-

acid-long peptide and used in vivo as a nontoxic

HCl salt. The IL-1beta nonapeptide reproduces im-

munostimulatory and adjuvant effects of the whole

mature IL-1beta, but does not possess any of the IL-

1beta inﬂammatory, vasoactive, tumor-promoting, and

systemically toxic effects, nor can it synergize with

tumor necrosis factor alpha or other molecules in in-

ducing toxicity and shock. Thus it acts as a promising

adjuvant (Boraschi and Tagliabue, 1999).

IL-12 has also been investigated as a promising

adjuvant. Its main effect is to drive Th-cell differen-

tiation throughout a T-helper type-1 response, thus

inducing interferon gamma (IFNgamma) and favor-

ing IgG2a. These properties make IL-12 a candidate

adjuvant for vaccination against cancer and infective

diseases. But, experience of some toxicity in humans

has hampered its further development into clinical

applications, which, however, are still possible if re-

stricted to local administration. Gene transfer has been

proposed to be the preferred approach to obtain local

release of cytokine (Rodolfo and Colombo, 1999).

Synthetic oligodeoxynucleotides containing CpG

motifs [immunostimulatory sequences (ISS)] have

been described as potent adjuvants of type 1 immune

responses when co-administered with protein or pep-

tide vaccines. ISS causes a rapid release of IL-12

and IFN-gamma in sera from treated mice. This data

provide a ﬁrst evidence for the ability of ISS to induce

an anti-CHO (polysaccharide) type 1-like immune re-

sponse and demonstrate that ISS have the potential

to increase host antibody response against both the

CHO and the protein component of a conjugated vac-

cine (von Hunolstein et al., 2000). Addition of CpG

ODN (Oligodeoxynucleotides) to hepatitis B vaccine

greatly increases the seroconversion rate and the titers

of antibody against HBsAg (anti-HBs). This is the ﬁrst

demonstration of CpG DNA in a great ape and the res-

ults have important implications for the vaccination of

humans against HBV and other diseases (Davis et al.,

2000).

Studies have indicated that professional APCs in

the periphery, such as dendritic cells and macro-

phages, play an important role in initiating DNA

vaccine-speciﬁc immune responses. To engineer the

immune response induced by DNA vaccines in vivo

the modulatory effects of codelivering growth factor

genes for the hematopoietic APCs along with DNA

vaccines was investigated. Speciﬁcally, the effects

on the antigen-speciﬁc immune responses follow-

ing the codelivery of the gene expression cassettes

for macrophage-colony stimulating factor (M-CSF),

granulocyte-colony stimulating factor (G-CSF), and

granulocyte macrophage-colony stimulating factor

(GM-CSF) along with HIV-1 DNA immunogen con-

structs were studied. It was observed that coimmuniz-

ation with GM-CSF increased the antibody response

and resulted in a signiﬁcant enhancement of lymph-

oproliferative response. Furthermore, among all coin-

jection combinations, M-CSF coinjections resulted in

a high level of CTL enhancement. This enhancement

of CTL responses observed from the coinjection with

M-CSF was CD8

T cell dependent and was associ-

ated with the presence of CD11c

cells at the site of

injection and with the antigen-speciﬁc induction of the

beta-chemokine MIP-1beta, suggesting a role for this

chemokine in CTL induction. These results suggest

that hematopoietic growth factors should be further

studied as potential adjuvants for in vivo modulators

of immune responses (Kim, 2000).

In the study that is being carried in the authors’

laboratory, monomeric lysine has been observed to

augment the Ig responses irrespective of the type and

size of the antigens. Heat killed Mycobacterium tuber-

culosis was observed to give a massive augmentation

of Ig responses reproducibly in the presence of the

monomeric aminoacid. BCG strains were also remark-

ably potentiated in their ability to induce humoral

immunity. Few other antigens are being studied at

the moment along with characterisation of the elicited

humoral and cellular types of immunity including

characterisation of the antibodies and cells (mostly

CD4

and CD8

) generated.

Antimicrobial peptides

Many peptides have antimicrobial activity. They en-

compass a wide variety of structural motifs. The ma-

jority of these peptides are cationic and amphipathic

but there are also hydrophobic alpha-helical peptides,

which possess antimicrobial activity. In addition, some

beta-sheet peptides have antimicrobial activity and

even antimicrobial alpha-helical peptides which have

been modiﬁed to possess a beta-structure retain part

of their antimicrobial activity. There are also anti-

microbial peptides, which are rich in certain speciﬁc

amino acids (for example shepherin I and shepherin

II, glycine- and histidine-rich peptides) (Park et al.,

2000). There are antimicrobial peptides which are

lipopeptides (for example Daptomycin) (Hodinka et

al., 1987). In spite of the structural diversity, a com-

mon feature of the cationic antimicrobial peptides is

that they all have an amphipathic structure which al-

lows them to bind to the membrane interface (Epand

and Vogel, 1999). Indeed, most antimicrobial peptides

interact with membranes and may be cytotoxic as a

result of disturbance of the bacterial inner or outer

membranes (Epand and Vogel, 1999). Alternatively, a

necessary but not sufﬁcient property of these peptides

may be their ability to pass through the membrane to

reach a target inside the cell (Epand and Vogel, 1999).

The interaction of these peptides with biological mem-

branes is not just a function of the peptide but is also

modulated by the lipid components of the membrane.

It is not likely that this diverse group of peptides has

a single mechanism of action, but interaction of the

peptides with membranes is an important requirement

for most, if not all, antimicrobial peptides (Epand and

Vogel, 1999).

It has been seen that thiazole and oxazole contain-

ing amino acids like alanine, valine, proline, leucine

and alanine and some peptides containing the 5-ring

heterocyclic backbone modiﬁcations show moderate

antibacterial activity in vitro against various Gram-

positive (Staphylococcus aureus, Bacillus cereus,etc.)

and Gram-negative (Escherichia coli, Protens vul-

garis, etc.) bacteria, fungi (Candida albicans), and

yeast (Stanchev et al., 1999).

The derivatives of cholic acid with basic aminoacids

also show signiﬁcant antimicrobial activity, especially

marked when L-arginine is the condensed aminoacid

(Bellini et al., 1979).

Macrocyclic peptides possess speciﬁc and potent

antimicrobial activity that is salt dependent (Tam et al.,

1999). It has been suggested that their initial interac-

tions with the microbial surface may be electrostatic,

an effect commonly found in defensin antimicrobial

preptides. They have an end to end cyclic structure

with a cystine-knot motif. It has been suggested that

this might provide a useful template for design of

novel peptide antimicrobials (Tam et al., 1999).

Nisin, an amphiphilic peptide, shows a strong

antimicrobial activity against various Gram-positive

bacteria. Its activity results from permeabilization of

bacterial membranes, causing efﬂux of cytoplasmic

compounds. It has been suggested that pore formation

of nisin involves translocation of the C-terminal region

of the molecule across the membrane (Van Kraaij et

al., 1998).

A family of aminoacyl alkyl citrate compounds

called viridiofungins, which are novel squalene syn-

thase inhibitors, have broad spectrum fungicidal activ-

ity but lack antibacterial activity. Although the com-

pounds inhibit squalene synthase, the ﬁrst commit-

ted step in ergosterol biosynthesis, results presented

show that inhibition of fungal growth is not related to

inhibition of ergosterol synthesis (Onishi et al., 1997).

Novel pseudopeptides, corresponding to a mem-

brane active depsipeptide, show more resistance to

serum proteases than peptides and similar antimi-

crobial activities, without hemolytic activity. The

pseudopeptides were found to be active against cur-

rent drug resistant fungi and pathogenic fungi isolated

from patients, and also have a strong synergism with

current antifungal drugs against Candida albicans.

The leakage assays suggest that the pseudopeptides

act on the lipid membrane of pathogenic cells. Thus

it is believed that they would have advantage over the

peptide(s) as candidate(s) for novel antifungal(s) (Oh

et al., 1999).

Peptoids, which differ from peptides in having

a N-substituted rather than alpha-carbon-substituted

glycine units, have been evaluated for antimicrobial

activity. It has been seen in vitro that the peptoid

CHIR29498 and some of its analogues were active

in the range of 3 to 12 µmml

−1

against a panel

of gram-positive and gram-negative bacteria which

included isolates which were resistant to known an-

tibiotics. Beta-Galactosidase and propidium iodide

leakage assays indicate that the membrane is the most

likely target of activity. Positional isomers of an active

peptoid were also active, consistent with a mode of

action, such as membrane disruption, that does not re-

quire a speciﬁc ﬁt between the molecule and its target

(Goodson et al., 1999).

Calprotectin, a protein in neutrophil cytoplasm and

abscess ﬂuids, appears to inhibit microbial growth

through competition for zinc. It has been suggested

that calprotectin’s antimicrobial activity may be re-

lated to certain histidine-based zinc-binding sequences

(Loomans et al., 1998).

A novel 26-residue proline-rich immune-inducible

peptide, Metchnikowin, has been characterised from

Drosophila, which exhibits both antibacterial (gram-

positive) and antifungal activities (Levashina et al.,

1995).

The antibacterial effect of o-carboranylalanine (o-

Cba), a highly lipophilic analog of phenylalanine,

against some species is comparable to that of the

widely used agricultural antibiotic, streptomycin. This

carborane-containing amino acid is more toxic to

Gram-positive bacteria, than to gram negative ones.

Compared to the commercial fungicide, prochloraz, o-

Cba is found to be weakly toxic against various fungi

(Oros et al., 1999).

Two novel phenylalanine-rich antimicrobial pep-

tides, Styelin A and Styelin B, puriﬁed from the hemo-

cytes of Styela clava, are effective against a panel of

gram negative and gram positive bacterial pathogens

of humans, usually acting with minimal inhibitory

concentrations of <1.5 µgml

−1

(<0.5 µM), even in

the presence of 100 mM NaCl. The presence of anti-

microbial peptides (Styelins) in tunicate hemocytes is

evidence that such molecules are ancient mediators of

host defense within the vertebrate lineage (Lee et al.,

1997).

L-methionine has long been known to prevent re-

infection with chronic urinary tract infection. The

therapeutic result is essentially due to its inﬂuence on

bacterial cytoadherence (Funfstuck et al., 1997).

Dermaseptins s1, s2, s3, s4, and s5, a family

of cationic (lysine-rich), amphipathic antifungal pep-

tides of 28–34 residues, share a similar spectrum of

lytic activity against the ﬁlamentous fungi that are

responsible for opportunistic lethal infections that fol-

low the immunodeﬁciency syndrome or the use of

immunosuppressive agents. They exhibit marked dif-

ferences in their potencies to arrest the growth of

gram-positive and gram-negative pathogenic bacteria

and yeasts (Mor et al., 1994). The mechanisms of

antimicrobial actions of magainin 2, buforin II and

poly L-lysine against various Escherichia coli strains

have been studied. Poly L-lysine inhibits BL21, AD

434 and GroE

/DnaK

growth without lysing the

cell. Magainin 2 has a pore-forming activity on BL

21 and AD 434 membrane but could not inhibit

the GroE

/DnaK

growth in a nutrient-rich medium.

Buforin II, which kills BL21 and AD 434 without cell

membrane damage, lysed GroE

/DnaK

to death.

Once they were introduced into the cell by electropor-

ation, all three peptides were able to inhibit cell growth

at concentrations of 10 times lower than their MICs

(Liang and Kim, 1999). The antimicrobial activity of

racemomycin compounds tended to be stronger with

increase in the number of beta-lysine moieties in the

molecule (Inamori et al., 1990).

Novel unnatural amino acids with more positively

charged and bulky side chain group than lysine residue

were synthesised. The incorporation of this amino

acid increased the resistance of the peptide against

serum protease more than three times without a de-

crease in the antimicrobial activity. It has been sug-

gested that the amino acids synthesized in this study

could be used not only as a novel building block for

combinatorial libraries of antimicrobial peptides, but

also for structure-activity relationship studies about

antimicrobial peptides (Oh and Lee, 1999).

An amphiphilic, cationic peptide composed of

eight leucines and six lysines was synthesized by solid

phase peptide synthesis (SPPS) and tested against E.

coli O157:H7 grown in TSB. The peptide was bacter-

icidal and bacteriostatic at concentrations of 50 and

25 µgml

−1

, respectively. An inhibitory effect was

also observed against stationary phase cells. Intra-

cellular K

and ATP depletion were also observed.

These results suggest that the peptide increased the

cell membrane permeability but it did not lyse the cells

(Appendini and Hotchkiss, 1999).

It has been reported that a new group of diastereo-

mers of short-model peptides (12 amino acids long)

composed of leucine and lysine with varying ratios,

possess several properties that make them potentially

better than native or de novo-designed all L-amino

acid antimicrobial peptides. Preliminary studies have

revealed that modulating the hydrophobicity and pos-

itive charges of these diastereomers is sufﬁcient to

confer antibacterial activity and cell selectivity (Hong

et al., 1999).

Oxalysine, a novel anti-fungal antibiotic, isolated

from Streptomyces roseoviridofuscus showed a good

activity against Candida parapsilosis when compared

to Amphotericin-B and 5-Fluorocytosine. It was found

that oxalysine 0.4 mmol L

−1

did not signiﬁcantly in-

hibit their incorporations into protein and/or DNA, but

strongly inhibited the incorporation of (

C)-adenine

into RNA (Zhang et al., 1993).

L-lysine works as a good anti viral agent for herpes

simplex. Supplementation of l-lysine is one of the best

options available for the treatment of herpes simplex

virus infections, especially oral forms. L-lysine is also

much cheaper than antiviral drugs such as Acyclovir.

L-lysine supplementation works by tilting the balance

between lysine and arginine heavily in favor of lysine.

This ameliorates herpes outbreaks because the herpes

virus depends on the presence of arginine for its rep-

lication. Lysine, in the amount of 1–3 g per day, is

effective in inhibiting the recurrence of herpes simplex

infections in some individuals (Flodin, 1997).

