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The furan or pyran related hetero cycles play basic role in structural units of nucleic acids (NA) and polysaccharides (PS), significantly predetermining their functional specifics. Some of such properties, in great relevancy for medicine, can be imitated through mimicry of polymers synthetic. Particularly, a formation of similar cycloisomeric chains is possible in process of free-radical cyclocopolymerization of divinyl ether (DVE) and maleic anhydride (MA). The products yielded (DVEMA) of general formula [DVE(MA)-alt-MA]n become precursors for a broad family of water-soluble derivatives capable of wide spectrum of bioactivity, including induction of interferon, immune-stimulated and direct antiviral protection. In this connection, the knowledge: what is content of different heterocyclic isomers in backbone of the preparations and what their partial contributions in promotion of the certain bioactivities observed, are in great importance. Available experimental data (NMR, IR, etc.), controversial for interpretations, didn't elucidate a required estimation of the DVEMA isomerism. The current work represents an independent exploration of the problem via quantum chemistry-based analysis of kinetic (activation barriers) and thermodynamic (enthalpies) priorities in competition between variable isomerism within the chain synthesis. The system is considered in maximal range of hypothetically allowable variations of two levels for double regioselective bifurcations: there are four competitive ways, each of which involves a sequence of four type elementary reactions for a diverse-isomeric formation of chain units. A genesis of six chiral centers (62 stereoisomers permitted) per every of the four part ways was accounted in view for up to 256 isomeric variations in total. The required time-minimized but precisely accurate computations were conducted via B3LYP/6-31G(d), M06-2X/6-311+G(d), M06-2X/6-31+G(2df,p) techniques, which were preselected through model test-systems. As a result, the mechanisms, crucial points and factors for the process-permitted regulation of isomeric content of DVEMA were studied in details. The narrow enough set of most probable enantiomers within highly competitive 5-exo- and 6-endo- ring closing sub-ways was revealed. The results obtained are very actual for an adequate modeling (docking / molecular dynamics) of DVEMA derivatives in their interactions with biopolymer targets, in search for purposed advancement of current background in design and synthesis of highly effective agents for combined antiviral protection (against HIV, flu, herpes, and other infections).Geterotsikly, rodstvennye furanu ili piranu, igraiut bazovuiu rol' v strukturnykh zven'iakh nukleinovykh kislot (NK) i polisakharidov (PS), v sushchestvennoĭ mere opredeliaia ikh funktsional'nye osobennosti. Riad takikh osobennosteĭ, imeiushchikh prakticheskoe znachenie dlia meditsiny, udaetsia imitirovat' s pomoshch'iu mimikrii polimerov sinteticheskikh. Formirovanie skhodnykh tsiklo-izomernykh struktur vozmozhno, v chastnosti, v protsesse svobodno-radikal'noĭ tsiklosopolimerizatsii divinilovogo éfira (DVÉ) i maleinovogo angidrida (MA). Obrazuiushchiesia produkty (DVÉMA) obshcheĭ formuly [DVÉ(MA)-cher-MA]n sluzhat prekursorami obshirnogo semeĭstva vodorastvorimykh proizvodnykh s shirokim spektrom biologicheskoĭ aktivnosti, vkliuchaia induktsiiu interferona, immunostimuliruiushchuiu i priamuiu protivovirusnuiu zashchitu. V sviazi s étim vazhno znat', kakovo sootnoshenie razlichnykh geterotsiklicheskikh izomerov v ostove étikh soedineniĭ i kakova ikh rol' v realizatsii tekh ili inykh vidov nabliudaemoĭ aktivnosti. Imeiushchiesia éksperimental'nye dannye (IaMR, IK i dr.), vvidu neodnoznachnosti ikh interpretatsii, ne pozvoliali priĭti k iasnoĭ otsenke izomernogo sostava DVÉMA. Poétomu v dannoĭ rabote provedeno nezavisimoe kvantovo-khimicheskoe issledovanie s analizom kineticheskikh (énergiia aktivatsii) i termodinamicheskikh (éntal'piia) prioritetov konkurentnoĭ izomerizatsii tsepi v protsesse sinteza. Rassmotrena sistema reaktsiĭ v maksimal'nom diapazone gipoteticheski dopustimykh variatsiĭ dvukh urovneĭ parno-razvetvlennoĭ regioselektivnosti: chetyre konkuriruiushchikh marshruta v posledovatel'nostiakh chetyrekh tipov élementarnykh reaktsiĭ formirovaniia zven'ev tsepi, razlichnykh po strukturnoĭ izomerii. Po kazhdomu iz marshrutov uchten poshagovyĭ genezis shesti khiral'nykh tsentrov (64 stereoizomerov) – do 256 izomernykh variatsiĭ summarno. Ékonomichnyĭ rezhim tochnykh raschetov obespechivalsia metodami B3LYP/6-31G(d), M06-2X/6-311+G(d), M06-2X/6-31+G(2df,p), proshedshimi predvaritel'nuiu selektsiiu na model'nykh test-sistemakh. V rezul'tate detal'no izuchen mekhanizm, kriticheskie tochki i faktory vozmozhnoĭ reguliatsii izomernogo sostava DVÉMA. Ustanovlen dostatochno uzkiĭ interval naibolee veroiatnykh énantiomerov v riadu vysoko konkurentnykh sub-marshrutov 5-ékzo- i 6-éndo- tsiklizatsii. Poluchennye rezul'taty neobkhodimy dlia adekvatnogo modelirovaniia (doking/molekuliarnaia dinamika) proizvodnykh DVÉMA vo vzaimodeĭstviiakh s biopolimernymi misheniami v tseliakh razvitiia imeiushchegosia zadela po dizaĭnu i sintezu vysokoéffektivnykh agentov kombinirovannoĭ protivovirusnoĭ zashchity (ot VICh, grippa, gerpesa i dr. infektsiĭ).

