Leptospirosis pulmonary haemorrhage syndrome is associated with linear deposition of immunoglobulin and complement on the alveolar surface

Leptospirosis is a zoonotic infection associated with severe diseases such as leptospirosis pulmonary haemorrhage syndrome (LPHS). The cause of pulmonary haemorrhage is unclear. Understanding which mechanisms and processes are involved in LPHS will be important in treatment regimens under development for this life-threatening syndrome. In the present study, we evaluated 30 lung specimens from LPHS patients and seven controls using histology and immunohistochemistry (detection of IgM, IgG, IgA and C3) in order to describe the pathological features associated with this syndrome. Immunoglobulin deposits were detected on the alveolar surface in 18/30 LPHS patients. Three staining patterns were observed for the immunoglobulins and C3 in the lung tissues of LPHS patients: AS, delicate linear staining adjacent to the alveolar surface, which was indicative of a membrane covering the luminal surface of type I and II pneumocyte cells; S, heterogeneous staining which was sporadically distributed along the alveolar septum; and IA, weak, focal intra-alveolar granular staining. Human LPHS is associated with individual and unique histological patterns that differ from those of other causes of pulmonary haemorrhage. In the present study, it was found that the linear deposition of immunoglobulins (IgA, IgG and IgM) and complement on the alveolar surface may play a role in the pathogenesis of pulmonary haemorrhage in human leptospirosis.

TGF-beta and mesenchymal hepatic involvement after visceral leishmaniasis

The liver involvement in the human visceral leishmaniasis (VL) has been related to parasitism and activated Kupffer cells with further occasional fibrotic alterations, especially after long-term disease without treatment. However, fibrotic alterations have been reported after therapy, whose clinical finding is the persistence of hepatomegaly. Fibrotic involvement of the liver after therapy was never well understood, and the aim of this study was to evaluate this finding through ultrastructural and morphometric analysis. A case-control study was performed with 20 patients (15 cases and five controls). Cases included patients with persistent hepatomegaly (residual) after treatment of VL submitted to liver biopsy to exclude other causes of liver enlargement, including serum tests of viral hepatitis. The material was evaluated by electron microcopy allowing ultrastructural with morphometric analysis of medium portion of hepatic lobule. Narrow sinusoidal lumen and prominent Kupffer cells were found with insignificant alterations of hepatocytes, pit, and endothelial cells. On ultrastructural analysis, the enlargement of the space of Disse was due to fibrous collagen, increase of number of Ito cells, and nonfibrous extracellular matrix that were associated with Kupffer cells enlargement. Immunohistochemistry showed an intense expression of TGF-beta in patients with VL. These findings suggest a production of TGF-beta by Kupffer cells that resulted in the characteristic fibrotic involvement of the liver. Residual hepatomegaly in visceral leishmaniasis could result from sustained Kupffer cell activation with perihepatocytic fibrosis.

Pathogenic role of anti-endothelial cell antibodies in autoimmune rheumatic diseases

Anti-endothelial cells antibodies have been detected in numerous autoimmune and inflammatory diseases, including systemic lupus erythematous, rheumatoid arthritis, vasculitis and sarcoidosis. Anti-endothelial cells antibodies bind to endothelial cell antigens and induce endothelial damage. Their effects on the endothelial cell have been considered responsible, at least in part, by the vascular injury which occurs in these pathological conditions. Lupus (2009) 18, 1233-1238.

Induction of CRP3/MLP expression during vein arterialization is dependent on stretch rather than shear stress

Aims Cysteine- and glycine-rich protein 3/muscle LIM-domain protein (CRP3/MLP) mediates protein-protein interaction with actin filaments in the heart and is involved in muscle differentiation and vascular remodelling. Here, we assessed the induction of CRP3/MLP expression during arterialization in human and rat veins. Methods and results Vascular CRP3/MLP expression was mainly observed in arterial samples from both human and rat. Using quantitative real time RT-PCR, we demonstrated that the CRP3/MLP expression was 10 times higher in smooth muscle cells (SMCs) from human mammary artery (h-MA) vs. saphenous vein (h-SV). In endothelial cells (ECs), CRP3/MLP was scarcely detected in either h-MA or h-SV. Using an ex vivo flow through system that mimics arterial condition, we observed induction of CRP3/MLP expression in arterialized h-SV. Interestingly, the upregulation of CRP3/MLP was primarily dependent on stretch stimulus in SMCs, rather than shear stress in ECs. Finally, using a rat vein in vivo arterialization model, early (1-14 days) CRP3/MLP immunostaining was observed predominantly in the inner layer and later (28-90 days) it appeared more scattered in the vessel layers. Conclusion Here we provide evidence that CRP3/MLP is primarily expressed in arterial SMCs and that stretch is the main stimulus for CRP3/MLP induction in veins exposed to arterial haemodynamic conditions.