Cell ﬁxatives

Most laboratories use formalin, glutaraldehyde, eth-

anol, paraformaldehyde and Bouin’s liquid for tissue

ﬁxation. Coagulative ﬁxatives are less popular. How-

ever, problems with formalin ﬁxation comprise delay

of ﬁxation and variations in the duration of the ﬁx-

ation mainly (Ezaki et al., 2000). Quantiﬁcation of

DNA extracted by microdissection of tissue sections

can be used for qualitative PCR analysis. Formalin

ﬁxation, before microdissection signiﬁcantly dimin-

ishes the amount of extractable DNA and may lead

to less reliable results, even of qualitative PCR ana-

lysis (Serth et al., 2000). Also a major artifact induced

by formaldehyde ﬁxation is the masking of tissue

antigens due to cross-linking of protein amino acid

residues (Ezaki et al., 2000). Bouin’s ﬂuid has been

shown to affect PCR analysis. The performance of

PCR has been shown to decrease for samples ﬁxed

in Bouin’s liquid for longer than 6 hr or after 48 hr

of incubation in a vacuum inﬁltration processor (in

which Bouin’s liquid-ﬁxed and formalin-ﬁxed samples

are mixed (Camilleri-Bruet et al., 2000). Glutaral-

dehyde affects lipids adversely. Classical procedure

i.e.glutaraldehyde ﬁxation followed by epoxy resin

embedding, results in the loss of 73–91% of the tissue

lipids (Maneta-Peyret et al., 1999).

But there is a better side to glutaraldehyde as a cell

ﬁxative. When gelatin ﬁlms were treated with glutaral-

dehyde (GA) solution at 60

◦

C, free aldehyde groups

were introduced in the ﬁlm. The bonding strength of

GA-crosslinked gelatin ﬁlms (GA gelatin ﬁlms) with

biological tissue was assessed using porcine skins. It

was found that bonding strength increased with in-

creasing aldehyde content in the ﬁlm (Matsuda et al.,

1999).

Ninety-ﬁve percent (95%) ethanol is the standard

cytological ﬁxative used in many laboratories. Com-

mercially available ethanol is expensive and not freely

available in some institutions. Methanol, a tissue de-

hydrant, is also known to be a cytological ﬁxative.

Methanol has been shown to be as effective as ethanol

for ﬁxation of smears and cheaper (Kumarasinghe et

al., 1997).

To establish a quantitative method for analysis of

gene expressions in small areas of tissue after par-

afﬁn embedding, preliminary validation experiments

with RT-PCR and Western blotting were performed

using methacarn-ﬁxed rodent tissues and a cultured

PC12 cell line. It was shown that in addition to its ad-

vantages for immunohistochemistry, methacarn-ﬁxed

parafﬁn-embedded tissue has beneﬁts for analysis of

both RNAs and proteins in the cells of histologically

deﬁned areas (Shibutani et al., 2000).

Adequate preservation of the cells and matrix

of mineralising tissues remains difﬁcult, as organic

components and initial mineral deposits may be lost

during conventional processing for electron micro-

scopy. An attempt has been made to decrease the pro-

cessing time by using microwave irradiation (MWI).

Rat molar tooth germs were ﬁxed using standard ﬁx-

atives in a microwave oven. Under electron micro-

scopy, differentiating ameloblasts and odontoblasts,

plasma membranes, mitochondria, rough endoplas-

mic reticulum, the Golgi complex, together with all

other cytoplasmic organelles showed excellent preser-

vation, especially microtubules, microﬁlaments and

coated vesicles. Crystal-like mineral deposits were

conspicuously present in relation to dentine matrix

vesicles and collagen ﬁbrils as well as in enamel mat-

rix. Thus this promises to be a fast and effective

method when mineralised tissue is involved (Massa

LF, Arana-Chavez et al., 2000). Even when applied in

immunohistochemistry, MWI greatly shortens the ﬁx-

ation, processing, and immunolabeling times without

compromising the quality of ultrastructural preserva-

tion and the speciﬁcity of labeling (Rangell and Keller,

2000).

Integral immunohistochemical analysis of immune

responses in frozen sections requires that, in addition

to constitutively expressed membrane CD markers,

less stable determinants should be reliably visualized.

Fixation with pararosaniline, as compared to acet-

one, resulted in better morphology of all tissues.

Out of a number of determinant-tissue combinations,

staining sensitivity and intensity were markedly in-

creased for selected determinant-tissue combinations,

e.g., for IL-4 in human spleen and CD40 in human

and mouse spleen. These data show that pararosanil-

ine is a useful alternative to acetone, resulting in

superior morphology and speciﬁc staining for selec-

ted determinant-tissue combinations (Schrijver et al.,

2000).

Acetone ﬁxation at –18

◦

C with subsequent em-

bedding in methyl-/butylmethacrylate is a reliable

method for the routine processing of bone marrow

biopsies. This method allows good conventional his-

tological visualization of morphological details, which

is comparable with other ﬁxation procedures. The es-

sential advantage of this method is that a wide range

of monoclonal antibodies and polyclonal antisera can

be used for immunohistochemical investigations for

diagnostic and scientiﬁc purposes. The addition of

5% polyethylene glycol 400 to the acetone minim-

izes freeze-related artefacts (Hantschick and Stosiek,

1998).

Poly-D-lysine (PDL) and Poly-L-Lysine (PLL) are

synthetic molecules which have been used to enhance

cell attachment to plastic and glass surfaces (McKee-

han, 1984). For many anchorage-dependent cells, the

nature of the culture substrate has a major effect on cell

growth and the requirement for serum proteins. Tissue

culture plastic has a net negative surface charge, which

is produced by plasma treatment of the polystyrene

(LaRocca and Barker, 1996). Many researchers have

shown that serum-free or reduced serum cultures can

be dramatically improved by coating the culture sur-

face with positively charged polymers, i.e., PDL and

PLL (Yavin and Yavin, 1974; McKeehan and Ham,

1976). Poly-lysine surface treatment improves adhes-

ive properties by altering the charge on the vessel

surface from negative to positive. In addition to pro-

moting cell adhesion, Poly-lysine also enhances the

adsorption of serum or extracellular matrix proteins to

the culture substrate (McKeehan, 1984).

While both PDL and PLL are being widely used,

PDL may be preferred for some cell types and applic-

ations because PDL, unlike PLL, is not broken down

by the proteases released by cells in culture (Banker

and Goslin, 1991). As PDL and PLL are synthetic

molecules, they do not stimulate biological activity in

the cells cultured on them. In addition, they do not

introduce impurities carried by natural polymers (Ham

and McKeehan, 1979).

Hybridoma

With rising costs in medical treatment, prevention of

diseases have become more important than the cure.

Hence the trend is towards early diagnosis so that ther-

apy is initiated at the earliest. The ﬁeld of diagnostics

has over the years developed and honed itself into

one of the most important sectors of clinical medi-

cine. These techniques rely heavily on the ability of

monoclonal antibodies (MAbs) for their speciﬁty and

afﬁnity for the antigens.

Since the advent of hybridoma technology in 1975

(Köhler and Milstein, 1975), MAbs have rapidly be-

come one of the most important humanised animal cell

products. They have rapidly made inroads into dia-

gnosis, research, therapy, and puriﬁcation processess

(Chua et al., 1994). However, production of MAbs

is cost intensive. This is mainly due to the expens-

ive media requirements, low maximum cell densities,

relatively low productivities and elaborate and high-

end downstream processing. This problem is further

compounded by the fact that hybridoma cell lines are

very unstable during prolonged cell culture, frequently

giving rise to non-producer cells (Chua et al., 1994).

There are two major approaches to obtain higher

yield of MAbs in culture. High density culture/large

scale culture of hybridomas constitute one approach

and the other is the enhancement of individual cellular

productivity (Sugahara et al., 1991).

Last couple of decades, optimisations have been

directed mostly to reactor modiﬁcations (Martens et

al., 1992; Lu et al., 1995), entrapment of cells (Lee

and Palsson, 1993), media modiﬁcations and modula-

tion of addition/feeding so that metabolite formation

is limited and kept to the minimum (Chua et al., 1994;

Bibila and Robinson, 1995; Bushell et al., 1994; Hiller

et al., 1994; Jo et al., 1992, 1993; Kurokawa et al.,

1994). Also, quite a few unique additives have been at-

tempted and this has enabled the bioprocess engineers

to scale up bioreactors and achieved greater productiv-

ity both through stand-alone and coupled approaches

(Hiller et al., 1994; Murray et al., 1996; Sugahara et

al., 1994).

In high-density culture there is a two-pronged ap-

proach to enhance antibody yield:

(1) Increasing the speciﬁc antibody secretion rate per

cell in culture.

(2) To increase cell culture time, cell concentra-

tion and hybridoma longevity thereby obtaining a

higher yield.

A model of cellular stoichiometry based on estim-

ated cell composition, product proﬁle, micronutrient

and ATP demand etc has been used to design nutri-

ent feeds. It has been shown that the use of nutrient

feeds in the form of concentrated complete media

eliminates the labour and time associated with the

identiﬁcation of the limiting nutrients and the optim-

isation of the feeding strategy (Bibila and Robinson,

1995). Supplementation with complete concentrated

medium has also shown to increase culture longev-

ity (Jo et al., 1992, 1993). The beneﬁcial effects of

concentrated medium is offset by the high cost of for-

mulation and build up of high concentration of toxic

by-products like NH

. Another aspect of using con-

centrated medium is the enhancement in osmolarity of

suspending ﬂuid which is harmful to the cells (Bibila

et al., 1994a).

Physico-chemical stimulation of hybridma cells in

culture is one of the varoius approaches of enhan-

cing MAb production. Of these, chemical additives

have been tried extensively (Xie and Wang, 1994a, b;

Maeda, 1992; Sugahara et al., 1997, 1998; Voigt and

Zintl, 1999) along with physical manipulations (Chua

et al., 1994; Sanfeliu et al., 1996; Ker-hwa Ou and

Patterson, 1997).

Production of MAbs has been hyperstimulated by

high osmolarity in eRDF medium. It was shown that

the medium eRDF, with or without serum, could stim-

ulate MAb production better than the basal RPMI,

DMEM media (Chua et al., 1994). It has been shown

that in the presence of high concentrations of glucose

and glutamine the antibody secretion rate decreases

as compared to low concentration of glucose (0.2 g

−1

) and glutamine (0.1 g l

−1

) where there was an

increase of three to fourfold in production of MAbs

in culture (Kurokawa et al., 1994). Dichloroacetate

has been shown to enhance the growth phase by 20 hr

thereby achieving a higher viable cell count leading to

enhanced MAb yield (Murray et al., 1996).

Number of serum-free media have been developed

and their effects on the growth and production of

MAbs have been studied (Moore and Hood, 1993).

Completely protein free media have also been de-

veloped (Fike et al., 1991). The physiological changes

that take place during the adaptation of cells to low

serum concentrations and serum-free media are of in-

terest because downstream processing becomes much

easier as the contaminating proteins are absent and the

metabolite build-up is also relatively low. Additions

of amino acids cysteine with methionine, trytophan

and isoleucine with valine and vitamin B12 has been

shown to result in signiﬁcant increases in viable cell

concentrations (Hiller et al., 1994).

Basic proteins and poly-basic amino acids have

been used to increase the yield of IgM MAbs from

human-human hybridomas. Addition of histones H1,

H2A and H2B enhanced the Ig productivity by 3.2-,

2.6- and 2.8-fold, respectively. Poly-basic amino acids

(poly L-lysine) have also been shown to enhance the

production whereas poly L-arginine did not (Sugahara

et al., 1994). Stimulation with step changes of lysine

added media showed a marked increase in viable cell

density and simultaneous increase in MAb titre. This

was observed with the cell line AE9D6 and the cells

could be kept viable for over 1400 hr in the fed-batch

culture (Datta et al., 1997a, b, c, d, 1999, 2000, 2001).

Subsequent work based on lysine mediated cel-

lular expansion in high-density culture of hybridoma

cells has resulted in few marked improvements in

the area of high-value-low-volume biochemical pro-

duction (mostly MAbs) where partitioned co-product

ion of more than one species of MAbs was possible

with much simplication of down-stream processing

(manuscript in preparation).

Adoptive immune therapy and

immunomodulation

Adoptive immune therapy involves the passive trans-

fer of antigen-primed T cells to initiate an immune

response. The T cells are extracted from the host,

‘taught’ to recognise the foreign antigen and then

transferred back into the host to initiate an effective

immune response.

An important application of this recent and mostly

experimental mode of therapy is against cancer. Tu-

mour immunology consists of two essential concepts:

immune surveillance, which speciﬁes the host immune

reactions against tumour cells, and tumour immune

escape, which refers to the tumour-cell evasion pro-

cess against the host immune system (Sheu et al.,

1999). Cytotoxic T cells have an important role to

play in the immune response against tumour cells

(Toes et al., 1994). Adoptive T-cell therapy involves

the passive transfer of antigen-reactive T cells to a

tumour-bearing host in order to initiate tumour re-

jection. Both CD4

and CD8

cells are capable of

initiating tumour rejection after adoptive transfer. Sev-

eral different culture methods have been reported that

permit in vitro expansion of immune T cells while re-

taining tumour speciﬁcity in this ﬁeld (Li and Chang,

1999; Chang and Shu, 1996). Future directions in

this ﬁeld involve the selective isolation and expansion

of subpopulations of T cells critical to initiating tu-

mour rejection, and the use of molecular techniques to

generate effector T cells (Markiewicz and Gajewski,

1999).

