Antigenic variation of H1N1, H1N2 and H3N2 swine influenza viruses in Japan and Vietnam

The antigenicity of the influenza A virus hemagglutinin is responsible for vaccine efficacy in protecting pigs against swine influenza virus (SIV) infection. However, the antigenicity of SIV strains currently circulating in Japan and Vietnam has not been well characterized. We examined the antigenicity of classical H1 SIVs, pandemic A(H1N1)2009 (A(H1N1)pdm09) viruses, and seasonal human-lineage SIVs isolated in Japan and Vietnam. A hemagglutination inhibition (HI) assay was used to determine antigenic differences that differentiate the recent Japanese H1N2 and H3N2 SIVs from the H1N1 and H3N2 domestic vaccine strains. Minor antigenic variation between pig A(H1N1)pdm09 viruses was evident by HI assay using 13 mAbs raised against homologous virus. A Vietnamese H1N2 SIV, whose H1 gene originated from a human strain in the mid-2000s, reacted poorly with post-infection ferret serum against human vaccine strains from 2000-2010. These results provide useful information for selection of optimal strains for SIV vaccine production.     

Brain-derived neurotrophic factor-mediated retrograde signaling required for the induction of long-term potentiation at inhibitory synapses of visual cortical pyramidal neurons

High-frequency stimulation (HFS) induces long-term potentiation (LTP) at inhibitory synapses of layer 5 pyramidal neurons in developing rat visual cortex. This LTP requires postsynaptic Ca²⁺ rise for induction, while the maintenance mechanism is present at the presynaptic site, suggesting presynaptic LTP expression and the necessity of retrograde signaling. We investigated whether the supposed signal is mediated by brain-derived neurotrophic factor (BDNF), which is expressed in pyramidal neurons but not inhibitory interneurons. LTP did not occur when HFS was applied in the presence of the Trk receptor tyrosine kinase inhibitor K252a in the perfusion medium. HFS produced LTP when bath application of K252a was started after HFS or when K252a was loaded into postsynaptic cells. LTP did not occur in the presence of TrkB-IgG scavenging BDNF or function-blocking anti-BDNF antibody in the medium. In cells loaded with the Ca²⁺ chelator BAPTA, the addition of BDNF to the medium enabled HFS to induce LTP without affecting baseline synaptic transmission. These results suggest that BDNF released from postsynaptic cells activates presynaptic TrkB, leading to LTP. Because BDNF, expressed activity dependently, regulates the maturation of cortical inhibition, inhibitory LTP may contribute to this developmental process, and hence experience-dependent functional maturation of visual cortex.     

Use of loop-mediated isothermal amplification of the IS900 sequence for rapid detection of cultured Mycobacterium avium subsp. paratuberculosis

We evaluated the usefulness of loop-mediated isothermal amplification (LAMP) in detecting specific gene sequences of Mycobacterium avium subsp. paratuberculosis (MAP). A total of 102 primer sets for LAMP was designed to amplify the IS900, HspX, and F57 gene sequences of MAP. Using each of two primer sets (P-1 and P-2) derived from the IS900 fragment, it was possible to detect MAP in a manner similar to that used with nested PCR. The sensitivity of LAMP with P-1 was 0.5 pg/tube, which was more sensitive than nested PCR. When P-2 was used, 5 pg/tube could be detected, which was the same level of sensitivity as that for nested PCR. LAMP with P-1 was specific. Although only 2 Mycobacterium scrofulaceum strains out of 43 non-MAP mycobacterial strains were amplified, the amplification reaction for these strains was less efficient than for MAP strains, and their products could be distinguished from MAP products by restriction digestion. LAMP with P-2 resulted in very specific amplification only from MAP, the same result obtained with nested PCR. Our LAMP method was highly specific, and the white turbidity of magnesium pyrophosphate, a by-product of the LAMP reaction, allowed simple visual detection. Our method is rapid, taking only 2 h, compared with 4 h for nested PCR. In addition, the LAMP method is performed under isothermal conditions and no special apparatus is needed, which makes it more economical and practical than nested PCR or real-time PCR. These results indicate that LAMP can provide a rapid yet simple test for the detection of MAP.     

