Orgaran in the prevention of deep vein thrombosis in stroke patients.

Venous thrombo-embolism is a common complication in patients with acute ischaemic stroke. Without prophylaxis, deep vein thrombosis occurs in 60-75% of patients with dense hemiplegia, usually in the paralyzed limb, and 1-2% suffer fatal pulmonary embolism. Orgaran (Org 10172, low-molecular-weight heparinoid) has been evaluated for the prevention of deep vein thrombosis in patients with acute ischaemic stroke in two studies. In a double-blind study, 75 patients were randomized to receive Orgaran (50 patients) in a loading dose of 1,000 anti-Xa units intravenously followed by 750 anti-Xa units subcutaneously 12-hourly or placebo (25 patients). Deep vein thrombosis occurred in 2 of 50 (4%) in the Orgaran group and 7 of 25 (28%) in the placebo group (p = 0.005). The corresponding rates for proximal deep vein thrombosis were 0 and 16%, respectively (p = 0.01). There was one major haemorrhage in the treated group and one minor haemorrhage in the placebo group. In the second study, the safety and efficacy of Orgaran was compared with unfractionated heparin in the prevention of deep vein thrombosis in a double-blind randomized trial. Eighty-seven patients with marked lower limb paralysis secondary to stroke were randomized to receive Orgaran (45 patients) in a dose of 750 anti-factor Xa units subcutaneously 12-hourly or unfractionated heparin (42 patients) in a dose of 5,000 units subcutaneously 12-hourly. Venous thrombosis occurred in 4 of 45 (8.9%) of the Orgaran group and 13 of 42 (31%) in the unfractionated heparin group (2p = 0.014). The corresponding rates for proximal vein thrombosis were 4.4 and 11.9%, respectively (2p = 0.255).(ABSTRACT TRUNCATED AT 250 WORDS)


Studies of Org 10172 in patients with acute ischemic stroke. TOAST Study Group.

This report presents data from pilot studies of Org 10172 in the management of patients with acute ischemic stroke. The studies have established a potentially optimal dosage and treatment regimen and have provided information for the development of a large randomized trial. The trial of Org 10172 in acute stroke treatment is now underway in the United States.


Protection by albumin against the pro-oxidant actions of phenolic dietary components.

Synthetic and natural phenolic compounds are increasingly used in food preservation. Carnosol and carnosic acid (active components of rosemary extract), flavonoids (morin, quercetin, fisetin, myricetin), other plant phenolics (gossypol) and propyl gallate may protect lipids against oxidative damage but have the potential to increase damage to non-lipid constituents of foods, such as carbohydrates and DNA. Thus, in the presence of ferric EDTA and H2O2, they can form highly reactive hydroxyl radicals that can degrade the sugar deoxyribose and/or accelerate DNA degradation by means of a ferric-bleomycin complex. Human and bovine serum albumin afford considerable protection against damage to deoxyribose and DNA mediated by the above reactions. It is suggested that, given the fortification of foods with iron and EDTA and the use of phenolic substances as 'antioxidant' food additives, the addition of albumin might afford some protection.


A study of uric acid pretreatment for the protection of rat gastric mucosa against toxic damage.

Uric acid was evaluated for its potential to protect the gastric mucosa against the injuries caused by 80% ethanol, 0.6 m-HCl and 0.2 M-NaOH in rats. Uric acid at doses of 50, 100 or 300 mg/kg body weight provided dose-dependent protection against the ulcerogenic effects of all three agents. Other effects caused by ethanol only were studied. Serum uric acid concentrations were statistically significantly increased by both uric acid and ethanol treatments. Treatments of rats by gavage with 1 ml 80% ethanol was found to cause depletion of stomach-wall mucus, to lower the concentrations of protein, nucleic acids and non-protein sulphydryl groups in the stomach wall, and to cause histopathological lesions, including necrosis, erosions, congestion and haemorrhage, of the stomach wall. Treatment with uric acid, at doses of 50, 100 or 300 mg/kg body weight, by gavage, provided some measure of protection against all of these effects, and the protection was generally dose dependent. The protective effects of uric acid against damage to the gastric-wall mucosa may be mediated through its effects on mucus production and non-protein sulphydryl concentrations, and/or its free-radical scavenging properties.


p185 HER2/neu epitope mapping with murine monoclonal antibodies.

In order to obtain further information on the biological role of the HER2/neu oncoprotein, 7 new monoclonal antibodies (MAbs) were produced against the p185HER2 extracellular domain. These MAbs, together with two others previously produced, were used to investigate the p185HER2 expression in breast carcinomas and compare the recognized antigenic determinants. The 7 reagents (MGR4,5,6,7,8,9 and 10), were shown to define five distinct epitopes. Three of these MAbs (MGR5,7,10), as well as one previously produced (MGR2), recognize the same epitope (Epitope-1) which seems, therefore, to be highly immunogenic for the murine immune system. Epitope-2 recognized by the MGR4 MAb, appears to be closely related to epitope-1 due to a cross inhibition between MGR4 and MGR10, but not MGR2. Epitope-2 is the only one of the 5 also present on the product of the neu oncogene, the rat analogue of the human HER2/neu gene. None of the reagents against epitope-1 and epitope-2 were found to mediate receptor internalization, whereas MGR6 as well as a previously produced MAb (MGR3), both of which define epitope-3 and MGR8 which defines epitope-4, were found to do so. Epitope-4 like the neu-specific peptide recognized by the reference c-neu Ab3 MAb, was detectable on all p185HER2 positive breast cancer, independently from the quantitative content of the oncoprotein, at variance with the other 4 epitopes whose availability on p185HER2 for the relevant MAbs varied with the degree of overexpression. Epitope-5, recognized by the MGR9 MAb, on the contrary to the other epitopes, was prevalently localized at the basal membrane level of the tumor nodule.


Characterization and nucleotide sequences of the variable regions of a monoclonal antibody against alpha-fetoprotein.

alpha-Fetoprotein (AFP) is a well-known tumor marker of hepatocellular carcinoma (HCC). Monoclonal antibodies against AFP possessing specific binding ability to HCC are potential candidates for immunoscintigraphy and immunotherapy. A new monoclonal antibody against AFP (0325-6-9) was isolated. Its specificity and targeting tumor ability were characterized by enzyme-linked immunosorbent assay (ELISA), cell immunostain and complement killing. These results suggest that 0325-6-9 is specific to hepatoma cells. The nucleotide sequences of variable regions of 0325-6-9 were determined by M13 dideoxynucleotide sequencing method. With the information of nucleotide sequence, this antibody then could be modified by recombinant technology for its usage in in vivo diagnosis and immunotherapy.


[Binding of LPS to CD14].

The 53 kDa glycoprotein CD14 (cluster of differentiation No. 14, defined by monoclonal antibodies of the 1st Leukocyte Typing Workshop) is expressed on monocyte surfaces and was identified 1990 as an endotoxin receptor (1). The binding of endotoxin to CD14 was mediated by a LPS-binding protein (LBP) present in serum.


Preliminary studies on the interaction of TNF alpha and IFN gamma with alpha 2-macroglobulin.

The binding of 125I-labelled recombinant human TNF alpha and IFN gamma to isolated human blood alpha 2-macroglobulin has been investigated using molecular sieving procedures and non-denaturing PA gel electrophoresis in combination with autoradiography. These studies revealed that both cytokines readily bind to the electrophoretically fast form of alpha 2M generated by methylamine or protease treatment of this protein. PAGE/SDS gel investigations indicated that TNF alpha bound non-covalently while the IFN gamma interaction was covalent in nature. Preliminary competition studies also indicate that cold TNF alpha and IL-2 are more effective than cold IFN gamma at inhibiting the binding of labelled IFN gamma to alpha 2M. Bioassays revealed that "native" alpha 2M or its derivatives at 2 mg/ml concentration did not impair the antiproliferative effects of TNF alpha and IFN gamma on susceptible bladder tumour cell lines. Furthermore they did not interfere in the induction of Class II antigen expression by IFN gamma on inducible cell lines or in a 2-site ELISA assay for TNF.


Role of the endothelial adhesion molecule VCAM in murine cardiac allograft rejection.

Murine heterotopic cardiac isografts (C57B1/6----C57B1/6) undergo transient, non-destructive inflammation that is characterized by the acquisition of microvascular endothelial reactivity with the antibody MECA 32. Cardiac allografts (C57B1/6----DBA/2) undergo destructive inflammation that is characterized by the acquisition of reactivity with the antibody M/K-2, in addition to MECA 32. M/K-2 recognizes the murine endothelial adhesion molecule, VCAM-1. Hence, there appear to be antigen-dependent and antigen-independent forms of graft inflammation. Treatment of cardiac allograft recipients with 200 micrograms/day M/K-2 antibody retarded graft loss by only a few days, and did not interfere significantly with leukocytic infiltration, as detected by limiting dilution analysis of graft-reactive CTL, despite the fact that large amounts of M/K-2 could be detected on graft microvascular endothelia and in the peripheral blood as rejection progressed. These data indicate that VCAM is apparently not essential for the leukocytic infiltration and subsequent rejection of cardiac allografts, and is not involved in leukocytic infiltration of murine cardiac isografts.


Two chicken monoclonal antibodies specific for heterophil Hanganutziu-Deicher antigens.

Two chicken monoclonal antibodies (MAbs), HU/Ch2-7 and HU/Ch6-1, against heterophil Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (NeuGc) at a terminal carbohydrate were established by cell fusions using chicken B cell lines lacking thymidine kinase and spleen cells from chickens immunized with II3NeuGc alpha-LacCer (HD3). The reactivities of these MAbs against several gangliosides including NeuGc-containing glycosphingolipids were examined by a thin-layer chromatography/immunostaining method. MAb HU/Ch2-7 (IgG) reacted strongly with HD3 and IV3NeuGc alpha-nLc4Cer (HD5) and weakly with VI3NeuGc alpha-nLc6Cer (HD7) and 4-O-acetyl-HD3. HU/Ch6-1 (IgG) reacted with HD3 and HD5, but did not react with the other HD antigens. The reactivities of these MAbs against HD antigen were greatly reduced by pre-treatment of the antigen with neuraminidase. These MAbs did not react with N-acetylneuraminic acid-containing gangliosides (GM1 and GM3). These results indicate that these two chicken MAbs are directed toward the antigenic epitope containing the NeuGc.


Immunological characterization of an immunomodulatory epitope in S-antigen/arrestin with a sequence motif common to tumor necrosis factor alpha.

