Evaluation of risk factors for nephrotoxicity associated with high-dose vancomycin in Japanese patients.

Introduction: Considering the physique of the Japanese population, the standard daily vancomycin dose of 2 g/day and doses ≥ 3 g/day are high in terms of dose per body weight. Studies have reported that administering high-dose vancomycin to achieve a high target trough concentration has been associated with nephrotoxicity. The risk of high-dose vancomycin-associated nephrotoxicity is believed to be exceptionally high for Japanese patients because of their relatively low body weights, but data on the population is lacking. In this retrospective study, we aimed to evaluate risk factors associated with nephrotoxicity in Japanese patients treated with vancomycin. Methods: We examined the medical records of 107 Japanese patients who received vancomycin (3 to 4 g/day). They were divided into two groups based on the presence or absence of nephrotoxicity, and their demographics and clinical characteristics were compared. Results : The incidence of nephrotoxicity in patients receiving high-dose vancomycin was 13%. Age (≥ 60 years) and concurrent use of piperacillin/tazobactam were independent risk factors for vancomycin-associated nephrotoxicity (P = 0.027 and 0.017, respectively). Conclusions : We conclude that the nephrotoxicity risk of high-dose vancomycin in Japanese patients is not excessively high when administered within the confines of a therapeutic drug-monitoring program. However, special care must be taken with patients who are older or on concurrent piperacillin/tazobactam therapy.

Surgical treatment for pyogenic vertebral osteomyelitis using iodine-supported spinal instruments: initial case series of 14 patients.

Reports have detailed the increasing use of spinal instrumentation in the treatment of pyogenic vertebral osteomyelitis, with the aims of achieving a lower pseudoarthrosis rate and restoring spinal alignment. However, controversy remains over the use of instrumentation in the presence of active infection because of concerns about increased bacterial adherence and biofilm formation on the metallic implant surface. Fourteen consecutive patients were followed who were diagnosed as having pyogenic vertebral osteomyelitis and underwent surgery with spinal instrumentation with iodine-containing surfaces that could be directly supported to existing titanium implants. Bone-cage interfaces and implant-related complications after surgery were evaluated. The white blood cell (WBC) count and C-reactive protein (CRP) level were analyzed during the follow-up period. To confirm the influence of iodine release from the implant, thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4) were also examined. The infection subsided in all 14 patients. Both WBC counts and CRP levels returned to normal ranges by the final follow-up. One patient showed a lucent area around the screw and two patients showed lucencies inside the cage. However, no cage dislocations, cage migrations, or screw pull-outs were noted, and all patients' FT3, FT4, and TSH levels were within normal ranges during the follow-up period. We demonstrated the efficacy of iodine-supported titanium implants in the management of pyogenic vertebral osteomyelitis. No cytotoxicity or adverse effects were noted in this series.

Characterization of mesenchymal progenitor cell populations from non-epithelial oral mucosa.

OBJECTIVES: The characteristics of cell populations extracted from oral mucosal non-epithelial tissues and their ability to differentiate were evaluated in vitro as a potential source of cells for mandibular and corneal regeneration. MATERIALS AND METHODS: Oral mucosal non-epithelial cells (OMNECs) were extracted from tissue samples and were studied by flow cytometry and RT-PCR. Cells differentiating into osteoblasts, adipocytes, chondrocytes, neurocytes, or keratocytes were characterized by RT-PCR and cell staining. RESULTS: OMNECs expressed CD44, CD90, CD105, CD166, and STRO-1 antigens, which are markers for mesenchymal stem cells. In addition, Oct3/4, c-Myc, Nanog, KLF4, and Rex, which are expressed by embryonic or pluripotent stem cells, were detected by RT-PCR. Expression of CD49d, CD56, and PDGFRα, proteins closely associated with the neural crest, was observed in OMNECs, as was expression of Twist1, Sox9, Snail1 and Snail2, which are early neural crest and neural markers. Specific differentiation markers were expressed in OMNECs after differentiation into osteoblasts, adipocytes, chondrocytes, or keratocytes. CONCLUSIONS: Populations of OMNECs may contain both mesenchymal stem cells and neural crest origin cells and are a potential cell source for autologous regeneration of mandibular or corneal stroma.

Glibenclamide attenuates brain edema associated with microglia activation after intracerebral hemorrhage.

