In vivo klotho gene transfer ameliorates angiotensin II-induced renal damage

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses the expression of multiple aging-associated phenotypes. This gene is predominantly expressed in the kidney. Recent studies have shown that expression of renal klotho gene is regulated in animal models of metabolic diseases and in humans with chronic renal failure. However, little is known about the mechanisms and the physiological relevance of the regulation of the expression of the klotho gene in the kidney in some diseased conditions. In the present study, we first investigated the role of angiotensin II in the regulation of renal klotho gene expression. Long-term infusion of angiotensin II downregulated renal klotho gene expression at both the mRNA and protein levels. This angiotensin II-induced renal klotho downregulation was an angiotensin type 1 receptor-dependent but pressor-independent event. Adenovirus harboring mouse klotho gene (ad-klotho, 3.3x10(10) plaque forming units) was also intravenously administered immediately before starting angiotensin II infusion in some rats. This resulted in a robust induction of Klotho protein in the liver at day 4, which was still detectable 14 days after the gene transfer. Ad-klotho gene transfer, but not ad-lacZ gene transfer, caused an improvement of creatinine clearance, decrease in urinary protein excretion, and amelioration of histologically demonstrated tubulointerstitial damage induced by angiotensin II administration. Our data suggest that downregulation of the renal klotho gene may have an aggravative role in the development of renal damage induced by angiotensin II, and that induction of the klotho gene may have therapeutic possibilities in treating angiotensin II-induced end organ damage.     

Localization of type IV collagen alpha chain in the myocardium of dilated and hypertrophic cardiomyopathy

A total of 6 alpha chains [alpha 1 (IV) to alpha 6 (IV)] have been identified in type IV collagen. We examined the localization of these chains in the myocardium of patients with dilated (DCM) and hypertrophic (HCM) cardiomyopathy. The localization of alpha 1 (IV)-alpha 6 (IV) in biopsy specimens of 5 patients with DCM and 4 with HCM was examined using immunohistochemistry with monoclonal antibodies. Both alpha 1 (IV) and alpha 2 (IV) immunostaining formed thin homogeneous outlines around myocytes in control hearts. In the DCM specimens, alpha 1 (IV) and alpha 2 (IV) immunostaining formed thick and irregular patterns around myocytes. Staining for alpha 1(IV) and alpha 2 (IV) was also observed in some enlarged intercellular spaces. In 3 DCM hearts, moderate staining for alpha 1 (IV) and alpha 2 (IV) was observed in small replacement fibrotic lesions. In large replacement fibrotic lesions, no alpha 1 (IV) or alpha 2 (IV) staining was observed. In the HCM specimens, alpha 1 (IV) and alpha 2 (IV) staining formed thick homogeneous patterns around myocytes. In the enlarged intercellular spaces, no alpha 1 (IV) or alpha 2 (IV) staining was observed. No labeling for alpha 3 (IV)-alpha 6 (IV) was observed in any heart examined. In conclusion, the present results demonstrate that type IV collagen consisting of alpha 1 and alpha 2 chains appears in the fibrotic lesions of DCM, indicating its contribution to the development of fibrotic changes in the myocardium of DCM patients. In contrast, type IV collagen was restricted to the myocyte membrane in the HCM hearts. Fibrotic processes in the intercellular spaces may differ between DCM and HCM hearts.     

Denosumab treatment effects on structural damage, bone mineral density, and bone turnover in rheumatoid arthritis: a twelve-month, multicenter, randomized, double-blind, placebo-controlled, phase II clinical trial

OBJECTIVE: RANKL is essential for osteoclast development, activation, and survival. Denosumab is a fully human monoclonal IgG2 antibody that binds RANKL, inhibiting its activity. The aim of this multicenter, randomized, double-blind, placebo-controlled, phase II study was to evaluate the effects of denosumab on structural damage in patients with rheumatoid arthritis (RA) receiving methotrexate treatment. METHODS: RA patients received subcutaneous placebo (n = 75), denosumab 60 mg (n = 71), or denosumab 180 mg (n = 72) injections every 6 months for 12 months. The primary end point was the change from baseline in the magnetic resonance imaging (MRI) erosion score at 6 months. RESULTS: At 6 months, the increase in the MRI erosion score from baseline was lower in the 60-mg denosumab group (mean change 0.13; P = 0.118) and significantly lower in the 180-mg denosumab group (mean change 0.06; P = 0.007) than in the placebo group (mean change 1.75). A significant difference in the modified Sharp erosion score was observed as early as 6 months in the 180-mg denosumab group (P = 0.019) as compared with placebo, and at 12 months, both the 60-mg (P = 0.012) and the 180-mg (P = 0.007) denosumab groups were significantly different from the placebo group. Denosumab caused sustained suppression of markers of bone turnover. There was no evidence of an effect of denosumab on joint space narrowing or on measures of RA disease activity. Rates of adverse events were comparable between the denosumab and placebo groups. CONCLUSION: Addition of twice-yearly injections of denosumab to ongoing methotrexate treatment inhibited structural damage in patients with RA for up to 12 months, with no increase in the rates of adverse events as compared with placebo.     

