Expression and regulation of αB-crystallin in the kidney in vivo and in vitro

αB-crystallin, a major component of the mammalian eye lens, is a small heat shock protein and molecular chaperone that is also abundant in the mammalian kidney. The present study aimed to characterize more closely the intrarenal expression and regulation of αB-crystallin in vivo and in vitro. In normal rat kidney, the expression of αB-crystallin mRNA and protein were both close to the detection limit in cortex, but increased steeply from the outer to the inner medulla where αB-crystallin constitutes approximately 2% of total tissue protein. Immunohistochemistry disclosed papillary collecting duct cells and thin limbs as the major sites for intrapapillary αB-crystallin immunoreactivity. In rats subjected to sucrose diuresis for 3 days, αB-crystallin mRNA expression was reduced by 27 and 46% in outer and inner medulla, respectively. In agreement with the results obtained in vivo, in Madine–Darby canine kidney cells, αB-crystallin mRNA and protein were induced significantly by elevating the medium osmolality to 500 mosm/kg H₂O by the addition of NaCl and raffinose, and also by urea. The NaCl-induced increase in αB-crystallin expression was concentration-dependently blunted by SP600125, a specific JNK inhibitor. Overexpression of αB-crystallin in 293 cells resulted in increased tolerance to acute osmotic stress. These results indicate that αB-crystallin may be regulated by papillary interstitial tonicity in a JNK-dependent process. Moreover, the high abundance of αB-crystallin in the renal medulla may be important for cell survival in an environment characterized by extreme interstitial solute concentrations as present during antidiuresis.     

Adiponectin/T-cadherin and apelin/APJ expression in human arteries and periadventitial fat: implication of local adipokine signaling in atherosclerosis?

IntroductionAdipose tissue is considered an endocrine organ, producing bioactive peptides, called adipokines. Adipokines produced by periadventitial fat have been implicated in the pathogenesis of vascular disease, including atherosclerosis. Adiponectin has established antiatherogenic actions, while the role of T-cadherin as an adiponectin receptor is not fully elucidated. The apelinergic system, consisting of apelin and its APJ receptor, is a mediator of various cardiovascular functions and may also be involved in the atherosclerotic process. We investigated the protein expression of adiponectin, T-cadherin, apelin and APJ in human aortas, coronary vessels, and the respective periadventitial adipose tissue and correlated their expression with the presence of atherosclerosis and clinical parameters.MethodsImmunohistochemistry for adiponectin, T-cadherin, apelin, and APJ was performed on human aortic and coronary artery samples including the periadventitial adipose tissue. Aortic and coronary atherosclerotic lesions were assessed using the american heart association (AHA) classification.ResultsAdiponectin immunostaining, of varied intensity, was detected only in adipocytes, while T-cadherin was localized to vascular smooth muscle cells (VSMCs) and endothelial cells. Apelin immunostaining was detected in adipocytes, VSMCs, endothelial cells, and foam cells in atherosclerotic lesions, while APJ was found in VSMCs and endothelia. Periadventitial adiponectin and VSMC T-cadherin expression were negatively correlated with atherosclerosis in both sites, as was VSMC apelin expression. Several other — depot specific — associations were observed.ConclusionsOur results suggest a possible role for T-cadherin as a mediator of antiatherogenic adiponectin actions, while they support the putative antiatherogenic profile for apelin and its APJ receptor in human arteries. Further research is absolutely necessary to confirm these notions.SummaryPeriadventitial adipose tissue adipokines are implicated in vascular physiology and pathology. Adiponectin/T-cadherin and apelin/APJ immunoreactivity is detected in human aortas and coronary arteries. Adiponectin/T-cadherin and apelin/APJ expression patterns were found to be inversely associated with human aortic and coronary atherosclerosis.     

