Preadipocyte Recruitment in Stromal Vascular Cultures After Depletion of Committed Preadipocytes by Immunocytotoxicity

Glucocorticoids or the glucocorticoid analog dexamethasone (DEX) enhances the differentiation of preadipocytes in the presence of insulin and influences preadipocyte proliferation. The purpose of the present study was to determine if DEX can induce the recruitment of preadipocytes. Using monoclonal antibodies for complement-mediated cytotoxicity, preadipocytes were removed from porcine stromal vascular (S-V) cell cultures. Our experiments demonstrated for the first time that after removal of preadipocytes by cytotoxicity, preadipocytes or fat cells could be induced by DEX or DEX plus insulin but not by insulin alone. However, many more fat cells were induced (258 ± 15/unit area) when DEX was added with fetal bovine serum (FBS) followed with insulin treatment, compared to DEX with insulin (21.3 ± 5.1/ unit area) after removal of preadipocytes. Immunocyto-chemistry with AD-3, a preadipocyte marker, showed that DEX with FBS for 3 days after seeding (i.e., the proliferation phase) produced many more preadipocytes (AD-3 positive, 223 ± 45/unit area) than FBS alone (10.5 ± 1.4/unit area). Bromodeoxyuridine (BrdU) incorporation assays demonstrated that the efficiency of DEX with FBS (i.e., during proliferation) was mitosis dependent. Accordingly, we conclude that: porcine S-V cultures contain preadipocytes at different stages of differentiation and that DEX induced early preadipocyte differentiation depends on mitosis.     

Forming a Complex with MHC Class I Molecules Interferes with Mouse CD1d Functional Expression

CD1d molecules are structurally similar to MHC class I, but present lipid antigens as opposed to peptides. Here, we show that MHC class I molecules physically associate with (and regulate the functional expression of) mouse CD1d on the surface of cells. Low pH (3.0) acid stripping of MHC class I molecules resulted in increased surface expression of murine CD1d on antigen presenting cells as well as augmented CD1d-mediated antigen presentation to NKT cells. Consistent with the above results, TAP1-/- mice were found to have a higher percentage of type I NKT cells as compared to wild type mice. Moreover, bone marrow-derived dendritic cells from TAP1-/- mice showed increased antigen presentation by CD1d compared to wild type mice. Together, these results suggest that MHC class I molecules can regulate NKT cell function, in part, by masking CD1d.     

Vaccinia virus infection induces dendritic cell maturation but inhibits antigen presentation by MHC class II

Vaccinia virus (VV) infection is known to inhibit dendritic cells (DC) functions in vitro. Paradoxically, VV is also highly immunogenic and thus has been used as a vaccine. In the present study, we investigated the effects of an in vivo VV infection on DC function by focusing on early innate immunity. Our data indicated that DC are activated upon in vivo VV infection of mice. Splenic DC from VV-infected mice expressed elevated levels of MHC class I and co-stimulatory molecules on their cell surface and exhibited the enhanced potential to produce cytokines upon LPS stimulation. DC from VV-infected mice also expressed a high level of interferon-beta. However, a VV infection resulted in the down-regulation of MHC class II expression and the impairment of antigen presentation to CD4 T cells by DC. Thus, during the early stage of a VV infection, although DC are impaired in some of the critical antigen presentation functions, they can promote innate immune defenses against viral infection.     

Incidence and levels of fumonisin contamination in Colombian corn and corn products

A survey was conducted to determine the levels of fumonisins B1 and B2 in corn and corn-based products available in Colombia for human and animal consumption. A total of 120 samples were analyzed by acetonitrile-water extraction, cleanup with a strong-anion-exchange column, and liquid chromatography with o-phthaldialdehyde-2-mercaptoethanol derivatization and fluorescence detection. The samples of corn and corn-based products for animal intake were taken at different feed manufacturing plants, whereas the samples used for human foods where purchased from local retail stores. The number of positive samples for fumonisin B1 was 20.0% higher in corn and corn-based products for animal intake (75.0%) than in corn and corn-based products for human consumption (55.0%). The levels of fumonisin B1 were also higher in corn and corn-based products for animal intake (mean = 694 μg/kg; range = 32–2964 μg/kg), than in corn and corn-based products for human intake (mean = 218 μg/kg; range = 24–2170 μg/ kg). The incidence and levels of fumonisin B2 were lower than those for fumonisin B1. Corn and corn-based products for animal consumption had an incidence of fumonisin B2 of 58.3%, with a mean value of 283 μg/kg, and a range of 44–987 μg/kg. The incidence of fumonisin B2 in corn-based products for human intake was 35.0%, with a mean value of 118 μg/kg and a range of 21–833 μg/kg. The highest incidence and levels of fumonisins were found in samples of hominy feed, with concentrations ranging from 86 to 2964 μg/kg fumonisin B1 and 57 to 987 μg/kg fumonisin B2.     

Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Swine Influenza H1N1 Virus Induces Acute Inflammatory Immune Responses in Pig Lungs: a Potential Animal Model for Human H1N1 Influenza Virus

Pigs are capable of generating reassortant influenza viruses of pandemic potential, as both the avian and mammalian influenza viruses can infect pig epithelial cells in the respiratory tract. The source of the current influenza pandemic is H1N1 influenza A virus, possibly of swine origin. This study was conducted to understand better the pathogenesis of H1N1 influenza virus and associated host mucosal immune responses during acute infection in humans. Therefore, we chose a H1N1 swine influenza virus, Sw/OH/24366/07 (SwIV), which has a history of transmission to humans. Clinically, inoculated pigs had nasal discharge and fever and shed virus through nasal secretions. Like pandemic H1N1, SwIV also replicated extensively in both the upper and lower respiratory tracts, and lung lesions were typical of H1N1 infection. We detected innate, proinflammatory, Th1, Th2, and Th3 cytokines, as well as SwIV-specific IgA antibody in lungs of the virus-inoculated pigs. Production of IFN-γ by lymphocytes of the tracheobronchial lymph nodes was also detected. Higher frequencies of cytotoxic T lymphocytes, γδ T cells, dendritic cells, activated T cells, and CD4⁺ and CD8⁺ T cells were detected in SwIV-infected pig lungs. Concomitantly, higher frequencies of the immunosuppressive T regulatory cells were also detected in the virus-infected pig lungs. The findings of this study have relevance to pathogenesis of the pandemic H1N1 influenza virus in humans; thus, pigs may serve as a useful animal model to design and test effective mucosal vaccines and therapeutics against influenza virus.Swine influenza is a highly contagious, acute respiratory viral disease of swine. The causative agent, swine influenza virus (SwIV), is a strain of influenza virus A in the Orthomyxoviridae family. Clinical disease in pigs is characterized by sudden onset of anorexia, weight loss, dyspnea, pyrexia, cough, fever, and nasal discharge (21). Porcine respiratory tract epithelial cells express sialic acid receptors utilized by both avian (α-2,3 SA-galactose) and mammalian (α-2,6 SA-galactose) influenza viruses. Thus, pigs can serve as “mixing vessels” for the generation of new reassortant strains of influenza A virus that may contain RNA elements of both mammalian and avian viruses. These “newly generated” and reassorted viruses may have the potential to cause pandemics in humans and enzootics in animals (52).Occasional transmission of SwIV to humans has been reported (34, 43, 52), and a few of these cases resulted in human deaths. In April 2009, a previously undescribed H1N1 influenza virus was isolated from humans in Mexico. This virus has spread efficiently among humans and resulted in the current human influenza pandemic. Pandemic H1N1 virus is a triple reassortant (TR) virus of swine origin that contains gene segments from swine, human, and avian influenza viruses. Considering the pandemic potential of swine H1N1 viruses, it is important to understand the pathogenesis and mucosal immune responses of these viruses in their natural host. Swine can serve as an excellent animal model for the influenza virus pathogenesis studies. The clinical manifestations and pathogenesis of influenza in pigs closely resemble those observed in humans. Like humans, pigs are also outbred species, and they are physiologically, anatomically, and immunologically similar to humans (9, 23, 39, 40). In contrast to the mouse lung, the porcine lung has marked similarities to its human counterpart in terms of its tracheobronchial tree structure, lung physiology, airway morphology, abundance of airway submucosal glands, and patterns of glycoprotein synthesis (8, 10, 17). Furthermore, the cytokine responses in bronchoalveolar lavage (BAL) fluid from SwIV-infected pigs are also identical to those observed for nasal lavage fluids of experimentally infected humans (20). These observations support the idea that the pig can serve as an excellent animal model to study the pathogenesis of influenza virus.Swine influenza virus causes an acute respiratory tract infection. Virus replicates extensively in epithelial cells of the bronchi and alveoli for 5 to 6 days followed by clearance of viremia by 1 week postinfection (48). During the acute phase of the disease, cytokines such as alpha interferon (IFN-α), tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-12, and gamma interferon (IFN-γ) are produced. These immune responses mediate both the clinical signs and pulmonary lesions (2). In acute SwIV-infected pigs, a positive correlation between cytokines in BAL fluid, lung viral titers, inflammatory cell infiltrates, and clinical signs has been detected (2, 48).Infection of pigs with SwIV of one subtype may confer complete protection from subsequent infections by homologous viruses and also partial protection against heterologous subtypes, but the nature of the immune responses generated in the swine are not fully delineated. Importantly, knowledge related to host mucosal immune responses in the SwIV-infected pigs is limited. So far only the protective virus-specific IgA and IgG responses in nasal washes and BAL fluid, as well as IgA, IgG, and IgM responses in the sera of infected pigs, have been reported (28). Pigs infected with H3N2 and H1N1 viruses have an increased frequency of neutrophils, NK cells, and CD4 and CD8 T cells in the BAL fluid (21). Pigs infected with the pandemic H1N1 virus showed activated CD4 and CD8 T cells in the peripheral blood on postinfection day (PID) 6 (27). Proliferating lymphocytes in BAL fluid and blood and virus-specific IFN-γ-secreting cells in the tracheobronchial lymph nodes (TBLN) and spleen were detected in SwIV-infected pigs (7). Limited information is available on the mucosal immune responses in pig lungs infected with SwIV, which has a history of transmission to humans.In this study, we examined the acute infection of SwIV (strain SwIV OH07) in pigs with respect to viral replication, pathology, and innate and adaptive immune responses in the respiratory tract of these pigs. This virus was isolated from pigs which suffered from respiratory disease in Ohio, and the same virus was also transmitted to humans and caused clinical disease (43, 55). Interestingly, like pandemic H1N1 influenza virus, SwIV also infects the lower respiratory tract of pigs. Delineation of detailed mucosal immune responses generated in pig lungs during acute SwIV OH07 infection may provide new insights for the development of therapeutic strategies for better control of virus-induced inflammation and for the design and testing of effective vaccines.     

