Evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis.

OBJECTIVE: A new loop-mediated isothermal amplification (LAMP) test kit, including a simple DNA extraction device for the detection of Mycobacterium tuberculosis complex, was developed for commercial use and evaluated for its usefulness in diagnosing tuberculosis (TB). DESIGN: The LAMP test was performed using untreated and N-acetyl-L-cysteine (NALC) NaOH-treated sputum specimen. The efficiency of the kit was compared with other conventional laboratory examinations, including other nucleic acid amplification (NAA) tests. RESULTS: The sensitivity of LAMP using raw sputum (direct LAMP) in smear- and culture-positive specimens was 98.2% (95%CI 94.9-99.4), while the sensitivity in smear-negative, culture-positive specimens was 55.6% (95%CI 43.4-68.0). The diagnostic sensitivity of direct LAMP for the diagnosis of individuals with TB was 88.2% (95%CI 81.4-92.7). The sensitivity values of direct LAMP were slightly, but not statistically significantly lower than those of Cobas Amplicor MTB and TRC Rapid MTB, while the sensitivity of the LAMP test using NALC-NaOH treated sputum was significantly lower than other NAA tests (P < 0.05) for smear-negative, culture-positive specimens. The new commercial version of the LAMP kit was easy to handle and yielded results within 1 h of receiving sputum specimens. CONCLUSIONS: This test is considered a promising diagnostic tool for TB, even for peripheral laboratories with limited equipment, such as those in developing countries.

[Evaluation of the BACTEC MGIT 960 system for drug susceptibility testing of Mycobacterium tuberculosis isolates compared with the proportion method on solid media].

The methods most widely used for susceptibility testing against anti-tuberculosis drug (AST) are the proportion method on Löwenstein-Jensen egg (L-J), Ogawa egg or Middle-brook agar media, and BACTEC TB 460 system. Recently, drug concentrations have been established for AST using the automated BACTEC MGIT 960 system (aMGIT). We have evaluated the BACTEC MGIT 960 SIRE kit for AST of Mycobacterium tuberculosis to isoniazid, rifampin, streptomycin and ethambutol. Also we compared the results with the proportion methods on Middlebrook 7H10 agar (7H10), L-J and Ogawa egg, and the manual MGIT system (mMGIT). Overall concordance rates among aMGIT and the proportion method on 7H10 or Ogawa media were 98.3% and 96.9% for 4 first-line drugs, respectively. Rates were particularly high for isoniazid and rifampin between aMGIT and 7H10 (efficiency of 100%). On the other hand, overall concordance rates among two egg media, L-J and Ogawa were 99.9%. Agreement between aMGIT and mMGIT was high for the AST to isoniazid and rifampin, but lower for the AST to ethambutol (90.9%), which relates to a lower specificity of mMGIT. The mean times to aMGIT and mMGIT results of susceptibility were 7 and 6 days, respectively, contrasted with 3 weeks in 7H10 and 4 weeks in L-J and Ogawa, indicating that both MGIT systems have the potential to consistently meet the turnaround time suggested by Centers for Disease Control and Prevention (CDC) of the United States. These results demonstrate that the fully automated BACTEC MGIT 960 SIRE system for AST is useful for rapid diagnosis of drug resistant tuberculosis.

Convenient dioxin measuring method using an efficient sampling train, an efficient HPLC system and a highly sensitive HRGC/LRMS with a PTV injector.

An efficient sampling method for dioxins from flue gas, an efficient automatic purification and fractionation method by a new HPLC system using a sulfuric acid-silica/silica column, a Nitro column and a PGC column, and sensitive determination method by an economical HRGC/LRMS using a programmable temperature vaporization (PTV) injector were developed. It was confirmed that the dioxins could be easily collected by the proposed simple sampling train consisting of only two bottles, and the extracted sample could be purified sufficiently and separated as mono-ortho PCBs, non-ortho PCBs and PCDDs/PCDFs completely with the proposed HPLC system. The peak areas of GC/MS were increased linearly with the injection volume up to 40 microl by the PTV technique, and the sensitivity could be increased to 20 times higher than usual. This convenient measuring method can drastically reduce operation time, consumption of hazardous solvent and cost.

[The effect of immunization with herpes simplex virus glycoprotein D fused with interluekin-2 against murine herpetic keratitis].

