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Infectious microorganisms (parasites, bacteria, viruses) which caused dramatic epidemics in human populations throughout the centuries, as well as their natural reservoirs and possible vectors, have not disappeared... Plague, cholera, malaria, tuberculosis, rickettsioses are still quite present in some areas of the world. Furthermore new infectious diseases caused by emerging pathogens have recently appeared dramatically, such as AIDS. Eradication of the most severe and lethal infectious diseases remains nevertheless an ambitious but not utopian objective, if one considers the spectacular advances made, more or less recently, in the field of specific vaccinations. For instance, smallpox has been eradicated around 1980. Present efforts are mainly concentrated on poliomyelitis, the eradication of which was hoped to take place in year 2005, but which is still present in a few areas. Eradication of measles, with the presently available vaccine, is a reasonable objective. Eradication of malaria appears to be much more difficult since this infection involves a complex parasite and an insect vector, the mosquito. And, as far as AIDS is concerned, intensive research has not yet led to the conception of an effective vaccine.

A new 7,8-methylenedioxy analogue (4) of (+)-porothramycin B (2) and its water-soluble sodium bisulfite derivative (15) have been synthesized in high yields and have been shown to exhibit high cytotoxic activities against several tumor cell lines. The new pyrrolo[2,1-c][1,4]benzodiazepine 4 was as effective against the resistant cell lines as against the doxorubicin-sensitive cell lines tested.

A series of 1-cyano and 2-cyanohexahydroindolizino[8,7-b]indole derivatives was prepared by 1,3-dipolar cycloaddition of acrylonitrile with ylides derived from 3,4-dihydro-beta-carboline and its 6-methoxy, 6-benzyloxy, 9-methyl and 9-benzyl analogues. The products, together with their reduced 1- or 2-aminomethyl derivatives, were evaluated for cytotoxic activity in L1210 cancer cells. Compounds derived from 6-benzyloxy or 9-benzyl-3,4-dihydro-beta-carboline were found to be the most active, with IC(50)'s in the 2-50 microM range. Of these, two compounds, the 1- and 2-cyano 8-benzyloxyindolizino[8,7-b]indole derivatives 20a and 20c, respectively, were found by cytometric flux analysis to stop cancer cell growth at the G(2)M and 8N (>G(2)M) stage of the cell cycle. These two compounds also showed no loss of cytotoxic activity in K562R cancer cells resistant to doxorubicin.

In the first part of this review, AIDS, prion diseases, Hantavirus and arbovirus infections, Ebola hemorrhagic fever, legionellosis, hepatitis C, enterotoxigenic Escherichia coli infections, Lyme disease, tuberculosis have provided alarming examples of emerging or reemerging infectious diseases. In this second part, the stress is placed on the reemergence of diphtheria and of serious streptococcal infections, on bartonelloses, Chlamydia infections, fungal infections, while malaria and cholera are still prevalent in several areas. The increasing resistance of too many pathogens to antimicrobial agents is a major source of concern, directly related to the challenge of nosocomial infections. An infectious cause has been demonstrated (or strongly suspected) for various diseases and the scope of infectiology keeps widening, while the threat of bioterrorism cannot be neglected. The causes of the emergence or reemergence of infectious diseases are multiple and diverse, often in direct relation with human activities (population migrations, changes in husbandry or farming practices, worldwide exchanges of goods and foods, inadequate uses of antibiotics) but also with climatic variations in several areas. The challenge represented by this unexpected comeback of infections to the forefront of human and animal pathology can only be met with a significant improvement of hygienic practices, cessation of certain dangerous behaviors and also, of course, with the development of novel antimicrobial molecules (acting on original targets) as well as of a whole series of new specific vaccines.

Gossypol, gossypolone, reduced gossypol and new Schiff's bases of racemic gossypol and gossypolone were extracted or synthesized. Their cytotoxic activities on KB human cancer cells were determined. Gossypolone and the ethylamine derivative of gossypolone were the most active compounds (IC(50) in the micromolar range in both cases). The cytotoxicity of gossypol and gossypolone was increased when the tests were performed in the absence of serum and decreased when catalase as well as mannitol were added to the culture medium.

