The variability of Vipera ammodytes ammodytes venoms from Croatia--biochemical properties and biological activity.

Vipera ammodytes ammodytes venom has been used for many years in Croatia for immunization of horses and production of specific therapeutic anti-venoms. The neutralizing effectiveness of anti-venoms is directly dependent on the properties of the snake venom used for immunization. Therefore, appropriate characterization of the whole venom is necessary prior to use in the immunization procedure. In the course of such analyses, the variability in biochemical properties and biological activity was observed in venoms collected from snakes originating from different parts of Croatia. The venom pools also differed with respect to time of snake collection (1992-2003). Analyses of three samples of whole venom pools were carried out revealing differences in lethal activity (LD50), minimum haemorrhagic dose (MHD), minimum necrotizing dose (MND), phospholipase A2 activity and in anticomplementary activity. SDS-PAGE electrophoretic patterns were similar, but not identical, for all tested venom pools with respect to the number of protein bands detected, but intensity of particular components differed. Preliminary immunogenicity testing in terms of determination of specific antibodies revealed similar immunogenicity and high cross-reactivity for three samples tested.

A novel ELISA for determination of polysaccharide specific immunoglobulins.

A novel ELISA using Nunc CovaLink microtiter plates has been developed for the determination of polysaccharide specific antibodies in mice sera. Glucuronoxylomannan (GXM), a major capsular polysaccharide of Cryptococcus neoformans serotype B, was immobilised on CovaLink plates by covalent linkage of CNBr activated hydroxyl groups of the sugar and free bnd NH(2) groups of the plates. The binding characteristics of GXM to CovaLink and to conventional polystyrene ELISA plate (Costar) were compared. The differences were observed in quality of standard curve with anti-GXM MoAb 2H1 (R(2) values were 0.9468 and 0.9872 for Costar and CovaLink plates, respectively) and in absorbance values of sera of immunised mice which were 2.5 times higher on CovaLink than on Costar plates. Negative control was low and having the same value on both the plates. Using the novel ELISA we have analysed the influence of immunomodulatory peptidoglycan monomer on humoral immune response to GXM in mice. The treatment with the conjugate of the immunomodulator and GXM resulted in the increase of GXM-specific IgGs in mice sera. Finally, the method has been successfully modified for the determination of dextran-specific antibodies in mice sera, indicating that the described procedure could be applied for other polysaccharides that have &z.sbnd;OH groups available for CNBr activation.

Synthesis of novel adamantylacetyl derivative of peptidoglycan monomer--biological evaluation of immunomodulatory peptidoglycan monomer and respective derivatives with lipophilic substituents on amino group.

Novel synthetic analogue of immunomodulatory peptidoglycan monomer 1 (PGM), (adamant-1-yl)-CH2CO-PGM (2), was prepared by acylation of epsilon-amino group of diaminopimelic acid with symmetrical (adamant-1-yl)-acetic acid anhydride in the presence of triethylamine. The product was isolated by gel filtration on Sephadex G-25, followed by ion exchange chromatography on SP-Sephadex C-25. The susceptibility of (adamant-1-yl)-CH2CO-PGM to hydrolysis with N-acetylmuramyl-L-alanine amidase was demonstrated, and the product of hydrolysis, (adamant-1-yl) CH2CO-pentapeptide 3, was characterized. Both 2 and 3 are water soluble and non-pyrogenic compounds. Immunomodulatory activity of PGM (adamant-1-yl)-CH2CO-PGM and structurally related derivative Boc-Tyr-PGM was compared in experiments in vivo, in mice, using ovalbumin (OVA) as an antigen. All three tested compounds exhibited comparable immunostimulating effects with respect to the induction of anti-ovalbumin immunoglobulin G. The results of evaluation of biological activity show that the substitution of free amino group in the parent peptidoglycan molecule with bulky lipophilic substituents did not affect the susceptibility to hydrolysis with N-acetylmuramyl-L-alanine amidase and did not alter markedly the immunostimulating activity. The results also indicate that the free amino group in the peptide chain is not a necessary requirement in the mechanism of immunostimulation of tested immunomodulators.

Immune, endocrine, and psychological responses in civilians displaced by war.