OBJECTIVE: Glibenclamide, Sulfonylurea receptor 1 antagonist, reduces brain edema after cerebral hemorrhage. However, the effects of glibenclamide on microglial activation and inflammatory cell infiltration after cerebral hemorrhage are unclear. The present study investigated the effect of glibenclamide on microglial activation and inflammatory cell infiltration in a rat cerebral hemorrhage model. METHODS: A collagenase intracerebral injection model was used to cause cerebral hemorrhage in rats. After injury, glibenclamide was continuously administered at 1.0µL/h for 24hours. We evaluated hematoma volume, brain edema, expression of ABCC8, galectin-3 and CD11b, and anti-Iba-1 antibody staining. RESULTS: Glibenclamide significantly reduced water content. Meanwhile, glibenclamide significantly reduced expression of galectin-3 and CD11b in the cerebral cortex and putamen on the bleeding side. Immunohistochemical staining confirmed that glibenclamide attenuated activation of microglia around the hematoma. CONCLUSIONS: Glibenclamide reduced microglial activation and infiltration of inflammatory cells, resulting in amelioration of cerebral edema.

Human immunodeficiency virus type 1 neutralizing antibodies accelerate clearance of cell-free virions from blood plasma.

The concentration of human immunodeficiency virus type 1 (HIV-1) particles in blood plasma is very predictive of the subsequent disease course in an infected individual; its measurement has become one of the most important parameters for monitoring clinical status. Steady-state virus levels in plasma reflect a balance between the rates of virions entering and leaving the peripheral blood. We analyzed the rate of virus clearance in the general circulation in rhesus macaques receiving a continuous infusion of cell-free particles in the presence and absence of virus-specific antibodies. Here we show, by measuring virion RNA, particle-associated p24 Gag protein and virus infectivity, that the clearance of physical and infectious particles from a primary, dual-tropic virus isolate, HIV-1DH12, is very rapid in naive animals, with half-lives ranging from 13 to 26 minutes. In the presence of high-titer HIV-1DH12-specific neutralizing antibodies, the half-life of virion RNA was considerably reduced (to 3.9-7.2 minutes), and infectious virus in the blood became undetectable. Although physical virus particles were eliminated extravascularly, the loss of virus infectivity in the blood reflected the combined effects of extravascular clearance and intravascular inactivation of HIV-1 infectivity due to antibody binding.

Neutralizing antibody directed against the HIV-1 envelope glycoprotein can completely block HIV-1/SIV chimeric virus infections of macaque monkeys.

Virus-specific antibodies protect individuals against a wide variety of viral infections. To assess whether human immunodeficiency virus type 1 (HIV-1) envelope-specific antibodies confer resistance against primate lentivirus infections, we purified immunoglobulin (IgG) from chimpanzees infected with several different HIV-1 isolates, and used this for passive immunization of pig-tailed macaques. These monkeys were subsequently challenged intravenously with a chimeric simian-human immunodeficiency virus (SHIV) bearing an envelope glycoprotein derived form HIV-1DH12, a dual-tropic primary virus isolate. Here we show that anti-SHIV neutralizing activity, determined in vitro using an assay measuring loss of infectivity, is the absolute requirement for antibody-mediated protection in vivo. Using an assay that measures 100% neutralization, the titer in plasma for complete protection of the SHIV-challenged macaques was in the range of 1:5-1:8. The HIV-1-specific neutralizing antibodies studied are able to bind to native gp120 present on infectious virus particles. Administration of non-neutralizing anti-HIV IgG neither inhibited nor enhanced a subsequent SHIV infection.

Four cases of postrenal renal failure induced by renal stone associated with rotavirus infection.

Rotavirus (RV) is a common pathogen that causes acute gastroenteritis in childhood. Some cases with RV infection also have prerenal renal failure induced by dehydration associated with vomiting and diarrhea. Here, we report 4 patients with RV infection who developed postrenal renal failure induced by urinary tract obstruction with uroammoniac calculi or crystals. The patients did not have metabolic disorders or abnormalities of the urinary tract, and increased urinary excretion of uric acid was not recognized at discharge. In addition, no abnormalities in the uric acid transporter (URAT1) were found in any of the patients. Uric acid stone formation was considered to have originated from the low pH caused by dehydration and the increase of urinary uric acid excretion from damaged cells.

Characterization of cerebral salt wasting after subarachnoid hemorrhage model induced by endovascular puncture.

