Early Conversion From Calcineurin Inhibitor- to Everolimus-Based Therapy Following Kidney Transplantation: Results of the Randomized ELEVATE Trial.

In a 24-month, multicenter, open-label, randomized trial, 715 de novo kidney transplant recipients were randomized at 10-14 weeks to convert to everolimus (n = 359) or remain on standard calcineurin inhibitor (CNI) therapy (n = 356; 231 tacrolimus; 125 cyclosporine), all with mycophenolic acid and steroids. The primary endpoint, change in estimated glomerular filtration rate (eGFR) from randomization to month 12, was similar for everolimus versus CNI: mean (standard error) 0.3(1.5) mL/min/1.732 versus -1.5(1.5) mL/min/1.732 (p = 0.116). Biopsy-proven acute rejection (BPAR) at month 12 was more frequent under everolimus versus CNI overall (9.7% vs. 4.8%, p = 0.014) and versus tacrolimus-treated patients (2.6%, p < 0.001) but similar to cyclosporine-treated patients (8.8%, p = 0.755). Reporting on de novo donor-specific antibodies (DSA) was limited but suggested more frequent anti-HLA Class I DSA under everolimus. Change in left ventricular mass index was similar. Discontinuation due to adverse events was more frequent with everolimus (23.6%) versus CNI (8.4%). In conclusion, conversion to everolimus at 10-14 weeks posttransplant was associated with renal function similar to that with standard therapy overall. Rates of BPAR were low in all groups, but lower with tacrolimus than everolimus.

Pancreas transplantation with enteroanastomosis to native duodenum poses technical challenges--but offers improved endoscopic access for scheduled biopsies and therapeutic interventions.

To facilitate endoscopic access for rejection surveillance and stenting of the pancreas, we have abandoned the duodenojejunostomy (DJ) in favor of duodenoduodenostomy (DD) in pancreas transplantation (PTx). From September 2012 to September 2013 we performed 40 PTx with DD; 20 solitary-PTx (S-PTx) and 20 simultaneous pancreas and kidney transplantation (SPK). We compared the outcomes with results from 40 PTx-DJ (10 S-PTx and 30 SPK) from the preceding era. The DD-enteroanastomoses were performed successfully. Endoscopic pancreas biopsies (endoscopic ultrasound examination [EUS]) yielded representative material in half of the cases. One exocrine fistula was treated by endoscopic stenting. PTxs-DD were associated with a higher rate of thrombosis compared to PTx-DJ (23% vs. 5%) and reoperations (48% vs. 30%), as well as inferior graft survival (80% vs. 88%). Time on waiting list, HLA A + B mismatches and reoperations were associated with graft loss. Only recipient age remained an independent predictor of patient death in multivariate analysis. PTx-DD showed a higher rate of thrombosis and inferior results, but facilitated a protocol biopsy program by EUS that was feasible and safe. Given that technical difficulties can be solved, the improved endoscopic access might confer long-term benefits, yet this remains to be proven.

Improved patient survival with simultaneous pancreas and kidney transplantation in recipients with diabetic end-stage renal disease.

AIMS/HYPOTHESIS: We aimed to determine whether simultaneous pancreas and kidney (SPK) transplantation would improve patient and kidney graft survival in diabetic end-stage renal disease (ESRD) compared with kidney transplantation alone (KTA). METHODS: Follow-up data were retrieved for all 630 patients with diabetic ESRD who had received SPK or KTA at our centre from 1983 to the end of 2010. Recipients younger than 55 years of age received either an SPK (n = 222) or, if available, a single live donor kidney (LDK; n = 171). Older recipients and recipients with greater comorbidity received a single deceased donor kidney (DDK; n = 237). Survival was analysed by the Kaplan-Meier method and in multivariate Cox regression analysis adjusting for recipient and donor characteristics. RESULTS: Patient survival was superior in SPK compared with both LDK and DDK recipients in univariate analysis. Follow-up time (mean ± SD) after transplantation was 7.1 ± 5.7 years. Median actuarial patient survival was 14.0 years for SPK, 11.5 years for LDK and 6.7 years for DDK recipients. In multivariate analyses including recipient age, sex, treatment modality, time on dialysis and era, SPK transplantation was protective for all-cause mortality compared with both LDK (p = 0.02) and DDK (p = 0.029) transplantation. After the year 2000, overall patient survival improved compared with previous years (HR 0.40, 95% CI 0.30, 0.55; p < 0.001). Pancreas graft survival also improved after 2000, with a 5 year graft survival rate of 78% vs 61% in previous years (1988-1999). CONCLUSIONS/INTERPRETATION: Recipients of SPK transplants have superior patient survival compared with both LDK and DDK recipients, with improved results seen over the last decade.

