Hantavirus infection induces the expression of RANTES and IP-10 without causing increased permeability in human lung microvascular endothelial cells

Sin Nombre virus (SNV) and Hantaan virus (HTN) infect endothelial cells and are associated with different patterns of increased vascular permeability during human disease. It is thought that such patterns of increased vascular permeability are a consequence of endothelial activation and subsequent dysfunction mediated by differential immune responses to hantavirus infection. In this study, the ability of hantavirus to directly induce activation of human lung microvascular endothelial cells (HMVEC-Ls) was examined. No virus-specific modulation in the constitutive or cytokine-induced expression of cellular adhesion molecules (CD40, CD54, CD61, CD62E, CD62P, CD106, and major histocompatibility complex classes I and II) or in cytokines and chemokines (eotaxin, tumor necrosis factor alpha, interleukin 1beta [IL-1beta], IL-6, IL-8, MCP-1, MIP-1alpha, and MIP-1beta) was detected at either the protein or message level in hantavirus-infected HMVEC-Ls. Furthermore, no virus-specific enhancement of paracellular or transcellular permeability or changes in the organization and distribution of endothelial intercellular junctional proteins was observed. However, infection with either HTN or SNV resulted in detectable levels of the chemokines RANTES and IP-10 (the 10-kDa interferon-inducible protein) in HMVEC-Ls within 72 h and was associated with nuclear translocation of interferon regulatory factor 3 (IRF-3) and IRF-7. Gamma interferon (IFN-gamma)-induced expression of RANTES and IP-10 could also be detected in uninfected HMVEC-Ls and was associated with nuclear translocation of IRF-1 and IRF-3. Treatment of hantavirus-infected HMVEC-Ls with IFN-gamma for 24 h resulted in a synergistic enhancement in the expression of both RANTES and IP-10 and was associated with nuclear translocation of IRF-1, IRF-3, IRF-7, and NF-kappaB p65. These results reveal a possible mechanism by which hantavirus infection and a TH1 immune response can cooperate to synergistically enhance chemokine expression by HMVEC-Ls and trigger immune-mediated increases in vascular permeability.     

The blood supply of the reverse temporalis muscle flap: anatomic study and clinical implications

Although the reverse temporalis muscle flap has been used clinically, the exact vascular connection between the superficial and deep temporal vessels has not been clearly defined. The purpose of this study was to investigate the vascular territory of the reverse temporalis muscle supplied by the superficial temporal vessels. Six cadaver heads were studied using a colored lead oxide injection through the superficial temporal artery. The specimens were examined macroscopically and radiographically. The reverse temporalis muscle flap was then applied to a clinical case presenting with traumatic anterior skull base defect communicating with the nasal cavity. The cadaver specimens demonstrated that the superficial temporal artery formed an average 1.3 +/- 0.2 cm in width of dense vascular zone, which was located within 1.8 cm below the superior temporal line. The dense vascular network further perfused the anterior and posterior deep temporal arteries and the muscular branch of the middle temporal artery to supply the temporalis muscle. The mean perfused area of the temporalis muscle was 83 percent, ranging from 79 to 89 percent, in five cadaver heads. One cadaver revealed only 55 percent of perfused area in the absence of the muscular branch of the middle temporal artery. The consistent area without perfusion was located in the distal third of the posterior portion of the reverse temporalis muscle. In clinical cases, the reverse temporalis muscle flap was used successfully to obliterate the anterior skull base defect without evidence of muscle flap necrosis. The exact blood supply to the distal third of the posterior portion of the reverse temporalis muscle flap needs to be investigated further in vivo. Particular attention was paid to the inclusion of the muscular branch of the middle temporal artery in this flap to augment the blood supply to the temporalis muscle.     

