A promoter polymorphism in the gene encoding interleukin-12 p40 (IL12B) is associated with mortality from cerebral malaria and with reduced nitric oxide production.

Interleukin-12 (IL-12) is an important regulatory cytokine in infection and immunity. Administration of IL-12 may reduce complications of severe malaria in rodents. Polymorphisms in IL12B, the gene encoding the IL-12 p40 subunit, influence the secretion of IL-12 and susceptibility to Type 1 diabetes. We therefore investigated whether IL12B polymorphisms may affect the outcome of severe malaria. Homozygosity for a polymorphism in the IL12B promoter was associated with increased mortality in Tanzanian children having cerebral malaria but not in Kenyan children with severe malaria. Furthermore, homozygotes for the IL12B promotor polymorphism had decreased production of nitric oxide, which is in part regulated by IL-12 activity. These studies suggest that IL12B polymorphisms, via regulation of IL-12 production, may influence the outcome of malaria infection in at least one African population.

Novel Salmonella enterica serovar Typhimurium protein that is indispensable for virulence and intracellular replication.

Upon contact with host cells, the intracellular pathogen Salmonella enterica serovar Typhimurium promotes its uptake, targeting, and survival in intracellular niches. In this process, the bacterium evades the microbicidal effector mechanisms of the macrophage, including oxygen intermediates. This study reports the phenotypic and genotypic characterization of an S. enterica serovar Typhimurium mutant that is hypersusceptible to superoxide. The susceptible phenotype is due to a MudJ insertion-inactivation of a previously undescribed Salmonella gene designated sspJ that is located between 54.4 and 64 min of the Salmonella chromosome and encodes a 392-amino-acid protein. In vivo, upon intraperitoneal injection of 10(4) to 10(7) bacteria in C3H/HeN and 10(1) to 10(4) bacteria in BALB/c mice, the mutant strain was less virulent than the wild type. Consistent with this finding, during the first hour after ingestion by macrophage-like J774 and RAW264.7 cells in vitro, the intracellular killing of the strain carrying sspJ::MudJ is enhanced fivefold over that of wild-type microorganisms. Wild-type salmonellae displayed significant intracellular replication during the first 24 h after uptake, but sspJ::MudJ mutants failed to do so. This phenotype could be restored to that of the wild type by sspJ complementation. The SspJ protein is found in the cytoplasmic membrane and periplasmic space. Amino acid sequence homology analysis did reveal a leader sequence and putative pyrroloquinoline quinone-binding domains, but no putative protein function. We excluded the possibility that SspJ is a scavenger of superoxide or has superoxide dismutase activity.

Nitric oxide synthase type 2 promoter polymorphisms, nitric oxide production, and disease severity in Tanzanian children with malaria.

Nitric oxide (NO) plays an important role in host resistance to infection with a variety of organisms. Two recent reports from Gabon and Gambia identified associations of malaria disease severity with the inducible NO synthase (NOS2) promoter G-954C and short allele (<11 repeats) pentanucleotide microsatellite polymorphisms, respectively. It was postulated that there would be a correlation of these polymorphisms with malaria disease severity and with measures of NO production in our cohort of Tanzanian children with malaria. In Tanzanian children, 15% were heterozygous or homozygous for the G-954C polymorphism, and 13% had the short-allele microsatellite polymorphism. There was no significant correlation of either polymorphism with disease severity or with measures of NO production and NOS2 expression. Black and white Americans differed significantly in the frequencies of these polymorphisms. The various association of these gene polymorphisms with malaria severity in different populations underscores the complexity of host resistance to malaria.

Nitric oxide, malaria, and anemia: inverse relationship between nitric oxide production and hemoglobin concentration in asymptomatic, malaria-exposed children.

The cause of the anemia associated with chronic, intermittent, asymptomatic, low-level parasitemia in children in malaria-endemic endemic areas is not well understood. Nitric oxide (NO) decreases erythropoiesis, and it is likely an important mediator of anemia of chronic disease. Production of NO is decreased in acute uncomplicated and cerebral malaria, but it is increased in asymptomatic Tanzanian children (with or without parasitemia). We hypothesized that chronic overproduction of NO in these asymptomatic children contributes to the anemia associated with subclinical/subpatent malaria. In 44 fasting, asymptomatic, malaria-exposed, Tanzanian children, NO production (measured using fasting urine NOx excretion) was inversely associated with hemoglobin concentration (P = 0.03, controlling for age and gender). Using multiple linear regression, hemoglobin concentration was negatively associated with parasitemia (P = 0.005). After controlling for age and parasitemia, NO was no longer an independent predictor of anemia. One of the mechanisms of parasite-related anemia in such children may be through the adverse hematologic effects of parasite-induced NO production.

