Biomedical subjects

M O Labeta

Publications and source records attributed to M O Labeta.

16 recordsLinked to original sources

PubMed2003

Evidence of expression of endotoxin receptors CD14, toll-like receptors TLR4 and TLR2 and associated molecule MD-2 and of sensitivity to endotoxin (LPS) in islet beta cells.

CD14, a GPI-linked membrane protein, is a component of the lipopolysaccharide (LPS) receptor complex, one of the pattern-recognizing receptors (PRR) expressed by myeloid lineage cells. Here we report that CD14, the functionally linked toll-like receptor molecules, TLR2 and TLR4, and the associated molecule MD-2 are expressed in endocrine cells of the human pancreatic islets. CD14 expression in human pancreatic islets was determined by immunofluorescence staining of tissue sections and primary cultures, and confirmed by flow cytometry of dispersed normal islets and SV40-transformed islet cells (HP62). The latter cells synthesized and secreted CD14 in response to lipopolysaccharide (LPS) in a time- and dose-dependent manner. Reverse transcription polymerase chain reaction (RT-PCR)-Southern was positive for CD14, TLR2, TLR4 and MD-2 in human pancreas, purified islets and HP62 cells. In vitro experiments using rat islets (also positive for CD14 by RT-PCR) and HP62 cells showed that LPS regulates glucose-dependent insulin secretion and induces inflammatory cytokines [interleukin (IL)-1alpha, IL-6 and tumour necrosis factor (TNF)-alpha]. The functional expression of CD14 and associated molecules in islet beta cells adds a new pathway that islet cells may follow to adjust their function to endotoxaemia situations and become vulnerable to the inflammatory events that occur during diabetogenic insulitis.

Adolescent↗

PubMed2001

The lipopolysaccharide co-receptor CD14 is present and functional in seminal plasma and expressed on spermatozoa.

CD14 is a 54 000-molecular weight (MW) glycolipid-anchored membrane glycoprotein, expressed on myeloid cells, which functions as a member of the lipopolysaccharide (LPS) receptor complex. Soluble forms of CD14 have been reported in plasma, cerebrospinal fluid, amniotic fluid and breast milk. In plasma and breast milk, soluble CD14 has been implicated as a regulator of T- and B-cell activation and function. Expression of CD14 in the male reproductive system has not previously been investigated. We here show that soluble CD14 is present in seminal plasma at levels comparable to those in serum. Spermatozoa expressed CD14 on their membranes, as demonstrated by fluorescence microscopy and flow cytometry. Post-vasectomy, the levels of seminal plasma CD14 (spCD14) were much reduced, implying an origin distal to the point of transection of the vas deferens. Ultracentrifugation analyses demonstrated that spCD14 was not associated with lipid complexes, indicating that it lacks the glycolipid anchor. Purified spCD14 mediated activation by LPS of CD14-negative cells. These findings suggest that CD14 may play a hitherto unexplored role in immune defence and cell activation in the male reproductive tract.

Blotting, Western↗

PubMed2000

Elevated serum concentrations of soluble CD14 in HIV- and HIV+ patients with tuberculosis in Africa: prolonged elevation during anti-tuberculosis treatment.

Data are limited regarding serum concentrations of soluble CD14 (sCD14), a marker of macrophage activation, in patients with active tuberculosis (TB) and during drug treatment. In this study, concentrations of sCD14 were measured in serum samples obtained from 105 African subjects who were categorized into one of four groups: persons with pulmonary TB alone (TB+HIV-, n = 30), pulmonary TB and HIV co-infection (TB+HIV+, n = 20), or HIV infection alone (TB-HIV+, n = 25), and healthy controls (TB-HIV-, n = 30). Mean total sCD14 was significantly increased in serum of patients with newly diagnosed pulmonary TB (mean = 6.6 g/ml, s.d. = 1.6 g/ml) compared with healthy controls (mean = 3.1 g/ml, s.d. = 0.6 g/ml; P < 0.0001), and this elevation comprised proportionate increases in the alpha (2.1-fold greater, P < 0.0001) and beta (2.0-fold greater, P < 0. 0001) forms of sCD14. Total sCD14 was also increased in serum of HIV-infected patients (mean = 4.1 g/ml, s.d. = 1.9 g/ml; P < 0.01), but the highest concentrations were observed in patients with pulmonary TB and HIV co-infection (mean = 8.7 g/ml, s.d. = 3.1 g/ml; P < 0.0001). Analysis of serum samples prospectively collected from TB+HIV-patients during the first 3 months of successful anti-TB treatment demonstrated steep reductions in mean concentrations of the acute-phase protein, C-reactive protein, and the soluble lymphocyte activation marker, sCD25. In contrast, levels of sCD14 increased during the first month of treatment and slowly declined thereafter. These data indicate that the serum concentration of sCD14 is not a sensitive index of response to anti-TB treatment and suggest that cellular activation resolves more slowly in the macrophage pool compared with the lymphocyte pool during anti-TB treatment.