To endow chitosan with solubility in the whole pH-range without loss of functionality of the amino groups, the cationic polysaccharide was exhaustively alkylated yielding N-[(2-hydroxy-3-trimethylammonium) propyl]chitosan chloride (QCht). Each alkylated unit of QCht contained both quaternary amino group and secondary amino group. Recently we demonstrated that QCht forms with nucleic acids of soluble polyelectrolyte complexes stable at physiological conditions and capable of cell transfection in vitro . In the current work, the anionic counterpart of QCht was hydrolyzed copolymer of divinyl ether and maleic anhydride (DIVEMA) which is known to possess some anti-tumor and immune stimulating activity and use as a drug carrier in anti-tumor drug delivery systems. According to the potentiometry data and ζ-potential measurements, almost all carboxylic groups of DIVEMA were able to form ion pairs with QCht. In aqueous and water–salt solutions, formation of either soluble or insoluble complexes was controlled by pH, ionic strength, a ratio of the oppositely charged groups, and degree of polymerization of the chains following general rules revealed on studying polyelectrolyte complexes of polycarboxylic acids. These findings evidence plausible advantages of the complex formation as the non-covalent modification that imparts to both polyelectrolytes of the ability for reversible soluble–insoluble transformation under enzyme-friendly conditions.

Purpose Subureteral injection of bulking agents in the endoscopic treatment of vesicoureteral reflux is widely accepted therapy with high success rates. Although the grade of vesicoureteric reflux and experience of surgeon is the mainstay of this success, the characteristics of augmenting substances may have an effect particularly in the long term. In this retrospective study, we aimed to evaluate the clinical outcomes of the endoscopic treatment of vesicoureteric reflux (VUR) with two different bulking agents: Dextranomer/hyaluronic acid copolymer (Dx/HA) and Polyacrylate polyalcohol copolymer (PPC). Materials and Methods A total 80 patients (49 girls and 31 boys) aged 1-12 years (mean age 5.3 years) underwent endoscopic subureteral injection for correction of VUR last six years. The patients were assigned to two groups: subureteral injections of Dx/HA (45 patients and 57 ureters) and PPC (35 patients and 45 ureters). VUR was grade II in 27 ureters, grade III in 35, grade IV in 22 and grade V in 18 ureters. Results VUR was resolved in 38 (66.6%) of 57 ureters and this equates to VUR correction in 33 (73.3%) of the 45 patients in Dx/HA group. In PPC group, overall success rate was 88.8% (of 40 in 45 ureters). Thus, Thus, this equates to VUR correction in 31 (88.5%) of the 35 patients. Conclusions Our short term data show that two different bulking agent injections provide a high level of reflux resolution and this study revealed that success rate of PPC was significantly higher than Dx/HA with less material.

The discovery of interferon (IFN), as an antiviral substance, by Isaacs and Lindenmann in 1957 [1] attracted the attention of a number of investigators, including Pieter De Somer at the Rega Institute in Leuven (Belgium) and Tom Merigan at Stanford University (California). De Somer’s original attempts were aimed at determining the mode of action of IFN [2], whereas Merigan’s work focused on trying to understand the role of IFN in human viral diseases [3], by use of the mouse as an animal model. Of pioneering importance was the demonstration by Merigan [4] in 1967 that IFN could be induced in mice by a synthetic polyanion called maleic divinyl ether copolymer (or pyran copolymer). In further experiments, pyran copolymer was also shown to induce IFN in humans [5], although the serum IFN titers obtained in humans were considerably lower than those in mice and, moreover, were accompanied by a considerable

Copolymer of divinyl ether and maleic anhydride (DIVEMA) is known to possess some anti-tumor and immune-stimulating activity and use as a drug carrier in anti-tumor drug delivery systems. Samples of DIVEMA of different degrees of polymerization were synthesized and characterized. Interaction of the hydrolyzed water-soluble DIVEMA polyanions with poly(N-ethyl-4-vinylpyridinium) cations (PEVP) has been studied. According to the potentiometry data, almost all carboxylic groups of the polyanions were able to form ion pairs with PEVP. In aqueous and water–salt solutions, formation of either soluble or insoluble polyelectrolyte complexes occurred depending on pH, ratio of the oppositely charged groups, and degree of polymerization of PEVP and/or DIVEMA. The phase separations followed general rules revealed by studying mixtures of PEVP and polycarboxylic acids. However in the case of DIVEMA, a significant broadening of the region for insoluble complexes at the expense of the region of soluble complexes was established. The data obtained demonstrate plausible advantages of the complex formation as the non-covalent modification of the polymeric carrier that endow DIVEMA with the ability for reversible soluble–insoluble transformation, in particular at physiological pH and ionic strength.

A protective effect of the SOD (superoxide dismutase)-DIVEMA (divinyl ether and maleic anhydride) conjugate on I-R (ischemia-reperfusion) liver injury was demonstrated. Twenty minutes of normothermic hepatic ischemia was induced by clamping the portal triad of Sprague-Dawley rats. Five minutes before the end of ischemia, SOD, SOD-DIVEMA, or NaCl (0.9%) was given intravenously. Using intravital fluorescence microscopy, hepatic microvascular perfusion was analyzed before ischemia and repeatedly during the 120-min reperfusion period. SOD-DIVEMA significantly restored the sinusoidal perfusion rate (control, 98.0 +/- 0.5; NaCl, 65.5 +/- 7. 7; SOD, 81.5 +/- 8.2; SOD-DIVEMA, 95.8 +/- 0.7%) and reduced the number of leukocytes stagnant in acini (control, 4.4 +/- 0.9; NaCl, 36.6 +/- 6.3; SOD, 27.7 +/- 6.8; SOD-DIVEMA, 12.3 +/- 3.3 cells/lobule) and adherent in postsinusoidal venules (control, 55.0 +/- 24; NaCl, 417 +/- 63; SOD, 253 +/- 58; SOD-DIVEMA, 40.0 +/- 14 cells/mm2). In addition, SOD-DIVEMA maintained postischemic hepatocellular integrity. The SOD-DIVEMA-treated group revealed higher serum SOD enzyme activity compared to the SOD group after 120 min of reperfusion (SOD-DIVEMA, 33.0 +/- 5.9; SOD, 8.6 +/- 3.1 U/ml). The beneficial effect of SOD-DIVEMA was most prominent after 120 min of reperfusion, indicating a longer intravascular half-life of SOD-DIVEMA.

