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.

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.

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)

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.

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.

Safety, tolerability, and effect on quality of life of a mixture of amino acids and other small molecules in cancer patients.

We performed two clinical studies to evaluate the safety, tolerability, and effect on quality of life of a product containing a mixture of amino acids, vitamins, and other small molecules. In the first one period, open-label, multiple-dose study, the safety and tolerability of a 1-week administration was evaluated in 24 healthy volunteers. In the second one period, open-label, multiple-dose, single-arm study, we investigated the safety, tolerability, and effect on quality of life of a 4-week administration in 50 cancer patients. The safety assessment included the monitoring of adverse events, changes in physical status, and clinical laboratory tests. Changes in quality of life were measured with the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire Core 30 version 3.0 (EORTC QLQ-C30). We have found that administration of the investigated product is safe and well tolerated in healthy individuals and in cancer patients. Administration of the product to cancer patients significantly improved their quality of life (EORTC QLQ-C30 global health status score: baseline: 24.17 ± 9.2; end of treatment: 47.08 ± 14.56, p<0.001). To evaluate the anticancer activity of the investigated product in humans, a randomized, blinded, combination clinical trial should be conducted.

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.

Absence of Nkx2-3 homeodomain transcription factor induces the formation of LYVE-1-positive endothelial cysts without lymphatic commitment in the spleen.

In contrast to peripheral lymph nodes possessing lymphatic and blood vasculature, the spleen in both humans and rodents is largely devoid of functioning lymphatic capillaries. Here it is reported that in mice lacking homeodomain transcription factor Nkx2-3, the spleen contains an extensive network of lymphocyte-filled sacs lined by cells expressing LYVE-1 antigen, a marker associated with lymphatic endothelium cells (LECs). Real-time quantitative PCR analyses of Nkx2-3 mutant spleen revealed a substantial increase of LYVE-1 and podoplanin mRNA levels, without the parallel increase of mRNA for VEGFR-3 (vascular endothelial growth factor receptor Type 3) and Prox1 (Prospero homeobobox protein 1), two markers specific for LECs. Although these structures express VEGFR-2/flk-1, they lack Prox1 protein, indicating their non-LEC endothelial origin. The LYVE-1(+) structures are bordered with ER-TR7(+) fibroblastic reticular cells with small clusters of macrophages expressing MARCO and sialoadhesin. Short-term cell-tracing studies using labeled lymphocytes indicate that these LYVE-1(+) cysts are largely excluded from the systemic circulation. Cells expressing LYVE-1 glycoprotein as putative precursors for such structures are detectable in the spleen of late-stage embryos, and the formation of LYVE-1(+) structures is independent from the activity of lymphotoxin ß-receptor. Thus the splenic vascular defects in Nkx2-3 deficiency include the generation of LYVE-1(+) cysts, comprised of endothelial cells without being committed along the LEC lineage.

Osteochondral integration of multiply incised pure cartilage allograft: repair method of focal chondral defects in a porcine model.

BACKGROUND: A focal cartilage lesion has limited capacity to heal, and the repair modalities used at present are still unable to provide a universal solution. Pure cartilage graft implantation appears to be a simple option, but it has not been applied widely as cartilage will not reattach easily to the subchondral bone. HYPOTHESIS: We used a multiple-incision technique (processed chondrograft) to increase cartilage graft surface. We hypothesized that pure cartilage graft with augmented osteochondral fusion capacity may be used for cartilage repair and we compared this method with other repair techniques. STUDY DESIGN: Controlled laboratory study. METHODS: Full-thickness focal cartilage defects were created on the medial femoral condyle of 9-month-old pigs; defects were repaired using various methods including bone marrow stimulation, autologous chondrocyte implantation, and processed chondrograft. After the repair, at weeks 6 and 24, macroscopic and histologic evaluation was carried out. RESULTS: Compared with other methods, processed chondrograft was found to be similarly effective in cartilage repair. Defects without repair and defects treated with bone marrow stimulation appeared slightly irregular with fibrocartilage filling. Autologous chondrocyte implantation produced hyalinelike cartilage, although its cellular organization was distinguishable from the surrounding articular cartilage. Processed chondrograft demonstrated good osteochondral integration, and the resulting tissue appeared to be hyaline cartilage. CONCLUSION: The applied cartilage surface processing method allows acceptable osteochondral integration, and the repair tissue appears to have good macroscopic and histologic characteristics. CLINICAL RELEVANCE: If further studies confirm its efficacy, this technique could be considered for human application in the future.

Investigation of sensory neurogenic components in a bleomycin-induced scleroderma model using transient receptor potential vanilloid 1 receptor- and calcitonin gene-related peptide-knockout mice.