Angiotensin II Binding to Angiotensin I-Converting Enzyme Triggers Calcium Signaling

Angiotensin (Ang) I-converting enzyme (ACE) is involved in the control of blood pressure by catalyzing the conversion of Ang I into the vasoconstrictor Ang II and degrading the vasodilator peptide bradykinin. Human ACE also functions as a signal transduction molecule, and the binding of ACE substrates or its inhibitors initiates a series of events. In this study, we examined whether Ang II could bind to ACE generating calcium signaling. Chinese hamster ovary cells transfected with an ACE expression vector reveal that Ang II is able to bind with high affinity to ACE in the absence of the Ang II type 1 and type 2 receptors and to activate intracellular signaling pathways, such as inositol 1,4,5-trisphosphate and calcium. These effects could be blocked by the ACE inhibitor, lisinopril. Calcium mobilization was specific for Ang II, because other ACE substrates or products, namely Ang 1-7, bradykinin, bradykinin 1-5, and N-acetyl-seryl-aspartyl-lysyl-proline, did not trigger this signaling pathway. Moreover, in Tm5, a mouse melanoma cell line endogenously expressing ACE but not Ang II type 1 or type 2 receptors, Ang II increased intracellular calcium and reactive oxygen species. In conclusion, we describe for the first time that Ang II can interact with ACE and evoke calcium and other signaling molecules in cells expressing only ACE. These findings uncover a new mechanism of Ang II action and have implications for the understanding of the renin-Ang system. (Hypertension. 2011;57:965-972.) . Online Data Supplement

Gangliosides Are Important for the Preservation of the Structure and Organization of RBL-2H3 Mast Cells

Gangliosides are known to be important in many biological processes. However, details concerning the exact function of these glycosphingolipids in cell physiology are poorly understood. in this study, the role of gangliosides present on the surface of rodent mast cells in maintaining cell structure was examined using RBL-2H3 mast cells and two mutant cell lines (E5 and D1) deficient in the gangliosides, GM(1) and the alpha-galactosyl derivatives of the ganglioside GD(1b). The two deficient cell lines were morphologically different from each other as well as from the parental RBL-2H3 cells. Actin filaments in RBL-2H3 and E5 cells were under the plasma membrane following the spindle shape of the cells, whereas in D1 cells, they were concentrated in large membrane ruffles. Microtubules in RBL-2H3 and E5 cells radiated from the centrosome and were organized into long, straight bundles. The bundles in D1 cells were thicker and organized circumferentially under the plasma membrane. The endoplasmic reticulum, the Golgi complex, and the secretory granule matrix were also altered in the mutant cell lines. These results suggest that the mast cell-specific alpha-galactosyl derivatives of ganglioside GD(1b) and GM(1) are important in maintaining normal cell morphology. (J Histochern Cytochem 58:83-93, 2010)

DC-SIGN (CD209) gene promoter polymorphisms in a Brazilian population and their association with human T-cell lymphotropic virus type 1 infection

This study evaluated four polymorphisms located in the DC-SIGN (CD209) gene promoter region (positions -336, -332 -201 and -139) in DNA samples from four Brazilian ethnic groups (Caucasians, Afro-Brazilian, Asians and Amerindians) to establish the population distribution of these single-nucleotide polymorphisms (SNPs) and correlated DC-SIGN polymorphisms and infection in samples from human T-cell lymphotropic virus type 1 (HTLV-1)-infected individuals. To identify CD209 SNPs, 452 bp of the CD209 promoter region were sequenced and the genotype and allelic frequencies were evaluated. This is the first study to show genetic polymorphism in the CD209 gene in distinct Brazilian ethnic groups with the distribution of allelic and genotypic frequency. The results showed that -336A and -139A SNPs were quite common in Asians and that the -201T allele was not observed in Caucasians, Asians or Amerindians. No significant differences were observed between individuals with HTLV-1 disease and asymptomatic patients. However, the -336A variant was more frequent in HTLV-1 -infected patients [HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), 80%; healthy asymptomatic HTLV-1 carriers, 90 %] than in the control group (70 %) [P=0.0197, odds ratio (OR)=2.511, 95 % confidence interval (CI)=1.218-5.179). In addition, the -139A allele was found to be associated with protection against HTLV-1 infection (P=0.0037, OR=0.3758, 95% CI=0.1954-0.7229) when the HTLV-1 -infected patients as a whole were compared with the healthy-control group. These observations suggest that the -139A allele may be associated with HTLV-1 infection, although no significant association was observed among asymptomatic and HAM/TSP patients. In conclusion, the variation observed in SNPs -336 and -139 indicates that this lectin may be of crucial importance in the susceptibility/transmission of HTLV-1 infections.

