ANCA vasculitis and IgA nephropathy linked to silica exposure.

There is a recognized association between silica exposure and Antineutrophil cytoplasmic antibodies (ANCA)-associated vasculitis (AAV); however, no clear association between silica exposure and Immunoglobulin A (IgA) nephropathy. We describe the case of a 26-year-old male stonemason who presents with hilar lymphadenopathy, haematuria and acute kidney injury related to silica exposure, AAV and IgA nephropathy. He was asymptomatic on presentation; urinalysis revealed glomerular haematuria (>1000 red blood cells/L) and proteinuria (protein-to-creatinine ratio 84 mg/mmol). ANCA anti-myeloperoxidase serology was strongly positive. Mediastinal lymph node biopsy revealed multiple necrotizing granulomas with silica inclusions, and renal biopsy demonstrated crescentic glomerulonephritis and mesangial IgA staining. The patient was treated with cyclophosphamide and high-dose prednisolone with subsequent improvement in renal function. To our knowledge, this is the first report of both ANCA vasculitis and IgA nephropathy in the setting of silica exposure. This case highlights the relevance of occupational exposures in renal disease, and the immune-stimulatory effect of silica.

Amustaline (S-303) treatment inactivates high levels of Chikungunya virus in red-blood-cell components.

BACKGROUND AND OBJECTIVES: Chikungunya virus (CHIKV) infections have been reported in all continents, and the potential risk for CHIKV transfusion-transmitted infections (TTIs) was demonstrated by the detection of CHIKV RNA-positive donations in several countries. TTIs can be reduced by pathogen inactivation (PI) of blood products. In this study, we evaluated the efficacy of amustaline and glutathione (S-303/GSH) to inactivate CHIKV in red-blood-cell concentrates (RBCs). MATERIAL AND METHODS: Red-blood-cells were spiked with high level of CHIKV. Infectious titres and RNA loads were measured before and after PI treatment. Residual CHIKV infectivity was also assessed after five successive cell culture passages. RESULTS: The mean CHIKV titres in RBCs before inactivation was 5·81 ± 0·18 log10 50% tissue culture infectious dose (TCID50 )/mL, and the mean viral RNA load was 10·49 ± 0·15 log10 genome equivalent (GEq)/mL. No CHIKV TCID was detected after S-303 treatment nor was replicative CHIKV particles and viral RNA present after five cell culture passages of samples obtained immediately after S-303 treatment. CONCLUSION: Chikungunya virus was previously shown to be inactivated by the PI technology using amotosalen and ultraviolet A light for the treatment of plasma and platelets. This new study demonstrates that S-303/GSH can inactivate high titres of CHIKV in RBCs.

Detection of Zika virus RNA in semen of asymptomatic blood donors.

OBJECTIVES: Zika virus (ZIKV) transmission through semen donation has never been reported but the risk is supported by the detection of ZIKV in semen and the demonstration of ZIKV sexual transmission. The potential impact of ZIKV on assisted reproductive procedures should be evaluated. METHODS: We tested longitudinally collected semen samples provided by asymptomatic blood donors who tested positive for ZIKV RNA in plasma during ZIKV outbreaks in Puerto Rico and Florida in 2016. RESULTS: Five of the 14 (35.7%) asymptomatic blood donors provided semen samples that tested positive for ZIKV RNA, with ZIKV RNA loads ranging from 8.03 × 103 to 2.55 × 106 copies/mL. Plasma collected at the same time as the semen tested negative for ZIKV RNA for most ZIKV RNA-positive semen collections; all corresponding plasma samples tested positive or equivocal for anti-ZIKV IgG antibodies and all except one tested positive for ZIKV IgM antibodies. The rate of detection of ZIKV RNA in semen in asymptomatic donors is not significantly different from the rate previously reported for symptomatic patients. CONCLUSIONS: Our results that show a high percentage of detection of ZIKV RNA in the semen of asymptomatic men confirm that ZIKV is a new threat for reproductive medicine and should have important implications for assisted reproductive technology. We recommend that semen donations from men at risk for ZIKV infection should be tested for ZIKV RNA, regardless of symptoms of ZIKV infection.

Incidence of West Nile virus infection in the Dallas-Fort Worth metropolitan area during the 2012 epidemic.