Some monoclonal antibodies (mAbs) to retinal S-antigen recognize a phylogenetically conserved epitope (S2) in the N-terminal part of the protein. These antibodies have been shown to inhibit the induction of experimental autoimmune uveoretinitis by S-antigen in rats. Using Pepscan method, we localized this epitope on the amino acid (aa) residues 40-50, i.e., PVDGVVLVDPE (peptide S2). MAb binding was confirmed by ELISA, competition-ELISA and dot blot. Other S-antigen peptides with homologies to epitope S2 and peptides exhibiting the pathogenic and T-cell proliferation inducing sites did not bind these mAbs. Epitope S2 displays an immunological crossreactivity with human tumor necrosis factor (TNF) alpha. Recent results indicate that both peptide S2 and a peptide from human TNF alpha (aa residues 31-53) containing the common sequence motif GVxLxD induce TNF alpha production in monocytes. We analyzed the fine structure of the common epitope by studying mAb binding in an amino acid residue exchange experiment.


In situ islet T cell receptor variable region gene usage in the nonobese diabetic mouse.

Several features of the genetics and immunopathology of diabetes in the nonobese diabetic (NOD) mouse, which spontaneously develops type I diabetes, are shared with the human disease. Immunohistochemical studies support the concept that T lymphocytes are the major components of inflammatory cells in the pancreatic islets and these cells may play a critical role in the destruction of the beta cells leading to diabetes. Therefore, we examined whether particular TCR-beta variable region genes were utilized by in situ islet T cells at different stages (4 - 5, 7, 14 - 15 and 16 weeks of age) of the disease process. Dot-blot hybridization was performed using RNA prepared from isolated islets, thymus, spleen, peripheral blood leukocytes and axillary lymph nodes of 10 to 15 mice pooled for each data point. Ten different TCR V-beta probes were used for the analyses. Limited usage of islet V-beta genes was observed only at the early prediabetic stage (4 - 5 weeks old) of the disease. At later stages of the disease (7 - 16 weeks old), no preferential usage of TCR genes was observed in the islets compared to those of peripheral lymphoid organs. These data suggest that only certain types of T cells bearing particular TCR V-beta genes may be responsible for initiating and perpetuating infiltration of immune cells into the islets and these particular T cells are only identified at the very early stages of the autoimmune process.


New antigenic clusters on human thyroglobulin defined by an expanded panel of monoclonal antibodies.

Twenty-seven hybridomas secreting monoclonal antibodies (mAb) directed against new antigenic clusters on human thyroglobulin (hTg) were obtained by fusion of the mouse myeloma P3-X63-Ag8 653 with spleen cells from BALB/c mice immunized with a mixture of hTg and six anti-hTg mAb with the aim of masking the corresponding antigenic clusters previously reported. Fourteen mAb were selected, produced in ascitic fluid and characterized. All these mAb were of the IgG1 subclass. Five new antigenic clusters on the hTg molecule were defined by the 14 mAb, extending the initial antigenic map of hTg to 11 clusters. These mAb were used in an attempt to probe the interaction between hTg and the autoantibodies from patients with Hashimoto's thyroiditis who do not recognize antigenic cluster II, a cluster whose recognition by anti-hTg autoantibodies is significantly associated with thyroid disorders.


Role of the mononuclear phagocyte as an antigen-presenting cell for human gamma delta T cells activated by live Mycobacterium tuberculosis.

gamma delta T cells, both human and murine, have been found to be highly responsive to mycobacterial antigens. However, the role and function of gamma delta T cells in the immune response to Mycobacterium tuberculosis remain largely unknown. In earlier studies, we demonstrated that monocytes infected with live M. tuberculosis were particularly effective inducers of human peripheral blood gamma delta T cells. The present studies were performed to further characterize the interaction between human mononuclear phagocytes, gamma delta T cells, and live M. tuberculosis, in comparison with CD4+ T cells. First, we found that resting gamma delta T cells expanded in vitro by live M. tuberculosis were specific for M. tuberculosis, and that heat killing and washing the mycobacteria removed the antigen(s) for gamma delta T cells. In contrast, the heat-killed mycobacteria retained significant antigenicity for CD4+ T cells. Second, live M. tuberculosis-expanded gamma delta T cells from healthy tuberculin-positive donors did not respond significantly to the antigens in M. tuberculosis culture filtrate, including the 65- and 71-kDa mycobacterial heat shock proteins. Third, the activation of gamma delta T cells by live mycobacteria was dependent on antigen-presenting cells, and mononuclear phagocytes were found to be very efficient antigen-presenting cells both for resting peripheral blood gamma delta T cells and for activated expanded gamma delta T cells. The mononuclear phagocyte carried the necessary costimulatory factors necessary for gamma delta T-cell proliferation. Fourth, the antigen repertoire and HLA requirements for CD4+ memory T cells and those for gamma delta T cells appear to be quite distinct from each other. CD4+ T cells recognized both soluble protein antigens and whole organisms in a class II major histocompatibility complex-restricted manner, whereas gamma delta T cells appeared to recognize only constituents associated with the whole organism and were not restricted by class I or class II major histocompatibility complex molecules. Finally, the assay system described to expand and purify responding CD4+ and gamma delta T cells after stimulation with live M. tuberculosis represented a simple approach to the direct comparison of these two T-cell populations in the interaction with mononuclear phagocytes infected with M. tuberculosis. Such studies provide insight not only into the relative roles of human CD4+ and gamma delta T cells in the human immune response to intracellular bacterial pathogens such as M. tuberculosis but also into the basic biologic role of human gamma delta T cells in antimicrobial immunity.


Synthetic peptides representing epitopes of outer membrane protein F of Pseudomonas aeruginosa that elicit antibodies reactive with whole cells of heterologous immunotype strains of P. aeruginosa.

By using the published amino acid sequence for mature outer membrane protein F of Pseudomonas aeruginosa, a computer-assisted analysis was performed to identify sites with potential as surface-exposed, antigenic regions located throughout the length of the protein molecule. Synthetic peptides 13 to 15 amino acid residues in length were synthesized for 10 such regions. Mice were immunized with each of the 10 synthetic peptides conjugated to keyhole limpet hemocyanin. An enzyme-linked immunosorbent assay (ELISA) of the antisera was performed by using each of the synthetic peptides as the ELISA antigen to verify that immunoglobulin G (IgG) antibodies capable of reacting with the peptide used as immunogen were elicited by each peptide. Each of the antipeptide antisera was screened for the presence of IgG antibodies that could bind to the surface of intact cells of strains representing the seven heterologous Fisher-Devlin immunotypes of P. aeruginosa by use of an ELISA with whole cells of the various strains as the ELISA antigen. Three peptides elicited antibodies capable of reacting with whole cells of all seven immunotype strains. Peptide 10, corresponding to amino acid residues 305 to 318, elicited whole-cell-reactive antibodies at high titers. Peptide 9, corresponding to amino acid residues 261 to 274, elicited whole-cell-reactive antibodies at more intermediate titers. Peptide 7, corresponding to amino acid residues 219 to 232, elicited such antibodies only at low titers. The carboxy-terminal portion of the mature protein appears to be the immunodominant portion. In particular, peptides 10 (NATAEGRAINRRVE) and 9 (TDAYNQKLSERRAN) appear to have potential for use as immunogens in a synthetic vaccine for immunoprophylaxis against infections caused by P. aeruginosa. Antisera from mice immunized with either peptide 9 or 10 mediated opsonophagocytic uptake by human polymorphonuclear leukocytes of wild-type cells of P. aeruginosa but exhibited no opsonic activity against a protein F-deficient mutant of P. aeruginosa.


Suppression of C3H/HeJ cell activation by lipopolysaccharide endotoxin.

Earlier studies in our laboratory showed that the lipopolysaccharide (LPS) of Salmonella typhi, which fails to activate B lymphocytes of C3H/HeJ mice, can suppress proliferation and polyclonal antibody synthesis by these cells when they are stimulated by polyclonal activators. In order to determine what stage of the cell cycle was blocked, resting B cells from C3H/HeJ spleens were activated by using different mitogens in the presence of inhibitory concentrations of LPS and analyzed by flow cytometry, using acridine orange to stain DNA and RNA. LPS was found to inhibit the progression of cells into the G1 stage of the cell cycle. Furthermore, [3H]uridine uptake studies showed that RNA synthesis is inhibited during the early phase of activation. These results indicate that inhibition by LPS of the signalling process occurs during a critical period of the cell cycle when the cells become susceptible to the inhibitory effects of LPS. To examine whether LPS acts only on B cells or whether it can suppress other immunocompetent cells from C3H/HeJ mice, studies were carried out on activated thymocytes and macrophages. LPS was found to inhibit thymocyte proliferation stimulated by concanavalin A or the combination of phorbol myristate acetate and ionomycin. Prostaglandin E2 synthesis by macrophages was also blocked by LPS. Thus, LPS is a potent inhibitor of the functioning of the major immunocompetent cells of C3H/HeJ mice.


Identification of a beta 1 integrin on Mycobacterium avium-Mycobacterium intracellulare.

Mycobacterium avium-Mycobacterium intracellulare (MAI) is an opportunistic intracellular pathogen responsible for the highest incidence of disseminated bacterial infection in patients with AIDS. Treatment of the infection is extremely difficult and has shown limited efficacy. A critical event in the initiation of a variety of bacterial infections involves the adherence of bacteria to host cell surfaces. In the present study, we have shown that MAI organisms bind avidly to extracellular matrix proteins such as laminin, collagen I, and fibronectin in an in vitro attachment assay. Immunoblot analysis of a sonicate of MAI with polyclonal antibodies against different integrin receptors indicated that the sonicate cross-reacts with polyclonal antibodies against a human laminin-binding integrin, alpha 3 beta 1, and a human fibronectin-binding integrin, alpha 5 beta 1, although it is reactive with only the beta 1 subunit in the case of both antisera. Antibodies against the alpha 3 beta 1 and alpha 5 beta 1 integrins specifically inhibited the binding of MAI to laminin, collagen I, and fibronectin by 70 to 97%, depending on the ligand, suggesting that the attachment of MAI to these extracellular matrix proteins may be mediated by a beta 1 integrin. Furthermore, the attachment of MAI to laminin, collagen I, and fibronectin was found to be cation dependent. MAI may use this and other beta 1-containing integrins to adhere and penetrate through basement membrane structures that underlie host cell linings. An understanding of the mechanism of attachment and a definition of the adhesive molecules on the surface of MAI may open up new approaches to the prevention of serious infection caused by this organism.


Borrelia burgdorferi HSP70 homolog: characterization of an immunoreactive stress protein.

