Susceptibility of mice to invasive aspergillosis correlates with delayed cell influx into the lungs.

Ubiquitous fungus Aspergillus fumigatus (A. fumigatus) is involved in invasive pulmonary aspergillosis (IPA), a frequent infection in immunocompromized patients. Genetic differences are likely to play a role predisposing to IPA. This study was aimed to compare six genetically different mouse strains in their susceptibility to IPA and to determine possible mechanisms involved in the pathogenesis of this infection. Immunosuppressed BALB/c and C57BL/6 mice infected with A. fumigatus conidia were more resistant to IPA than DBA/1, DBA/2, CBA, and A/Sn strains. Phagocytosis of A. fumigatus conidia by blood polymorphonuclear neutrophils (PMN) or bone marrow derived dendritic cells showed no difference between strains. All IPA susceptible strains demonstrated decreased PMN influx into the lungs during infection compared with resistant strains. Flow cytometry analysis of the composition of lung infiltrating cells showed that IPA susceptible mice had a decreased number of phagocytes before the infection. After infection the numbers of Gr-1(+)CD11b(+) PMN cells in the lungs of immunosuppressed mice increased from 10-20% to 50-60% while the percentage of CD11(+)F4/80(+) resident macrophages was unchanged. Among susceptible strains DBA/2 and A/Sn have a defect in C5 component of complement. Injection of normal serum into complement deficient but not into complement sufficient CBA or DBA/1 mice significantly improved their survival. We showed that complement replacement significantly increased PMN homing to the lungs of complement deficient mice. Thus, defect in complement system can predispose to IPA. Our results demonstrated that early influx of PMN into the lungs of mice is important for the resistance to IPA.

Effects of conidia of various Aspergillus species on apoptosis of human pneumocytes and bronchial epithelial cells.

Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species.

Haptoglobin is present in human endometrium and shows elevated levels in the decidua during pregnancy.

Assuming that haptoglobin, by virtue of its immunomodulatory properties, could be a regulatory factor during reproduction, its presence in the human uterus was determined. Protein extracts from endometrial tissue samples of pregnant and non-pregnant women were analysed by the immunoblot technique and the intensities of specific bands were quantified. Bands corresponding to haptoglobin were identified in tissue samples obtained from both sources. Protein, purified by high-performance liquid chromatography and monitored by Western blot analysis for its haptoglobin identity, was used for amino-terminal sequencing. Sequencing of the 42 kDa protein identified it as the beta chain of haptoglobin. Immunohistochemistry was used to corroborate the findings and to visualize the distribution of haptoglobin in the tissue. The intensity of the 42 kDa band derived from decidua graviditatis was significantly higher than the intensity of bands derived from non-pregnant endometrium in the proliferative phase (P < 0.01) and in the secretory phase (P < 0.05). Immunohistochemical staining with anti-human haptoglobin antibody elicited strong signals in the decidua graviditatis and weaker signals in the normal endometrium, with the latter showing menstrual cycle-dependent variation. Moderate staining of stroma and a lack of staining of epithelium in the proliferative phase contrasted with the strong staining of stroma and moderate level of staining of epithelium observed in the secretory phase. Haptoglobin in the uterus may exert several functions such as the known binding of haemoglobin, but could also be involved in the multi-factorial mechanism protecting the fetus from a maternal allograft-like immune response.

TNF-induced haptoglobin release from human neutrophils: pivotal role of the TNF p55 receptor.

Haptoglobin (Hp), TNF-alpha, and neutrophils are parts of a highly interactive ensemble participating in inflammatory processes. Hp is taken up by neutrophils, stored within a cytoplasmic granular compartment, and is secreted during phagocytosis by those cells. In the present study, the effects of TNF-alpha on the release of Hp from human neutrophils were investigated. Incubation of neutrophils with TNF-alpha induced the release of Hp from cells in a time- and concentration-dependent manner as revealed by Western blot analysis and immunofluorescence. The release of Hp induced by TNF-alpha was not due to nonspecific lysis of the cells. TNF-alpha is a highly pleiotropic cytokine that mediates its effects by binding to two distinct receptors (p55 and p75). Administration of TNF-alpha mutants binding specifically either to the p55 or to the p75 TNF receptors showed that there is a preference of TNF-alpha for the p55 receptor in the mediation of Hp release by neutrophils. A stimulated release of Hp was also induced by the chemotactic tripeptide fMLP. The TNF-alpha-induced release of Hp from neutrophils was inhibited by erbstatin, a tyrosine kinase inhibitor. These findings suggest that TNF-alpha may promptly increase the level of Hp at sites of infection or injury, leading to the modulation of the acute inflammatory response.

