Novel mutations in COL4A3, COL4A4, and COL4A5 in Chinese patients with Alport Syndrome.

Alport syndrome (AS) is a clinically and genetically heterogeneous, progressive nephropathy caused by mutations in COL4A3, COL4A4, and COL4A5, which encode type IV collagen. The large sizes of these genes and the absence of mutation hot spots have complicated mutational analysis by routine polymerase chain reaction (PCR)-based approaches. Here, in order to design a rapid and effective method for the genetic diagnosis of AS, we developed a strategy by utilizing targeted capture associated with next-generation sequencing (NGS) to analyze COL4A3, COL4A4, and COL4A5 simultaneously in 20 AS patients. All the coding exons and flanking sequences of COL4A3, COL4A4, and COL4A5 from the probands were captured followed by HiSeq 2500 sequencing. Candidate mutations were validated by classic Sanger sequencing and quantitative (q)PCR. Sixteen patients (16/20, 75%) showed X-linked inheritance, and four patients (4/20, 20%) showed autosomal recessive inheritance. None of the individuals had autosomal-dominant AS. Fifteen novel mutations, 6 known mutations, and 2 novel fragment deletions were detected by targeted capture and NGS. Of these novel mutations, 12, 3, and 2 mutations were detected in COL4A5, COL4A4, and COL4A3, respectively. A comparison of the clinical manifestations caused by different types of mutations in COL4A5 suggested that nonsense mutations and glycine substitution by an acidic amino acid are more severe than the other missense mutations. Pathogenic mutations were detected in 20 patients. These novel mutations can expand the genotypic spectrum of AS. Our results demonstrated that targeted capture and NGS technology are effective in the genetic diagnosis of AS.

HLA-DRB1*15:01 and HLA-DRB3*02:02 in PLA2R-Related Membranous Nephropathy.

Idiopathic membranous nephropathy (MN) is associated with HLA; however, the HLA allele involved remains unknown. To identify the HLA risk alleles associated with phospholipase A2 receptor (PLA2R)-related MN in the Chinese population, we sequenced the entire MHC region in DNA samples from 99 patients with PLA2R-related MN, 50 patients with PLA2R-unrelated MN, and 100 healthy subjects. Two HLA risk alleles, HLA-DRB1*15:01 and HLA-DRB3*02:02, independently and strongly associated with an increased risk of PLA2R-related MN. After adjusting for HLA-DRB1*15:01 and HLA-DRB3*02:02, no other alleles showed significant association with PLA2R-related MN. A replication study in an independent cohort of 293 participants with PLA2R-related MN and 285 healthy controls validated these findings. In a joint analysis, a multivariate logistic regression model confirmed that HLA-DRB1*15:01 (odds ratio [OR], 24.9; 95% confidence interval [95% CI], 15.3 to 42.6; P=2.3×10-35) and HLA-DRB3*02:02 (OR, 17.7; 95% CI, 11.0 to 30.3; P=8.0×10-29) independently and strongly associated with PLA2R-related MN. As many as 98.7% of patients with PLA2R-related MN, compared with 43.9% of control subjects, carried at least one HLA risk allele. Subjects with either risk allele had higher odds of developing PLA2R-related MN than those without a risk allele (OR, 98.9; 95% CI, 44.4 to 281.7; P=2.5×10-23). These HLA risk alleles also associated with the age at disease onset in patients with PLA2R-related MN. In conclusion, our findings provide clear evidence that the HLA-DRB1*15:01 and HLA-DRB3*02:02 alleles independently and strongly associate with PLA2R-related MN in the Chinese population.

Characterization of the whole genome from a human parechovirus type 3 detected from the serum of a child with sepsis in Beijing, China / 病毒学报

Human parechovirus type 3 (HPeV3) is an important pathogen of severe sepsis. HPeV3 is a non- enveloped, single-stranded, positive-sense RNA virus with a linear and continuous genomic RNA. The complete genome of a HPeV3 (BJ-C3174) strain was analyzed from the serum specimen from a child with sepsis hospitalized in Beijing, China, in 2012. The whole genome of BJ-C3174 was 7329 nucleotides (nt) in length excluding a poly (A) tail. One large open reading frame (ORF) of 6531 nt encoding a putative polyprotein precursor of 2177 amino acids (aa) was flanked by a 5' untranslated region (UTR) of 709 nt and 3' UTR of 91 nt. Phylogenetic analysis showed that BJ-C3174 belonged to HPeV3 and was closest to the HPeV3 strain BONN-2 from Germany. Compared with HPeV1-8 reference strains, BJ-C3174 shared the highest similarities with BONN-2 in full length and in each of the gene segments of the genome. The nucleotide and predicted amino acid identities of the whole genome between BJ-C3174 and BONN-2 were 99.3% and 99.8%, respectively, which were higher than those compared with HPeV3 prototype. Recom- bination of the gene segment with other HPeVs types was not identified.