Differential expression and subcellular localization for subunits of cAMP-dependent protein kinase during ram spermatogenesis.

The expression of mRNAs for the RI alpha, RII alpha, and C alpha subunits of cAMP-dependent protein kinase has been studied in different ram germ cells. The sizes of the specific RI alpha, RII alpha, and C alpha mRNAs, observed in germ cells were 1.6, 2.0, and 2.6 kb, respectively. RI alpha and C alpha mRNAs were mainly expressed in primary spermatocytes. A postmeiotic expression predominating in early spermatids was unique to RII alpha mRNA. The location of RI, RII alpha, and C subunits in well-defined organelles of ram spermatids and epididymal sperm was assessed by immunogold electron microscopy. In spermatids, RI, RII alpha, and C were essentially present in the forming acrosome and, to a lesser extent, in the nucleus. During sperm epididymal maturation, the protein kinases disappeared from the acrosome and were detected in a variety of sperm functional areas, such as the tip of the acrosome, the motility apparatus, and the membrane network. The present study on subunits of cAMP-dependent protein kinase supports the concept that specific functions are attached to the different subunits in that it shows differential expression and differential subcellular localization in germ cells.

Transcriptional profiling reveals monocyte-related macrophages phenotypically resembling DC in human intestine.

The tissue dendritic cell (DC) compartment is heterogeneous, and the ontogeny and functional specialization of human tissue conventional DC (cDC) subsets and their relationship with monocytes is unresolved. Here we identify monocyte-related CSF1R+Flt3- antigen presenting cells (APCs) that constitute about half of the cells classically defined as SIRPα+ DCs in the steady-state human small intestine. CSF1R+Flt3- APCs express calprotectin and very low levels of CD14, are transcriptionally related to monocyte-derived cells, and accumulate during inflammation. CSF1R+Flt3- APCs show typical macrophage characteristics functionally distinct from their Flt3+ cDC counterparts: under steady-state conditions they excel at antigen uptake, have a lower migratory potential, and are inefficient activators of naïve T cells. These results have important implications for the understanding of the ontogenetic and functional heterogeneity within human tissue DCs and their relation to the monocyte lineage.

Minimally invasive kidney transplantation: the first experience.

Minimally invasive procedures in recent years have gained widespread acceptance. Within the field of transplantation, laparoscopic living donor nephrectomy (LLDN), requiring a 6- to 10-cm incision, is now considered the optimal procedure. According to recent MEDLINE searches, no minimally invasive technique has been reported for kidney transplantation. Considering the rapid evolution of minimally invasive surgery during the last decade, there is little reason to believe that kidney transplantation in future will be excluded from this development. A novel minimally invasive technique for kidney transplantation (MIKT) is presented, restricted to a 7- to 9-cm incision and minimal dissection/tissue trauma. The kidney is meticulously prepared on the back table and placed in a fitted lateral, retroperitoneal pouch. All three anastomoses are performed with the kidney in its final "in situ" position, and ureter reimplantation is done by extravesical technique. Twenty-one patients have been transplanted by MIKT and followed in a prospective manner, along with a matched control group subjected to conventional kidney transplantation. Our results indicate that MIKT may be executed safely and quickly. Beneficial effects on postoperative pain/analgesia, recovery, and complications are suggested by this first MIKT experience. The technical solutions of MIKT are per se not unique. However, the incision is minimal and not larger than the one required for LLDN. Minimally invasive surgery seems particularly attractive in the immunosuppressed population, and even more so with the recent introduction of potent antiproliferative drugs.

Biphasic response to 3',5'-cyclic adenosine monophosphate (cAMP) at the messenger ribonucleic acid level for a regulatory subunit of cAMP-dependent protein kinase.