Terpene synthases are widely distributed in bacteria

Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes.Some 50,000 terpenoid metabolites, including monoterpenes, sesquiterpenes, and diterpenes representing nearly 400 distinct structural families, have been isolated from both terrestrial and marine plants, liverworts, and fungi. In contrast, only a relatively minor fraction of these widely occurring metabolites has been identified in prokaryotes. The first study of bacterial terpenes grew out of an investigation of the characteristic odor of freshly plowed soil reported in 1891 by Berthelot and André (1). Berthelot and André noted that a volatile substance apparently responsible for the typical earthy odor of soil could be extracted from soil by steam distillation. Their attempts to assign a structure to the isolated odor constituent failed;, however, when the neutral alcohol resisted oxidative degradation or other conventional chemical modification. The first modern studies of volatile bacterial terpenes were carried out some 75 years later by Gerber and Lechevalier (2) and Gerber (3–7), who speculated that the characteristic odor of cultures of Actinomycetales microorganisms, which are widely distributed in soil, might be caused by volatile terpenes. In addition to determining the structure of Berthelot’s geosmin, shown to be a C₁₂ degraded sesquiterpene alcohol (and giving it its name, which means earth odor) (2, 3), Gerber (4) also isolated and determined the structures of the methylated monoterpene 2-methylisoborneol as well as several other cyclic sesquiterpenes produced by streptomycetes (5–7). In subsequent years, numerous volatile terpenes have been detected in streptomycetes (8–16). The three most commonly detected streptomycetes terpenoids, geosmin, and 2-methylisoborneol and the tricyclic α,β-unsaturated ketone albaflavenone (Fig. 1) are well-known as volatile odoriferous microbial metabolites. The two terpene alcohols are, in fact, the most frequently found secondary metabolites in actinomycetes (8, 11, 17), filamentous Cyanobacteria (18–20), and Myxobacteria (21), and they are also produced by a small number of fungi (22–24). The production of 2-methylisoborneol is associated with a characteristic scent, whereas albaflavenone, which was first isolated from cultures of a highly odoriferous Streptomyces albidoflavus species, is best described as earthy and camphor-like (25).Open in a separate windowFig. 1.The structures of the major known terpenes produced by bacteria.Cyclic monoterpene, sesquiterpene, and diterpene hydrocarbons and alcohols are formed by variations of a universal cyclization mechanism that is initiated by enzyme-catalyzed ionization of the universal acyclic precursors geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) to form the corresponding allylic cations. These parental branched, linear isoprenoid precursors are themselves synthesized by mechanistically related electrophilic condensations of the 5-carbon building blocks dimethylallyl diphosphate and isopentenyl diphosphate. The several thousand known or suspected terpene synthases from plants and fungi have a strongly conserved level of overall amino acid sequence similarity, thus making possible the application of local alignment methods, such as the widely used BLAST algorithm, for the discovery of genes encoding presumptive terpene synthases from plant and fungal sources. Despite the relatively high level of overall sequence conservation, however, assignment of the actual biosynthetic cyclization product of each fungal or plant terpene synthase has remained beyond the reach of available bioinformatic methods. The discovery and biochemical characterization of bacterial terpene synthases represent an even greater challenge, because unlike the plant and fungal enzymes, bacterial terpene synthases not only exhibit no significant overall amino acid sequence similarity to those from plants and fungi but typically display relatively low levels of mutual sequence similarity. To address this challenge, we recently described the successful application of an alternative genome mining strategy for the discovery of previously unidentified bacterial terpene synthases based on the use of hidden Markov models (HMMs) and protein families database (Pfam) searching methods (26). These initial efforts identified a large number of previously unrecognized bacterial terpene synthase candidates, including the discovery of the previously unidentified synthase for the methylated monoterpene 2-methylisoborneol, and led to the heterologous expression of the relevant genes that produce 2-methylisoborneol and 2-methylenebornane from 2-methylgeranyl diphosphate (27). We subsequently refined and expanded the set of HMM parameters using as a reference set exclusively the group of newly predicted bacterial terpene synthases in distinction to the original HMM model (PF03936), which had been based on plant terpene synthases. Using these newly refined parameters, we then succeeded in identifying a previously unrecognized ortholog of 2-methylisoborneol synthase in the cyanobacterium Pseudanabaena limnetica str. Castaic Lake (28). Application of this second generation HMM model allowed, in total, the discovery of 140 predicted terpene synthases of bacterial origin.We now report the development of a third generation HMM model trained by the previously identified 140 bacterial terpene synthases that has expanded the number of predicted bacterial terpene synthases to 262 from within the most complete set of predicted proteins incorporated in the most recent collection of public databases and in-house draft genome sequences of streptomycete microorganisms. Among the newly identified gene sequences, a subset selected by phylogenetic analysis has been expressed in a specially engineered heterologous Streptomyces host, and the resultant terpenes have been identified and structurally characterized.     