PURPOSE: To evaluate the effect of vaccination with fusion protein(gD-IL-2) consisting of herpes simplex type1(HSV-1) glycoprotein D(gD) and human interleukin-2(IL-2), and the effect of plasmid DNA vaccine encoding gD-IL-2 against murine herpetic keratitis. METHODS: Plasmid containing gD-IL-2(pHDL-neol) was constructed, and gD-IL-2 peptide was purified. BALB/c mice were injected twice hypodermally or subconjunctivally with 1 microgram/0.1 ml of gD-IL-2 peptide, or twice subconjunctivally with 90 micrograms/0.05 ml of gD-IL-2 plasmid DNA. Neutralizing antibody titer and delayed-type hypersensitivity (DTH) against HSV-1 were measured. Immunized mice were challenged with CHR3 strain of HSV-1 via the cornea. The clinical picture of epithelial and stromal keratitis was scored. RESULTS: Stromal keratitis was inhibited in gD-IL-2 peptide- or gD-IL-2 DNA-immunized mice, but epithelial keratitis was not. It was confirmed that plasmid gD-IL-2 elicited significant serum virus neutralizing titer and DTH response. CONCLUSION: Vaccination with gD-IL-2 was effective against herpetic keratitis.

Preventive effect of local plasmid DNA vaccine encoding gD or gD-IL-2 on herpetic keratitis.

PURPOSE: The goal of this study was to evaluate the effectiveness of a local plasmid DNA vaccine encoding herpes simplex virus (HSV) type 1 glycoprotein D (gD) or gD-interleukin (IL)-2 (chimeric gene of gD and human IL-2) in preventing murine herpetic keratitis. METHODS: Plasmids containing gD (pHSDneo1), gD-IL-2 (pHDLneo1), or vaccine vector (pHSGneo) were injected subconjunctivally with BALB/c mice on days 0 and 7 (90 microgram x 2). Immunization was indicated by positive virus-neutralizing antibody titer, swollen pinna (due to delayed-type hypersensitivity [DTH] reaction), and release of (51)Cr from splenic and/or local cytotoxic effector cells on day 28. In another group of the immunized mice, corneas were challenged with HSV-1 (CHR3 strain, 10 microliter of 3 x 10(6) plaque-forming units [PFU]/ml). Mice were evaluated for clinical signs of epithelial or stromal keratitis on days 1 through 8 and days 10 and 14 or measured on days 2, 4, or 6 for viral titers in the eyes, trigeminal ganglia, and brain. RESULTS: All gD-DNA-injected mice obtained specific immunity. Furthermore, gD-IL-2-DNA elicited a higher DTH reaction and more vigorous cytotoxic effector cell activity. Stromal keratitis scores were lower for all immunized mice compared with control mice, although the difference in epithelial keratitis scores was not statistically significant. Viral titers in eyes, trigeminal ganglia, and brains were suppressed in all immunized mice. CONCLUSIONS: Local immunization with plasmid DNA encoding gD or gD-IL-2 induces humoral and cellular immunity against HSV-1 and inhibits development of stromal keratitis. gD-IL-2 DNA induces greater cell-mediated immunity than gD DNA alone. A plasmid encoding gD-IL-2 is therefore a promising candidate for a vaccine against HSV-1.

Forced expression of terminal deoxynucleotidyl transferase in fetal thymus resulted in a decrease in gammadelta T cells and random dissemination of Vgamma3Vdelta1 T cells in skin of newborn but not adult mice.

The repertoire of lymphocyte receptor genes encoded in a germline is further diversified by a number of processes, including the template-independent addition of nucleotides (N regions) by means of terminal deoxynucleotidyl transferase (TdT). Normally, mouse gammadelta T cells in the early fetal thymus, whose T-cell receptor (TCR) genes lack N regions and are encoded by Vgamma3-Jgamma1 and Vdelta1-Ddelta2-Jdelta2 with canonical junctions (invariant Vgamma3Vdelta1), are thought to be the precursors of dendritic epidermal T cells (DETC). We generated mutant mice whose endogenous TdT promoter was replaced with the lck promoter through homologous recombination. These mutant mice expressed TdT in fetal thymus, had abundant N regions and infrequent canonical junctions in gamma and delta rearrangements, and showed a decreased number of gammadelta T cells. Various Vgamma3Vdelta1 T cells, most of which had N regions in their TCR genes, were found to disseminate in the skin of newborn mutant mice, whereas normal numbers of DETCs with the invariant Vgamma3Vdelta1 rearrangement were observed in adult mutants. These data demonstrate that the regulation of TdT expression during fetal development is important for the generation of gammadelta T cells, and that Vgamma3Vdelta1 T cells, which have various junctional sequences in their TCR genes, randomly disseminate in skin, but invariant Vgamma3Vdelta1 T cells have a great advantage for proliferation in skin.

[Participation of pharmacists in the medical team at home].

This report reviews drug consultations at patients' home and collaboration of pharmacists with medical teams over 4 years. The participation of pharmacists is necessary for home care service, because of their pharmaceutical expertise and the demands from the medical team and patients at home.