Structure and function of Norrin in assembly and activation of a Frizzled 4–Lrp5/6 complex

Norrin is a cysteine-rich growth factor that is required for angiogenesis in the eye, ear, brain, and female reproductive organs. It functions as an atypical Wnt ligand by specifically binding to the Frizzled 4 (Fz4) receptor. Here we report the crystal structure of Norrin, which reveals a unique dimeric structure with each monomer adopting a conserved cystine knot fold. Functional studies demonstrate that the novel Norrin dimer interface is required for Fz4 activation. Furthermore, we demonstrate that Norrin contains separate binding sites for Fz4 and for the Wnt ligand coreceptor Lrp5 (low-density lipoprotein-related protein 5) or Lrp6. Instead of inducing Fz4 dimerization, Norrin induces the formation of a ternary complex with Fz4 and Lrp5/6 by binding to their respective extracellular domains. These results provide crucial insights into the assembly and activation of the Norrin–Fz4–Lrp5/6 signaling complex.     

Evaluation of the in vitro and in vivo proinflammatory activities of gold (+) and gold (−) nanoparticles

Objective and design

The aim of this study was to determine potential effects of gold (+) and gold (?) nanoparticles, AuNP(+) and AuNP(?), on neutrophil biology.

Material or subjects

Freshly isolated human neutrophils were used for the in vitro aspects and CD-1 mice were used in the in vivo murine air pouch model of acute neutrophilic inflammation.

Treatment

Human neutrophils were treated with the indicated concentrations of AuNP(+) or AuNP(?) in vitro and mice received 100 or 500 µg/ml AuNP(+) or AuNP(?) into air pouches.

Methods

Cellular uptake of AuNP by neutrophils was confirmed by transmission electron microscopy and the ability of the NP to modulate apoptosis, gelatinase activity, and chemokine production and chemotaxis was determined by cytology, zymography, ELISArray, antibody array, and ELISA and by a micro-chemotaxis chamber, respectively. In vivo, exudates were harvested after 6 h to determine the leukocyte infiltration to detect the production of several cytokines by an antibody array approach and ELISA. One-way analysis of variance was used for statistical analysis.

Results

AuNP possess proinflammatory activities in vitro and induce mainly a neutrophil influx in vivo, albeit at different degrees.

Conclusions

AuNP(+) and AuNP(?) should be added as new candidates into a growing list of NP having proinflammatory activities by themselves.

     

Persistence of TEM-52/TEM-92 and SHV-12 extended-spectrum β-lactamases in clinical isolates of Enterobacteriaceae in Italy

Extended-spectrum β-lactamases (ESBLs) belonging to the TEM and SHV families were investigated in 583 ESBL-producing Enterobacteriaceae collected at the clinical microbiology laboratories of 11 teaching Italian hospitals. By molecular analysis TEM-type and SHV-type ESBLs were confirmed on 154 and 74 isolates, respectively. High variability was found among TEM-types β-lactamases with the following variants: TEM-5, TEM-6, TEM-12, TEM-15, TEM-24, TEM-26, TEM-29, TEM-52, TEM-92, TEM-134, and TEM-149. Among SHV variants, SHV-2a, SHV-5, SHV-12, and SHV-28 have been detected. The most widespread variants are TEM-52/92 and SHV-12.     

Usefulness of Negative and Weak–Diffuse Pattern of SDHB Immunostaining in Assessment of SDH Mutations in Paragangliomas and Pheochromocytomas

This is a confirmatory study about usefulness of SDHB and SDHA immunostaining in assessment of SDH mutations in paragangliomas and pheochromocytomas. Paraganglioma/pheochromocytoma syndrome (PGL/PCC syndrome) consists of different entities, associated with germline mutations in five different genes: SDHD, SDHAF2, SDHC, SDHA and SDHB. It has been suggested that negative immunostaining of SDHB can be taken as an indicator of the presence of a mutation in one of the five SDH genes. We have performed SDHB and SDHA immunohistochemical staining in a series of paragangliomas and pheochromocytomas from 64 patients. The patients had been previously checked for mutations in SDHD, SDHC and SDHB, but also for mutation in RET and VHL. All 14 patients with SDH mutations (9 with SDHB and 5 with SDHD mutations) exhibited negative or weak–diffuse SDHB staining pattern in tumour tissue, whereas cells of the 23 RET mutated and 8 VHL mutated tumours showed a positive SDHB immunostaining. Sixteen of the patients that did not exhibit a mutation in any gene showed positive SDHB immunostaining in tumour tissue, while only three of the patients without mutation exhibited negative staining. All patients exhibited positive pattern of SDHA immunostaining. The results confirm the value of SDHB immunohistochemical status in assessment of germline mutations in PGL/PCC syndrome.     