OBJECTIVES: The objectives of this study were to assess the influence of trauma caused by forced expulsion from home in a war-ravaged region on the psychological, hormonal, and immune responses in displaced persons and to analyze the relationships between psychometric, hormonal, and immunologic variables. METHODS: Participants were 20 displaced and 14 control women. Psychosomatic response was evaluated using the COR-NEX2 test. Serum concentrations of cortisol, prolactin, endorphin, thyroxine, and triiodothyronine were measured by radioimmunoassay. Immunophenotyping and lymphocyte proliferation were determined by flow cytometry, and phagocyte functions (i.e., ingestion and antibody-dependent cytotoxicity) against 51Cr-labeled sheep red blood cells were assessed through radioactivity uptake and release, respectively. RESULTS: In comparison with control women, displaced women had higher COR-NEX2 test scores; higher serum cortisol, prolactin, and endorphin levels; an increase in activated phenotype within all three measured cell populations (i.e., B, T, and natural killer cells); as well as an enhanced proportion of proliferating lymphocytes in freshly isolated samples. However, the phytohemagglutinin-stimulated proliferative response, estimated as the stimulation index, was lower in displaced women. A complex pattern of relations between psychological, hormonal, and immune responses was observed. CONCLUSIONS: Chronic psychological stress elicited multiple, predominantly stimulatory influences on immune functions.

Comparative study of the effects of peptidoglycan monomer and structurally related adamantyltripeptides on humoral immune response to ovalbumin in the mouse.

Peptidoglycan monomer, GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDAP(omega NH2)-D-Ala-D-Ala (PGM) originating from Brevibacterium divaricatum and synthetic adamantyltripeptides, diastereoisomers of D,L-(adamant-2-yl)-Gly-L-Ala-D-isoGln (AdTP1 and AdTP2) exhibit immunomodulating activity. An experimental model in the mouse has been established with suboptimal amounts of ovalbumin (OVA) as the immunogen, and parallel testing of adjuvant activity of these three immunomodulators was carried out in Balb/c, C57B16 or CBA mice. Tested compounds (100 or 200 micrograms/mouse) mixed with OVA in saline (50 micrograms/mouse) were administered s.c. Anti-OVA was assayed by ELISA in the sera of mice taken 7 days after the boosters (given on days 14 and 28). The treatment with PGM and one of the diastereoisomers, AdTP2, resulted in significantly higher increase in anti-OVA IgG levels (stimulation index up to 46) with respect to controls and groups treated with AdTP1. The effect of AdTP2 treatment was comparable to that of PGM in most experiments after the first booster, but after the second booster PGM exhibited markedly better effect. PGM and AdTP2 also induced markedly higher levels of IgG1 and IgG2 anti-OVA subclasses than detected in controls and AdTP1 treated mice, indicating that these two immunomodulators might upregulate both Th1-like and Th2-like immune responses.

Preparation of novel conjugates involving immunomodulating peptidoglycan monomer.

Peptidoglycan monomer, the disaccharide pentapeptide beta-D-Glcp-N-Ac-(1-->4)-D-Murp-N-Ac-L-Ala-D-mesoA2pm- (epsilonN H2)-D-Ala-D-Ala (PGM) is an immunomodulator. PGM and/or its derivative N-tert-butyloxycarbonyl-L-tyrosyl peptidoglycan monomer (Boc-Tyr-PGM) were coupled to two polysaccharides: the glucuronoxylomannan (GXM) from Cryptococcus neoformans, type B, solubilized by ultrasonic irradiation (MW 12-400 kDa) and to the dextran FP 70 (MW 70 kDa). Both polysaccharides were activated by CNBr. Initially, unprotected PGM was coupled via its amino group to GXM. The reactions yielded 42%-52% of the conjugate, containing only 0.18%-0.31% of PGM. In another approach Boc-Tyr-PGM (having its amino group blocked) was reacted via its free carboxyl group. Both CNBr-activated polysaccharides were first coupled to adipic acid dihydrazide (ADH) and then subsequently coupled to Boc-Tyr-PGM. The dextran conjugate (approximately 80% yield ) contained 6.3% of Boc-Tyr-PGM. The isolation of GXM conjugate required several modifications and it was obtained in lower yield (approximately 30%) but contained 13.7% of Boc-Tyr-PGM. Both conjugates were water soluble and apyrogenic and suitable for further testing of biological activity.