The gene encoding an immunoreactive Borrelia burgdorferi HSP70 homolog was isolated and characterized. The predicted amino acid sequence of this spirochetal protein confirms that this gene encodes a member of the HSP70 family of proteins. Although there appears to be a single copy of this gene on the spirochetal chromosome, two distinct transcripts hybridizing to the hsp70 probe are detected in RNA isolated from B. burgdorferi. The amount of spirochetal HSP70 RNA transcripts is shown to be thermally regulated. Antibodies in the serum of three Lyme arthritis patients and cloned T-cell lines isolated from one patient with Lyme arthritis recognize the expressed recombinant HSP70, indicating that it is an immunologically important spirochetal antigen. Antibodies in a rabbit antiserum, as well as antibodies in the serum of two of three Lyme arthritis patients examined, bound to expressed truncated recombinant HSP70s with 250 amino acids deleted from either the amino or carboxy terminus of the protein. However, antibodies in the serum of three Lyme arthritis patients, which were reactive with spirochetal HSP70, did not cross-react with human HSP70 proteins.


Mapping of Candida albicans oligomannosidic epitopes by using monoclonal antibodies.

Six monoclonal antibodies (MAbs) from various laboratory sources (EB-CA1, EB-CA2, H5, AF1, C6, and 5B2), reacting with the polysaccharidic moieties of Candida albicans mannoproteins, were used for epitope mapping by an enzyme-linked immunosorbent assay (ELISA) with neoglycolipids and by Western blotting (immunoblotting) of a C. albicans germ tube extract. The ELISA involved neoglycolipids constructed from three families of oligomannosides released by sequential depolymerization of C. albicans phosphopeptidomannan by acid hydrolysis (NGLH), beta-elimination (NGLO), and acetolysis (NGLA). All of the MAbs exhibited low reactivities against NGLO. MAbs EB-CA1, EB-CA2, and H5 reacted mainly against NGLA, and MAbs C6 and AF1 recognized mainly NGLH, whereas MAb 5B2 reacted with both families of neoantigens. When this method was compared with Western blotting, strong reactivity to NGLA was associated with the presence of epitopes shared by high-molecular-weight mannoproteins, whereas strong reactivity to NGLH was associated with a reactivity to a family of 14- to 18-kDa antigens. The reactivity of MAb 5B2 was associated with both high-molecular-weight mannoproteins and the 14- to 18-kDa antigens. In relation to the present knowledge about the structure of the C. albicans phosphopeptidomannan oligomannosidic repertoire, these results provide preliminary data concerning the molecular basis of the recognition of mannopyranosyl sequences by MAbs and their distribution among C. albicans mannoproteins.


Inhibition of bactericidal activity of anticapsular antibody by nonspecific antibodies reactive with surface-exposed antigenic determinants on Actinobacillus pleuropneumoniae.

In an attempt to understand the mechanism of serum resistance in Actinobacillus pleuropneumoniae, in the present study we examined various interactions among the bacterial surface constituents, serum antibodies, and complement. Analysis of swine sera revealed the presence of anticapsular antibodies in convalescent-phase sera but not in preimmune sera. Both types of sera contained antibodies which reacted with each of 14 polypeptides present in saline extracts of the bacteria. Absorption of the preimmune sera with intact bacteria depleted antibodies to two of the polypeptides (27 and 32 kDa) and high-molecular-weight (greater than 97.4,000) components which did not stain with Coomassie blue. Data derived from complement consumption and C3-binding experiments indicated that the organism was capable of initiating complement activation and binding C3 during incubation in preimmune and immune sera. Experiments designed to evaluate the bactericidal effectiveness of anticapsular antibody revealed that the purified antibody was bactericidal only when preimmune sera absorbed with intact bacteria were used as a source of complement. The bactericidal effects of anticapsular antibody and absorbed preimmune sera were inhibited in a dose-dependent manner by heat-inactivated preimmune sera and immunoglobulin G derived from the sera. The inhibitory activity of the preimmune sera was neutralized by preincubating the sera with column fractions of the saline extract which contained either the 27- or the 32-kDa polypeptide. These results indicate that serum resistance in A. pleuropneumoniae 4074 could be related to inhibition of the bactericidal action of anticapsular antibody by nonspecific antibodies which recognize surface-exposed epitopes on the polypeptides.


Characterization of a major hemagglutinin protein from Mycoplasma gallisepticum.

Mycoplasma gallisepticum cell membranes were used to immunize mice to produce monoclonal antibodies to cell surface proteins. Three monoclonal antibodies were chosen for further characterization. All three reacted in immunoblots with an M. gallisepticum protein band of M(r) approximately 67,000 (designated pMGA). By using immunoelectron microscopy, pMGA was shown to be located on the cell surface. When M. gallisepticum whole cells were treated with up to 250 micrograms of trypsin per ml for 30 min, the only major protein lost from the cell surface as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western immunoblot transfer was pMGA. Two of the pMGA-specific monoclonal antibodies inhibited hemagglutination of chicken erythrocytes by M. gallisepticum S6, suggesting a role for pMGA in the attachment of M. gallisepticum to chicken erythrocytes. Sequencing the amino terminus of pMGA yielded 17 amino acids with no significant homology with the Mycoplasma pneumoniae attachment protein P1 or any other protein in the GenBank, Swiss-Prot, and EMBL data bases.


Bacterial motility is a colonization factor in experimental urinary tract infection.

In an experimental urinary tract infection of the mouse, colonization of the urinary bladder by isogenic strains of Salmonella enterica serovar Typhimurium was found to depend on the motility of the bacteria. Strains were obtained by genetic recombination between a highly motile O-6,7 and a poorly motile O-4,5,12 strain. The O antigen did not interfere with the colonization, whereas motility did; flagellated and motile O-6,7 and O-4,5,12 bacteria colonized the bladder equally well.


Hyaluronan (hyaluronic acid) and hyaluronectin in the extracellular matrix of human breast carcinomas: comparison between invasive and non-invasive areas.

We performed quantitative determination of the distribution of hyaluronan (hyaluronic acid, HA) and the HA-binding protein, hyaluronectin (HN), 2 components of the extracellular matrix of tumor desmoplasia, within 71 human breast carcinomas. Results showed that HA and HN were more elevated in tumoral than in non-tumoral adjacent tissue, and that the peripheral invasive area of tumors contained increased levels of HA and HN as compared with the central non-invasive area (p less than 10(-3) and p less than 10(-5) respectively). HN and HA levels of 61 ductal carcinomas were related to the histological grade of tumors; no significant difference was found between grades for HA; HN was found to be significantly lower in grade III than in grade II tumors (p less than 0.01). HA and HN rates were correlated in grade I and grade II tumors and were not correlated in grade III. Mean percentage of HA saturation level by HN for whole tumors was found to be less than 4%, indicating that HA is essentially free of proteins and could be used as a target for cancer diagnosis or therapy.


Enhancement of hepatitis-B surface-antigen expression by 5-azacytidine in a hepatitis-B-virus-transfected cell line.

The human hepatoblastoma-derived cell line HB611 secretes hepatitis-B surface antigen (HBsAg) and hepatitis-B e antigen (HBeAg) into the medium. Hepatitis-B-virus (HBV) DNA integrated into the cellular genome was found to be hypermethylated. When the cells were treated with 5-azacytidine for 3 days, the level of HBsAg in the medium increased, while the level of HBeAg remained constant. The level of alpha-fetoprotein (AFP) decreased with the 5-azacytidine treatment. Southern blot analysis of DNA digested with HpaII or MspI showed that 5-azacytidine treatment resulted in hypomethylation of the integrated HBV DNA, suggesting that 5-azacytidine increased HBsAg production in the cells through hypomethylation of the HBV genomic DNA.


Protein-kinase-C inhibitor calphostin C reduces B16 amelanotic melanoma cell adhesion to endothelium and lung colonization.

We recently reported that the Ca(2+)- and phospholipid-dependent protein kinase, protein kinase C (PKC), was involved in rat Walker carcinosarcoma cell adhesion to large-vessel endothelium. We extended our studies to explore the role of this kinase in the adhesion to small-vessel endothelium and lung colonization of murine B16 amelanotic melanoma (B16a). Subpopulations of B16a cells, which differ in lung-colonization potentials, were isolated by centrifugal elutriation from solid tumors. In this study, we demonstrate that cells from a high metastatic sub-population (HM340), when compared with cells from a low metastatic sub-population (LM180), exhibit elevated levels of total cellular as well as membrane-bound PKC. The increase in PKC in cells from the HM340 correlates positively to their increased ability to adhere to murine pulmonary-microvessel endothelial-cell monolayer, and to form pulmonary colonies in syngeneic mice. Calphostin C, a potent and selective PKC inhibitor, decreases in a dose-dependent manner the adhesion to endothelium and the lung colonization of cells from both the low and the high metastatic sub-populations with IC50 at sub-micromolar concentrations. In conclusion, our results suggest that PKC may be a key element in regulating tumor-cell metastasis and that PKC inhibitors may be anti-metastatic agents.


Gamma-D-glutamylaminomethyl sulfonic acid (GAMS) distinguishes subtypes of glutamate receptor in the chick cochlear nucleus (nuc. magnocellularis).

Because kainic acid (KA) is more potent than other excitatory amino acids (EAAs) in affecting synaptic transmission in the cochlear nucleus, previous reports have concluded that primary afferent neurotransmission to the cochlear nucleus in birds and mammals is mediated by KA-preferring non-N-methyl-D-aspartate (non-NMDA) EAA receptors. Since this conclusion is at odds with a number of studies suggesting that rapid excitatory neurotransmission in the CNS is mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-preferring non-NMDA receptors, we re-examined the pharmacology of synaptic transmission between the cochlear nerve and nucleus magnocellularis (NM) in chickens, using bath application of drugs and recording of field potentials evoked in NM by electrical stimulation of the cochlear nerve in vitro. A series of EAA agonists produced complete, concentration-dependent and reversible suppression of postsynaptic responses: the order of potency was domoic acid (DO) greater than KA greater than AMPA much greater than quisqualic acid much greater than L-glutamic acid (Glu). Three quinoxalinedione antagonists of non-6-nitro-7-sulphamobenzo[f]quinoxaline-2,3-dione NMDA receptors also produced complete, concentration-dependent and reversible suppression of postsynaptic responses in NM without affecting the presynaptic action potential; the half-maximal inhibitory concentrations (IC50's) were 2.7 +/- 0.4 microM for 6-nitro-7-sulphamobenzo[f]quinoxaline-2,3-dione (NBQX), 5.3 +/- 0.1 microM for 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and 10.6 +/- 1.2 microM for 6,7-dinitroquinoxaline-2,3-dione (DNQX).(ABSTRACT TRUNCATED AT 250 WORDS)


Surgery for ovarian cancer.

Surgery is a critical component of the multimodality approach to ovarian cancer. Surgery confirms the diagnosis and establishes the stage of disease, especially important when the tumor appears to be limited to the ovaries. In advanced disease, surgical cytoreduction improves response to chemotherapy and survival. Second-look laparotomy provides a means for assessing response to therapy, predicting prognosis, and determining the need for further therapy. Surgery can also provide palliation of intestinal obstruction.


A modified method for the differential staining of spermatozoa using alcoholic acidic silver nitrate.