Many chemical entities, either from natural sources or prepared by synthesis, are known to exert stimulating activities on various functions of the immune system, such as antibody production, resistance to infections, rejection of malignant cells, etc. In this review, the origin, chemical structures and main activities of several immunostimulants are described, with special emphasis on their present or potential medical usefulness. An attempt is made to envisage the future of this type of pharmacological agents, excluding however from the presentation the endogenous modulators of the immune system (cytokines), the production and activities of which are influenced by the immunostimulants themselves.

The non-enveloped picornaviruses, which are particularly resistant to physicochemical inactivation, include the aetiological agents of poliomyelitis, hepatitis A and E and infectious common cold (rhinovirus). In this work we used human rhinovirus type 5 (RV-5) cultivated in VERO cells to study the photoinactivating effects of several phthalocyanines and naphthobenzoporphyrazines. Free RV-5 was photoinactivated by aluminium trisulphonated naphthobenzoporphyrazine at 5 x 10(-8) M concentration. This photosensitizer was also active on replicating virus when the infected VERO cells were treated with 5 x 10(-6) M concentration followed by a very short illumination period. On the other hand, the ZnPc(3-MeO-Py)4 phthalocyanine, which possesses four positive charges, does not photoinactivate free rhinovirus, but this molecule protects VERO cells against RV-5 infection when added to the cultures before virus inoculation, in the presence or absence of subsequent illumination, and may therefore be considered as an antiviral agent in itself.

In the novel field of immunopharmacology, one finds two main classes of potential drugs: immunosuppressive agents (autoimmune disorders, transplantations) and immunostimulants. The latter aim at stimulating the humoral and cell-mediated functions of the immune system, in order to treat various immunodeficiency states and to increase defenses against infectious agents and cancer cells. Some immunostimulants (the so-called adjuvants) enhance the immunogenicity of vaccines. Immunostimulants act directly on various cell populations of the immune system and modulate the production and activity of molecular mediators of the latter (cytokines). From a chemical perspective, one must distinguish between natural macromolecular immunostimulants obtained by extraction or genetic engineering and compounds prepared by chemical synthesis. In the latter class, we find many molecules the structures of which derive from those of natural substances (eucaryotic and procaryotic peptides) and a large number of purely synthetic compounds. The latter belong to a wide variety of chemical families and their mechanisms of action are not always perfectly elucidated. Presently, whereas natural macromolecular immunostimulants (bacterial extracts, cytokines, growth factors) are being increasingly used in human medicine, only a small number of synthetic immunostimulants are beyond the stage of preclinical evaluation or phase I clinical studies.

In man, humoral immunity against influenza viruses (induced by presently available vaccines) is exerted by local and circulating antibodies against two surface antigens of the virions: hemagglutinin (HA) and neuraminidase (NA). Within each major antigenic type of influenza virus (A, B and C) these antibodies are strictly specific of the HA and NA characterizing variable sub-types, which appear in the course of time from an epidemic to the next one. There exists however a cell-mediated facet of immunity: study of the experimental infection of laboratory mice with mouse-adapted viruses has shown that this cellular immunity is type-specific and therefore covers all the sub-types of a given type of virus. This heterologous immunity is primarily mediated by cytotoxic T lymphocytes (CTL) which recognize an internal nucleoprotein (NP) common to all the sub-types. These CTL are able to lyse influenza virus-infected cells and contribute to local production of interferon in the course of infection. Such mechanisms likely play a major role in the natural resistance of the host to this infection. Thanks to the molecular characterization of the NP protein, it is now possible to conceive the design of vaccines endowed with a wider spectrum than those presently used; in association with the latter, it should become possible in the future to stimulate efficiently both facets of anti-influenza immunity.

In this paper we report that a human erythroleukemia cell line made 100 times more resistant than the parental line to the cytostatic activity of doxorubicin and spontaneously 500-1000 times more resistant to the cytostatic activity of an unrelated drug, namely taxol, exhibits on the other hand unchanged susceptibility to the photosensitizing activity of a sulfonated phthalocyanine.