CD44 differentially activates mouse NK T cells and conventional T cells

NK T (NKT) cells are an important component of the innate immune system and recognize the MHC class I-like CD1d molecule. NKT cells possess significant immunoregulatory activity due to their rapid secretion of large quantities of pro- and anti-inflammatory cytokines following CD1d-dependent stimulation. Because the innate immune system is programmed to respond to a multitude of diverse stimuli and must be able to quickly differentiate between pathogenic and endogenous signals, we hypothesized that, apart from stimulation via the TCR (e.g., CD1d-dependent activation), there must be multiple activation pathways that can be triggered through other cell surface receptors on NKT cells. Therefore, we analyzed the ability of CD44, a structurally diverse cell surface receptor expressed on most cells, to stimulate murine NKT cells, compared with conventional T cells. Stimulation of CD44 through Ab cross-linking or binding to its natural ligands hyaluronan and osteopontin induced NKT cells to secrete cytokines, up-regulate activation markers, undergo morphological changes, and resist activation-induced cell death, whereas conventional T cells only exhibited changes in morphology and protection from activation-induced cell death. This CD44-specific stimulation of NKT cells correlated with their ability to bind hyaluronan. Thus, fundamental differences in CD44 function between these lymphocyte subsets suggest an important biological role for CD44 in the innate immune response.     

Cytomegalovirus seropositivity is associated with glucose regulation in the oldest old. Results from the Leiden 85-plus Study

ABSTRACT: BACKGROUND: Cytomegalovirus (CMV) infection has been reported to contribute to the pathogenesis of type 1 diabetes and post-transplantation diabetes. However, CMV infection has not been evaluated as a possible risk factor for type 2 diabetes. Our aim was to investigate potential associations between CMV seropositivity, CMV IgG antibody level and glucose regulation in the oldest old. RESULTS: CMV seropositive subjects were more likely to have type 2 diabetes (17.2% vs 7.9%, p = 0.016), had a higher level of HbA1c (p = 0.014) and higher non-fasting glucose (p = 0.024) in the oldest olds. These associations remained significant after adjustment for possible confounders. CMV IgG antibody level was not significantly associated with glucose regulation (all p > 0.05). CONCLUSIONS: In the oldest old, CMV seropositivity is significantly associated with various indicators of glucose regulation. This finding suggests that CMV infection might be a risk factor for the development of type 2 diabetes in the elderly.     

Comparative Analysis of the glg Operons of Pectobacterium chrysanthemi PY35 and Other Prokaryotes

A chromosomal region of Pectobacterium chrysanthemi PY35 that contains of genes for glycogen synthesis was isolated from a cosmid library. The operon consists of glycogen branching enzyme (glgB), glycogen debranching enzyme (glgX), ADP-glucose pyrophosphorylase (glgC), glycogen synthase (glgA), and glycogen phosphorylase (glgP) genes. Gene organization is similar to that of Escherichia coli. The purified ADP-glucose pyrophosphorylase (GlgC) was activated by fructose 1,6-bisphosphate and inhibited by AMP. The constructed glgX::Omega mutant failed to integrate into the chromosome of P. chrysanthemi by marker exchange. Phylogenetic analysis based on the 16S rDNA and the amino acid sequence of Glg enzymes showed correlation with other bacteria. gamma-Proteobacteria have the glgX gene instead of the bacilli glgD gene in the glg operon. The possible evolutionary implications of the results among the prokaryotes are discussed.