Adoptive transfer of polyclonal, tumour-speciﬁc,

IFN -gamma-producing CD4

T cells [T helper

type-1 (Th1) cells] have been used successfully

against disseminated lymphomas. A single injection

of 0.5 × 10

A20-speciﬁc Th1 cells has been shown to

eradicate disseminated A20 lymphomas and provide

lifelong protection without inducing autoimmune dis-

ease (Egeter et al., 2000). Cytotoxic T cell immuno-

therapy has also been tried against invasive cancers.

A 27-year-old woman with systemic chemoresistant

and radioresistant metastatic disease secondary to a re-

currence of human papillomavirus (HPV)-18 infected

cervical adenocarcinoma of the uterine cervix received

adoptive transfer of peripheral blood T cells stim-

ulated with HPV18 E7-pulsed autologous dendritic

cells (DC). The patient received two infusions of cyto-

toxic tumour speciﬁc T cells at two weeks intervals,

and in-vivo distribution of the T cells was followed by

111-oxine labeling and serial gamma camera imaging.

Persistent accumulation of radioactivity in the lungs,

which harbored extensive metastatic disease, was de-

tected upto 120 hr. after the infusion (Santin et al.,

2000). In vitro CTL induction, however, is difﬁcult

in patients with advanced cancer. But once the cells

are induced successfully, some favourable clinical ef-

fects are seen by the adoptive transfer of such cell

populations (Soda et al., 1999).

Viral infections are also being combated by adopt-

ive transfer of T cells. However, immunocytotherapy

for persistent viral infections has proven successful in

animal models but less effective in humans. Adoptive

transfer of T cells has been shown to effectively purge

virus from all tissues. But, maintenance of CD8

T cell effective functions after adoptive transfer is

directly proportional to the amount of co-transfered,

virus-speciﬁc CD4

T cells (Berger et al., 2000).

Infectious bronchitis virus (IBV) infection and associ-

ated illness may be dramatically modiﬁed by passive

transfer of immune T lymphocytes. As determined

by respiratory illness and viral load, transfer of syn-

geneic immune T lymphocytes protected chicks from

challenge infections (Seo et al., 2000). In vitro gen-

erated primary antigen speciﬁc Tc1 effective cells,

producing high amounts of IFN-γ , or resting Tc1

memory cells, generated from these effectors, were

protective aginst lethal pulmonary inﬂuenza infection

in mice. Highly activated CD62Llow Tc1 effectors

accumulated in the lung with rapid kinetics and most

efﬁciently reduced the pulmonary viral titre early dur-

ing infection (Cerwenka et al., 2000). Prophylactic

administration of Ebstein Barr virus speciﬁc cytotoxic

T lymphocytes (EBV-CTL) early after bone marrow

transplantation (BMT) in humans appears to provide

the most effective protection against the development

of EBV-associated lymphoproliferative disease. Ad-

ministration of EBV-CTLs before the onset of the

EBV-DNA peak has been shown to stabilise virus

titres within two to three logs above the normal levels.

Administration of two to four infusions of 10

EBV-

speciﬁc cytotoxic T lymphocytes CTLs/m

starting

from the time of maximal virus load resulted in a 2-

to 3-log decrease of virus titres (Gustafsson et al.,

2000). Thus adoptive immune therapy appears to have

an important say in the therapy against viral infections.

Another important application is against parasites.

Experiments involving adoptive transfer of T cells

have been successful in controlling malaria and toxo-

plasma infections in murine models. Adoptive transfer

of CD8

T-cell splenocytes from Neospora caninum-

infected mice was found to be protective against

challenge with toxoplasma. The CD8

T-cells from

Neospora-infected mice proliferated to both neospora

and toxoplasma antigens in vitro and secreted substan-

tial quantities of gamma IFN when pulsed with the

parasite antigen (Kasper and Khan, 1998). In a mur-

ine model, it has been demonstrated that the adoptive

transfer of intraepithelial lymphocytes (IEL) obtained

from inbred mice at day 11 postinfection is able to

protect against a virulent challenge by toxoplasma in

syngenic recipients. A puriﬁed CD8alpha/beta

IEL

population was isolated from infected mice at day

11 postinfection. These cells were able to protect

naive mice by adoptive transfer against a lethal para-

site challenge. RNA analysis by reverse-transcriptase

PCR revealed that primed CD8alpha/beta

IEL pro-

duce signiﬁcant message for IFN-gamma, an essential

cytokine for host protection against toxoplasmosis

(Buzoni-Gatel et al., 1997).

CD4

T cells seems to play an important role in

malaria infection. Cultured CD4+ T-cells that produce

interferon gamma and IL-2, but not IL-4, in response

to stimulation with the rodent parasite plasmodium

berghei have been found to reduce but not elimin-

ate parasites in vivo after adoptive transfer (Hirun-

petcharat and Good, 1998). In vitro cultured CD4

T-cells, generated following immunisation with dead

blood stage Plasmodiium yoelii parasites were found

to mediate protection against homologous challenge

infection in reconstituted nude mice. P. yoelii-speciﬁc

T cell line cells produced IFN-gamma after in vitro

stimulation with speciﬁc antigen, and were protective

when adoptively transferred into athymic nude mice

(Amante and Good, 1997).

Infectious diseases like leprosy have been com-

bated by adoptively transferred T cells, although in

a murine model. The model used was severe com-

bined immunodeﬁciency (SCID) mice which lack both

T and B cells. Cells from a known responder to my-

cobacterial antigens and from a non responder were

activated by M. leprae cell wall antigens. The cells

were harvested and co-injected with fresh M. leprae

into the right hind foot pads of SCID mice. After three

months, there was no growth of M. leprae in the foot

pads of mice co-injected with cells from the mycobac-

terial antigen responder, while growth was uninhibited

in mice receiving cells from the non-responder (Con-

verse et al., 1995). Dendritic cells (DC) are being

tried to generate an effective immune response against

mycobacterium tuberculosis infection. Conditionally

immortalised dendritic cell line (tsDC) were infec-

ted with Mycobacterium tuberculosis. These activated

DCs were shown to be capable of eliciting antigen-

speciﬁc T cell responses and potent anti-mycobacterial

protective immunity in a murine model of experi-

mental tuberculosis infection (Tascon et al., 2000). To

overcome the difﬁculty in generating speciﬁc CTLs,

stable artiﬁcial antigen-presenting cells (AAPCs) are

being developed that can be used to stimulate T cells

of any patient of a given Human Leukocyte Anti-

gen (HLA) type. Mouse ﬁbroblasts are retrovirally

transduced with a single HLA-peptide complex along

with the human accessory molecules B7.1, ICAM-1,

and LFA-3. These AAPCs consistently elicit strong

stimulation and expansion of HLA-restricted CTLs

(Latouche and Sadelain, 2000). Works are underway

as for the role(s) of lysine and its analogues, acting

as ‘stimulating’ co-stimulatory molecules in antigen

presentation process (unpublished data). This seems

interesting given the fact that the amino acid does

act as a molecular bridge connecting cells and their

growth factors (Datta and Kundu, 1999; Datta et al.,

2000a, 2001). In a simultaneous study being carried

out heat-killed MTb Ag showed varied types of im-

mune reactions when administered with the amino

acid given subcutaneously over a deﬁnite immuniza-

tion regime. One of the main considerations of this

immunomodulation capacity – both humoral and cell

mediated – of the amino acid was whether it was injec-

ted separately or premixed with antigen (unpublished

data).

Thus the important elements of adoptive im-

mune therapy and immunomodulation are generation

of stable antigen speciﬁc cells and their expansion.

Lysine has been postulated to act as a non-speciﬁc

bridging molecule. It holds a deﬁnite prospect in

bridging the presented antigen to the T cell and thus

ensure efﬁcient priming of the T-cell against the anti-

gen. Also, once antigen speciﬁc T-cells are created, the

amino acid can possibly be used to bring about rapid

cell proliferation expansion in-vitro. Thus a large bank

of primed cells seems to be a distinct possibility using

this molecule.

Tissue engineering

Tissue engineering is an emerging ﬁeld focused on the

creation of living tissues and organs for use in tissue

repair and transplantation. Tissue engineering involves

the fabrication of new functional living tissue using

living cells which are seeded onto biocompatible scaf-

folds which can be natural, man-made or composite

of both and grown under physiological conditions to

produce human biointeractive implants. Tissue engin-

eering is still in it’s embryonic stage with a reasonable

degree of challenges ahead. The scientiﬁc challenge

here lies in understanding the cells themselves, includ-

ing their behaviour in an altered environment, their

mass transfer requirements and their interaction with

the immediate milieu and the fabrication of compatible

materials to provide scaffolding and templates. Large

cell banks composed of universal cells that would be

immunologically transparent to any individual are re-

quired. A viable alternative is to have autologous cells.

Development of immunologically inert cells requires

manipulation of these cells which still poses a great

challenge. Advances in genetic manipulations might

be required. These cells could be either differenti-

ated ones or stem cell reservoirs which could then

be signalled to differentiate into different lineages for

structural application. These then need to be expanded

to appropriate cell number before transfer to templates

where further remodelling is expected to take place. A

proper substrate for cell survival and differentiation is

required. One of the strategies here is to use biocom-

patible implants composed of extracellular matrix mo-

lecules seeded with autologous cells or heterologous

cells in conjunction with immunosuppressing drugs.

Addition of growth and differentiation factors to these

matrices as well as agonists and antagonists that fa-

vour cell matrix interactions can potentially increase

the rate of successful tissue replacement. Collagen,

either alone or in combination with other materials,

is an important natural biomaterial that is used in

a variety of tissue-engineering applications. The ad-

hesiveness of collagen may be spatially controlled to

allow controlled localization or redistribution of cells

(Myles et al., 2000). Among one of the many ap-

praoches involving different tissues, a biocompatible

heterogeneous hydrogel of poly [N-(2-hydroxypropyl)

methacrylamide] (PHPMA) matrix has been studied

for it’s neuroinductive and neuroconductive proper-

ties. It has the potential to repair tissue defects in the

central nervous system by replacing lost tissue and by

promoting the formation of a histotypic tissue mat-

rix that facilitates and supports regenerative axonal

growth (Woerly et al., 1999).

Recently, tissue-engineering strategies have in-

cluded cell and gene therapy. ‘Cell therapy’, use of

cells to deliver active factors, is an emerging tech-

nique in the treatment of neurodegenerative disease.

The method entails encapsulation into a hollow ﬁber

device of discrete numbers of cell-containing micro-

carriers. Proliferation control is attained by embedding

cell-containing microcarriers in nonmitogenic hydro-

gels. In one of the approaches, ability to control dose

released over a several-fold range was demonstrated

with encapsulated PC-12 cells delivering neurotrans-

mitters and C2C12 mouse myoblast cells delivering

neurotrophic factors (CNTF) (Li et al., 1999).

Tissue-engineering advances will be interdepend-

ent with advances in gene therapy techniques to restore

function at a cellular level. The combination of tissue-

engineering strategies with gene therapy approaches

might allow transfection of diseased tissues with des-

ignated cDNA to eliminate inherent or acquired de-

fects. Identiﬁcation of the growth factors capable of

directing tissue development and of the technique to

be used for their delivery would aid in the engineering

of human tissue (Amiel et al., 2000).

Tissue engineering strategies are being applied to

replace various tissues and organs in various systems

of the body. Tissue engineering of musculoskeletal tis-

sues is a rapidly advancing ﬁeld. In bone, technology

has centred on bone graft substitute materials and the

development of biodegradable scaffolds (Boyan et al.,

1999). A composite matrix, containing esteriﬁed hya-

luronic acid and gelatine, has been shown to facilitate

the osteochondral differentiation of culture-expanded,

bone marrow-derived mesenchymal progenitor cells.

Thus, this composite matrix is useful for in vitro tis-

sue engineering for repair of chondral and osseous

defects (Angele et al., 1999). Important ongoing re-

search is being aimed at tissue-engineering cartilage

for surgical repair of tracheal defects (Van Veenendaal

et al., 2000). Myoblast transplantation to date has

been limited to the cellular level of replacement. It

has been suggested that myoblast-polyglycolic acid

constructs may be useful in deﬁning the application

of tissue engineering for future skeletal muscle trans-

plantation. A study was conducted to tissue engineer

three-dimensional vascularized skeletal muscle using

isolated myoblasts attached to synthetic biodegradable

polymer for tissue replacement in the enhancement

of muscle regeneration. In this study, organisation of

neomuscle strands surrounded by vascularized tissue

composed of degrading polymer and fusing myoblasts

demonstrated the ability of myoblast constructs to sur-

vive, reorganise and regenerate tissue-like structures

(Saxena et al., 1999). An attempt has been made to

culture ligament tissues in vitro by seeding human

anterior cruciate ligament and medial collateral liga-

ment cells onto synthetic biodegradable polymer ﬁber

scaffolds. In the present study, mechanical stimulus

and transforming growth factor enhanced prolifera-

tion whereas inﬂammatory agents had a negative effect

(Lin et al., 1999).

Near term products, including injectable human

matrix for contour defects and tissue-engineered car-

tilage, are proving to be important tools for plastic and

reconstructive surgery (Naughton and Mansbridge,

1999). The development of tissue-engineered fat equi-

valents for reconstructive and augmentation purposes

will be most welcome by nearly every surgical dis-

cipline and prove to be especially useful for plastic

surgeons (Katz et al., 1999).

Improvement of cell culture conditions and func-

tional cell expansion strategies in hepatic tissue en-

gineering may permit cell/tissue banking and the gen-

eration of liver tissue equivalents for transplantation.

Stimulatory effects of pancreatic islets on hepatocytes

in co-culture for continuous hepatotrophic stimulation

has been investigated in this context (Kauffman et al.,

1999).