Differential silver staining patterns have been demonstrated in mammalian spermatozoa, using an aqueous silver nitrate reagent. In the present study, mouse, rabbit, and human spermatozoa were stained using a modification of the earlier method. In the modified method, an alcoholic acidic silver nitrate stain, with subsequent differentiation in alcoholic ammonia, was used. This method enhanced the intensity of staining of the head, mid-piece, and tail. In particular, marked differentiation of the acrosomal, subacrosomal, and postacrosomal regions was obtained, which facilitated determination of acrosomal integrity. Moreover, background interference was reduced, yielding better clarity of the stained smears. The staining was carried out in the cold. This modified technique offers an advantage in the assessment of sperm morphological anomalies and membrane and acrosomal integrity, and is a simple, reliable, and useful method for the evaluation of sperm function.


Purification of a low molecular weight calf pineal peptide controlling DNA transcription in vitro.

A low molecular factor showing high specific activity in the control DNA of transcription in vitro was isolated from aqueous ultrafiltered calf pineal gland extracts. The active factor was purified by means of Gel filtration on Sephadex G-25 and G-10, thin layer chromatography on aluminum sheet cellulose and high performance liquid chromatography using a Supelcosil LC 318 reverse phase column. The purified pineal factor was characterized as a peptide of low molecular weight (of about 1200 Dalton) containing glutamic acid, leucine, glycine, threonine and alanine in their approximate molar ratio, referred to glycine taken as 1: glycine 1, threonine 1, leucine 1, alanine 6, glutamic acid 2. Studies of the aminoacid sequence by N-terminal analysis using the automated Edman degradation procedure, were unsuccessful, suggesting the presence of a blocked NH2 group. The purified peptide appears to be different from peptide factors till now isolated from pineal gland.


Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity.

Exposure of bovine aortic or capillary endothelial cells to basic FGF (bFGF) for 1 h resulted in an approximately sixfold increase in plasminogen activator (PA) activity by 18 h that returned nearly to basal levels by 36 h. We hypothesized that the decrease in PA activity following bFGF stimulation was mediated by transforming growth factor beta (TGF-beta) formed from its inactive precursor. Conditioned medium collected from endothelial cells 36 h after a 1-h exposure to bFGF, but not control medium, inhibited basal levels of PA activity when transferred to confluent monolayers of bovine aortic endothelial cells. Antibody to TGF-beta neutralized the inhibitory activity of this conditioned medium, indicating that the medium contained active TGF-beta. Northern blot analysis and quantitation of acid activatable latent TGF-beta in conditioned medium demonstrated that bFGF exposure did not increase the amount of transcription or secretion of latent TGF-beta by the endothelial cells. Both aprotinin, an inhibitor of plasmin, and anti-urokinase type PA IgG blocked the generation of active TGF-beta in cultures exposed to bFGF. These results demonstrated that plasmin generated by uPA activity is required for the activation of latent TGF-beta in endothelial cell cultures treated with bFGF. Activation of TGF-beta by endothelial cells exposed to bFGF appears to limit both the degree and duration of PA stimulation. Thus, in bFGF-stimulated endothelial cell cultures, PA levels are controlled by a negative feedback loop: PA, whose expression is stimulated by bFGF, contributes to the formation of TGF-beta, which in turn opposes the effects of bFGF by limiting PA synthesis and activity. These studies suggest a role for TGF-beta in reversing the invasive stage of angiogenesis and contributing to the formation of quiescent capillaries.


Identification of a peptide sequence involved in homophilic binding in the neural cell adhesion molecule NCAM.

The neural cell adhesion molecule NCAM is capable of mediating cell-cell adhesion via homophilic interactions. In this study, three strategies have been combined to identify regions of NCAM that participate directly in NCAM-NCAM binding: analysis of domain deletion mutations, mapping of epitopes of monoclonal antibodies, and use of synthetic peptides to inhibit NCAM activity. Studies on L cells transfected with NCAM mutant cDNAs using cell aggregation and NCAM-covasphere binding assays indicate that the third immunoglobulin-like domain is involved in homophilic binding. The epitopes of four monoclonal antibodies that have been previously shown to affect cell-cell adhesion mediated by NCAM were also mapped to domain 3. Overlapping hexapeptides were synthesized on plastic pins and assayed for binding with these monoclonal antibodies. One of them (PP) reacted specifically with the sequence KYSFNY. Synthetic oligopeptides containing the PP epitope were potent and specific inhibitors of NCAM binding activity. A substratum containing immobilized peptide conjugates also exhibited adhesiveness for neural retinal cells. Cell attachment was specifically inhibited by peptides that contained the PP-epitope and by anti-NCAM univalent antibodies. The shortest active peptide has the sequence KYSFNYDGSE, suggesting that this site is directly involved in NCAM homophilic interaction.


Identification of vimentin in rat peritoneal mast cells and its phosphorylation in association with histamine release.

Stimulation of rat peritoneal mast cells with histamine releasers, such as compound 48/80 and substance P, caused a similar pattern of protein phosphorylations: the molecular weights of the two major phosphorylated proteins were 45 kDa and 59 kDa. When rat mast cells permeabilized with beta-escin were exposed to Ca2+ at concentrations higher than 0.6 microM, phosphorylated proteins of identical molecular weight were also detected. By a radioimmunoprecipitation assay using anti-vimentin mouse monoclonal antibody, the 59 kDa protein was identified as vimentin, one of the intermediate cytoskeletal proteins. Moreover, it became apparent that the phosphoamino acid in phosphorylated vimentin was a serine residue. Sequential changes in vimentin phosphorylation were similar to that of histamine release elicited by histamine releasers: phosphorylation took place within 5 s of stimulation and reached a maximum within 10 s. When permeabilized mast cells were treated with calphostin C, a specific protein kinase C inhibitor, phosphorylation was markedly inhibited. Fluorescence images of mast cells stained with FITC-labelled anti-vimentin antibody showed filamentous structures surrounding the granules in the cytoplasm. However, after exposure to compound 48/80, the filamentous structures promptly disappeared and a dim fluorescence was observed homogeneously in the cell indicating that a rapid depolymerization of vimentin had taken place. From the present study, it became clear that when rat peritoneal mast cells were stimulated, vimentin was rapidly phosphorylated by protein kinase C and this phosphorylation process seems to be related to histamine release.


CD44H regulates tumor cell migration on hyaluronate-coated substrate.

CD44 is a broadly distributed cell surface glycoprotein expressed in different isoforms in various tissues and cell lines. One of two recently characterized human isoforms, CD44H, is a cell surface receptor for hyaluronate, suggesting a role in the regulation of cell-cell and cell-substrate interactions as well as of cell migration. While CD44H has been shown to mediate cell adhesion, direct demonstration that CD44H expression promotes cell motility has been lacking. In this work we show that a human melanoma cell line, stably transfected with CD44H, displays enhanced motility on hyaluronate-coated surfaces while transfectants expressing an isoform that does not bind hyaluronate, CD44E, fail to do so. Migration of CD44H-expressing transfectants is observed to be blocked by a soluble CD44-immunoglobulin fusion protein as well as by anti-CD44 antibody, and to depend on the presence of the cytoplasmic domain of CD44. However, cells expressing CD44H cytoplasmic deletion mutants retain significant binding capacity to hyaluronate-coated substrate. Taken together, our results provide direct evidence that CD44H plays a major role in regulating cell migration on hyaluronate-coated substrate.


Structural and histochemical studies of Golgi complex differentiation in salivary gland cells during Drosophila development.

Morphological alterations in the Golgi complex (GC) and changes in the distribution of acid phosphatase (AcPase), thiamine pyrophosphatase (TPPase), complex carbohydrates and reduced osmium tetroxide compounds in this organelle were studied in the salivary gland cells of Drosophila during larval and prepupal development. The morphology and the AcPase, TPPase and complex carbohydrates cytochemical patterns of the Golgi complex varied characteristically during cell differentiation. At the early 3rd instar period the Golgi complex consisted mainly of vesiculated cisternae, and AcPase activity was observed in all cisternae but not in the secretory granules. As development proceeded to the late 3rd instar the Golgi complex displayed its typical appearance, consisting of four to six cisternae, and only the two to three cisternae towards the trans-face as well as the trans-Golgi network and some of the immature secretory granules exhibited AcPase reactivity. In the course of a 'wave' of production of the 'glue' secretory granules proceeding proximally through the gland, the number of AcPase positive cisternae changed correspondingly. After secretion of the 'glue' secretory granules, the size of the Golgi complex decreased and almost all cisternae displayed AcPase reactivity. The detection of TPPase activity presented some specificity problems, since staining was observed not only in the GC cisternae but in the endoplasmic reticulum (ER) and microvilli. The reaction products were seen in a few GC vesicles during the early 3rd instar and in the trans side of the organelle at the end of the 3rd instar. During production of the secretory granules, every GC cisterna was intensely stained. These results agree with previous findings suggesting that AcPase and TPPase in secretory cells may be primarily involved in the processing of exportable proteins. The vicinal (vic)-glycol groups of the complex carbohydrates were detected using the periodic acid/thiocarbohydrazide/silver proteinate (PA-TCH-SP) technique. During synthesis of the 'glue' secretory granules, the reaction products were observed over the GC cisternae and the trans-Golgi network, with increasing intensity from the cis to the trans side of the organelle. No PA-TCH-SP staining was observed over the GC cisternae during the early 3rd instar. Following discharge of the 'glue' secretory granules, all GC cisternae displayed uniform PA-TCH-SP staining. After OsO4 impregnation, the reaction products were observed mainly in ER and mitochondria and rarely in the GC. In numerous cells, only the mitochondria were stained, while in many cases the ER of neighboring cells exhibited differential staining.(ABSTRACT TRUNCATED AT 400 WORDS)


Determination of protein pattern in embryonic cavities of human early pregnancies: a means to understand materno-embryonic exchanges.

Exocoelomic and amniotic fluids were obtained by selective puncture under ultrasound guidance in normal human pregnancies between 5 and 13 weeks of gestation. Evaluation of the protein patterns in the exocoelomic fluid showed qualitative and quantitative changes with advancing gestation. During the second month of gestation, three electrophoretic bands were found with mobility compatible with albumin, alpha 1-globulin and beta-globulin and composed of at least eight proteins including: pre-albumin, albumin, alpha-fetoprotein (alpha-FP), alpha 1-protease inhibitor, haptoglobin, ceruloplasmin, transferrin and immunoglobulin-G, as revealed by immunoblotting. Protein patterns obtained between 9 and 13 weeks were comparable in exocoelomic fluid and in maternal serum except for the presence of alpha-FP in the alpha 1-globulin band. At the same gestational age, protein electrophoresis of amniotic fluid revealed four bands corresponding to albumin, alpha-FP, haptoglobin and transferrin. Creatinine levels were significantly lower (P less than 0.01) in amniotic fluid than in exocoelomic fluid, and alpha-FP levels were similar in both exocoelomic and amniotic fluids. These results suggest that the exocoelomic fluid is a transudate of the maternal serum except for the presence of high levels of alpha-FP, that amniotic and exocoelomic cavities are separated by a non-permeable membrane and that the secondary yolk sac plays an important role in early protein synthesis and transfer.