In the present study we have examined the effect of long-term stimulation with (Bu)2cAMP on mRNA levels for the hormone responsive regulatory subunit (RII beta) of cAMP-dependent protein kinase in cultured rat Sertoli cells. The effects of the same treatment on two other mRNAs [androgen binding protein (ABP) and cellular retinol binding protein (cRBP)], shown to be regulated by cAMP, were examined simultaneously. The addition of (Bu)2cAMP (0.1 mM) to primary Sertoli cell cultures, for 14 and 24 h, caused a 50- to 60-fold stimulation in the steady state levels of mRNA for RII beta. During the same period of stimulation, we also observed a significant increase (2- to 3-fold) in the mRNA levels for ABP, and a 80% decrease in the mRNA levels for cRBP. Continued stimulation for 36 and 48 h was associated with a significant time-dependent decrease in the mRNA level for RII beta, in spite of the continuous presence of (Bu)2cAMP (0.1 mM) in the medium. This reduced response by long term stimulation with (Bu)2cAMP appears to be specific for RII beta, since mRNA for ABP remained elevated and mRNA for cRBP remained depressed during the entire period of cAMP stimulation. Our data demonstrate the presence of a biphasic type of regulation at the mRNA level, specific for the regulatory subunit RII beta of cAMP-dependent protein kinase. This response may be analogous to the desensitization mechanisms observed at other levels of the cAMP signalling pathway. For proteins constituting part of the signal transduction pathway this type of biphasic regulation, may be particularly important in maintaining homeostasis in the cell.

Molecular cloning, complementary deoxyribonucleic acid structure and predicted full-length amino acid sequence of the hormone-inducible regulatory subunit of 3'-5'-cyclic adenosine monophosphate-dependent protein kinase from human testis.

In this study, we report the isolation and characterization of a full-length cDNA clone for the hormone-inducible regulatory subunit RII beta (formerly called RII51) of type II cAMP-dependent protein kinase from a human testis cDNA library. The cloned cDNA demonstrated tissue-specific expression of RII beta mRNA in human tissues, with the highest mRNA levels in testis and ovary. The isolated human cDNA clone was 3.3 kilobases (kb) in length and contained 166 base pairs (bp) of G/C-rich 5'-noncoding sequence, an open reading frame of 1254 bp and an A/T-rich 3'-nontranslated region containing 1836 bp followed by an 89 nucleotide long poly(A)-tail. The predicted protein contains 418 amino acids including the start methionine, and the estimated mol wt of human RII beta is 53,856. The nucleotide sequence within the open reading frame and the predicted amino acid sequence of human RII beta are highly conserved compared with partial rat RII beta sequences, displaying 91% and 97% similarity, respectively. Codon preference analysis of the cloned cDNA sequence indicated that the two cAMP-binding domains and the hinge region are highly conserved through evolution, whereas the dimerization domain displayed a codon preference pattern indicative of appearance at a later stage of evolution. The isolated human cDNA detected an FSH- and cAMP-inducible mRNA of 3.2 kb in rat Sertoli cells, thus confirming that the cloned cDNA represents the hormone-inducible regulatory subunit of cAMP-dependent protein kinase. This is the first report documenting the isolation of a full-length cDNA clone for the RII beta of cAMP-dependent protein kinase.

Molecular cloning of a tissue-specific protein kinase (C gamma) from human testis--representing a third isoform for the catalytic subunit of cAMP-dependent protein kinase.

Two different mammalian genes for the catalytic subunit (C) of cAMP-dependent protein kinase have previously been characterized (C alpha, C beta). In the present study, we report the molecular cloning of a third isoform of C, from a human testis cDNA library, as well as the isolation of human cDNAs for C alpha and C beta. This third form of C, which we will designate C gamma, is clearly derived from a distinct gene and shows a tissue-specific expression. A close evolutionary relation between C gamma and C alpha was suggested by nucleotide homologies (86% inside the open reading frame, 81% in the 3'-untranslated region). Thus, the C gamma cDNA cross-hybridized with the 2.8 kilobase (kb) C alpha mRNA, present at high levels in most human tissues, as well as with a 1.8 kb C gamma-specific mRNA, which was only found at detectable levels in human testis. However, at the amino acid level, C alpha and C beta showed a close relationship (93% homology), whereas C gamma diverged significantly from both C alpha (83%) and C beta (79%). Taken together with the tissue-specific expression of C gamma, this suggests a pressure on C gamma during evolution, acting to modulate it in a functionally specific way. Certain amino acid substitutions make C gamma a distinct member of the cAMP-dependent subfamily of protein kinases, and suggest that C gamma may be distinct in its protein substrate specificity or its interaction with the different regulatory subunits.

