Dual-probe assay for rapid detection of drug-resistant Mycobacterium tuberculosis by real-time PCR

Mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in Mycobacterium tuberculosis. For rapid detection of drug-resistant M. tuberculosis in clinical specimens, a simple and applicable method is needed. Eight TaqMan minor groove binder (MGB) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). The target of six MGB probes was the rpoB gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpoB gene, and one probe was designed as a tuberculosis (TB) control outside the rpoB gene hot-spot. We also designed probes to examine codon 315 of katG and codon 306 of embB for mutations associated with resistance to isoniazid and ethambutol, respectively. Our system was M. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. Detection limits in direct and preamplified analyses were 250 and 10 fg of genomic DNA, respectively. The system could detect mutations of the rpoB, katG, and embB genes in DNAs extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary TB. This system was much faster (3 h from DNA preparation) than conventional drug susceptibility testing (3 weeks). Results from the dual-MGB-probe assay were consistent with DNA sequencing. Because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drug-resistant M. tuberculosis in clinical laboratories.     

Costimulation through OX40 is crucial for induction of an alloreactive human T-cell response

The alloreactive immune response is a series of events initiated by the interaction of T cells with allogeneic dendritic cells (DCs), involving alloantigen recognition and costimulatory signals. In this study, we investigated the role of OX40 in alloreactivity in vitro. We first demonstrated that anti-OX40 ligand (anti-OX40L) monoclonal antibody (mAb) could markedly suppress the mixed leucocyte reaction (MLR) of peripheral blood mononuclear cells (PBMC). To further define the contribution of the OX40/OX40L system to the MLR, we set up a co-culture system of CD4+ T cells and allogeneic monocyte-derived dendritic cells (DCs). After 2 days, OX40 expression was induced on CD4+ T cells and this induction was strongly inhibited by the addition of cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)-Fc fusion protein, suggesting that the expression of OX40 during alloreaction is dependent on CD28 signalling. Next we examined the effects of anti-OX40L mAb, CTLA-4-Fc fusion protein and anti-human leucocyte antigen (HLA)-DR mAb on the proliferative response of CD4+ T cells to allogeneic DCs. The proliferation of T cells was almost completely suppressed by anti-OX40L mAb, which was comparable with that of CTLA-4-Fc. Measurement of interleukin-2 (IL-2) production in the culture supernatants showed that suppression of a proliferative response was at least in part ascribed to reduced IL-2 production. Furthermore, purified OX40L- allogeneic DCs could induce considerable proliferation of CD4+ T cells, which was suppressed by anti-OX40L mAb. These results suggest that the OX40/OX40L system is crucial for induction of the allogeneic T-cell response and the OX40/OX40L system is subsequent to and dependent on CD28 signalling, but is crucial for the end outcome of the human alloreactive T-cell response.     

Isolation of an influenza C virus introduced into Japan by a traveler from Malaysia

An influenza C virus was isolated from a Japanese traveler who had visited Malaysia in April 1999. Phylogenetic analysis indicated that the genome composition of this virus was distinct from that of any other strain isolated in Japan. The possibility that a genetically unique influenza C virus was introduced into Japan by a traveler is shown.     

Mixed growth hormone cell- prolactin cell pituitary adenoma with acromegaly: α-subunit most growth hormone cells

A 51 -year-old woman with mixed growth hormone (GH) cell-prolactin (PRL) cell pituitary adenoma is presented. She had clinical signs due to hypersecretion of GH and PRL. Resected tissue was studied immunohistochemically and morphologically. The serial sections revealed that GH and α-subunit were co-localized in most cells, while GH and PRL were localized in different cells.     

Conjugation of acetaldehyde with cysteinylglycine,the first metabolite in glutathione breakdown byγ-glutamyltranspeptidase

Glutathione and its metabolites were examined for reactivity to acetaldehyde. When acetaldehyde was incubated with glutathione alone, there was only a slight decrease of acetaldehyde, while an apparently equimolar reaction between acetaldehyde and free sulfhydryl was observed with the addition of -glutamyltranspeptidase. Cysteinylglycine, the first metabolite in the glutathione breakdown by -glutamyltranspeptidase, showed a rapid and equimolar reactivity to acetaldehyde and such was comparable to the reaction seen withl-cysteine ord-penicillamine. In light of the chemical structure, cysteinylglycine probably conjugates with acetaldehyde to form thiazolidinecarboxylic acid derivatives, 2-methyl-thiazolidine-4-carbonyl-glycine, and if so, the alteration of glutathione metabolism by acetaldehyde during ethanol intoxication warrants further attention.     

Expression of cytoplasmic TFF2 is a marker of tumor metastasis and negative prognostic factor in gastric cancer

Trefoil factor family 2 (TFF2) is a small peptide constitutively expressed in the gastric mucosa, where it plays a protective role in restitution of gastric mucosa. TFF2 has also been shown to be expressed in some gastric cancers, but its role in tumor metastasis and patient prognosis has not been examined. In this study, we examined TFF2 expression at both the mRNA and protein levels and correlated these results with the clinicopathologic characteristics and prognosis of gastric cancer patients. Among the 144 curatively resected samples, 43 (30%) were positive for TFF2. TFF2 expression was preferentially observed in the infiltrating tumor cells sparing the superficial cells. Significantly increased expression of TFF2 was noted in large tumors of the diffuse type. An increased prevalence of TFF2 expression was also found in tumors with advanced T and N stage and in patients with lymphatic and venous invasion. Accordingly, patients with TFF2-expressing tumors had a significantly worse disease-free survival, and in multivariate analysis, this finding remained significant as an independent prognostic factor. Taken together, our results suggest that TFF2 expression may play a role in gastric cancer invasion and as such could be a useful target for therapeutic intervention.     