The use of fibrin sealant for embryo transfer: development and clinical studies.

Many embryo transfers after in-vitro fertilization may fail because of expulsion of the embryos from the uterus. Approximately 5-8% of pregnancies resulting from embryo transfer are ectopic. The aim of our study was to find a technique to avoid ectopic pregnancies and to improve the pregnancy rate. We used a two-component fibrin sealant which also contains a fibrinolysis inhibitor (aprotinin) at various concentrations. After gaining experience with mouse embryos, the sealant was used in human embryo transfer with great success. The results of a pilot study encouraged us to perform a prospective randomized study on 546 patients (270 with fibrin sealant, 276 conventional embryo transfers). There were 47 (17.0%) orthotopic pregnancies and 6 (2.2%) ectopic pregnancies in the control group, whereas there were 51 (18.9%) intrauterine and no ectopic pregnancies in the treatment group. The difference in ectopic pregnancies was statistically significant (P less than 0.05). With regard to the aprotinin concentration, there was a trend towards better results with 100-150 kIU (28.5% clinical pregnancies) in comparison to 250-300 kIU (19.2%) or no aprotinin (20.4%) (not significant). Further improvements of the technique may raise the pregnancy rate when fibrin sealant is used. As shown in our prospective randomized study, ectopic pregnancies may be completely avoided.


Immunodominant regions within the hepatitis C virus core and putative matrix proteins.

The complete amino acid sequences of hepatitis C virus (HCV) core (residues 1 to 115) and putative matrix (residues 116 to 190) proteins were synthesized as 18-residue-long peptides with an 8-amino-acid overlap. The peptides were assayed with 50 human serum samples with antibodies to HCV (anti-HCV) and 46 serum samples without anti-HCV, as determined by several commercial assays. Immunodominant regions were defined within residues 1 to 18, 11 to 28, 21 to 38, 51 to 68, and 101 to 118. The peptides that covered these regions were recognized by 40 of 50 (80%), 42 of 50 (84%), 36 of 50 (72%), 34 of 48 (68%), and 36 of 48 (72%) of the anti-HCV positive serum samples, respectively. Two anti-HCV negative serum samples were each repeatedly reactive with one peptide, but both were found to be negative by confirmatory anti-HCV assays. Four serum samples that were confirmed to be positive for anti-HCV in commercial assays did not recognize any of the peptides that cover the HCV core-matrix regions. Ninety-two percent of anti-HCV-positive serum samples reacted with a combination of peptides covering residues 1 to 18 and 11 to 28. Testing of peptides that contain the reported genotypic variations of the HCV core within the regions at residues 1 to 18, 51 to 68, and 101 to 118 showed that a change from Thr-110 to Asn-110 decreased the reactivities of eight serum samples. In conclusion, we found that human antibodies to the HCV core-matrix protein(s) are mainly directed to linear determinants and can easily be reproduced by using short synthetic peptides. We also found that such antibodies develop in more than 90% of HCV-infected people.


Rapid identification of fibronectin, vitronectin, laminin, and collagen cell surface binding proteins on coagulase-negative staphylococci by particle agglutination assays.

Seventeen strains of ten different species of coagulase-negative staphylococci were shown to interact with collagen, laminin, fibronectin, and vitronectin immobilized on latex beads. Different species of coagulase-negative staphylococci have different capacities to agglutinate proteins. Cells of 18 strains of Staphylococcus haemolyticus reacted more strongly than did cells of 18 Staphylococcus epidermidis strains with proteincoated latex beads, although no significant difference in cell surface hydrophobicity or charge could be shown. The cell surface receptors of S. haemolyticus were more heat and protease resistant than were Staphylococcus aureus receptors. Strains of Staphylococcus saprophyticus isolated from urinary tract infections showed a high capacity to adhere to laminin. The ability to agglutinate fibronectin and collagen was common among coagulase-negative staphylococci isolated from other infections; 55% (31 of 56) and 63% (35 of 56) agglutinated fibronectin and/or collagen. S. haemolyticus and S. epidermidis bound to both N-terminal (29-kDa) and C-terminal (120-kDa) fragments of fibronectin.


Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.


Molecular epidemiology of Pseudomonas cepacia determined by polymerase chain reaction ribotyping.

Traditional ribotyping detects genomic restriction fragment length polymorphisms by probing chromosomal DNA with rRNA. Although it is a powerful method for determining the molecular epidemiology of bacterial pathogens, technical difficulties limit its application. As an alternative, polymorphisms were sought in the 16S-23S spacer regions of bacterial rRNA genes by use of the polymerase chain reaction (PCR). Chromosomal DNA from isolates of Pseudomonas cepacia was used as a template in the PCR with oligonucleotide primers complementary to highly conserved sequences flanking the spacer regions of the rRNA genes. Length polymorphisms in the amplified DNA distinguished unrelated isolates of P. cepacia. Isolates of P. cepacia previously implicated in person-to-person transmission were shown to have identical amplification patterns. These data demonstrate the utility of this new PCR ribotyping method for determining the molecular epidemiology of bacterial species.


Development of a polymerase chain reaction-probe test for identification of Alloiococcus otitis.

A rapid polymerase chain reaction test was developed for specific identification of the human middle ear pathogen Alloiococcus otitis. Primers for the enzymatic amplification reaction were designed from highly specific sequences within the 16S rRNA gene. In addition, a confirmatory test based on hybridization of the polymerase chain reaction products to a specific internal probe was developed.


Cortical afferents to behaviorally defined regions of the inferior temporal and parahippocampal gyri as demonstrated by WGA-HRP.

The inferior temporal gyrus in the monkey appears to be unique among the many extrastriate visual cortices in its importance for normal performance of delayed match-to-sample, a visual memory task. However, the anatomical pathway providing visual information to this portion of the temporal lobe remains unclear. In this study, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) was injected into the anterior inferior temporal gyrus and heavy projections were found to arise in cytoarchitectural area TF of the parahippocampal gyrus, as well as moderate projections in more posterior portions of inferior temporal gyrus and perirhinal and entorhinal cortices. Subsequently, WGA-HRP was injected into area TF, resulting in retrogradely labeled cells primarily located in the portions of area TF adjacent to the injection and also in the occipitotemporal sulcus including the ventral portion of the prestriate visual area V4. Moderate projections were found to originate from the dorsal region of area V4 in the lunate sulcus, portions of the caudal parietal lobe, the posterior bank of caudal superior temporal sulcus, and area OPT located at the tip of the superior temporal sulcus. The middle temporal gyrus, foveal prestriate cortex, and area TEO, a transitional area between temporal and occipital visual areas, were all free from retrogradely labeled cells. These latter areas are included in the well-established anatomical system that is known to carry visual information from striate cortex through prestriate to eventually reach dorsal portions of inferotemporal cortex which is coincident with the temporal lobe visual area TE. It is suggested here that there is an additional ventral pathway into area TE as well, which includes projections through portions of the prestriate cortex, occipitotemporal sulcus, and parahippocampal gyrus, ultimately reaching the anterior inferior temporal gyrus, an area that may be specialized to hold visual information over brief periods of time.


Retinal growth and cell addition during embryogenesis in the teleost, Haplochromis burtoni.

Neurogenesis of the developing embryonic retina is described for the African cichlid fish, Haplochromis burtoni, from 4 days post fertilization until all cell phenotypes are generated (day 7). Cell addition and differentiation both begin at the same absolute location which later becomes the central retina. As observed in most other vertebrates, cones and ganglion cells differentiate first, followed by amacrine and bipolar cells. Rod photoreceptors, which are added late, differentiate last. Changes in retinal thickness, retinal stretching, cell size, and cell density were measured during development. From day 4 through 7, there is an increase in retinal thickness largely due to the expansion of the inner plexiform layer (IPL) and outer nuclear layer (ONL). The inner nuclear layer (INL) decreases in thickness and there is a transient decrease in the density of cells in the scleral portion of the INL. Cells increase in size in the ganglion cell layer (GCL) and the vitread INL, decrease in size in the sclerad INL, and remain the same in the ONL. Changes in the density of the cell layers were observed: the density of ONL cells increased, the density of GCL cells decreased, and INL cells increased then decreased. From day 4 to day 6, eye growth is entirely due to cell addition because no retinal stretching was observed in the ONL or the horizontal layer. During this same developmental period, the pattern and rate of neurogenesis were measured in the differentiated portion of the retina by means of 3H-thymidine labeling. A small number of cell divisions within the differentiated INL precede the onset of cell divisions in the ONL. The number of 3H-thymidine labeled cells within the INL increases at a low rate consistent with an asymmetric pattern of cell division characteristic of stem cells. In contrast, cell divisions in the ONL increase exponentially, consistent with a symmetric pattern of cell division characteristic of progenitor cells. Double-label experiments (3H-thymidine and a rod specific opsin antibody) show that some of the symmetrically dividing cells in the ONL express the rod specific opsin within 2 days, suggesting that these dividing cells are rod progenitors. Although we do not hae conclusive evidence, these developmental processes support the hypothesis that stem cells within the INL could be the source of rod precursors in the embryonic teleost retina.


NADPH diaphorase in the spinal cord of rats.

To identify spinal neurons that may synthesize nitric oxide, cells and fibers histochemically stained for NADPH diaphorase (a nitric oxide synthase) were studied in the spinal cord of rats. The histochemical reaction gave an image similar to the best Golgi impregnations, staining cells down to their finest processes. Transverse, horizontal, and parasagittal 50 and 100 microns sections were used to follow dendritic and axonal arborizations of stained neurons. Major cell groups were identified in the superficial dorsal horn and around the central canal (at all spinal levels), and in the intermediolateral cell column (at thoracic and sacral levels). Scattered positive cells were also found in deeper dorsal horn, ventral horn, and white matter. In some cases, axons of cells in the dorsal horn could be traced into the white matter; many of these cells resembled neurons projecting to various supraspinal targets. Stained cells in the intermediolateral column, which sent their axons into the ventral root, were presumed to be preganglionic autonomic neurons. Dense plexes of fibers were stained in laminae I and II and in the intermediolateral column. A large number of NADPH diaphorase-positive neurons in the spinal cord appear to be involved in visceral regulation. Fibers of the intermediolateral system had a special relationship with vasculature, suggesting that nitric oxide may help to couple neural activity with regional blood flow in the spinal cord. The abundance of NADPH diaphorase-positive neurons and fibers in the superficial dorsal horn suggests that nitric oxide may also be involved in spinal sensory processing.