Effects of age and parasitemia on nitric oxide production/leukocyte nitric oxide synthase type 2 expression in asymptomatic, malaria-exposed children.

Age appears to influence not only the acquisition of clinical immunity to malaria but also the susceptibility to and clinical manifestations of severe malaria. Asymptomatic malaria-exposed Tanzanian children have high production of nitric oxide (NO) and universal expression of leukocyte NO synthase type 2 (NOS2), which may protect against disease. To determine the effects of age and parasitemia on NO production, we measured urine and plasma NO metabolites and leukocyte NOS2 expression in 45 fasting, asymptomatic, malaria-exposed children of different ages, stratifying parasitemia by thick film and polymerase chain reaction (PCR) analysis. Although NO production was significantly higher in thick film-positive children than in thick film-negative children, after adjusting for age and gender, we were unable to detect a difference in NO production in thick film-negative children between those who were PCR positive and PCR negative. The relationship between age and NO production was determined using a generalized additive model adjusted for the effects of gender and parasitemia. Production of NO using all three measures was highest in infancy, decreasing after the first year of life, and then increasing again after 5 years of age. This pattern of age-related NO production is the reverse of the pattern of age-related morbidity from cerebral malaria in coastal Tanzanian children. Elevated production of NO in both infants and older children may be related to age per se and malaria infection respectively, and may be one of the mediators of the anti-disease immunity found most commonly in these two age groups.

Comparison of oral and tympanic temperatures in a Veterans Administration outpatient clinic.

BACKGROUND: To examine the accuracy of noncontact tympanic (NCT) temperatures in outpatients, we conducted a prospective study comparing NCT temperature with temperatures obtained by oral mercury thermometers. METHODS: The study included 410 patients in whom oral and NCT temperatures were obtained. RESULTS: Mean oral temperature was 36.47 +/- 0.44 degrees C and mean NCT temperature was 36.36 +/- 0.49 degrees C. On paired-sample, two-sided t-testing, oral temperature differed significantly from NCT temperature, with a P-value < 0.0001. The difference between simultaneous oral and NCT temperatures was > or = 1 degree F; in 63 cases, oral temperature was higher than NCT temperature. CONCLUSION: We conclude that NCT temperature measurement is not reliable in an internal medicine outpatient clinic setting.

Structure and biological activities of acapsular Cryptococcus neoformans 602 complemented with the CAP64 gene.

The extracellular polysaccharide capsule of Cryptococcus neoformans is a well-recognized virulence factor. Strain 602 is an acapsular clinical isolate of unknown serotype which has been widely used in studies of virulence and host-parasite interactions. In previous studies, strain 602 was compared with genetically unrelated strains of various serotypes because the wild-type equivalent of strain 602 was not available. We created an encapsulated strain, TYCC38-602, by transforming strain 602 with the CAP64 gene which was isolated from a serotype D strain. Serological tests and chemical analysis of the major polysaccharide capsule of TYCC38-602 indicated that strain 602 was originally derived from a serotype A strain. Restoration of the ability to produce a capsule enabled strain 602 to cause fatal infection in mice, whereas the acapsular strain 602 remained avirulent. Capsule-restored yeast cells of strain 602 activated the human complement system and bound C3 fragments in a manner that is characteristic of encapsulated cryptococci. In addition, the capsule in TYCC38-602 masked the ability of the organism to induce tumor necrosis factor alpha and subsequent nitric oxide synthase production in primed macrophage-like cells. These results indicate that the lack of capsule in strain 602 is the reason for its inability to cause fatal infection. Moreover, the acapsular phenotype accounts for differences in various biological activities of strain 602 compared to encapsulated strains. The results also indicate that the gene product of CAP64 does not contribute to serotype specificity of capsules in C. neoformans.

Nitric oxide in Tanzanian children with malaria: inverse relationship between malaria severity and nitric oxide production/nitric oxide synthase type 2 expression.

Nitric oxide (NO)-related activity has been shown to be protective against Plasmodium falciparum in vitro. It has been hypothesized, however, that excess NO production contributes to the pathogenesis of cerebral malaria. The purpose of this study was to compare markers of NO production [urinary and plasma nitrate + nitrite (NOx)], leukocyte-inducible nitric oxide synthase type 2 (NOS2), and plasma TNF-alpha and IL-10 levels with disease severity in 191 Tanzanian children with and without malaria. Urine NOx excretion and plasma NOx levels (corrected for renal impairment) were inversely related to disease severity, with levels highest in subclinical infection and lowest in fatal cerebral malaria. Results could not be explained by differences in dietary nitrate ingestion among the groups. Plasma levels of IL-10, a cytokine known to suppress NO synthesis, increased with disease severity. Leukocyte NOS2 antigen was detectable in all control children tested and in all those with subclinical infection, but was undetectable in all but one subject with cerebral malaria. This suppression of NO synthesis in cerebral malaria may contribute to pathogenesis. In contrast, high fasting NOx levels and leukocyte NOS2 in healthy controls and asymptomatic infection suggest that increased NO synthesis might protect against clinical disease. NO appears to have a protective rather than pathological role in African children with malaria.

