Sensitive and rapid detection of herpes simplex virus and varicella-zoster virus DNA by loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react rapidly and efficiently, with a high specificity, under isothermal conditions. We used a LAMP assay for the detection of herpes simplex virus type 1 (HSV-1), herpes simplex virus type 2 (HSV-2), and varicella-zoster virus (VZV). The virus specificities of primers were confirmed by using 50 HSV-1, 50 HSV-2, and 8 VZV strains. The assay was performed for 45 min at 65 degrees C. The LAMP assay had a 10-fold higher sensitivity than a PCR assay. An analysis of nucleotide sequence variations in the target and primer regions used for the LAMP assay indicated that 3 of 50 HSV-1 strains had single nucleotide polymorphisms. No HSV-2 or VZV strains had nucleotide polymorphisms. Regardless of the sequence variation, there were no differences in sensitivity with the HSV-1-specific LAMP assay. To evaluate the application of the LAMP assay for clinical diagnosis, we tested clinical samples from 40 genital herpes patients and 20 ocular herpes patients. With the LAMP assay, 41 samples with DNA extraction and 26 direct samples without DNA extraction were identified as positive for HSV-1 or HSV-2, although 37 samples with DNA extraction and just one without DNA extraction were positive by a PCR assay. Thus, the LAMP assay was less influenced than the PCR assay by the presence of inhibitory substances in clinical samples. These observations indicate that the LAMP assay is very useful for the diagnosis of HSV-1, HSV-2, and VZV infections.     

Analysis of class switch recombination and somatic hypermutation in patients affected with autosomal dominant hyper-IgM syndrome type 2

Autosomal recessive form of hyper-IgM syndrome type 2 (AR-HIGM2) is secondary to mutations affecting both alleles of AICDA gene encoding activation-induced cytidine deaminase, characterized by defects of immunoglobulin class switch recombination (CSR) and somatic hypermutation (SHM) in most of the patients. We herein report the immunological phenotype of seven patients carrying a single heterozygous R190X mutation in AICDA. Variable defect in in vivo CSR inherited as an autosomal dominant (AD) trait strongly suggests that this heterozygous AICDA mutation causes HIGM (AD-HIGM2). In AD-HIGM2 B cells, CSR was consistently found impaired in vitro. However, in contrast to AR-HIGM2, the CSR-induced double-stranded DNA breaks in the switch region of IgM heavy chain gene were detected. The SHM frequency in V regions of IgM heavy chain gene in B cells was normal in all (but one patient). The characteristics of the AD-HIGM2 phenotype indicate that the AID C-terminal region may be involved in DNA repair machinery required for CSR.     

Suppression of development of diabetes in NOD mice by lactate dehydrogenase virus infection

It has been reported that lactate dehydrogenase virus (LDV) selectively infects a subpopulation of macrophages, thereby affecting the immune system. We studied the effects of LDV infection on the development of diabetes in non-obese diabetic (NOD) mice. Five-week-old female NOD mice were infected with LDV (10(8) ID50/mouse) and observed until 23 weeks of age. None of the 21-LDV-infected mice developed diabetes, whereas 10/14 (71.4%) uninfected mice did. Although the subpopulations of T cells and the percentage of Mac1-positive cells in the NOD murine spleen and the number of harvested peritoneal macrophages were unaffected by LDV infection, the proportions of Ia-positive peritoneal macrophages were significantly decreased in LDV-infected compared with uninfected mice (1.1 +/- 0.2%, 6.5 +/- 2.9%; P < 0.01). In LDV-infected NOD mice, insulitis of the same grade as that seen in uninfected NOD mice was observed. In another experiment, 3, 5, 10 or 16-week-old female NOD mice were infected with LDV. None of the mice infected with LDV at 3, 5 or 10 weeks of age developed diabetes and only one of six infected at 16 weeks of age did. These findings indicate that LDV infection suppresses the development of diabetes in female NOD mice by reducing the capacity of Ia-positive macrophages, and suggest that the development of human type 1 diabetes may be suppressed by certain viral infections.     

Quantitative Analysis of Mycobacterial and Propionibacterial DNA in Lymph Nodes of Japanese and European Patients with Sarcoidosis

The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.     

Liver metastasis from rectal cancer with prominent intrabile duct growth

Intrabiliary growth of liver metastases from colorectal cancer has rarely been studied. A surgically resected case of a metastatic liver tumor with prominent intrabiliary growth derived from rectal cancer is reported. The patient was a 62-year-old man who had received a low anterior resection for rectal cancer in March 2000. He was re-admitted due to obstructive jaundice in January 2003, and was diagnosed with hepatic malignancy in segment II of the liver with an intrabiliary tumor extending from the intrahepatic bile duct of segment II to the common hepatic duct. He underwent a left hepatectomy, a partial resection of segment VI, and an extrahepatic bile duct resection with reconstruction of the biliary tract. In the resected specimen, there were whitish tumors of 3 cm and 1.5 cm in diameter in segments II and VI, respectively, and an intrabiliary tumor originating from the main tumor in segment II extended to the common hepatic duct. Both the liver tumors and the intrabiliary tumor consisted of a well- to moderately differentiated adenocarcinoma, which showed the same histological features as the rectal cancer. The immunohistochemical findings strongly supported that these tumors, including the intrabiliary growth, were liver metastasis from the rectal cancer. The intrabiliary invasion and growth of metastatic liver tumors has generally been overlooked, notwithstanding their frequently observed biological behavior. The present case is informative, and further investigation into this type of metastatic liver tumor may be warranted.     

Effects of ortho-substituents on the living polymerization of phenylacetylenes by MoOCl4-based catalysts

Polymerization of phenylacetylenes (PAs) having various ortho-substituents were examined by using MoOCl₄—n-Bu₄Sn—EtOH (mole ratio 1:1:1) as catalyst. Phenylacetylenes with no or sterically small ortho-substituents did not polymerize in a living fashion. On the other hand, in the polymerization of phenylacetylenes having medium-sized substituents (e.g., CH₃, Cl, Br, and iPr), the number-average molecular weights M_n of polymers increased in direct proportion to monomer consumption, while the polydispersity ratios were 1,2– 1,3, which is in favor of living polymerization. Further, monomers having bulky ortho-groups (CF₃ and Me₃Ge) exhibited excellent living nature with small polydispersity ratios of ≈ 1,1. Thus, it is concluded that not the electronic but the steric effect of the ortho-substituent is important to achieve living polymerization.     

Regulation of B cell fates by BCR signaling components

Recent results obtained in mice harboring cytoplasmic mutations of Igalpha and/or Igbeta have reinforced the concept that the strength of BCR signaling is important for ensuring appropriate developmental outcomes as well as antigen-specific responses. To establish the optimal signaling intensity and duration, the BCR utilizes positive and negative regulatory molecules. Studies are beginning to reveal how these molecules maintain immunological homeostasis and tolerance.