Accumulation of γ/δ T Cells in Human Dysgerminoma and Seminoma: Roles in Autologous Tumor Killing and Granuloma Formation

The precise biological function of a subset of T cells bearing γ/δ T cell receptor (TCR) remains poorly understood. The present study demonstrated the presence of γ/δ T cells in tumor-infiltrating lymphocytes (TIL) and peripheral blood lymphocytes (PBL) of human patients with dysgerminoma and seminoma when determined by flow cytometry and in situ immunohistochemical staining. TIL contained a high percentage of γ/δ T cells, ranging from 17.3 to 35.1%. γ/δ T cells often accumulated within the granulomatous inflammation of tumor tissues. The majority of γ/δ T cells were Vγ9/Vδ2+ cells. Freshly isolated PBL, TIL and purified γ/δ T cells showed autologous tumor killing (ATK) activity, which could be inhibited by monoclonal antibodies (mAb) against Vδ2. Furthermore, two γ/δ T cell clones established from TIL showed cytotoxicity against autologous and allogeneic dysgerminoma, while they had low or no lytic effects on other cell types including carcinomas of ovary and tumor cell lines such as K562, Daudi and Molt-4. Lysis of autologous tumor cells by the clone was inhibited completely by anti-Vδ2 mAb and partially by mAb against CD3, LFA-1α and ICAM-1 molecules, while it was resistant to anti-CD8, anti-HLA-ABC and anti-HLA-DR mAb. Supernatants produced by γ/δ T cell clones induced adhesion, aggregation and increased DNA synthesis of monocytes and some characteristics of activated macrophages or epithelioid cells. Tumor necrosis factor (TNF)-α, granulocyte-macrophage colony stimulating factor (GM-CSF) and interferon (IFN)-γ were detected in the supernatants of γ/δ T cell clone. These results suggest that γ/δ T cells accumulating in dysgerminoma and seminoma exhibit ATK activity through Vγ9/δ2 TCR and these γ/δ T cells also play a role in the formation of granulomatous inflammation, which is associated with human dysgerminoma and seminoma.     

Effects of pioglitazone and/or simvastatin on circulating TNFα and adiponectin levels in insulin resistance

The current study investigated the effects of 14-day pioglitazone (PIO) and/or simvastatin (SIM) treatments on serum adiponectin (Adp) and TNFα levels (markers of adipocyte dysfunction), as well as on metabolic perturbations that arise from prolonged (8 week) consumption of a high fructose (HFD; 60%) diet in a rat model of pre-diabetic insulin resistance. The HFD induced a deranged lipid profile that was associated with adipose tissue hypertrophy, increased ratios of visceral and epididymal fats to body weight, and fatty liver. These perturbations were associated with hypo-adiponectinemia (50.8%) and increased serum TNFα (6.5-fold) levels. Treatment with PIO ameliorated the altered blood and hepatic glucose metabolism via an Adp-dependent mechanism; PIO also mitigated the changes in blood TNFα and led to a hyperelevation of Adp levels. SIM amended hepatic and overall lipid metabolism, regulated TNFα, but failed to alter the glucose intolerance or significantly impact on the HFD-altered Adp levels. Coadministration of SIM + PIO was superior in improving overall metabolic parameters compared to each monotherapy. Cotreatment was optimal in reestablishing insulin resistance, most efficacious in improving serum lipid profiles, normalizing percentage ratios of epididymal and visceral fats to body weight, and augmenting Adp/reducing TNFα levels relative to that in the HFD group or with HFD + each drug alone. The results here show that use of either monotherapy or a combined SIM + PIO approach might, from a clinical perspective, provide an ability to delay progression to Type 2 diabetes and its associated inflammatory/cardiovascular effects.     