Rhinoviruses are the main etiologic agents of infectious common colds, which represent about 40% of the acute respiratory infections in man. The antigenic diversity of rhinoviruses precludes any prevention by vaccination. Within the last 20 years, efforts have therefore concentrated on chemoprophylaxis or chemotherapy with antiviral agents. Interferons (alpha and beta) administered intranasally at high doses, exerted a significant prophylactic activity in volunteers inoculated with rhinoviruses or in the course of epidemics in families. By contrast, the therapeutic activity of interferons is almost nonexistent, which greatly limits their practical interest. Various synthetic chemicals exert a marked and selective inhibitory effect on the replication of various serotypes of rhinoviruses in cell cultures. In the absence of an animal model of rhinovirus infection, in vivo studies of these molecules have been performed in human volunteers, chiefly at the Common Cold Research Unit in Salisbury (Great Britain). 3 synthetic compounds (an imidazothiazole, a benzimidazole derivative and a piperazinyl-pyridazine) have exerted a significant prophylactic activity, especially marked with the latter. None of these compounds, however, was active when administered after the infectious challenge. The search for selective anti-rhinovirus compounds is still going on; it will probably be facilitated through a combination of structural studies of rhinovirus capsids (or of their cellular receptors) with computer-assisted design of synthetic molecules. The recent observation of the influence of psychological factors on the propensity of rhinovirus-infected subjects to develop clinically apparent common cold must be taken into account in future studies.

Infection of mice with subtype A0 OR A2 human influenza viruses, by a non-respiratory route causing no lethality, renders the animals markedly resistant to subsequent respiratory challenge with a strain differing from the first one through its haemagglutinin and neuraminidase antigens. This state of heterotypic immunity which appears rapidly (5 days) after the first infection, manifests itself during the second infection by a much reduced mortality, by less extensive lung lesions than in the control mice and by a final drop in lung virus titre (while in controls this titre stays at a high level until death) associated with a rapid rise of serum antibody levels against the haemagglutinin of the challenge virus and the "soluble" antigen common to type A strains. The development of this state of heterotypic immunity is dependent on the capacity of the first virus inoculated to replicate actively in the mouse. The role played by cell-mediated immunity in this phenomenon is evidenced by the fact that both the induction and the expression of this state of heterotypic resistance may be abolished by treatment of the mice with anti-thymocyte serum, while they are not affected by cyclophosphamide. Furthermore, in the mouse infected with an influenza A0 or A2 virus, it has been possible to demonstrate completely cross-reactive delayed type hypersensitivity reactions against the virions or their products. The fact that heterotypic immunity is not demonstrable between influenza viruses of type A and B favors the hypothesis that an antigen (matrix or nucleoprotein) common to all A subtypes--but different in B type strains--plays a role in these reactions of cross-protection.

A synthetic compound, 2-[(1,5,10,10a-tetrahydro-3H-thiazolo[3,4b]isoquinolin-3-ylidene) amino]-4-thiazoleacetic acid (S), 44 081 R.P., inhibits the multiplication of rhinoviruses in cell cultures. Of the 69 rhinovirus strains and serotypes that have been studied, 39% were inhibited at a concentration of 7 micrograms/ml, far below that which affects cellular metabolism (250 micrograms/ml). Preliminary data indicate that the compound inhibits some early events of virus replication but that some cellular functions are also involved in its mechanism of action. Despite its antiviral activity in vitro, the compound, when self-administered intranasally as a 0.2% solution to volunteers from the day before to 5 days after inoculation with a human rhinovirus strain, had no significant effect on rhinorrhea, clinical score, or laboratory evidence of infection.

Delipidated human casein was digested with trypsin and the enzymatic digest was fractionated on Sephadex G-50. The peptidic fractions were assayed for immunomodulating activity in two in vitro models. Fractions corresponding to molecular weights in the range of 2,000 +/- 600 were found to possess stimulating activity in these models.