Bladder regeneration is another important area

where extensive tissue engineering studies are carried

out. Currently, two techniques for the induction of

bladder regeneration are being researched. The ﬁrst,

in vivo technique, involves the use of a biodegrad-

able scaffold that the host bladder can use to remodel

and regenerate. This technique takes advantage of the

cell’s natural ability to heal or regenerate itself back

to a normal state. The second technology, the in vitro

technique, involves establishment of primary cell cul-

tures from the host’s native bladder. These cells are

seeded on a biodegradable scaffold to create a com-

posite graft that is then transplanted back into the host

for continuation of the regeneration process (Kropp

and Cheng, 2000). Tissue engineering for neointest-

ine is also being studied. It has been shown that

anastromosis between tissue-engineered intestine and

native small bowel has a moderately high patency rate

and a positive effect on maintenance of the size of

the neointestine and development of the neomucosa

(Kaihara et al., 1999). An attempt has been made to

tissue engineer branched or bifurcated hybrid vascular

prosthesis using bovine smooth muscle cells and type

I collagen with minimal reinforcement by a knitted

fabric mesh made of segmented polyester. A branched

hybrid graft with mesh reinforcement is expected to

be applicable to arterial replacement in a branching

region (Kobashi and Matsuda, 1999). A new type of

a biodegradable nerve graft conduit material, the am-

nion tube, has been developed. The amnion tube is

a potential ideal nerve conduit material secondary to

its unique characteristics: it contains important neuro-

tropic factors, is biodegradable, provokes a very weak

immune response, is semiﬂexible, is readily available,

and is easily manufactured into different sizes and dia-

meters (Mohammad et al., 2000). Tissue engineering

for bioprosthetic and mechanical valves and valved

conduits poses quite a challenge because of its inab-

ility to grow, repair or remodel. An attempt to evaluate

the feasibility of creating 3-leaﬂet, valved, pulmon-

ary conduits from autologous ovine vascular cells

and biodegradable polymers with tissue-engineering

techniques has been tried (Stock et al., 2000).

Surface wound healing is another important area

studied in the context of tissue engineering. Tissue-

engineered skin implants have shown efﬁcacy in a

variety of wound care applications. Research efforts

to modify cultured autologous skin transplants for

large full-thickness burn wounds and in chronic ulcers

are shifting from multilayered differentiated grafts

(‘sheet’ grafts) toward smaller units of basal undiffer-

entiated single cell suspensions in a transport medium

and subconﬂuently covered static carriers (Voigt et al.,

1999). A novel living skin replacement (LSR) biother-

apy concept, combining the elements of cell therapy

along with those of tissue engineering took advant-

age of the biodegradable microspheres, onto which

donor skin epidermal and dermal cells could be at-

tached and expanded in vitro for subsequent direct

application down to the deepest recesses of the wound

bed. This novel approach presents a number of ad-

vantages over existing therapies including facilitated

cell manipulations, ease of storage and transportation,

rapid clinical intervention due to the elimination of

any surgical suturing or stapling, and a more natural

three-dimensional tissue remodelling and anatomical

compliance (LaFrance and Armstrong, 1999).

It is evident that the basic principle in tissue en-

gineering is seeding appropriate number of cells after

proper expansion on an appropriate substrate. This

requires good cell proliferation to provide the appro-

priate cell number before seeding onto substrate and

good cell viability. It has been observed that L-lysine

HCl supports good cell proliferation both in-vivo and

in-vitro, both in anchorage dependent and independent

cells and it would be interesting to study any positive

effects it might have in rapid expansion of autolog-

ous cells for development of bioengineered soft tissue

prostesis. Autologous ﬁbroblast layered bioprosthetic

heart valve (aortic) development is in progress in the

authors laboratory involving a new approach of ﬁbre

renewal in situ.

Gene therapy and DNA vaccination

Gene therapy has attracted much interest since the

ﬁrst submissions of phase I clinical trials in the early

1990s, for the treatment of inherited genetic diseases.

Preliminary results were very encouraging and promp-

ted many investigators to submit protocols for phase

I and phase II clinical trials for the treatment of in-

herited genetic diseases and cancer. Also in 1990,

the ﬁrst gene therapy clinical trial for the treatment

of patients with melanoma (Rosenberg et al., 1990)

was conducted. The results of this study indicated

that retroviral-mediated gene transfer in patients was

safe. This ﬁnding prompted the submission of many

other protocols for gene therapy clinical trials to treat

patients affected by cancer, primarily in the area of

melanoma (Osanto et al., 1993; Mahvi et al., 1997),

followed by ovarian carcinoma (Deshane et al., 1997),

sarcoma (Mahvi et al., 1997), brain tumor (Kun et

al., 1995), and lung cancer (Nguyen et al., 1996).

There is also a strong interest in beginning gene ther-

apy clinical trials for the treatment of patients with

AIDS, cardiopathies, and neurologic diseases. Indeed,

gene transfer technology has already been applied in

the phase I and phase II trials for the AIDS vaccine

programs, which have recently begun in the U.S.A.

(Haynes, 1996; Weber, 1996). As already anticipated,

the spectrum ranges from the treatment of inherited

or acquired genetic disorders to cancer, AIDS, car-

diopathies, and neurologic diseases. This is strongly

encouraging to the pursuit of gene therapy programs

in medicine (stem cell).

There are a wide variety of vectors used to de-

liver DNA or oligonucleotides into mammalian cells,

either in vitro or in vivo. The most common vector sys-

tems are based on retroviruses (Cournoyer and Caskey,

1993; Gilboa, 1990; Kohn et al., 1989; Miller, 1990;

Temin, 1986), adeno-associated virus (AAV) (Berns

et al., 1975; Cheung et al., 1980; Podsakoff et al.,

1994), adenovirus (Karlsson et al., 1985; Yamada et

al., 1985), herpes simplex virus (HSV) (Glorioso et

al., 1995), cationic liposomes (Thierry et al., 1992;

Benne et al., 1992; Ropert et al., 1993). The stage of

development of vectors and their variety are still not

sufﬁcient to be efﬁciently applied in therapy.

Among the other popular techniques for gene de-

livery, particle-mediated bombardment with a device

called the gene gun has become an important devel-

opmental tool. This instrument has been used in nu-

merous examples of wound gene therapy with growth

factors or their receptors in the last decade. Among

the advantages of particle-mediated bombardment are

ease and speed of preparation of the delivery vehicle,

the stability of the DNA preparation, the absence of

(viral) antigens, the ability to target the projectiles to

different tissue depths and areas, and the rapid shed-

ding of both particles and DNA if they are targeted

to the epidermis. Clinical application of the techno-

logy remains limited by the relatively low efﬁciency

of the method, the potential tissue damage created by

impact of the particles, and the coverage area (Kun

et al., 1995). The ﬁeld of gene therapy is at present

actively involved in the challenging task of improving

the design of vector systems for in vivo applications.

DNA vaccines have been used to stimulate pro-

tective immunity against many infectious pathogens,

malignancies, and autoimmune disorders in animal

models. Since their inception in 1950, there have been

many research works done on the ability of DNA

vaccines to induce strong immune responses against

proteins from infectious agents such as malaria (Hoff-

man et al., 1997; Kalinna, 1997; Wang et al., 1998;

Strugnell et al., 1997; Kaufmann, 1995; Lowrie et al.,

1997), rabies virus (Xiang et al., 1994), hepatitis B

virus (HBV) (Davis et al., 1994; Tacket et al., 1999),

HSV (Kriesel et al., 1996), Ebola virus (Xu et al.,

1998), and HIV (Boyer et al., 1997, 1999; Wang et

al., 1993a, b). The strategy of most of these investig-

ations is relatively simple: A DNA plasmid encoding

a desired protein is injected into the muscle or skin

of an animal, where it thereupon enters host cells

and directs the synthesis of its polypeptide antigen.

Once the plasmid-antigen is processed and presen-

ted by transfected host cells, a cellular and humoral

immune response against the antigen is provoked. Ge-

netic immunization exhibits many advantages over

traditional vaccines that use live-attenuated or killed

pathogen, proteins, or synthetic peptides: (a) Im-

munogenicity: can induce both humoral and cellular

immune responses at low effective dosages (micro-

grams) in animal models. (b) Safety: Unability to

revert into virulence unlike live vaccines and efﬁcacy

does not require the use of toxic treatments unlike

some killed vaccines. (c) Engineering: Plasmid vec-

tors are simple to manipulate and can be tested rapidly.

Also, combination approaches can be easily adapted.

(d) Manufacture: Conceptually low cost and reprodu-

cible large-scale production and isolation. Also can

be produced at high frequency in bacteria and eas-

ily isolated. (e) Stability: More temperature-stable

than conventional vaccines and has a long shelf-life.

(f) Mobility: Ease of storage and transport (Shedlock

and Weiner, 2000).

Another facet of DNA vaccine technology focuses

on immune-related diseases, such as autoimmunity

and cancer (Chen et al., 1999). By manipulating the

balance of T helper (Th) 1 and 2 lymphocytes using

DNA plasmid immunization, many of the pathogenic

qualities of autoimmune disease may be potentially

addressed (Ramshaw et al., 1997). DNA immunization

has also proven as an effective candidate in the ﬁght

against certain cancers. The growth of human tumor

cells that produce and secrete a target protein has been

retarded or inhibited by a DNA vaccine construct en-

coding a subunit of that target protein (Geissler et al.,

1997).

The most popular method of administering DNA

vaccines has been parenterally, which includes needle

injection into muscle or skin and gas-powered, DNA-

covered particle bombardment using a ‘gene-gun’

(g.g.) (Shedlock and Weiner, 2000). Noninvasive

methods of plasmid delivery involve the topical ap-

plication of plasmid to the skin or mucosae. The

induction of antigen-speciﬁc immune responses has

been shown following the application of a plasmid

solution to various mucosal surfaces including in-

tranasal (Klavinskis et al., 1999; Hamajima et al.,

1998), oral (Etchart et al., 1997), and intravaginal

(Bagarazzi et al., 1998; Wang et al., 1997). Forms

of delivery targeting the skin, including i.d. injection,

g.g. bombardment, and topical application, have been

shown to elicit a humoral response primarily, charac-

terized by a rapid progression to a Th2-type response,

associated with the production of an IgA and IgG1

antibody isotype (Fan et al., 1999; Boyle et al., 1997).

Conversely, injection into muscle results in the induc-

tion of a strong cellular-mediated response, or Th1

type, that primes antigen-speciﬁc cytolytic T lympho-

cytes (CTLs) and is associated with the production of

IgG2a antibody (Sin et al., 1999). DNA vaccines eli-

cit strong and long-lasting humoral and cell-mediated

immune responses in many animal models. At the

cellular level, the efﬁcacy of DNA vaccination de-

Figure 2. Schematic diagram depicting the biochemical roles of lysime.

pends on the interaction between their polypeptide

products and the two major groups of cells that medi-

ate immunity: lymphocytes and APCs. The codelivery

of plasmids encoding costimulatory molecules is a

method theorized to improve the antigen-presenting

capabilities of transfected host cells. Another strategy

currently under development is the coadministration of

cell-surface molecules that induce cellular apoptosis.

Theoretically, this technique targets vaccine antigen to

the cross-priming pathway by delivering antigen asso-

ciated with apoptotic cells to DCs and thereby guiding

immune responses toward a Th1 phenotype (Chatter-

goon et al., 2000). Recently, expression of fusion

proteins from a DNA vaccine is an attractive means

of modulating an antigen-speciﬁc immune response

without the use of potentially toxic chemical adjuvants

(Shedlock and Weiner, 2000). Combination vaccin-

ation strategies are also being targeted nowadays.

Many heterologous prime-boost strategies use DNA

vaccines, recombinant virus, and protein in different

assortments, which ‘prime’ the immune system to the

vaccine antigen ﬁrst, followed by a subsequent ‘boost’

immunization, which enhances the preliminary re-

sponse. Strategies gaining the most attention are DNA

vaccine-priming followed by protein or recombinant

virus-boosting (Sin et al., 1999). Currently, many

promising techniques of immune enhancement are be-

ing developed that modulate the intensity and direction

of responses, such as the use of genetic adjuvants and

DNA vectors of greater immunostimulation (Shedlock

and Weiner, 2000).

Due to the hazards associated with viral vectors

nonviral DNA complexes show promise as alternative

and attractive gene delivery vectors for treating ge-

netic diseases. Nonviral DNA complexes are typically

formed by combining DNA with various condens-

ing/complexing agents such as lipids, polyelectrolytes,

polymers, polypeptides, and surfactants in solution.

DNA/poly-L-lysine polyplex formation kinetics are

probed by time-resolved multiangle laser light scat-

tering (TR-MALLS), which yields the time evolution

of the supramolecular complex mass and geomet-

ric size. Primary polyplexes whose geometric size

is smaller than individual DNA molecules in solu-

tion are formed very rapidly upon mixing DNA and

poly-L-lysine. Over time, these primary polyplexes

aggregate into larger structures whose ultimate size is

determined primarily by the relative concentrations of

DNA and poly-L-lysine (Lai and Van Zanten, 2001).

Polylysine (pLy) has been used as a DNA carrier in

nonviral gene delivery systems because it forms com-

plexes with plasmid DNA via charge interaction, and

condenses it into a compact structure. It has been re-

cently shown that cross-linking nuclear localization

sequences (NLSs) to pLy can enhance transfection by

conferring speciﬁc recognition by the cellular nuc-

lear import ‘receptor’, the NLS-binding importin al-

pha/beta heterodimer. A clear correlation has been in-

dicated between the pLy-DNA structure, importin al-

pha/beta recognition, and gene transfer efﬁciency, thus

underlining the importance of using pLy-DNA at the

optimal Ly/Nucleotide ratio (Chan et al., 2000). Poly-

L-lysine has been shown to be a major contributor for

gene transfer to hematopoietic progenitor cells, medi-

ating the initial vector-cell binding. Human progenitor

cell lines are poorly transduceable with recombinant

adenovirus vectors. This new poly-L-lysine-modiﬁed,

adenovirus-based vector could overcome virus trop-

ism restrictions and consistently achieve very high

transduction efﬁciency (>90%) in cells otherwise re-

fractory to adenovirus gene transfer. Polylysine-based

adenovirus vectors may have promise for situations

in which high-efﬁciency gene transfer with transient

high level transgene expression in hematopoietic cells

is needed, such as leukemia vaccine protocols or for

purging strategies in leukemia cell contaminated stem

cell preparations (Schwarzenberger et al., 2001).