Afferent projections to the mammillary complex of the rat, with special reference to those from surrounding hypothalamic regions.

To better understand the functional organization of the mammillary nuclei, we investigated the afferents to this nuclear complex in the rat with iontophoretically injected wheat germ agglutinin conjugated to horseradish peroxidase. Particular attention was paid to tracing local hypothalamic afferents to these nuclei. Injections into the medial mammillary nucleus (MMN) revealed strong projections from the subicular region, and weaker projections from the prefrontal cortex, medial septum, and the nucleus of the diagonal band of Broca. Other descending subcortical projections to the MMN arise from the anterior and the lateral hypothalamic area, the medial preoptic area, and the bed nucleus of the stria terminalis. Ascending afferents to the MMN were found to originate in the raphe and various tegmental nuclei. Following all injections into the MMN, labelled neurons were found in nuclei surrounding the mammillary body. The lateral and posterior subdivisions of the tuberomammillary nucleus projected mainly to the pars medianus and pars medialis of the MMN. The dorsal and ventral premammillary nuclei projected to the pars lateralis of the MMN. The supramammillary nucleus at rostral level had a small projection to the pars medialis and lateralis of the MMN. However, the most obvious projection from this nucleus was to the pars posterior of the MMN, chiefly from the lateral part of the caudal supramammillary nucleus. Injections into the lateral mammillary nucleus revealed inputs from the presubiculum, parasubiculum, septal region, dorsal tegmental nucleus, dorsal raphe nucleus, and periaqueductal gray. In addition, the lateral mammillary nucleus was found to receive a moderate projection from the medial part of the supramammillary nucleus and stronger projections from the lateral part of the caudal supramammillary nucleus. A very light projection was also seen from the lateral and posterior subdivisions of the tuberomammillary nucleus. These findings add to our knowledge of the extensive and complex connectivity of the mammillary nuclei. In particular, the local connections we have demonstrated with the supramammillary and tuberomammillary nuclei indicate the existence of significant local circuits as well as circuits involving more distant brain regions such as the septal nuclei, subiculum, prefrontal cortex, and brain stem tegmentum.


A dopaminergic projection to the rat mammillary nuclei demonstrated by retrograde transport of wheat germ agglutinin-horseradish peroxidase and tyrosine hydroxylase immunohistochemistry.

The presence and distribution of dopaminergic neurons and terminals in the hypothalamus of the rat were studied by tyrosine hydroxylase (TH) immunohistochemistry. Strongly labelled TH-immunoreactive neurons were seen in the dorsomedial hypothalamic nucleus, periventricular region, zona incerta, arcuate nucleus, and supramammillary nucleus. A few TH-positive neurons were also identified in the dorsal and ventral premammillary nucleus, as well as the lateral hypothalamic area. TH-immunoreactive fibres and terminals were unevenly distributed in the mammillary nuclei; small, weakly labelled terminals were scattered in the medial mammillary nucleus, while large, strongly labelled, varicose terminals were densely concentrated in the internal part of the lateral mammillary nucleus. A few dorsoventrally oriented TH-positive axon bundles were also identified in the lateral mammillary nucleus. A dopaminergic projection to the mammillary nuclei from the supramammillary nucleus and lateral hypothalamic area was identified by double labelling with retrograde transport of wheat germ agglutinin-horseradish peroxidase and TH-immunohistochemistry. The lateral mammillary nucleus receives a weak dopaminergic projection from the medial, and stronger projections from the lateral, caudal supramammillary nucleus. The double-labelled neurons in the lateral supramammillary nucleus appear to encapsulate the caudal end of the mammillary nuclei. The medial mammillary nucleus receives a very light dopaminergic projection from the caudal lateral hypothalamic area. These results suggest that the supramammillary nucleus is the principal source of the dopaminergic input to the mammillary nuclei, establishing a local TH-pathway in the mammillary complex. The supramammillary cell groups are able to modulate the limbic system through its dopaminergic input to the mammillary nuclei as well as through its extensive dopaminergic projection to the lateral septal nucleus.


Effect of clinical and subclinical mastitis on lipid composition of teat canal keratin.

Two experiments were conducted to investigate lipid composition of teat canal keratin when different conditions of bacterial colonization and quarter inflammation were present. In Experiment 1, 11 multiparous cows with subclinical mastitis (bacteria present but no visible inflammation) in at least one quarter were selected for study. Quarters that were sampled and found negative for bacterial growth were classified as control. In Experiment 2, 10 multiparous cows with clinical mastitis in one or more quarters were selected. Milk samples from inflamed quarters were cultured to identify mastitis pathogens; these quarters were classified as clinical; all other quarters were classified as control. Teat canal keratin was collected from all quarters just before an a.m. milking, samples were weighed, and lipid determinations were conducted by TLC. Keratin from subclinical quarters compared with keratin from control quarters did not differ in either neutral lipid or fatty acid composition. Total lipid was significantly higher in keratin from teats of clinical quarters than in keratin from control quarters (27.8 vs. 21.5 microgram/mg). Neutral lipid composition of keratin was similar between teats from clinical quarters and teats from control quarters. In Experiment 2, quarter foremilk samples were also obtained to determine lipid composition. The FFA in milk from clinical quarters contained fewer short-chain fatty acids, whereas polyunsaturated fatty acids were significantly higher in milk from clinical quarters.


The effect of inhaled allergen on circulating basophils in atopic asthma.

There is increasing evidence for the role of basophils in the allergen-induced late asthmatic response (LAR). To study the effect of inhaled allergen on basophil function in subjects with asthma, ex vivo basophil spontaneous histamine release (SHR) in peripheral blood and plasma histamine was measured before and 2, 5, 10, and 15 minutes, and 2, 4, 6, and 8 hours after allergen bronchial challenge (allergen study day) in six subjects with atopic asthma. Allergen inhalation induced an early response and LAR consisting of a mean (+/- SD) 32.5% (+/- 7.9%) and 28.8% (+/- 7.7%) fall in FEV1, respectively. As a control for the effects of bronchoconstriction, on another occasion, methacholine challenge was performed to produce a mean 33.4% (+/- 3.4%) fall in FEV1 during the early response and no LAR, and blood was obtained to measure basophil histamine release (HR) and plasma histamine. There was a small, but significant (p less than 0.05), rise in median SHR from 4.6% to 6.1% of total basophil histamine after allergen but not after methacholine inhalation. HR remained high after allergen inhalation during the 8 hours of study, whereas it demonstrated a steady, significant, decrease between 4 to 8 hours after methacholine inhalation. No significant changes in plasma histamine were recorded on either allergen or methacholine study days. On a third occasion, SHR was measured after challenge with physiologic saline to control for any effects of methacholine on SHR, and a decrease in HR was recorded during the day similar to HR observed after methacholine challenge. These studies suggest an enhancing effect of inhaled allergen on SHR.(ABSTRACT TRUNCATED AT 250 WORDS)


Skin-derived aeroallergen-specific T-cell clones of Th2 phenotype in patients with atopic dermatitis.

T-cell lines have been derived from aeroallergen-induced eczematous patch test sites of patients with atopic dermatitis (AD). Biopsy specimens were obtained 24 hours after allergen application to the skin, and only in vivo-activated T cells were propagated. From one patient, the T-cell lines were subcloned at 0.3 cells per well. With allergen-induced interleukin (IL) production, all clones tested (n = 13) were found to be specific for the allergen, producing IL-4 and granulocyte-macrophage-colony-stimulating factor. No IL-2 or interferon-gamma was found. The allergen-specific proliferative response of these clones, although the response was dependent on exogenous IL-2 or IL-4, also proved that all clones were allergen specific. Under optimal stimulation (aCD3 plus phorbol myristate acetate), 15% of the clones appeared to be of Th0 phenotype and 70% of Th2 phenotype. In 15% of the clones, IL-4 was produced in the absence of IL-2, IL-5, or interferon-gamma. Supernatants of all clones tested induced IgE production by B cells from normal non-atopic donors. The T-cell lines of the other patient demonstrated similar results; allergen-specific proliferation was dependent on exogenous IL-2 or IL-4 and stimulation with aCD3 plus phorbol myristate acetate demonstrated that the T cells in these lines were of the Th2 phenotype. In conclusion, our data reveal that, in AD, percutaneous sensitization to aeroallergens may occur and indicate that allergen-specific Th2 type T cells may be responsible for the high levels of (specific) IgE found in 80% of patients with AD.


Chronic Pneumocystis carinii infection of the liver. A case report and review of the literature.

Pneumocystis carinii infection of the liver is being reported with increasing frequency in patients with acquired immune deficiency syndrome (AIDS). The clinical picture typically resembles hepatitis. We report such an occurrence in a patient with persistent elevation of alkaline phosphatase and gamma-glutamyl transpeptidase with relatively normal transaminases who was found to have P. carinii on antemortem liver biopsy. The differential diagnosis of abnormal alkaline phosphatase and gamma-glutamyl transpeptidase in patients with AIDS should include P. carinii.


Anti-HCV positive hepatocellular carcinoma in cirrhosis. Prevalence, risk factors and clinical features.

Recent reports indicate that hepatitis C virus (HCV) may play a role in the pathogenesis of hepatocellular carcinoma in cirrhotics. Using an ELISA test, we evaluated the prevalence of anti-HCV antibodies in 97 patients with hepatocellular carcinoma (HCC) in cirrhosis and in a group of 223 patients, including: 49 patients with HBsAg-positive chronic liver disease (CLD), 42 with alcoholic CLD, 110 with cryptogenic CLD and 22 with post-transfusional HBsAg-negative CLD. All diagnoses were histologically confirmed. Overall, anti-HCV-positive HCC were 64% of the total, with no statistically significant difference with respect to CLD (60.9%). The prevalence of anti-HCV was higher in cryptogenic HCC (80%) than in HBsAg-positive (60%) or alcoholic HCC (42.8%) (p less than 0.005). When HCC and cirrhosis of similar putative etiology were considered, anti-HCV prevalence was significantly higher in HCC than in cirrhosis only in the groups of patients with alcoholic liver damage (60% in HCC vs. 38% in cirrhosis, p less than 0.005). In HBsAg-positive patients, anti-HCV prevalence was twice as high in HCC than in CLD, but the difference was not statistically significant. Overall, anti-HCV prevalence in HCC was significantly higher than in alcoholic or HBsAg-positive CLD (p less than 0.001 and p less than 0.01, respectively) but lower than in cryptogenic CLD (p less than 0.001). Association between anti-HCV and anti-HBc was significantly more prevalent in patients with CLD than in those with HCC.(ABSTRACT TRUNCATED AT 250 WORDS)


Cytokeratin of apparent high molecular weight in livers from griseofulvin-fed mice.