The peptides LAla-DGlu (L-alanyl-D-glutamic acid), LAla-DGlu[LLA2pm(Gly)]NH2, i.e. N2-(L-alanyl-D-gamma-isoglutaminyl)-N6-glycyl-LL-2,6-diaminopimelic acid, and LAla-DGlu[LLA2pm(Gly)-D-Ala], i.e. N2-(L-alanyl-D-gamma-glutamyl)-N6-glycyl-LL-alpha-2,6-diaminopimelyl-D-alanine, extracted from a Streptomyces strain and their immunostimulating synthetic lauroyl derivatives were studied by 1H NMR spectroscopy at 270 MHz. The chemical shift analysis confirms (a) the sequence of these compounds, (b) the occurrence of a gamma bond between Glu (or GluNH2) and A2pm, (c) the presence of a carboxamide group on the backbone of the tetrapeptide LAla-DGlu[LLA2pm(Gly)]. Temperature and pH studies strongly suggest that the tetrapeptide and pentapeptide exist under several folded conformations characterized by a proximity between the NH3 group of Gly and the Glu (or GluNH2) residue. THe similarity between the chemical shifts of the corresponding protons in LAla-DGlu, lauroly-LAla-DGlu and muramyl-L:Ala-DGluNH2 shows that the coupling of the dipeptide with a lauroyl or a muramyl group does not induce significant changes in the structure of the peptide moiety. In the lipopeptide series the presence of the lauroyl chain leads to amphipathic substances which form, micelles in aqueous solution as shown by the large increase of the proton linewidths. The immunostimulating properties of this kind of peptide manifest themselves at very low concentration and only after their coupling with a sugar or lipid moiety. These features can be interpreted by the formation of mixed complexes between the immunoadjuvants and cell membrane constituents.

The tetrasaccharide-heptapeptide (TH), when injected with mineral oil, exerted a strong adjuvant effect. It stimulated B and T cells, but did not increase the phagocytic activity of the reticulo-endothelial system. While BCG exerted significant preventive effect on the growth of sarcoma 180, leucosarcomatosis an EHRLICH ascitic tumor, TH, at the doses used, was devoid of such activity.

CD-1 or OF-1 mice were inoculated intravenously with 1 mg per mouse (i.e. about 10(6) live bacilli) of Pasteur Institute BCG and challenged 15 to 31 days later with the following viruses introduced by various routes: encephalomyocarditis, murine hepatitis, type 1 and 2 herpes simplex, foot-and-mouth disease and A0 and A2 influenza viruses. In most cases, BCG-inoculated mice exhibited a significantly higher resistance to these lethal infections than control mice (overall survival in control: 18%; in BCG-inoculated mice: 41%). Enhancement of resistance by BCG was especially marked in infections with encephalomyocarditis, herpes simplex type 1 and influenza A2 viruses. Intercurrent infection of BCG-inoculated mice with non lethal doses of viruses did not abrogate their resistance towards subsequent challenge with lethal doses of an unrelated virus. The possible mechanisms of this enhancing effect of BCG on host's resistance are discussed in the light of the known effects of this immunostimulating agent on the various facets of the immune response and of the respective roles the latter play in the defence against virus infections.

Spleen cells from mice inoculated with partially purified preparations of interferon (Sp. Act. 1 X 10(7) i.u./mg protein, 0.2 ml i. v./mouse) were stimulated in vitro with phytohemagglutinin, concanavalin A or lipopolysaccharide. After 2 days of stimulation, the incorporation of 3H-thymidine into TCA-insoluble radioactivity was inhibited 50-90% when compared with cells from animals inoculated with mock interferon. Maximal inhibition, with optimal doses of lectins was obtained when interferon was;inoculated 18 hours before. This effect of interferon on DNA synthesis was preceeded by inhibition of the incorporation of 3H-uridine into TCA-insoluble material. When cells were pretreated in vitro with interferon for 24 hours and subsequently stimulated with PHA, RNA synthesis was inhibited by 30-40%, whatever was the dose of the mitogen. The synthesis of 4S tRNA, 18S and 28S ribosomal RNAs were inhibited to the same degree by interferon. The incorporation of methyl groups into cytoplasmic sRNA was unaltered.