Modiﬁcation of poly-L-lysine and poly-L-ornithine

by the covalent attachment of palmitoyl and methoxy-

poly (ethylene glycol) (mPEG) residues produces a

new class of amphiphilic polymers-PLP and POP, re-

spectively. These amphiphilic amino acid based poly-

mers have been found to assemble into polymeric

vesicles in the presence of cholesterol. In vitro biolo-

gical testing revealed that POP and PLP vesicle-DNA

complexes are about one to 2 orders of magnitude less

cytotoxic than the parent polymer-DNA complexes

although more haemolytic than the parent polymer-

DNA complexes. The polymeric vesicles condense

DNA at a polymer: DNA weight ratio of 5:1 or greater

and the polymeric vesicle-DNA complexes improved

gene transfer to human tumor cell lines in comparison

to the parent homopolymers despite the absence of re-

ceptor speciﬁc ligands and lysosomotropic agents such

as chloroquine (Brown et al., 2000).

Thus it is seen that lysine can be employed to

build an intracellular DNA delivery system, which in

addition to being efﬁcient is also non-toxic.

Finally the biochemical roles and applications of

lysine are summarised in Figure 2.

References

Aaregawa T (1999) bFGF increases regional myocardial blood ﬂow

and salvages myocardium in the infarcted border zone in a

rabbit model of acute myocardial infarction. Angiology 50(6):

487–495.

Allison AC (1999) Squalene and squalane emulsions as adjuvants.

Methods 19: 87–93.

Amante FH & Good MF (1997) Prolonged Th1-like response

generated by a Plasmodium yoelii-speciﬁc T cell clone allows

complete clearance of infection in reconstituted mice. Parasite

Immunol 19: 111–126.

Andreasen CB & Frank DE (1999) The effects of ascorbic acid on

in vitro heterophil function. Avian Dis 43: 656–63.

Aoki M (2000) Angiogenesis induced by HGF in non infarcted

myocardium and infarcted myocardium: upregulation of essen-

tial transcription factor for angiogenesis. Gene Ther 7: 417–427.

Appendini P & Hotchkiss JH (1999) Antimicrobial activity of a

14-residue peptide against Escherichia coli O157:H7. J Appl

Microbiol 87: 750–756.

Ashcroft GS, Greenwell-Wild T, Horan MA, Wahl SM & Ferguson

MW (1999a) Topical estrogen accelerates cutaneous wound heal-

ing in aged humans associated with an altered inﬂammatory

response. Am J Pathol 155: 1137–1146.

Ashcroft GS, Yang X, Glick AB, weinstein M, Lelterio JL, Mizel

DE, Anzan OM, Greenwell-Wild T, Wahl SM, Deng C & Roberts

AB (1999b) Mice lacking Smad3 show accelerated wound heal-

ing and an impaired local inﬂammatory response. Nat Cell Biol

1: 260–266.

Bagarazzi ML, Boyer JD, Ugen KE, Javadian MA, Chattergoon M,

Shah A, Bennett M, Ciccarelli R, Carrano R, Coney L & Weiner

DB (1998) Safety and immunogenicity of HIV-1 DNA constructs

in chimpanzees. Vaccine 16: 1836–1841.

Baichey G, Penkoya R & Deliiski T (1995) The treatment of slowly

healing wounds with collagen and growth factors. Khururgiia

(Soﬁia) 48: 17–19.

Banker G & Goslin K (1991) Culturing Nerve Cells (pp. 65). MIT

Press, Cambridge.

Barnett PV, Pullen L, Williams L & Doel TR (1996) Interna-

tional bank for foot-and-mouth disease vaccine: Assessment of

Montanide ISA 25 and ISA 206, two commercially available oil

adjuvants. Vaccine 14: 1187–1198.

Bellini AM, Vertuani G, Quaglio MP & Cavazzini G (1979) Bile

acid derivatives with antimicrobial activity. Farmaco 34: 967–

978.

Bennet CF, Chiang MY, Chan H et al. (1992) Cationic lipids en-

hance cellular uptake and activity of phosphothioate antisense

oligonucleotides. Mol Pharmacol 41: 1023–1033.

Berger DP, Homann D & Oldstone MB (2000) Deﬁning parameters

for successful immunocytotherapy of persistent viral infection.

Virology 266: 257–263.

Berns KI, Pinkerton TC, Thomas GF et al. (1975) Detection of

adeno-associated virus (AAV)-speciﬁc nucleotide sequences in

DNA isolated from latently infected Detroit 6 cells. Virology 68:

556–560.

Berstad AK, Andersen SR, Dalseg R, Dromtorp S, Holst J, Namork

E, Wedege E & Haneberg B (2000) Inactivated meningococci

and pertussis bacteria are immunogenic and act as mucosal ad-

juvants for a nasal inactivated inﬂuenza virus vaccine. Vaccin 18:

1910–1919.

Bibila TA & Robinson DK (1995) In pursuit of the optimal fed-

batch process for monoclonal antibody production. Biotechnol

Prog 11, 1–13.

Bibila TA, Glazomitsky K, Ranucci CS et al. (1994) Monoclonal

antibody process development using medium concentrates. Bio-

technol Prog 10: 87–96.

Bickford PC, Shukitt-Hale B & Joseph J (1999) Effects of aging on

cerebellar noradrenergic function and motor learning: Nutritional

interventions. Mech Ageing Dev 111: 141–154.

Blaszyk M & Holley RA (1998) Interaction of monolaurin, eugenol

and sodium citrate on growth of common meat spoilage and

pathogenic organisms. Int J Food Microbiol 39: 175–183.

Boraschi D & Tagliabue A (1999) Interleukin-1 and interleukin-1

fragments as vaccine adjuvants. Methods 19: 108–113.

Boyer JD, Chattergoon MA, Ugen KE, Shah A, Bennett M, Cohen

A, Nyland S, Lacy KE, Bagatazzi ML, Higgins TJ, Baine Y, Cic-

carelli RB, Ginsberg RS, MacGregor RR & Weiner DB (1999)

Enhancement of cellular immune response in HIV-1 seropositive

individuals: A DNA-based trial. Clin Immunol 90: 100–107.

Boyer JD, Ugen KE, Wang B, Agadjanyan M, Gilbert L, Bagarazzi

ML, Chattergoon M, Frost P, Javadian A, Williams WV, Refaeli

Y, Ciccarelli RB, McCallus D, Coney L & Weiner DB (1997)

Protection of chimpanzees from high-dose heterologous HIV-1

challenge by DNA vaccination. Nat Med 3: 526–532.

Boyle JS, Silva A, Brady JL & Lew AM (1997) DNA immunization:

Induction of higher avidity antibody and effect of route on T cell

cytotoxicity. Proc Natl Acad Sci USA 94: 14626–14631.

Breitbart AS, Mason JM, Urmacher C, Barcia M, Grant RT, Per-

golizzi RG & Grande PA (1999) Gene enhanced tissue engin-

eering: Applications for wound healing using cultured dermal

ﬁbroblasts transduced retrovirally with PDGF-B gene. Ann Plast

Surg 43: 632–639.

Brown MD, Schatzlein A, Brownlie A, Jack V, Wang W, Tetley

L, Gray AI & Uchegbu IF (2000) Preliminary characterization

of novel amino acid based polymeric vesicles as gene and drug

delivery agents. Bioconjug Chem 11: 880–891.

Bushell ME, Bell SL, Scott MF, Spier RE, Wardell JN & Sanders

PG (1994) Enhancement of monoclonal antibody yield by hy-

bridoma fed-batch culture resulting in extended maintenance of

viable cell populations. Biotechnol Bioeng 44: 1099–1106.

Buzoni-Gatel D, Lepage AC, Dimier-Poisson IH, Bout DT &

Kasper LH (1997) Adoptive transfer of gut intraepithelial lymph-

ocytes protects against murine infection with Toxoplasma gondii.

J Immunol 158: 5883–5889.

Camilleri-Bruet S, Devez F, Tissier F, Ducruit V, Le Tourneau A,

Diebold J, Audouin J & Molina T (2000) Quality control and

sensitivity of polymerase chain reaction techniques for the as-

sessment of immunoglobulin heavy chain gene rearrangements

from ﬁxed- and parafﬁn-embedded samples. Ann Diagn Pathol

4: 71–76.

Cerwenka A, Morgan TM & Dutton RW(1999) Naive, effector, and

memory CD8 T cells in protection against pulmonary inﬂuenza

virus infection: Homing properties rather than initial frequencies

are crucial. J Immunol 163: 5535–5543.

Chan CK, Senden T & Jans DA (2000) Supramolecular structure

and nuclear targeting efﬁciency determine the enhancement of

transfection by modiﬁed polylysines. Gene Ther 7: 1690–1697.

Chang AE & Shu S (1996) Current status of adoptive immunother-

apy of cancer. Crit Rev Oncol Hematol 22: 213–28.

Chattergoon MA, Kim JJ, Yang JS, Robinson TM, Lee DJ,

Dentchev T, Wilson DM, Ayyavoo V & Weiner DB (2000)

Targeted antigen delivery to antigen-presenting cells includ-

ing dendritic cells by engineered Fas-mediated apoptosis. Nat

Biotechnol 18: 974–979.

Chen C, Ji H, Suh KW, Choti MA, Pardoll DM & Wu T (1999)

Gene gun-mediated DNA vaccination induces antitumor im-

munity against human papillomavirus type 16 E7-expressing

murine tumor metastases in the liver and lungs. Gene Ther 6:

1972–1981.

Chen LH, Boissonneault GA & Glauert HP (1988) Vitamin C,

vitamin E and cancer (review). Anticancer Res 8: 739–748.

Cheung AK, Hoggan MD, Hauswirth WW et al. (1980) Integra-

tion of the adeno-associated virus genome into cellular DNA in

latently infected human Detroit 6 cells. J Virol 33: 739–748.

Cho YW, Cho YN, Chung SH, Yoo G & Ko SW (1999) Water

soluble chitin as wound healing accelerator. Biomaterials 20:

2139–2145.

Chua FK, Yap MG & Oh SK (1994) Hyper-stimulation of mono-

clonal antibody production by high osmolarity stress in eRDF

medium. J Biotechnol 37: 265–75.

Chua FKF, Yap MGS & Oh SKW (1994) Hyperstimulation of

monoclonal antibody production by high osmolarity stress in

eRDF medium. J Biotechnol 37: 265–275.

Converse PJ, Haines VL, Wondimu A, Craig LE & Meyers WM

(1995) Infection of SCID mice with Mycobacterium leprae and

control with antigen-activated ‘immune’ human peripheral blood

mononuclear cells. Infect Immun 63: 1047–54.

CorreaR,BlancoB,DelRioM,VictorV,GuayerbasN,Med-

ina S & De la Fuente M (1999) Effect of a diet supplemented

with thioproline on murine macrophage function in a model of

premature ageing. Biofactors 10: 195–200.

Cournoyer D & Caskey CT (1993) Gene therapy of the immune

system. Annu Rev Immunol 11: 297–329.

Cynober LA (1999) The use of alpha-ketoglutarate salts in clinical

nutrition and metabolic care. Curr Opin Clin Nutr Metab Care 2:

33–37.

Datta D (2000) Essential amino acid lysine and its analogues

support faster and qualitatively better healing of wound.

www.pharma-transfer.com (Balantyne-Ross Publications; In-

vited submission).

Datta D & Kundu PK (1999) Do we have a healer molecule? In:

Gordon Conference on Wound Repair.

Datta D, Kundu PK & Senthilkumar M (1997) Studies on high dens-

ity culture of hybridoma cells (AE9D6) In-Vitro Artif Organs 21:

534 (Blackwell Publications).

Datta D, Kundu PK & Senthilkumar M (1997b) Effect of L-

lysine on the growth and secretion of hybridoma cell (AE9D6).

Biotechnology Technique 11: 617–620.

Datta D, Kundu PK, Siva Prasad N, Electricwalla SE & Varma R

(1997c) Getting higher yields of monclonal antibodies in culture.

Ind J Physiol Pharm 42: 155–171.

Datta D, Kundu PK, Siva Prasad N & Varma R (1997d) Monoclonal

antibodies: A brief overview of high-density culture, down-

stream processing and recovery. Ind J Exp Biol 36: 125–135.

Datta D, Kundu PK, Biswas S, Dasgupta S, Bhinge A & Chandran

V (2000a) Effect of cationic amino acid L-Lysine and its poly-

mers on the growth and secretion of hybridoma cell line OKT3.

Hybridoma 19: 339–346.

Datta D, Vidya C & Bhinge A (2000b) Cationic amino acid lysine

and it’s analogues (C-6 to C-10) in wound repair and angiogen-

esis. Proceedings of 1st World Congress in wound repair, Sept

2000, Sydney Australia.

Datta D, Kundu PK, Dasgupta S & Fulzele K (2001) Effect of addi-

tion of proton carriers in culture medium on growth and secretion

of hybridoma cell line OKT3. Ind J Physiol Pharm (In press).

Davis HL, Suparto II, Weeratna RR, Jumintarto, Iskandriati DD,

Chamzah SS, Ma’ruf AA, Nente CC, Pawitri DD, Krieg AM,

Heriyanto, Smits W & Sajuthi DD (2000) CpG DNA overcomes

hyporesponsiveness to hepatitis B vaccine in orangutans. Vaccine

18: 1920–1924.