The 2012 West Nile virus (WNV) epidemic was the largest since 2003 and the North Texas region was the most heavily impacted. We conducted a serosurvey of blood donors from four counties in the Dallas-Fort Worth area to characterize the epidemic. Blood donor specimens collected in November 2012 were tested for WNV-specific antibodies. Donors positive for WNV-specific IgG, IgM, and neutralizing antibodies were considered to have been infected in 2012. This number was adjusted using a multi-step process that accounted for timing of IgM seroreversion determined from previous longitudinal studies of WNV-infected donors. Of 4971 donations screened, 139 (2·8%) were confirmed WNV IgG positive, and 69 (1·4%) had IgM indicating infection in 2012. After adjusting for timing of sampling and potential seroreversion, we estimated that 1·8% [95% confidence interval (CI) 1·5-2·2] of the adult population in the Dallas-Fort Worth area were infected during 2012. The resulting overall estimate for the ratio of infections to reported WNV neuroinvasive disease (WNND) cases was 238:1 (95% CI 192-290), with significantly increased risk of WNND in older age groups. These findings were very similar to previous estimates of infections per WNND case, indicating no change in virulence as WNV evolved into an endemic infection in the United States.

Mechanisms of xylanase-induced nitric oxide and phosphatidic acid production in tomato cells.

The second messenger nitric oxide (NO), phosphatidic acid (PA) and reactive oxygen species (ROS) are involved in the plant defense response during plant-pathogen interactions. NO has been shown to participate in PA production in response to the pathogen-associated molecular pattern xylanase in tomato cell suspensions. Defense responses downstream of PA include ROS production. The goal of this work was to study the signaling mechanisms involved in PA production during the defense responses triggered by xylanase and mediated by NO in the suspension-cultured tomato cells. We analyzed the participation of protein kinases, guanylate cyclase and the NO-mediated posttranslational modification S-nitrosylation, by means of pharmacology and biochemistry. We showed that NO, PA and ROS levels are significantly diminished by treatment with the general protein kinase inhibitor staurosporine. This indicates that xylanase-induced protein phosphorylation events might be the important components leading to NO formation, and hence for the downstream regulation of PA and ROS levels. When assayed, a guanylate cyclase inhibitor or a cGMP analog did not alter the PA accumulation. These results suggest that a cGMP-mediated pathway is not involved in xylanase-induced PA formation. Finally, the inhibition of protein S-nitrosylation did not affect NO formation but compromised PA and ROS production. Data collectively indicate that upon xylanase perception, cells activate a protein kinase pathway required for NO formation and that, S-nitrosylation-dependent mechanisms are involved in downstream signaling leading to PA and ROS.

Phosphatidic acid production in chitosan-elicited tomato cells, via both phospholipase D and phospholipase C/diacylglycerol kinase, requires nitric oxide.

Nitric oxide (NO) and the lipid second messenger phosphatidic acid (PA) are involved in plant defense responses during plant-pathogen interactions. NO has been shown to be involved in the induction of PA production in response to the pathogen associated molecular pattern (PAMP) xylanase in tomato cells. It was shown that NO is critical for PA production induced via phospholipase C (PLC) in concerted action with diacylglycerol kinase (DGK) but not for the xylanase-induced PA via phospholipase D (PLD). In order to study whether this is a general phenomenon during PAMP perception or if it is particular for xylanase, we studied the effect of the PAMP chitosan in tomato cell suspensions. We observed a rapid NO production in tomato cells treated with chitosan. Chitosan induced the formation of PA by activating both PLD and PLC/DGK. The activation of either phospholipase-mediated signaling pathway was inhibited in cells treated with the NO scavenger cPTIO. This indicates that NO is required for PA generation via both the PLD and PLC/DGK pathway during plant defense response in chitosan elicited cells. Responses downstream PA were studied. PLC inhibitors neomycin and U73122 inhibited chitosan-induced ROS production. Differences between xylanase and chitosan-induced phospholipid signaling pathways are discussed.

Inhibition of hyperacute transplant rejection by soluble proteins with the functional domains of CD46 and FcgammaRII.

BACKGROUND: Recombinant soluble forms of complement regulatory molecules, including the human complement regulatory protein CD46 (rsCD46), have been shown to inhibit hyperacute transplant rejection (HAR) and protect against complement-mediated inflammatory tissue damage. Similarly, recombinant soluble forms of the immunoglobulin receptor FcgammaRII (rsFcgammaRII) can attenuate antibody-mediated inflammatory responses. We have produced and tested the function of novel recombinant chimeric proteins that incorporate the functional domains of both CD46 (membrane cofactor protein, MCP) and the low affinity human IgG receptor FcgammaRII (CD32). METHODS: Two recombinant soluble chimeric proteins (CD46:FcR and FcR:CD46) were designed and produced using a human cell expression system. Their ability to protect cells against complement-mediated lysis (through the CD46 domain) and bind human IgG (through the Fc receptor domain) was assessed in vitro. They were also tested in vivo in the rat reverse passive Arthus reaction and a murine model of hyperacute cardiac transplant rejection. RESULTS: In vitro, the functional domains of the chimeric proteins each retained their activity. In vivo, the serum half-life of the recombinant chimeric proteins in mice was more than either rsCD46 or rsFcgammaRII. In the rat reverse passive Arthus reaction, intradermal injection of each recombinant protein substantially reduced inflammatory skin edema (>50%) and polymorphonuclear neutrophil infiltration (>90%). In the hyperacute rejection model, i.v. treatment with FcR:CD46 prevented complement-mediated rejection, macroscopic bruising, edema, and thrombosis more effectively than rsCD46. CONCLUSIONS: CD46/FcgammaRII bifunctional proteins have an improved ability to control complement-mediated hyperacute graft rejection and have therapeutic potential in other conditions involving antibody-mediated inflammation.

