Absence of Nkx2-3 homeodomain transcription factor reprograms the endothelial addressin preference for lymphocyte homing in Peyer's patches.

Although the homing of lymphocytes to GALT has been extensively studied, little is known about how high endothelial venules (HEVs) within Peyer's patches (PPs) are patterned to display dominantly mucosal addressin cell adhesion molecule 1 (MAdCAM-1). In this study, we report that Nkx2-3-deficient mice show gradual loss of MAdCAM-1 in PPs postnatally and increased levels of mRNA for peripheral lymph node addressin (PNAd) backbone proteins as well as enhanced expression of MECA79 sulfated glycoepitope at the luminal aspect of HEVs, thus replacing MAdCAM-1 with PNAd. Induction of PNAd in mutant PPs requires lymphotoxin ß receptor activity, and its upregulation needs the presence of mature T and B cells. Furthermore, treatment with MECA-79 anti-PNAd mAb in vivo effectively blocks lymphocyte homing to mutant PPs. Despite the replacement of MAdCAM-1 by PNAd in HEV endothelia, lymphocytes could efficiently home to PPs in mutant mice. We conclude that although Nkx2-3 activity controls the addressin balance of HEVs in GALT, the general HEV functionality is preserved independently from Nkx2-3, indicating a substantial plasticity in the specification of GALT HEV endothelium.

A mixture of amino acids and other small molecules present in the serum suppresses the growth of murine and human tumors in vivo.

Previously we have hypothesized that the small molecules which are selectively accumulated in cancer cells might participate in a non-immunological antitumor surveillance mechanism. We demonstrated earlier that a mixture of experimentally selected substances ("active mixture", AM: L-arginine, L-histidine, L-methionine, L-phenylalanine, L-tyrosine, L-tryptophan, L-ascorbate, D-biotin, pyridoxine, riboflavin, adenine, L(-)malate) possesses a selective toxic effect in vitro on a variety of tumor cell lines, and we have shown that the AM selectively induces apoptosis of cancer cells in vitro. To explore the in vivo significance of our earlier findings we examined the antitumor effect of AM in Colon 26 murine colorectal adenocarcinoma, B16 murine melanoma, MXT murine mammary carcinoma, S180 murine sarcoma, P388 murine lymphoid leukemia, HL-60 human promyeloid leukemia, PC-3 human prostate carcinoma, and HT-29 human colon carcinoma tumor models. Treatment of tumor bearing mice with AM inhibited the growth of the tumors investigated, with an inhibitory effect ranging from 40 to 69%. The AM had a comparable antitumor effect with 5-fluorouracil and cisplatin in the Colon-26 tumor model, and combined treatment with AM and 5-fluorouracil or cisplatin resulted in an enhanced tumor growth inhibitory effect. The AM induced apoptosis through the mitochondrial pathway and induced G1 arrest in PC-3 cells and increased the number of apoptotic cells in PC-3 xenografts. These findings suggest that the AM might offer an interesting perspective in the treatment of cancer and in combination with other treatments may offer hope for a more effective cancer therapy.

Transcription factor Nkx2-3 controls the vascular identity and lymphocyte homing in the spleen.

The vasculature in the spleen and peripheral lymph nodes (pLNs) is considerably different, which affects both homing of lymphocytes and antigenic access to these peripheral lymphoid organs. In this paper, we demonstrate that in mice lacking the homeodomain transcription factor Nkx2-3, the spleen develops a pLN-like mRNA expression signature, coupled with the appearance of high endothelial venules (HEVs) that mediate L-selectin-dependent homing of lymphocytes into the mutant spleen. These ectopic HEV-like vessels undergo postnatal maturation and progressively replace MAdCAM-1 by pLN addressin together with the display of CCL21 arrest chemokine in a process that is reminiscent of HEV formation in pLNs. Similarly to pLNs, development of HEV-like vessels in the Nkx2-3-deficient spleen depends on lymphotoxin-ß receptor-mediated signaling. The replacement of splenic vessels with a pLN-patterned vasculature impairs the recirculation of adoptively transferred lymphocytes and reduces the uptake of blood-borne pathogens. The Nkx2-3 mutation in BALB/c background causes a particularly disturbed splenic architecture, characterized by the near complete lack of the red pulp, without affecting lymph nodes. Thus, our observations reveal that the organ-specific patterning of splenic vasculature is critically regulated by Nkx2-3, thereby profoundly affecting the lymphocyte homing mechanism and blood filtering capacity of the spleen in a tissue-specific manner.

