Canagliflozin Improves Erythropoiesis in Diabetes Patients with Anemia of Chronic Kidney Disease.

Background: We evaluated the erythropoietic effects of canagliflozin, a sodium-glucose cotransporter 2 inhibitor, in type 2 diabetes patients with anemia of chronic kidney disease. Methods: Nine diabetes patients were enrolled and administered 100 mg canagliflozin once a day for 12 weeks. The patients received fixed doses of conventional antidiabetic drugs and renin-angiotensin system inhibitors for 8 weeks before enrollment; these drugs were continued during the study. Endpoints were changes in erythropoiesis parameters, including erythrocyte and reticulocyte count, hemoglobin, hematocrit, and serum erythropoietin (EPO) concentration from baseline to 12 weeks. All variables were measured every 2 weeks. Results: Serum EPO concentration increased by 38 [15-62]% (P = 0.043) between baseline and 2 and 4 weeks. Reticulocyte count transiently increased at 2 weeks. Erythropoiesis occurred after 2 weeks of canagliflozin treatment. Erythrocyte count (from 386 ± 36 × 104/µL to 421 ± 36 × 104/µL; P = 0.0009), hemoglobin (from 11.8 ± 0.6 g/dL to 12.9 ± 1.1 g/dL; P = 0.0049), and hematocrit (from 37.1 ± 2.3% to 40.4 ± 3.2%; P = 0.002) increased from baseline to study completion. Although there were no significant changes in transferrin saturation, serum ferritin levels were decreased (P = 0.003). Conclusions: Canagliflozin treatment led to an improvement in erythropoiesis in patients with impaired kidney function. The effect on erythropoiesis appeared to be due to an EPO production-mediated mechanism and might be independent of glycemic control; however, further studies are needed to clarify this since the present study had a small sample size and no comparator group.

Glutamine protects intestinal barrier function of colon epithelial cells from ethanol by modulating Hsp70 expression.

In this study, we investigated the protective effect of glutamine on barrier dysfunction induced by ethanol, by using human epithelial colorectal adenocarcinoma cells (Caco-2). Our results show that addition of glutamine to culture medium significantly improved the disruption of integrity caused by ethanol, which was associated with increased expression of heat shock protein 70 (Hsp70). Ethanol exposure moderately activates heat shock factor 1 (HSF1), which was characterized by increased DNA-binding activity and phosphorylation status of HSF1. Remarkably, glutamine treatment enhanced ethanol-mediated expression of Hsp70 and activation of HSF1. Up-regulation of Hsp70 by pretreatment with heat stress also promoted recovery from the ethanol-induced barrier dysfunction. Taken together, these observations indicate that glutamine protects the intestinal barrier function in Caco-2 cells, in part by modulating HSF1-mediated Hsp70 expression.

Conditional ablation of CD205+ conventional dendritic cells impacts the regulation of T-cell immunity and homeostasis in vivo.

Dendritic cells (DCs) are composed of multiple subsets that play a dual role in inducing immunity and tolerance. However, it is unclear how CD205(+) conventional DCs (cDCs) control immune responses in vivo. Here we generated knock-in mice with the selective conditional ablation of CD205(+) cDCs. CD205(+) cDCs contributed to antigen-specific priming of CD4(+) T cells under steady-state conditions, whereas they were dispensable for antigen-specific CD4(+) T-cell responses under inflammatory conditions. In contrast, CD205(+) cDCs were required for antigen-specific priming of CD8(+) T cells to generate cytotoxic T lymphocytes (CTLs) mediated through cross-presentation. Although CD205(+) cDCs were involved in the thymic generation of CD4(+) regulatory T cells (Tregs), they maintained the homeostasis of CD4(+) Tregs and CD4(+) effector T cells in peripheral and mucosal tissues. On the other hand, CD205(+) cDCs were involved in the inflammation triggered by Toll-like receptor ligand as well as bacterial and viral infections. Upon microbial infections, CD205(+) cDCs contributed to the cross-priming of CD8(+) T cells for generating antimicrobial CTLs to efficiently eliminate pathogens, whereas they suppressed antimicrobial CD4(+) T-cell responses. Thus, these findings reveal a critical role for CD205(+) cDCs in the regulation of T-cell immunity and homeostasis in vivo.

Chromosome analysis by spectral karyotyping of spermatozoa from an oligoasthenozoospermic carrier of a 10; 21 reciprocal translocation.