Synthesis of acylated methyl beta-D-xylopyranosides and their enzymic deacylations by rabbit serum esterases.

Selective pivaloylations of methyl beta-D-xylopyranoside have been studied under various reaction conditions. Partially pivaloylated products were submitted to additional acetylations. The structures were established by 1H NMR spectroscopy. Representatives of acylated methyl beta-D-xylopyranosides (acyl being pivaloyl, acetyl, or a combination of both) were submitted to hydrolysis catalyzed by rabbit serum and esterases isolated from rabbit serum.

Psychological condition hormone levels in war trauma.

Psychological and hormonal responses to various degrees of war-related traumatic experience were analysed in 91 subjects. Their psychological responses (psychosomatic, personality traits, etc.) were evaluated by the COR-NEX2 test. Based on test results, the subjects were classified into three groups: G1 = normal, G2 = moderate, and G3 = severe response. The distribution of subjects in the three groups was related to the intensity and duration of stress that they had been exposed to. Serum levels of cortisol, prolactin, beta-endorphin, thyroxin and triiodothyronine were analysed in all subjects. The levels of cortisol and prolactin were significantly decreased in subjects expressing a severe psychological response, while the level of prolactin correlated with COR-NEX2 test scores. Although relations to other intervening variables are to be investigated, our results indicated that endocrine changes, following trauma, were not random, but rather related to stress-induced psychological responses, and not to trauma per se.

Endogenously labeled thyroid hormones (131I-T3/T4) in sera of patients with differentiated thyroid carcinoma.

Patients with differentiated thyroid carcinoma (DTC), who have undergone thyroidectomy and radioablation with iodine (131I), are usually monitored with ultrasonography (US) of the neck, 131I whole body scan (WBS), and determination of thyroglobulin (Tg) concentrations in serum. A chromatographic method (CHROM), designed for detection of in vivo labeled thyroid hormones (131I-T3/T4) that circulate in the body after administration of 131I for a WBS, may be used in monitoring of these patients. This study included 35 patients with DTC. Ultrasonography, WBS, Tg, and CHROM method were performed for each of them. One patient was followed-up 6 times, 11 were monitored twice, and the remaining group of 23 patients was examined only once (51 diagnostic tests). We found CHROM results to be in disagreement with the WBS and/or Tg findings in three patients out of 35 at the time of the first visit. In one of them WBS was negative, while Tg and CHROM findings were positive. In this patient the local metastases were proven by US with fine needle biopsy (FNB). In another patient (without signs of thyroid tissue remnant, determined by WBS, Tg, and US) only CHROM was positive. Finally, in the third patient, a thyroid remnant was proven positive on WBS and US (negative CHROM and Tg). We registered an additional peak (peak 4) in chromatograms of 12 out of 35 patients. This component could be tentatively characterized as 3,3'-diiodo-L-thyronine (131I-3,3'-T2). Although this preliminary study included a small number of patients, we show that the CHROM method can be useful as an additional test in monitoring of patients with DTC, especially those with discordant WBS and Tg results.

Catalytic properties of rabbit serum esterases hydrolyzing esterified monosaccharides.