HBV and HCV infection in Japanese dental care workers

Protective measures against occupational exposure to the hepatitis B virus (HBV) and hepatitis C virus (HCV) must be taken in order to prevent infection in dental care workers. To determine the best way to protect these workers, our study examined viral hepatitis infection in dental care workers in regions with a high prevalence of HCV infections in Japan. In total, 141 dental care workers (including dentists, dental hygienists and dental assistants) were enrolled. After a questionnaire to elicit demographic information was administered by an oral surgeon, hepatitis B surface antigen (HBsAg), antibody to HBs (anti-HBs), antibody to hepatitis B core antigen (anti-HBc) and antibody to HCV (anti-HCV) were measured. When necessary, HBeAg, anti-HBe, levels of HBV DNA, anti-HBc IgM and HCV RNA in serum were measured. Of the dental care workers included, 68 (48.2%) had been immunized with a HBV vaccine. Only 9 wore a new pair of gloves for each new patient being treated, 36 changed to a new pair only after the old gloves were torn and 24 did not wear any gloves at all. No one was positive for HBsAg or anti-HCV, though 73 (51.8%) and 17 (12.1%) workers were respectively positive for anti-HBs and anti-HBc. The positive rate of anti-HBc varied directly with worker age and experience. Of the 68 workers immunized with HBV vaccine, 51 (75%) were positive for anti-HBs. Of the 63 workers who were not so immunized, 17 (27%) were positive for anti-HBs and 15 of these were also positive for anti-HBc. Immunized workers were more protected against HBV infection than non-immunized workers, indicating that HBV vaccine was a useful measure for protection against the infection. The anti-HBc positive rate was significantly higher among dental care workers than general blood donors, suggesting that frequency of exposure to HBV was greater in dental care workers. HBV vaccination should be made compulsory for all dental care workers who handle sharp instruments.     

Novel mutations in the cytochrome P450 2C19 gene: a pitfall of the PCR-RFLP method for identifying a common mutation

CYP2C19 is a clinically important enzyme involved in the metabolism of therapeutic drugs such as (S)-mephenytoin, omeprazole, proguanil, and diazepam. Individuals can be characterized as either extensive metabolizers (EM) or poor metabolizers (PM) on the basis of CYP2C19 enzyme activity. The PM phenotype occurs in 2–5% of Caucasian populations, but at higher frequencies (18–23%) in Asians. CYP2C19*2 and CYP2C19*3, which are single-nucleotide polymorphisms of CYP2C19, are the main cause of PM phenotyping in homozygotes or compound heterozygotes. We report two novel mutations in the CYP2C19 gene identified by direct sequencing and subcloning procedures. One of these mutations was considered to be CYP2C19*3 by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). This result suggests that mutations classed as CYP2C19*3 might include other mutations. Further studies are needed to clarify the relationship between these novel mutations and enzyme activity.The DDBJ accession number of the novel mutation is AB113829     

TCRzeta mRNA splice variant forms observed in the peripheral blood T cells from systemic lupus erythematosus patients

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown etiology. Tyrosine phosphorylation and protein expression of the T-cell receptor ζ chain (ζ) have been reported to be significantly decreased in SLE T cells. In addition, ζ mRNA with alternatively spliced 3′ untranslated region (ζmRNA/as-3′UTR) is detected predominantly in SLE T cells, and aberrant ζ mRNA accompanied by the mutations in the open reading frame including ζ mRNA lacking exon7 (ζmRNA/exon7-) is observed in SLE T cells. These ζ mRNA splice variant forms exhibit a reduction in the expression of TCR/CD3 complex and ζ protein on their cell surface due to the instability of ζ mRNA splice variant forms as well as the reduction in interleukin (IL)-2 production after stimulating with anti-CD3 antibody. Data from cDNA microarray showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-β2, were down-regulated in the MA5.8 cells transfected with the ζ mRNA splice variant forms. Another 16 genes were up-regulated and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus-receptor-related 2, syndecan-1, and granzyme A.     

Follicular lymphoma frequently originates in the salivary gland

The aim of the present study was to examine the clinicopathological presentations of follicular lymphomas (FL) of the salivary glands, as compared to mucosa-associated lymphoid tissue (MALT) lymphomas. A total of 27 primary salivary gland lymphomas were examined: 6 FL (five, grade 1; one, grade 2); 19 MALT lymphomas; and two diffuse large B-cell lymphomas. The FL patients ranged in age from 24 to 73 years, with a mean of 49 years, which was younger than that of MALT patients (mean: 64 years; P < 0.05). Four of the six FL arose from the submandibular gland, which was the origin of only five out of a total of 19 MALT lymphomas. One FL patient was in clinical stage (CS) IE, two in CS IIE, and two in CS III and IV. As regards the MALT lymphoma patients, 13 (68%) were in CS IE and five (26%) in CS IIE. None of the FL patients had clinical diagnosis of autoimmune disease but eight MALT lymphoma patients had autoimmune disease. The present study found a relatively high incidence of FL in the salivary glands. The observed differences in age of onset, background of autoimmune disease, and lesion site suggests that the pathogenesis of FL may differ from that of MALT lymphoma.