Livers from mice fed griseofulvin (GF) for 12 months contain abnormal cytokeratin (CK) intermediate filaments (IFs) that aggregate to form Mallory bodies (MBs). Sections from control and GF-treated livers were extracted with Triton X-100. IF-enriched fractions were analyzed by gel electrophoresis and IF proteins were identified by Western blotting using anti-CK 55 (TROMA 1) and CK 49 (TROMA 2), and the anti-'universal' IF known as aIF. Western blotting on control and GF-treated livers with TROMA 1 and TROMA 2 obtained with gels loaded with 2 micrograms of protein revealed the presence of CK 55 and CK 49. In contrast, when a larger amount of protein was loaded on the gel (10 or 12 micrograms) the results were different. In the control we detected bands corresponding to CK 55 and CK 49 plus a faint band at approx. 97 kDa. In the GF-treated animals, we detected a more pronounced reaction with the 97 kDa band and in addition we now detected several higher molecular weight bands which reacted with anti-CK antibodies. These results demonstrated that the induced changes in CKs in the GF liver are associated with the increase of apparent high molecular weight CK. These apparent high molecular weight CKs could be obtained by a pathological cross-linking of the preexisting liver CK monomers in different combinations such as CK 55-55, CK 49-49 and/or with other proteins.


Increased transforming growth factor-beta 1 gene expression in human liver disease.

We recently demonstrated that transforming growth factor-beta 1 stimulates collagen synthesis in hepatic cells in vitro, and that the synthesis of this cytokine is markedly increased in two rodent models of hepatic fibrosis. In the present study, we investigated the association of transforming growth factor-beta 1 (TFG-beta 1) gene expression in human liver disease. Sixteen patients with active liver disease had percutaneous liver biopsies performed for diagnostic purposes. Total RNA was extracted from an unused portion of each biopsy and then subjected to hybridization analysis with the following human cDNA clones: albumin, pro alpha 1 (I) collagen, and TGF-beta 1. Surgical liver biopsy specimens from two patients without hepatic disease were used as controls. When compared to controls, the patients with active liver disease had a 19% decrease in albumin, a 97% increase in type I collagen, and a 120% increase in transforming growth factor-beta 1 mRNA levels. Moreover, steady-state levels of TGF-beta 1 and procollagen mRNAs were significantly correlated. Nuclear run-on assays showed that livers from two patients with fibrosis had TGF-beta 1 transcription rates that were more than 2-fold higher than rates in control livers. These findings indicate that transforming growth factor-beta 1 gene expression is significantly enhanced in man during active liver disease.


Paracentesis with Dextran 70 vs. paracentesis with albumin in cirrhosis with tense ascites. Results of a randomized study.

Forty-one patients with cirrhosis and tense ascites were randomized to receive daily paracentesis of 5 liters associated with Dextran 70 as volume expander (6 g for each 1000 ml of ascites removed) (group I = 20 patients) or paracentesis with albumin (6 g for each 1000 ml of ascites) (group II = 21 patients). The basal clinical features, laboratory data, and plasma renin activity were similar in both groups. The volume of ascites removed was 12.9 +/- 4.4 and 10.9 +/- 3.7 liters in group I and II, respectively (n.s.). No significant changes were observed in liver and renal function tests, KPTT, platelet count, factor VIII, serum electrolytes or plasma renin activity 24 and 96 h after the last paracentesis in both groups, except for a decrease in bilirubin in group I and a transient increase of serum albumin in group II. Four patients developed complications in each group, mainly hyponatremia, while one patient in each group developed renal impairment. One patient from group I died with hepatic encephalopathy. Moreover, the probability of survival and readmission to the hospital because of tense ascites were similar in both groups of patients during the follow-up. The treatment cost with Dextran 70 was 15.50 dollars vs. 364.30 dollars with albumin for each patient treated. These results indicate that repeated large volume paracentesis associated with Dextran 70 is as effective and safe as paracentesis associated with albumin in cirrhotic patients with tense ascites. However, due to its reduced cost, paracentesis with Dextran 70 may be considered the treatment of choice in cirrhotic patients with tense ascites without liver cancer and renal failure.


Serological response and detection of viraemia in acute hepatitis C virus infection.

The serological response during acute hepatitis C virus (HCV) infection was examined by enzyme-linked immunosorbent assay (ELISA) in sequential serum samples from 13 haemophiliacs following their first exposure to factor VIII concentrates contaminated with HCV. The commercially available C100-3 peptide and a new 22 kDa recombinant protein (p22) encoded by the nucleocapsid region of the viral genome were used for antibody detection, whilst a nested polymerase chain reaction (PCR) method was used for the detection of viraemia. In addition, eight sporadic cases of acute HCV infection were studied. The results in haemophiliacs demonstrated that seroconversion to the C100-3 antigen occurred in only one-third of the patients within 12 weeks of disease onset, but all of the patients had a diagnostic serological response to p22 during this phase of the disease. The new test was positive in all the sporadic cases at a time when the commercially available test was negative. Although PCR offers a sensitive method for the detection of recent HCV infection, the complex methodology makes it unsuitable for diagnostic laboratories. The new ELISA test with p22 may therefore have a useful diagnostic role in acute disease.


c-erbB-2 oncogene expression in hepatocellular carcinoma and cholangiocarcinoma.

The c-erbB-2 proto-oncogene encodes a transmembrane protein which is homologous to the epidermal growth factor receptor. This protein can be localized immunohistochemically in formalin-fixed paraffin-embedded material using a monoclonal antibody NCL-CB11; positive membrane staining correlates with gene amplification and protein overexpression in breast cancer. Using this technique we have shown that only 2/26 (8%) of hepatocellular carcinomas, 0/10 (0%) of cholangiocarcinomas and 0/2 (0%) hepatoblastomas overexpressed c-erbB-2 as evidenced by membrane staining. Moreover c-erbB-2 mRNA was not detected in seven hepatocellular carcinomas examined by Northern blot analysis. c-erbB-2 overexpression is, therefore, unlikely to be contributing to the malignant phenotype in hepatocellular carcinoma and cholangiocarcinoma.


Hepatitis C viremia and anti-HCV antibodies in alcoholics.

We determined serum hepatitis C status using a RIBA2 kit and a sensitive PCR procedure in 62 chronic alcoholics, 36 of whom had anti-HCV antibodies (Ab) detectable in an ELISA1 assay. Anti-HCV antibodies were detected in 22 patients using RIBA2. HCV RNA was detected by means of PCR in 18 patients who were RIBA2 positive and in none who were RIBA2 negative. Liver biopsies, available for 12 HCV RNA-positive patients, revealed histological features of purely alcohol-related lesions in seven and mixed alcohol-viral lesions in five. These results indicate that HCV replication is maintained in most alcoholics who score positive for anti-HCV Ab in the RIBA2 test, and that HCV viremia can be associated with histological features typical of alcoholic liver disease.


Involvement of CD28 in MHC-unrestricted cytotoxicity mediated by a human natural killer leukemia cell line.

NK cells and certain CTL can recognize and lyse targets without restriction by the MHC. NK cells do not express CD3/TCR complexes and the membrane receptors participating in MHC-unrestricted cytotoxicity are largely unknown. We demonstrate that YT2C2, a human NK leukemia cell line, expresses the CD28 differentiation Ag and can spontaneously lyse both murine and human cell lines expressing B7, a B cell- activation Ag that is a ligand for CD28. The participation of CD28/B7 interactions in MHC-unrestricted cytotoxicity mediated by YT2C2 cells was demonstrated by correlation of target sensitivity with levels of B7 expression, inhibition of cytotoxicity by anti-CD28 or anti-B7 mAb, and by making both murine and human cell lines susceptible to YT2C2-mediated lysis by genetic transfection with expression vectors containing B7 cDNA. However, CD28/B7 interactions alone were insufficient to initiate cytotoxicity. mAb inhibition experiments and selection of CD54- (intercellular adhesion molecule-1) deficient B cell targets indicated that CD11a/18 (lymphocyte function-associated Ag-1) also cooperated in CD28/B7-dependent cytotoxicity. The requirement for both CD28/B7 and lymphocyte function-associated Ag-1/intercellular adhesion molecule-1 interactions in YT2C2-mediated MHC-unrestricted cytotoxicity was confirmed by demonstrating that efficient lysis of murine L cells required cotransfection with both B7 and intercellular adhesion molecule-1. These findings support the concept that MHC-unrestricted cytotoxicity may not be due to a unique receptor, but may result from interactions between an appropriate array of "adhesion" molecules with their ligands.


Differential function of intestinal intraepithelial lymphocyte subsets.

It has been proposed that intestinal intraepithelial lymphocytes (I-IEL) perform immune surveillance of the epithelial layer (1) and regulate mucosal humoral responses to exogenous Ag (2). To better understand the functional potential of this unique population, purified murine I-IEL were analyzed phenotypically and functionally. Initial studies determined that I-IEL could be distinguished based on several phenotypic characteristics including: TCR (TCR-alpha beta vs TCR-gamma delta); Thy-1, CD45R/B220, CD5, and CD8 (CD8 alpha alpha vs CD8 alpha beta) expression. Using anti-TCR mAb, individual I-IEL subsets were activated and examined functionally. Both TCR-alpha beta and TCR-gamma delta I-IEL were found to synthesize an array of lymphokines that included IL-2, IL-3, and IL-6 but not IL-4 or IL-5. Additionally, a number of lymphokines were detected that directly influence epithelial function (IFN-gamma, TNF-alpha, and TGF-beta 1). However, the majority of the I-IEL function was localized within the Thy-1+, CD45R/B220- I-IEL subset. In addition those TCR-alpha beta I-IEL expressing the CD8 alpha beta heterodimer were more easily activated. Thus, a subset of I-IEL have the capacity to respond to TCR-mediated stimuli. The functional activities of these cells may influence both local immune cell populations as well as epithelial differentiation.


Restoration of T cell receptor-mediated signal transduction by transfection of CD45 cDNA into a CD45-deficient variant of the Jurkat T cell line.

The TCR is a multimeric structure comprised of distinct Ag recognition and signal transduction components. Although none of the molecules that make up the TCR possess intrinsic protein tyrosine kinase (PTK) activity, stimulation of T cells via the TCR results in the rapid appearance of newly tyrosine phosphorylated proteins in cell lysates. Evidence suggests ligation of the TCR induces activation of a PTK that may be a member of the src family. One early consequence of this TCR-mediated PTK activation is the phosphorylation of the gamma 1 isoform of phospholipase C. This phosphorylation event is associated with increased enzymatic activity resulting in the hydrolysis of phosphatidylinositol 4,5 bisphosphate into two second messengers, inositol 1,4,5 trisphosphate and diacylglycerol. Recently, our laboratory and others have isolated mutant T cells that lack surface expression of CD45, the major surface tyrosine phosphatase expressed on lymphoid cells. Stimulation of the TCR on these cells fails to result in the expected activation events. We demonstrate that reconstitution of surface expression of the 180-kDa isoform of CD45 by gene transfer into a CD45-deficient mutant of the Jurkat T cell leukemic line restores the ability of the TCR to couple fully to its signal transduction machinery. These results support the role of CD45 tyrosine phosphatase activity in regulating the TCR-activated PTK.