Rabbit serum and one enzyme fraction isolated from rabbit serum by column chromatography (Fraction II) were used as catalysts in regioselective hydrolysis of radiolabelled pivaloylated monosaccharides (Piv = Me3CCO). The hydrolysis of 14C-labelled methyl 2-O-pivaloyl-(2-MP)-, 6-O-pivaloyl (6-MP)-, 2,6-di-O-pivaloyl-(2,6-DP) alpha-D- glucopyranosides and methyl 2-acetamido-2-deoxy-3,6- di-O-pivaloyl-(3,6-DPNAc) alpha-D-glucopyranosides, was studied, as well as that of the non-sugar substrates butyrylthiocholine, thiophenylbutyrate, phenylacetate and paraoxon. The specific activities of 2,6-DP, 3,6-DPNAc, butyrylthiocholine and thiophenylbutyrate were higher in Fraction II than in native sera, while those of phenylacetate and paraoxon were lower. Inhibition studies were done using the substrates mentioned and five different inhibitors, namely bis(p-nitrophenyl phosphate) (BNPP), eserine, paraoxon, HgCl2 and EDTA. The hydrolysis of 2,6-DP and 3,6-DPNAc was not inhibited by HgCl2 and only slightly by EDTA. Paraoxon, eserine and BNPP were progressive inhibitors of the hydrolysis of the two sugar substrates, and the pattern of inhibition resembled closely the inhibition of butyrylthiocholine and thiophenylbutyrate hydrolysis. This result applied to both, native serum and Fraction II. It was concluded that esterases in rabbit serum which hydrolyze pivaloylated sugar substrates belong to the category of serine esterases. Kinetic parameters (KM and Vmax), effects of temperature and pH on activity of esterases from Fraction II were also determined for the hydrolysis of sugar substrates.

Effects of L-(adamant-2-yl)glycyl-L-alanyl-d-isoglutamine on the antitumor action of cyclophosphamide, 5-fu, Cisplatin and dacarbazine on advanced carcinomas of the mouse.

A new biological response modifier, L-(adamant-2-yl)glycyl-L-alanyl-D-isoglutamine hydrochloride (AdTP), recently synthesized and characterized for antitumor, antiviral and immunomodulating properties was studied in comparison to the peptidoglycan monomer (PGM) isolated from Brevibacterium divaricatum to test the effects of their use concomitant to that of anticancer cytotoxic drugs such as cyclophosphamide, 5-fluorouracil (5-FU), cisplatin and 4-(3,3-dimethyl-1-triazeno)-5-carboxamide (dacarbazine). The experiments, performed using both Lewis lung carcinoma and MCa mammary carcinoma of CBA mouse, indicated that: a) the cytotoxic drugs, used at the maximum tolerated doses, caused different degrees of reduction of the tumors; b) the same drugs reduced lung metastases with greater efficacy when treatments were applied at the early stages of metastasis formation; c) AdTP, similarly to PGM confirmed its ability to increase some immunological parameters of lymphocytes obtained from the spleens of the treated mice; d) AdTP increased the effects of 5-FU on lung metastases but failed to show any increase of life expectancy with any treatment performed. These data indicate that AdTP, although increasing some functional responses of lymphocytes in vitro, does not improve the therapeutic activity of cyclophosphamide, cisplatin, 5-FU and dacarbazine in the experimental models presently used.

Multiple changes of immunologic parameters in prisoners of war. Assessments after release from a camp in Manjaca, Bosnia.

OBJECTIVE: To assess immune reactivity in men just released from a war prisoner camp. PARTICIPANTS: Random sample of 29 men from a group of 764 liberated detainees in war prisoner camp in Bosnia, 15 matched healthy control subjects, and pre-war historical control subjects. MAIN OUTCOME MEASURES: Report on immune reactivity parameters, such as lymphocyte immunophenotypes, natural killer cell and phagocyte function, serum cytokines, and hormones. RESULTS: Compared with control subjects, detainees had significantly lowered red blood cell count, hemoglobin mass concentration, hematocrit, total serum proteins, and albumin level, while the percentage and count of monocytes and non-segmented neutrophils were increased. Flow cytometry revealed a significant increase in percentage of activated lymphocytes, activated T lymphocytes, Tc/s lymphocytes, B lymphocytes, and total HLA-DR lymphocytes. The absolute counts of activated lymphocytes and activated T lymphocytes were also significantly increased. The percentages of naive Th/i lymphocytes and the ratio of CD4:CD8 lymphocytes were decreased. The in vitro natural killer cell cytotoxic activity and phagocytic functions of ingestion and digestion were significantly depressed. Serum interferon, serum cortisol, and prolactin were also significantly lowered. Serum tumor necrosis factor was increased. CONCLUSIONS: Alterations in the main parameters of the immune system and depression of important immune effector functions may have resulted from the psychological stress, physical deprivation, and malnutrition experienced by these war camp prisoners during their detainment.

Determination of foetal calf serum in vaccine preparations by immunoradiometric assay and enzyme-linked immunosorbent assay.