OBJECTIVE: Along with their classic afferent function (nociception), capsaicin-sensitive transient receptor potential vanilloid 1 (TRPV1) receptor-expressing sensory nerve terminals exert local and systemic efferent activities. Activation of TRPV1 causes sensory neuropeptide release, which modulates the inflammation process. The aim of the present study was to examine the role of this modulatory role of TRPV1 receptor and that of calcitonin gene-related peptide (CGRP) in bleomycin-induced scleroderma, using transgenic mice. METHODS: Cutaneous sclerosis was induced with daily subcutaneous injections of bleomycin for 30 days. Control groups were treated with phosphate buffered saline (PBS). TRPV1 receptor gene-deficient (TRPV1(-/-)) mice and CGRP-knockout (CGRP(-/-)) mice and their wild-type (WT) counterparts were investigated. A composite sclerosis score was calculated on the basis of thickening, leukocyte infiltration, and the amount/orientation of collagen bundles. Dermal thickness and the number of alpha-smooth muscle actin (alpha-SMA)-positive cells were also determined. The quantity of the collagen-specific amino acid hydroxyproline was measured by spectrophotometry. RESULTS: Bleomycin treatment induced marked cutaneous thickening and fibrosis compared with that observed in control mice treated with PBS. The composite sclerosis score was 18% higher, dermal thickness was 19% higher, the number of alpha-SMA-positive cells was 47% higher, and the amount of hydroxyproline was 57% higher in TRPV1(-/-) mice than in their WT counterparts. Similarly, the composite sclerosis score was 47% higher, dermal thickness was 29% higher, the number of alpha-SMA-positive cells was 76% higher, and the amount of hydroxyproline was 30% higher in CGRP(-/-) mice than in the respective WT groups. CONCLUSION: These results suggest that activation of the TRPV1 receptor by mediators of inflammation induces sensory neuropeptide release, which might exert protective action against fibrosis. We confirmed the protective role of CGRP in the development of cutaneous sclerosis.

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.

Distinct roles of lymphotoxin-beta signaling and the homeodomain transcription factor Nkx2.3 in the ontogeny of endothelial compartments in spleen.

The formation of peripheral lymphoid tissues is indispensable for the efficient recognition and elimination of external antigens by lymphoid and accessory cells of the adaptive immune system. The spleen is structurally arranged around various vascular beds with distinct endothelial phenotypes. Using immunohistochemistry, we investigated the postnatal developmental characteristics of the marginal sinus and its relationship with various red-pulp sinus subsets. We also determined the importance of the lymphotoxin beta receptor (LT beta R) and the role of the Nkx2.3 transcription factor for the formation of the splenic vasculature. Both the administration of soluble LT beta R-Ig fusion protein to neonates and the deletion of LT beta R or downstream signaling components (RelB and p52) of the NF-kappaB family inhibited the phenotypic maturation of marginal sinus but had no effect on the vascular compartmentalization of the red pulp. The integrity of the marginal sinus and the proper vascular segregation of the red pulp appeared to be controlled by Nkx2.3, as Nkx2.3-deficient mice exhibited an abnormal distribution of IBL-7/1(hi)/IBL-9/2(-) sinuses and a lack of IBL-7/1(lo)/IBL-9/2(+) vessels. Our data suggest that phenotypic heterogeneity among different vascular elements within distinct anatomical regions of the spleen differentially depends on developmental factors such as lymphotoxin signaling or Nkx2.3, whereas the marginal sinus is controlled by both pathways.

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.

Species-specific restriction of cell surface expression of mouse MARCO glycoprotein in murine cell lines.

The MARCO (macrophage receptor with collagenous structure) glycoprotein belongs to the scavenger receptor type family of pattern-recognition molecules produced by a subset of marginal zone macrophages in the spleen. Stimulation with LPS leads to its appearance on macrophages located at other tissue compartments. In the present work, we report its in vitro expression by various cell lines using transient and stable (lentiviral) gene delivery aimed at investigating the signaling properties of this receptor and its analysis using a novel rat monoclonal antibody against the SRCR-domain of mouse MARCO. When trying to establish stable mouse MARCO-transfectants using lentiviral transduction and other methods, we consistently found that MARCO accumulated intracellularly in various murine host cells. In contrast, such a phenomenon was not observed in non-murine cell lines. Our observations indicate the presence of an unexpected limitation of the in vitro expression of mouse MARCO glycoprotein in murine cell lines. We believe that the failure to express MARCO on the cell surface of the many murine cell lines is likely due to the absence of endoplasmic reticulum molecular chaperones needed for the correct folding and assembly of the trimeric MARCO molecule.

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.