An internally and externally validated nomogram for predicting the risk of irinotecan-induced severe neutropenia in advanced colorectal cancer patients

Background:

In Asians, the risk of irinotecan-induced severe toxicities is related in part to UGT1A1*6 (UGT, UDP glucuronosyltransferase) and UGT1A1*28, variant alleles that reduce the elimination of SN-38, the active metabolite of irinotecan. We prospectively studied the relation between the UGT1A1 genotype and the safety of irinotecan-based regimens in Japanese patients with advanced colorectal cancer, and then constructed a nomogram for predicting the risk of severe neutropenia in the first treatment cycle.

Methods:

Safety data were obtained from 1312 patients monitored during the first 3 cycles of irinotecan-based regimen in a prospective observational study. In development of the nomogram, multivariable logistic regression analysis was used to test the associations of candidate factors to severe neutropenia in the first cycle. The final nomogram based on the results of multivariable analysis was constructed and validated internally using a bootstrapping technique and externally in an independent data set (n=350).

Results:

The UGT1A1 genotype was confirmed to be associated with increased risks of irinotecan-induced grade 3 or 4 neutropenia and diarrhoea. The final nomogram included type of regimen, administered dose of irinotecan, gender, age, UGT1A1 genotype, Eastern Cooperative Oncology Group performance status, pre-treatment absolute neutrophil count, and total bilirubin level. The model was validated both internally (bootstrap-adjusted concordance index, 0.69) and externally (concordance index, 0.70).

Conclusions:

Our nomogram can be used before treatment to accurately predict the probability of irinotecan-induced severe neutropenia in the first cycle of therapy. Additional studies should evaluate the effect of nomogram-guided dosing on efficacy in patients receiving irinotecan.     

Gas gangrene in the deep spaces of the head and neck visualized on computed tomography images

Cellulitis accompanied by gas gangrene is a rapidly-spreading and potentially fatal infection. Here, we present a case of gas gangrene in the deep spaces of the head and neck in an elderly woman, diagnosed by computed tomography (CT). An 86-year-old woman with Alzheimer’s disease, hypertension, hyperlipidemia, and osteoporosis was referred to our institute by her local dentist. The patient exhibited trismus caused by severe swelling in the left submandibular area. CT images of the head and neck area showed swelling of the cervical tissue with air in the parapharyngeal and masticator spaces. She was treated with antibiotics, followed by drainage. Although the therapy was continued, the patient died from a cardiac complication on hospital day 42. Our case highlights the usefulness of CT for diagnosing gas gangrene in the deep spaces of the head and neck in a woman with Alzheimer’s disease.     

The accuracy of bone tunnel position using fluoroscopic-based navigation system in anterior cruciate ligament reconstruction

Purpose

The first purpose of this study was to examine whether fluoroscopic-based navigation system contributes to the accuracy and reproducibility of the bone tunnel placements in single-bundle anterior cruciate ligament (ACL) reconstruction. The second purpose was to investigate the application of the navigation system for double-bundle ACL reconstruction.

Methods

A hospital-based case–control study was conducted, including a consecutive series of 55 patients. In 37 patients who received single-bundle ACL reconstruction, surgeries were performed with this system for 19 knees (group 1) and without this system for 18 knees (group 2). The positioning of the femoral and tibial tunnels was evaluated by plain sagittal radiographs. In 18 patients who received double-bundle ACL reconstruction using the navigation system (group 3), the bone tunnel positions were assessed by three-dimensional computed tomography (3D-CT). Clinical assessment of all patients was followed with the use of Lysholm Knees Score and IKDC.

Results

Taking 0% as the anterior and 100% as the posterior extent, the femoral tunnels were 74.9?±?3.0% in group 1 and 71.5?±?5.8% in group 2 along Blumensaat’s line, and the tibial tunnels were 42.3?±?1.4% in group 1 and 42.5?±?4.6% in group 2 along the tibia plateau. The bone tunnel positions in group 1 were located significantly closer to the position planned preoperatively and varied less in both femur and tibial side, compared with those without navigation (group 2). (Femur: P?P?Conclusion The fluoroscopic-based navigation system contributed to the more reproducible placement of the bone tunnel during single-bundle ACL reconstruction compared with conventional technique. Additionally, this device was also useful for double-bundle ACL reconstruction.

Level of evidence

Case–control study, Therapeutic study, Level III.