Mitochondria, glycogen, and lipid droplets in skeletal muscle during testosterone treatment and strength training: a randomized, double-blinded, placebo-controlled trial.

Low testosterone levels in aging men are associated with insulin resistance. Mitochondrial dysfunction, changes in glycogen metabolism, and lipid accumulation are linked to insulin resistance in skeletal muscle. In this randomized, double-blinded, placebo-controlled study, we investigated the effects of six-month testosterone replacement therapy (TRT) and strength training (ST) on mitochondrial, glycogen, and lipid droplet (LD) content in skeletal muscle of aging men with subnormal bioavailable testosterone (BioT) levels. Mitochondrial, glycogen, and LD volume fractions in muscle biopsies were estimated by transmission electron microscopy. Insulin sensitivity (insulin-stimulated Rd) and body composition were assessed by euglycemic-hyperinsulinemic clamp and dual X-ray absorptiometry, respectively. TRT significantly increased total testosterone levels, BioT, and lean body mass (LBM) (p < 0.05), whereas percent body fat decreased (p < 0.05), and insulin sensitivity was unchanged. Baseline mitochondrial volume fraction correlated inversely with percent body fat (ρ = -0.43; p = 0.003). Δ-mitochondrial fraction correlated positively with Δ-total testosterone (ρ = 0.70; p = 0.02), and Δ-glycogen fraction correlated inversely with Δ-LBM (ρ = -0.83; p = 0.002) during six-month TRT, but no significant changes were observed in mitochondrial, glycogen, and LD volume fractions during TRT and ST. In conclusion, in this exploratory small-scale study, the beneficial effects of six-month TRT on total testosterone, LBM, and percent body fat were not followed by significant changes in fractions of mitochondria, glycogen, or lipid in skeletal muscle of aging men with lowered testosterone levels. Six-month ST or combined three-month ST+TRT did not change intramyocellular mitochondria, glycogen, and LD fractions compared to placebo. However, further studies with a larger sample size are needed.

Reduction of calprotectin and phosphate during testosterone therapy in aging men: a randomized controlled trial.

OBJECTIVES: To investigate the effect of testosterone treatment on biomarkers calprotectin, fibroblast growth factor 23 (FGF23), soluble Klotho, phosphate, calcium, parathyroid hormone, creatinine and estimated glomerular filtration rate. DESIGN: Randomized, double-blinded, placebo-controlled study. SETTING: Odense Androgen Study-the effect of Testim and training in hypogonadal men. PARTICIPANTS: Men aged 60-78 years old with a low normal concentration of free of bioavailable testosterone <7.3 nmol/L and waist circumference >94 cm recruited from 2008 to 2009 (N = 48) by advertisement. INTERVENTION: Participants were randomized to receive 5-10 g gel/50-100 mg testosterone (Testim®, Ipsen, France) or 5-10 g gel/placebo. RESULTS: The plasma levels of calprotectin and phosphate were significantly reduced in the group receiving testosterone therapy (gel) compared to the placebo group (p < 0.05). Testosterone treatment did not have any significant effect on plasma levels of FGF23 or soluble Klotho. The reduction in phosphate levels was inversely associated with bioavailable testosterone. CONCLUSION: Compared to the placebo group, 6 months of testosterone therapy (gel) reduced calprotectin and phosphate levels suggesting decreased inflammation and decreased cardiovascular risk.

Strength training and testosterone treatment have opposing effects on migration inhibitor factor levels in ageing men.