We investigated the efficacy of a simple syngeneic tumor vaccine to induce specific antitumor immunity in female C57Bl/6 mice. Tumor vaccine was prepared by mixing irradiated B-16 melanoma tumor cells with the pleiotropic biological response modifier-maleic anhydride divinyl ether (MVE-2). Experimental animals were pretreated with the vaccine in order to prevent the development of intraperitoneal (i.p.) B-16 melanoma tumors after inoculation of viable tumor cells. More than 40% of prevaccinated animals challenged i.p. with 5 x 10(5) viable tumor cells were completely protected from tumor development and remained tumor-free 100 days after tumor cell inoculation. The percentage of tumor-free animals (survivors) rose to as much as 90% when the application of tumor vaccine was repeated two weeks after the first vaccination (i.e. one week after the inoculation of viable tumor cells). The induced antitumor response depended predominantly upon macrophage function, since vaccinated animals which were depleted of peritoneal macrophages died within the same time range as animals in the control group. Also, tumor-type specificity of the vaccine was confirmed by the fact that the animals vaccinated with B-16 melanoma vaccine were not protected from the development of another type of tumor. In conclusion, comparison of the experimental data with the data from the literature suggests that our simple tumor vaccine may be as effective as genetically engineered tumor vaccines. At the same time, this kind of vaccine is easier to control and thus safer to apply in humans when compared to genetically engineered vaccines.

Bovine erythrocyte superoxide dismutase (SOD) was conjugated with the copolymer of divinyl ether and maleic anhydride, known as DIVEMA or pyran copolymer. The DM–SOD conjugate was resistant against the proteolytic enzymes in serum, and showed a prolonged half-life in vivo after intravenous injection. DM–SOD was applied for the treatment of rat lung edema caused by the re-expansion after the left bronchus has been clipped for 3 days. The anti-inflammatory effect was evaluated by the inhibition rate of the water content due to the edema. Neither SOD nor DIVEMA reduced the increase in water content in the lung, whereas DM–SOD could effectively protect against the increase in water content such that the water content of the re-expanded lung was the same as that of the control right lung. Using electron microscopic observation the difference between the SOD and DM–SOD treatment in the water swelling of the interalveolar septum was clearly indicated. Observation of the surface of the endothelial wall in the blood vessel of the lung showed more a drastic difference. The leukocyte attachment on the endothelial layer observed following the treatment with SOD was not observed following that with DM–SOD. This observation indicates that DM–SOD had stopped the inflammation process at the first step of leukocyte adhesion, which should be followed by the rolling and expansion of the leukocytes to initiate the normal inflammation process.

We employed the Rauscher murine leukemia virus (RMuLV) as a murine retrovirus model of AIDS, to test biological response modifiers (BRM) and antiviral agents for potential therapeutic activity against the human immunodeficiency virus (HIV). We examined the relationship between the augmentation of natural killer (NK) cell activity and antiviral efficacy of a series of BRM, most of which are known inducers of interferon, in this model. Poly [I,C]-LC, MVE-2, and CL 246,738, but not Ampligen, soluble glucan, or 7-thia-8-oxoguanosine, consistently produced antiviral activity. In addition, the combination of suboptimal doses of oral 3'-azido-3'-deoxythymidine (AZT) (in drinking water) and poly [I,C]-LC produced a synergistic antiviral effect. With all the BRM tested, a consistent pattern emerged, namely that antiviral activity always correlated with the augmentation of splenic NK cell activity in infected animals. For instance, poly [I,C]-LC boosted NK activity much more in infected mice treated therapeutically (treatment initiated after infection) than prophylactically (treatment initiated before infection), and it had greater antiviral activity therapeutically than prophylactically. For the BRM tested, antiviral activity did not occur without augmentation of NK activity in infected mice. In contrast, augmentation of NK activity in uninfected mice bore no relationship to antiviral activity. Furthermore, elimination of NK cells by treating mice with anti-asialo GM1 abolished the antiviral activity of poly [I,C]-LC. Although splenic NK activity was ablated by anti-asialo GM1, serum interferon levels were not affected by this treatment. These results point to a causal connection between the augmentation of NK cell activity and the antiviral efficacy of these BRM in this murine AIDS model. NK cells thus appear to play a key role in resistance to this retrovirus, as has been suggested for HIV.

Highly reactive hydroxyl radical (OH) is generated during reaction of Cu,Zn-SOD (SOD)1 with H202, its own enzymatic reaction product (SOD plus 0;-). During inactivation of Cu,Zn-SOD with H202, Cu2+ liberated from the SOD catalyzes the formation of OH from H202. The OH thus generated will react with a,-protease inhibitor (a,-PI) if it is available in the milieu. To circumvent OH generation, we prepared eight different polymer-SOD conjugates and compared their stability against H202 treatment. Furthermore, the effect of each polymer-SOD conjugate on a,-PI in the presence of H202 was tested. Most polymer-SOD conjugates, except poly(styrene-co-maleic acid butylate; SMA)-SOD and poly(ethylene glycol; PEG)-SOD, showed higher stability than native SOD against inactivation by H202. The residual activity of divinylether maleic acid/anhydride copolymer (pyran copolymer)-conjugated SOD was 87% of the initial activity after incubation with 0.1 mM H202 for 2 h, whereas that of native SOD was 49%. Antiprotease activity of ca,-PI was completely abrogated when the inhibitor was treated with native SOD and 3.0 mM H202, but there was no change with pyran copolymer-SOD after 3 h of incubation with H202. Our results suggest that pyran copolymer captured loosely bound Cu2+ during the reaction of SOD with H202, thus resulting in suppression of both 0OH-mediated inactivation of SOD and suppression of activity of a,-PI. PEG showed little Cu2+-binding activity; no appreciable protection of SOD and a,-PI against OH generation was observed when Cu,Zn-SOD was conjugated with PEG. Circular dichroism and electron spin resonance studies of Cu,Zn-SOD treated with H202 indicated that the peptide conformation as well as the copper ligand of Cu,Zn-SOD can be stabilized not only by pyran copolymer per se but also by conjugation of Cu,Zn-SOD with some copolymers such as pyran.

Blocking conditions that are optimal for the detection of surface antigens on resident peritoneal macrophages (PMϕ) by flow cytometry are not ideal for elicited or activated PMϕ. A blocking step of 10% goat serum can be used routinely to detect the F4/80 and Mac-1 antigens on resident PMϕ. In contrast, high concentrations (33–50% each) of combined goat and mouse sera were required to reduce nonspecific binding and to improve the detection of the F4/80 antigen on PMϕ elicited by thioglycollate broth (TG) or activated by maleic anhydride divinyl ether copolymer (MVE-2). However, even low concentrations of goat serum masked the expression of the Mac-2 antigen on TG and MVE-2 PMϕ. Thus, within a given elicited or activated PMϕ population, different blocking conditions may be necessary to detect different surface antigens optimally. In addition to blocking, the use of isotypic controls that match the monoclonal antibody isotypes was found to be necessary for the optimal detection of antigen expression on TG and MVE-2 PMϕ. © 1994 Wiley-Liss, Inc.

