Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.

Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the TUBB4B isotype that specifically perturbed centriole and cilium biogenesis. Distinct TUBB4B variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.

Systemic inflammation early after kidney transplantation is associated with long-term graft loss: a cohort study.

Background: Early graft loss following kidney transplantation is mainly a result of acute rejection or surgical complications, while long-term kidney allograft loss is more complex. We examined the association between systemic inflammation early after kidney transplantation and long-term graft loss, as well as correlations between systemic inflammation scores and inflammatory findings in biopsies 6 weeks and 1 year after kidney transplantation. Methods: We measured 21 inflammatory biomarkers 10 weeks after transplantation in 699 patients who were transplanted between 2009 and 2012 at Oslo University Hospital, Rikshospitalet, Norway. Low-grade inflammation was assessed with predefined inflammation scores based on specific biomarkers: one overall inflammation score and five pathway-specific scores. Surveillance or indication biopsies were performed in all patients 6 weeks after transplantation. The scores were tested in Cox regression models. Results: Median follow-up time was 9.1 years (interquartile range 7.6-10.7 years). During the study period, there were 84 (12.2%) death-censored graft losses. The overall inflammation score was associated with long-term kidney graft loss both when assessed as a continuous variable (hazard ratio 1.03, 95% CI 1.01-1.06, P = 0.005) and as a categorical variable (4th quartile: hazard ratio 3.19, 95% CI 1.43-7.10, P = 0.005). In the pathway-specific analyses, fibrogenesis activity and vascular inflammation stood out. The vascular inflammation score was associated with inflammation in biopsies 6 weeks and 1 year after transplantation, while the fibrinogenesis score was associated with interstitial fibrosis and tubular atrophy. Conclusion: In conclusion, a systemic inflammatory environment early after kidney transplantation was associated with biopsy-confirmed kidney graft pathology and long-term kidney graft loss. The systemic vascular inflammation score correlated with inflammatory findings in biopsies 6 weeks and 1 year after transplantation.

Osteocyte-Like Cells Differentiated From Primary Osteoblasts in an Artificial Human Bone Tissue Model.

In vitro models of primary human osteocytes embedded in natural mineralized matrix without artificial scaffolds are lacking. We have established cell culture conditions that favored the natural 3D orientation of the bone cells and stimulated the cascade of signaling needed for primary human osteoblasts to differentiate into osteocytes with the characteristically phenotypical dendritic network between cells. Primary human osteoblasts cultured in a 3D rotating bioreactor and incubated with a combination of vitamins A, C, and D for up to 21 days produced osteospheres resembling native bone. Osteocyte-like cells were identified as entrapped, stellate-shaped cells interconnected through canaliculi embedded in a structured, mineralized, collagen matrix. These cells expressed late osteoblast and osteocyte markers such as osteocalcin (OCN), podoplanin (E11), dentin matrix acidic phosphoprotein 1 (DMP1), and sclerostin (SOST). Organized collagen fibrils, observed associated with the cell hydroxyapatite (HAp) crystals, were found throughout the spheroid and in between the collagen fibrils. In addition to osteocyte-like cells, the spheroids consisted of osteoblasts at various differentiation stages surrounded by a rim of cells resembling lining cells. This resemblance to native bone indicates a model system with potential for studying osteocyte-like cell differentiation, cross-talk between bone cells, and the mineralization process in a bonelike structure in vitro without artificial scaffolds. In addition, natural extracellular matrix may allow for the study of tissue-specific biochemical, biophysical, and mechanical properties. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Chronic Kidney Disease from Polyvinylpyrrolidone Deposition in Persons with Intravenous Drug Use.