Probing Serum Albumins and Cyclodextrins as Binders of the Mycotoxin Metabolites Alternariol-3-Glucoside, Alternariol-9-Monomethylether-3-Glucoside, and Zearalenone-14-Glucuronide.

Mycotoxins are toxic metabolites of molds. Chronic exposure to alternariol, zearalenone, and their metabolites may cause the development of endocrine-disrupting and carcinogenic effects. Alternariol-3-glucoside (AG) and alternariol-9-monomethylether-3-glucoside (AMG) are masked derivatives of alternariol. Furthermore, in mammals, zearalenone-14-glucuronide (Z14Glr) is one of the most dominant metabolites of zearalenone. In this study, we examined serum albumins and cyclodextrins (CDs) as potential binders of AG, AMG, and Z14Glr. The most important results/conclusions were as follows: AG and AMG formed moderately strong complexes with human, bovine, porcine, and rat albumins. Rat albumin bound Z14Glr approximately 4.5-fold stronger than human albumin. AG-albumin and Z14Glr-albumin interactions were barely influenced by the environmental pH, while the formation of AMG-albumin complexes was strongly favored by alkaline conditions. Among the mycotoxin-CD complexes examined, AMG-sugammadex interaction proved to be the most stable. CD bead polymers decreased the mycotoxin content of aqueous solutions, with moderate removal of AG and AMG, while weak extraction of Z14Glr was observed. In conclusion, rat albumin is a relatively strong binder of Z14Glr, and albumin can form highly stable complexes with AMG at pH 8.5. Therefore, albumins can be considered as affinity proteins with regard to the latter mycotoxin metabolites.

The individual and combined effects of ochratoxin A with citrinin and their metabolites (ochratoxin B, ochratoxin C, and dihydrocitrinone) on 2D/3D cell cultures, and zebrafish embryo models.

Ochratoxin A and citrinin are nephrotoxic mycotoxins produced by Aspergillus, Penicillium, and/or Monascus species. The combined effects of ochratoxin A and citrinin have been examined in more studies; however, only limited data are available regarding the co-exposure to their metabolites. In this investigation, the individual toxic effects of ochratoxin A, ochratoxin B, ochratoxin C, citrinin, and dihydrocitrinone were tested as well as the combinations of ochratoxin A with the latter mycotoxins were examined on 2D and 3D cell cultures, and on zebrafish embryos. Our results demonstrate that even subtoxic concentrations of certain mycotoxins can increase the toxic impact of ochratoxin A. In addition, typically additive effects or synergism were observed as the combined effects of mycotoxins tested. These observations highlight that different cell lines (e.g. MDBK vs. MDCK), cell cultures (e.g. 2D vs. 3D), and models (e.g. in vitro vs. in vivo) can show different (sometimes opposite) impacts. Mycotoxin combinations considerably increased miR-731 levels in zebrafish embryos, which is an early marker of the toxicity on kidney development. These results underline that the co-exposure to mycotoxins (and/or mycotoxin metabolites) should be seriously considered, since even the barely toxic mycotoxins (or metabolites) in combinations can cause significant toxicity.

Construct validity evaluation of the European Scleroderma Study Group activity index, and investigation of possible new disease activity markers in systemic sclerosis.