Protein kinases are important enzymes in solid tumour and leukaemia pathologies. Their structures are well understood at the atomic level and their key role in the progression of certain cancers makes them valuable targets for anti-cancer therapy. Through medicinal chemical approaches, we developed an efficient preparative stereospecific synthesis of N12, N13 pyran-bridged indolocarbazoles that opens access to functional diversity within this previously challenging series. We focused upon the indolocarbazole class of chemical inhibitors, which includes S27888, an inhibitor we previously identified. We used biochemical and cell-based assays to identify small molecule inhibitors of Checkpoint kinase 1 (Chk1), a serine/threonine protein kinase that is activated when cancer cells are treated with genotoxic agents. These compounds show a promising inhibitory profile on Chk1. Furthermore, these compounds are active against FLT3, which is a tyrosine kinase that is frequently activated in human leukaemias. These data suggest that this chemical class may provide a source of therapeutic compounds for a broad range of human cancers.

Protein microarray technology provides a useful approach for the simultaneous serodetection of various antibodies in low sample volumes. To implement functional protein microarrays, appropriate surface chemistry must be designed so that both the protein structure and the biological activity can be retained. In the current study, two surface chemistries for protein microarrays and immunofluorescent assays were developed. Glass slides were functionalized with N-hydroxysuccinimide (NHS) ester via a monofunctional silane or maleic anhydride-alt-methyl vinyl ether (MAMVE) copolymer to allow covalent grafting of histone proteins. Analytical performance of these microarrays was then evaluated for the detection of anti-histone autoantibodies present in the sera of patients suffering from a systemic autoimmune disease, namely systemic lupus erythematosus (SLE), and the results were compared with those of the classical enzyme-linked immunosorbent assay (ELISA) and Western blot. The detection limit of our MAMVE copolymer microarrays was 50-fold lower than that of the classical ELISA. Furthermore, 100-fold less volume of biological samples was required with these miniaturized immunoassays.

The question of whether interferon alpha/beta is the common mechanism of antiviral action of synthetic immunomodulators was investigated in B6C3F1 mice infected with Semliki Forest virus. Mice were treated with various concentrations of normal sheep serum or potent anti-alpha/beta interferon antiserum, inoculated with the immunomodulators, and infected 24 hours later with virus. Three patterns emerged. The antiviral action of the pyrimidinone (ABMP) and the oral interferon inducer (CL246,738) appeared to be mediated primarily by interferon alpha/beta; their protective ability was almost completely abrogated by treatment with low levels of anti-alpha/beta interferon antiserum. The antiviral action of two other immunomodulators, a mismatched polyribonucleotide (Ampligen) and a polyanionic copolymer (MVE-2) at least partially involved interferon. Activity of these compounds was reduced, but not consistently eliminated by treatments with high doses of antiserum. The antiviral activity of another polyribonucleotide, polyriboinosinic-cytidylic acid complexed with lysine carboxymethylcellulose (poly ICLC), was not affected by treatment with even the highest amount of antiserum (two injections of 100,000 neutralizing units each). Almost complete protection by poly ICLC was observed despite the fact that this high concentration of antiserum, when given alone, caused a decrease in natural resistance to Semliki Forest virus infection. Taken together, these results indicate that induction of interferon alpha/beta does not appear to be the major common mechanism of antiviral activity among these diverse synthetic immunomodulators.

Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice.Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freund's complete adjuvant (FCA).Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe.For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.

The aim of this study was to develop as effective as possible autologous tumor vaccine which would be at the same time easy to produce, highly controllable, and without undesired side effects on normal tissue. Therefore, irradiated autologous - syngeneic B-16 tumor cells admixed with a non-specific immunomodulator MVE-2 (a polymer fraction of 1,2-co-polymer of divinyl ether and maleic anhydride) were used for subcutaneous (s.c.). or intraperitoneal (i.p.) prevaccination of experimental mice. Compared to the control mice, a statistically significant delay in tumor development of s.c. tumors was achieved in prevaccinated mice (p

To enhance the therapeutic usefulness of antitumor cytokines in vivo, we synthesized bioconjugated tumor necrosis factor-alpha (TNF-alpha) with divinyl ether and maleic anhydride copolymer (DIVEMA), which has intrinsic antitumor activity as a synthetic biological response modifier. The degree of modification could be controlled by the addition of 2,3-dimethylmaleic anhydride (DMMAn), which binds to amino groups of TNF-alpha by changing the pH. In addition, the specific activity of DIVEMA-TNF-alpha was hardly decreased in vitro. DIVEMA-TNF-alpha showed a marked antitumor effect compared to native TNF-alpha without any side effects such as sudden death, body-weight reduction, and decrease in platelet count on mice bearing solid tumors. These results suggest that DIVEMA is a useful polymeric modifier for-bioconjugation of TNF-alpha in order to increase its antitumor activity, and multifunctionally bioconjugated TNF-alpha may be a potentiated antitumor agent for therapeutic use.

Numerous clinical trials have been performed during the last 5 years in multiple sclerosis patients. Some of the results have been encouraging. However, clinical benefit remains limited. Corticosteroids are indicated during the course of severe relapses but have not proven any long term benefit. Immunosuppressive agents may be of some help during very active stages of the disease. Results of interferon beta-1b trial in relapsing multiple sclerosis have shown a moderate decrease in the frequency of relapses. The same effect has recently been reported with interferon beta-1a. In addition, an effect on disability progression have been suggested with the latter interferon. In France, interferon beta-1b is now authorized in the relapsing forms of the disease. Initial results with copolymer I also suggest an effect on the frequency of relapses. Despite these major therapeutical efforts, further trials, possibly using new therapeutical approaches, are still needed.