Growth disturbance in fetal liver hematopoiesis of Mll-mutant mice.

The MLL (ALL-1, HRX) gene is frequently involved in chromosomal translocations in acute leukemia and has homology with Drosophila trithorax, which controls homeobox gene expression and embryogenesis. To elucidate the function of Mll, we generated mice with a mutated Mll locus. Mice with a homozygous mutation were embryonic lethal and died at embryonic day 11.5 to 14.5, showing edematous bodies and petechiae. Histological examination revealed that hematopoietic cells were decreased in the liver of homozygous embryos, although they were composed of erythroid, myeloid, monocytic, and megakaryocytic cells with normal differentiation. Colony-forming assays using cells from fetal livers and yolk sacs showed that the number of colonies was markedly reduced and many of the colonies delayed to be recognized in Mllmu/mu embryos, although some of the colonies from Mllmu/mu embryos developed similarly with that from Mll+/+ and Mll+/mu embryos, suggesting the delayed onset of the proliferation of hematopoitic precursors. These data show that the hematopoietic precursors were greatly reduced in mutant mice, and suggest that Mll functions as a regulator of the growth of hematopoietic precursors.

Structure and function of a new STAT-induced STAT inhibitor.

The signalling pathway that comprises JAK kinases and STAT proteins (for signal transducer and activator of transcription) is important for relaying signals from various cytokines outside the cell to the inside. The feedback mechanism responsible for switching off the cytokine signal has not been elucidated. We now report the cloning and characterization of an inhibitor of STAT activation which we name SSI-1 (for STAT-induced STAT inhibitor-1). We found that SSI-1 messenger RNA was induced by the cytokines interleukins 4 and 6 (IL-4, IL-6), leukaemia-inhibitory factor (LIF), and granulocyte colony-stimulating factor (G-CSF). Stimulation by IL-6 or LIF of murine myeloid leukaemia cells (M1 cells) induced SSI-1 mRNA expression which was blocked by transfection of a dominant-negative mutant of Stat3, indicating that the SSI-1 gene is a target of Stat3. Forced overexpression of SSI-1 complementary DNA interfered with IL-6- and LIF-mediated apoptosis and macrophage differentiation of M1 cells, as well as IL-6 induced tyrosine-phosphorylation of a receptor glycoprotein component, gp130, and of Stat3. When SSI-1 is overexpressed in COS7 cells, it can associate with the kinases Jak2 and Tyk2. These findings indicate that SSI-1 is responsible for negative-feedback regulation of the JAK-STAT pathway induced by cytokine stimulation.

Potent ectopic bone-inducing activity of bone morphogenetic protein-4/7 heterodimer.

We have purified and characterized recombinant Xenopus bone morphogenetic proteins (xBMPs): homodimers of xBMP-4, 7 and heterodimers (xBMP-4/7) produced by a baculovirus expression system. Highly purified xBMPs had homogeneous NH2-termini predicted from a consensus motif, Arg-X-X-Arg, while they possessed diverse sugar chains. Implantation of xBMPs together with pure collagen carrier in rats induced new bone formation in a dose-dependent manner. The xBMP-4/7 heterodimer showed the strongest activity, with an effective dose of 1-30 micrograms, while more than 10 micrograms of xBMP-4 or 7 homodimer was required for a significant effect. Histological examination revealed that xBMP-4/7 implants showed intramembranous ossification without chondrogenesis. In primary cultures of rat bone marrow stromal cells, xBMP-4/7 induced alkaline phosphatase 3-fold more strongly than xBMP-7 and 20-fold more than xBMP-4. These results suggest that the heterodimeric form of BMP would generate the strongest signal triggering osteogenic differentiation of osteoprogenitor cells in adult tissues.

Efficient expression of a heterodimer of bone morphogenetic protein subunits using a baculovirus expression system.

Recombinant baculoviruses as expression vectors for Xenopus bone morphogenetic protein (xBMP)-2, 4 and 7 were generated. The conditioned medium of insect cells infected with the virus for xBMP-2 or 4 showed strong alkaline phosphatase-inducing activity in a mouse osteoblastic cell line, MC3T3-E1, although a large portion of the activity remained in the infected cells. In contrast, xBMP-7 was preferentially secreted into the medium, but had only weak activity. Conditioned media following simultaneous inoculation with the viruses for xBMP-7 and for xBMP-2 or 4 showed a remarkably increased level of activity. The increased activity was clearly separated from other peaks derived from single infection on a cation-exchange column and was found to arise from the disulfide-linked heterodimer consisting of xBMP-4 and 7 subunits by immunoblot analysis. The heterodimer also augmented osteocalcin production and parathyroid hormone-sensitivity more strongly than the homodimers. These results suggest that our expression system provides a convenient source of heterodimeric BMP with high osteogenic differentiation-inducing activity.

