Inhibition of cytokine-induced inducible nitric oxide synthase expression by glucagon and cAMP in cultured hepatocytes.

Addition of lipopolysaccharide plus interferon gamma, tumour necrosis factor alpha and interleukin 1 beta to cultured hepatocytes resulted in the induction of inducible nitric oxide synthase (iNOS) activity as measured by NO3(-)+NO2- formation, the conversion of L-[U-14C]arginine into citrulline and Western blotting of the iNOS protein. The inclusion of 1 microM glucagon during the induction period significantly decreased the effect of the cytokines on iNOS activity, the major effect being at the level of the total amount of protein, rather than alterations in substrate supply or covalent modification of the existing protein. In contrast, 1 microM insulin was without effect. The effect of glucagon was mediated via cAMP and could be mimicked by the presence of either dibutyryl cAMP or forskolin to activate adenylate cyclase directly. It was rapid in onset and long-lived, a 30 min pretreatment period protecting the cells from the induction of NO synthesis over the next 21 h in the presence of cytokines. Addition of glucagon at any time point up to 9 h after treatment of the cells with lipopolysaccharide plus the cytokines resulted in a significant inhibition of iNOS activity, glucagon being most potent when added during the first 3 h.

Effect of multiple cytokines plus bacterial endotoxin on glucose and nitric oxide production by cultured hepatocytes.

Treatment of cultured hepatocytes with a combination of cytokines, including tumour necrosis factor-alpha, interferon-gamma and interleukin-1 beta, plus lipopolysaccharide resulted in a time-dependent induction of nitric oxide (NO) synthase (as measured by NO2- (+) NO3- production) and inhibition of hepatic gluconeogenesis and glycogen breakdown. The inhibition of glucose release was comparable with the observed following treatment of rats with lipopolysaccharide or treatment of isolated hepatocytes with artificial NO donors. In addition, this effect was also evident with all substrates tested that enter the gluconeogenic pathway below the level of phosphoenolpyruvate carboxykinase, suggesting that this combination of cytokines may underlie the inhibition of gluconeogenesis observed in endotoxic shock. The maximal inhibition of glucose output required the presence of all the cytokines plus lipopolysaccharide, whereas the induction of NO synthase was independent of the lipopolysaccharide when the cytokines were employed. Inclusion of interferon-gamma was essential to obtain a maximal response for either parameter. Inclusion of 1 mM N(G)-monomethyl-L-arginine in the incubation abolished the increase in NO2- (+) NO3- observed with the complete cytokine mixture and various combinations; however, it failed to prevent the inhibition in glucose output, indicating that mechanisms other than NO underlie the cytokine-induced inhibition of glucose release.

Human parainfluenza virus type 1 immunization of infant mice protects from subsequent Sendai virus infection.

Human parainfluenza virus type 1 (hPIV-1) infections are a common cause of "croup" and hospitalizations among young children, yet no vaccine is yet available. Sendai virus (mouse PIV-1) is the closest known homologue of hPIV-1. Here we address the possibility of using a xenotropic, nonpathogenic PIV as a vaccine in infants, by assessing the efficacy of hPIV-1 vaccination of infant mice against a subsequent challenge with Sendai virus. hPIV-1 was administered intranasally to mice age 3-6 days and shown by serum antibody ELISA and elispot analysis to elicit virus-specific IgM and isotype-switched antibody-forming cells (AFC). The response was completely cross-reactive between hPIV-1 and Sendai virus. Mice were challenged with Sendai virus 6-8 weeks later and generated AFC and serum antibody responses composed of IgM, as well as IgG and IgA, unlike challenged, age-matched controls. The high IgM response among AFC was not seen in mice primed as adults with hPIV-1 and challenged with Sendai virus. The hPIV-1 priming of infant mice afforded protection, as the majority of these mice survived the lethal Sendai virus challenge, as did all adult primed animals. These data support the notion that the unmodified xenotropic Sendai virus might function effectively in human infants as a vaccine against hPIV-1.

T cell receptor repertoire of CD4+ and CD8+ T cell subsets in the allogeneic bone marrow transplant recipient.

Allogeneic bone marrow transplantation (BMT) has become a therapy of choice for the treatment of certain malignancies and hematopoietic disorders. However, immunodeficiencies following BMT continue to cause significant morbidity and mortality. We have compared the T cell receptor (TCR) repertoire of BMT patients and healthy control individuals by staining peripheral blood mononuclear cells with fluorochrome-labeled TCR-specific antibodies. Several patients exhibited a biased pattern of TCR expression atypical of the healthy controls, yet no particular TCR bias characterized all patients. For example, we found that 2%-8% of T cells from healthy individuals expressed the V beta 19 TCR. One BMT patient exhibited V beta 19 expression on more than 60% of peripheral T cells, while additional patients expressed V beta 19 on less than 1% of T cells. The patients with the most extreme skewing of TCR types suffered from graft-versus-host disease. The causes of skewed TCR V beta expression patterns in BMT patients are not fully understood, yet some researchers have suggested that an oligoclonal expansion of CD8+ T cell populations may be largely responsible. To test this hypothesis, we examined the TCR V beta repertoire of CD4+ and CD8+ T cell populations. We found that biased V beta expression characterized both CD4+ and CD8+ T cell populations, sometimes within a single individual. Thus, therapies directed toward CD8+ T cells alone may not fully correct repertoire abnormalities following BMT.