Neutrophil migration induced by IL-1 beta depends upon LTB4 released by macrophages and upon TNF-alpha and IL-1 beta released by mast cells

In the present study, we investigate whether mast cells and macrophages are involved in the control of IL-1 beta-induced neutrophil migration, as well as the participation of chemotactic mediators. IL-1 beta induced a dose-dependent neutrophil migration to the peritoneal cavity of rats which depends on LTB4, PAF and cytokines, since the animal treatment with inhibitors of these mediators (MK 886, PCA 4248 and dexamethasone respectively) inhibited IL-1 beta-induced neutrophil migration. The neutrophil migration induced by IL-1 beta is dependent on mast cells and macrophages, since depletion of mast cells reduced the process whereas the increase of macrophage population enhanced the migration. Moreover, mast cells or macrophages stimulated with IL-1 beta released a neutrophil chemotactic factor, which mimicked the neutrophil migration induced by IL-1 beta. The chemotactic activity of the supernatant of IL-1 beta-stimulated macrophages is due to the presence of LTB4, since MK 886 inhibited its release. Moreover, the chemotactic activity of IL-1 beta-stimulated mast cells supernatant is due to the presence of IL-1 beta and TNF-alpha, since antibodies against these cytokines inhibited its activity. Furthermore, significant amounts of these cytokines were detected in the supernatant. In conclusion, our results suggest that neutrophil migration induced by IL-1 beta depends upon LTB4 released by macrophages and upon IL-1 beta and TNF alpha released by mast cells.

Volume-activated chloride channels in mice Leydig cells

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume- activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of similar to 75 mOsm. These currents display the typical biophysical signature of volume- activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(-)>Cl(-)>F(-)). Staurosporine (200 nM) did not block the activation of I(Cl), swell. The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 mu M), SITS (200 mu M), ATP (500 mu M), pyridoxalphosphate-6- azophenyl-2`,4`-disulfonate (PPADS; 100 mu M), and Suramin (10 mu M) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(d)(0) and fractional distance of the binding site (delta) of 334 mu M and 47%, 880 mu M and 35%, 2,100 mu M and 49%, 188 mu M and 27%, and 66.5 mu M and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that ICl, swell in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Immunohistochemical study of stromal and vascular components of tonsillar polyps: high endothelial venules as participants of the polyp`s lymphoid tissue

Tonsillar polyps are nonneoplastic lesions usually composed of variable amounts of lymphoid and vascular and connective tissues. All of them are generally assumed to be hamartomatous proliferations, but the profile of vascular and connective components has yet to be explored. The vascular system of the tonsils is complex and includes highly specialized structures (i.e., high endothelial venules (HEVs)) involved in lymphocyte homing into lymphoid tissues. In 14 tonsillar polyps and 26 control tonsils, an immunohistochemical study was performed using CD34 (blood vessels and HEVs), MECA-79 (HEVs), D2-40 (lymphatic vessels), Ki-67, collagens I and III, fibronectin, and tenascin-C. The polyps showed increased total lymphatic area, whereas the number of blood vessels and lymphatics and the blood vascular area did not differ significantly from those of control tonsils. Rare Ki-67+ endothelial cells were found. In the polyps, we detected, possibly for the first time, HEVs amid lymphoid tissue, and that the amount of the latter correlated positively with HEV density. The polyps also presented lesser amounts of fibronectin and collagens I and III than in normal tonsils, which were distributed in a disorganized fashion. Tenascin-C expression was uncommon in the polyps and control tonsils. Tonsillar polyps are composed of disorganized connective tissue and lymphatic channels which can be considered hamartomatous proliferations. However, the lymphoid component is possibly reactive due to its relationship with the HEVs. The highly differentiated phenotype of the HEVs and their complex biology are not in agreement with what would be expected for a component of hamartomatous nature.

Intravascular papillary endothelial hyperplasia: Report of 4 cases with immunohistochemical findings

Intravascular papillary endothelial hyperplasia (IPEH) is a benign endothelial proliferation, usually intravascular, that may mimic angiosarcoma. In this report, four new cases of IPEH involving the oral region are described. The affected sites were the lower lip, labial comissure and the submandibular region. After clinical evaluation, the complete removal of the lesions showed a circumscribed and soft mass. Histologically, the major feature was a reactive proliferation of endothelial cells composed of small papillary structures with hypocellular and hyalinized cores arising in an organized thrombus. Immunohistochemical staining for CD34 was strongly positive in endothelial cells. Vimentin and laminin immunolabelling were also consistent with a vascular origin. In order to verify the proliferative potential of the lesions, the Ki-67 antibody was used, revealing low percentage of labeled cells (<20%). No immunoreactivity for GLUT-1 was observed. Since the complete removal is curative, no additional treatment was necessary, and no signs of recurrence had been observed until now. Due to the particular features of IPEH, it is important for pathologists and clinicians to become familiar with this lesion. Additionally, the specific histological arrangement, including the absence of cellular pleomorphism, mitotic activity and necrosis, represents a guide to help in the differential diagnosis. Moreover, the vascular origin and the proliferative index should be assessed by immunohistochemistry in order to provide an accurate diagnosis.