Tumor necrosis factor mutants with selective cytotoxic activity.

Tumor necrosis factor (TNF-alpha) has a cytotoxic or cytostatic effect when tested with various malignant cell lines. Clinical trials in cancer patients, however, revealed high systemic toxicity of TNF-alpha. The existence of two types of receptor may partially explain the pleiotropic activity of TNF-alpha. The purpose of this study was to characterize the relative cytotoxic activity of TNF-alpha and TNF mutants on the mouse fibrosarcoma L929 cells in a standard cytotoxicity test, on human larynx carcinoma HEp-2 cells, and on human monoblastoid leukemic cells U937. TNF mutants were obtained by site-directed mutagenesis. The purity of TNF-alpha was established by capillary electrophoresis. TNF-alpha and TNF mutants were analysed by Western blot analysis using monoclonal antibodies against TNF-alpha. The results show that TNF mutants can recognize the different TNF-receptors (TNF-R) selectivity. It is generally believed that activation of TNF-R75 is responsible for the systemic toxicity of TNF-alpha. Hence, the development of TNF mutants, binding selectively to TNF-R55, could lead to new option for an anticancer treatment that would be devoid of the deleterious effect of TNF-alpha.

Immunoblot detection of decreased antibodies to haptoglobin-like protein in the serum of infertile women with or without endometriosis.

The purpose of this study was to find out differences in the levels of antibodies to distinct antigens in the serum of fertile versus infertile patients with and without endometriosis and to identify these antigens. Blood was collected from 61 patients undergoing laparoscopy for pelvic pain, infertility, or tubal ligation. Serum antibodies against serum antigens with apparent molecular masses of 22 kDa and 18 kDa were assessed by immunoblot analysis. Gel filtration, HPLC DEAE ion-exchange chromatography, NH2-terminal sequencing, and double immunodiffusion were used to characterize and identify these proteins. The relative amount of antibodies reacting with 22- and 18-kDa proteins detected in a standard preparation of antigens was significantly lower in the serum of infertile patients with endometriosis (0.20 +/- 0.05 and 0.57 +/- 0.10) and without endometriosis (0.21 +/- 0.06 and 0.53 +/- 0.08) compared to that of control fertile women without endometriosis (0.53 +/- 0.08 and 1.09 +/- 0.13). After purification by chromatography, the NH2-terminal amino acid sequence of the proteins in the 22- and 18-kDa range was identical through 20 amino acids with the alpha chain of the human haptoglobin. Double immunodiffusion implied immunochemical identity between commercial human haptoglobin and the purified proteins. We conclude that infertile patients with and without endometriosis show reduced serum levels of antibodies against a haptoglobin-like protein. These results would indicate an alteration of the immune system or changes in the levels of these antigens in infertility and/or endometriosis.

Determination of binding site of anti-tumour necrosis factor-alpha monoclonal antibody using hybrid and mutant proteins.

In order to map the immunogenic epitope for the monoclonal antibody E7H2 on the human tumour necrosis factor (hTNF-alpha) molecule, a number of chimeric proteins were developed by in-frame joining segments of the human genes encoding TNF-alpha and lymphotoxin (TNF-beta) as well as by coupling appropriate coding regions for human and mouse TNF-alpha. High level expression of these chimeric genes was achieved in Escherichia coli by placing the coding sequences under control of either E. coli trp-promoter or a tandem of bacteriophage T7 constitutive promoters A2 and A3. As revealed by Western blot analysis with monoclonal antibody E7H2 directed against human TNF-alpha, the region involved in the binding of this antibody includes sequence ValGluLeuArg in the N-terminal part of the TNF-alpha molecule.

Decreased amounts of antibodies to 22 and 18 kDa antigens in the peritoneal fluid of patients with endometriosis. LE Group d'Investigation en Gynecologie.