OBJECTIVES: To evaluate the construct validity of the European Scleroderma Study Group (EScSG) activity index and to propose modifications if necessary. METHODS: One hundred and thirty-one consecutive patients were investigated and re-evaluated 1 year later. Modified Rodnan skin score (MRSS), skin ulcers and joint contracture numbers, hand anatomic index (HAI), BMI, spirometry, carbon monoxide diffusing capacity (DL(CO)), left ventricular ejection fraction, pulmonary arterial hypertension, HAQ Disability Index (HAQ-DI), patient skin self-assessment questionnaire and several biomarkers were recorded, in addition to the data required for the EScSG activity index. Statistical analysis was performed by categorical principal component analysis (CATPCA). RESULTS: The EScSG activity index appeared in the same dimension as the HAQ-DI, ulcer score and joint contractures, MRSS, patient-reported skin score and HAI by CATPCA. Parameters of lung involvement appeared in another dimension. We constructed a 12-point activity index that was equally associated with both dimensions, by adding the forced vital capacity/DL(CO), change in DL(CO), change in the ulcer scores, HAQ-DI and patient-reported skin score. Biomarkers including vascular endothelial growth factor, soluble P-selectin glycoprotein ligand-1, CRP and albumin were related to both the EScSG and the 12-point index, though they did not improve the total variance of the model. CONCLUSION: The construct validity of the EScSG activity index is good, though the lung-related disease activity may not be sufficiently represented. Further validation steps may be required for both the EScSG and our 12-point activity index.

Naturally occurring and disease-associated auto-antibodies against topoisomerase I: a fine epitope mapping study in systemic sclerosis and systemic lupus erythematosus.

Auto-antibodies against topoisomerase I (topo I) are frequently detected in sera of systemic sclerosis (SSc) patients. Anti-topo I auto-antibodies are considered to be associated with the diffuse cutaneous form of systemic sclerosis (dcSSc). However, anti-topo I auto-antibodies are also detected in limited cutaneous systemic sclerosis (lcSSc) and systemic lupus erythematosus (SLE). In this study, we compared the epitope specificity of anti-topo I auto-antibodies present in sera of dcSSc, lcSSc and SLE patients. We have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with IgG purified from patients' sera. Regions of topo I selected from the library were expressed as recombinant fusion proteins and were tested with ELISA and western blot. We unexpectedly found that antibodies against a fragment of topo I [fragment F4 [amino acid (AA)] 451-593] could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than SSc and SLE. Using sera of dcSSc, lcSSc and SLE patients, we showed that the pattern of recognized epitopes is different between these patient groups. Fragment F4 was recognized by all patients. Fragment F1 (AA 5-30) was recognized by 9 of 34 dcSSc patients. Fragment F8 (AA 350-400) was recognized by four of eight SLE patients. Analysis of clinical data revealed a significant difference between the F1-negative and F1-positive groups of SSc patients in age and in the duration of the disease. According to our results, the newly identified fragments F1 and F8 could represent characteristic epitopes for dcSSc and SLE, respectively.

Characterisation of eGFP-transgenic BALB/c mouse strain established by lentiviral transgenesis.

Lentiviral technology is a powerful tool for the creation of stable transgenic animals. However, uncertainties have remained whether constitutive promoters resist long-term silencing. We used concentrated HIV-1 based lentiviral vectors to create stable transgenic BALB/c mice by perivitelline injection. In our vectors eGFP expression was driven by the human EF1alpha promoter. The established transgenic animals were analyzed for eGFP expression by in vivo fluorescence imaging, PCR, histology and flow-cytometry. eGFP expression showed even distribution without mosaicism; however, tissue-dependent differences of eGFP expression were observed. Up to the sixth generation only one newborn showed eGFP inactivation. eGFP + transgenic bone marrow cells efficiently provided long-term haemopoietic repopulation in radiation chimeras, regenerating all bone marrow-derived lineages with eGFP + cells with distinct eGFP expression profiles. The established eGFP + BALB/c mouse strain is expected to be extremely useful in various immunological experiments.