There is ample evidence that protection against group B streptococcal (GBS) disease, both in experimental animals and in humans, is related to the presence of specific antibodies and complement. However, until now the possibility of increasing resistance to GBS infection by potentiating natural cell-mediated immunity in the host, has not been explored. In this study we examine the effect of administering in vivo MVE-2 (a polymer fraction of 1,2-co-polymer of divinyl ether and maleic anhydride) and inactivated Candida albicans (CA) cells on mouse resistance to the reference strain type Ia 090 GBS (GBS-090) lethal infection. MVE-2 and CA, respectively a synthetic and a microbial biological response modifier (BRM), are strong inducers and activators of natural resistance effectors, such as natural killer (NK) cells, macrophages and polymorphonuclear cells (PMN). The results showed that MVE-2 protected 100% CD-1 mice from a systemic lethal challenge with GBS-090 (5 x 10(3) microorganisms/mouse) when administered 3 days before infection at dose of 50 mg kg-1. CA treatment, in five doses (CA-5d) over 14 days protected 100% mice when administered at 2 x 10(7) cells/mouse and when the last CA injection was given 1 day before the GBS-090 challenge. Instead, when the GBS-090 challenge was performed by intraperitoneal route, protection was obtained with CA-5d treatment but not with MVE-2. The possibility that MVE-2 or CA stimulated a rapid production of specific antibodies against GBS-090 infection was excluded by the ELISA assay. Evidence exists that NK cells do not play a primary role as effectors in the MVE-2 and CA conferred protection since the strong reduction in NK activity, due to in vivo administration of anti-asialo GM1 antibodies before GBS-090 infection, did not influence the BRM-induced protection. Besides, high NK activity levels, induced by in vivo rhIL-2 administration, did not protect the mice against GBS-090 infection. Both studies on in vivo clearance and in vitro microbicidal activity, showed that, after 1 h, immunopotentiated effectors were unable to kill GBS-090, but were highly effective against GBS type VI. These results seem to indicate that intracellular GBS-090 killing is a slow process requiring more than 1 h. This study demonstrates that it is possible to increase resistance to GBS-090 lethal infection by BRMs, by potentiating the antibody-independent microbicidal activity of the phagocytes.

The underlying cellular mechanisms for the antitumor effects of biological response modifiers (BRMs) have not been clearly resolved. We have investigated this issue in the Lewis lung (3LL) peritoneal carcinomatosis model in which treatment with the BRM MVE-2 slows tumor growth and enhances survival. MVE-2 is a potent inducer of cytotoxic macrophages (mφs); however, the in vivo tumoricidal properties of these mφs remain to be firmly established. To directly establish that mφs were at least in part responsible for the in vivo efficacy of MVE-2, a novel method of obtaining highly enriched mφ suspensions was developed which gave high purity, satisfactory yield, and excellent viability without affecting antitumor activity. Using the 3LL peritoneal carcinomatosis model and adoptive transfer techniques, we directly demonstrate that the majority of antitumor activity was associated with the adherent cell fraction enriched for mφs. Histological observations supported this conclusion, indicating that MVE-2 treatment initially activated cells associated with nonspecific immunity, retarding tumor growth in the ascites long enough for a multifaceted immune response to develop. J. Leukoc. Biol. 56: 41–51; 1994.

A variety of biological response modifiers (BRMs) have provided antiviral protection to immunocompetent mice, and this prompted us to determine their efficacy against murine cytomegalovirus (MCMV) infection in immunocompromised mice-including the profoundly immunocompromised SCID mice and C57B1/6 and B6D2F1 aged mice. SCID mice showed a marked decrease (>20-fold) in resistance to MCMV, while there was a slight decrease (3-fold) in aged mice. In BRM antiviral protection studies, SCID mice were almost completely protected against MCMV infection by the pleiotropic immunomodulators, MVE-2 and pICLC, but much less by the more selective CSF-1. pICLC-induced IFN and NK cell cytotoxicity were maintained in SCID mice, suggesting that pleiotropic immunomodulatory effects may be required for antiviral protection in such a profoundly immunocompromised model. pICLC also effectively protected aged mice against lethal MCMV infection and effectively induced IFN. These results emphasize the potential for BRM treatment in immunocompromised hosts.

The effects of two immunomodulators were investigated in severe combined immunodeficient mice reconstituted with human peripheral blood lymphocytes (hu-PBL-SCID mice). Both immunomodulators, maleic anhydride divinyl ether (MVE-2) and 4-imino-1,3-diazobicyclo-(3.1.0)-hexan-2- one (imexon), have been previously studied by us in retrovirus-infected mice. To determine the effects of these compounds as they may function in humans, 24 SCID mice were each reconstituted with 20 x 10(6) ficoll-purified lymphocytes from a single donor. Five weeks after reconstitution, the mice received 16 mg/kg/day of MVE-2 intraperitoneally (i.p.) on days 0, 7, and 14 or 110 mg/kg/day of imexon i.p. daily for 14 days. Spleens were removed and splenocytes labeled with monoclonal antibodies for T- and B-cell enumeration as determined by flow cytometry 24 h after final treatment. Imexon-treated mice demonstrated a slight increase in total T cells and T cell subsets compared to control mice. T helper/T suppressor cell ratios in imexon-treated mice were brought to a normal 3:2 ratio compared to placebo-treated mice. Human immunoglobulin levels were markedly increased in imexon-treated mice. MVE-2-treated hu-PBL-SCID mice had significantly reduced numbers of total T cells compared to controls. The T-cell population results using human cells in SCID mice were similar to the effects of these immunomodulators on murine cells in immunologically competent mice.

In contrast with other macrophage (MO) populations, there is little information on the antiviral resistance in vitro of isolated liver MO (Kupffer cells, KC). We have demonstrated that the KC exhibits marked intrinsic resistance to infection in vitro with herpes simplex virus type 1 (HSV-1). Liver and peritoneal MO (PMO) were harvested from untreated mice (naive), from mice treated with drug vehicle, or from mice treated with either a synthetic nonspecific immunomodulator, maleic anhydride divinyl ether copolymer (MVE-2), or with MO colony-stimulating factor-1 (CSF-1). The studies revealed that resident KC, isolated by two different methods, are equally as nonpermissive for infection with HSV-1 as are resident PMO. When infected with HSV-1, resident KC showed no cytopathic effect, and no infectious virus was produced. Intravenous (i.v.) treatment of mice with MVE-2 (50 mg/kg 3 days before cell harvest) increased the number of KC recovered. However, neither i.v. treatment with MVE-2 nor CSF-1 (20,000 U daily for 4 days) had any pronounced effect on the permissiveness of KC or PMO to infection with HSV-1. These data support a role for KC in host antiviral resistance, and indicate that KC intrinsic antiviral resistance is maintained during immunotherapy with MVE-2 or CSF-1.

