J. Russell Lindsey

Helicobacter pylori-Induced Mucosal Inflammation Is Th1 Mediated and Exacerbated in IL-4, But Not IFN-γ, Gene-Deficient Mice

To elucidate the pathogenesis of Helicobacter pylori-associated gastritis, we studied immune responses of C57BL/6J wild-type (WT), SCID, and gene deficient (IFN-γ−/− and IL-4−/−) mice following infection with a pathogenic isolate of H. pylori (SPM326). During early infection in WT mice, mononuclear and polymorphonuclear cells accumulated in the gastric lamina propria, and the numbers of cells in the inflamed mucosa expressing IFN-γ, but not IL-4, mRNA rose significantly (p < 0.005), consistent with a local Th1 response. Splenic T cells from the same infected WT mice produced high levels of IFN-γ, no detectable IL-4, and low amounts of IL-10 following in vitro H. pylori urease stimulation, reflecting a systemic Th1 response. Infected C57BL/6J SCID mice did not develop gastric inflammation despite colonization by many bacteria. Infected C57BL/10J and BALB/c mice also did not develop gastric inflammation and displayed a mixed Th1/Th2 splenic cytokine profile. These data imply a major role for the Th1 cytokine IFN-γ in H. pylori-associated gastric inflammation in C57BL/6J mice. Compared with WT animals, infected IL-4−/− animals had more severe gastritis and higher levels of IFN-γ production by urease-stimulated splenocytes (p < 0.01), whereas IFN-γ−/− mice exhibited no gastric inflammation and higher levels of IL-4 production by stimulated splenocytes. These findings establish C57BL/6J mice as an important model for H. pylori infection and demonstrate that up-regulated production of IFN-γ, in the absence of the opposing effects of IL-4 (and possibly IL-10), plays a pivotal role in promoting H. pylori-induced mucosal inflammation.

Angiogenesis in Mice with Chronic Airway Inflammation: Strain-Dependent Differences

Chronic inflammation is associated with blood vessel proliferation and enlargement and changes in vessel phenotype. We sought to determine whether these changes represent different types of angiogenesis and whether they are stimulus dependent. Chronic airway inflammation, produced by infection with Mycoplasma pulmonis, was compared in strains of mice known to be resistant (C57BL/6) or susceptible (C3H). Tracheal vascularity, assessed in whole mounts after Lycopersicon esculentum lectin staining, increased in both strains at 1, 2, 4, and 8 weeks after infection, but the type of vascular remodeling was different. The number of vessels doubled in tracheas of C57BL/6 mice, with corresponding increases of capillaries and venules. In contrast, neither the number nor the length of vessels changed in C3H mice. Instead, vessel diameter and endothelial cell number doubled, and the proportion of venules doubled with a corresponding decrease of capillaries. Although the infection had no effect on baseline plasma leakage, in both strains it potentiated the leakage produced by substance P. We conclude that the same stimulus can result in blood vessel proliferation or enlargement, depending on the host response. Endothelial cells proliferate in both cases, but in one case new capillaries form whereas in the other capillaries convert to venules.

Neuronal GM1 Gangliosidosis in a Siamese Cat with β-Galactosidase Deficiency

A juvenile Siamese cat with severe, progressive motor disability was shown to have extensive neuronal degeneration caused by accumulation of GM1 ganglioside. Tissues from brain and kidney were markedly deficient in β-galactosidase activity. The disease in this cat is thought to be inherited as an autosomal recessive trait, and is strikingly similar to juvenile GM1 gangliosidosis of children.

Amyloid-β Deposition in Skeletal Muscle of Transgenic Mice: Possible Model of Inclusion Body Myopathy

Inclusion body myopathy is a progressive muscle disorder characterized by nuclear and cytoplasmic inclusions and vacuolation of muscle fibers. Affected muscle fibers contain deposits of congophilic amyloid, amyloid-beta immunoreactive filaments, and paired helical filaments, all of which are pathological hallmarks of Alzheimers disease in brain. Accumulations of amyloid-beta and its precursor are thought to play important roles in the pathogenesis of both inclusion body myopathy and Alzheimers disease. Overexpression of mutant forms of beta protein precursor in transgenic mice by neuron-specific promoters has been reported to cause amyloid deposits in the brain. Here we report that overexpression in transgenic mice of the signal plus 99-amino acid carboxyl-terminal sequences of beta protein precursor, under the control of a cytomegalovirus enhancer/beta-actin promoter, resulted in vacuolation and increasing accumulation of the 4-kd amyloid-beta and the carboxyl-terminus in skeletal muscle fibers during aging. These deposits in transgenic muscle only rarely showed Congo red birefringence. Thus, overexpression of part of beta protein precursor in transgenic mice led to development of some of the characteristic features of inclusion body myopathy. These mice may be a useful model of inclusion body myopathy, which shares a number of pathological markers with Alzheimers disease.