Increased chondrocyte death after steroid and local anesthetic combination.

BACKGROUND: Hyaline articular cartilage has limited repair and regeneration capacity. Intraarticular administration of glucocorticoid and local anesthetic injections play an important role in the therapy of osteoarthritis. Glucocorticoids and anesthetics reportedly enhance apoptosis in chondrocytes, but effects of the combined use of glucocorticoids and local anesthetics are unknown. QUESTIONS/PURPOSES: We asked whether glucocorticoid and local anesthetic agents combined had any synergistic effects on chondrocyte apoptosis. METHODS: Cell viability and apoptosis/necrosis assessment of human articular chondrocytes were performed in vitro (chondrocyte cell cultures) and ex vivo (osteochondral specimens) using flow cytometry and TUNEL analysis, respectively. RESULTS: Glucocorticoids and local anesthetics induce apoptosis in chondrocytes at various rates. When used in combination, the percentage of dead chondrocytes was increased in in vitro chondrocyte cell cultures and osteochondral ex vivo specimens. CONCLUSIONS: We observed a time-dependent decrease in chondrocyte viability after concurrent steroid and local anesthetic exposure. CLINICAL RELEVANCE: The combination of glucocorticoids and local anesthetics has an adverse effect on articular chondrocytes, and it raises a question regarding whether concomitant administration should be used in treating osteoarthritis.

Effect of Bitis gabonica and Dendroaspis angusticeps snake venoms on apoptosis-related genes in human thymic epithelial cells.

BACKGROUND: Certain environmental toxins permanently damage the thymic epithelium, accelerate immune senescence and trigger secondary immune pathologies. However, the exact underlying cellular mechanisms and pathways of permanent immune intoxication remain unknown. The aim of the present study was to demonstrate gene expressional changes of apoptosis-related cellular pathways in human thymic epithelial cells following exposure to snake venom from Bitis gabonica and Dendroaspis angusticeps. METHODS: Snake venoms were characterized by analytical methods including reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, then applied on human thymic epithelial cells (1889c) for 24 h at 10 µg/mL (as used in previous TaqMan Array study). Gene expressional changes restricted to apoptosis were assayed by TaqMan Array (Human Apoptosis Plate). RESULTS: The most prominent gene expressional changes were shown by CASP5 (≈ 2.5 million-fold, confirmed by dedicated quantitative polymerase chain reaction) and CARD9 (0.016-fold) for B. gabonica, and BIRC7 (6.46-fold) and CASP1 (0.30-fold) for D. angusticeps. CONCLUSION: The observed apoptotic environment suggests that pyroptosis may be the dominant pathway through which B. gabonica and D. angusticeps snake venoms trigger thymic epithelial apoptosis following envenomation.

Active mixture of serum-circulating small molecules selectively inhibits proliferation and triggers apoptosis in cancer cells via induction of ER stress.

Genetic and epigenetic regulation as well as immune surveillance are known defense mechanisms to protect organisms from developing cancer. Based on experimental evidence, we proposed that small metabolically active molecules accumulating in cancer cells may play a role in an alternative antitumor surveillance system. Previously, we reported that treatment with a mixture of experimentally selected small molecules, usually found in the serum (defined 'active mixture', AM), selectively induces apoptosis in cancer cells and significantly inhibits tumor formation in vivo. In this study, we show that the AM elicits gene expression changes characteristic of endoplasmic reticulum (ER) stress in HeLa, MCF-7, PC-3 and Caco-2 cancer cells, but not in primary human renal epithelial cells. The activation of the ER stress pathway was confirmed by the upregulation of ATF3, ATF4, CHAC1, DDIT3 and GDF15 proteins. Mechanistically, our investigation revealed that eIF2α, PERK and IRE1α are phosphorylated upon treatment with the AM, linking the induction of ER stress to the antiproliferative and proapoptotic effects of the AM previously demonstrated. Inhibition of ER stress in combination with BBC3 and PMAIP1 knockdown completely abrogated the effect of the AM. Moreover, we also demonstrated that the AM induces mIR-3189-3p, which in turn enhances the expression of ATF3 and DDIT3, thus representing a possible new feedback mechanism in the regulation of ATF3 and DDIT3 during ER stress. Our results highlight small molecules as attractive anticancer agents and warrant further evaluation of the AM in cancer therapy, either alone or in combination with other ER stress inducing agents.