High level, cell-specific expression of ornithine decarboxylase transcripts in rat genitourinary tissues.

We evaluated transcript levels for the rate-limiting enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC), in rat tissues by Northern blotting and in situ hybridization histochemistry, using a rat cDNA probe. ODC transcripts were expressed at a high level, relative to levels in other tissues, in the kidney and testis of the adult rat; maximal levels of transcripts in these tissues occurred after sexual maturation had taken place, i.e. between 20 and 150 days of age. In situ hybridization histochemistry revealed high level expression in the kidney, testis, prostate, and seminal vesicles of the male rat; this high level expression was limited to certain cell types: kidney, S3 cells of the proximal convoluted tubule; prostate and seminal vesicles, glandular or luminal epithelial cells; and testis, early spermatogenic cells. High level expression of ODC mRNA disappeared from the prostate and seminal vesicle epithelial cells after castration and reappeared with testosterone treatment; in contrast, levels of kidney ODC mRNA were essentially unchanged by castration and were similar in male and female adult rats. We conclude that high level ODC mRNA expression occurs in specific cell types in the adult rat, where it appears to be regulated by both androgen-dependent and independent mechanisms.

Should kidneys from older cadaveric donors be age-matched to the recipient?

Persistent shortage of kidneys for transplantation has forced most transplant centers to include procurement and use of kidneys from older donors. It is not clear whether the optimal use of these kidneys involve age-matching to the recipient. The aim of this study was to evaluate the clinical outcome of older cadaveric kidneys (>60 years), transplanted to young recipients (<50 years) and older recipients (>60 years). From 1989 through 2002, 252 first kidney grafts were procured from donors above the age of 60; 149 of the recipients to these grafts were above 60 years and 45 recipients were below 50. Minimum follow-up time was 12 months. Variables for waiting time to transplantation, DR mismatches, PRA, dialysis prior to transplantation, episodes of acute rejection, number of steroid-resistant rejections, creatinine levels, cold ischemia time, and causes of graft loss did not differ between the two groups. There was no significant difference in graft survival for young and older recipients receiving kidney from donors above 60 years of age. Graft survival at 1 year for young recipients was 90% and for older recipients 93% (NS). Five-year graft survival was 72% and 79%, respectively (NS). However, there was a significant positive effect on long-term graft survival if the donor kidney was less than 50 years. From our data, there is no evidence that age-matching of older donors has any beneficial effect on graft survival in kidney transplantation.

Laparoscopic ventral hernia repair: outcomes in primary versus incisional hernias: no effect of defect closure.

PURPOSE: Supposing divergent aetiology, we found it interesting to investigate outcomes between primary (PH) versus incisional (IH) hernias. In addition, we wanted to analyse the effect of defect closure and mesh fixation techniques. METHODS: 37 patients with PH and 70 with IH were enrolled in a prospective cohort-study, treated with laparoscopic ventral hernia repair (LVHR) and randomised to ± transfascial sutures. In addition, we analysed results from a retrospective study with 36 PH and 51 IH patients. Mean follow-up time was 38 months in the prospective study and 27 months in the retrospective study. RESULTS: 35 % of PH's and 10 % of IH's were recurrences after previous suture repair. No late infections or mesh removals occurred. Recurrence rates in the prospective study were 0 vs. 4.3 % (p = 0.55) and the complication rates were 16 vs. 27 % (p = 0.24) in favour of the PH cohort. The IH group had a mesh protrusion rate of 13 vs. 5 % in the PH group (p = 0.32), and significantly (p < 0.01) larger hernias and adhesion score, longer operating time (100 vs. 79 min) and admission time (2.8 vs. 1.6 days). Closure of the hernia defect did not influence rate of seroma, pain at 2 months, protrusion or recurrence. An overall increased complication rate was seen after defect closure (OR 3.42; CI 1.25-9.33). CONCLUSIONS: With PH, in comparison to IH treated with LVHR, no differences were observed regarding recurrence, protrusion or complication rates. Defect closure (raphe), when using absorbable suture, did not benefit long-term outcomes and caused a higher overall complication rate. (ClinicalTrials.gov number: NCT00455299).