Evaluation of serum specific IgM detection in diagnosis of respiratory viral infections in children / 中华儿科杂志

<p><b>OBJECTIVE</b>The present study was designed to explore the practical application of the rapid etiological diagnosis by detecting specific IgM antibody against common respiratory viruses in children with acute lower respiratory infections (ALRI).</p><p><b>METHOD</b>Clinical specimens including nasopharyngeal aspirates and serum of acute phase from hospitalized children were collected from 207 infants and children with acute lower respiratory infections from March 2009 to September 2010. Seven common respiratory virus antigens were identified from the collected nasopharyngeal aspirates by direct immunofluorescence assay (DFA). ELISA was used to detect specific IgM antibody against RSV, ADV, IFVA, IFVB and PIV, while indirect immunofluorescence assay (IFA) was used to detect specific IgM antibody against RSV, ADV, IFVA, IFVB, PIV1, PIV2 and PIV3 in collected acute phase serum.</p><p><b>RESULT</b>The overall positive rates to detect viral antigen by using DFA, ELISA and IFA was 67.6%, 57.5% and 39.6%, respectively. The consistent rate of ELISA and IFA versus accepted DFA were 21.7% and 31.4%, respectively. The average days from onset of the symptoms to blood sample collection for those with the consistent results by ELISA and DFA were 12.0 d for ADV, 9.6 d for PIV2, 9.5 d for IFV, and 5.3 d for RSV, respectively, and by IFA and DFA were 15.0 d for PIV3, 9.2 d for ADV, and 7.4 d for RSV, respectively. Among all age groups, the consistent rate of serum viral IgM and antigen detections was highest in children younger than 3 years old.</p><p><b>CONCLUSION</b>Although there were differences between serum IgM antibody and viral antigen detections, specific IgM antibody detection was of value in early and rapid etiological diagnosis of pediatric ALRI, especially for young children. It could provide serologic evidence of respiratory virus infection. The diagnostic rate of pathogen could be improved if it was used in combination with viral antigen diagnostic methods.</p>

Triptolide reduces proteinuria in experimental membranous nephropathy and protects against C5b-9-induced podocyte injury in vitro.

Membranous nephropathy is a major cause of nephrotic syndrome in adults where podocyte injuries were found to mediate the development of proteinuria. Triptolide, a major active component of Tripterygium wilfordii Hook F, has potent immunosuppressive, anti-inflammatory and antiproteinuric effects. To study its antiproteinuric properties, we established an experimental rat model of passive Heymann nephritis and a C5b-9 injury model of podocytes in vitro. Treatment or pretreatment with triptolide markedly reduced established proteinuria as well as the titer of circulating rat anti-rabbit IgG antibodies in these nephritic rats, accompanied by a reduction in glomerular C5b-9 deposits. Expression of desmin, a marker of podocyte injury, diminished after triptolide treatment, whereas quantitative analysis of mean foot process width showed that effacement of foot processes was substantially reversed. In in vitro studies we found that triptolide deactivated NADPH oxidase, suppressed reactive oxygen species generation and p38 mitogen-activated protein kinase, and restored RhoA signaling activity. Triptolide did not interfere with the formation of C5b-9 on the membrane of podocytes. Thus, triptolide reduces established heavy proteinuria and podocyte injuries in rats with passive Heymann nephritis, and protects podocytes from C5b-9-mediated injury.

Rhein improves renal lesion and ameliorates dyslipidemia in db/db mice with diabetic nephropathy.

Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is purified from rhubarb (Rheum officinale), a widely used traditional Chinese herb. In our previous studies, rhein was shown to be effective in ameliorating diabetic renal pathological changes and attenuating hyperlipidemia. Statins have also been proven to ameliorate renal pathological changes associated with diabetic nephropathy (DN) through lipid-dependent and -independent mechanisms. We here study the protective and regulatory effects of rhein on renal injury and dyslipidemia in db/db mice with DN, using simvastatin as the control, and provide information on the mechanisms by which rhein protects against renal damage from DN. The results indicated that urinary albumin excretion (UAE) was reduced after 8 weeks of treatment in the rhein group, and 12 weeks in the simvastatin group. The morphometric analysis revealed that levels of extracellular matrix (ECM) significantly decreased in the rhein group after the full treatment course, but not in the simvastatin group. The more powerful effects of rhein on decreasing transforming growth factor-beta1 (TGF-beta1) and fibronectin immunohistochemistry expression in renal tissue were also observed. And the plasma levels of cholesterol (Chol), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and ApoE all decreased in both the rhein and the simvastatin groups. Together, our data suggested that both rhein and simvastatin regulate dyslipidemia. The powerful effect of rhein in renal protection is due to its widespread effects. Rhein is a new drug that can decrease lipid levels and protect against DN progression in a different fashion with simvastatin.

Triptolide protects podocytes from puromycin aminonucleoside induced injury in vivo and in vitro.

Extracts of Tripterygium wilfordii Hook F have been used to treat glomerulonephritis for more than 30 years in China with dramatic antiproteinuric effects. Triptolide, a diterpene triepoxide, is one of the major active components of these extracts. To clarify its antiproteinuric effects we induced podocyte injury by puromycin aminonucleoside. Triptolide effectively reduced the proteinuria induced by puromycin in nephrotic rats without reducing the glomerular filtration rate. The antiproteinuric effect was associated with improvement in the foot process effacement, a decrease in the podocyte injury marker desmin as well as the restoration of nephrin and podocin expression and distribution. In cultured mouse podocytes triptolide pretreatment prevented the puromycin-induced disruption of the actin cytoskeleton and microfilament-associated synaptopodin while protecting nephrin and podocin expression. Triptolide suppressed reactive oxygen species generation and p38 mitogen-activated protein kinase activation while restoring RhoA signaling activity. These results show that triptolide ameliorates puromycin aminonucleoside-mediated podocyte injury in vivo and in vitro.

[The expression, renaturation, purification and preliminary identification of human IgE Cepsilon3-Cepsilon4].

AIM: To gain recombinant protein Cepsilon3-Cepsilon4 of IgE Fc (E34). METHODS: We cloned the gene coding human IgE Cepsilon3-Cepsilon4 (E34) and constructed an expression vector pET28a(+)-E34. The target protein was expressed as inclusion body in E.coli BL-21. Following renaturation and purification through a CM sephorose FF column, the soluble protein was acquired, and its binding ability to murine anti-hIgE mAb was identified by Western blot and ELISA. RESULTS: The cloned E34 gene was sequenced and proved by SDS-PAGE to be the same as reported sequence. SDS-PAGE analysis showed the relative molecular mass of E34 protein obtained was correct as predicted. Western blot and ELISA data revealed that it owned the epitope of binding to murine anti-hIgE mAb. CONCLUSION: The expression vector pET28a(+)-E34 has been successfully constructed and the target protein E34 recognized specifically by murine anti-hIgE mAb is obtained.

[Establishment of a cell model targeted to NFAT signal transduction pathway for preliminary screening of FK506-like immunosuppressants].

To screen NFAT antagonistic drugs and research signal transduction pathway related to NFAT. Four recombinant vectors were constructed. Each consists of three tandem copies of the human IL-2 distal NFAT-AP1 binding site in the context of the minimal IL-2 enhancer, either the sequence from -326 - +46 or the sequence from -89 - +46 (containing only the TATA box), driving a luciferase reporter gene or a destabilized enhanced green fluorescence protein (d2EGFP) reporter gene, respectively. Transient transfection of Jurkat cells was achieved by electroporation with 5 - 10 microg of the above plasmid and one pulse at 200V, 65ms. Plasmid pEFBos-mNFAT1 constitutively expressing murine full length NFAT1 protein was used for transient cotransfection. The results showed that neither of non-stimulation nor PMA or ionomycin stimulation alone could activate the reporter gene except PMA plus ionomycin costimulation. Furthermore, overexpressed murine NFAT1 augmented the activation of either IL-2 promoter or NFAT-AP1 enhancer drived reporter gene compared to the endogenous did. However, the reporter gene expression was nearly completely inhibited by pretreatment for 1h with FK506 at 5 microg/mL and then stimulation for 6-12h with PMA plus ionomycin in the presence of FK506. These findings indicated that such a transient Jurkat cell model offered a potential platform for preliminary screening of FK506 or CsA-like immunosuppressive agents.