Identification of Further Components of an Anticancer Defense System Composed of Small Molecules Present in the Serum.

BACKGROUND: Earlier we assumed that small molecules selectively accumulated in cancer cells might have a role in a defense system capable of killing cancer cells. We reported earlier that an experimentally selected mixture of substances present in the serum ("active mixture," AM) shows a selective toxic effect in vitro and in vivo on various cancer cells. In this study we investigated additional compounds found in the serum to further expand our knowledge of this defense system. MATERIALS AND METHODS: The cell proliferation was detected by WST-1 assay. The mRNA level of the examined genes was measured by quantitative real-time polymerase chain reaction. RESULTS: We identified 34 additional compounds (l-amino acid metabolites, phenolic acids, d-amino acids, keto acids, etc.), which when applied in a per se nontoxic concentration are able to enhance the effect of AM. The combination of the mixture of these newly identified substances (new mixture, NM) with AM produced a significantly higher cancer cell growth inhibitory effect than NM or AM applied alone on HeLa, MCF-7, PC-3, Caco-2, HepG2, and 4T1 cancer cell lines, and more efficiently induced the expression of certain proapoptotic genes in HeLa cells. Any given combinations of the individual compounds of AM and NM always produced an increased effect compared with AM alone. CONCLUSIONS: The newly identified compounds significantly enhance the anticancer effect of AM. The components of AM and NM together may form part of a defense system capable of killing cancer cells and are worthy of further investigation.

Detailed analyses of antibodies recognizing mitochondrial antigens suggest similar or identical mechanism for production of natural antibodies and natural autoantibodies.

Because of their endosymbiotic evolutionary origin, proteins compartmentalized into mitochondria represent an interesting transition from prokaryotic foreign to essential self molecules. We investigated the presence of naturally occurring antibodies (nAbs) recognizing mitochondrial inner membrane enzymes. Epitope mapping analysis of a mitochondrial inner membrane enzyme, citrate synthase (CS) by synthetic overlapping peptides and phage display libraries using sera from healthy individuals and from patients having systemic autoimmune disease revealed CS recognizing nAbs with IgM isotype. We analyzed cross-reactive epitopes on human CS, bacterial CS, and various standard autoantigens. We have found that the fine epitope pattern on CS is different under physiological and pathological conditions. Moreover sera affinity purified on CS cross reacts with nucleosome antigen, which cross-reactivity could be mapped to a short epitope on human CS. These data indicate that in theory, nAbs "specific" for a given self antigen could fulfill the function of participating in innate defense mechanisms and at the same time recognize a target antigen in a systemic autoimmune disease. Thus, at the level of recognized epitopes there is a possible link between the innate like part and the adaptive-autoimmune arm of the humoral immune system.

Complex organizational defects of fibroblast architecture in the mouse spleen with Nkx2.3 homeodomain deficiency.