Expression of Bcl-2 and Survivin in uterine cervical carcinogenesis and correlation between the expression and infection of HPV_(16/18)

Carcinomaofthecervixisthesecondleading causeofdeathamongwomenworldwide.Each year,anestimated500000casesarenewlydiag nosed[1].Manystudiesshowedinfectionofhu manpapillomavirus(HPV)wasoneofthemost importantetiologicfactorsforcervicalcarcinoma[24].Morethan95%ofallcervicalcarcinomas havebeenfoundtobeassociatedwithHPV(ma inlytypes16and18)[5].Thestudiesinmolec ularoncologyrecentlyshowedthatitresultedin occurrenceanddevelopmentofcervicalcarcinomasthatcarcinogenicfactorsmadeproto oncogene activatean…     

Diverse roles of TGF-β receptor II in renal fibrosis and inflammation in vivo and in vitro

TGF-β1 binds receptor II (TβRII) to exert its biological activities but its functional importance in kidney diseases remains largely unclear. In the present study, we hypothesized that TβRII may function to initiate the downstream TGF-β signalling and determine the diverse role of TGF-β1 in kidney injury. The hypothesis was examined in a model of unilateral ureteral obstructive (UUO) nephropathy and in kidney fibroblasts and tubular epithelial cells in which the TβRII was deleted conditionally. We found that disruption of TβRII inhibited severe tubulointerstitial fibrosis in the UUO kidney, which was associated with the impairment of TGF-β/Smad3 signalling, but not with the ERK/p38 MAP kinase pathway. In contrast, deletion of TβRII enhanced NF-κB signalling and renal inflammation including up-regulation of Il-1β and Tnfα in the UUO kidney. Similarly, in vitro disruption of TβRII from kidney fibroblasts or tubular epithelial cells inhibited TGF-β1-induced Smad signalling and fibrosis but impaired the anti-inflammatory effect of TGF-β1 on IL-1β-stimulated NF-κB activation and pro-inflammatory cytokine expression. In conclusion, TβRII plays an important but diverse role in regulating renal fibrosis and inflammation. Impaired TGF-β/Smad3, but not the non-canonical TGF-β signalling pathway, may be a key mechanism by which disruption of TβRII protects against renal fibrosis. In addition, deletion of TβRII also enhances NF-κB signalling along with up-regulation of renal pro-inflammatory cytokines, which may be associated with the impairment of anti-inflammatory properties of TGF-β1.     

Immunohistochemical localization of α- and βA-subunits of inhibin/activin in human normal endocrine cells and related tumors of the digestive system

 Activin A and inhibin A, first isolated from the ovary, are dimeric proteins able to modulate pituitary FSH secretion. Inhibin A is a heterodimer composed of one α-subunit and one βA-subunit (α-βA), while activin A is a homodimer of the βA-subunit (βA-βA). Their identification in several tissues has suggested that they have numerous physiological functions, acting as either paracrine or autocrine factors. The aim of this study was to evaluate the expression of activin A and inhibin A in normal endocrine cells and in 70 endocrine tumours from different sites in the gastro-entero-pancreatic system, using specific monoclonal antibodies directed against the α- and βA-subunits of inhibin/activin. Immunoreactivity for the βA-subunit, but not for the α-subunit, was observed in normal G, EC, and GIP cells of the antrum and duodenum, and in pancreatic A cells. βA-subunit expression was observed in G cell and A cell tumours, and in a few insulinomas and ileal EC cell carcinoids. The α-subunit was found in rare cells in 7 of the 70 tumours and was colocalized with the βA-subunit in only 1 tumor. Specific types of endocrine cells from the gut and pancreas appear to produce only activin A, a possible paracrine or autocrine modulator. Activin A is mainly produced by tumours derived from endocrine cells that normally express it. Received: 17 July 1998 / Accepted: 30 July 1998     

Natural Infections of Angiostrongylus vasorum and Crenosoma vulpis in Dogs in Germany (2007–2009)