Effect of PTK/ZK on the Angiogenic Switch in Head and Neck Tumors

Transformation of small avascular masses of tumor cells into rapidly progressive cancers is triggered by the angiogenic switch, a process that involves vascular endothelial growth factor (VEGF) signaling. We have shown that VEGF enhances the survival and angiogenic potential of endothelial cells by activating the Bcl-2-CXCL8 signaling axis. The purpose of this study was to evaluate the effect of a small-molecule inhibitor of VEGF receptors (PTK/ZK) on the initial stages of head and neck tumor angiogenesis. In vitro, PTK/ZK blocked head and neck tumor cell (OSCC3 or UM-SCC-17B)-induced Bcl-2 and CXCL8 expression in endothelial cells. Oral administration of PTK/ZK decreased xenograft head and neck tumor microvessel density, and inhibited Bcl-2 and CXCL8 expression in tumor-associated endothelial cells. Analysis of these data demonstrates that PTK/ZK blocks downstream targets of VEGF signaling in endothelial cells, and suggests that PTK/ZK may inhibit the angiogenic switch in head and neck tumors. Abbreviations: HDMEC, human dermal microvascular endothelial cells; VEGF, vascular endothelial growth factor; CXCL8, CXC ligand-8; PTK/ZK, PTK787/ZK222584.

Human osteoblastic cell response to a Ca- and P-enriched titanium surface obtained by anodization

The aim of the present study was to evaluate the in vitro osteogenic potential of subcultured human osteoblastic cells derived from alveolar bone on a titanium (Ti) surface produced by an anodized alkali treatment (BSP-AK). Primary osteoblastic cells were subcultured on BSP-AK and machined Ti discs (control) and grown for periods of up to 21 days under osteogenic conditions. Morphologic and biochemical methods were used to assess important parameters of in vitro bone-like tissue formation. Although no major differences were observed between the BSP-AK and the control Ti surface in terms of cell attachment and mineralized matrix formation, a significant increase in cell population, ALP activity, and collagen content was detected in cultures on BSP-AK surface. Our results demonstrate that human osteoblastic cells are sensitive to the BSP-AK-modified Ti surface during the transitional stage between the end of the proliferative phase and the onset of the differentiation /matrix maturation ones. Together with the good mechanical properties exhibited by the Ca- and P- coating, our findings suggest that BSP-AK treatment could be useful for the development of a new surface for dental and orthopedic implants. (c) 2008 Wiley Periodicals, Inc.J Biomed Mater Res 88A: 841-848, 2009

Human papillomavirus type 6b virus-like particles are able to activate the Ras-MAP kinase pathway and induce cell proliferation

The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the alpha6 integrin. The alpha6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. sere we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is Lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in fell proliferation, suggesting involvement of the Ras-MAP kinase pathway, Indeed, VLP binding results in rapid phosphorylation of the beta4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein She to beta4, Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c-myc mRNA was observed at 60 min, These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

Corneal endothelial response to induced myopia in the chicken

Purpose: To investigate the role of corneal endothelial surface enlargement in the chicken myopia model in inducing corneal endothelial changes. Methods: Lid suture was performed on one eye of 1-day-old cockerels. Five chickens were killed at 1 week, and four chickens killed at each of 3 weeks, 6 weeks, and 10 weeks postnatal. The endothelial morphology was obtained by flat mounting the endothelial surface and the subsequent digitisation. Comparisons were undertaken between the control unsutured eye and the lid-sutured eye endothelium, and between the central endothelial areas compared to the peripheral endothelial areas in both the myopic and the normal corneas. Calculation of the contribution to the endothelial change by hypertrophy and mitosis were calculated using Bahn's formula. Results: Total endothelial surface area increased significantly over time in the myopic model compared to control eyes but the mean cell area of endothelial cells remained the same for both the enlarged myopic endothelial surface area and in the normal controls. Sampling from the central and the peripheral corneal endothelial surface also disclosed no difference. The mean cell area did increase steadily with age but was the same for both normal and myopic corneas. Conclusions: It would appear that there are equal contributions from hypertrophy and mitosis in the myopic group and the normal corneal group with a slightly increasing trend towards mitotic activity in the myopic corneal endothelial layer.