The current results provide direct evidence for a role of tissue macrophages (Mφ) in natural immunity and support the use of immunomodulators to enhance antiviral resistance in immunocompromised individuals. In this study, macrophages (Mφ) in the spleen and liver were eliminated by intravenous (i.v.) injection of the drug dichloromethylene diphosphonate (DMDP) encapsulated in liposomes. The effect of this depletion system on peritoneal Mφ, peripheral blood leukocytes, splenic natural killer (NK) activity, and natural and immunomodulator-induced host resistance was then assessed. Barrier-maintained CD-1 female mice were inoculated i.v. either with DMDP liposomes, free liposomes (containing no DMDP), or saline on day -2 or on days -3 and -1 before cell population analysis or infection. Single or double treatment with DMDP liposomes had no effect on peritoneal Mφ as indicated by no changes in total number, differential counts, or ectoenzyme patterns. Double treatment with DMDP liposomes caused a marked leukocytosis in blood, primarily of lymphocytes and polymorphonuclear leukocytes (PMN), and a transient depression of spontaneous and interferon-inducible splenic NK activity. The effects on host resistance to i.v. infection with Listeria monocytogenes or herpes simplex virus type 2 (HSV-2) indicated that i.v. treatment with DMDP liposomes significantly reduced natural resistance to these microorganisms as evidenced by increased mortality and decreased median survival time. When DMDP liposomes-treated mice were given the immunomodulator maleic anhydride divinyl ether copolymer (MVE-2) intraperitoneally the day before infection with HSV-2, the immunosuppressive effect of DMDP liposome treatment was significantly reversed.

The effects of anticalculus dentifrices were compared with other commercially available dentifrices in in vitro models of dentin sensitivity. Changes in the hydraulic conductance of dentin discs were measured with and without a smear layer before and after treatment and also after a post-treatment acid etch. The capacity of dentifrices to occlude open dentinal tubules in vitro was also assessed by scanning electron microscopy (SEM). There was good correlation (R = 0.98) between our test and values reported in the literature. Tartar control dentifrices gave reductions in fluid flow rates through the dentin discs comparable to those obtained with Promise, Sensodyne, Thermodent and Denquel. Additionally, tartar control dentifrices did not remove microcrystalline debris (smear layers) from the surfaces of dentin in vitro. These results were confirmed by SEM. Thus, according to the hydrodynamic theory of dentin sensitivity, these in vitro results suggest that pyrophosphate-containing dentifrices should reduce dentinal sensitivity.

Neocarzinostatin (NCS) was conjugated with divinyl ether-maleic acid anhydride copolymer (pyran copolymer), and its therapeutic effect was compared with that of NCS. The conjugated NCS (pyran-NCS) with a molecular weight of about 23,000, exhibited in vitro cytotoxic activity against eight cell lines and bone marrow cells that was similar to the cytotoxic activity of NCS on a molar basis. Furthermore, both drugs had similar effects against a multidrug-resistant Chinese hamster ovary cell line (CHR C5) and its parent cell line (AUXB1) in vitro. However, pharmacological analysis showed that pyran-NCS had reduced accumulation in the spleen, and most important was three times less hematotoxic in vivo compared with NCS. Also, pyran-NCS had a 1.7-fold higher 50% lethal dose (LD50). Antitumor activity of pyran-NCS and NCS was tested against two different forms of Meth A tumor. In a solid tumor model, pyran-NCS and NCS suppressed tumor growth at three-fourths of the LD50 to 12.8 and 19.0% of the control tumor as evaluated on day 28, respectively (P less than 0.025). In an ascitic tumor model, the percentage increase in the median life span caused by pyran-NCS and NCS was more than 400 and 150% on day 60, respectively. Pyran-NCS is more effective than NCS because the reduced acute toxicity permits an increased drug dosage.

Polyanionid copolymer of divinyl ether and maleic anhydride (DIVEMA) with narrow molecular weight distribution was synthesized and tested of its antitumor activity. DIVEMA showed a significant antitumor activity against colon 26 adenocarcinoma and FSaI fibrosarcoma transplanted in syngenic mice. Furthermore, DIVEMA was used as a polymeric drug carrier of antitumor drugs to reduce side effects and enhance the antitumor activity of the drugs. Adriamycin and neocarzinostatin were attached covalently to DIVEMA and the polymeric conjugates showed higher antitumor activity than the corresponding mother drugs against P 388 leukemic mice.

The purpose of the present investigation was to determine if RES activity was altered after the infusion (bolus i.v. injection over approximately 3-5 min) of hydroxyethylstarch (HES). RES function was determined by vascular clearance of 51Chromium-labeled sheep erythrocytes and the subsequent uptake into the liver, spleen, lungs, and thymus at 1 hr, 3 hr, 6 hr, 1 day, 3 days, and 7 days post infusion. Infusion with the low doses of HES (20 and 40 ml/kg) produced changes in vascular clearance which were comparable to physiological saline. Infusion with 80 ml/kg HES produced a biphasic response with a modest suppression of vascular clearance (i.e., 151% increase in half life) and hepatic phagocytosis (50%) during the first 6 hours after injection, followed by recovery at 24 hours and a stimulation in hepatic uptake (42%) after 3 days. These effects by HES were compared to those produced by infusion with 80 ml/kg HSA, a comparable colloid and with 80 ml/kg pyran copolymer, a positive control.

