Helicobacter pylori with a truncated lipopolysaccharide O chain fails to induce gastritis in SCID mice injected with splenocytes from wild-type C57BL/6J mice.

The goal of this study was to determine whether Helicobacter pylori lipopolysaccharide (LPS) O-chain polysaccharide contributes to gastritis in a mouse model. C57BL/6J or C57BL/6-Prkdc(scid) (severe combined immunodeficient [SCID]) mice were inoculated with H. pylori strain SS1 or SS1::0826kan, in which a beta-1,4-galactosyltransferase (HP0826), an LPS biosynthetic enzyme, had been disrupted. H. pylori strain SS1::0826kan expresses truncated LPS lacking O chain. Recipient SCID mice were given C57BL/6J splenocytes by intraperitoneal injection. Bacterial colonization, gastric lesions (gastritis, neutrophilic infiltration, and gastric epithelial metaplasia), cellular (delayed-type hypersensitivity) and humoral immune responses to H. pylori sonicate, and gastric gamma interferon (IFN-gamma) mRNA expression were quantified. Recipient SCID mice colonized by H. pylori strain SS1 developed extensive gastritis with loss of normal fundic gland morphology. In contrast, gastric mucosa of recipient SCID mice colonized by H. pylori strain SS1::0826kan was not statistically distinguishable from that of uninfected recipient mice. Delayed-type hypersensitivity and humoral immune responses were detected in infected mice inoculated with wild-type SS1, but not with SS1::0826kan. IFN-gamma transcription was lower in mice infected with SS1::0826kan than in mice infected with SS1. In this model of rapidly progressive gastritis due to H. pylori, the O chain contributed to the extent of gastritis and to the host immune response. These data support a role for H. pylori LPS O chain in direct induction of the host immune response leading to gastritis and gastric damage and are in contrast to protein antigens, such as urease and cag products which do not contribute to gastritis in mice.

California residential air exchange rates and residence volumes.

UNLABELLED: Air exchange rate data from two residential indoor air quality studies are presented. In the first investigation, over 500 residences in Southern California were sampled for three one-week periods from 1984 to 1985. Those data provided seasonal information for a broad range of residential characteristics in a large metropolitan area. In the second study, a probability sample of nearly 300 residences were sampled for a two-day period during the winter of 1991-1992 throughout the state of California. Air exchange rate is summarized by season, geographic area, and appliance type. Residence volumes are presented by cooking and heating appliance. The data approximately followed lognormal distributions. IMPLICATIONS: Indoor air quality and human exposure models often require estimates of air exchange rate and residence volumes. Application of those models to California residences can be improved by using the data distributions provided in this manuscript. Data distributions presented for heating and cooking appliances are useful for modeling the impact of indoor sources specific for those appliance types. Measured air exchange rate is also useful for modeling energy use for heating and cooling in residences.

Isolation of tetranucleotide repeat polymorphisms flanking the BRCA1 gene.

Large pools of cosmids from the BRCA1 region of human chromosome 17 were screened for tetranucleotide repeat polymorphisms by hybridizing shotgun subcloned pools with a mixture of 25 oligonucleotides. Identified subclones were PCR amplified and directly sequenced to design PCR primers for short tandem repeat polymorphism (STRP) analysis of family DNAs. With the identification of the BRCA1 gene and the observation that most mutations in this > 100-kb gene are unique, haplotyping and linkage analysis may play a significant role in diagnosis and carrier detection of BRCA1-associated breast and ovarian cancers. We report the characterization of 15 new STRPs flanking the BRCA1 locus.

Cytogenetic and molecular analysis of inv dup(15) chromosomes observed in two patients with autistic disorder and mental retardation.

A variety of distinct phenotypes has been associated with supernumerary inv dup(15) chromosomes. Although different cytogenetic rearrangements have been associated with distinguishable clinical syndromes, precise genotype-phenotype correlations have not been determined. However, the availability of chromosome 15 DNA markers provides a means to characterize inv dup(15) chromosomes in detail to facilitate the determination of specific genotype-phenotype associations. We describe 2 patients with an autistic disorder, mental retardation, developmental delay, seizures, and supernumerary inv dup(15) chromosomes. Conventional and molecular cytogenetic studies confirmed the chromosomal origin of the supernumerary chromosomes and showed that the duplicated region extended to at least band 15q13. An analysis of chromosome 15 microsatellite CA polymorphisms suggested a maternal origin of the inv dup(15) chromosomes and biparental inheritance of the two intact chromosome 15 homologs. The results of this study add to the existing literature which suggests that the clinical phenotype of patients with a supernumerary inv dup(15) chromosome is determined not only by the extent of the duplicated region, but by the dosage of genes located within band 15q13 and the origin of the normal chromosomes 15.