Interaction of CD46 with measles virus: accessory role of CD46 short consensus repeat IV.

To define further the accessory role(s) of the CD46 (membrane cofactor protein) short consensus repeat (SCR) III and IV domains in the interaction of CD46 with measles virus (MV), chimeric proteins were generated by substituting domains from the structurally related protein decay accelerating factor (DAF, CD55): x3DAF (exchange of CD46 SCR III) and x4DAF (exchange of SCR IV). Transfected CHO cell lines that stably expressed these chimeric proteins were compared for MV binding and infection. Compared with wild-type CD46 (I-II-III-IV), a significant decrease in MV binding was observed with x4DAF. Despite this limited binding, these cells were still capable of supporting virus entry. In a quantitative fusion assay, no significant differences in fusion were observed as a result of the exchange of either CD46 SCR III or IV. However, the down-regulation of cell surface CD46 typically observed following MV infection was abolished with x4DAF, as was the redistribution of CD46 on the cell surface. Thus, CD46 SCR IV appears to be required for optimal virus binding and receptor down-regulation, although importantly, in spite of these functional limitations, x4DAF can still be used for MV entry.

Chimeric CD46/DAF molecules reveal a cryptic functional role for SCR1 of DAF in regulating complement activation.

Chimeric proteins using membrane cofactor (CD46) and decay accelerating factor (DAF or CD55) were generated to further investigate the functional domains involved in the regulation of human serum complement. Following activation of the classical pathway, the isolated substitution of CD46 SCR III (x3DAF) exhibited a modest regulatory activity comparable to that of CD46. The isolated substitution of CD46 SCR IV (x4DAF), and the combined CD46 SCR III+IV substitutions (x3/4DAF) were essentially as efficient as DAF. No regulation of C3b deposition was observed with the combined CD46 SCR I+II substitutions (x1/2DAF). When tested after activation of the alternative pathway, both the x3DAF and x3/4DAF chimeras failed to regulate C3b deposition, while the x4DAF chimera still displayed some activity. In contrast to that observed following classical pathway activation, the x1/2DAF chimera exhibited a similar efficiency to wild type CD46 and DAF in controlling C3b deposition. Using SCR specific antibodies, the regulatory activity of the x1/2DAF chimera against the alternative pathway was mapped to the first three distal SCR (i.e. DAF 1, DAF 2 and CD46 III). These data demonstrate that several combinations of SCR domains from two related complement regulators can result in functional molecules, and reveal a novel and cryptic functional role for DAF SCR1.

An immunological approach to the evaluation of welfare in Holstein Frisian cattle.

Clinical immunological and haematological parameters, along with clinical conditions and growth rate, were studied in 413 male Holstein Frisian calves introduced into a large centre for genetic selection in different seasons of the year. Abnormalities were revealed by the laboratory tests in the great majority of calves after transportation stress, a general tendency to the restoration of physiological values being evident thereafter. Laboratory parameters were highly correlated with disease conditions: with three exceptions only, animals showed altered laboratory parameters some days before the occurrence of clinical symptoms. Eighteen per cent of animals showed altered laboratory parameters with no obvious clinical signs of disease; yet they experienced a reduced weight gain. Results suggest that clinical immunological and haematological parameters could be the foundation of a new, large-scale, robust approach to the control of welfare in cattle, which should be integrated preferably by a further range of records and measures.

Transgenic expression of a CD46 (membrane cofactor protein) minigene: studies of xenotransplantation and measles virus infection.