Antagonist of growth hormone-releasing hormone induces apoptosis in LNCaP human prostate cancer cells through a Ca2+-dependent pathway.

Antagonists of growth hormone-releasing hormone (GHRH) exert antiproliferative effects directly on cancer cells, which are mediated by the tumoral GHRH receptors. However, the signal transduction pathways involved in antiproliferative effect of GHRH antagonists have not yet been elucidated. We used flow cytometry to investigate whether GHRH antagonist JV-1-38 can induce changes in the cytosolic free Ca2+ concentration leading to apoptosis in LNCaP human prostate cancer cells. JV-1-38 evoked prompt Ca2+ signal in a dose-dependent way (1-10 microM) and induced early stage of apoptosis in LNCaP human prostate cancer cells at a concentration effective in suppression of cell proliferation (10 microM) peaking after 3 h. Unexpectedly, agonist GHRH(1-29)NH2, which elevates cytosolic free Ca2+ concentration in pituitary somatotrophs at nanomolar concentrations, failed to induce Ca2+ signal or apoptosis even at a 10-fold higher concentration (100 microM). However, agonist GHRH(1-29)NH2 inhibited JV-1-38-induced Ca2+ signals in a dose-dependent way without affecting the antagonist-induced apoptosis. Peptides unrelated to GHRH did not induce Ca2+ signals in LNCaP human prostate cancer cells. EDTA (10 mM) or nifedipine (10 microM) significantly reduced the Ca2+ signal and early stage of apoptosis induced by JV-1-38, supporting the view that the increase in intracellular Ca2+ in response to JV-1-38 occurs primarily through extracellular Ca2+ entry through voltage-operated Ca2+ channels. In conclusion, GHRH antagonists activate tumoral GHRH receptors and are able to induce apoptosis in LNCaP human prostate cancer cells through a Ca2+-dependent pathway. Treatment with GHRH antagonists may offer a new approach to the therapy of prostate and other hormone-sensitive cancers.

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.

Use of cyclic peptide phage display library for the identification of a CD45RC epitope expressed on murine B cells and their precursors.

The alternative splicing and variable expression of the exons near to the N-terminus of the leukocyte common antigen (L-CA, CD45) result in distinct extracellular isoforms expressed by cells with different functional and developmental properties. Here we report the tissue reactivity pattern and epitope specificity of a novel rat monoclonal antibody (IBL-8) against a restricted epitope of mouse CD45. We found that this mAb reacts with an epitope displayed by B cells and their precursors (both in newborn spleen and adult bone marrow). Moreover, peripheral CD8-positive T cells were also recognised at an intermediate intensity, whereas the CD4 T cell subset was weakly reactive. The epitope of this mAb was determined with M13 filamentous phages that display cysteine constrained nonapeptides on their coat proteins. The isolated bacteriophages expressing the putative epitope showed an isoform-specific inhibition of the binding of exon-specific mAbs. Deduced amino acid sequence data of these phages indicate that the epitope recognised by the IBL-8 mAb lies at the 136-144 region of the mouse CD45 molecule within its C exon, with a TAFP consensus sequence at its centre.

Expression of growth hormone-releasing hormone and its receptor splice variants in human prostate cancer.

Antagonists of GHRH inhibit the growth of various human tumors, including prostate cancer, but the tumoral receptors mediating the antiproliferative effect of GHRH antagonists have not been clearly identified. Recently, we demonstrated that human cancer cell lines express splice variants (SVs) of receptors for GHRH, of which SV1 exhibits the greatest similarity to the pituitary GHRH receptors. In this study we investigated the expression of GHRH and SVs of GHRH receptor and the binding characteristics of the GHRH receptor isoform in 20 surgical specimens of organ-confined and locally advanced human prostatic adenocarcinomas. The mRNA expression of GHRH and SVs of GHRH receptor was investigated by RT-PCR. The affinity and density of receptors for GHRH were determined by ligand competition assays based on binding of (125)I-labeled GHRH antagonist JV-1-42 to tumor membranes. Twelve of 20 tumors (60%) exhibited specific, high affinity binding for JV-1-42, with a mean dissociation constant (K(d)) of 0.81 nmol/liter and a mean maximal binding capacity of 185.2 fmol/mg membrane protein. The mRNA of SV1 was detected in 13 of 20 (65%) prostate cancer specimens and was consistent with the presence of GHRH binding. RT-PCR analyses also revealed the expression of mRNA for GHRH in 13 of 15 (86%) prostatic carcinoma specimens examined. The presence of GHRH and its tumoral receptor SVs in prostate cancers suggests the possible existence of an autocrine mitogenic loop. The antitumor effects of GHRH antagonists in prostate cancer could be exerted in part by interference with this local GHRH system.