In order to assess the occurrence and regional geographical distribution of Angiostrongylus vasorum and Crenosoma vulpis in Germany, faecal samples of 810 dogs with clinical symptoms of respiratory and circulatory disease, bleeding disorder and/or neurological signs were collected from September 2007 to March 2009. The zinc chloride/sodium chloride flotation and Baermann funnel technique were used to examine the samples for presence of lungworm larvae. Infections with lungworms were diagnosed in 105 (13.0%) of the examined dogs. A. vasorum and C. vulpis were found in 60 (7.4%) and 49 (6.0%) faecal samples, respectively. 33 A. vasorum- and 12 C. vulpis-positive dogs were located in Baden-Württemberg, 13 and 12 in North Rhine-Westphalia, 3 and 4 in Bavaria, 1 and 7 in Rhineland-Palatinate, 7 and 4 in Saarland, 1 and 2 in Saxony, respectively. In Brandenburg only 2 dogs with A. vasorum and in Hesse a total of 5 dogs with C. vulpis were detected. In Mecklenburg-Western Pomerania, Lower Saxony and Thuringia only 1 dog with C. vulpis was detected at a time. 4 dogs were coinfected with A. vasorum and C. vulpis. These surprisingly high prevalence rates indicate that both parasites are endemic in Germany.     

Expression and Localization of α-amylase in the Submandibular and Sublingual Glands of Mice

In the major salivary glands of mice, acinar cells in the parotid gland (PG) are known to be the main site for the production of the digestive enzyme α-amylase, whereas α-amylase production in the submandibular gland (SMG) and sublingual gland (SLG), as well as the cell types responsible for α-amylase production, has been less firmly established. To clarify this issue, we examined the expression and localization of both the mRNA and protein of α-amylase in the major salivary glands of male and female mice by quantitative and histochemical methods. α-amylase mRNA levels were higher in the order of PG, SMG, and SLG. No sexual difference was observed in α-amylase mRNA levels in the PG and SLG, whereas α-amylase mRNA levels in the female SMG were approximately 30% those in the male SMG. Using in situ hybridization and immunohistochemistry, signals for α-amylase mRNA and protein were found to be strongly positive in acinar cells of the PG, serous demilune cells of the SLG, and granular convoluted tubule (GCT) cells of the male SMG, weakly positive in seromucous acinar cells of the male and female SMG, and negative in mucous acinar cells of the SLG. These results clarified that α-amylase is produced mainly by GCT cells and partly by acinar cells in the SMG, whereas it is produced exclusively by serous demilune cells in the SLG of mice.     

Is PET/CT essential in the diagnosis and follow-up of temporal arteritis?

The increasing availability and improvement of imaging techniques are deeply influencing diagnosis and work-up of patients affected with vasculitis, particularly those with large vessel vasculitis (LVV). Fluorodeoxyglucose (¹⁸F-FDG) positron emission tomography (PET), especially when integrated with computed tomography (CT), is taking hold as a useful diagnostic technique to examine the aorta and the other large vessels in giant cell arteritis (GCA) with concomitant large vessel involvement (LV-GCA). In this paper we examined the progresses performed in this field in the last twenty years and the evidence available so far according to two different points of view (‘pros’ and ‘cons’), in order to give a comprehensive answer to a still open question about the role of PET/CT in the diagnosis and follow-up of GCA.     

The influence of surface microroughness and hydrophilicity of titanium on the up-regulation of TGFβ/BMP signalling in osteoblasts

The topography of titanium implants has been identified as an important factor affecting the osseointegration of surgically placed dental implants. Further modification to produce a hydrophilic microrough titanium implant surface has been shown to increase osseointegration compared with microrough topology alone. This study aimed to determine possible molecular mechanisms to explain this clinical observation by examining differences in the whole genome mRNA expression profile of primary human osteoblasts in response to sand-blasted acid-etched (SLA) and hydrophilic SLA (modSLA) titanium surfaces. A decrease in osteoblast proliferation associated with the titanium surfaces (modSLA > SLA > control) correlated with an increase in expression of the osteogenic differentiation markers BSPII and osteocalcin. Pathway analysis demonstrated that a number of genes associated with the TGFβ?BMP signalling cascade (BMP2, BMP6, SP1, CREBBP, RBL2, TBS3, ACVR1 and ZFYVE16) were significantly differentially up-regulated with culture on the modSLA surface. BMP2 was shown to have the largest fold change increase in expression which was subsequently confirmed at the protein level by ELISA. Several other genes associated with the functionally important mechanisms relevant to bone healing, such as Wnt signalling (CTNNA1, FBX4, FZD6), angiogenesis (KDR), osteoclastogenesis (HSF2, MCL1) and proteolysis (HEXB, TPP1), were also differentially regulated. These results suggest that chemical (hydrophilic) modification of the SLA surface may result in more successful osseointegration through BMP signalling.     