BACKGROUND AND OBJECTIVES: Persons with intravenous drug use have a higher risk of developing CKD compared with the general population. In Norway, deposits of polyvinylpyrrolidone have been observed in kidney biopsies taken from persons with opioid addiction and intravenous drug use since 2009. Polyvinylpyrrolidone is an excipient commonly used in pharmaceuticals, and the polyvinylpyrrolidone deposits observed in these patients were caused by intravenous injection of a specific oral methadone syrup containing very high molecular weight polyvinylpyrrolidone. Here, we present the clinicopathologic findings from 28 patients with CKD associated with polyvinylpyrrolidone deposition in the kidney. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The 28 patients and their kidney biopsies were included when polyvinylpyrrolidone deposition was recognized, either retrospectively or at the time of diagnostic evaluation. Biopsies were taken between 2009 and 2016. We collected laboratory parameters and clinical data from digital patient charts. For each kidney biopsy, the glomerular volume, extent of polyvinylpyrrolidone deposition, and tubulointerstitial area with tubular atrophy were assessed quantitatively. RESULTS: All patients (mean age: 37 years) had CKD (mean eGFR: 33 ml/min per 1.73 m2) and normal urine protein or non-nephrotic-range proteinuria. Biopsies showed moderate to severe tubular atrophy (mean extent: 65%) and interstitial infiltrates of vacuolated macrophages containing polyvinylpyrrolidone (mean share of biopsy area: 1.5%). Underperfused and ischemic glomeruli were common findings. In 22 samples, ultrastructural investigation revealed polyvinylpyrrolidone-containing vacuoles in the mesangial or endothelial cells of glomeruli. At the last follow-up, most patients had stable or improved eGFR. Two patients had developed kidney failure and underwent hemodialysis. CONCLUSIONS: Intravenous injection of a specific oral methadone syrup caused polyvinylpyrrolidone deposition in the kidney in persons with opioid addiction and intravenous drug use. Kidney biopsy findings suggested an association between polyvinylpyrrolidone deposition and tubular atrophy.

International consensus guideline for reporting transmission electron microscopy results in the diagnosis of primary ciliary dyskinesia (BEAT PCD TEM Criteria).

Primary ciliary dyskinesia (PCD) is a heterogeneous genetic condition. European and North American diagnostic guidelines recommend transmission electron microscopy (TEM) as one of a combination of tests to confirm a diagnosis. However, there is no definition of what constitutes a defect or consensus on reporting terminology. The aim of this project was to provide an internationally agreed ultrastructural classification for PCD diagnosis by TEM.A consensus guideline was developed by PCD electron microscopy experts representing 18 centres in 14 countries. An initial meeting and discussion were followed by a Delphi consensus process. The agreed guideline was then tested, modified and retested through exchange of samples and electron micrographs between the 18 diagnostic centres.The final guideline a) provides agreed terminology and a definition of Class 1 defects which are diagnostic for PCD; b) identifies Class 2 defects which can indicate a diagnosis of PCD in combination with other supporting evidence; c) describes features which should be included in a ciliary ultrastructure report to assist multidisciplinary diagnosis of PCD; and d) defines adequacy of a diagnostic sample.This tested and externally validated statement provides a clear guideline for the diagnosis of PCD by TEM which can be used to standardise diagnosis internationally.

Effects of marine n-3 fatty acid supplementation in renal transplantation: A randomized controlled trial.

Marine n-3 fatty acids (FAs) may exert beneficial effects on inflammation, fibrosis, and endothelial function, which could preserve renal graft function. In this randomized controlled trial, 132 Norwegian renal transplant recipients received either 2.6 g of marine n-3 FAs or olive oil (control) daily for 44 weeks, in addition to standard care. Thirty patients did not complete the trial. The primary endpoint was change (Δ) in measured glomerular filtration rate (mGFR) during follow-up. We found no significant difference in Δ mGFR between the marine n-3 FA group and controls (6.7 vs 3.8 mL/min per 1.73 m2 , P = .15). Significant beneficial effects from marine n-3 FA supplementation were, however, seen in secondary endpoints plasma triglycerides, plasma high-sensitivity C-reactive protein, and brachial artery flow-mediated dilation. In the per-protocol population, the renal graft indices percent interstitial fibrosis and Chronic Allograft Damage Index also were significantly lower in the marine n-3 FA group. The cumulative incidence of adverse events did not differ between the marine n-3 FA group (n = 218) and controls (n = 240). In conclusion, marine FA supplementation did not improve renal function compared with controls, but was safe, lowered plasma triglyceride and high-sensitivity C-reactive protein levels, and improved endothelial function (Clinical.Trials.gov identifier NCT01744067).

Machine learning for intraoperative prediction of viability in ischemic small intestine.