Deshane J, Siegal GP, Wang M et al. (1997) Transductional efﬁ-

ciency and safety of an intraperitoneally delivered adenovirus

encoding an anti-erbB-2 intracellular single-chain antibody for

ovarian cancer therapy. Gynecol Oncol 64: 378–385.

Di Luigi L, Guidetti L, Pigozzi F, Baldari C, Casini A, Nordio M

& Romanelli F (1999) Acute amino acids supplementation en-

hances pituitary responsiveness in athletes. Med Sci Sports Exerc

31: 1748–1754.

Dupuis M, McDonald DM & Ott G (1999) Distribution of adjuvant

MF59 and antigen gD2 after intramuscular injection in mice.

Vaccine 18: 434–439.

Egeter O, Mocikat R, Ghoreschi K, Dieckmann A & Rocken M

(2000) Eradication of disseminated lymphomas with CpG-DNA

activated T helper type 1 cells from nontransgenic mice. Cancer

Res 60: 1515–1520.

Epand RM & Vogel HJ (1999) Diversity of antimicrobial peptides

and their mechanisms of action. Biophys Acta 1462: 11–28.

Etchart NR, Buckland R, Liu MA, Wild TF & Kaiserlian D (1997)

Class I-restricted induction by mucosal immunization with naked

DNA encoding measles virus haemagglutinin. J Gen Virol 78:

1577–1580.

Ezaki T (2000) Antigen retrieval on formaldehyde-ﬁxed parafﬁn

sections: it’s potential drawbacks and optimization for double

immunostaining. Micron 31: 639–49.

Fan JJ & Chen JH (1999) Inhibition of aﬂatoxin-producing fungi by

Welsh onion extracts. J Food Prot 62: 414–417.

Fan H, Lin Q, Morrissey GR & Khavari PA (1999) Immunization

via hair follicles by topical application of naked DNA to normal

skin. Nat Biotechnol 19: 870–872.

Fike RM, Pfohl JL, Epstein DA, Jayme DW & Weiss S (1991) Hy-

bridoma growth and monoclonal antibody production in protein-

free hybridoma medium. BioPharm 3: 26–29.

Flodin NW (1997) The metabolic roles, pharmacology, and toxico-

logy of lysine. J Am Coll Nutr 16: 7–21.

Funfstuck R, Straube E, Schildbach O & Tietz U (1997) Prevention

of reinfection by L-methionine in patients with recurrent urinary

tract infection. Med Klin 92: 574–581.

Geissler M, Wands G, Gesien A, Monte S, Bellet D & Wands JR

(1997) Genetic immunization with the free human chorionic gon-

adotropin beta subunit elicits cytotoxic T lymphocyte responses

and protects against tumor formation in mice. Lab Investig 76:

859–871.

Gilboa E (1990) Retroviral gene transfer. Applications to human

gene therapy. Prog Clin Biol Res 352: 301–311.

Glorioso JC, De Luca NA & Fink DJ (1995) Development and ap-

plication of herpes simplex virus vectors for human gene therapy.

Annu Rev Microbiol 49: 675–710.

Goodman DL, McDonnell JT, Nelson HS, Vaughan TR & Weber

RW (1990). Chronic urticaria exacerbated by the antioxid-

ant food preservatives, butylated hydroxyanisole (BHA) and

butylated hydroxytoluene (BHT). J Allergy Clin Immunol 86 (4

Pt 1): 570–575.

Goodson B, Ehrhardt A, Ng S, Nuss J, Johnson K, Giedlin M,

Yamamoto R, Moos WH, Krebber A, Ladner M, Giacona MB,

Vitt C & Winter J (1999) Characterization of novel antimicrobial

peptoids. Antimicrob Agents Chemother 43: 1429–1434.

Grizard D & Barthomeuf C (1999) Non-digestible oligosacchar-

ides used as prebiotic agents: Mode of production and beneﬁcial

effects on animal and human health. Reprod Nutr Dev 39:

563–88.

Grzybowski J, Oldak E & Janiak MK (1999) Local application of

Granulocyte-colony stimulating factor, Granulocyte-macrophage

colony stimulating factor and Epidermal growth factor in treat-

ment of wounds. Postepy Hig Med Dosw 53: 75–86.

Gupta RK, Chang AC & Siber GR (1998) Biodegradable polymer

microspheres as vaccine adjuvants and delivery systems. Dev

Biol Stand 92: 63–78.

Gustafsson A, Levitsky V, Zou JZ, Frisan T, Dalianis T, Ljungman

P, Ringden O, Winiarski J, Ernberg I & Masucci MG (2000)

Epstein-Barr virus (EBV) load in bone marrow transplant re-

cipients at risk to develop posttransplant lymphoproliferative

disease: prophylactic infusion of EBV-speciﬁc cytotoxic T cells.

Blood 95: 807–814.

Ham RG & McKeehan WL (1979) Methods in enzymology, In: W

Jakoby and IH Pastan (eds.), Cell Culture, Vol. LVIII, (p. 49),

Academic Press Limited, London.

Hamajima K, Sasaki S, Fukushima J, Kaneko T, Xin KQ, Kudoh I

& Okuda K (1998) Intranasal administration of HIV-DNA vac-

cine formulated with a polymer, carboxymethylcellulose, aug-

ments mucosal antibody production and cell-mediated immune

response. Clin Immunol Immunopathol 88: 205–210.

Haneberg B, Dalseg R, Oftung F, Wedege E, Hoiby EA, Haugen

IL, Holst J, Andersen SR, Aase A, Meyer Naess L, Michaelsen

TE, Namork E & Haaheim LR (1998) Towards a nasal vaccine

against meningococcal disease, and prospects for its use as a

mucosal adjuvant. Dev Biol Stand 92: 127–133.

Hansen G, Yeung VP, Berry G, Umetsu DT & DeKruyff RH

(2000) Vaccination with heat-killed Listeria as adjuvant reverses

established allergen-induced airway hyperreactivity and inﬂam-

mation: role of CD8+ T cells and IL-18. J Immunol 164:

223–30.

Hantschick M & Stosiek P (1998) Cold acetone ﬁxation and methac-

rylate embedding. A suitable method for routine processing of

bone marrow biopsies. Pathol Res Pract 194: 111–121.

Haynes BF (1996) HIV vaccines: Where we are and where we are

going. Lancet 348: 933–937.

Heimbach J, Rieth S, Mohamedshah F, Slesinski R, Samuel-

Fernando P, Sheehan T, Dickmann R & Borzelleca J (2000)

Safety assessment of iron EDTA [sodium iron (Fe(

)) ethylene-

diaminetetraacetic acid]: Summary of toxicological, fortiﬁcation

and exposure data. Food Chem Toxicol 38: 99–111.

Hiller GW, Clark DS & Blanch HW (1994) Transient responses

of hybridoma cells in continuous culture to step changes in

amino acid and vitamin concentrations. Biotechnol Bioeng 44:

303–321.

Hirunpetcharat C & Good MF (1998) Deletion of Plasmodium

berghei-speciﬁc CD4

T cells adoptively transferred into recip-

ient mice after challenge with homologous parasite. Proc Natl

Acad Sci USA 95: 1715–1720.

Hodinka RL, Jack-Wait K, Wannamaker N, Walden TP & Gilligan

PH (1987) Comparative in vitro activity of LY146032 (daptomy-

cin), a new lipopeptide antimicrobial. Eur J Clin Microbiol 6:

100–103.

Hong J, Oren Z & Shai Y (1999) Structure and organization

of hemolytic and nonhemolytic diastereomers of antimicrobial

peptides in membranes. Biochemistry 38: 16963–16973.

Inamori Y, Amino H, Tsuboi M, Yamaguchi S & Tsujibo H

(1990) Biological activities of racemomycin-B, beta-lysine rich

streptothricin antibiotic, the main component of Streptomyces

lavendulae OP-2. Chem Pharm Bull (Tokyo) 38: 2296–2298.

Isner JM (1999) Manipulating angiogenesis against vascular dis-

eases. Hosp Pract (Off Ed) 34: 69–74, 76, 79–80.

Ivashkin VT, Tel’nykh IuV, Kuznetsov EN & Kolokol’nikova OA

(1999) Use of biologically active food additive cardiohels in

the treatment of patients with ischemic heart disease. Klin Med

(Mosk) 77: 34–36.

Jaschke E, Zabernigg A & Gattringer C (1999) Recombinant hu-

man Granulocyte-macrophage colony stimulating factor applied

locally in low doses enhances healing and prevents recurrence of

chronic venous ulcers. Int J Dermatol 38: 380–386.

Jenkins TC (2000) Feeding oleamide to lactating Jersey cows 1.

Effects on lactation performance and milk fatty acid compositio.

J Dairy Sci 83: 332–337.

Jennings R, Simms JR & Heath AW (1998) Adjuvants and delivery

systems for viral vaccines-mechanisms and potential. Dev Biol

Stand 92: 19–28.

Jia C, Chen B & Arnold F (1998) The effect of ultrapure hyaluronic

acid with different molecular weights on the healing of porcine

full thickness skin wound. Chung Kuo Hsiu Fu Chung Chien Wai

Ko Tsa Chih 12: 197–200.

Jo EC, Park HJ, Kim D & Moon HM (1992) Step fortiﬁcations

of nutrients in mammalian cell culture. Biotechnol Bioeng 42:

1218–1226.

Jo EC, Park HJ, Kim D & Moon HM (1993) Repeated fed-

batch culture of hybridoma cells in nutrient-fortiﬁed high-density

medium. Biotechnol Bioeng 42: 1229–1237.

Junienez PA & Rampy MA (1999) Keratinocyte growth factor – 2

accelerates wound healing in incisional wounds. J Surg Res 81:

238–242.

Karlsson S, Van Doren K, Schweiger SG et al. (1985) Stable gene

transfer and tissue-speciﬁc expression of a human globin gene

using adenoviral vectors. EMBO J 5: 2377–2385.

Kasper LH & Khan IA (1998) Antigen-speciﬁc CD8

T cells pro-

tect against lethal toxoplasmosis in mice infected with Neospora

caninum. Infect Immun 66: 1554–1560.

Ker-hwa Ou S, Patterson PH (1997) A more efﬁcient and econom-

ical approach for monoclonal Antibody production. J Immunol

209: 105–108.

Kim JJ, Nottingham LK, Tsai A, Lee DJ, Maguire HC, Oh J,

Dentchev T, Manson KH, Wyand MS, Agadjanyan MG, Ugen

KE & Weiner DB (1999) Antigen-speciﬁc humoral and cellular

immune responses can be modulated in rhesus macaques through

the use of IFN-gamma, IL-12, or IL-18 gene adjuvants. J Med

Primatol 28: 214–223.

Kim JJ, Yang JS, Lee DJ, Wilson DM, Nottingham LK, Mor-

rison L, Tsai A, Oh J, Dang K, Dentchev T, Agadjanyan MG,

Sin JI, Chalian AA & Weiner DB (2000) Macrophage colony-

stimulating factor can modulate immune responses and attract

dendritic cells in vivo. Hum Gene Ther 11: 305–321.

Klavinskis LS, Barnﬁeld C, Gao L & Parker S (1999) Intranasal im-

munization with plasmid DNA-lipid complexes elicits mucosal

immunity in the female genital and rectal tracts. J Immunol 162:

243–262.

Kohler G & Milstein (1975) Continuous cultures of fused cells se-

creting antibody of predeﬁned speciﬁcity. Nature 256: 495–497.

Kohn DB, Anderson WF & Blaese MB (1989) Gene therapy for

genetic diseases. Cancer Invest 7: 179–192.

Kolsarici N & Candogan K (1995) The effects of potassium sorbate

and lactic acid on the shelf-life of vaccum-packed chicken meats.

Poult Sci 74: 1884–1893.

Kornowski R et al. (2000) Delivery strategies to achieve therapeutic

myocardial angiogenesis. Circulation 101: 454–458.

Kovarik J & Siegrist CA (1998) Optimization of vaccine responses

in early life: The role of delivery systems and immunomodulat-

ors. Immunol Cell Biol 76: 222–236.

Kratz G, Back M, Amander C & Larun O (1998) Immobilised hep-

arin accelerates the healing of human wounds in vivo. Scand J

Plast Reconstr Surg hand Surg 32: 381–385.

Kreider RB (1999) Dietary supplements and the promotion of

muscle growth with resistance exercise. Sports Med 27: 97–110.

Krishnan L, Dicaire CJ, Patel GB & Sprott GD (2000) Archaeo-

some vaccine adjuvants induce strong humoral, cell-mediated,

and memory responses: comparison to conventional liposomes

and alum. Infect Immun 68: 54–63.

Krup OC, Kroll I, Bose G & Falkenberg FW (1999) Cytokine

depot formulations as adjuvants for tumor vaccines. Liposome-

encapsulated IL-2 as a depot formulation. J Immunother 22:

525–538.

Kumarasinghe MP, Constantine SR & Hemamali RL (1997) Meth-

anol as an alternative ﬁxative for cytological smears. Malays J

Pathol 19: 137–140.

Kun LE, Gajjar A, Muhlbauer M et al. (1995) Stereotactic injection

of herpes simplex thymidine kinase vector producer cells (PA

317-G1Tk1SvNa.7) and intravenous ganciclovir for the treat-

ment of progressive or recurrent primary supratentorial pediatric

malignant brain tumors. Hum Gene Ther 6: 1231–1255.

Kundu PK, Bharmal SE, Siva Prasad N & Datta D (1999) Scaled up

culture of stimulated hybridoma cells AE9D6. Indian Chemical

Engineer 41: 113–116.

Kurokawa H, Park YS, Iijima S & Kobayashi T (1994) Growth char-

acteristics in fed-batch culture of hybridoma cells with control

of glucose and glutamine concentrations. Biotechnol Bioeng 44:

95–110.