Conversion of β-alanine in vivo and in isolated liver tissue

Summary -alanine was incubated with chopped liver tissue of rats in oxygen for 160 minutes at 37°C. It was established that a certain part of added -alanine (about 12%) disappears, causing an excessive production of urea and of a small quantity of -alanine.In parenteral administration of -alanine an increased urinary excretion of this amino acid is observed in B₆-deficient rats in which the process of -alanine transformation is disturbed.     

Role of Interferon-γ in GVHD and GVL

Interferon （IFN）-γ, a potent proinflammatory cytokine produced by multiple types of cells （e.g., activated T, NK and NKT cells）, plays important and complex roles in both innate and adaptive immune responses. There may be a correlation between the IFN-γ level and GVHD severity in patients receiving allogeneic hematopoietic cell transplantation. However, such a correlation may just reflect the presence of large numbers of activated T cells, and does not necessarily imply a harmful role of IFN-γ in the pathogenesis of GVHD. There has been increasing experimental evidence that IFN-γ is not required and may even inhibit GVHD. Paradoxically, IFN-γ facilitates graft-versus-leukemic （GVL） effects. Thus, IFN-γ blockade is likely deleterious in patients after allogeneic hematopoietic cell transplantation, and not beneficial as previously suggested.     

Expression and localization of E-cadherin and β-catenin in uterine carcinosarcoma

This study was designed to analyze the subcellular localization of E-cadherin and β-catenin both of which play a critical role in cell–cell adhesion in uterine carcinosarcoma (UCS). We performed an immunohistochemical reaction analysis of the subcellular localization of E-cadherin and β-catenin proteins in 46 cases of UCSs consisting of 28 UCSs with heterologous sarcoma and 18 UCSs with homologous sarcoma and compared their clinicopathological features. In most UCSs, membranous expression of E-cadherin and β-catenin was completely lost in sarcomatous components, but it was preserved in carcinomatous components. Nuclear β-catenin expression was observed significantly more frequently in sarcomatous components (31/46, 67.4%) than in carcinomatous components (22/46, 47.8%; P = 0.0025). In sarcomatous components, nuclear β-catenin expression was found significantly more frequently in heterologous sarcoma (23/28, 82.1%) than in homologous sarcoma (8/18, 44.4%; P = 0.0279). The stage was the only independent prognostic significant factor. These results suggest that reduced membranous expression of E-cadherin and β-catenin may contribute to the biphasic morphology of UCS. Furthermore, although the precise mechanism is unclear, nuclear β-catenin expression in sarcomatous components may also be associated with biphasic morphology and heterologous sarcomatous differentiation.     

Role of α2/δ subunit in the development of morphine-induced rewarding effect and behavioral sensitization

Previous data demonstrate that L-type voltage-gated calcium channel (VGCC) blockers, which bind to α₁ subunits of VGCC to suppress Ca²⁺ entry into cells, inhibit the development of psychological dependence on drugs of abuse, suggesting the upregulation of L-type VGCC in the development of psychological dependence. However, there are few available data on changes of the auxiliary subunit α₂/δ modifying L-type VGCC under such conditions. We therefore investigated here the role of α₂/δ subunits of VGCCs in the brain of mouse after repeated treatment with morphine. The treatment with morphine increased α₂/δ subunit expression in the frontal cortex and the limbic forebrain of mice showing rewarding effect and sensitization to hyperlocomotion by morphine. The morphine-induced behavioral sensitization and place preference were also suppressed by gabapentin, which binds to an exofacial epitope of the α₂/δ auxiliary subunits of VGCCs. These findings indicate that the upregulation of α₂/δ subunit as well as α₁ subunits of VGCC in the frontal cortex and the limbic forebrain plays a critical role in development of morphine-induced rewarding effect and behavioral sensitization following neuronal plasticity.