The capacity of secondary lymphoid organs to provide suitable tissue environment for mounting immune responses is dependent on their compartmentalized stromal constituents, including distinct fibroblasts. In addition to various members of the tumor necrosis factor/lymphotoxin beta family as important morphogenic regulators of peripheral lymphoid tissue development, the formation of stromal elements of spleen is also influenced by the Nkx2.3 homeodomain transcription factor in a tissue-specific fashion. Here we extend our previous work on the role of Nkx2.3-mediated regulation in the development of spleen architecture by analyzing the structure of reticular fibroblastic meshwork of spleen in inbred Nkx2.3-deficient mice. Using immunohistochemistry and dual-label immunofluorescence we found both distributional abnormalities, manifested as poor reticular compartmentalization of T-zone and circumferential reticulum, and developmental blockade, resulting in the absence of a complementary fibroblast subpopulation of white pulp. The disregulated distribution of fibroblasts was accompanied with an increased binding of immunohistochemically detectable complement factor C4 by T-cell zone-associated reticular fibroblasts, distinct from follicular dendritic cells with inherently high-level expression of bound C4. These data indicate that the impact of Nkx2.3 gene deficiency on fibroblast ontogeny within the spleen extends beyond its distributional effects, and that the formation of various white pulp fibroblast subsets is differentially affected by the presence of Nkx2.3 activity, possibly also influencing their role in various immune functions linked with complement activation and deposition.

A possible new bridge between innate and adaptive immunity: Are the anti-mitochondrial citrate synthase autoantibodies components of the natural antibody network?

Natural antibody (nAb) producing B-1 B cells are considered an intermediate stage of evolution between innate and adaptive immunity. nAbs are immunoglobulins that are produced without antigen priming. nAbs can recognize foreign targets and may serve in the first line of immune defense during an infection. Natural autoantibodies (nAAbs) present in the serum of both healthy humans and patients suffering from systemic autoimmune diseases recognize a set of evolutionarily conserved self-structures. Because of their endosymbiotic evolutionary origin, proteins compartmentalized into mitochondria represent an interesting transition from prokaryotic foreign (non-self) to essential (self) molecules. We investigated the possible overlap in recognized epitopes of innate and self-reactive nAbs and surveyed changes in physiological autoreactivity under pathological autoimmune conditions. Epitope mapping analysis of a mitochondrial inner membrane enzyme, citrate synthase (CS) (EC 2.3.3.1) by synthetic overlapping peptides and phage display libraries using sera from healthy individuals and from patients having systemic autoimmune disease revealed CS recognizing nAAbs with IgM isotype. We analyzed cross reactive epitopes on human CS, bacterial CS, and various standard autoantigens. The anti-CS nAAbs by participating in the nAb network, could function in innate defense mechanisms and at the same time recognize a target antigen (nucleosome) in a systemic autoimmune disease. Thus, at the level of recognized epitopes there is a possible new link between the innate like component and the adaptive-autoimmune arm of the humoral immune system.

Low glucocorticoid receptor (GR), high Dig2 and low Bcl-2 expression in double positive thymocytes of BALB/c mice indicates their endogenous glucocorticoid hormone exposure.

Several studies have shown that of the four major thymocyte subsets, the CD4/CD8 double positive (DP) thymocytes are the most sensitive to in vivo glucocorticoid hormone (GC)-induced apoptosis. Our aim was to analyse fine molecular differences among thymocyte subgroups that could underlie this phenomenon. Therefore, we characterised the glucocorticoid hormone receptor (GR) expression of thymocyte subgroups both at the mRNA and protein levels by real-time PCR and flow cytometry, and correlated these features to their apoptotic sensitivity. We also investigated the time-dependent effects of the GC agonist dexamethasone (DX) with or without GC antagonist (RU486) treatments on GR mRNA/protein expression. We also analysed the expression of two apoptosis-related gene products: dexamethasone-induced gene 2 (Dig2) mRNA and Bcl-2 protein. We found that DN thymocytes had the highest GR expression, followed by CD8 single positive (SP), CD4 SP and DP thymocytes in 4-week-old BALB/c mice, both at the mRNA and protein levels, respectively. In DP cells, the Dig2 expression was significantly higher, while the Bcl-2 expression was significantly lower than in DN, CD4 SP and CD8 SP thymocytes. Single high dose DX treatment caused time-dependent depletion of DP thymocytes due to their higher apoptosis rate, which could not be abolished with RU486 pretreatment. After a single high dose DX treatment, there was a transient, significant increase of the GR mRNA and protein level of unsorted thymocytes after 8 and 16 h, followed by a significant decrease at 24 h, respectively. The time-dependent GR expression changes after DX administration could not be inhibited by the GC antagonist RU486. Twenty-four hours after exposure to high dose DX the DN, CD4 SP and CD8 SP cells showed a significant decrease of GR mRNA and protein expression, whereas the DP thymocytes, showed no significant alteration of GR mRNA or protein expression. The kinetical analysis of GR expression and apoptotic marker changes upon single high dose GC analogue administration revealed a two-phase process in thymocytes: early events, within 4-8 h, include GR upregulation and early apoptosis induction, while the late events appear most prominently at 16-20 h, when the GR is already downregulated and apoptotic cell ratio reaches its peak, with marked DP cell depletion. The low GR, high Dig2 and low Bcl-2 expression, coupled with the absence of homologous downregulation of GR after exogenous GC analogue treatment, could contribute to the high GC sensitivity of DP thymocytes. The downregulated GR and Bcl-2 together with the upregulated Dig2 level in DP cells indicates the significance of intrathymic GC effects at this differentiation stage. Since GR expression changes and apoptotic events could not be completely inhibited by GC antagonist, we propose the involvement of non-genomic GR mechanisms in these processes.