Surfactant protein A mediates mycoplasmacidal activity of alveolar macrophages.

Mycoplasma pneumoniae is a leading cause of pneumonia and exacerbates other respiratory diseases in humans. We investigated the potential role of surfactant protein (SP) A in antimycoplasmal defense using alveolar macrophages (AMs) from C57BL/6NCr (C57BL) mice, which are highly resistant to infections of Mycoplasma pulmonis. C57BL AMs, activated with interferon (IFN)-γ and incubated with SP-A (25 μg/ml) at 37°C, produced significant amounts of nitric oxide (⋅ NO; nitrate and nitrite production = 1.1 μM ⋅ h-1 ⋅ 105AMs-1) and effected an 83% decrease in mycoplasma colony-forming units (CFUs) by 6 h postinfection. Preincubation of AMs with the inducible nitric oxide synthase inhibitor N G-monomethyl-l-arginine abolished ⋅ NO production and SP-A-mediated killing of mycoplasmas. No decrease in CFUs was seen when IFN-γ-activated macrophages were infected with mycoplasmas in the absence of SP-A despite significant ⋅ NO production (nitrate and nitrite production = 0.6 μM ⋅ h-1 ⋅ 105AMs-1). These results demonstrate that SP-A mediates killing of mycoplasmas by AMs, possibly through an ⋅ NO-dependent mechanism.Mycoplasma pneumoniae is a leading cause of pneumonia and exacerbates other respiratory diseases in humans. We investigated the potential role of surfactant protein (SP) A in antimycoplasmal defense using alveolar macrophages (AMs) from C57BL/6NCr (C57BL) mice, which are highly resistant to infections of Mycoplasma pulmonis. C57BL AMs, activated with interferon (IFN)-gamma and incubated with SP-A (25 micrograms/ml) at 37 degrees C, produced significant amounts of nitric oxide (.NO; nitrate and nitrite production = 1.1 microM.h-1.10(5) AMs-1) and effected an 83% decrease in mycoplasma colony-forming units (CFUs) by 6 h postinfection. Preincubation of AMs with the inducible nitric oxide synthase inhibitor NG-monomethyl-L-arginine abolished .NO production and SP-A-mediated killing of mycoplasmas. No decrease in CFUs was seen when IFN-gamma-activated macrophages were infected with mycoplasmas in the absence of SP-A despite significant .NO production (nitrate and nitrite production = 0.6 microM.h-1.10(5) AMs-1). These results demonstrate that SP-A mediates killing of mycoplasmas by AMs, possibly through an .NO-dependent mechanism.

Variations in the surface proteins and restriction enzyme systems of Mycoplasma pulmonis in the respiratory tract of infected rats

Restriction and modification (R–M) systems are generally thought to protect bacteria from invasion by foreign DNA. This paper proposes the existence of an alternative role for the phase‐variable R–M systems encoded by the hsd loci of Mycoplasma pulmonis. Populations of M. pulmonis cells that arose during growth in different environments were compared with respect to R–M activity and surface antigen production. When M. pulmonis strain X1048 was propagated in laboratory culture medium, > 95% of colony‐forming units (cfu) lacked R–M activity and produced the variable surface protein VsaA. Mycoplasmas isolated from the nose of experimentally infected rats also lacked R–M activity and produced VsaA. In contrast, the cell population of mycoplasmas isolated from the lower respiratory tract of the infected rats was more complex. The most dramatic results were obtained for mycoplasmas isolated from the trachea. At 14 days postinfection, 38% of mycoplasma isolates produced a Vsa protein other than VsaA, and 34% of isolates had active restriction systems. These data suggest that differences in selection pressures in animal tissues affect the surface proteins and the R–M activity of the mycoplasmal cell population. We propose that variations in the production of R–M activity and cell surface proteins are important for the survival of the mycoplasma within the host.

Cyclophosphamide Decreases Nitrotyrosine Formation and Inhibits Nitric Oxide Production by Alveolar Macrophages in Mycoplasmosis