BACKGROUND: The beneficial effects of testosterone treatment (TT) are debated. METHODS: Double-blinded, placebo-controlled study of six months TT (gel) in 54 men aged 60-78 with bioavailable testosterone (BioT) <7.3 nmol/L and waist >94 cm randomized to TT (50-100 mg/day, n = 20), placebo (n = 18), or strength training (ST) (n = 16) for 24 weeks. Moreover, the ST group was randomized to TT (n = 7) or placebo (n = 9) after 12 weeks. OUTCOMES: Chemokines (MIF, MCP-1, and MIP-1 α ) and lean body mass (LBM), total, central, extremity, visceral, and subcutaneous (SAT) fat mass established by DXA and MRI. Results. From 0 to 24 weeks, MIF and SAT decreased during ST + placebo versus placebo, whereas BioT and LBM were unchanged. TT decreased fat mass (total, central, extremity, and SAT) and increased BioT and LBM versus placebo. MIF levels increased during TT versus ST + placebo. ST + TT decreased fat mass (total, central, and extremity) and increased BioT and LBM versus placebo. From 12 to 24 weeks, MCP-1 levels increased during TT versus placebo and MCP-1 levels decreased during ST + placebo versus placebo. CONCLUSION: ST + placebo was associated with decreased MIF levels suggesting decreased inflammatory activity. TT may be associated with increased inflammatory activity.

Attenuation of the beta-catenin/TCF4 complex in colorectal cancer cells induces several growth-suppressive microRNAs that target cancer promoting genes.

Aberrant activation of the Wnt signaling pathway is causally involved in the formation of most colorectal cancers (CRCs). Although detailed knowledge exists regarding Wnt-regulated protein-coding genes, much less is known about the possible involvement of non-coding RNAs. Here we used TaqMan Array MicroRNA Cards, capable of detecting 664 unique human microRNAs (miRNAs), to describe changes of the miRNA transcriptome following disruption of beta-catenin/TCF4 activity in DLD1 CRC cells. Most miRNAs appeared to respond independent of host gene regulation and proximal TCF4 chromatin occupancy as inferred from expression microarray and ChIP-chip data. A module of miRNAs induced by abrogated Wnt signaling in vitro was downregulated in two independent series of human primary CRCs (n=76) relative to normal adjacent mucosa (n=34). Several of these miRNAs (miR-145, miR-126, miR-30e-3p and miR-139-5p) markedly inhibited CRC cell growth in vitro when ectopically expressed. By using an integrative approach of proteomics and expression microarrays, we found numerous mRNAs and proteins to be affected by ectopic miR-30e-3p levels. This included HELZ and PIK3C2A that were directly repressed by several miRNA binding sites as confirmed by luciferase reporter assays in combination with mutational analyses. Finally, small interfering RNA-mediated downregulation of PIK3C2A, but not HELZ, was sufficient on its own to restrict CRC cell growth. Collectively, our study demonstrates that multiple miRNAs are upregulated as a consequence of forced attenuation of Wnt signaling in CRC cells, and some of these miRNAs inhibit cell growth with concomitant suppression of several growth-stimulatory cancer-related genes.

Dysregulation of the transcription factors SOX4, CBFB and SMARCC1 correlates with outcome of colorectal cancer.

The aim of this study was to identify deregulated transcription factors (TFs) in colorectal cancer (CRC) and to evaluate their relation with the recurrence of stage II CRC and overall survival. Microarray-based transcript profiles of 20 normal mucosas and 424 CRC samples were used to identify 51 TFs displaying differential transcript levels between normal mucosa and CRC. For a subset of these we provide in vitro evidence that deregulation of the Wnt signalling pathway can lead to the alterations observed in tissues. Furthermore, in two independent cohorts of microsatellite-stable stage II cancers we found that high SOX4 transcript levels correlated with recurrence (HR 2.7; 95% CI, 1.2-6.0; P=0.01). Analyses of approximately 1000 stage I-III adenocarcinomas, by immunohistochemistry, revealed that patients with tumours displaying high levels of CBFB and SMARCC1 proteins had a significantly better overall survival rate (P=0.0001 and P=0.0275, respectively) than patients with low levels. Multivariate analyses revealed that a high CBFB protein level was an independent predictor of survival. In conclusion, several of the identified TFs seem to be involved in the progression of CRC.

Kidney and pancreas transplantation in the United States, 1997-2006: the HRSA Breakthrough Collaboratives and the 58 DSA Challenge.