Adult↗

PubMed1995

Prevention of experimental endotoxin shock by a monocyte activator.

In patients with polytrauma or major surgery, severe bacterial infections leading to septic shock and multiorgan failure are still a major cause of death. Prevention of septic shock in patients at risk would be an alternative to treatment of patients with overt septic shock. We therefore conducted a trial with the monocyte activator muramyl tripeptide phosphatidylethanolamine (MTP-PE) in an experimental pig model. Liposome encapsulated MTP-PE (50 micrograms/kg of body weight) or liposomes alone were given intravenously at 72 or 24 h before endotoxemia was induced by lipopolysaccharide (LPS), simultaneously with the induction of endotoxin shock, or 1 h thereafter. Pretreatment with MTP-PE at 72 and 24 h before endotoxemia was induced resulted in a reduction of endotoxin shock-induced mortality from 81.8% (9 of 11 animals) in the control group to 8.3% (1 of 12 animals) of the MTP-PE-pretreated animals (P < 0.001). The administration of MTP-PE 24 h before the induction of endotoxin shock was more effective (P < 0.01) than administration of MTP-PE 72 h before endotoxemia was induced (P = 0.05). The pretreated animals did not develop fever or cardiovascular complications, and pulmonary function was significantly improved. Furthermore, the alpha-form of the soluble CD14 LPS receptor in pig serum showed a marked decrease in LPS-treated animals, and this decrease was reduced by MTP-PE pretreatment at 24 h before endotoxemia was induced. When MTP-PE was given simultaneously with the induction of septic shock or 1 h thereafter, it did not influence either mortality or morbidity. In conclusion, pretreatment of pigs with MTP-PE improves several parameters of endotoxin shock and it reduces mortality. Patients with high risk of developing septic complications might benefit from a pretreatment with this monocyte-activating substance.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1994

CD14 is expressed and functional in human B cells.

The human B cell line RPMI 8226 exhibits variable staining with the CD14 antibody My4. We have isolated three stable clones from this line with clones 1 and 2 being My4 positive and clone 3 My4 negative. Similar to previous results in monocytes, immunoprecipitation with the My4 antibody revealed a 54-kDa cell surface molecule, analysis of supernatants showed soluble CD14, and Northern blotting demonstrated a 1.4-kb transcript in clones 1 and 2, but not in clone 3, which suggests that the My4 antibody detects CD14 in clones 1 and 2. This CD14 molecule was functional in that lipopolysaccharide stimulation induced interleukin (IL)-6 and IL-10 in clones 1 and 2 but not in clone 3. Furthermore, the My4 antibody was capable of blocking these responses at the transcript and protein levels. Finally, peripheral blood B cells were highly purified by cell sorting (> 98% CD19 positive). These cells produced IL-6 in response to lipopolysaccharide, and this response was blocked by anti-CD14 antiserum. Thus, our findings demonstrated that human B cells can express functionally active CD14.

Antigens, CD↗

PubMed1994

The antibody MY4 recognizes CD14 on porcine monocytes and macrophages.