A simple and sensitive immunoradiometric assay (IRMA) for detection of foetal calf serum proteins in vaccine preparations has been developed. In this two-site sandwich assay the same preparation of polyclonal anti-FCS immunoglobulin (IgG) was used for coating the solid phase (as a capture antibody) and in a labelled form (I-125 labelled) for detection. The developed assay allows precise and accurate quantification of FCS proteins (down to 10 ng/ml) in vaccine preparations and has been used for screening of FCS residues (a) during production of measles vaccine, and (b) in various different commercial vaccine preparations. An enzyme-linked immunosorbent assay (ELISA) was also developed based on the same assay two-site sandwich principle, where anti-FCS was directly labelled with horse-radish peroxidase. ELISA was comparable in sensitivity and accuracy with IRMA.

Modification of the growth of mca mammary-carcinoma of cba mouse by new adamantylpeptides.

Two new adamantyl-peptides, D-(2-adamantyl)-glycyl-L-alanyl-D-isoglutamine hydro-chloride (Isomer-1) and L-(2-adamantyl)-glycyl-L-alanyl-D-isoglutamine hydrochloride (Isomer-2), were tested in the model of the MCa mammary carcinoma of the CBA mouse. These compounds, and particularly Isomer-2 showed to be more effective in reducing lung metastases than primary tumor growth and to be active in conditions in which a low number of metastases are formed, a condition that is seen when the tumor is implanted s.c. or when its growth is slowed down by a lower inoculum size. The apparent lack of any correlation between the effect of Isomer-2 on tumor growth and metastasis formation and that on the response to ConA of in vitro IL-2 selected spleen lymphocytes seems to suggest that the dose and treatment schedule chosen might not be the most appropriate. In fact, the immunopotentiation observed a 2nd drug injection is virtually lost at the end of treatment and this fact might explain the limited effect on tumor growth, and could probably account for the lack of macroscopical effects on lung metastases and on survival time.

A competitive radioimmunoassay for peptidoglycan monomer.

The preparation and characterization of the essential components to be used in the radioimmunoassay of peptidoglycan monomer (PGM) is described. In order to raise the anti-peptidoglycan monomer antibodies 14C-labelled peptidoglycan monomer-bovine serum albumin conjugate was prepared by the coupling of 14C-peptidoglycan monomer to bovine serum albumin in the presence of glutaraldehyde in 0.1 M NaHCO3 at pH 8.3. The prepared conjugate elicited anti-PGM response in rabbits. A synthetic analog of peptidoglycan monomer, Boc-L-tyrosyl-peptidoglycan monomer was prepared by condensation of unprotected peptidoglycan monomer and N-hydroxysuccinimidester of Boc-L-tyrosine in the presence of triethylamine and the obtained disaccharide-hexapeptide was labelled with Na125I. This compound exhibited the ability of binding to anti-peptidoglycan monomer antibodies. The prepared compounds, namely anti-PGM antibodies and 125I-labelled Boc-L-tyrosyl-peptidoglycan monomer, were used as essential components in competitive radioimmunoassay for peptidoglycan monomer determination in mammalian and human sera and plasma, respectively.

Partial purification of esterases from rabbit serum and their use in regioselective deacylations of sugars.

14C-Labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside was used as a substrate for the detection of esterase activity in the isolation of esterase II from rabbit serum. On treatment of methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside with rabbit serum and esterase II, the 6-O-pivaloyl group was removed selectively. Likewise, the 6-O-pivaloyl group was removed selectively from methyl 2-acetamido-2-deoxy-3,4,6-tri-O-pivaloyl-alpha-D-glucopyranoside.

Immunophenotypization of cells involved in local immune response and serum antibodies in cephalosporin-treated mice.