Growth in the number of active patients on the kidney transplant waiting list has slowed. Projections based on the most recent 5-year data suggest the total waiting list will grow at a rate of 4138 registrations per year, whereas the active waiting list will increase at less than one-sixth that rate, or 663 registrations per year. The last 5 years have seen a small trend toward improved unadjusted allograft survival for living and deceased donor kidneys. Since 2004 the overall number of pancreas transplants has declined. Among pancreas recipients, those with simultaneous kidney-pancreas transplants experienced the highest pancreas graft survival rates. In response to the ongoing shortage of deceased donor organs, the US Health Resources and Services Administration launched the Organ Donation Breakthrough Collaborative in September 2003 and the Organ Transplantation Breakthrough Collaborative (OTBC) in October 2005. The 58 DSA Challenge is prominent among the goals adopted by the OTBC. Its premise: were each of the 58 existing donation service areas to increase the number of kidney transplants performed within their boundaries by 10 per month, an additional 7000 transplants over current annual levels would result. Such an increase could potentially eliminate the national kidney transplantation waiting list by 2030.

Determinants of discard of expanded criteria donor kidneys: impact of biopsy and machine perfusion.

We examined factors associated with expanded criteria donor (ECD) kidney discard. Scientific Registry of Transplant Recipients (SRTR)/Organ Procurement and Transplantation Network (OPTN) data were examined for donor factors using logistic regression to determine the adjusted odds ratio (AOR) of discard of kidneys recovered between October 1999 and June 2005. Logistic and Cox regression models were used to determine associations with delayed graft function (DGF) and graft failure. Of the 12,536 recovered ECD kidneys, 5139 (41%) were discarded. Both the performance of a biopsy (AOR = 1.21, p = 0.02) and the degree of glomerulosclerosis (GS) on biopsy were significantly associated with increased odds of discard. GS was not consistently associated with DGF or graft failure. The discard rate of pumped ECD kidneys was 29.7% versus 43.6% for unpumped (AOR = 0.52, p < 0.0001). Among pumped kidneys, those with resistances of 0.26-0.38 and >0.38 mmHg/mL/min were discarded more than those with resistances of 0.18-0.25 mmHg/mL/min (AOR = 2.5 and 7.9, respectively). Among ECD kidneys, pumped kidneys were less likely to have DGF (AOR = 0.59, p < 0.0001) but not graft failure (RR = 0.9, p = 0.27). Biopsy findings and machine perfusion are important correlates of ECD kidney discard; corresponding associations with graft failure require further study.

Pediatric transplantation in the United States, 1996-2005.

Solid organ transplantation is accepted as a standard lifesaving therapy for end-stage organ failure in children. This article reviews trends in pediatric transplantation from 1996 to 2005 using OPTN data analyzed by the Scientific Registry of Transplant Recipients. Over this period, children have contributed significantly to the donor pool, and although the number of pediatric donors has fallen from 1062 to 900, this still accounts for 12% of all deceased donors. In 2005, 2% of 89,884 candidates listed for transplantation were less than 18 years old; in 2005, 1955 children, or 7% of 28,105 recipients, received a transplant. Improvement in waiting list mortality is documented for most organs, but pretransplant mortality, especially among the youngest children, remains a concern. Posttransplant survival for both patients and allografts similarly has shown improvement throughout the period; in most cases, survival is as good as or better than that seen in adults. Examination of immunosuppressive practices shows an increasing tendency across organs toward tacrolimus-based regimens. In addition, use of induction immunotherapy in the form of anti-lymphocyte antibody preparations, especially the interleukin-2 receptor antagonists, has increased steadily. Despite documented advances in care and outcomes for children undergoing transplantation, several considerations remain that require attention as we attempt to optimize transplant management.

Kidney and pancreas transplantation in the United States, 1995-2004.

This article examines OPTN/SRTR data on kidney and pancreas transplantation for 2004 and the previous decade, and discusses recent changes in kidney-pancreas (KP) allocation policy and emerging issues in kidney donation after cardiac death (DCD). Although the number of kidney donors continues to increase, new waiting list registrations again outpaced the number of kidney transplants performed, rising by 11% between 2003 and 2004 and contributing to a 1-year increase of 8% in the number of patients active on the waiting list. DCD has increased steadily since 2000; 39% more DCD transplants were performed in 2004 than 2003. Both deceased donor and living donor kidney graft survival rates remain excellent and are improving. The number of people living with a functioning kidney transplant doubled between 1995 and 2004, to 101,440 with a functioning kidney-alone and 7213 with a functioning KP. Health care providers in all settings are more likely to be exposed to these transplant recipients. Patient survival following simultaneous pancreas-kidney (SPK) transplantation is excellent and has improved incrementally since 1995; death rates in the first year fell from 60 per 1000 patient-years at risk in 2001 to 45 in 2003. The number of solitary pancreas transplants increased dramatically in 2004.