CD46 (membrane cofactor protein) is a human cell-surface regulator of activated complement and a receptor for the measles virus. A CD46 transgenic mouse line with an expression pattern similar to that of human tissues has been produced, to develop an animal model of (i) the control of complement activation by complement regulators in hyperacute rejection of xenografts, and (ii) measles virus infection. The mouse line was made using a CD46 minigene that includes promoter sequence and the first two introns of genomic CD46, which was coinjected into mouse ova with chicken lysozyme matrix attachment region DNA. A high level of CD46 expression in homozygotic transgenic mice was obtained with spleen cells having approximately 75% of the level found on human peripheral blood mononuclear cells. CD46 was detected in all tissues examined by immunohistochemistry, radioimmunoassay and Western blotting, showing that these mice were suitable for transplantation and measles virus infection studies. It also indicated that the transgene included the important regulatory elements of the CD46 promoter. Transgenic spleen cells were significantly protected in vitro from human complement activated by either the classical or alternative pathways and from alternative pathway rat complement. Furthermore, transgenic mouse hearts transplanted to rats regulated complement deposition in an in vivo model of antibody-dependent hyperacute xenograft rejection. Similar to human lymphocytes, transgenic lymphoblasts could be infected in vitro with measles virus; infected cells expressed viral proteins and produced infectious viral particles. The data demonstrate the suitability of this minigene for obtaining high-level CD46 expression sufficient for enhanced resistance of transgenic cells to complement attack and for obtaining wide tissue distribution of CD46, analogous to human tissues and, therefore, useful for comparative studies.

Ultrasound changes in the wrist and hand in hemodialysis patients.

Dialysis related amyloidosis (DRA) is a common complication of chronic dialysis, but diagnosis can be difficult. Hand involvement, as carpal tunnel syndrome (CTS) or particularly amyloid hand, can affect dexterity. To investigate the prevalence and extent of hand involvement, ultrasound evaluation of the carpal tunnel and tendons of the hand was performed in 25 chronic hemodialysis patients. Clinical symptoms of CTS were present in 18/50 wrists (36%); symptomatic patients had undergone hemodialysis for a significantly longer period than those without symptoms (16 +/- 2.9 years vs 5.3 +/- 3.0 years). The ultrasound indicators suggestive of DRA in the wrist which correlated with CTS were 1) increased carpal tunnel depth (15 +/- 2.3 mm vs 11 +/- 1.5 mm), 2) increased displacement of flexor tendons from the radius (3.4 +/- 2.4 mm vs 11 +/- 0.6 mm), and 3) presence of hypoechoic masses, erosions and fluid collections (16% of wrists). There was no relationship between symptoms and either flexor retinaculum thickness or median nerve surface area index. Five patients (20%) had severe stiffness of their fingers; ultrasound revealed thickening of the tendons of the hand in all these cases. Ultrasound technology allows the non-invasive examination of the wrist and hand for DRA and may be valuable in diagnosis and planning surgery.

Clinical features in two patients with IgA glomerulonephritis and thin-basement-membrane disease.

BACKGROUND: Both IgA glomerulonephritis (IgA gn) and thin basement membrane disease (TBMD) are common forms of glomerulonephritis. Patients with these conditions may present with identical clinical features, but higher urinary RBC counts, heavier proteinuria, and impaired renal function are more common in patients with IgA gn. Because IgA gn and TBMD are common, some patients will have both diseases. SUBJECTS: We describe the clinical features of two individuals with both Iga gn and TBMD, and compare them with the clinical and laboratory characteristics in patients with TBMD (n=15) or IgA gn (n=32) alone. RESULTS: IgA gn was found in two individuals of the 110 with TBMD who were studied. They both had haematuria with >/100 000 RBC/ml and proteinuria >0.2/day (one had more than 1 g/day). These features were more consistent with Iga gn than TBMD alone. However, both individuals had normal serum creatinine and creatinine clearance at presentation. Additional clinical features were macroscopic haematuria in one and hypertension in both. CONCLUSIONS: IgA deposits are not uncommon in patients with TBMD, and these patients have clinical features that resemble those seen in IgA gn rather than TBMD. Patients with both IgA gn and TBMD do not necessarily have the worse prognosis noted in some patients with Iga gn.

Correlation between e/anti e system and evolution of B virus chronic hepatitis in pediatric patients.

The authors have performed a longitudinal study of 118 children affected with B virus chronic hepatitis. Our first observation revealed 92 children with HBeAg positive (26 CPH, 66 CAH), 22 children with anti HBe positive (6CPH, 15 CAH, 1 cirrhosis), 4 children (CAH) with e/anti-e negative. A correlation between the severity of clinical forms and the behaviour of the e/anti-e system was not observed. Seroconversion was observed during the follow up period in 37 of 92 subjects in an average time of 59.83 +/- 32 months, time rather prolonged in patients under immunosuppressive therapy. To compare the clinical progress and the evolution of CPH and CAH respectively, always with regard to the e/anti-e system, statistically significant differences did not result. Only anti HBe positive recovered subjects, inclusive of seroconverted patients and those anti HBe from the first observation, showed significant results to the statistical analysis. Still, seroconversion corresponds frequently to a stable improvement of hepatitis. On the contrary evolution into cirrhosis was observed in 5 patients that had anti HBe antibodies.