Differential regulation of messenger ribonucleic acids for specific subunits of cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase by cAMP in rat Sertoli cells.

In the present study we have examined the effects of FSH, forskolin, and (Bu)2cAMP on messenger RNA (mRNA) levels for all known subunits of cAMP-dependent protein kinase in rat Sertoli cells, using newly developed complementary DNA (cDNA) probes. mRNAs for the three regulatory subunits [RI alpha, RII51, (RII beta), and RII54 (RII alpha)] and the catalytic subunit C alpha were shown to be present in cultured rat Sertoli cells, whereas mRNAs for the subunits designated RI beta and C beta were below the level of detection. A high-levelled, concentration-dependent increase in a 3.2 kilobase mRNA for RII51 was observed when cultured immature Sertoli cells were incubated with increasing concentrations of (Bu)2cAMP (10(-6) to 5 X 10(-3) M) for 16 h. Densitometric scanning indicated a maximal stimulation by (Bu)2cAMP of 30- to 40-fold. Incubation with forskolin (100 microM) and FSH (200 ng/ml) gave rise to a smaller but significant increase in mRNA for RII51. When cultured Sertoli cells were incubated in the presence of 10(-4) M (Bu)2cAMP for varying time periods, there was a biphasic regulation of mRNA for RII51. (Bu)2cAMP caused an initial increase in mRNA for RII51 with maximal levels obtained after 10-16 h, after which a time-dependent decrease was observed. For the other three subunits present in Sertoli cells (RI alpha, RII54, and C alpha) a smaller but significant stimulation by (Bu)2cAMP and forskolin (2-4 fold) was seen. The functional implications of these changes in mRNA levels for the different subunits of cAMP-dependent protein kinase have not yet been revealed. However, our data clearly demonstrate differential regulation of the various subunits of cAMP-dependent protein kinase in Sertoli cells. Furthermore, these results document the presence of distinct adaptational changes taking place at the level of cAMP-dependent protein kinase in response to long term elevation of cAMP.

Human testis cDNA for the regulatory subunit RII alpha of cAMP-dependent protein kinase encodes an alternate amino-terminal region.

Phosphorylations catalyzed by cAMP-dependent protein kinase are essential for sperm motility, and type II cAMP-dependent protein kinase in mature sperm has been shown to be firmly bound to the flagellum via the regulatory subunit, RII. The present study documents high-levelled expression of a human, testis-specific RII alpha mRNA (2.0 kb) analogous to the rat mRNA which is induced in haploid germ cells [(1988) FEBS Lett. 229, 391-394]. We report the molecular cloning of a full-length human cDNA corresponding to this unique testis mRNA, and the presence of an alternative amino-terminal region (amino acids 45-75) of the predicted RII alpha protein (404 amino acids) compared with the previously published mouse and rat sequences. However, this alternate region is also shown to be present in RII alpha mRNA (7.0 kb) of human somatic cells. Our data indicate the divergent amino-terminal sequence to be due to species differences, suggesting an active evolutionary pressure on this particular region, which could be involved in subcellular attachment of RII alpha and thereby localization of kinase activity to certain targets within the cell.

A unique mRNA species for a regulatory subunit of cAMP-dependent protein kinase is specifically induced in haploid germ cells.

Cyclic AMP (cAMP) and its action by way of cAMP-dependent protein kinase is important for sperm motility. Previous studies on germ cells have demonstrated a selective decrease in the amount of type I cAMP-dependent protein kinase during spermatid development, and that type II was the major form present in elongating spermatids and in mature sperm. This would indicate activation of a gene in haploid germ cells, encoding a regulatory subunit of type II protein kinase. However, haploid expression of such a gene has so far not been shown. In the present study we demonstrate high-levelled expression of a unique mRNA species for a specific regulatory subunit of type II cAMP-dependent protein kinase at late stages of spermatogenesis, i.e. during spermatid elongation.

Differences in the rate of molar wear between monkeys raised on different diets.