Accumulated evidence implicates immunological alterations in endometriosis. The purpose of this study was to look for variations in antibodies to distinct antigens in peritoneal fluid of women with and without endometriosis. Peritoneal fluid was aspirated from 17 women undergoing laparoscopy for tubal ligation and 37 patients complaining of symptoms of pain and /or infertility. Peritoneal fluid antibodies to a standard preparation of peritoneal fluid antigens were detected by Western blot analysis using peroxidase-labelled anti-human immunoglobulin G antibodies specific to the Fc region. Antibodies to distinct antigens were quantified by estimating the ratio of the relative optical density between samples and a standard amount of antibodies. Marked changes were found in the antibody detection to two antigens having apparent molecular weights of 22 and 18 kDa. The intensity of the antibody signal was significantly weaker in the peritoneal fluid from endometriosis patients (0.36 +/- 0.06 and 0.46 +/- 0.06) compared with that in women without endometriosis (0.62 +/- 0.08 and 0.75 +/- 0.06). It was also weaker in patients without endometriosis presenting with infertility (0.36 +/- 0.07 and 0.47 +/- 0.08), but only the 18 kDa antigen result was significant. After adjusting for infertility, the P values for the 18 and 22 kDa bands were 0.03 and 0.28 (not significant) respectively in the group of endometriosis patients. These changes were not related to the phase of the menstrual cycle. These data suggest an alteration in the immune response to two distinct antigens in the peritoneal fluid from women with endometriosis and infertility. Further evaluation of these two antigens and their antibodies would be of interest to help understand endometriosis and its associated infertility.

Decreased amounts of antibodies to 22 and 18 kDa antigens in the peritoneal fluid of patients with endometriosis

Accumulated evidence implicates immunological alterations in endometriosis. The purpose of this study was to look for variations in antibodies to distinct antigens in peritoneal fluid of women with and without endometriosis. Peritoneal fluid was aspirated from 17 women undergoing laparoscopy for tubal ligation and 37 patients complaining of symptoms of pain and/or infertility. Peritoneal fluid antibodies to a standard preparation of peritoneal fluid antigens were detected by Western blot analysis using peroxidase-labelled anti-human immunoglobulin G antibodies specific to the Fc region. Antibodies to distinct antigens were quantified by estimating the ratio of the relative optical density between samples and a standard amount of antibodies. Marked changes were found in the antibody detection to two antigens having apparent molecular weights of 22 and 18 kDa. The intensity of the antibody signal was significantly weaker in the peritoneal fluid from endometriosis patients (0.36 ± 0.06 and 0.46 ± 0.06) compared with that in women without endometriosis (0.62 ± 0.08 and 0.75 ± 0.06). It was also weaker in patients without endometriosis presenting with infertility (0.36 ± 0.07 and 0.47 ± 0.08), but only the 18 kDa antigen result was significant. After adjusting for infertility, the P values for the 18 and 22 kDa bands were 0.03 and 0.28 (not significant) respectively in the group of endometriosis patients. These changes were not related to the phase of the menstrual cycle. These data suggest an alteration in the immune response to two distinct antigens in the peritoneal fluid from women with endometriosis and infertility. Further evaluation of these two antigens and their antibodies would be of interest to help understand endometriosis and its associated infertility. Keywords: antibodies/antigens/endometriosis/peritoneal fluid/Western blot analysis

Development of an enzyme immunoassay for the measurement of human tumour necrosis factor-alpha (hTNF-alpha) using bispecific antibodies to hTNF-alpha and horseradish peroxidase.

The cytokine tumour necrosis factor-alpha (TNF-alpha) is involved in several pathological processes, and human recombinant TNF-alpha (hrTNF-alpha) is available for testing in preclinical and clinical research. The purpose of this work was the creation of tetradomas producing bispecific anti-hTNF-alpha/anti-HRP (horseradish peroxidase) antibodies and the development of a rapid and sensitive solid-phase enzyme immunoassay. Monoclonal antibodies obtained against hrTNF-alpha could recognize both natural and recombinant hTNF-alpha. The four chosen hybridomas produced IgG1 with an affinity constant of the order of 10(-9) M. Three of them recognized different epitopes. The clone selected for fusion with the hybridoma producing anti-HRP antibodies secreted antibodies against portion 30-50 of the hTNF-alpha N-terminal amino acid residues as found by Western-blot analysis with mutant and chimaeric proteins. The tetradoma producing bispecific anti-hTNF-alpha/anti-HRP antibodies was identified using a fluorescence-activated cell sorter. Bispecific antibodies were isolated by hydroxyapatite chromatography. A sandwich ELISA was developed: one of the monoclonal anti-TNF-alpha antibodies was absorbed to the solid phase as the catcher and was detected by a bispecific anti-hTNF-alpha/anti-HRP antibody. The detection limit of the assay was 1 ng/ml. With such ELISA, the level of hTNF-alpha could be conveniently estimated in different samples containing either natural or recombinant hTNF-alpha in an experimental environment or in clinical trials.