Cerebral salt wasting (CSW) frequently occurs concomitantly with an aneurysmal subarachnoid hemorrhage (SAH). CSW induces excessive natriuresis and osmotic diuresis, and reduces the total volume of blood. We previously reported that a rat model with SAH induced by endovascular puncture (EP) exhibited CSW. Therefore, we investigated the relationship between the spread of bleeding in the subarachnoid space and the intensity of CSW. We also investigated the development of CSW in different SAH models. SAH was induced by EP or by 0.3 mL of blood injection (BI) into the cisterna magna. To evaluate the occurrence of CSW, urine was cumulatively collected at the onset of SAH to 6 h later and analyzed for sodium (Na) excretion. SAH was classified from grade 1 (no bleeding) to grade 4 (severe bleeding) based on the spread of bleeding in the subarachnoid space. In the EP model (SAH grade > 2) as the SAH grade increased, the volume of urine and Na excretion also significantly increased. Although the BI model rats exhibited SAH of grade 4, the volume of urine and Na excretion did not change. Therefore, our conclusion is that the spread of bleeding in the subarachnoid space may not cause CSW.

A suppressive oligodeoxynucleotide inhibits ocular inflammation.

Synthetic oligodeoxynucleotides (ODN) expressing 'suppressive' TTAGGG motifs down-regulate a variety of proinflammatory and T helper type 1 (Th1)-mediated pathological immune responses. The ability of the archetypal suppressive ODN A151 to inhibit ocular inflammation was examined in two murine models: experimental autoimmune uveitis, induced by immunization with a retinal antigen, interphotoreceptor retinoid-binding protein (IRBP) and adoptively transferred ocular inflammation, induced by transferring Th1 cells specific to hen egg lysozyme (HEL) into recipient mice that express HEL in their eyes. A151 treatment suppressed the inflammation in both models. In addition, A151 inhibited IRBP-specific cytokine production and lymphocyte proliferation in mice immunized with IRBP. These findings suggest that suppressive ODN affects both afferent and efferent limbs of the immunopathogenic process and may be of use in the treatment of autoimmune ocular inflammation.

Correlation of autophagy type in podocytes with histopathological diagnosis of IgA nephropathy.

OBJECTIVE: IgA nephropathy (IgA-N) frequently leads to progressive renal failure, thus estimation of the degree of progression is important for patient management. Autophagy is a mechanism that facilitates clearance of waste products to preserve renal function. The aim of this study was to assess autophagy in podocytes in children with progressive IgA-N at initial diagnosis by electron microscopy and investigate the relationship between the types of autophagy and severity of the disease. METHODS: Renal biopsies from 16 children with established progressive IgA-N were examined by light and transmission electron microscopy with reference to autophagy types in the podocytes and histopathological diagnosis of IgA-N. RESULTS: Two autophagy types were found. Type I rarely transformed to autophagic vacuoles and did not dissolve, thus possibly impairing cell function. However, type II frequently transformed to autophagosomes and autophagic vacuoles thus facilitating protein and lipid clearance. Of the 16 children studied, 8 (50%) with type I autophagy at initial diagnosis showed focal proliferative glomerulosclerosis (GN) of mild type (3 cases, 37.5%), mild/moderate type (2 cases, 25%) and moderate type (3 cases, 37.5%). In contrast, the remaining 8 children with type II autophagy at initial diagnosis showed focal proliferative GN of mild type in 7 (87.5%) and mild/moderate type in 1 (12.5%) case. CONCLUSION: In IgA-N children, the occurrence of type I autophagy is correlated with histopathologically more progressive disease, possibly reflecting a tendency to a poorer prognosis.

Characterization of L-cysteine desulfhydrase from Prevotella intermedia.

INTRODUCTION: Hydrogen sulfide is responsible for lysis of red blood cells and is a major compound for oral malodor. To clarify the production mechanism of hydrogen sulfide in Prevotella intermedia, we found an L-cysteine desulfhydrase gene (lcs) homologue on the genome database of P. intermedia ATCC25611 and characterized its gene product. METHODS: The lcs gene homologue cloned into pGEX6p-1 vector was expressed in Escherichia coli and purified. Lcs activity was assayed by detection of the reaction products (hydrogen sulfide and pyruvate) or its derivatives from L-cysteine. Site-directed mutagenesis was used to convert an amino acid of the Lcs molecule. RESULTS: The purified lcs gene product catalysed the degradation of L-cysteine to pyruvate, ammonia, and hydrogen sulfide, indicating that the protein is L-cysteine desulfhydrase. The enzyme required pyridoxal 5'-phosphate as a cofactor, and it was highly active at pH 7.0 and completely inhibited by ZnCl(2). The K(m) and V(max) of the enzyme were 0.7 mm and 4.2 micromol/min/mg, respectively. Replacement of Tyr-59, Tyr-118, Asp-198, and Lys-233 with any of the amino acids resulted in the complete disappearance of Lcs activity, implying that these amino acids are essential for enzyme activity. In addition, hydrogen sulfide produced by this enzyme lysed sheep red blood cells and modified hemoglobin. CONCLUSION: These results show the enzymatic properties of L-cysteine desulfhydrase from P. intermedia ATCC25611 and also suggest that the Lcs enzyme, which produces hydrogen sulfide from L-cysteine, is closely associated with the pathogenesis of P. intermedia.