Kyriahides TR, Tain JW & Bomstein P (1999) Accelerated wound

healing in mice with a disruption of thrombospondin 2 gene. J

Invest Dermatol 113: 782–787.

LaRocca P & Barker S (1996) Tissue culture surface treatments. The

Cell/Line 6: 1–6.

Laham RJ et al. (1999) Local perivascular delivery of basic ﬁbro-

blast growth factor in patients undergoing coronary bypass

surgery: results of a phase I randomized, double-blind, placebo-

controlled trial. Circulation 100: 1865–1871.

Laham RJ et al. (2000) Intrapericardial delivery of FGF2 induces

neo-vascularisation in a porcine model of chronic myocardial

ischemia. J Pharmacol Exp Ther 292: 795–802.

Lai E & van Zanten JH (2001) Monitoring DNA/poly-L-lysine

polyplex formation with time-resolved multiangle laser light

scattering. Biophys J 80: 864–873.

Lansdown AB, Sampson B, Laupattarakasam P & Vultivirojana A

(1997) Silver aids healing in the sterile skin wounds: Experi-

mental studies in Laboratory rat. Br J Dermatol 137: 728–735.

Lee IH, Cho Y & Lehrer RI (1997) Styelins, broad-spectrum anti-

microbial peptides from the solitary tunicate, Styela clava. Comp

Biochem Physiol Biochem Mol Biol 118: 515–521.

Lee LY et al. (2000). Focal angiogen therapy using intramyocar-

dial delivery of an adenovirus vector coding for VEGF121. Ann

Thorac Surg Jan 69: 14–23.

Lee GM & Palsson BO (1993) Stability of antibody productiv-

ity is improved when hybridoma cells are entrapped in calcium

alginate beads. Biotechnol Bioeng 42: 1131–1135.

Levashina EA, Ohresser S, Bulet P, Reichhart JM, Hetru C &

Hoffmann JA (1995) Metchnikowin, a novel immune-inducible

proline-rich peptide from Drosophila with antibacterial and anti-

fungal properties. Eur J Biochem 233: 694–700.

Li J et al. (2000) PR39, a peptide regulator of angiogenesis. Nat

Med 6: 49–55.

Li Q & Chang AE (1999) Adoptive T-cell immunotherapy of cancer.

Cytokines Cell Mol Ther 5: 105–117

Li Q, Normolle DP, Sayre DM, Zeng X, Sun R, Jiang G, Redman

BD & Chang AE (2000) Immunological effects of BCG as an

adjuvant in autologous tumor vaccines. Clin Immunol 94: 64–72.

Liang JF & Kim SC (1999) Not only the nature of peptide but also

the characteristics of cell membrane determine the antimicrobial

mechanism of a peptide. J Pept Res 53: 518–522.

Liechty KW, Nesbit M, Herlyn M, Radu A, Adzick NS &

Crombleholme TM (1999) Adenoviral-mediated overexpression

of PDGF-B corrects ischemic impaired wound healing. J Invest

Dermatol 113: 375–383.

Loomans HJ, Hahn BL, Li QQ, Phadnis SH & Sohnle PG (1998)

Histidine-based zinc-binding sequences and the antimicrobial

activity of calprotectin. J Infect Dis 177: 812–814.

Lu GZ, Thompson BG, Suresh MR & Gray MR (1995) Cultivation

of hybridoma cells in an inclined bioreactor. Biotechnol Bioeng

45, 176–186.

Luengo JM, Revilla G, Lopez MJ, Villanueva JR & Martin JF (1980)

Inhibition and repression of homocitrate synthase by lysine in

Penicillium chrysogenum. Bacteriol 144: 869–876.

Maeda M, Murakami H, Ohta H & Tajima M (1992) Stimulation

of IgM production in human-human hybridoma HB4C5 cells by

chitosan. Biosci Biotechnol Biochem 56: 427–431.

Magovern CJ (1996) Direct in vivo gene transfer to canine myocar-

dium using a replication deﬁcient adenovirus vector. Ann Thorac

Surg 62: 425–433.

Mahvi DM, Sondel PM, Yang NS et al. (1997) Phase I/IB study

of immunization with autologous tumor cells transduced with

the GM-CSF gene by particle-mediated transfer in patients with

melanoma or sarcoma. Hum Gene Ther 8: 875–891.

Malinda KM, Sidhu GS, Mani H, Banaudha K, Maheshwari RK,

Goldstein AL & Kleinman HK (1999) Thymosin beta 4 acceler-

ates wound healing. J Invest Dermatol 113: 364–368.

Maneta-Peyret L, Compere P, Moreau P, Gofﬁnet G & Cassagne C

(1999) Immunocytochemistry of lipids: Chemical ﬁxatives have

dramatic effects on the preservation of tissue lipids. Histochem J

31: 541–547.

Markiewicz MA & Gajewski TF (1999) The immune system as anti-

tumor sentinel:molecular requirements for an anti-tumor immune

response. Crit Rev Oncog 10: 247–260.

Martens DE, De Gooijer CD, Beuvery EC & Tramper J (1992)

Effect of serum concentration on hybridoma viable cell density

and production of monoclonal antibodies in CSTRs and on shear

sensitivity in air-loop reactors. Biotechnol Bioeng 39: 891–897.

Marty HH & Risau W (1999) Angiogenesis in Ischemic disease.

Thromb Haemost 82 (Suppl 1): 44–52.

Masalova OV, Shepelev AV, Atanadze SN, Parnes ZN, Roman-

ova VS, Vol’pina OM, Semiletov IuA & Kushch AA (1999)

Immunostimulating effect of water-soluble fullerene derivatives-

perspective adjuvants for a new generation of vaccine. Dokl

Akad Nauk 369: 411–413.

Massa LF & Arana-Chavez VE (2000) Ultrastructural preserva-

tion of rat embryonic dental tissues after rapid ﬁxation and

dehydration under microwave irradiation. Eur J Oral Sci 108:

74–77.

Matsuda S, Iwata H, Se N & Ikada Y (1999) Bioadhesion of gelatin

ﬁlms crosslinked with gluteraldehyde. J Biomed Mater Res 45:

20–27.

Maxwell AJ, Anderson BE & Cooke JP (2000) Nutritional ther-

apy for peripheral arterial disease: A double-blind, placebo-

controlled, randomized trial of HeartBar. Vasc Med 5: 11–9.

McKeehan WL & Ham RG (1976) Stimulation of clonal growth of

normal ﬁbroblasts with substrata coated with basic polymers. J

Cell Biol 71: 727–734.

McKeehan WL (1984) Methods for Preparation of Media, Sup-

plements, and Substrata for Serum-free Animal Cell Culture

(p. 209), A.R. Liss, New York.

Medina E, Talay SR, Chhatwal GS & Guzman CA (1998)

Fibronectin-binding protein I of Streptococcus pyogenes is a

promising adjuvant for antigens delivered by mucosal route. Eur

J Immunol 28: 1069–1077.

Meng WC, Leung KL, Ho RL, Leung TW & Lau WY (1999)

Prospective randomized control study on the effect of branched-

chain amino acids in patients with liver resection for hepatocel-

lular carcinoma. Aust N Z J Surg 69: 811–815.

Miller AD (1990) Retrovirus packaging cells. Hum Gene Ther 1:

5–14.

Montville TJ & Chen Y (1998) Mechanistic action of pediocin and

nisin: Recent progress and unresolved questions. Am J Contact

Dermat Appl Microbiol Biotechnol 50: 511–519.

Moore GE & Hood DB (1993) Modiﬁed RPMI 1640 culture

medium. In Vitro Cell Dev Biol 29A: 265–267.

Mor A, Hani K & Nicolas P (1994) The vertebrate peptide antibiot-

ics dermaseptins have overlapping structural features but target

speciﬁc microorganisms. J Biol Chem 269: 31635–31641.

Morein B, Villacres-Eriksson M, Sjolander A & Bengtsson KL

(1996) Novel adjuvants and vaccine delivery systems. Vet Im-

munol Immunopathol 54: 373–384.

Moulin V, Lawny F, Barritault D & Caruelle JT (1998) Platelet re-

lease treatment improves skin healing in diabetic rats through

endogenous growth factor secretion. Cell Mol Biol (Noisy-le-

grand) 44: 961–971.

Mowad CM (2000) Allergic contact dermatitis caused by parabens:

2 case reports and a review. Am J Contact Dermat 11: 53–56.

Oikawa S, Nishino K, Oikawa S, Inoue S, Mizutani T & Kawan-

ishi S (1998) Oxidative DNA damage and apoptosis induced by

metabolites of butylated hydroxytoluene. Biochem Pharmacol

56: 361–370.

Mrsny RJ (1998) Adjuvants and delivery issues related to immun-

ization: A survey of the recent patent literature. J Drug Target 6:

243–249.

Murray K, Gull K & Dickson AJ (1996) Dichloroacetate increases

cell and antibody yield in batch cultures of a hybridoma cell line.

Biotechnol Bioeng 49: 377–382.

Nguyen DM, Spitz FR, Yen N et al. (1996) Gene therapy for lung

cancer: Enhancement of tumor suppression by a combination

of sequential systematic cisplatin and adenovirus-mediated p53

gene transfer. J Thorac Cardiovasc Surg 112: 1372–1376.

Oh JE & Lee KH (1999) Synthesis of novel unnatural amino acid

as a building block and its incorporation into an antimicrobial

peptide. Bioorg Med Chem 7: 2985–2990.

Oh JE, Hong SY & Lee KH (1999) The comparison of char-

acteristics between membrane-active antifungal peptide and its

pseudopeptides. Bioorg Med Chem 7: 2509–2515.

Onishi JC, Milligan JA, Basilio A, Bergstrom J, Curotto J, Huang

L, Meinz M, Nallin-Omstead M, Pelaez F, Rew D, Salvatore

M, Thompson J, Vicente F & Kurtz MB (1997) Antimicrobial

activity of viridiofungins. J Antibiot (Tokyo) 50: 334–338.

Ono I, Pateshita T & Inoue M (1999) Effects of a collagen matrix

containing basic ﬁbroblast growth factor on wound contraction.

J Biomed Mater Res 48: 621–630.

Oros G, Ujvary I & Nachman RJ (1999) Antimicrobial activity of

o-carboranylalanine. Amino Acids 17: 357–368.

Osanto S, Brouwenstyn N, Vaessen N et al. (1993) Immunization

with interleukin-2 transfected melanoma cells. A phase I-II study

in patients with metastatic melanoma. Hum Gene Ther 4: 323–

330.

Ozturk SS & Palsson BO (1991) Physiological changes during

the adaptation of hybridoma cells to low serum and serum-free

media. Biotechnol Bioeng 37: 25–46

Pandit A, Ashar R & Feldman D (1999) The effect of TGF-b de-

livered through a collagen scaffold on wound healing. J Invest

Surg 12: 89–100.

Park CJ, Park CB, Hong SS, Lee HS, Lee SY & Kim SC

(2000) Characterization and cDNA cloning of two glycine- and

histidine-rich antimicrobial peptides from the roots of Shep-

herd’s purse, Capsella bursa-pastoris. Plant Mol Biol 44: 187–

197.

Parke DV & Lewis DF (1992) Safety aspects of food preservatives.

Food Addit Contam 9: 561–577.

Pedersen KL, Pedersen SN, Christiansen LB, Korsgaard B & Bjer-

regaard P (2000) The preservatives ethyl-, propyl- and butylpa-

raben are oestrogenic in an in vivo ﬁsh assay. Pharmacol Toxicol

86: 110–113.

Piper PW (1999) Yeast superoxide dismutase mutants reveal a pro-

oxidant action of weak organic acid food preservatives. Free

Radic Biol Med 27: 1219–1227.

Podsakoff G, Wong Jr KK & Chatterjee S (1994) Efﬁcient gene

transfer into nondividing cells by adeno-associated virus-based

vectors. J Virol 68: 5656–5666.

Ramshaw IA, Fordham SA, Bernard CC, Maguire D, Cowden WB

& Willenborg DO (1997) DNA vaccines for the treatment of

autoimmune disease. Immunol Cell Biol 75: 409–413.

Rangell LK & Keller GA (2000) Application of microwave techno-

logy to the processing and immunolabeling of plastic-embedded

and cryosections. J Histochem Cytochem 48: 1153–1160.

Rasik AM, Raghubir R, Gupta A, Shukla A, Dubey MP, Srivast-

ava S, Jain HK & Kulshrutha DK (1999) Healing potential

of Calotropis procera on dermal wounds in Guinea pigs. J

Ethnopharmacol 68: 261–266.

Ray EC, Landis ME & Miller VM (1999) Effects of dietary L-

arginine on the reactivity of canine coronary arteries. Vasc Med

4: 211–217.

Robinson PH, Chalupa W, Sniffen CJ, Julien WE, Sato H, Fujieda

T, Watanabe K & Suzuki H (1999) Inﬂuence of postruminal sup-

plementation of methionine and lysine, isoleucine, or all three

amino acids on intake and chewing behavior, ruminal fermenta-

tion, and milk and milk component production. J Anim Sci 77:

2781–2792.

Rodolfo M & Colombo MP (1999) Interleukin-12 as an adjuvant for

cancer immunotherapy. Methods 19: 114–120.

Ropert C, Malvy C & Couvreur P (1993) Inhibition of the Friend ret-

rovirus by antisense oligonucleotides encapsulated in liposomes:

mechanism of action. Pharm Res 10: 1427–1433.

Rosenberg SA, Aebersold P, Cornetta K et al. (1990) Gene transfer

into humans: immunotherapy of patients with advanced melan-

oma, using tumor-inﬁltrating lymphocytes modiﬁed by retroviral

gene transduction. N Engl J Med 323: 570–578.

Ruszczak Z & Schwartz RA (1999) Collagen uses in dermatology –

An update. J Dermatol 199: 285–289.