Cytogenetic analysis of germ-line cells prior to intracytoplasmic sperm injection (ICSI) treatment is thought to be necessary for infertile males with an identified chromosomal abnormality. We analyzed the chromosomal karyotype of human spermatozoa from an oligoasthenozoospermic carrier of a reciprocal translocation t(10; 21). Cytogenetic analysis of 39 spermatozoa was performed by spectral karyotyping (SKY) and by ICSI into mouse oocytes. The motile morphologically normal spermatozoa were injected into mouse oocytes. Of these spermatozoa, 38 (97.4%) were activated. Twenty-one (53.8%) of the activated oocytes formed two pronuclei. Metaphase chromosome spreads from 13 spermatozoa were analyzed. Only one spermatozoon was normal and 2 spermatozoa exhibited balanced translocation. Nine and one spermatozoa showed abnormalities related and unrelated to the translocation, respectively. The numbers of normal/balanced spermatozoa were lower than those in previous reports analyzing reciprocal translocations using a previously described technique involving penetrated golden hamster oocytes. After genetic counseling with the carrier and his partner, ICSI treatment was performed. Healthy female and male infants were delivered at 37 weeks gestation via a Caesarean section. The female infant was a carrier of the reciprocal translocation and the male infant was confirmed normal on prenatal diagnosis at 16 weeks gestation. For genetic counseling prior to ICSI treatment, the incidence of unbalanced type spermatozoa after swim-up or Percoll gradient treatment should be investigated and discussed with couples having fertility problems related to oligozoospermia autosomal structural abnormalities.

Surface expression of a C-terminal alpha-helix region in heat shock protein 72 on murine LL/2 lung carcinoma can be recognized by innate immune sentinels.

Surface expression of Hsp70 members has been previously reported on human tumor cell lines. Here we examined how the inducible mouse Hsp72 can be expressed on the surface of two types of murine tumor cell lines in response to non-lethal heat shock. Exposure to 42 degrees C for 2h led to the intracellular production of Hsp72 for both murine LL/2 lung carcinoma and B16 melanoma cells. Flow cytometric analyses showed that living LL/2 carcinoma, but not B16 melanoma, transported a fraction of inducible Hsp72 to the cell-surface membrane. Induction of the surface expression of Hsp72 occurred upon non-lethal heat shock only when Hsp72 expression was forced to be elevated in B16 transfectants. Hsp72 expressed on the LL/2 cell surface was detected by the monoclonal antibody that recognized the epitope of 504-617 amino acid residues, but not by another antibody with the epitope of 122-264 residues. When we analyzed the binding of recombinant full-length Hsp72 to mouse splenocytes, significant binding was observed for innate immune cells such as CD11b(+)-, CD11c(+)-, or NK1.1(+)-cells. The recombinant variants obtained by truncation of the C-terminal helical region of Hsp72 exhibited more robust binding to these innate immune cells in a similar fashion, however, further deletion offered less binding to those immunocytes. Two fragment variants lacking the N-terminal nucleotide-binding domain were found to extensively bind to peritoneal macrophages. Taken together with these results, it thus follows that the sentinels in an innate immune system, macrophages, dendritic cells and NK cells, can be involved in the surveillance of functionally aberrant cells through the recognition of a specific C-terminal structure of Hsp70 as a danger signal in living cells.

Systolic dysfunction in urban Japan.

BACKGROUND: Heart failure (HF), which can be caused by left ventricular systolic dysfunction (LVSD), is a growing problem in developed countries with a large aging population. The aim of the present study was to characterize outpatients with LVSD in the adult population (45-84 years) in an urban Japanese community (Niigata City), and delineate their characteristics in comparison with those in a rural one (Sado). METHODS AND RESULTS: Over a 5-year period, 1,297 patients (67% males) with LVSD (defined as ejection fraction < or =50%) were extracted from 87,953 echocardiography records available in 15 hospitals in Niigata City. The proportion of LVSD increased progressively with age (p-for-trend <0.0001), reaching 1-2% in those aged > or =75 years. The prevalence of comorbidities was noticeable (47% had hypertension, 41% myocardial ischemia, 34% atrial fibrillation, 33% previous hospitalization because of congestive HF, 27% cerebral stroke). In comparison with Sado, Niigata patients were younger, with a higher prevalence of comorbidities (hypertension, diabetes, dyslipidemia, and cerebral stroke). CONCLUSIONS: As the proportion of LVSD cases increases progressively with age, it is expected to simulate a future epidemic. The differences between patients' characteristics and disease patterns in urban and rural communities may favor individually tailoring preventive strategies for HF in these areas.