DNA extraction and PCR amplification method suitable for fresh, herbarium-stored, lichenized, and other fungi

This paper presents a DNA extraction method suitable for fresh, herbarium-stored, lichenized and other fungal specimens. The method is fast and reliable, comparatively inexpensive and is suitable for obtaining PCR amplification quality DNA from large numbers of samples in a short time. The method has been tested with over 300 samples ofAscochyta, Phyllosticta, Ramalina, Parmelia andPhysconia. Amplifiable fungal DNA was extracted from pure cultures, leaf lesions, whole thalli and dissected only-fungal sections of lichenized fungi. In addition, the method has proved suitable for use with herbarium specimens of both lichenized and non-lichenized fungi, stored as dried pure cultures or dried infected plant material.     

Involvement of Ca2+ channels in endothelin-1-induced MAP kinase phosphorylation, myosin light chain phosphorylation and contraction in rabbit iris sphincter smooth muscle

The purpose of the present study was to investigate the role and type of Ca2+ channels involved in the stimulatory effects of endothelin-1 (ET-1) on the Ca2+-dependent functional responses, p42/p44 MAP kinase phosphorylation, 20-kDa myosin light chain (MLC) phosphorylation and contraction, in rabbit iris sphincter, a nonvascular smooth muscle. ET-1 induced inositol phosphates production, MAP kinase phosphorylation, MLC phosphorylation (MLC20-P plus MLC20-2P) and contraction in a concentration-dependent manner with EC50 values of 71, 8, 6 and 25 nM, respectively. ET-1-induced MAP kinase phosphorylation, MLC phosphorylation and contraction were not significantly affected by nifedipine (1-60 microM), an L-type Ca2+ channel blocker, or by LOE 908 (1-100 microM), a blocker of Ca2+-permeable nonselective cation channels. However, SKF96365, a receptor-operated Ca2+ channel (ROCC) blocker, inhibited MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 28, 30 and 42 microM, respectively. 2-APB, a store-operated Ca2+ channel (SOCC) blocker, inhibited ET-1-induced MLC phosphorylation and contraction in a concentration-dependent manner with IC50 values of 12.7 and 19 microM, respectively, but was without effect on MAP kinase phosphorylation. The combined effects of submaximal concentrations of SKF96365 and 2-APB on ET-1-induced MLC phosphorylation and contraction were not additive, implying that their inhibitory actions could be mediated through a common Ca2+ entry channel. PD98059, a MAP kinase inhibitor, had no effect on ET-1-induced MLC phosphorylation and contraction, suggesting that these ET-1 effects in the rabbit iris muscle are MAP kinase-independent. In conclusion, the present study demonstrated for the first time that in rabbit iris sphincter (a) ET-1, through the ETA receptor, stimulates MAP kinase phosphorylation, MLC phosphorylation and contraction in a concentration-dependent manner, (b) that these Ca2+-dependent functional responses are not significantly affected by nifedipine or LOE908, and (c) that ET-1-induced MLC phosphorylation and contraction are inhibited by SKF96365 and 2-APB, suggesting that these effects are mainly due to store- and/or receptor Ca2+ entry.     

Inorganic salts influence IAA ionization and adventitious rhizogenesis in Pongamia pinnata

The basal cut end of coppice shoot cuttings of Pongamia pinnata was treated for 24 h with 0 (water treated control) or 5.0 mmol/L of KMnO4, KCI, and KH2PO4 or 2.5 mmol/L of K2HPO4 and K2SO4. Inorganic salts of P, S, Cl and Mn significantly influenced IAA ionization and adventitious rhizogenesis. P and S salts had lower IAA ionization potential, but more pronounced effect on adventitious rhizogenesis than Cl and Mn salts. The linear regression analysis also established negative correlations between salt induced IAA ionization with various characteristics of adventitious rhizogenesis such as sprouting (r = -0.83, p < 0.05), rooting (r = -0.82, p < 0.05), root number (r = -0.95, p < 0.01), and root length (r = -0.80, p < 0.1). The implication of IAA ionization in adventitious rhizogenesis has been discussed and the possible role of inorganic salts therein suggested.     