Addition of hTNF alpha potentiates cytotoxicity of taxol in human ovarian cancer lines.

Some reports indicate that the cytotoxic activity of hTNFa and taxol might be enhanced when combined with certain chemotherapeutic agents. To assess if taxol might modulate the action of hTNF alpha, we have studied the cytotoxic action of hTNF alpha in combination with taxol on two ovarian carcinoma cell lines, the SKOV3 and SKVLB. These two cell lines are resistant to the action of hTNF alpha. The SKVLB cells overexpressed the multidrug resistant gene (MDR-1) which confers a resistance to taxol. We observed that the combination of low concentration of hTNF alpha with taxol could increase the cytotoxic activity of taxol in SKOV3 cells. Furthermore, a four-hour pretreatment with taxol before adding hTNF alpha to the culture has shown a significant increase in their cytotoxic activity. This increase was not observed at the same level in SKOV3 cells pretreated with hTNF alpha. However, the phenomena was not observed in SKVLB cells. This indicates that taxol could alter the mechanism of hTNF alpha-resistance.

[Modeling antigenic determinants of the hepatitis A virus using synthetic peptides]. / Modelirovanie antigennykh determinant virusa gepatita A s pomoshch'iu sinteticheskikh peptidov.

Monoclonal and polyclonal anti-hepatitis A (HAV) antibodies were used to search for peptides mimicking the antigenic determinants of HAV. Synthetic peptides VP1 115-139, VP1 117-139, VP1 126-139, VP2 69-99, VP2 80-99, VP3 45-57, VP3 137-150, were shown to bind the anti-HAV antibodies in ELISA. Peptides VP1 115-139, VP1 117-139, VP2 69-99 were utilized to produce the antipeptide antibodies. Mice were immunized with the free peptides or with their conjugates with ovalbumin. Only the free VP2 69-99 caused formation of HAV binding antibodies.

A 56- to 54-kilodalton non grata signal in immunoblot analysis using the horseradish peroxidase chemiluminescence system.

The major advantages of the horseradish peroxidase chemiluminescence (HRP-CL) immunodetection method in Western blot analysis are its high sensitivity, nonradioactive detection, economy of the primary antibody, and speed of detecting the signal. However, we observed a strong and reproducible signal that was detected regardless of the primary antibody and of the cell type used. This signal, present at 56-54 kilodaltons (kDa), is generated in absence of any primary antibody and seems to be an intrinsic reaction of the HRP-labeled second antibody anti-immunoglobulin with an unidentified cellular protein. The use of dry milk throughout all the steps of the procedure abolishes this signal. For those interested in one of the numerous proteins migrating at or close to 56-54 kDa, the question therefore arises as to which signals generated by the HRP-CL in this region are bona fide and which are non grata pseudo signals.

[Synthesis of an artificial gene coding for thymosin alpha1 and its expression in Escherichia coli as a hybrid with human tumor necrosis factor]. / Sintez iskusstvennogo gena, kodiruiushchego timozin alpha1, i ego ékspressiia v Escherichia coli v sostave gibridov s faktorom nekroza opukholei cheloveka.

Chemical-enzymatic synthesis and cloning in Escherichia coli of an artificial gene encoding the immunoactive peptide thymosin alpha 1 have been carried out. Recombinant plasmids were constructed which contain fusion genes coding for hybrids of human tumour necrosis factor (TNF) and thymosin alpha 1 as N- or C-terminal part of the hybrid protein. In the C-terminal hybrid protein, TNF and thymosin alpha 1 are linked through a methionine residue, thus allowing for thymosin alpha 1 to be cleaved off the rest of the hybrid protein with cyanogen bromide. In case of the N-terminal hybrid protein, the linker between the thymosin alpha 1 and TNF sequences is the acid-labile dipeptide Asp-Pro. Expression of the hybrid genes in E. coli and properties of the recombinant proteins were studied. The N-terminal hybrid protein was secreted into periplasmic space, in contrast with the C-terminal hybrid protein, which formed insoluble aggregates inside bacterial cells. Procedures for the isolation of both hybrid proteins were developed. The N-terminal hybrid protein displayed full biological activity in the cytotoxic assay on the mouse fibroblast L-929 whereas the C-terminal hybrid protein proved to be much less active. Treatment of the hybrid protein TNF-thymosin alpha 1 with cyanogen bromide lead to a mixture of two polypeptides, from which thymosin alpha 1 was purified to homogeneity by simple chromatographic procedures.