We have used a murine renal adenocarcinoma of spontaneous origin (Renca) inplanted in the peritoneal cavity to study the therapeutic potential of biological response modifiers (BRMs) used alone or in conjunction with chemotherapy. This tumor model is therapeutically challenging since following intraperitoneal (i.p.) injection, the tumor grows progressively with hemorrhagic ascites, abdominal metastases to lymph nodes, liver, spleen, most serous membranes, and, in some animals, metastases to extra-abdominal sites (lungs). In the absence of therapy, death invariably occurs within 36 +/- 2 days. The tumor is efficiently lysed in 4 hours by peritoneal cells isolated from mice treated with BRMs. Both MVE-2 and rIL-2 significantly increased the survival time of tumor-bearing mice, but only treatment with MVE-2 led to definite cures of i.p. Renca. A single i.p. injection of MVE-2 cured 20% of the tumor-bearing mice, while repeated i.p. administration of this drug at 12 day intervals cured 70% of i.p. Renca-bearing mice. Combined therapy with doxorubicin hydrochloride and a single dose of MVE-2 cured 90% of tumor-bearing animals. The superior therapeutic efficiency of MVE-2 compared to that of the rIL-2 may be due to its ability, after i.p. inoculation, to generate and maintain high levels of cytotoxic effector cell activity for an elevated period of time within the peritoneal cell population. Additionally, MVE-2 augments effector cell activity in the liver, lungs, spleen, and blood and may therefore more efficiently interfere with metastasis formation in those compartments. The additive effects of MVE-2 and the chemotherapeutic agent suggest that more effective therapy may be achieved by the combination of immunotherapy with BRMs with chemotherapeutic drugs.

Many naturally occurring polyanionic materials are known, each of which possesses its own set of physiological properties. Recently, a variety of synthetic polyanions have become available, and their physiological properties have been compared with the naturally occurring polyanions. Probably the most widely investigated synthetic polyanion is the 1:2 regularly alternating cyclo-copolymer (DIVEMA) (Eq. 1) of divinyl ether (DVE) and maleic anhydride (MA), first discovered in these laboratories in 1951 [1], This material has been investigated extensively, both from the standpoint of its chemical structure [2] and its antitumor activity [3]. Although certain aspects of its structure remain to be determined [4], it has been shown to possess a wide spectrum of biological activity [5] and is an interferon inducer [3]. Many structural modifications of DIVEMA have been synthesized which also possess antitumor activity and interferon-inducing capability [6].

Natural and synthetic polyanions are known to have a broad spectrum of physiologic and pharmacologic properties. Some polyanions can activate in vitro nucleic acid biosynthesis in isolated nuclei, and induce varying degrees of nuclear swelling and chromatin decondensation. This in vitro phenomenon has an in vivo analog in gene activation. Pyran, a copolymer of divinyl ether and maleic anhydride, is a particularly interesting polyanion because of its antitumor, antiviral, and immunostimulating activities. Although the mechanism of its activities is unknown, its polyanionic character suggests that it may be considered as a model “gene regulator.” The present investigation focused upon the interaction of Pyran with nuclei isolated from normal mouse spleens. Small angle light scattering was used to monitor nuclear swelling, and the conditions required to demonstrate swelling were established. It was shown that Pyran-induced nuclear swelling results from the ability of this polyanion to form a complex with Mg 2+ . It was also shown by nephelometric titration methods and small angle light scattering that the interaction of Pyran and Mg 2+ resulted in the precipitation of “microspheres.” These results have led to a working hypothesis relating to the possible molecular mechanism of the pharmacologic action of Pyran copolymer.

The effect of four immunomodulators (BCG, Corynebacterium parvum, pyran copolymer, and levamisole) on the cellular arm of antibody-dependent cellular cytotoxicity (ADCC) was investigated in mice with 51Cr-labeled chicken erythrocytes employed as targets. All these drugs, except levamisole, stimulated the effector cells of ADCC in the spleen, but the kinetics of their effect differed. Stimulation of the effector cells of ADCC peaked on day 15 after injection of BCG and C. parvum and on day 7 after injection of pyran, which was less efficient in this respect than the two bacterial immunostimulants. The increase in ADCC activity caused by BCG and C. parvum was eliminated by treatment with carbonyl iron of the splenocyte suspensions.

The degree of inhibition of mammalian DNA-dependent RNA polymerases I and II and Moloney leukemia virus RNA-dependent DNA polymerase by pyran copolymer was dependent on the concentration of the divalent cation cofactor in the reaction mixture. Inhibition was completely blocked by an excess of divalent cations. It was concluded that pyran inhibited these enzymes by complexing with the essential divalent cation cofactor.

The polyanionic immunomodulatory polymer, maleic vinyl ether anhydride (MVE-2), enhances antitumor macrophage activity and causes heavy proteinuria. The effects of this compound on renal function and renal morphology were investigated in a rat model. Rats were given daily intravenous infusions of MVE-2, 100 mg/kg, over 2–4 hr on each of three consecutive days. Renal function and morphology in MVE-2-infused rats were examined by standard techniques and compared to control rats given saline. On the average, MVE-2 rats had a significant reduction in inulin clearance to 62% of control values. MVE-2 rats developed heavy proteinuria 1–3 days after the first infusion (mean ± 1 SEM, 387 ± 91 mg/24 hr). By light microscopy, the only finding was intratubular protein casts; glomeruli were normal. Immunoflorescence showed no deposition of antibody, complement, or fibrin. Electron microscopy revealed foot process effacement, epithelial cell vacuolization, and subepithelial ring-shaped structures; no immunecomplex deposits were present. MVE-2 rats had no increase in the number of glomerular Ia(+) cells. To examine further the mechanism of MVE-2 nephropathy, the ability of MVE-2 to induce proteinuria in animals pretreated with radiation (750 rad), methylprednisolone (MP) or cyclosporine (CyA) was determined. Animals pretreated with radiation or MP had significantly less proteinuria after MVE-2 treatment compared to animals receiving no immunosuppressive therapy, while CyA pretreated rats developed heavy proteinuria. These results are compatible with the hypothesis that MVE-2 induces proteinuria via an effect on steroid- and radiation-sensitive cells, perhaps related to production of circulating factors which alter glomerular permeability.

Propionibacterium granulosum KP-45, glucan and pyran copolymer stimulated the elimination of 75Selenomethionine-labelled 3LL tumor cells from murine lungs, as measured 4 hr after intravenous injection of these cells into 16- to 25-week-old B6DF1 mice. This effect was most pronounced 4 to 6 days following intravenous administration of the above biological response modifiers and disappeared 6 to 8 days later. Intraperitoneal injection of all three agents produced only insignificant stimulation results. Spontaneous clearance of 3LL cells from lungs of 8-week-old B6DF1 mice was significantly more effective than in animals over 16 weeks old. Cyclophosphamide suppressed the elimination of tumor cells from lungs in both young and older mice and neutralized the stimulatory effect of P. granulosum KP-45 and glucan. The results suggest that the effector cells responsible for the clearance of radiolabelled 3LL cells from lungs of B6DF1 mice are at least similar to natural killer (NK) lymphocytes.