The in vivo potency of cefodizime (HR 221), tiprotimod (a new synthetic thiazole derivative of HR 221, HBW 538) and cefotaxime to modulate the initiation of immune response in the draining lymph node (LN) after subcutaneous injection of SRBC was evaluated. The timing and sequence of events in the regional LN was investigated by immunophenotypization of node cells with monoclonal antibodies, and the systemic reaction was estimated as primary antibody response to SRBC. From the results it is possible to conclude that: (1) subcutaneous administration of a small dose (2.5-3.0 mg/kg) of cephalosporins, together with antigen, enhanced primary antibody production and persistence; (2) the increase in serum antibodies was preceded by a change in percentage of L3T4+ cells within the regional (popliteal) lymph node. In comparison to antigen alone, cephalosporins (during early immune response) increased the percentage of L3T4+ cells; (3) LN cellularity was strongly enhanced by cephalosporins; (4) cefotaxime influenced the kinetics of the cellularity and the L3T4/Lyt-2 index differently than cefodizime and HBW 538. HBW 538 had either a similar or stronger effect than cefodizime, as judged by the adjuvant effect on antibody production and the appearance of L3T4+ cells during the immunizing period.

Comparison of immunogenicity of recombinant and plasma derived hepatitis B antigen in guinea-pigs.

Four different preparations of hepatitis B antigen (HBsAg) were tested in parallel with respect to their ability to elicit anti-HBs in guinea-pigs. We compared the effect of low doses (5 micrograms single dose and 30 micrograms total amount) of human plasma derived and recombinant HBsAg, in purified forms or prepared as vaccines, respectively. The highest titre of anti-HBs developed was detected in guinea-pigs immunized with plasma derived HBsAg, followed the by response in animals immunized with plasma derived vaccine. Purified recombinant antigen and recombinant vaccine exhibited poor immunogenicity compared with plasma derived antigen preparations. The increase in the dose of recombinant antigen (150 micrograms total amount) resulted in markedly improved response in two out of ten animals. Anti-HBs immunoglobulin fractions from pooled sera within each group of animals were isolated, partially purified, coupled to polystyrene beads and subsequently used in immunoradiometric assay with monoclonal 125I-anti-HBs. Only the anti-HBs isolated from pooled sera of animals immunized with purified plasma derived antigen met the requirements for high sensitivity and antigen specificity in such assay. Comparable properties were exhibited by the anti-HBs obtained from one guinea-pig immunized with a larger amount of recombinant antigen. With these two anti-HBs immunoglobulin preparations, amounts as low as 1 ng of HBsAg could be detected.

Enzymatic deacylations of esterified saccharides--III. Comparison of de-esterifications by serum esterases from different sources.

1. 14C-labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside (1) was used as a substrate for esterases from rabbit, guinea pig, mouse, donkey, pig, horse, sheep and human sera. 2. Stepwise de-esterification of the diester substrate 1 occurred with rabbit, guinea pig and mouse serum. Data on time-course experiments and kinetic data are reported. 3. The use of donkey, pig, horse, sheep and human serum led to the migration of the 2-O-pivaloyl group in substrate 1 to the position 4- in the sugar molecule, followed by stepwise de-esterifications of both 1 and the newly formed methyl 4,6-di-O-pivaloyl-alpha-D-glucopyranoside (4). A report is given on the time-course experiments.

In vivo and in vitro modulation of NK and ADCC activities of mouse spleen cells by peptidoglycan monomer (PGM).

The ability of peptidoglycan monomer (PGM), an immunomodulator obtained from Brevibacterium divaricatum, to modulate NK and ADCC activities of spleen cells was tested in mice with constitutively weak (C57B1) or strong NK activity (CBA, C3H). In weak reactors, i.v. injection of PGM enhanced the NK and ADCC activity as effectively as a known stimulator, poly I.C, and the dynamics of the response was comparable. In strong reactors, PGM caused two peaks of the ADCC response, and one peak of NK stimulation (after an initial decline), whereas poly I.C caused more or less persistent stimulation of both activities. Incubation of spleen cells with PGM was generally less effective than the treatment in vivo. No alteration of NK activity was recorded at high effector-to-target ratio (E:T), and at low ones, PGM caused suppression. This was true both for weakly and strongly reactive cells. ADCC was either unchanged (CBA spleen cells), stimulated (C3H), or suppressed at high E:T and enhanced at low E:T (C57B1). PGM apparently activates an interlocked regulatory mechanism, and the final outcome probably depends on relative concentrations of regulatory and effector cells and on their per cell activities. Hence the effect varied with the time interval between treatment and assay, with strain-related constitutive reactivity, and with the E:T ratio.