Ryan EJ, McNeela E, Murphy GA, Stewart H, O’Hagan D, Pizza

M, Rappuoli R & Mills KH (1999) Mutants of Escherichia coli

heat-labile toxin act as effective mucosal adjuvants for nasal de-

livery of an acellular pertussis vaccine: Differential effects of the

nontoxic AB complex and enzyme activity on Th1 and Th2 cells.

Infect Immun 67: 6270–6280.

Sadelain M & Latouche JB (2000) Induction of human cyto-

toxic T lymphocytes by artiﬁcial antigen-presenting cells. Nat

Biotechnol 18: 405–409.

Sanfeliu A, Cairo JJ, Casas C, Sola C & Godia F (1996) Analysis of

nutritional factors and physical conditions affecting growth and

monoclonal antibody production of the hybridoma KB-26.5 cell

line. Biotechnol Prog 12: 209–216.

Santin AD, Hermonat PL, Ravaggi A, Bellone S, Cowan C, Kor-

ourian S, Pecorelli S, Cannon MJ & Parham GP (2000) Develop-

ment, characterization and distribution of adoptively transferred

peripheral blood lymphocytes primed by human papillomavirus

18 E7-pulsed autologous dendritic cells in a patient with meta-

static adenocarcinoma of the uterine cervix. Eur J Gynaecol

Oncol 21: 17–23.

Scannell AG, Hill C, Buckley DJ & Arendt EK (1997) Determ-

ination of the inﬂuence of organic acids and nisin on shelf-life

and microbiological safety aspects of fresh pork sausage. J Appl

Microbiol 83: 407–412.

Schlecht S, Wiesmuller KH, Jung G & Bessler WG (1993)

Lipopeptides as natural adjuvants for vaccines from Gram-

negative bacteria. Naturwissenschaften 80: 9–17.

Schrijver IA, Melief MJ, van Meurs M, Companjen AR & Laman

JD (2000) Pararosaniline ﬁxation for detection of co-stimulatory

molecules, cytokines, and speciﬁc antibody. J Histochem Cyto-

chem Jan 48(1): 95–103.

Schwarzenberger P, Huang W, Oliver P, Osidipe T, Theodossiou C

& Kolls JK (2001) Poly-L-lysine-based molecular conjugate vec-

tors: a high efﬁciency gene transfer system for human progenitor

and leukemia cells. Am J Med Sci 321: 129–136.

Seo SH, Pei J, Briles WE, Dzielawa J & Collisson EW (2000) Ad-

optive transfer of infectious bronchitis virus primed alphabeta T

cells bearing CD8 antigen protects chicks from acute infection.

Virology 269: 183–189.

Serth J, Kuczyk MA, Paeslack U, Lichtinghagen R & Jonas U

(2000) Quantitation of DNA extracted after micropreparation of

cells from frozen and formalin ﬁxed tissue sections. Am J Pathol

156: 1189–1196.

Shedlock DJ & Weiner DB (2000) DNA vaccination: Antigen

presentation and the induction of immunity. J Leukocyte Biol 68:

793–806.

Sheu BC, Hsu SM, Ho HN, Lin RH & Huang SC (1999) Tu-

mor immunology-when a cancer cell meets the immune cells.

J Formos Med Assoc 98: 730–735.

Shibutani M, Uneyama C, Miyazaki K, Toyoda K & Hirose M

(2000) Methacarn ﬁxation: A novel tool for analysis of gene

expressions in parafﬁn-embedded tissue specimens. Lab Invest

80: 199–208.

Sidhu GS, Singh AK, Banaudha KK, Gaddipati JP, Patuaik GK &

Maheshwari RK (1999a) Arnebin-1 accelerates normal and hy-

drocortisone induced impaired wound healing. J Invest Dermatol

113: 773–781.

Sidhu GS, Mani H, Gaddipati JP, Singh AK, Seth P, Banaudha

KK, Patnaik GK & Maheshwari RK (1999b) Curcumin en-

hances wound healing in streptozotocin induced diabetic rats and

genetically diabetic mice. Wound Repair Regen 7: 362–374.

Sin JI, Bagarazzi M, Pachuk C & Weiner DB (1999) DNA priming-

protein boosting enhances both antigen-speciﬁc antibody and

Th1-type cellular immune responses in a murine herpes simplex

virus-2 gD vaccine model. DNA Cell Biol 18: 771–779.

Singh M & O’Hagan D (1999) Advances in vaccine adjuvants. Nat

Biotechnol 17: 1075–1081.

Soda H, Koda K, Yasutomi J, Oda K, Takiguchi N, Saito N & Na-

kajima N (1999) Adoptive immunotherapy for advanced cancer

patients using in vitro activated cytotoxic T lymphocytes. J Surg

Oncol 72: 211–217

Sreter L (1999) The role of nutrition in the pathogenesis and

prevention of oncologic diseases. Orv Hetil 140: 2275–2283.

Stanchev M, Tabakova S, Videnov G, Golovinsky E & Jung G

(1999) Synthesis and antimicrobial activity in vitro of new amino

acids and peptides containing thiazole and oxazole moieties.

Arch Pharm (Weinheim) 332: 297–304.

Sugahara T, Shirahata S, Akiyohsi K, Isobe T, Okuyama T &

Murakami H (1991) Immunoglobulin production stimulating

factor-IIa (IPSF IIα) is a glyceraldehyde-3-phosphate dehydro-

genase like protein. Cytotechnology 6: 115–120.

Sugahara T, Sasaki T & Murakami H (1994) Enhancement of im-

munoglobulin productivity of human-human hybridoma HB4C5

cells by basic proteins and poly-basic amino acids. Biosci Bi-

otech Biochem 58: 2212–2214.

Sugahara T, Furutani H & Sasaki T (1997) Alcohol dehydrogenase-I

from horse liver stimulates immunoglobulin production by hu-

man hybridoma and human peripheral blood lymphocytes. Mol

Cell Biochem 173: 113–9.

Sugahara T, Shimizu S, Abiru M, Matsuoka S & Sasaki T (1998)

A novel function of enolase from rabbit muscle; an immuno-

globulin production stimulating factor. Biochim Biophys Acta

1380: 163–176.

Takahashi K, Onodera K & Akiba Y (1999) Effect of dietary xylitol

on growth and inﬂammatory responses in immune stimulated

chickens. Br Poult Sci 40: 552–4.

Talwar M, Moyana TN, Bharadwaj B & Tan LK (1996) The effect

of synthetic analogue of PGE2 on wound healing in rats. Ann

Clin Lab Sci 26: 451–457.

Tam JP, Lu YA, Yang JL & Chiu KW (1999) An unusual structural

motif of antimicrobial peptides containing end-to-end macro-

cycle and cystine-knot disulﬁdes. Proc Natl Acad Sci USA 96:

8913–8918.

Tascon RE, Soares CS, Ragno S, Stavropoulos E, Hirst EM &

Colston MJ (2000) Mycobacterium tuberculosis-activated dend-

ritic cells induce protective immunity in mice. Immunology 99:

473–480.

Tawil NJ, Connors D, Gies D, Bennet S, Gruskin E & Mustoe T

(1999) Stimulation of wound healing by positively charged dex-

tran beads depends upon clustering of beads and cells in close

proximity to the wound. Wound Repair Regen 7: 389–399.

Temin HM (1986) Retrovirus vectors for gene transfer: Efﬁcient

integration into and expression of exogenous DNA in verteb-

rate cell genomes. In: R Kucherlapati (ed.), Gene Transfer

(pp. 144–187) Plenum Press, New York.

Thierry AR & Dritschilo A (1992) Intracellular availability of

unmodiﬁed, phosphorothioates and liposomal encapsulated oli-

godeoxynucleotides for antisense activity. Nucleic Acids Res 20:

5691–5698.

Thierry AR, Rahman A & Dritschilo A (1993) Overcoming mul-

tidrug resistance in human tumor cells using free and lipo-

somally encapsulated antisense oligodeoxynucleotides. Biochem

Biophys Res Commun 190: 952–960.

Thurner B, Haendle I, Roder C, Dieckmann D, Keikavoussi P,

Jonuleit H, Bender A, Maczek C, Schreiner D, von den Driesch

P, Brocker EB, Steinman RM, Enk A, Kampgen E & Schuler

G (1999) Vaccination with mage-3A1 peptide-pulsed mature,

monocyte-derived dendritic cells expands speciﬁc cytotoxic T

cells and induces regression of some metastases in advanced

stage IV melanoma. J Exp Med 190: 1669–1678.

Tjomsland O (1999) Transmyocardial laser treatment – A new

treatment modality in pronounced angina pectoris. Tidsskr Nor

Laegeforen 119: 3597–3601.

Toes RE, Offringa R, Feltkamp MC, Visseren MJ, Schoenberger

SP, Melief CJ & Kast WM (1994) Tumor rejection antigens and

tumor speciﬁc cytotoxic T lymphocytes. Behring Inst Mitt 94:

72–86.

Tomita S et al. (1999) Autologous transplantation of bone marrow

cells improves damaged heart function. Circulation 100 (Suppl

II): 247–256.

Trengove NJ, Stacey MC, Mac Auley S, Bennet N, Gibson J,

Burslem F, Murphy G & Schultz G (1999) Analysis of acute

and chronic wound environments: the role of proteases and their

inhibitors. Wound Repair Regen 7: 442–452.

Tripp MW, Hoagland TA, Dahl GE, Kimrey AS & Zinn SA (1998)

Methionine and somatotropin supplementation in growing beef

cattle. J Anim Sci 76: 1197–203.

Ulrich JT & Myers KR (1995) Monophosphoryl lipid A as an ad-

juvant. Past experiences and new directions. Pharm Biotechnol

6: 495–524.

Van Kraaij C, Breukink E, Noordermeer MA, Demel RA, Siezen RJ,

Kuipers OP & De Kruijff B (1998) Pore formation by nisin in-

volves translocation of its C-terminal part across the membrane.

Biochemistry 37: 16033–16040.

Van Slooten ML, Storm G, Zoephel A, Kupcu Z, Boerman O, Crom-

melin DJ, Wagner E & Kircheis R (2000) Liposomes containing

interferon-gamma as adjuvant in tumor cell vaccines. Pharm Res

17: 42–48.

Vogel FR (1998) Adjuvants in perspective. Dev Biol Stand 92: 241–

248.

Voigt A & Zintl F (1999) Hybridoma cell growth and anti-

neuroblastoma monoclonal antibody production in spinner ﬂasks

using a protein-free medium with microcarriers. J Biotechnol 68:

213–226.

Von Hunolstein C, Teloni R, Mariotti S, Recchia S, Oreﬁci G &

Nisini R (2000) Synthetic oligodeoxynucleotide containing CpG

motif induces an anti-polysaccharide type 1-like immune re-

sponse after immunization of mice with haemophilus inﬂuenzae

type b conjugate vaccine. Int Immunol 12: 295–303.

Wang B, Ugen KE, Srikantan V, Agadjanyan MG, Dang K, Re-

faeli Y, Sato A, Boyer J, Williams WV & Weiner DB (1993)

Gene inoculation generates immune responses against human

immunodeﬁciency virus type 1. Proc Natl Acad Sci USA 90:

4156–4160.

Wang B, Boyer JD, Srikantan V, Coney L, Carrano R, Phan C,

Merva M, Dang K, Agadjanyan MG, Ugen KE, Williams MV &

Weiner DB (1993a) DNA inoculation induces neutralizing im-

mune responses against human immunodeﬁciency virus type 1

in mice and non-human primates. DNA Cell Biol 12: 799–805

Wang B, Dang K, Agadjanyan MG, Srikantan V, Li F, Ugen KE,

Boyer J, Merva M, Williams WV & Weiner DB (1997) Mucosal

immunization with a DNA vaccine induces immune responses

against HIV-1 at a mucosal site. Vaccine 15: 821–825.

WangS,LiuX,FisherK,SmithJG,ChenF,ToberyTW,Ulmer

JB, Evans RK & Caulﬁeld MJ (2000) Enhanced type I immune

response to a hepatitis B DNA vaccine by formulation with

calcium- or aluminum phosphate. Vaccine 18: 1227–1235.

Weber J (1996) Distinguishing between response to HIV vaccine

and response to HIV. Lancet 350: 230–231.

Woodworth JC, Goodband RD, Nelssen JL, Tokach MD & Musser

RE (2000) Added dietary pyridoxine, but not thiamin, improves

weanling pig growth performance. J Anim Sci 78: 88–93.

Xie L & Wang DIC (1994a) Stoichiometric analysis of animal cell

growth and it’s application in medium design. Biotechnol Bioeng

43: 1164–1174.

Xie L & Wang DIC (1994b) Fed-batch cultivation of animal cells us-

ing different design concepts and feeding strategies. Biotechnol

Bioeng 43: 1175–1189.

Yamada M, Lewis JA & Grodzicker T (1985) Overproduction of

the protein product of a nonselected foreign gene carried by an

adenovirus vector. Proc Natl Acad Sci USA 82: 3597–3571.

Yamakawa K, Karasawa T, Ohta T, Hayashi H & Nakamura S

(1998) Inhibition of enhanced toxin production by Clostridium

difﬁcle in biotin-limited conditions. J Med Microbiol 47: 767–

771.

Yavin E & Yavin Z (1974) Attachment and culture of dissociated

cells from rat embryo cerebral hemispheres on polylysine-coated

surface. J Cell Biol 62: 540–546.

Zhang H, Gu X, Wang Q & Becker JM (1993) Preliminary exper-

iments on the mode of action of oxalysine on Candida albicans

and C. parapsilosis. Wei Sheng Wu Hsueh Pao 33: 411–417.

Zhou J, Song Y & Wang Y (1997) The study of epidermal growth

factor in acceleration of skin wound healing. Chung Kuo HsinFu

Chung Chien Wai Ko Tsa Chih 11: 267–268.