Several monoclonal antibodies directed against the human CD14 antigen have been established. We now report that the antibody My4, but not LeuM3, reacts with porcine monocytes. Among porcine peripheral blood mononuclear cells (PBMC), 14.6% of the cells stain with the CD14 antibody My4, which is similar to the percentage obtained with the antiporcine monocyte antibody 74-22-15. Two-colour immunofluorescence reveals that My4 and 74-22-15 antigens are coexpressed on the same cells, and cell sorter-purified My4+ cells exhibit the morphology of monocytes. Whole blood analysis (which also shows staining of granulocytes) reveals that the average percentage of My4+ monocytes amongst all leucocytes is 5.8% with 580 cells/microliters. Furthermore, porcine peritoneal macrophages (PM) and alveolar macrophages (AM), both stain for My4, with a four-fold lower level on AM. Treatment of cells with phosphatidylinositol-specific phospholipase C decreases My4 staining, but does not affect staining with antibody 74-22-15. Immunoprecipitation with the My4 antibody from surface labelled pig mononuclear cells demonstrates a 54 kDa band similar to human CD14, and Western blotting with pig serum demonstrates two bands similar to the alpha and beta forms of human soluble CD14. Finally, the My4 antibody is capable of blocking lipopolysaccharide- (LPS)-induced interleukin-6 production in isolated PBMC. These data show that the My4 antibody recognizes genuine CD14 on porcine monocytes and macrophages.

Animals↗

PubMed1993

Release from a human monocyte-like cell line of two different soluble forms of the lipopolysaccharide receptor, CD14.

Lipopolysaccharide (LPS) stimulates mononuclear phagocytes to synthesize and secrete immunoregulatory and inflammatory molecules such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-alpha (TNF-alpha). LPS forms complexes with either the serum protein termed LPS-binding protein or a serum factor, septin. These complexes are more stimulatory than LPS alone. The myeloid differentiation antigen CD14 is known to be the receptor for such complexes. In the present study, by using a monocytic cell line, we demonstrate the release of two different soluble forms of CD14 (sCD14) which are secreted by different mechanisms. We show that the two sCD14 forms differ in their electrophoretic mobility, two-dimensional gel electrophoretic patterns, sensitivity to endoglycosidases and peptide maps. One of the sCD14 molecules, apparent molecular mass 48 kDa, was found in supernatants of both surface iodinated and [35S]methionine biosynthetically labeled cells. The other sCD14 molecule (56 kDa) was found labeled only in supernatants of [35S]methionine-labeled cells. Furthermore, purified 48 kDa sCD14 enhanced the LPS-induced TNF-alpha and IL-6 release by the monocytic cells suggesting that a cell-surface signal transducer molecule may be involved in signaling. The data suggest a possible novel role for sCD14 in the monocyte response to LPS.

Antigens, CD↗

PubMed1993

CD14 and tolerance to lipopolysaccharide: biochemical and functional analysis.

To evaluate the role of the high-affinity monocyte receptor for lipopolysaccharide (LPS), CD14, in the process of tolerance to LPS, the human monocytic cell line Mono-Mac-6 was cultured in the absence or presence of different amounts of LPS. The kinetics of CD14 modulation in these cells showed an initial 4-day period characterized by increased cell-surface expression, rate of biosynthesis (peaking at 48 hr) and release of its soluble forms (sCD14) which correlated with the amount of LPS in the culture. At this time, tolerance to LPS was already established, as measured by tumour necrosis factor-alpha (TNF-alpha) induction, it was LPS dose dependent and persisted up to 15 days. LPS also reduced the cell proliferation rate in a dose-dependent manner. After 8 days and up to 15 days, the CD14 biosynthesis, cell-surface expression and release of sCD14 inversely correlated with the level of LPS in the culture. The 48-hr LPS-pretreated cells showed a slightly decreased CD14 affinity for LPS, a relative high number of CD14 molecules per cells, and desensitization also to a phorbol 12-myristate 13-acetate (PMA) challenge. An anti-CD14 monoclonal antibody (mAb) protected the cells from tolerization when added at the beginning of culture, as revealed by challenge with LPS and PMA. The data indicate that in this model tolerization to LPS (1) precedes CD14 down-modulation, (2) operates by alteration of the receptor affinity for LPS and by a mechanism which affects a protein kinase C (PKC)-dependent signalling pathway, and (3) that CD14 plays a critical role in the establishment of tolerance to LPS. In addition, analysis of the data suggests the existence of a PKC-independent signalling pathway for LPS tolerization and a CD14-independent mechanism for establishing tolerance.