Bartonella henselae: etiology of pulmonary nodules in a patient with depressed cell-mediated immunity.

We describe an immunocompromised renal transplantation patient with opportunistic lung infection due to Bartonella henselae (formerly Rochalimaea henselae) and provide evidence suggesting transmission from a pet cat. Computed tomographic scans of the chest and lung biopsies provided material for diagnosis. The etiology was established by polymerase chain reaction and sequencing of a 16S ribosomal DNA segment from infected lung tissue. Histopathologic and serological evidence supported the molecular data. B. henselae was isolated from the blood of eight of the patient's many cats. The patient responded to prolonged therapy with doxycycline, and relapse did not occur during a 1-year follow-up. B. henselae joins a long list of pathogens that can cause lung infections in association with cell-mediated immunodeficiency states. Molecular methods are useful in diagnosis of this infection in light of the bacterium's fastidious growth characteristics. If an immunocompromised patient has lung nodules and a history of exposure to cats, B. henselae should be sought in biopsy specimens.

Cryptococcus neoformans fails to induce nitric oxide synthase in primed murine macrophage-like cells.

Nitric oxide (NO) is a microbiostatic gas generated by activated murine macrophages. Cytokine signals, gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) act synergistically to induce production of a macrophage nitric oxide synthase (NOS). A variety of intracellular pathogens, when recognized by macrophages primed with IFN-gamma, induce NOS by eliciting TNF-alpha secretion, which then functions as a positive autocrine signal. In cell culture assays, a murine macrophage cell line (J774), primed with IFN-gamma, was tested for NOS induction upon challenge with virulent Cryptococcus neoformans. C. neoformans failed to induce macrophage NOS as measured by nitrite production. This was true irrespective of the C. neoformans-to-J774 ratio. Other nonpathogenic Cryptococcus species likewise failed to induce NOS, yet Saccharomyces cerevisiae, Histoplasma capsulatum, and Candida albicans were efficient inducers of NOS. Conditions which promoted attachment and/or phagocytosis of C. neoformans did not lead to NOS induction (including opsonization with specific antibodies against C. neoformans). Assays for transcriptional repressors of NOS were negative. Tests for consumption of nitrite by measurement of additional products of NOS induction were negative. No TNF-alpha was detected by enzyme-linked immunosorbent assay in supernatants from C. neoformans-J774 cocultures. A mutant C. neoformans strain with a minimal, but visible, polysaccharide capsule also failed to induce NOS; however, several nonencapsulated mutants of C. neoformans did induce NOS. Failure of C. neoformans to act as an inducer of NOS may be related to the virulence of this pathogen in mice; C. neoformans is a unique example of a facultative intracellular pathogen which fails to induce NOS in primed macrophages. The mechanism appears to involve the failure of TNF-alpha secretion once the macrophage comes in contact with the fungus. The presence of the polysaccharide capsule appears to mask the signal necessary for TNF-alpha secretion and, ultimately, NOS induction.

Human immunodeficiency virus (HIV)-infected human blood monocytes and peritoneal macrophages have reduced anticryptococcal activity whereas HIV-infected alveolar macrophages retain normal activity.

Human immunodeficiency virus type 1 (HIV-1) infection causes immune dysfunction. Mononuclear phagocytes (MNP) are immune effector cells against some intracellular pathogens and reservoirs for HIV-1. This study determined effects of HIV-1 on MNP-mediated antifungal function. MNP from seronegative volunteers were inoculated with HIVBal or HIVIIIB. MNP were infected with an avirulent clone of Cryptococcus neoformans; 48 h later, MNP were lysed and yeasts were counted. Viral replication was determined by reverse transcriptase and by visualization of cytopathic effects. Monocytes and peritoneal macrophages exhibited reduced anticryptococcal activity 14 days after infection with HIVBal but retained normal activity when infected with HIVIIIB. Loss of anticryptococcal activity correlated with viral replication. Alveolar macrophages retained normal anticryptococcal activity whether infected with HIVBal or HIVIIIB. In vitro MNP-mediated antifungal activity may be altered by HIV-1 infection; this altered activity appears to depend on viral tropism, viral replication, and MNP tissue origin.

N omega -monomethyl-L-arginine inhibits nitric oxide production in murine cardiac allografts but does not affect graft rejection.