Experimental autoimmune encephalomyelitis is exacerbated by IL-1 alpha and suppressed by soluble IL-1 receptor.

To assess the role of IL-1 in the development of experimental autoimmune encephalomyelitis (EAE), the effects of in vivo treatment with IL-1 alpha or an IL-1 antagonist on the clinical course of EAE were evaluated. First, Lewis rats were immunized with guinea pig myelin in CFA and treated for 19 consecutive days with i.p. injections of recombinant human IL-1 alpha. Clinical signs of paralysis in the IL-1 alpha-treated groups were of longer duration and of greater severity compared to placebo injected controls. In addition, more weight loss was observed in the IL-1 alpha-treated groups compared to controls. This enhanced weight loss was not due to IL-1 alpha injections alone as CFA-treated rats injected with IL-1 alpha did not lose weight when compared to placebo injected, CFA-treated controls. Second, soluble mouse rIL-1R (sIL-1R), which binds both IL-1 alpha and IL-1 beta, was given as an IL-1 antagonist. Treatment of guinea pig myelin/CFA immunized rats with sIL-1R for 13 consecutive days significantly delayed the onset of EAE, reduced the severity of paralysis and weight loss, and shortened the duration of disease. Treatment with sIL-1R was most effective in reducing EAE if administered for 15 consecutive days immediately after immunization. Shortened 5-day treatment regimens spanning days 1 to 5, days 6 to 10, or days 11 to 15 after immunization were less effective in reducing EAE. These data suggest that IL-1 may initiate or promote inflammation within the central nervous system. In addition, specifically blocking the biological activity of IL-1 in vivo by soluble receptors may prove beneficial for the treatment of autoimmune or inflammatory diseases.

Bovine recombinant granulocyte-macrophage colony-stimulating factor enhancement of bovine neutrophil functions in vitro.

Neutrophils were purified from blood of dexamethasone-treated (0.04 mg/kg of body weight) and untreated calves. Cells were untreated (controls) or cultured in media containing 5 or 10 ng of bovine recombinant granulocyte-macrophage colony-stimulating factor (rbGM-CSF)/ml for 10 to 12 hours before being tested for various functions. Dexamethasone treatment of calves decreased luminol-dependent chemiluminescence, decreased phagocytosis of Pasteurella multocida and several Staphylococcus spp by various degrees, and decreased antibody-dependent cell-mediated cytotoxicity against bovine herpesvirus-infected cells by 26 to 32%. The percentage phagocytosis of coagulase-positive S aureus and S intermedius was higher than that of coagulase-negative S epidermidis for neutrophils from all calves. Culture of neutrophils with rbGM-CSF significantly increased (P less than 0.05) all of the aforementioned functions, compared with control neutrophils; however, rbGM-CSF-induced increases in function tended to be higher in neutrophils from dexamethasone-treated calves than in neutrophils from untreated calves.

Selective alteration of bovine neutrophil responses by recombinant bovine interleukin-1 beta.

The effects of recombinant bovine interleukin-1 beta (rBIL-1 beta) upon in vitro bovine neutrophil functions were determined. Exposure of peripheral blood neutrophils to various concentrations of rBIL-1 beta induced dose dependent suppression of the phagocyte's ability to migrate under agarose. Preincubation of neutrophils with rBIL-1 beta did not influence their ability to ingest radiolabelled Staphylococcus aureus nor did it induce hydrogen peroxide production or elastase release. However, pretreatment of phagocytes with rBIL-1 beta did result in a dose-dependent enhancement of opsonized zymosan-induced H2O2 production. In contrast, rBIL-1 beta had no effect upon the ability of opsonized zymosan-stimulated neutrophils to release elastase from primary granules. Pretreatment of neutrophils with rBIL-1 beta for as little as 15 min was sufficient to induce suppression of migration and enhancement of opsonized zymosan-induced H2O2 production. These results suggest rBIL-1 beta is capable of directly modulating selected neutrophil activities. In addition, rBIL-1 beta appears to augment the phagocyte's oxidative metabolic responses to subsequent stimulation by microbial antigens.