The purpose of this study was to make detailed comparisons of rates and patterns of tooth wear in 15 growing vervet monkeys raised on hard vs. soft diets. Dental impressions were taken every six to eight weeks over a four-year period. Cusp heights and areas of dentin exposure on the buccal cusps of the left mandibular first molar were measured from high-resolution epoxy casts, by use of a Reflex Measuring Microscope. Areas of dentin exposure were regressed against time (by use of least-squares regression) so that the course of tooth wear in animals from both diet groups could be charted. By use of a two-sample t test and the Mann-Whitney test, slopes of the regressions and changes in cusp height were compared between diet groups. In both comparisons, animals raised on the hard diet showed more rapid tooth wear than did animals raised on the soft diet. Analyses of other parameters indicate that this was probably because of differences in dietary consistency between the two groups.

Developmental effects of impaired breathing in the face of the growing child.

Craniofacial morphology and occlusal pattern are evaluated in 71 subjects having impaired breathing as diagnosed by an otolaryngologist, and in an equal number of controls. The impaired group demonstrate characteristic combinations of craniofacial deformities and malocclusions, with the younger individuals demonstrating a lesser expression of malocclusion progression and morphologic deformities. This suggests that early recognition of such facial patterns may be utilized to identify those breathing compromised individuals who have a likely tendency to develop certain types of malocclusion.

Class I and Class III malocclusion sub-groupings related to headform type.

Different headform types establish different lines of craniofacial growth resulting in anatomic sub-groupings of Classes I, II, and III with characteristic morphologic features. Several key basicranial and facial relationships are involved, with the nasal region particularly significant in group distinctions.

The use of specific dental school-taught restorative techniques by practicing clinicians.

In 1995, a survey requesting information about the utilization of certain prosthodontic techniques was mailed to 3,544 graduates of a midwestern dental school. Responses were received from 1,455 alumni, representing a 41 percent return rate. In general, the results are consistent with international and national trends and show significant disparity in the utilization rates of certain procedures between general dentists and prosthodontists, as well as a disconnect between what is taught in the undergraduate dental educational program and what is applied in practice. For example, while prosthodontists typically apply what was taught in their educational program, utilization rates of general dentists for the facebow was 29.64 percent; the custom tray 68.48 percent; border molding 58.67 percent; altered casts 24.10 percent; custom posts 49.29 percent; prefabricated posts 67.54 percent; and semi-adjustable articulators 50.64 percent. While no solutions to this disconnect are offered the authors do pose important questions that must be addressed by the dental educational community.

Targeting risk factors for impaired wound healing and wound complications after kidney transplantation.

BACKGROUND: Because of potent immunosuppression, impaired wound healing and complications are frequent features after kidney transplantation (KTx). OBJECTIVE: To investigate the incidence and nature of impaired wound healing and complications at a single transplantation center in Norway. PATIENTS: Of 226 patients who underwent KTx, 199 (87%) were followed up prospectively for 1 year (2005) via close and meticulous wound inspection. RESULTS: The study revealed a high rate of wound complications (200-250/y) in a high-volume center. Fifty-four patients (27%) experienced prolonged wound healing, defined as gaps, secretions, or wound complications, at 3 to 5 weeks posttransplantation, and 41 patients (21%) had impaired wound healing, defined as gaps, secretions, or wound complications after 5 weeks posttransplantation. In total, 50 patients (25%) required surgical or radiologic reintervention. Complications included lymphocele in 29 patients (14.6%), wound dehiscence in 16 (8.0%), bleeding or hematoma in 10 (5.0%), and infection in 9 (4.5%). Risk factors associated with wound complications included recipient older than 60 years, body mass index greater than 30, hemoglobin concentration less than 10 g/dL, albumin concentration less than 36 g/dL, duration of surgery more than 200 minutes, no subcutaneous sutures, and sirolimus or everolimus therapy. At nominal and logistic regression analysis, recipient older than 60 years, body mass index greater than 30, and no subcutaneous sutures were independent risk factors. CONCLUSION: Risk factor analysis and previous documentation suggest that wound complications might be counteracted using the following measures: subcutaneous sutures, predialysis transplantation, sealing or ligation of lymphatic trunks, prophylactic fenestration, reduction of corticosteroid load, and avoiding sirolimus/everolimus therapy.