Endogenous nitric oxide biosynthesis in mice receiving allogeneic heterotopic heart transplants was monitored as a function of time post-transplant. Nitric oxide production was measured by daily urine nitrate levels and by formation of paramagnetic heme-nitrosyl complexes in the cardiac tissue. Exogenous sources of urine nitrate and EPR signal were minimized by maintaining the animals on a low nitrite/nitrate diet. Urine nitrate peaked on postoperative day 7. A heme-nitrosyl EPR signal also appeared in the cardiac tissue on postoperative day 7 and remained unchanged in size until rejection on postoperative day 9 at which time the peak height of the signal nearly tripled. Some of the animals in the study were treated with the nitric oxide synthase inhibitor, N omega-monomethyl-L-arginine which caused marked inhibition of urinary nitrate excretion and prevented heme-nitrosyl complex formation in beating hearts. However, administration of the inhibitor did not increase graft survival time. Low intensity heme-nitrosyl signals were identified in inhibitor-treated allogeneic hearts after rejection. Syngeneic heart transplants did not induce urinary nitrate excretion nor EPR signal formation. These results show that cytokine induced high output nitric oxide synthesis from L-arginine is a prominent biochemical component of the cell-mediated immune response to cardiac allografts in mice. However, nitric oxide production was not essential for rejection of cardiac allografts mismatched at the major histocompatibility locus.

Nitric oxide produced during murine listeriosis is protective.

Nitric oxide (NO) has been shown to be important for intracellular microbiostasis in vitro. To determine the role of NO in immune function in vivo, groups of C57BL/6 mice were given a sublethal intravenous inoculum of Listeria monocytogenes EGD, and their urine was monitored daily for nitrate, the mammalian end product of NO metabolism. Urinary nitrate levels peaked at 5 to 10 times the basal level on days 5 to 6, when spleen and liver Listeria counts declined most steeply, and decreased thereafter, when spleens and livers were nearly sterile. Peritoneal macrophages explanted from Listeria-infected mice produced nitrite spontaneously, whereas macrophages from uninfected mice did not. The inducible NO synthase mRNA was detectable in the spleens of infected mice on days 1 to 4 of infection. When Listeria-infected mice were treated orally throughout the infection with NG-monomethyl-L-arginine (NMMA), a specific NO synthase inhibitor they showed no detectable rise in urinary nitrate excretion. Mean Listeria counts in the livers and spleens NMMA-treated mice were 1 to 3 orders of magnitude greater than counts in control mice on days 4 through 8 of infection. Compared with control mice, NMMA-treated mice also showed worse clinical signs of infection, namely, weight loss, hypothermia, decreased food and water intake, and decreased urine output. Histologically NMMA-treated mice had many more inflammatory foci in their livers and spleens than control mice. The histologic observation that mononuclear cells are present at sites of infection suggests that inhibiting NO production did not block the flux of macrophages into infected viscera. As controls for possible drug toxicity, a group of uninfected mice given NMMA orally showed no detrimental effects on weight, temperature, and food and water intake. These experiments demonstrate that inhibition of NO production in Listeria-infected mice results in an exacerbated infection and thus that NO synthesis is important for immune defense against Listeria infection in mice.

The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease: increased nitric oxide production and nitric oxide synthase expression in MRL-lpr/lpr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine.

MRL-lpr/lpr mice spontaneously develop various manifestations of autoimmunity including an inflammatory arthropathy and immune complex glomerulonephritis. This study examines the role of nitric oxide, a molecule with proinflammatory actions, in the pathogenesis of MRL-lpr/lpr autoimmune disease. MRL-lpr/lpr mice excreted more urinary nitrite/nitrate (an in vivo marker of nitric oxide production) than did mice of normal strains and MRL-(+/+) and B6-lpr/lpr congenic strains. In addition, MRL-lpr/lpr peritoneal macrophages had an enhanced capacity to produce nitric oxide in vitro as well as increased nitric oxide synthase activity, and certain tissues from MRL-lpr/lpr mice had increased expression of inducible nitric oxide synthase (NOS) mRNA and increased amounts of material immunoreactive for inducible NOS. Oral administration of NG-monomethyl-L-arginine, a nitric oxide synthase inhibitor, prevented the development of glomerulonephritis and reduced the intensity of inflammatory arthritis in MRL-lpr/lpr mice. By using interspecific backcross mice, the gene for inducible NOS (Nosi) was mapped to mouse chromosome 11. This chromosomal localization was different from those loci that we have previously demonstrated to be linked to enhanced susceptibility to renal disease in an MRL-lpr/lpr cross. However, the chromosomal location of the NOS gene was consistent with an insulin-dependent diabetes locus identified in an analysis of nonobese diabetic (NOD) mice. These results suggest that elevated nitric oxide production could be important in the pathogenesis of autoimmunity, and that treatments to block the production of nitric oxide or block its effects might be valuable therapeutically.