Monoclonal antibodies block murine IL-4 receptor function.

IL-4 is a cytokine which can induce B-lymphocyte proliferation, increase cell-surface Ia expression, and induce some activated B cells to differentiate and begin to secrete IgE. IL-4 binds specifically to a cell-surface receptor (IL-4R) on cells from a variety of lineages including T and B cells. In general both primary cells and in vitro cell lines express less than 5000 receptors per cell. Utilizing a subclone of the cytotoxic T cell line CTLL-2 expressing a high level of IL-4R, mAb against the murine IL-4R were prepared. Two mAb have been identified which have different properties. These antibodies, designated M1 and M2, recognize sequences specific to the murine IL-4R. Immunoprecipitation studies with M1 and M2 on CTLL-2 cells have identified the receptor as a Mr = 145,000 cell-surface protein. Similar results have been obtained with the recently isolated full length murine IL-4R cDNA expressed in COS-7 cells. In addition the antibodies are capable of inhibiting IL-4 binding. One antibody, M1, is also a potent inhibitor of IL-4-induced proliferation. These antibodies will be useful in dissecting a wide array of activities attributed to IL-4.

Functional studies of bovine alveolar neutrophils elicited with recombinant bovine IL-1 beta.

Bovine rIL-1 beta (rbIL-1 beta) was instilled intrabronchially into the lungs of steers to elicit harvestable alveolar neutrophils for functional analysis. Before instillation, bronchoalveolar lavage samples from the steers consisted of 96.4 +/- 1.5% (mean +/- SEM) macrophages, with the remaining cells neutrophils and occasional lymphocytes. Four hours after instillation of 1.0 and 10.0 nmol of IL-1, the lavage samples consisted of 96.3 +/- 0.8% and 91.0 +/- 5.7% neutrophils, respectively. Alveolar neutrophils elicited with rbIL-1 beta and challenged with the calcium ionophore, A23187, released similar amounts of leukotriene B4 (LTB4) and its nonenzymatic isomer LTB I, and significantly greater amounts of 5-hydroxyeicosatetraenoic acid and the nonenzymatic isomer LTB II, when compared with circulating neutrophils. The rbIL-1 beta did not, by itself, stimulate release of arachidonate metabolites from circulating neutrophils in quantities that were detectable by HPLC. Circulating neutrophils, preincubated with rbIL-1 beta and stimulated with A23187, released significantly greater amounts of 5-hydroxyeicosatetraenoic acid and total 5-lipoxygenase metabolites when compared with control cells not incubated with rbIL-1 beta. Incubation of circulating neutrophils with rbIL-1 beta and A23187 concurrently resulted in a significantly increased release of all 5-lipoxygenase metabolites of arachidonate. However, both the release of superoxide anion and bacterial killing by rbIL-1 beta-elicited bovine alveolar neutrophils did not differ from the values obtained for circulating neutrophils.

Adjuvanticity of recombinant bovine interleukin-1 beta: influence on immunity, infection, and latency in a bovine herpesvirus-1 infection.

Recombinant bovine interleukin-1 beta (rBoIL-1 beta) was administered to calves in conjunction with a bovine herpesvirus-1 (BHV-1) vaccine. All calves were immunized against BHV-1 and three groups received rBoIL-1 beta at 33, 100, or 330 ng/kg on days 1 and 15; control animals received physiological saline. All calves were challenged with BHV-1 on day 22. Total leukocytes were increased by rBoIL-1 beta, primarily by causing neutrophilia and monocytosis; CD4/CD8 ratios tended to be increased in rBoIL-1 beta-treated animals. Serum neutralizing antibody titers and cytotoxic responses against BHV-1-infected bovine kidney fibroblasts were increased and virus excretion was decreased in rBoIL-1 beta-treated calves. On days 58 and 59, control and 100 ng/kg rBoIL-1 beta-treated calves were injected with dexamethasone (.04 mg/kg). Virus excretion was less and clinical signs of BHV-1 infection were lower in rBoIL-1 beta-treated calves after dexamethasone injection. These data suggest that rBoIL-1 beta may be an effective adjuvant to BHV-1 immunization.