Identification of subsets of human T cells capable of enhanced transendothelial migration.

A critical step in immunologically mediated inflammation is the migration of T cells between endothelial cells of postcapillary venules and into the tissues. To determine whether specific cells are capable of transendothelial migration, T cells that had migrated through endothelial monolayers were retrieved and analyzed. To accomplish this, human umbilical vein endothelial cells (EC) were cultured to confluence on collagen gels and incubated with human T cells. T cells that were nonadherent to the EC, those that bound to the endothelium, and cells that had migrated through the endothelial monolayer and into the collagen were individually harvested and characterized. After a 4-h incubation with EC, T cells distributed themselves such that 77 +/- 2% were nonadherent, 13 +/- 2% were bound to EC, and 10 +/- 1% had migrated into the collagen. The CD4+ T cells that had migrated into the collagen were predominantly CD29bright/CD45RObright and CD45RA-. CD8+ T cells demonstrated a greater transendothelial migratory capacity than the CD4+ T cells. The migrated CD8+ T cells were mainly CD29bright but CD45RA+. Additional phenotypic analysis of the migrating cells indicated that they contained fewer cells that expressed L-selectin. Moreover the surface expression of CD7 was less dense in the T cells that had migrated than in the nonadherent T cells. Finally the T cells that migrated were not enriched for CD45RBdim T cells. Prolonging the incubation with EC to 36 h increased the number of T cells that migrated but did not alter the predominance of CD29bright T cells in the migrated population. Stimulation of EC with IL-1 or IFN-gamma also increased the number of adherent and migrating T cells, respectively, but did not alter the phenotype of the migrating cells. These results indicate that the capacity for transendothelial migration is an intrinsic ability of certain subpopulations of T cells and is related to their stage of differentiation as identified by their surface phenotype.


Receptor-mediated activation of human B lymphocytes in a nonphosphotyrosine-dependent manner.

The B cell AgR regulates two signal transduction pathways: the tyrosine kinase and the phosphatidylinositol (PtdIns) pathways. Stimulation of B cells with Ag or anti-Ig antibody results in a rapid increase in tyrosine phosphorylation of multiple substrates. The AgR also mediates the activation of phospholipase C-gamma 1 (PLC-gamma 1) thus producing the second messengers, inositol trisphosphate and diacylglycerol. Although the detailed relationship between these two signaling pathways remains unclear, it has recently become apparent that PLC-gamma 1 might be a target for the AgR-associated protein tyrosine kinase. To address the question of whether tyrosine kinase activity is essential for B cell activation, we studied early biochemical changes and later cellular events induced by ligation of the purinoceptor (P2R). Ligation of ATP to its receptor on B cells has been previously shown to elicit increases in cytosolic free Ca2+ and inositol phosphate production as well as induction of c-fos mRNA expression and increased expression of IL-2 and transferrin receptors. We show here that ATP in a wide range of concentrations did not increase protein tyrosine kinase activity. In contrast with the AgR, P2R did not mediate tyrosine phosphorylation of PLC-gamma 1, thus suggesting that it may use another phosphoinositide-specific PLC that does not require phosphorylation on tyrosine residues for its activation. The results were supported by experiments with a specific tyrosine kinase inhibitor, tyrphostin AG-126. Preincubation with this inhibitor blocked AgR but not P2R-mediated inositol phosphate production, cytosolic free Ca2+ changes, and IL-2 and transferrin receptor expression. The results indicate that the PtdIns pathway may be sufficient to induce activation of B cells and that the tyrosine phosphorylation pathway is not necessary for nonantigenic B cell activation.


Molecular cloning and characterization of the gene encoding mouse melanoma antigen by cDNA library transfection.

We have isolated a cDNA (H52) of 2.8-kb-long encoding an 80-kDa mouse melanoma Ag that is defined by a syngeneic anti-B16 melanoma mAb with an ability to block anti-melanoma cytotoxic T cell responses. H52 transfectants were brightly stained with the antibody, and the 80-kDa molecule was immunoprecipitated from the transfectants. Northern blot analysis showed that this transcript was detected in mouse melanoma cells of C57BL/6 and DBA/2 origin, C1300 A/J neuroblastoma, L cell (C3H) and EL-4 T lymphoma (C57BL/6), faintly in BW5147 (AKR) T lymphoma, but not in other tumors, such as S913 fibrosarcoma (C57BL/10), NIH3T3, 70 Z/3 pre-B lymphoma, and P3U1 plasmacytoma (BALB/c). Since the transcripts were not found in normal C57BL/6 tissues of fetus, newborn, and adult origin, the H52 expression is associated with transforming phenotypes. However, no tissue- or cell type-specific expression was observed. Nucleotide sequence analysis has clearly demonstrated that H52 cDNA encodes the full length of the env gene and long terminal repeat region of endogenous ecotropic murine leukemia provirus of AKV-type, which is defective in C57BL/6. The H52 envelope protein has several amino acid changes compared to those of AKV, one of which is in the env 14 peptide region preferentially associated with MHC molecule, suggesting the possible reason for the difference of antibody reactivity even in H52-positive tumors. We also demonstrate that CTL against H52 transfectant kills B16 melanoma. Thus, the above results are direct evidence that even the endogenous self molecule, when constitutively expressed, does act as a tumor Ag.


Oncostatin M is a differentiation factor for myeloid leukemia cells.

Oncostatin M (OSM) is a 28-kDa glycoprotein produced by stimulated macrophages and T lymphocytes that inhibits the proliferation of a number of different cell lines derived from solid tumors. Analysis of both amino acid sequence and gene structure has demonstrated that OSM is a member of a cytokine family that includes leukemia inhibitory factor (LIF), IL-6, and granulocyte colony-stimulating factor (G-CSF). We demonstrate that, like LIF, IL-6 and G-CSF, OSM can induce the differentiation of the myeloblastic M1 murine leukemia cells into macrophage-like cells. The morphologic and functional changes induced by OSM are more similar to those observed with LIF and IL-6 than those induced with G-CSF. OSM can also induce the differentiation of the histiocytic U937 human leukemia cells in the presence of granulocyte-macrophage CSF, a property shared with LIF and IL-6. In murine M1 cells, binding of labeled OSM is completely inhibited by excess LIF or OSM, reflecting the binding of OSM to the high affinity form of the murine LIF receptor. In contrast, the binding of labeled OSM to human U937 leukemia cells is inhibited by OSM, but the inhibition by LIF is significantly less. These results suggest that, in human leukemia cells, OSM may act through the LIF receptor and an OSM-specific receptor. The existence of an OSM-specific receptor was confirmed by both growth inhibition and competition binding assays on A375 human melanoma cells. The growth of human A375 cells was inhibited by OSM and IL-6 but not LIF or G-CSF. Neither LIF, G-CSF, nor IL-6 could compete with the binding of labeled OSM to A375 cells.


Components of the protein kinase C pathway induce Ia expression after injection into macrophages.

Macrophages are activated by a variety of microbial and cytokine stimuli. One feature of activation is the induction of class II Ag (Ia) on the cell surface. To understand the intracellular events that occur during activation, we investigated various agents with intracellular activities, and examined their effects on the induction of Ia. We first noted that several agents that activate protein kinase C (PKC) induced Ia, and that several inhibitors of PKC inhibited Ia induction by IFN-gamma. To directly test whether PKC induced Ia, we microinjected normal peritoneal macrophages with this enzyme and other intracellular mediators, then examined Ia expression. We observed that injection of PKC itself, or of other intracellular proteins thought to participate in the PKC pathway (Ras or phospholipase C gamma) strongly induced Ia expression. The Ia-inducing activity of transforming Ras protein was blocked by kinase inhibitor treatment of cells, suggesting that Ras signal transduction requires kinase activity. On the other hand, components of the protein kinase A pathway (phospholipase A2 and protein kinase A itself) did not induce Ia. Thus, the PKC pathway can control expression of macrophage surface Ia, possibly by regulating the genes of the MHC, and may play many other roles in the activation of macrophages.


Immunization with soluble protein-pulsed spleen cells induces class I-restricted cytotoxic T lymphocytes that recognize immunodominant epitopic peptides from Plasmodium falciparum and HIV-1.

CTL lines specific for two different proteins derived from the human pathogens, Plasmodium falciparum (malaria) circumsporozoite protein and HIV-1 reverse transcriptase, were obtained by immunizing mice with protein-pulsed syngeneic spleen cells. The lysis of the target cells was dependent on a class I MHC molecule and the accessory molecule CD8. Immunodominant epitopic peptides were identified previously in the two proteins using murine CTL derived after immunization with recombinant virus or sporozoites, or using CTL from HIV-1-infected patients. These peptides were also recognized by the CTL lines obtained after protein-pulsed spleen-cell immunization. A new CTL antigenic determinant was localized in HIV-1 reverse transcriptase to residues 514 to 528, a sequence that, if folded as an alpha-helix, would be strongly amphipathic. The determinant was tentatively narrowed, using overlapping peptides, to a core of at least nine residues, 515 to 523. This site was also recognized by CTL obtained by the two different methods of immunization. Therefore, extracellular proteins incubated with spleen cells can be processed and presented in vivo in the same way as intracellular proteins, resulting in recognition of the same epitopes in association with the same class I MHC molecules. The potential implications for vaccine development and for the understanding of class I-restricted Ag presentation are discussed.


A recombinant malaria protein that can induce Th1 and CD8+ T cell responses without antibody formation.

Inbred strains of mice were immunized with p190-3, a 38-kDa recombinant protein derived from p190, a major merozoite surface Ag of the malaria parasite Plasmodium falciparum. Ag-specific proliferative T cell responses were obtained in H-2b, H-2d, and H-2k mouse strains. Surprisingly, mice of the H-2b haplotype (e.g., C57BL/6) did not give a measurable antibody response to the recombinant protein administered in Freund's adjuvant, but CD8+/CD4- as well as CD4+/CD8- T cells specific for p190-3 could be obtained after in vivo priming and in vitro selection with Ag. Distinct epitopes of p190-3 recognized by the CD8+ and CD4+ T cells from C57BL/6 mice were identified. The CD8+ T cells could kill H-2b APC in the presence of the appropriate epitope-containing peptide. The p190-3-specific CD4+ cells isolated from C57BL/6 mice were of the Th1 type. In contrast, Th2 cells, but no CD8+ T cells were present in a p190-3-specific line from BALB/c mice, which give good antibody responses to p190-3.