Gene expression signatures for colorectal cancer microsatellite status and HNPCC.

The majority of microsatellite instable (MSI) colorectal cancers are sporadic, but a subset belongs to the syndrome hereditary non-polyposis colorectal cancer (HNPCC). Microsatellite instability is caused by dysfunction of the mismatch repair (MMR) system that leads to a mutator phenotype, and MSI is correlated to prognosis and response to chemotherapy. Gene expression signatures as predictive markers are being developed for many cancers, and the identification of a signature for MMR deficiency would be of interest both clinically and biologically. To address this issue, we profiled the gene expression of 101 stage II and III colorectal cancers (34 MSI, 67 microsatellite stable (MSS)) using high-density oligonucleotide microarrays. From these data, we constructed a nine-gene signature capable of separating the mismatch repair proficient and deficient tumours. Subsequently, we demonstrated the robustness of the signature by transferring it to a real-time RT-PCR platform. Using this platform, the signature was validated on an independent test set consisting of 47 tumours (10 MSI, 37 MSS), of which 45 were correctly classified. In a second step, we constructed a signature capable of separating MMR-deficient tumours into sporadic MSI and HNPCC cases, and validated this by a mathematical cross-validation approach. The demonstration that this two-step classification approach can identify MSI as well as HNPCC cases merits further gene expression studies to identify prognostic signatures.

Marriage and family therapists evaluate managed mental health care: a qualitative inquiry.

This study examined the experiences of 26 marriage and family therapists working in managed mental health care. A qualitative strategy was used to explore therapists' perspectives regarding practice in a managed care environment. Using an open-ended, semi-structured, mailed questionnaire four themes emerged from the data. These are the adaptations of clinical practice, issues of treatment duration/abandonment, effects of managed care on the therapeutic relationship, and issues of diagnosis. Recommendations are drawn from the findings and discussed.

Alternative case definitions of ventilator-associated pneumonia identify different patients in a surgical intensive care unit.

Diagnostic criteria that define ventilator-associated pneumonia (VAP) remain controversial. The purpose of this study was to evaluate common definitions of VAP and determine their relationship to each other and clinical treatment. This study prospectively evaluated several diagnostic criteria that define VAP in a cohort of 255 consecutive SICU patients ventilated for < 48 h. Definitions evaluated include the CDC definitions, the Johanson definitions which do not rely on culture data, the Physician's Probable diagnosis which relies on positive quantitative cultures, and the antibiotic treatment group. Forty-four patients (17%) received antibiotic treatment for VAP. Depending on the definition evaluated, criteria were met for a diagnosis of VAP from as low as 4% of patients by the Johanson definition to as high as 48% of patients by the CDC definition. There was poor agreement among the definitions in their ability to select the same patient as having VAP. Besides duration of mechanical ventilation and tube feeding, which were risk factors that predicted meeting the criteria for all groups, risk factors predicting VAP varied among the definitions. This study demonstrates that in a surgical ICU, the candidate definitions of pneumonia evaluated show little agreement. The particular case definition chosen to diagnose VAP will determine the incidence rate of pneumonia, the time to onset of pneumonia, and the risk factors of the type of patient treated.

Specific binding of alpha-macroglobulin to complement-type repeat CR4 of the low-density lipoprotein receptor-related protein.