Bovine recombinant interleukin-2 augments immunity and resistance to bovine herpesvirus infection.

The in vivo administration of bovine recombinant interleukin-2 (rIL-2) was evaluated in calves vaccinated and then challenged with bovine herpesvirus-1 (BHV-1). In Experiment 1, 24 calves were allotted to four groups: control; bovine rIL-2; BHV-1 vaccine (modified-live); and bovine rIL-2 + BHV-1 vaccine. Serum neutralizing antibody titers to BHV-1 were increased sixfold, and virus shedding was fourfold less in calves vaccinated and treated with rIL-2 (25 micrograms/kg, intramuscularly) when compared to calves that received vaccine only. Treatment with rIL-2 induced lymphokine-activated killer activity that was eliminated by pretreating effector cells with complement and a monoclonal antibody (B26A) specific for the sheep red blood cell receptor. The rIL-2 treatment in BHV-1-vaccinated calves increased the calves' ability to withstand a BHV-1 challenge. However, during treatment with rIL-2, calves developed diarrhea and mild fever that abated after IL-2 treatment was stopped. A second experiment was then conducted to determine a dose of rIL-2 that would enhance immunity to BHV-1 without causing adverse side effects. Twenty-five calves were allotted to five groups that received injections of rIL-2 at 0.0, 25.0, 2.5, 0.25, or 0.025 micrograms kg-1 day-1 for 5 days. All calves received a modified-live BHV-1 vaccine. Calves treated with 25.0 micrograms kg-1 day-1 showed similar adverse side effects as in the first experiment but all other calves were normal. Compared to control calves, those treated with 25.0, 2.5, and 0.25 micrograms kg-1 day-1 of rIL-2 had higher (P less than 0.05) serum antibody titers to BHV-1 and following challenge lower (P less than 0.05) BHV-1 titers in nasal secretions; additionally, clinical disease as evidenced by nasal and ocular discharge was less severe (P less than 0.05). In vitro cytotoxic responses against BHV-1-infected bovine kidney cells were increased (P less than 0.05) in calves treated with rIL-2 in a dose dependent manner. These data suggest that bovine rIL-2 at 2.5 to 0.25 micrograms/kg may be an effective adjuvant to immunization.

Bovine GM-CSF: molecular cloning and biological activity of the recombinant protein.

The lymphokine granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates the growth and differentiation of granulocytes and macrophages from bone marrow progenitors, and regulates biological functions expressed by mature cells of these lineages. In order to isolate a bovine GM-CSF cDNA, a cDNA library, generated from the BT2 bovine T cell line, was screened with a human GM-CSF cDNA probe. A cDNA clone was isolated with an insert of 783 bp, that would encode a protein of 143 amino acids, with a predicted mol. wt of 16,160. Bovine GM-CSF exhibits a high degree of sequence homology with mouse and human GM-CSF at both the nucleotide and amino acid levels. Comparison of GM-CSF amino acid sequences from the three species indicates that the bovine GM-CSF precursor contains a putative 17 amino acid signal sequence, cleavage of which would yield a 14,250 mol. wt protein. The cDNA was inserted into a mammalian expression vector and transfected into COS-7 monkey kidney cells. Biologically active bovine GM-CSF was secreted as judged by a bovine bone marrow proliferation assay. Bovine GM-CSF was weakly active in both human and mouse bone marrow proliferation assays. In contrast, human GM-CSF was weakly active on bovine but not murine mouse bone marrow cells and mouse GM-CSF was only active on murine bone marrow cells.

Cloning, sequence and expression of bovine interleukin 1 alpha and interleukin 1 beta complementary DNAs.

Interleukin 1 (IL-1) is a cytokine which mediates a variety of immunoregulatory and inflammatory activities. Using human IL-1 alpha and IL-1 beta probes, cDNAs for the corresponding bovine genes were isolated from an alveolar macrophage library. The open reading frames of the bovine IL-1 alpha and IL-1 beta cDNAs encode proteins of 268 and 266 amino acids, respectively, each with a predicted mol. wt of approx. 31,000. Both forms of bovine IL-1 exhibit a high degree of sequence homology with IL-1 gene products from other mammalian species. Based upon comparisons with human IL-1 amino acid sequences, the post-translationally processed, mature forms of bovine IL-1 would occur as 17-18,000 mol. wt proteins. Sequences encoding mature bovine IL-1 alpha and IL-1 beta were inserted into E. coli expression plasmids and biologically active proteins were synthesized as judged by the ability of the recombinant proteins to induce proliferation of bovine thymocytes. Both IL-1 alpha and IL-1 beta exist as single genomic copies. In addition, bovine IL-1 beta mRNA is approx. 10-fold more abundant than IL-1 alpha mRNA in stimulated alveolar macrophages.