Functional region of mouse heat shock protein 72 for its binding to lymphoid neoplastic P388D1 cells.

Extracellular heat shock proteins have been reported to participate in both innate and adaptive immune responses. We have found that recombinant mouse inducible heat shock protein 72 (Hsp72) bound to lymphoid neoplastic P388D1 cells. In the present study, we examined which region of mouse Hsp72 interacted with this cell line by using truncated variants that are sequentially lacking sections of the C-terminal region. The full-length mouse Hsp72 specifically bound to P388D1 cells, but not to mastocytoma P815 cells. Deletion of the C-terminal tail portion of mouse Hsp72 markedly decreased the binding to P388D1 cells and the sequential truncation of the C-terminal helical region led to a loss of binding activity. Specific binding was not observed for either the variant with a minimal substrate-binding structure or the ATP-binding domain alone. On the other hand, two truncated variants lacking the ATP-binding domain significantly bound to P388D1 cells. However, the variant lacking the substrate-binding domain did not show any binding to this cell line. These results suggest that the activity to bind P388D1 cells is attributable to the C-terminal region of mouse Hsp72 in combination with the substrate-binding domain. Interestingly, the binding of mouse Hsp72 to P388D1 cells was competed by the variant with the C-terminal flexible tail sequence, but not by the variant without that sequence. These competitive experiments imply that there may be at least two membrane receptors on P388D1 cells and also that both receptors may recognize the various structures in the C-terminal region of the Hsp70 family for regulation of innate immunity.

Role of the C-terminal region of mouse inducible Hsp72 in the recognition of peptide substrate for chaperone activity.

Here, we produced the C-terminal truncation variants of mouse inducible heat shock protein 72 (Hsp72) to elucidate the regulatory role of the C-terminal helical lid of Hsp70 for substrate recognition. All of the truncation variants containing the substrate binding domain bound a short-length peptide substrate CLLLSAPRR. When a large mass reduced carboxymethyl alpha-lactalbumin (RCMLA) as a substrate was used in gel filtration experiment, we observed the complex formation only for the truncation variants containing the long alpha-helix C in the helical lid. However, RCMLA binding occurred even for the variants lacking alpha-helix C when their C-terminal region was anchored onto a solid phase. Together with the finding that helix C is involved in the self-association of Hsp70, our present data suggest that the C-terminal region of Hsp70 modulates the substrate recognition and its kinetics may be substrate-mass dependent.

[An outbreak of pulmonary tuberculosis probably due to exogenous reinfection at a nursing home for the elderly].

In Japan and other countries where tuberculosis is not so common, people who were once infected with tuberculosis are thought to rarely suffer from the disease again due to exogenous reinfection. We experienced a mass outbreak of tuberculosis with 27 patients (including the source of infection) at a nursing home for the elderly. Epidemiological investigation suggested that the source of infection was an 82-year-old woman resident. For about 2 years before this outbreak, she had complained of a productive cough. At the time of the diagnosis of tuberculosis, chest radiography revealed a cavitary lesion and a smear of her sputum revealed organisms rated as Gaffky No. 8. Sputum culture was also positive (++++). Of the 27 patients, 19 (including the source) underwent restriction fragment length polymorphism (RFLP) analysis of isolates from the sputum. Eighteen patients showed an identical RFLP pattern, indicating that the infection had arisen from one source. Out of all patients, the source case of infection, 9 others with the same RFLP pattern, and other 3 who did not undergo RFLP analysis were admitted to our hospital. In 12 patients (3 men and 9 women excluding the source case) aged 80.6 years (range: 67-89 years), chest radiography disclosed tuberculous lesions, and smears, the polymerase chain reaction, and culture of sputum demonstrated Mycobacterium tuberculosis. As the prevalence of tuberculosis infection in Japanese aged 80 years at the time of the mass outbreak (1995) was presumed to be about 80%, the disease seemed to be caused by exogenous reinfection in most of these patients. All of the patients had senile dementia and other complications, and they were bedridden and undernourished. Anemia, hypoalbuminemia and lymphocytopenia were also observed in most of the cases. Malnutrition due to these complications appeared to be a possible risk factor of tuberculosis caused by exogenous reinfection.