Biological response modifiers are agents capable of affecting the host immune response toward tumors and include those biological substances produced by the human mammalian cell genome. Interferons and other lymphokines, tumor antigens, antibodies and agents which can activate or stimulate host immune responses are all included in this general category of agents. With the advent of genetic engineering and with monoclonal antibody technology, highly pure preparations of these biological substances can now be produced for tests of activity in preclinical models and in man. As the initial results accrue with highly purified preparations of interferon and with monoclonal antibodies alone or conjugated to toxic substances, the possibility of tumor specific therapy is becoming a reality. The development of preclinical models to predict for clinical activity remains a most important task to attempt to bring to clinical trials the substances most likely to be efficacious as anticancer agents. Early clinical results indicate that many of these agents can give responses in patients with clinically perceptible disease and Phase II activity studies are just beginning to define the range of clinical activity for a variety of biological response modifying agents. The development of biological response modifiers through preclinical testing and into clinical trials will be discussed in detail.

SummaryData are presented which show that protection of mice by endotoxin against viral infection is not mediated by interferon while that of poly(I:C) and pyran can be at least partially attributed to interferon induction. The results also suggest that circulating interferon does not protect against a virus administered intraperitoneally.

A single injection of mice with maleic anhydride divinyl ether (MVE-2) resulted in significantly augmented natural killer (NK) activity. However, multiple injections with MVE-2 led to a hyporesponsive to boosting of NK activity. Stimulation of prostaglandin E secretion of induction of suppressor macrophages (Mo) or lymphocytes were shown not to be responsible for the depressed NK cell responses. Rather the hyporesponsiveness to NK boosting was associated with a decreased number of large granular lymphocytes (LGLs). Percoll discontinuousdensity-gradient studies showed that the augmented NK activity of spleen cells after a single injection with MVE-2 was associated with an increase in the percentage of LGLs in the lowerdensity fractions (Fraction 1 and 2). In contrast, the NK activity and percentage of LGLs in the lower-density fractions were markedly decreased after multiple injections with MVE-2. Polyinosinic: polycytidylic acid stabilized with poly( l -lysine) in carboxymethylcellulose, another BRM capable of augmenting NK activity, was able to substantially augment NK activity in mice hyporesponsive to MVE-2 and this was accompanied by an increase in the percentage of LGLs in the lower-density Percoll fractions.

Administration of BCG and Corynebacterium parvum is known to cause depression of the hepatic microsomal enzyme system (HMES) in mice. In the present study we explored the effects on HMES of two chemical immuno-adjuvants, one of which (pyran copolymer) with peculiarly long-lasting biological activities. The two synthetic immunoadjuvants proved to be potent HMES inhibitors and, for pyran copolymer, peak levels of inhibition concurred with maximal macrophage activation. The inhibition was largely dose-dependent and could not be prevented by immunopharmacologic maneuvers that are known to block the C. parvum effect on HMES. The possibility is discussed that common mechanism(s) underlie the depression of HMES by immunoactive substances of both bacterial and chemical origin.

A sensitive potentiometric monitoring method was developed that permits the continuous measurement of the dissolution profiles of methyl vinyl ether-maleic anhydride-acid copolymers and salt derivatives. Three distinct rate periods were observed in the dissolution of the anhydride copolymer. The relative dissolution rate of the anhydride copolymer, expressed as percent anhydride dissolved, was independent of sample weight over the weight range studied. The acid form of the copolymer showed only one dissolution rate period, with dissolution being very rapid. The rapid initial pH decrease observed during the first stage of dissolution for a series of anhydride-acid copolymer powder samples correlated closely with the anhydride-acid ratio, permitting chemical characterization of the copolymer functionality simultaneously with the analysis of dissolution profiles. Similarly, the extent of copolymer alkaline salt conversion was inversely proportional to the initial maximum pH increase observed during the first stage of dissolution of these salts. Mechanisms of dissolution of copolymer powder materials are discussed and compared to the dissolution of compressed disks and films reported previously.

A weakly immunogenic, 3-methylcholanthrene-induced, subcutaneous fibrosarcoma syngeneic to inbred C3H/HeJ mice was used. Pyran copolymer was injected either directly into the tumor, ip, or iv as soon as tumors appeared or when tumors were 8 mm in diameter. One, three, or five doses of pyran copolymer at 10 or 20 mg/kg/dose were injected, with multiple doses being given every other day. Pyran copolymer injected intratumorally once, three times, or five times significantly retarded tumor growth and prolonged the survival times of the hosts. Of the other routes and doses, only pyran copolymer given three times iv significantly retarded tumor growth, but none of these significantly prolonged the survival times of the hosts. Pyran copolymer alone did not induce any complete regression of tumor. Local tumor irradiation with a single exposure to 2,000 rads of X-ray induced complete regressions in some mice, but a higher percentage of tumor cure was observed when tumor irradiation was followed by pyran copolymer treatment.

Tumor bearing patients have repeatedly been shown to develop states of protein (PD). PD has frequently been associated with depression of acquired immunity. In contrast, little is known about the effects of PD on natural killer (NK) cell cytotoxicity and on bone marrow cellularity and how modulation of these parameters by maleic anhydride divinyl ether copolymer (MVE-2) would be affected. BALB/c mice 6-10 weeks old were fed normal diets (18% protein) (ND mice) or protein deficient isocaloric diets (PD mice) for 35 days. On days 5, 9, 15, 23, and 35 assays were performed. Three days prior to each assay day, some groups received MVE-2 i.p. In PD mice spontaneous NK activity was reduced by some 80%. After MVE-2 the levels only rose to half the amount of activity of ND mice, although in both groups an increase of NK activity took place. BMC levels of PD mice were also strongly decreased. After MVE-2, increases of BMC in PD mice were quite weak and did not reach the level of normal unstimulated BMC. In PD mice we found body weight loss of 46%, reduction in albumin and total protein of 22% and complete disappearance of prealbumin after 5 days of diets. Preliminary results indicate that repletion with amino acids (NeoAminomel L12.5 o.K.H. Salvia, Boehringer Mannheim Co.) is able restore NK activities and BMC levels.