ABSTRACT We previously reported that congenic C57BL/6 inducible nitric oxide synthase−/− (iNOS−/−) mice infected withMycoplasma pulmonis developed higher bacterial numbers and lung lesion scores than C57BL/6 iNOS+/+ controls but had similar lung nitrotyrosine levels. The present studies investigated the role of inflammatory cells in nitrotyrosine formation during mycoplasmal infection. iNOS+/+ and iNOS−/−mice were injected with cyclophosphamide (CYP) and inoculated with 107 CFU of M. pulmonis. CYP pretreatment ofM. pulmonis-infected iNOS+/+ and iNOS−/− mice reduced polymorphonuclear cells (PMNs) within bronchoalveolar lavages (BALs) by 88 and 72%, respectively, and whole-lung myeloperoxidase levels by 80 and 78%, respectively, at 72 h postinfection but did not alter the number of alveolar macrophages (AMs) in BALs. CYP treatment also significantly decreased nitrate and nitrite (NOx) levels in BALs and plasma of infected iNOS+/+ mice, whereas neither CYP nor mycoplasmal infection altered NOx in iNOS−/− mice. CYP reduced lung nitrotyrosine levels in both iNOS+/+ and iNOS−/− mice to uninfected-control levels as shown by immunohistochemical staining and enzyme-linked immunosorbent assay and inhibited mycoplasmal killing by iNOS+/+ mice in vivo. CYP inhibited the production of gamma interferon-inducible NOx by iNOS+/+ AMs in vitro but did not alter the number of iNOS-positive AMs, as detected by immunocytochemistry. In addition, AMs from CYP-treated iNOS+/+ mice had significantly decreased ability to kill mycoplasmas in vitro. These results demonstrate that reactive species generated by inflammatory cells as well as PMN myeloperoxidase are important contributors to nitrotyrosine formation during mycoplasmal infection and that treatment with CYP decreases NO⋅ production by AMs and inhibits mycoplasmal killing.

Mycoplasmal and Other Bacterial Diseases of the Respiratory System

Publisher Summary This chapter discusses the diseases of the respiratory system in mice, such as murine respiratory mycoplasmosis (MRM), Klebsiellosis, Pasteurellosis, Chlamydial pneumonitis, and Corynebacteriosis. Mycoplasma pulmonis is a bacterium of the sterol-requiring family Mycoplasmataceae. Like other members of the class Mollicutes, it lacks a cell wall and has a single outer limiting membrane. Although it exhibits extreme pleomorphism, ultrastructural studies have shown it to be predominantly spherical in shape and 600–1500 nm in diameter. The mechanisms by which M. pulmonis causes disease are incompletely understood, although a wide range of possibilities have been advanced. The organism is an extracellular pathogen that attaches or adheres to host-cell membranes as an initial event in infection. Klebsiella pneumoniae is widely disseminated in nature, soil, and water, as well as in agricultural and forest products. It also occurs commonly in the intestinal tract of man and animals. Pathogenic serotypes apparently are much more restricted in prevalence. P. pneumotropica is an opportunist that plays a role in respiratory tract disease only under certain circumstances.

Suppression of immune response induction in Peyer's patch lymphoid cells from mice infected with mouse hepatitis virus

Abstract Multiple previous studies have demonstrated significant alterations of immunologic parameters associated with mouse hepatitis virus (MHV) infection, but effects of the virus on mucosal lymphoid cells have not been examined. Coincident with a natural outbreak of MHV at our institution, we noted alterations in immunoglobulin secretion by mature Peyers patch B cells under an inductive stimulus provided by dendritic cells and mitogen-activated T cells (DC-T). MHV was isolated from mice affected during the outbreak, and experimental infection of mice with the isolate consistently resulted in failures of immunoglobulin secretion by cocultures of Peyers patch DC-T and B cells. In subsequent experiments, MHV appeared to negatively affect DC-T more than B cells. Therefore, the effects of inapparent MHV infection on experimental mucosal immune responses can result from natural infection and can be experimentally reproduced.

Overproduction of perlecan core protein in cultured cells and transgenic mice

Heparan sulphate proteoglycan (HSPG) and amyloid P component are the only macromolecules consistently associated with all varieties of amyloid, irrespective of the type of amyloid protein, suggesting that HSPG may play a pathogenetic role in amyloid formation through a common mechanism. In the case of Alzheimers disease (AD), HSPG, such as perlecan, co‐accumulates with amyloid‐β protein (Aβ), a main constituent of amyloid plaques, and paired helical filaments (PHFs). Additionally, in vitro, HSPG accelerates both Aβ fibril and PHF formation and protects Aβ from degradation. Therefore, this study first established lines of P19 mouse embryonic carcinoma cells stably carrying an expression vector encoding the complete perlecan core protein (∼400 kD). In the cell lysates, overexpressed perlecan was identified as an ∼400 kD protein without glycosaminoglycan side‐chains, while in the media, secreted perlecan was mostly glycosylated, suggesting that the secretion and glycosylation of perlecan are coupled. Next, transgenic mice were produced using the same expression vector. Marked perlecan overexpression occurred in the cytoplasm of multiple tissues including the brain, heart, kidney, and pancreas, without a discernible increase of perlecan in extracellular matrices. The transgenic mice up to 18 months of age did not develop amyloid or AD‐like pathology in the brain or elsewhere, based on histochemical and immunohistochemical analyses. Thus, overproduction of perlecan core protein isinsufficient to lead to amyloidosis and AD‐like pathology. Copyright