Antigens, CD↗

PubMed1993

Cell-surface expression of human histocompatibility leucocyte antigen (HLA) class II-associated invariant chain (CD74) does not always correlate with cell-surface expression of HLA class II molecules.

The human histocompatibility leucocyte antigen (HLA) class II-associated invariant chain comprises at least four polypeptides. One of these is expressed as a membrane-bound subunit and has been designated as CD74. We investigated the expression of CD74 on B- and T-cell types utilizing monoclonal antibodies (mAb) specific for the c-terminus of CD74. All B cells tested expressed CD74 regardless of their HLA phenotypes. High and low CD74 expressors were provisionally assigned based on quantitative variations found in some haplotypes. A mutant cell with selective deletions of class II loci DR and DQ but with retention of DP and additional class II heterodimers synthesize normal amounts of CD74. In contrast, however, a mutant with a total class II deletion had diminished expression of CD74, suggesting that at least one of the class II isotypes is essential for full constitutive expression of CD74. Major histocompatibility complex (MHC) class II negative T-cell lines and CD3+ T cell isolated from peripheral blood did not express CD74. However, allostimulated T cells which express de novo induced class II determinants also did not express CD74. Taken together, these results show that CD74 is expressed on B cells and unexpectedly is not synthesized on de novo-induced class II positive T-cell clones.

Antigens, Differentiation, B-Lymphocyte↗

PubMed1989

Biochemical analysis of a novel H-2 class I-like glycoprotein expressed in five AKR-(Gross virus) derived spontaneous T cell leukemias.

The H-2 class I Ag profiles of five spontaneous AKR (H-2K) Gross virus leukemic cell lines were analyzed. A novel H-2 class I, "alloantigen"-like glycoprotein was immunoprecipitated and isolated from all the tumor cell lines using an H-2Dd-specific mAb 35-5-8. The novel Ag was also recognized in vitro by anti-H-2Dd-specific CTL. In addition, DNA from all the thymomas, but not the DNA from normal adult AKR thymic cells showed a transcribed gene detectable with an H-2Dd-specific oligonucleotide probe. The molecular profile of the novel antigen was further studied by two-dimensional gel electrophoresis and analyzed by a computer based image analyzer system and reverse-phase HPLC tryptic peptide mapping. Its molecular pattern was different from the syngeneic H-2Kk, H-2Dk, and the allogeneic H-2Dd gene products. The two-dimensional gel pattern of the novel H-2 class I molecule had a different overall structure reflected in isoelectric point, number, and distribution of polypeptide spots. The tryptic peptide map analysis showed six peaks exclusively identified with the novel Ag. The calculated degree of homology with the corresponding H-2Dd, H-Dk, and H-Kk peptides was 41, 56, and 51%, respectively. In addition, an unusual cell surface distribution of the novel Ag was observed in most of the leukemic lines. The removal of sialic acid residues by neuraminidase treatment facilitated the detection of the allodeterminants by anti-H-2Dd-specific mAb and CTL. Furthermore, we showed that in one AKR tumor line, 424, there is a close association of the novel Ag with the syngeneic class I molecules. Prior preclearance of the syngeneic class I molecules revealed the presence of the H-2Dd-like allospecificity. The genetic and molecular relationship between the expression of this novel class I-like glycoprotein and the recently sequenced Q5 gene is under current investigation.

AKR murine leukemia virus↗

PubMed1988

Solubilisation effect of Nonidet P-40, triton X-100 and CHAPS in the detection of MHC-like glycoproteins.