Bovine interleukin 2: cloning, high level expression, and purification.

We utilized a human IL2 probe to isolate bovine IL2 sequences from a lymph node cDNA library. Bovine IL2 was subsequently expressed in both bacteria and yeast. Using a rapid, two-step purification scheme, we have been able to isolate over 20 mg/l of homogenous bovine rIL2 secreted from the yeast. The availability of sizable quantities of bovine rIL2 should make it possible to ascertain potential therapeutic or prophylactic utility of this lymphokine in cattle.

Effect of dexamethasone on bovine leukocyte functions and bovine herpesvirus type-1 replication.

In an attempt to elucidate the mechanism whereby dexamethasone could reactivate bovine herpesvirus type-1 the effect of dexamethasone on virus replication and leukocyte functions was assessed. No effect was detectable on either virus yield or in vitro replication kinetics. In contrast, dexamethasone influenced several leukocyte functions thought to be of importance in antiviral defense and maintenance of latency. In vitro exposure of peripheral blood polymorphonuclear neutrophilic granulocytes of normal animals to dexamethasone depressed their migratory and cytotoxic activities, but had no effect on Fc- and complement receptor expression. Dexamethasone also depressed lectin-induced lymphocyte proliferation and interleukin-2 generation in a dose-dependent manner. When cows were treated repeatedly with dexamethasone and their leukocytes assayed, suppression of phytohemagglutinin-induced lymphocyte proliferation, interleukin-2 generation, natural cytotoxicity of mononuclear cells and polymorphonuclear neutrophilic granulocyte functions were observed. In contrast, concanavalin A induced lymphocyte proliferation was increased following treatment.

Influence of isoprinosine on bovine herpesvirus type-1 infection in cattle.

A study was conducted to determine the in vivo efficacy of isoprinosine (ISO) in calves infected with bovine herpesvirus type-1 (BHV-1). Calves were infected with BHV-1 on day 0 and received ISO daily for 14 days. Clinical signs of disease, shedding of BHV-1, lymphocyte proliferative responses to mitogens, interleukin-2 production, and alveolar macrophage bactericidal activity were monitored during the study. Rectal temperatures were increased (P less than 0.05) in BHV-1 and ISO-BHV-1 calves at days 3 to 7 postinfection (PI). Isoprinosine did not influence BHV-1 shedding in calves. Lymphocyte proliferative responses to phytohemagglutinin (PHA) were lower (P less than 0.01) in BHV-1 calves when compared to control or ISO calves at day 4 PI, but ISO did not ameliorate this effect. Interleukin-2 activity was greater (P less than 0.05) in ISO-BHV-1 calves on days 4 and 8 PI in PHA-stimulated lymphocytes and on day 8 PI in concanavalin A-stimulated lymphocytes when compared to control, ISO or BHV-1 calves. Isoprinosine treatment of BHV-1-infected calves tended to decrease alveolar macrophage bactericidal activity. These data suggest that ISO does not reverse BHV-1 suppression of lymphocyte proliferation, but may enhance IL-2 production in BHV-1 infected calves.

Cloning, sequence, and expression of bovine interferon-gamma.

Bovine interferon-gamma (IFN-gamma) sequences have been isolated by screening a cDNA library with a human IFN-gamma cDNA probe. The cDNA library was constructed from RNA isolated from concanavalin A-stimulated bovine lymph node cells. The open reading frame predicts that the bovine IFN-gamma precursor is composed of 166 amino acids with a predicted m.w. of 19,393. Alignment of the amino acid sequence with human IFN-gamma indicates that mature bovine IFN-gamma is composed of 143 amino acids with a predicted m.w. of 16,858. It has an amino acid homology of 63% with human IFN-gamma, and 47% with murine IFN-gamma. Biologically active bovine IFN-gamma was synthesized in an Escherichia coli expression system.