Characteristic intestinal microflora of specific pathogen-free mice bred in two different colonies and their influence on postnatal murine immunocyte profiles.

Cecal microflora of BALB/c mice originating from two different SPF-breeding colonies were compared. The analysis of cultivable bacteria in the ceca showed significantly higher numbers of total bacteria in BALB/cCrSlc (SLC mice) than in BALB/cA Jcl (JCL mice) (p<0.05), which were mainly based on higher numbers and occurrence of Peptococaceae. Bifidobacteria were detected only in SLC mice. Feeding an oligosaccharide, raffinose, to the mice also induced different shifts in the composition of cecal microflora and the concentration of cecal organic acids. In the second experiment, hysterectomy-derived (HD) SLC mice were fostered to SPF lactating SLC mothers, or SPF lactating JCL mice, together with the mother's own natural birth (NB) pups in each isolator. HD mice fostered to SLC-mothers showed significantly higher percentages of T-cell receptor alphabeta cells expressing a CD8alpha homodimer (p<0.05) and a CD8alphabeta heterodimer (p<0.001) in the intraepithelial lymphocytes (IEL) compared with HD mice fostered to JCL-mothers. IEL profiles of HD mice corresponded well to those of NB mice that were breast-fed by the same mothers. Differences in the ratio of B220(+)cells to Thy1.2(+)cells in the splenocytes were also observed as a trend between both HD mice fostered to SLC or JCL mothers (p=0.06). These results suggest that postnatal colonization of various characteristic intestinal microflora derived from SPF-breeding colonies results in differences in development of lymphocyte populations in the intestinal and systemic organs of mice.     

Blood flow restriction during low-intensity resistance exercise increases S6K1 phosphorylation and muscle protein synthesis.

Low-intensity resistance exercise training combined with blood flow restriction (REFR) increases muscle size and strength as much as conventional resistance exercise with high loads. However, the cellular mechanism(s) underlying the hypertrophy and strength gains induced by REFR are unknown. We have recently shown that both the mammalian target of rapamycin (mTOR) signaling pathway and muscle protein synthesis (MPS) were stimulated after an acute bout of high-intensity resistance exercise in humans. Therefore, we hypothesized that an acute bout of REFR would enhance mTOR signaling and stimulate MPS. We measured MPS and phosphorylation status of mTOR-associated signaling proteins in six young male subjects. Subjects were studied once during blood flow restriction (REFR, bilateral leg extension exercise at 20% of 1 repetition maximum while a pressure cuff was placed on the proximal end of both thighs and inflated at 200 mmHg) and a second time using the same exercise protocol but without the pressure cuff [control (Ctrl)]. MPS in the vastus lateralis muscle was measured by using stable isotope techniques, and the phosphorylation status of signaling proteins was determined by immunoblotting. Blood lactate, cortisol, and growth hormone were higher following REFR compared with Ctrl (P < 0.05). Ribosomal S6 kinase 1 (S6K1) phosphorylation, a downstream target of mTOR, increased concurrently with a decreased eukaryotic translation elongation factor 2 (eEF2) phosphorylation and a 46% increase in MPS following REFR (P < 0.05). MPS and S6K1 phosphorylation were unchanged in the Ctrl group postexercise. We conclude that the activation of the mTOR signaling pathway appears to be an important cellular mechanism that may help explain the enhanced muscle protein synthesis during REFR.     

Apoptosis Induction by the Binding of the Carboxyl Terminus of Human Immunodeficiency Virus Type 1 gp160 to Calmodulin

The role of calmodulin (CaM) in apoptosis induced by gp160 of human immunodeficiency virus type 1 was investigated with cells undergoing single-cell killing. These cells were found to express, under the control of an inducible promoter, wild-type gp160 or mutant gp160 devoid of various lengths of the carboxyl terminus. Immunoprecipitation accompanied by immunoblotting revealed binding of CaM to wild-type gp160 but not to mutant gp160 bearing a carboxyl terminus with a deletion spanning more than five amino acid residues. A significant coenzyme activity was detected in the CaM bound to gp160 even in the presence of a Ca²⁺ chelater, EGTA. The cells forming this gp160-CaM complex exhibited an elevated intracellular Ca²⁺ level followed by DNA fragmentation, which is a hallmark of apoptosis, and finally cell killing, while the cells not forming this complex did not show any significant elevation in Ca²⁺ level or DNA fragmentation. These results thus indicated that CaM plays a key role in gp160-induced apoptosis.     