We have analysed the differential solubilisation effect of three detergents on cell-membrane histocompatibility glycoproteins. Two nonionic detergents (Nonidet P-40 and Triton X-100) which are extensively used in the extraction of MHC proteins and a zwitterionic detergent (CHAPS) which is sulphobetaine derivative of cholic acid were used. An AKR (H-2k) derived spontaneous leukaemic cell line--424--was used as the experimental model. In this tumour cell line a class I-like antigen is expressed but not directly detected by cell-binding radioimmunoassay or immunoprecipitation from NP-40 or Triton X-100 solubilised glycoproteins. However, 46 kDa and 12 kDa bands consistent with the classical H-2 class I pattern were seen by SDS-PAGE after immunoprecipitation with the 34.5.8 anti-H-2Dd MoAb using CHAPS solubilised 424 glycoproteins. The H-2Dd-reactive molecule appears to be associated with at least one of the syngeneic class I specificities (H-2Kk, H-2Dk) and not accessible to react with the specific anti H-2Dd MoAb. The detergents NP-40 and Triton X-100 appear to be less efficient than CHAPS in breaking protein-protein interactions. This property of CHAPS permitted the adequate solubilisation of the novel antigen and its direct detection. The results of this study suggest that the alternative use of a non-denaturing zwitterionic detergent may contribute to the detection and characterisation of MHC-related, membrane-bound proteins of tumours and normal cells.

Animals↗

PubMed1988

A novel HLA class II molecule.

Molecular evidence has been obtained for a novel monomorphic HLA class II molecule distinct from HLA-DP/DQ/DR using a panel of lymphoblastoid cells which include HLA-loss mutants. The expression of this molecule was investigated using monomorphic affinity-purified mouse monoclonal antibodies (mAbs), including one of the IgG2a subclass designated EDU-1. This antibody reacts strongly in a cell-binding radioimmunoassay with HLA-DR and -DQ loss mutants derived from a lymphoblastoid parental cell. The EDU-1 mAb also reacted with a local panel of homozygous Epstein-Barr virus-transformed cell lines. The reactive molecules were further detected on allostimulated T-cell clones and various leukemic cells including those of myeloid origin which lack surface expression of HLA-DQ molecules. Thus the class II molecule described in this study corresponds to a monomorphic HLA class II determinant expressed on a variety of cells of different origin and HLA phenotypes. Moreover, this antigen structure is distinct from that of HLA-DP/DQ/DR as shown by direct immunoprecipitation, serial immunodepletion experiments, and two-dimensional gel electrophoresis. The molecule could be specified by new class II genes between DP and DQ. An alternative explanation for the genetic basis of the novel molecule is the existence of isotypic associations between alpha and beta chains of various class II molecules (DP, DX, DZ, and DO) but not DR and DQ as the mutant cells tested lack the latter genes.

Antibodies, Monoclonal↗

PubMed1987

Analytical studies of the binding parameters describing the interaction of HLA-DR epitopes with a specific monomorphic monoclonal antibody.

In this paper we present an analytical study of the binding parameters related to the interactions of the HLA-DR-specific monoclonal antibody (L243) and its reacting epitope as expressed on the cell surface of seven Epstein-Barr virus-transformed B cell lines. Scatchard, Sips and Langmuir equations were used to plot and analyse the data obtained from each reaction. A single affinity constant (K) value was derived at low and high concentrations of free antibody for each antibody-cell interaction tested and was of the order of 10(7) M-1. Similar heterogeneity indices (a) (close to 1.0) and K values were obtained for most of the cells. These results suggest that the reacting HLA-DR epitopes are homogeneously distributed and equally accessible to the antibody on all the cells tested. The average number of epitopes per cell was 3.4 X 10(6), SD 0.5 X 10(6) and were similar for all the cell lines. The analytical and experimental model presented here can be useful for studying quantitative and qualitative variations in the expression of MHC epitopes in oncogenesis and disease associations.

Antibodies, Monoclonal↗

PubMed1986

Structure of asymmetric non-precipitating antibody: presence of a carbohydrate residue in only one Fab region of the molecule.

The reactions between purified precipitating and non-precipitating anti-DNP sheep and rabbit antibodies and the antigens DNP-BSA and DNP-GABA-BSA have been studied by immunodiffusion, complement fixation and an inhibition test. Both antigens reacted identically with precipitating antibodies. On the contrary, non-precipitating antibodies did not precipitate and did not fix complement with DNP-BSA but were able to do so with DNP-GABA-BSA. A different behaviour with both antigens was also demonstrated by an inhibition test. The properties of these antibodies were also studied after treatment with endo-beta-N-acetylglucosaminidase H. Non-precipitating antibody was able to give precipitin bands in gel diffusion and to fix complement with DNP-BSA after treatment with the enzyme. The treated antibody was able to agglutinate sensitized erythrocytes. Studies by fluorescence quenching showed that the affinity for the ligand DNP-GABA was significantly increased after hydrolysis of the carbohydrate residue. The properties of precipitating antibody were not modified by the endoglycosidase. Affinity chromatography of the F(ab')2 and Fab fragments obtained from precipitating and non-precipitating antibodies was made with Con A-Sepharose. The Con A retained all the F(ab')2 and 50% of the Fab from non-precipitating antibody, which were subsequently eluted with alpha-methyl-D-mannoside. The fragments from precipitating antibody were not retained at all. It is concluded that the asymmetry of the non-precipitating antibody molecule is due to a carbohydrate moiety which is present in only one of the Fab regions. This carbohydrate affects the reaction between the combining site and the antigen, and renders the molecule functionally univalent.