Sandwich type ELISA and a fluorescent cytometric microbead assay for quantitative determination of hepatitis B virus X antigen level in human sera.

The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.

Determination of the fine epitope specificity of an anti-hepatitis B virus X protein monoclonal antibody using microanalytical and molecular biological methods.

The recombinant form of the 17kDa, highly hydrophobic and disulfide-bonded hepatitis B virus X protein (HBX) was used for developing a set of monoclonal antibodies (Mab). Our present goal was to determine the fine epitope specificity of our anti-HBX Mab. Based on computer analysis two sequences (amino acids 22-31 and 100-114) were predicted for possessing high immunogenity while the anti-HBX Mab did not recognized them. Limited proteolysis and mass spectroscopic analysis suggested another possible sequence (amino acids 14-26), which also proved to be negative using an immunoserological test. Subsequently, we performed a screen of a phage displayed random peptide library, by which we could localize the epitope to amino acids 88-93. This finding was confirmed using three overlapping fusion peptides spanning amino acids 77-142. Their testing in ELISA assigned the epitope to amino acids 77-95, which supports the result obtained by screening the phage displayed library. Our results suggest the necessity of a complex application of current molecular biological and immunological techniques in fine structure mapping. This approach will be useful to study the prognostic relevance of different antigenic sites on HBX during the development of chronic hepatitis and primary hepatocellular carcinoma.

Effect of Bitis gabonica and Dendroaspis angusticeps snake venoms on apoptosis-related genes in human thymic epithelial cells

Certain environmental toxins permanently damage the thymic epithelium, accelerate immune senescence and trigger secondary immune pathologies. However, the exact underlying cellular mechanisms and pathways of permanent immune intoxication remain unknown. The aim of the present study was to demonstrate gene expressional changes of apoptosis-related cellular pathways in human thymic epithelial cells following exposure to snake venom from Bitis gabonica and Dendroaspis angusticeps. Methods: Snake venoms were characterized by analytical methods including reversed phase high-performance liquid chromatography and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, then applied on human thymic epithelial cells (1889c) for 24 h at 10 μg/mL (as used in previous TaqMan Array study). Gene expressional changes restricted to apoptosis were assayed by TaqMan Array (Human Apoptosis Plate). Results: The most prominent gene expressional changes were shown by CASP5 (≈ 2.5 million-fold, confirmed by dedicated quantitative polymerase chain reaction) and CARD9 (0.016-fold) for B. gabonica, and BIRC7 (6.46-fold) and CASP1 (0.30-fold) for D. angusticeps. Conclusion: The observed apoptotic environment suggests that pyroptosis may be the dominant pathway through which B. gabonica and D. angusticeps snake venoms trigger thymic epithelial apoptosis following envenomation.(AU)