Age-related development of human memory T-helper and B-cell responses toward parainfluenza virus type-1.

Human parainfluenza-1 virus (hPIV-1) infections are a major cause of respiratory illness in young children. While children and adults are each susceptible to hPIV-1 infection, the clinical symptoms in adults are mild and hospitalizations are rare. One explanation for the differences in disease severity is that immune memory responses are simply inferior in children as compared to adults and cannot counter virus growth. Alternatively, it has been suggested that immune (particularly T-helper (TH) cell) responses toward respiratory viruses are superior in children versus older individuals, and that these responses contribute to, rather than protect from, disease symptoms. As a test of these possibilities, we analyzed hPIV-1-specific T-helper (TH) and B-cell memory responses among individuals of various ages, including children hospitalized with hPIV-1-induced croup. Experiments revealed: (1) hPIV-1-specific B-cell and class-II restricted TH-cell proliferative responses were present in all tested adults. (2) TH-cells responded to internal viral proteins as well as to the external glycoprotein, hemagglutinin-neuraminidase. (3) Immune responses were highly cross-reactive with Sendai virus. (4) Memory B-cell and TH-cell responses were extremely poor in young children, inclusive of children tested upon hospital entry for hPIV-1-induced croup. In total, results did not support the theory that naturally induced hPIV-specific memory responses cause respiratory illness. Rather, results showed a correlation between memory and a good clinical outcome and highlighted Sendai virus as a strong candidate for an hPIV-1 vaccine.

Viral cross-reactivity and antigenic determinants recognized by human parainfluenza virus type 1-specific cytotoxic T-cells.

To obtain information relevant to vaccination against human parainfluenza virus type 1 (hPIV-1), cytotoxic T-lymphocyte (CTL) responses to individual viral components were tested. The CD8-positive T-cell fraction was first enriched from human, adult PBL and grown for several passages in the presence of hPIV-1-infected stimulator cells. T-cell lines were then tested for CTL activity toward hPIV-1 and toward the related viruses hPIV-3 and Sendai virus (the murine parainfluenza type 1 virus). All tested cultures which responded to hPIV-1 also responded to hPIV-3 and Sendai virus, demonstrating sequence conservation between all three viruses among major antigenic determinants for CTL. Specificity for particular viral components was defined using recombinant vaccinia viruses expressing individual proteins from either mouse or human parainfluenza type 1 viruses. Strong CTL responses toward hemagglutinin-neuraminidase, phosphoprotein, and nucleoprotein (NP) were demonstrated. The testing of vaccinia constructs expressing truncated proteins then showed that there were multiple CTL determinants within NP. Several T-cell lines from one donor recognized an NP peptide (amino acids 321-336) conserved between the hPIV-1 and Sendai virus. In total, the results demonstrated that the human CTL response is directed to multiple determinants within several distinct hPIV-1 proteins.

Polyarteritis nodosa, hairy cell leukemia and splenosis.

Appendiceal vasculitis and splenosis in a young women provided pathologic evidence that a systemic necrotizing vasculitis preceded the onset of hairy cell leukemia by at least six years. As in other patients who have had both diseases, florid polyarteritis nodosa developed during the course of the malignancy. However, the antecedent vasculitis, as well as the coincidence of these rare diseases, indicate that systemic vasculitis does not necessarily result from hairy cell leukemia as previously reported and suggest that the two diseases might share a common pathogenesis or predisposing factor.

Pulmonary resection for localized lesions of cryptococcosis (torulosis): a review of eight cases.

Torulosis is an uncommon, but potentially lethal disease. The aim of this report is to indicate that resection of isolated pulmonary lesions due to torulosis is a safe procedure. Resection has proved useful in the definitive diagnosis and treatment of eight cases seen in this thoracic surgical unit.

Carcinoma of the oesophagus: preoperative irradiation followed by planned resection for lesions in the middle and lower thirds--an interim report.

Since 1968, at the Thoracic Surgery Unit in the Royal Perth Hospital, a combined approach of preoperative deep X-ray therapy and excisional surgery has been adopted for patients with potentially operable carcinoma of the oesophagus. This has produced an improvement in the survival time for middle third lesions when compared with the result previously obtained following unaided surgical excision. The method is described, and the early results are indicated.