Immunoglobulin G from anti-glucose-6-phosphate isomerase antibodies positive patient with rheumatoid arthritis induces synovitis in cynomolgus monkeys

Anti-glucose-6-phosphate isomerase (GPI) antibodies (Abs) solely induce arthritis in mice. High titers of anti-GPI Abs are found in some patients with rheumatoid arthritis (RA), but their pathogenic role remains elusive. The aim of this study was to evaluate the pathogenic role of anti-GPI Abs in cynomolgus monkeys. IgG fractions were separated from sera of anti-GPI Abs-positive RA patients and healthy subjects and directly injected into the metacarpophalangeal joints of 4 cynomolgus monkeys. At day 16, the joints were harvested and examined histologically and immunohistochemically. The expression of C5a receptor (C5aR) molecule in the synovium was quantified by real-time PCR using cDNA from monkey joints. In monkey joints, IgG including anti-GPI Abs resulted in recruitment of granulocytes and mononuclear cells, strong deposition of human IgG on the articular surface, and overexpression of C5aR, but no joint swelling. No infiltrated cells or IgG deposition were observed in monkeys injected with IgGs from healthy subjects. Our results suggest that IgG fraction from RA patients including anti-GPI Abs may play a crucial role in the generation of synovitis in monkeys, although the pathogenesis of anti-GPI Abs in RA patients is still uncertain.     

Association of natural tooth loss with genetic variation at the human matrix Gla protein locus in elderly women

Natural tooth loss represents a major medical issue within the elderly population, since it impairs masticatory function critical for oral intake of essential nutrition. Contribution of genetic factors has been implicated in the determination of natural tooth loss; degree of reduction in number of natural teeth remaining intact (NTI) varies among individuals; thus, heterogeneity in NTI might reflect genetic variation within the population. One candidate gene, the matrix Gla protein gene (MGP), has been implicated in the pathogenesis of bone loss through a repression of bone/tooth formation. We have investigated a possible association between the CA repeat polymorphism at the human MGP gene locus and the NTI in 458 elderly Japanese women. In 916 chromosomes tested, ten alleles of the polymorphic nucleotide repeat were observed (designated A1–A10), among which five alleles were regarded as major alleles to be tested for the association. Twenty-seven women who possessed an A6 allele (164 bp) had significantly higher NTI than the remaining participants (n=431), who did not carry an allele of that size (mean: 10.0 teeth vs 5.6 teeth; P=0.007, Mann-Whitney test). An eight-year longitudinal follow-up study of NTI suggested that the genetic variations at the MGP locus did not affect the rate of tooth loss in the elderly period. These results suggest that genetic variation at the MGP gene locus is associated with some determinants for tooth loss in elderly women.     

Axis development in avian embryos: the ability of Hensen's node to self-differentiate,as analyzed with heterochronic grafting experiments

A series of experiments consisting of transplantation of Hensen's nodes has been conducted to examine axis development in avian embryos. In the first group of experiments, Hensen's nodes from quail embryos were transplanted homotopically and either isochronically or heterochronically to chick embryos, and the structures derived form the grafted nodes were assessed. The grafted Hensen's nodes typically self-differentiated structures appropriate for their stages, and the host embryos developed normally; the structures formed from grafted tissue usually merged caudally with the comparable host structures. Thus, even when the stages of the donor and host tissues were significantly mismatched (e.g., stage 3 donors and stage 9 hosts or vice versa), the graft was unable to repattern the host's neuraxis, and the host was unable to respecify the types of structures derived from the graft. In the second group of experiments, Hensen's nodes from quail embryos were transplanted to sites located just lateral to Hensen's nodes of host chick embryos, thereby providing the potential for development of additional axes. A single axis always resulted in each case in which further development occurred, with the graft self-differentiating its typical stage-specific structures, all of which merged caudally with comparable host structures. A final group of experiments served principally as a control and tested the ability of a part of Hensen's node, when it was transplanted to the extraembryonic germ cell crescent, to organize an ectopic embryo. In these experiments, the entire thickness and length of each Hensen's node, but only the central one-third to one-half of its width, was transplanted to host blastoderms, yet ectopic embryos, complete with induced neuraxes, were formed. Therefore, a part of Hensen's node has the ability to function fully as an organizer when placed in a conducive environment. Collectively, these results provide further documentation of the strong ability of Hensen's node to self-differentiate, and they suggest that once morphogenetic movements are under way, neuraxial structures can form, and characteristic rostrocaudal patterning of the neuraxis can occur, without sustained influence from Hensen's node.     

Inhibition of proliferative responses of mouse spleen lymphocytes by bovine milk κ‐casein digests

The inhibitory effects of bovine milk κ‐casein and its enzymatic digests on the proliferative responses of mouse spleen lymphocytes induced or not induced by mitogens were studied with a colorimetric assay using 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT). κ‐Casein and its glyco‐macropeptide (residues 106–169) inhibited the lipopolysaccharide (LPS)‐induced proliferative response, but the inhibitory effect was lost significantly after neuraminidase and chymotrypsin digestions. In contrast, trypsin and pronase digestions of K‐casein increased inhibitory effects. The pronase digest inhibited the proliferative responses not only induced by LPS but also in the absence of mitogen or when induced by concanavalin A and phytohemagglutinin. The pronase digest seemed to possess weak cytotoxity for lymphocytes. The inhibitory peptide was a glycopeptide(s) having specific size, and the inhibitory effects were reduced significantly by neuraminidase and chymotrypsin digestions. Moreover, similar inhibitory effects on proliferation of lymphocytes were observed in pronase‐digested bovine milk αs₁‐casein and β‐casein. These findings suggest that some peptides produced from milk caseins by the action of gastrointestinal proteinases might contribute to down‐regulate the immune response of neonates.