Genes responsible for dextran-dependent aggregation of Streptococcus sobrinus strain 6715.

INTRODUCTION: Streptococcus sobrinus exhibits more significant dextran-dependent aggregation mediated by glucan-binding proteins than Streptococcus mutans. We have identified four glucan-binding protein C gene (gbpC) homologues designated as gbpC1, gbpC2, dblA and dblB in S. sobrinus in contrast to the single gene gbpC in S. mutans. We attempted to determine which gene is most responsible for the dextran-dependent aggregation of S. sobrinus. METHODS: We introduced mutation with a chemical mutagen, 1-methyl-3-nitro-1-nitrosoguanidine, into S. sobrinus strain 6715 and analysed the four gbpC homologous gene sequences in the parental strain 6715 and an obtained aggregation-negative mutant NUM-Ssg99. We also examined the localization of proteins encoded by these genes in the mutant NUM-Ssg99. RESULTS: The nucleotide sequences of the gbpC1, gbpC2 and dblA genes in NUM-Ssg99 were 100% identical to the homologous genes in parental strain 6715. In contrast, a truncated mutation was detected in the dblB gene and the mutant protein devoid of the LPXTG motif was confirmed by Western blot analysis to be released into the extracellular milieu. CONCLUSION: We conclude that the dblB gene among the four GbpC homologous protein genes is most responsible for aggregation in strain 6715.

Molecular analysis of digenic inheritance in Bartter syndrome with sensorineural deafness.

BACKGROUND: Bartter syndrome (BS) is a genetic disorder accompanied by hypokalaemic metabolic alkalosis. BS with sensorineural deafness (SND, OMIM602522) is a newly identified phenotype caused by mutations in the BSND gene that encodes barttin, a beta-subunit for chloride channel ClC-Ka and ClC-Kb and classified as type IV BS. Type IV BS features the most severe phenotype entailing life-threatening neonatal volume depletion and chronic renal failure developing during infancy. A recent report described a case of BS with SND from a consanguineous family who showed homozygous mutations in the CLCNKA and CLCNKB genes. This case indicated the possibility of the occurrence of digenic inheritance in BS with SND resulting from double mutations in the CLCNKA and CLCNKB genes. SUBJECT AND RESULTS: The current report concerns a 2-year-old girl from a non-consanguineous family with BS accompanied by SND. In our case, four loss-of-function mutations, consisting of mutations in both parental alleles in both CLCNKA and CLCNKB, were identified. The paternal allele had a nonsense mutation (Q260X) in CLCNKA and a splicing site mutation (IVS17+1 g>a) in CLCNKB. The maternal allele had a large deletion mutation (about 12 kbp) extending from CLCNKA to CLCNKB. Our case provides clear evidence that loss-of-function alleles in both alleles of both CLCNKA and CLCNKB results in a phenotype indistinguishable from that of mutations in BSND (type IV BS). CONCLUSIONS: Recent advances in genetics have resulted in a better understanding of many human inherited diseases, but most of them are monogenic disorders and more complex inheritance patterns remain unresolved. Our case provides clear evidence of digenic inheritance outside the scope of Mendelian inheritance disorders.

Detection of Paracoccidioides brasiliensis gp43 gene in sputa by loop-mediated isothermal amplification method.

The fungus Paracoccidioides brasiliensis is the pathogen of paracoccidioidomycosis (PCM), a systemic mycosis prevalent in Latin America. The loop-mediated isothermal amplification method (LAMP) was used in this study to detect the presence of P. brasiliensis in sputa samples from patients with chronic PCM, suspected PCM, and a negative control. The target P. brasiliensis gp43 gene was amplified in less than 4 hr in 11 of 18 sputa samples tested. The LAMP method had the advantage of speed and simplicity compared with the classic diagnostic methods such as the histopathological test or biological material culture and did not require sophisticated technical apparatus. It would be an important aid in cases where immediate treatment would mean patient survival, especially in immune-suppressed patients.