Acetylglucosaminidase↗

PubMedDate not supplied

Human B cells express membrane-bound and soluble forms of the CD14 myeloid antigen.

The expression of the myeloid differentiation antigen CD14 on the B lineage was analyzed. A CD14-specific monoclonal antibody was used to isolate the antigen from normal B, B-type chronic lymphocytic leukemia cells, and a representative Epstein-Barr virus-transformed B lymphoblastoid cell line (EBVLCL). A soluble form of this protein was detected in the culture supernatant of all the B cell types tested. The molecule expressed in the normal B and B-type chronic lymphocytic leukemia cells was identical in size to the 52,000 mol. wt monocyte-isolated CD14 glycoprotein. A 64,000 mol. wt antigen was isolated from the lymphoblastoid cell line. Similar 2-D gel electrophoretic patterns to that of the monocyte-derived CD14 were obtained from the normal B and B-type chronic lymphocytic leukemia cell-isolated molecules. These similarities were reflected in minor isoelectric point (pI) differences between the polypeptide spots (pI 4.8), in the first dimension, and identical molecular weight (52,000) in the second dimension. The EBVLCL-isolated polypeptide, when analyzed by 2-D gel electrophoresis, showed a pI identical to that of the myeloid antigen (pI 4.6). The isolated soluble form was of smaller (47,000 mol. wt, normal B and B-type chronic lymphocytic leukemia cells) or similar size (64,000 mol. wt, lymphoblastoid cell line) compared with their corresponding membrane-bound forms. Interestingly, two-colour immunofluorescence analysis showed that only two out of four CD14-specific mAb tested bound to the B cells. We conclude that the CD14 antigen is, in fact, expressed in the B lineage. Its cell surface expression and serum level in the prognosis of B-type chronic lymphocytic leukemia patients needs to be evaluated.

Antigens, CD↗

PubMedDate not supplied

Functional studies of H-2k-like epitopes on DTIC treated and untreated L1210 (H-2d) clones.

Following 'in vivo' treatment with 5-(3-3'-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), murine leukemic cells acquire new antigenic specificities not detectable on parental cells and responsible for the rejection of the tumour by syngeneic hosts. 'In vivo' and 'in vitro' experiments pointed out an immunological cross reactivity between DTIC treated and untreated lines. Furthermore, specific CTLs raised against DTIC treated L1210 tumour cells (H-2d) were cytotoxic for H-2k target cells. The aim of this study is to investigate whether the H-2k cross reactivity displayed by L1210/DTIC is related to the drug treatment rather than due to an antigen already present in the parental line and maintained after treatment. Cloned cells from L1210, obtained by limiting dilution 'in vitro', were recloned 'in vivo' and then treated with DTIC. Syngeneic and allogeneic CTLs raised 'in vitro' against parental and treated clones showed lytic activity against H-2k target cells. Treated and untreated clones were then checked for the presence of H-2k-like determinants using monoclonal antibodies. One of these, HB-53 (IgG2bKkDk) was highly positive with all the clones tested in binding assay using iodinated Fab anti-mouse Ig, fluorescence and FACS analysis. Others displayed a low reactivity against both treated and untreated clones without significant differences. After neuraminidase treatment of two clones (D and D/DTIC), the H-100.5 (anti H-2Kk)-reactive epitope was dramatically exposed on the DTIC tumour cells but not on the parental clones. These data suggest that the H-2k cross reactivity is related to the presence of a TAA that is maintained after treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