Diagnostic Value of the Amplicor PCR Assay for Initial Diagnosis and Assessment of Treatment Response for Pulmonary Tuberculosis

We evaluated the Amplicor PCR assay as an initial diagnostic tool on the basis of clinical diagnosis, and assessed this assay as a follow-up test for patients with pulmonary tuberculosis during chemotherapy. Of the 208 specimens from 155 patients who were bacteriologically and/or clinically diagnosed with active tuberculosis before chemotherapy, 144 were Amplicor PCR-positive (sensitivity, 69.2%), which was equal to the results of culturing. Among 89 specimens which showed positive results by smear and culturing, the Amplicor PCR assay detected 87 (97.8%), whereas among 55 specimens which showed smear-negative but culture-positive results, the Amplicor PCR assay detected 46 (83.6%)(P = 0.003). No false positive results were found in the two systems (specificity, 100%, 120/120). The Amplicor PCR assay was also evaluated as a follow-up test using 926 specimens from 207 patients receiving active tuberculosis chemotherapy. Among 433 specimens which showed Amplicor-PCR positive, 222 (51.3%) were culture-negative. On the other hand, among 233 culture-positive specimens, only 12 (5.2%) were Amplicor PCR-negative. Therefore, this assay is useful for the rapid diagnosis of tuberculosis. The duration of Amplicor PCR-positive after culture-negative conversion was significantly associated with the presence of cavitary lesion, smear-positive specimens before treatment, and smear-positive specimens with negative cultures during chemotherapy.     

Detection of HIV-Gag p24-Specific Antibodies in Sera and Saliva of HIV-1-Infected Adults and in Sera of Infants Born to HIV-1-Infected Mothers

Secretory immunoglobulin A (IgA) is known to play an important role in the mucosal defense against a variety of pathogens. Although the role of IgA antibodies during sexual transmission of HIV is not clear, HIV-specific IgA antibodies have been detected in various mucosal secretions of HIV-infected individuals. Using a monoclonal antibody against human IgA, we established an ELISA system to detect anti-HIV p24 IgA antibodies in sera and saliva. We have analyzed the levels of anti-HIV p24 IgG and IgA antibodies in sera and saliva of 107 and 119 adults, respectively, with HIV infection at different clinical stages, and in the sera of 13 infants born to HIV-infected mothers. The level of anti-HIV p24 IgA antibodies was lower in sera and higher in saliva as compared to that of anti-HIV p24 IgG antibodies. Where the percentage of HIV-specific serum antibody-positive cases decreased with disease progression, that of saliva antibody-positive cases increased in AIDS patients. Among the 13 infants born to HIV-infected mothers, 7 infants were HIV-p24-specific serum IgA positive. These sera were negative for anti-HIV p24 secretory IgA, suggesting that some infants develop their own immune responses against HIV infection. Thus, the detection of HIV-specific IgA antibodies, especially in saliva, could be a simple and reliable test for the diagnosis of HIV infection.     

Activation of macrophages by a laccase-polymerized polyphenol is dependent on phosphorylation of Rac1

Various physiologically active effects of polymerized polyphenols have been reported. In this study, we synthesized a polymerized polyphenol (mL2a-pCA) by polymerizing caffeic acid using mutant Agaricus brasiliensis laccase and analyzed its physiological activity and mechanism of action. We found that mL2a-pCA induced morphological changes and the production of cytokines and chemokines in C3H/HeN mouse-derived resident peritoneal macrophages in vitro. The mechanisms of action of polymerized polyphenols on in vitro mouse resident peritoneal cells have not been characterized in detail previously. Herein, we report that the mL2a-pCA-induced production of interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in C3H/HeN mouse-derived resident peritoneal cells was inhibited by treatment with the Rac1 inhibitor NSC23766 trihydrochloride. In addition, we found that mL2a-pCA activated the phosphorylation Rac1. Taken together, the results show that mL2a-pCA induced macrophage activation via Rac1 phosphorylation-dependent pathways.     

Newly developed selective immunoinactivation assay revealed reduction in adipose triglyceride lipase activity in peripheral leucocytes from patients with idiopathic triglyceride deposit cardiomyovasculopathy

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare and newly identified disease among patients requiring cardiac transplantation. TGCV is characterized by cardiomyocyte steatosis and triglyceride (TG)-deposit atherosclerosis, resulting from the abnormal intracellular metabolism of TG. TGCV is classified into primary and idiopathic types. Primary TGCV carries ultra-rare genetic mutations in the adipose triglyceride lipase (ATGL), a rate-liming enzyme that hydrolyzes intracellular TG in adipose and non-adipose tissues. Idiopathic TGCV, first identified among autopsied individuals with diabetes mellitus (DM) with severe heart diseases, shows no ATGL mutations and its causes and underlying mechanisms are still unknown. TGCV is difficult to diagnose in daily clinics, thereby demanding feasible diagnostic procedures. We aimed to develop an assay to measure ATGL activity using peripheral leucocytes. Human his6-ATGL was expressed in COS1 cells, purified to homogeneity, and used to raise a polyclonal antibody neutralizing TG-hydrolyzing activity of ATGL. We developed a selective immunoinactivation assay (SIIA) for the quantitation of ATGL activity in cell lysates of leucocytes by the antibody neutralizing ATGL activities. ATGL activity was measured in 13 idiopathic TGCV patients, with two patients with primary TGCV as the negative control. Healthy (non-DM) and DM controls without heart diseases were also subjected. The developed SIIA assay revealed significant reduction in ATGL activity in leucocytes from patients with idiopathic TGCV who did not carry ATGL mutations as compared with non-DM and DM controls. Thus, ATGL in leucocytes may be an important biomarker for the diagnosis of TGCV and our assay may provide insights into pathophysiology and elucidate the underlying mechanism of TGCV and related disorders.