From crude extracts of a Streptomyces strain exhibiting immunopotentiating effects, a tetrapeptide was isolated and its structure established as L Ala leads to D isoGlu leads to L, L Dap comes from Gly. This peptide was devoid of biological activity but its chemical coupling with lauric acid gave a substance endowed with adjuvant and immunostimulating properties. This substance and the corresponding synthetic lauroyltetrapeptide were as active in this respect as the muramyl-dipeptide, thus far considered as the minimal adjuvant-active structure of bacterial cell walls: the presence of a sugar moiety is therefore not a prerequisite for immunopotentiating activities.

Water-soluble substances have been extracted from two strains of Mycobacterium tuberculosis var. hominis: the native hydrosoluble part (polysaccharide and peptidoglycan), a substance in which the polysaccharide moiety is less abundant than in the latter, the acetylated peptidoglycan and, finally a tetrasaccharide-heptapeptide. All four types of substances, when they were injected together with Freund's incomplete adjuvant, exerted an adjuvant effect on the production of delayed-type hypersensitivity to ovalbumin in the guinea pig and on the production of anti-influenza virus antibodies in the rabbit. Injected intravenously in the mouse, they increased the number of antibody-producing cells in the spleen and enhanced the graft versus host reaction; no effect was seen on the phagocytic activity of the reticulo-endothelial system. By contrast with wax D, the water-soluble substances were devoid of arthritis-inducing activity in the rat. Altogether, these water-soluble substances seem to be endowed with at least some of the adjuvant activities of Freund's complete adjuvant and some of the immunostimulant activities of a live Mycobacterium like BCG.

A hexapeptide obtained from human casein by enzymatic digestion has been purified, sequenced and synthesized; its structure is: Val-Glu-Pro-Ile-Pro-Tyr. In vitro this hexapeptide stimulates the phagocytosis of opsonized sheep red blood cells by murine peritoneal macrophages. Administered intravenously to adult mice, it enhances the resistance to infection with Klebsiella pneumoniae.

Intraperitoneal treatments with D-glucosamine, an inhibitor of the glycosylation of the viral envelope, decreased the growth rate of tumors induced in quails or in chicks by Rous sarcoma virus and increased the survival of mice inoculated with human influenza virus.

31P High resolution nuclear magnetic resonance studies have been carried out on the P388D1 tumoral cell line and the BCG elicited alveolar rabbit macrophages both in sedimented cells and in perfused agarose-embedded cells. When the cells were sufficiently oxygenated, the phosphorylated sugars and ATP concentrations attained high levels. The intensity of the peak representing phosphorylated sugars varied inversely with ATP level when macrophagic cells were treated by NaF. The identities of the phosphorylated sugars were revealed by 1H and 31P NMR studies of the P 388D1 cells perchloric extracts.

A large number of peptides, in most cases of low or relatively low molecular weight, exert immunomodulating activities, i.e., they interact in vitro with various cell populations of the immune system and, in vivo, enhance or depress, according to the case, cell-mediated and humoral immune functions. Immunomodulating peptides include glycopeptides from the bacterial cell wall, natural acyloligopeptide ciclosporin, peptidic hormones from the thytide cyclosporine, peptidic hormones from the thymus, peptidic fragments of immunoglobulins and other plasma proteins, as well as peptides isolated from food proteins. An amazing diversity of structure exists among the various immunomodulating peptides. The molecular mechanisms of interaction between these peptides and the cells of the immune system remain, in most cases, to be elucidated. Possibilities of therapeutic applications exist for many of these immunomodulating peptides: one of them (ciclosporin) is widely used as an immunosuppressive drug, several others (glycopeptides, lipopeptides, tuftsin, thymic peptides) are under clinical investigation as immunostimulating and/or immunorestoring agents.

Ammonium 5-tungsto-2-antimoniate (HPA 23) protected micr partially or completely against two strains of encephalomyocarditis (EMC) virus and one strain of vesicular stomatitis (VSV) virus. The best protective effect was obtained with EMC strain VR 129 and VSV when a single i.p. injection of HPA 23 was administered shortly before virus inoculation. Mice protected by HPA 23 against EMC strain VR129 had virus titres in the blood and brain similar to those in untreated mice. A synergism between interferon and HPA 23 was observed in mice infected with EMC VR129. Our results demonstrate the in vivo activity of HPA 23 against two lethal viral infections and suggest that, at least in mice infected with EMC, death may not be related solely to virus multiplication.