Green tea polyphenol (-)-epigallocatechin gallate blocks epithelial barrier dysfunction provoked by IFN-gamma but not by IL-4

A characteristic of many enteropathies is increased epithelial permeability, a potentially pathophysiological event that can be evoked by T helper (Th)-1 (i.e., IFN-gamma) and Th2 (i.e., IL-4) cytokines and bacterial infection [e.g., enteropathogenic Escherichia coli (EPEC)]. The green tea polyphenol (-)-epigallocatechin gallate (EGCG) has immunosuppressive properties, and we hypothesized that it would ameliorate the increased epithelial permeability induced by IFN-gamma, IL-4, and/or EPEC. EGCG, but not the related epigallocatechin, completely prevented the increase in epithelial (i.e., T84 cell monolayer) permeability caused by IFN-gamma exposure as gauged by transepithelial resistance and horseradish peroxidase flux; EGCG did not alleviate the barrier disruption induced by IL-4 or EPEC. IFN-gamma-treated T84 and THP-1 (monocytic cell line) cells displayed STAT1 activation (tyrosine phosphorylation on Western blot analysis, DNA binding on EMSA) and upregulation of interferon response factor-1 mRNA, a STAT1-dependent gene. All three events were inhibited by EGCG pretreatment. Aurintricarboxylic acid also blocked IFN-gamma-induced STAT1 activation, but it did not prevent the increase in epithelial permeability. Additionally, pharmacological blockade of MAPK signaling did not affect IFN-gamma-induced epithelial barrier dysfunction. Thus, as a potential adjunct anti-inflammatory agent, EGCG can block STAT1-dependent events in gut epithelia and monocytes and prevent IFN-gamma-induced increased epithelial permeability. The latter event is both a STAT1- and MAPK-independent event.     

Mitochondria and autoimmunity in primary biliary cirrhosis

Primary biliary cirrhosis is an enigmatic autoimmune liver disease that predominantly affects women and is characterized by antimitochondrial antibodies and specific destruction of small bile ducts. Interestingly, patients with this disease not only have high titer antibodies to mitochondria, but also highly directed, liver-specific CD4 and CD8 cells directed at the same mitochondrial autoantigens. These mitochondrial autoantigens are all members of the 2-oxo dehydrogenase complex family and include the E2 component of pyruvate dehydrogenase as the major autoantigen. Moreover, the epitopes recognized by CD4, CD8 T cells and autoantibody, are all directed within the same region, namely the lipoyl domain of pyruvate dehydrogenase complex-E2. All cells in the body have mitochondria but there appear to be specific destruction of biliary cells. We believe that this specific destruction is secondary to a highly directed mucosal response that focuses on biliary cells because of the involvement of a polymeric immunoglobulin receptor, the presence of immunoglobulin A in mucosal secretions, and the unique apoptotic properties of biliary epithelium.     

Caveolin-1 null mice develop cardiac hypertrophy with hyperactivation of p42/44 MAP kinase in cardiac fibroblasts

Recently, development ofa caveolin-1-deficient (Cav-1 null) mouse model has allowed thedetailed analysis of caveolin-1's function in the context of awhole animal. Interestingly, we now report that the hearts ofCav-1 null mice are markedly abnormal, despite the fact that caveolin-1is not expressed in cardiac myocytes. However, caveolin-1 is abundantlyexpressed in the nonmyocytic cells of the heart, i.e., cardiacfibroblasts and endothelia. Quantitative imaging studies of Cav-1 nullhearts demonstrate a significantly enlarged right ventricular cavityand a thickened left ventricular wall with decreased systolic function.Histological analysis reveals myocyte hypertrophy withinterstitial/perivascular fibrosis. Because caveolin-1 is thought toact as a negative regulator of the p42/44 MAP kinase cascade, weperformed Western blot analysis with phospho-specific antibodies thatonly recognize activated ERK1/2. As predicted, the p42/44 MAP kinasecascade is hyperactivated in Cav-1 null heart tissue (i.e.,interstitial fibrotic lesions) and isolated cardiac fibroblasts. Inaddition, endothelial and inducible nitric oxide synthase levels aredramatically upregulated. Thus loss of caveolin-1 expression drivesp42/44 MAP kinase activation and cardiac hypertrophy.