The low-density lipoprotein receptor-related protein (LRP) is a large surface receptor that mediates binding and internalization of a large number of structurally and functionally unrelated ligands. The ligand binding sites are located in clusters of complement-type repeats (CR), where the general absence of mutual binding competition suggests that different ligands map to distinct sites. Binding of alpha(2)-macroglobulin-protease complexes to the LRP is mediated by the receptor binding domain (RBD) of alpha(2)-macroglobulin (alpha(2)M). To determine the major binding epitope(s) in the LRP, we generated a complete set of tandem CR proteins spanning the second cluster of CR domains, and identified a binding site for alpha(2)M in the N-terminal part of the cluster comprising CR3-CR6, using ligand blotting and surface plasmon resonance (SPR) analysis. The specific site involved in alpha(2)M recognition resides in the fourth CR domain, CR4, whereas another site is identified in CR5. An acidic epitope in CR4 is identified as important for binding alpha(2)M by mutagenesis and SPR analysis. The formation of the complex between the rat alpha(1)-macroglobulin RBD and CR domain pairs is characterized by analytical size-exclusion chromatography, which demonstrates a sufficiently strong interaction between the alpha(1)M RBD and CR34 or CR45 for the isolation of a complex.

The C-terminal N-glycosylation sites of the human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, adn -VI) are necessary for the expression of full enzyme activity.

The alpha1,3/4-fucosyltransferases are involved in the synthesis of fucosylated cell surface glycoconjugates. Human alpha1,3/4-fucosyltransferase III, -V, and -VI (hFucTIII, -V, and -VI) contain two conserved C-terminal N-glycosylation sites (hFucTIII: Asn154 and Asn185; hFucTV: Asn167 and Asn198; and hFucTVI: Asn153 and Asn184). In the present study, we have analyzed the functional role of these potential N-glycosylation sites, laying the main emphasis on the sites in hFucTIII. Tunicamycin treatment completely abolished hFucTIII enzyme activity while castanospermine treatment diminished hFucTIII enzyme activity to approximately 40% of the activity of the native enzyme. To further analyze the role of the conserved N-glycosylation sites in hFucTIII, -V, and -VI, we made a series of mutant genomic DNAs in which the asparagine residues in the potential C-terminal N-glycosylation sites were replaced by glutamine. Subsequently, the hFucTIII, -V, and -VI wild type and the mutants were expressed in COS-7 cells. All the mutants exhibited lower enzyme activity than the wild type and elimination of individual sites had different effects on the activity. The mutations did not affect the protein level of the mutants in the cells, but reduced the molecular mass as predicted. Kinetic analysis of hFucTIII revealed that lack of glycosylation at Asn185 did not change the Km values for the oligosaccharide acceptor and the nucleotide sugar donor. The present study demonstrates that hFucTIII, -V, and -VI require N-glycosylation at the two conserved C-terminal N-glycosylation sites for expression of full enzyme activity.

Identification of the minimal functional unit in the low density lipoprotein receptor-related protein for binding the receptor-associated protein (RAP). A conserved acidic residue in the complement-type repeats is important for recognition of RAP.

The low density lipoprotein receptor-related protein (LRP), a member of the low density lipoprotein receptor family, mediates the internalization of a diverse set of ligands. The ligand binding sites are located in different regions of clusters consisting of approximately 40 residues, cysteine-rich complement-type repeats (CRs). The 39-40-kDa receptor-associated protein, a folding chaperone/escort protein required for efficient transport of functional LRP to the cell surface, is an antagonist of all identified ligands. To analyze the multisite inhibition by RAP in ligand binding of LRP, we have used an Escherichia coli expression system to produce fragments of the entire second ligand binding cluster of LRP (CR3-10). By ligand affinity chromatography and surface plasmon resonance analysis, we show that RAP binds to all two-repeat modules except CR910. CR10 differs from other repeats in cluster II by not containing a surface-exposed conserved acidic residue between Cys(IV) and Cys(V). By site-directed mutagenesis and ligand competition analysis, we provide evidence for a crucial importance of this conserved residue for RAP binding. We provide experimental evidence showing that two adjacent complement-type repeats, both containing a conserved acidic residue, represent a minimal unit required for efficient binding to RAP.

Glycosylation of the N-terminal potential N-glycosylation sites in the human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI).