Immunotherapy of acute and recurrent herpes simplex virus type 2 infection with an adjuvant-free form of recombinant glycoprotein D-interleukin-2 fusion protein.

Previous studies demonstrated that the adjuvant-free form of a fusion protein consisting of a truncated herpes simplex virus type 1 (HSV-1) glycoprotein D and human interleukin-2 (tgD-IL-2) elicited superior protective immunity in mice. In this study, the immunotherapeutic efficacy of tgD-IL-2 against vaginal HSV-2 infection was investigated using a guinea pig model. Footpad injections of tgD-IL-2 (12.5 micrograms/dose) after the onset of primary lesions strongly suppressed recurrence in the chronic phase of infection; consequently, the number of days with lesions was reduced 65%. Continuous medication with 100 mg/kg/day acyclovir for 5 days failed to suppress recurrent infection. In a UV radiation-induced recurrence model, prophylactic tgD-IL-2 significantly suppressed both duration and severity of disease. A single injection of tgD-IL-2 plus acyclovir produced an additive effect on the suppression of the disease in the acute phase. These results suggest that tgD-IL-2 is a promising immunotherapeutic agent against HSV-2 genital infections.

Intranasal immunization against herpes simplex virus infection by using a recombinant glycoprotein D fused with immunomodulating proteins, the B subunit of Escherichia coli heat-labile enterotoxin and interleukin-2.

To establish a novel strategy of mucosal immunization against herpes simplex virus type 1 (HSV-1) infection, we studied the immune responses elicited by intranasal immunization with several forms of a recombinant glycoprotein D (gD) of HSV-1. A truncated gD (t-gD) co-administered with heat-labile enterotoxin B subunit (LTB) from Escherichia coli induced both a mucosal immune response involving secretion of anti-gD IgA and serum IgG production. The levels of these responses are comparable to those in mice which have recovered from intranasal HSV-1 infections. The fusion protein (t-gD-LTB), consisting of t-gD and LTB, induced the responses more efficiently than did co-administration of t-gD and LTB, although GM1 ganglioside binding activity was significantly reduced in t-gD-LTB. We found that another fusion protein, consisting of t-gD and human interleukin-2 (t-gD-IL-2), also elicited antibody responses comparable to those induced by t-gD-LTB. Immunity acquired by intranasal immunization with t-gD-IL-2 protected mice from intraperitoneal HSV-1 infections, whereas t-gD-LTB or t-gD alone failed to provide protection against infection. Even in a mouse strain that responded highly to subcutaneously administered gD, intranasally administered t-gD did not elicit antibody responses. The lack of response to gD was clearly abrogated by co-administration with IL-2, and administration of t-gD-IL-2 induced an excellent level of antibody responses in this strain. These results suggest that the IL-2 fusion strategy yields a new type of mucosal immunization, the mechanism of which differs from that speculated for the mucosal adjuvant activity of LTB.

Adjuvant-independent enhanced immune responses to recombinant herpes simplex virus type 1 glycoprotein D by fusion with biologically active interleukin-2.

A truncated herpes simplex virus (HSV) type 1 glycoprotein D (t-gD) gene was fused to the human interleukin-2 (IL-2) gene (t-gD-IL-2 gene) and introduced into mouse myeloma Sp2/0 cells. The gene product, t-gD-IL-2, secreted from the cells was immunoprecipitated with five monoclonal antibodies specific for native gD. Purified t-gD-IL-2 supported the growth of IL-2-dependent cells, with a specific activity almost comparable to that of recombinant human IL-2. Mice immunized with t-gD-IL-2 in an adjuvant-free form showed superior anti-HSV antibody responses, and were completely protected against HSV challenge, whereas immunization with t-gD adsorbed onto aluminum hydroxide (alum) partially failed to prevent the virus infection. The high immunogenicity of t-gD-IL-2 was due to the biological activity of the fused IL-2 rather than to a hapten-carrier effect of the IL-2 moiety, because mice primed with t-gD-IL-2 showed delayed-type hypersensitivity against stimulation with gD, but not against that with IL-2 antigen, and because a booster immunization with t-gD-IL-2 extensively augmented the response of anti-gD antibody, but not that of the anti-human IL-2 antibody. The serological half-life of IL-2 activity in mice injected with t-gD-IL-2 was prolonged to about four times that of rIL-2. However, when t-gD-IL-2 was co-administered with human albumin (HSA), the mouse anti-HSA antibody response was slightly enhanced.(ABSTRACT TRUNCATED AT 250 WORDS)