[The antigenic properties of hepatitis A virus particles possessing different physicochemical characteristics]. / Antigennye svoistva chastits virusa gepatita A, obladaiushchikh razlichnymi fiziko-khimicheskimi kharakteristikami.

A modified enzyme immunoassay based on adsorption of antihepatitis A virus (HAV) IgG-HRPO conjugate and monoclonal antibodies to HAV were used to investigate antigenic differences between mature HAV virions and subviral particles with different buoyant densities in CsCl produced in HAV-infected cells. The mature virions (1.34 g/cm3) appeared to have common antigenic determinants with subviral particles (1.20, 1.27, and 1.30 g/cm3) and possess some additional determinants. Nevertheless, both subviral particles and mature virions induced antibodies capable of neutralizing HAV infectivity in tissue culture.

[Hybrid human lymphokine genes. I. Design of recombinant plasmids coding hybrids of immune interferon and human tumor necrosis factor]. / Geny gibridnykh limfokinov cheloveka. I. Konstruirovanie rekombinantnykh plazmid, kodiruiushchikh gibridy immunnogo interferona i faktorov nekroza opukholei cheloveka.

Recombinant plasmids coding for hybrid proteins between human interferon gamma and human tumour necrosis factor alpha or beta have been constructed using site-directed mutagenesis. The genes were fused via a synthetic oligonucleotide linker coding for tetrapeptide Pro-Val-Gly-Pro. The fused genes were expressed in Escherichia coli under control of early promoters of bacteriophage T7. E. coli cells harbouring the plasmids with the hybrid genes gave rather high level of the fused proteins biosynthesis. The hybrid recombinant proteins proved to be unstable in E. coli cells.

[Expression in Escherichia coli of DNA coding for human tumor necrosis factor]. / Ekspressiia v Escherichia coli DNK, kodiruiushchei faktor nekroza opukholei cheloveka.

The variants of expression in Escherichia coli of artificial DNA coding for human tumor necrosis factor, an important immune modulator with selective cytotoxic action on a number of transformed cell lines have been described. The DNA was placed under control of either phage M13 promoter of gene for main coat protein or tandem of pair of E. coli tryptophane promoters. It has been shown that E. coli cells harbouring plasmids described with artificial TNF gene provide good level of protein biosynthesis. The protein has been purified by anion exchange chromatography near to homogeneity and used for preparation of monoclonals. As result three hybridomas effectively produced high affinity monoclonal anti-TNF antibodies have been obtained and characterized.

[Production in vivo of macrophage migration inhibition and stimulation factors in the inductive phase of the alloimmune response]. / Produktsiia in vivo faktorov podavleniia i stimuliatsii migratsii makrofagov v induktovnoi faze alloimmunnogo otveta.

Macrophage migration inhibition and stimulation factors were revealed in the serum of alloimmune mice by polyacrylamide gel electrophoresis. CBA mice were primed intravenously with 90 X 10(6) and challenged subsequently intravenously with 20 X 10(6) spleen cells of BALB/c mice. Macrophage migration inhibition and stimulation factors were revealed in pre- and post-albumin fractions, respectively, already on days 1 and 6 after alloimmunization. It is suggested that at an early phase of immune response the immunomediators with alternative functions whose local activity gradients predetermined the macrophage behaviour, are released into the serum of alloimmune animals.

[Nonidentity of Lyt phenotypes and the radiosensitivity of early and late producers of macrophage migration inhibitory factor in reaction to H-2 complex antigens]. / Neidentichnost' Lyt-fenotipov i radiochuvstvitel'nosti "rannikh" i "pozdnikh" produtsentov faktora podavleniia migratsii makrofagov pri reaktsii na antigeny kompleksa H-2.

MIF production induced at different times after intravenous immunization of mice with irradiated allogeneic splenic cells showed different sensitivity to the treatment with anti-Lyt-antibodies and to gamma-irradiation. The "early" MIF producers induced several hours after alloimmunization were sensitive to irradiation at a dose of 500 rad and to the treatment with anti-Lyt-1- and anti-Lyt-2-antibodies and complement, while the "late" MIF producers which appeared 21 days after alloimmunization were resistant to irradiation at doses of 500 and 1500 rad and to the treatment with anti-Lyt-2-antibodies but sensitive to anti-Lyt-1-antibodies. It is supposed that the "early" MIF producers of the Lyt-1+2+ phenotype are immature precursors of T cells which, in contradistinction to the "late" MIF producers of the Lyt-1+2+ phenotype, are activated and produce MIF without proliferation after a twofold contact with antigen.