     

Evidence for antibody-mediated enhancement of simian immunodeficiency virus (SIV) Gag antigen processing and cross presentation in SIV-infected rhesus macaques

By using the dominant simian immunodeficiency virus (SIV) Gag Mamu-A01 restricted major histocompatibility complex (MHC) class I epitope p11CM, we demonstrate antibody-mediated enhanced MHC class I cross presentation of SIV Gag. In vitro restimulation of peripheral blood mononuclear cells from SIV-infected rhesus macaques with recombinant full-length SIV Gag p55 plus p55 affinity-purified immunoglobulin G (p55 Gag/p55-IgG) led to the generation of markedly higher frequencies of p11CM specific precursor cytotoxic T lymphocytes (p-CTLs) compared with restimulation with (i) SIV Gag p55 alone or (ii) optimal concentrations of the p11CM peptide alone. These results, along with the finding that CD4 depletion abrogated the enhancement, suggest a prominent role for CD4(+) T cells. Testing for p-CTLs against other Mamu-A01-restricted SIV Gag epitopes suggested that this mechanism favored recognition of the dominant p11CM peptide, potentially further skewing of the CTL response. The p-CTL enhancing effect was also decreased or abrogated by pepsin digestion of the p55-specific IgG or by the addition of monoclonal antibodies to Fc receptor (FcR) II/III, suggesting that the effect was dependent on FcR-mediated uptake of the immune-complexed antigen. Finally, incubation of antigen-presenting cells with SIV Gag p55 immune complexes in the presence of lactacystin or of bafilomycin indicated that the mechanism of antibody-mediated enhancement of cross presentation required both the proteasomal and the endosomal pathways. These data demonstrate for the first time the cross presentation of antigens via immune complexes in lentiviral infection and indicate a heretofore-unrecognized role for antibodies in modulating the magnitude and potentially also the breadth of MHC class I-restricted antigen processing and presentation and CTL responses.     

Caveolin-1 null (-/-) mice show dramatic reductions in life span

Caveolae are 50-100 nm flask-shaped invaginations of the plasma membrane found in most cell types. Caveolin-1 is the principal protein component of caveolae membranes in nonmuscle cells. The recent development of Cav-1-deficient mice has allowed investigators to study the in vivo functional role of caveolae in the context of a whole animal model, as these mice lack morphologically detectable caveolae membrane domains. Surprisingly, Cav-1 null mice are both viable and fertile. However, it remains unknown whether loss of caveolin-1 significantly affects the overall life span of these animals. To quantitatively determine whether loss of Cav-1 gene expression confers any survival disadvantages with increasing age, we generated a large cohort of mice (n = 180), consisting of Cav-1 wild-type (+/+) (n = 53), Cav-1 heterozygous (+/-) (n = 70), and Cav-1 knockout (-/-) (n = 57) animals, and monitored their long-term survival over a 2 year period. Here, we show that Cav-1 null (-/-) mice exhibit an approximately 50% reduction in life span, with major declines in viability occurring between 27 and 65 weeks of age. However, Cav-1 heterozygous (+/-) mice did not show any changes in long-term survival, indicating that loss of both Cav-1 alleles is required to mediate a reduction in life span. Mechanistically, these dramatic reductions in life span appear to be secondary to a combination of pulmonary fibrosis, pulmonary hypertension, and cardiac hypertrophy in Cav-1 null mice. Taken together, our results provide the first demonstration that loss of Cav-1 gene expression and caveolae organelles dramatically affects the long-term survival of an organism. In addition, aged Cav-1 null mice may provide a new animal model to study the pathogenesis and treatment of progressive hypertrophic cardiomyopathy and sudden cardiac death syndrome.