Many different approaches have been used, over the last 15 years, for the design of potential immunostimulating drugs: Fractionation of crude natural substances (of eukaryotic or prokaryotic origin) already known to enhance immune functions, followed by chemical characterization and, in many cases, full synthesis of the active moiety: examples are provided by thymic hormones and the muramyldipeptide (MDP); Chemical modification of natural substances of known chemical structure in order to potentiate or change their biological activities or reduce their toxicity: murabutide, lipophilic MDP derivatives, lipopeptides (such as pimelautide), tuftsin analogs; Chemical synthesis (often without preconceived ideas about structure-activity relationship) of a great variety of molecules which are then screened in vitro and in vivo for immunopharmacological activity. The chemical structures and the biological profiles (in terms of possible primary cellular targets and mechanisms of immunostimulating activities) of representatives of class (b) and class (c) immunostimulants are reviewed in this paper.

The induction in mice of a sterile subcutaneous granuloma exerted no influence upon the mortality following their infection with herpes type 1, murine hepatitis or encephalomyocarditis viruses. Attempts to reproduce the resistance -- which has been found to occur as a result of the granulomatous reaction, in the case of bacterial, fungal or protozoa infections and tumour invasions -- by varying the route and timing of the virus inoculation or the strain of mice have failed. We conclude that it is not merely through their inflammatory properties that some non-specific immunostimulating substances enhance resistance against viral infection.

Type I (alpha, beta) and type II (gamma) murine interferons are able to potentiate each other with respect to the inhibition of encephalomyocarditis (EMC) virus and of herpes simplex virus type 1 (HSV-1) multiplication in a murine cell line (DBT). Examination of two double-stranded RNA-dependent enzymes in DBT cells, the 2-5A synthetase and the 67,000 MW protein phosphokinase indicates that mixed interferon preparations act synergistically at least with respect to an increase in the activity of the former enzyme. The results obtained with gamma interferons of different origin and of different specific activity suggest that interferon itself, rather than the lymphokines present in the interferon preparations, is responsible for the synergistic effect.

Relatively low molecular weight peptidoglycan fragments extracted from two strains of Mycobacterium tuberculosis var. hominis were chemically coupled with lauric acid. The fatty acid conjugates were compared with the native substances with respect to some immunopotentiating activities. In vitro, the mitogenic effect on murine spleen lymphocytes was significantly enhanced following conjugation. One of the lauric acid conjugates stimulated, upon intravenous administration in mice, the formation of antibody-producing cells in the spleen, while the native substance was devoid of such activity. In adjuvanticity tests performed in the guinea pig in the absence of mineral oil, the fatty acid conjugates generally exerted a higher adjuvant effect on antibody production or on delayed type hypersensitivity reactions than did the native preparations.

Water-soluble peptidoglycan fragments extracted from the cells of two strains of Mycobacterium tuberculosis var. hominis were chemically conjugated with lauric or with palmitic acid. The coupling reaction was confirmed by physicochemical procedures. The native and the fatty acid-conjugated substances were studied for their adjuvant activity in the induction of delayed type hypersensitivity (DTH) reactions in guinea-pigs and on the production of circulating antibodies in the rabbit. Palmitic acid conjugation of one of the substances increased its adjuvanticity on DTH in the presence of mineral oil; lauric or palmitic acid conjugation rendered the substances adjuvant-active on DTH in the absence of mineral oil. Lauric acid, but not palmitic acid conjugation conferred on both substances an adjuvant activity on antibody production in the absence of mineral oil. Furthermore, lauric acid conjugation of one of the substances led to the appearance of an in vitro mitogen-like activity for murine spleen lymphocytes. In conclusion, fatty acid conjugation exerted a significant modifying effect on the immuno-potentiating activities of these peptidoglycan fragments, and such a chemical procedure may lead to the development of substances exerting a full adjuvant activity without the need of injecting them in an oily vehicle.