Clock and ATF4 transcription system regulates drug resistance in human cancer cell lines.

The mechanisms underlying cellular drug resistance have been extensively studied, but little is known about its regulation. We have previously reported that activating transcription factor 4 (ATF4) is upregulated in cisplatin-resistant cells and plays a role in cisplatin resistance. Here, we find out a novel relationship between the circadian transcription factor Clock and drug resistance. Clock drives the periodical expression of many genes that regulate hormone release, cell division, sleep-awake cycle and tumor growth. We demonstrate that ATF4 is a direct target of Clock, and that Clock is overexpressed in cisplatin-resistant cells. Furthermore, Clock expression significantly correlates with cisplatin sensitivity, and that the downregulation of either Clock or ATF4 confers sensitivity of A549 cells to cisplatin and etoposide. Notably, ATF4-overexpressing cells show multidrug resistance and marked elevation of intracellular glutathione. The microarray study reveals that genes for glutathione metabolism are generally downregulated by the knockdown of ATF4 expression. These results suggest that the Clock and ATF4 transcription system might play an important role in multidrug resistance through glutathione-dependent redox system, and also indicate that physiological potentials of Clock-controlled redox system might be important to better understand the oxidative stress-associated disorders including cancer and systemic chronotherapy.

Coexistence of hERG current block and disruption of protein trafficking in ketoconazole-induced long QT syndrome.

BACKGROUND AND PURPOSE: Many drugs associated with acquired long QT syndrome (LQTS) directly block human ether-a-go-go-related gene (hERG) K(+) channels. Recently, disrupted trafficking of the hERG channel protein was proposed as a new mechanism underlying LQTS, but whether this defect coexists with the hERG current block remains unclear. This study investigated how ketoconazole, a direct hERG current inhibitor, affects the trafficking of hERG channel protein. EXPERIMENTAL APPROACH: Wild-type hERG and SCN5A/hNa(v) 1.5 Na(+) channels or the Y652A and F656C mutated forms of the hERG were stably expressed in HEK293 cells. The K(+) and Na(+) currents were recorded in these cells by using the whole-cell patch-clamp technique (23 degrees C). Protein trafficking of the hERG was evaluated by Western blot analysis and flow cytometry. KEY RESULTS: Ketoconazole directly blocked the hERG channel current and reduced the amount of hERG channel protein trafficked to the cell surface in a concentration-dependent manner. Current density of the hERG channels but not of the hNa(v) 1.5 channels was reduced after 48 h of incubation with ketoconazole, with preservation of the acute direct effect on hERG current. Mutations in drug-binding sites (F656C or Y652A) of the hERG channel significantly attenuated the hERG current blockade by ketoconazole, but did not affect the disruption of trafficking. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that ketoconazole might cause acquired LQTS via a direct inhibition of current through the hERG channel and by disrupting hERG protein trafficking within therapeutic concentrations. These findings should be considered when evaluating new drugs.

Role of prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein-specific signal peptidase II (LspA) in localization and physiological function of lipoprotein MsmE in Streptococcus mutans.

INTRODUCTION: To clarify the role that prolipoprotein diacylglyceryl transferase (Lgt) and lipoprotein-specific signal peptidase II (LspA) play in the physiological function of MsmE, we constructed lgt-deficient and lspA-deficient mutants of Streptococcus mutans 109c and examined the potential role of Lgt and LspA in membrane anchoring and growth in a melibiose medium of S. mutans. METHODS: The lgt-, lspA-, and msmE-deficient mutants of S. mutans 109c were constructed by double-crossover recombination of their respective genes. Localization of MsmE was demonstrated by Western blot analysis with an MsmE antiserum. The growth of S. mutans cells was examined in a Trypton medium containing melibiose or glucose. RESULTS: In the S. mutans lgt mutant, localization of the surface lipoprotein MsmE changed with the culture supernatant. The growth of the S. mutans lgt and lspA mutants was remarkably reduced in the melibiose medium; however, growth was recovered in the strains complemented with the lgt or the lspA gene. Therefore, lipid-modification by Lgt and subsequent signal peptide cleavage by LspA were crucial for membrane anchoring and the physiological function of MsmE in S. mutans. CONCLUSION: These results demonstrate that MsmE is required for melibiose metabolism in S. mutans and that modification by Lgt and LspA are important processes for the physiological function of MsmE.