Human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and hFucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced by glutamine. Western blotting analyses demonstrated that both sites in hFucTV were glycosylated, whereas in hFucTVI only one of the sites (Asn91) was glycosylated. All the single mutants and the hFucTVI N46Q/N91Q double mutant exhibited enzyme activities that did not differ considerably from the wt activities. However, the enzyme activity of the hFucTV N60Q/N105Q double mutant was reduced to approximately 40% of the wt activity. In addition, castanospermine treatment diminished the enzyme activity and hence trimming of the N-linked glycans are required for expression of full enzyme activity of both hFucTV and -VI. The present study demonstrates that both of the N-terminal potential N-glycosylation sites in hFucTV and one of the sites in hFucTVI are glycosylated. Individually, their glycosylation does not contribute considerably to expression of enzyme activity. However, elimination of both sites in hFucTV reduces the enzyme activity.

Lewis antigen mediated adhesion of freshly removed human bladder tumors to E-selectin.

PURPOSE: Twenty fresh surgical specimens of human bladder tumors were tested for their ability to adhere to recombinant P and E-selectin. The adhesion was correlated to immunological detection of carbohydrate structures. MATERIALS AND METHODS: A static titertray assay with immobilized selectins and appropriate controls was used for bladder tumor cell adhesion. On the same tumors expression of carbohydrate structures was examined by immunohistochemistry and Western blotting. RESULTS: No tumor bound to P-selectin. Nine tumors showed a high number of cells binding to E-selectin, 5 showed intermediate binding, and 6 showed only rare binding. The specificity of the binding was verified by inhibition with EDTA, by blocking antibodies to E-selectin, and by an acrylamide based sLe(x) (Galbeta1-4 [Fucalpha1-3]GlcNAc-) polymer. The binding was significantly more frequent (p <0.045) in superficial tumors than in invasive tumors. The binding property was correlated to the detection of carbohydrate structures in Western blots and tissue sections of the same tumors, using six different monoclonal antibodies: anti-sLe(a), anti-sLe(x), anti-Le(a), anti-Le(x) (two different clones) and anti-Le(b). Most blot-stainings were smeared indicating a mucin-type carrier molecule, but 115, 55 and 40 kDa bands carrying Le(a) and/or Le(b) epitopes were present in all tumors that bound. The Le(a) structure, as detected by blotting, was the only structure necessary for binding in the center of the wells (p <0.001), and was correlated to number of bound cells (p <0.006). A weaker correlation was found between Le(b) and number of bound cells (p <0.032), whereas it was remarkable that no correlation was found with Le(x) or sLe(x). Immunohistological staining of Le(a) on cell membranes correlated with frequent binding (p <0.003), whereas no correlation was found to secretor and Lewis genotypes. CONCLUSIONS: These data on clinical specimens indicate that Lewis antigen mediated E-selectin adhesion may play a role in the human bladder cancer disease.

Indications of immunological tolerance in kidney transplantation.

As a complication to immunosuppressive treatment in allotransplantation, malignant diseases such as post-transplant lymphoproliferative disorder (PTLD) may occur. The patient in the present case is a 21-year-old man transplanted at the age of 11 with a kidney from his mother and at the age of 15 with a kidney from his father. During the immunosuppressive treatment the patient developed PTLD resulting in the withdrawal of the immunosuppressive drugs. At the time of writing, the immunosuppressive drugs have been withdrawn for more than 3 years. We report the findings of a state of donor-specific tolerance occurring after transplantation. Post-transplant cells from the patient show a non-reactive response in mixed lymphocyte cultures (MLCs) to cells from both the mother and the father. We demonstrate a reduction in the mRNA expression of the Thl cytokines IL-2 and IFN-gamma in the very same MLCs. The expression of Th1 cytokine mRNA was measured semi-quantitatively using competitive reverse transcription-polymerase chain reaction (RT-PCR). The reduction in the Th1 cytokine mRNA expression is not seen in the MLCs with patient cells against cells from a paternal HLA-A, B and DR-matched individual, suggesting the influence of other allorecognition factors than HLA-A, B and DR. Detection in vitro of a lowered expression of Th1 cytokine mRNA supports the notion of these mRNAs as indicators of post-transplant tolerance. Further studies will reveal whether the cytokine mRNA measurements on short time stimulated lymphocytes can be used more generally as a monitoring parameter of tolerance in kidney transplantation.