OBJECTIVE: Evaluation of intestinal viability is essential in surgical decision-making in patients with acute intestinal ischemia. There has been no substantial change in the mortality rate (30%-93%) of patients with acute mesenteric ischemia (AMI) since the 1980s. As the accuracy from the first laparotomy alone is 50%, the gold standard is a second-look laparotomy, increasing the accuracy to 87%-89%. This study investigates the use of machine learning to classify intestinal viability and histological grading in pig jejunum, based on multivariate time-series of bioimpedance sensor data. APPROACH: We have previously used a bioimpedance sensor system to acquire electrical parameters from perfused, ischemic and reperfused pig jejunum (7 + 15 pigs) over 1-16 h of ischemia and 1-8 h of reperfusion following selected durations of ischemia. In this study we compare the accuracy of using end-point bioimpedance measurements with a feedforward neural network (FNN), versus the accuracy when using a recurrent neural network with long short-term memory units (LSTM-RNN) with bioimpedance data history over different periods of time. MAIN RESULTS: Accuracies in the range of what has been reported clinically can be achieved using FNN's on a single bioimpedance measurement, and higher accuracies can be achieved when employing LSTM-RNN on a sequence of data history. SIGNIFICANCE: Intraoperative bioimpedance measurements on intestine of suspect viability combined with machine learning can increase the accuracy of intraoperative assessment of intestinal viability. Increased accuracy in intraoperative assessment of intestinal viability has the potential to reduce the high mortality and morbidity rate of the patients.

Ischemia/reperfusion injury in porcine intestine - Viability assessment.

AIM: To investigate viability assessment of segmental small bowel ischemia/reperfusion in a porcine model. METHODS: In 15 pigs, five or six 30-cm segments of jejunum were simultaneously made ischemic by clamping the mesenteric arteries and veins for 1 to 16 h. Reperfusion was initiated after different intervals of ischemia (1-8 h) and subsequently monitored for 5-15 h. The intestinal segments were regularly photographed and assessed visually and by palpation. Intraluminal lactate and glycerol concentrations were measured by microdialysis, and samples were collected for light microscopy and transmission electron microscopy. The histological changes were described and graded. RESULTS: Using light microscopy, the jejunum was considered as viable until 6 h of ischemia, while with transmission electron microscopy the ischemic muscularis propria was considered viable until 5 h of ischemia. However, following ≥ 1 h of reperfusion, only segments that had been ischemic for ≤ 3 h appeared viable, suggesting a possible upper limit for viability in the porcine mesenteric occlusion model. Although intraluminal microdialysis allowed us to closely monitor the onset and duration of ischemia and the onset of reperfusion, we were unable to find sufficient level of association between tissue viability and metabolic markers to conclude that microdialysis is clinically relevant for viability assessment. Evaluation of color and motility appears to be poor indicators of intestinal viability. CONCLUSION: Three hours of total ischemia of the small bowel followed by reperfusion appears to be the upper limit for viability in this porcine mesenteric ischemia model.

Negative effect of zoledronic acid on tendon-to-bone healing.

Background and purpose - Outcome after ligament reconstruction or tendon repair depends on secure tendon-to-bone healing. Increased osteoclastic activity resulting in local bone loss may contribute to delayed healing of the tendon-bone interface. The objective of this study was to evaluate the effect of the bisphosphonate zoledronic acid (ZA) on tendon-to-bone healing. Methods - Wistar rats (n = 92) had their right Achilles tendon cut proximally, pulled through a bone tunnel in the distal tibia and sutured anteriorly. After 1 week animals were randomized to receive a single dose of ZA (0.1 mg/kg IV) or control. Healing was evaluated at 3 and 6 weeks by mechanical testing, dual-energy X-ray absorptiometry and histology including immunohistochemical staining of osteoclasts. Results - ZA treatment resulted in 19% (95% CI 5-33%) lower pullout strength and 43% (95% CI 14-72%) lower stiffness of the tendon-bone interface, compared with control (2-way ANOVA; p = 0.009, p = 0.007). Administration of ZA did not affect bone mineral density (BMD) or bone mineral content (BMC). Histological analyses did not reveal differences in callus formation or osteoclasts between the study groups. Interpretation - ZA reduced pullout strength and stiffness of the tendon-bone interface. The study does not provide support for ZA as adjuvant treatment in tendon-to-bone healing.

Kidney allograft subclinical rejection modulates systemic inflammation measured by C-reactive protein at 1 year after transplantation.

Kidney allograft inflammation is associated with proinflammatory modifications of peripheral blood mononuclear cells, suggesting that renal inflammation contributes to systemic inflammation. Thus, the aim of this study was to evaluate the relationship between subclinical inflammation in surveillance biopsies performed at 1 year and systemic inflammation assessed by C-reactive protein (CRP) levels at the time of biopsy. We analyzed 544 surveillance biopsies performed at 1 year that were classified as normal (n = 368), borderline (n = 148), or subclinical rejection (SCR) (n = 28). CRP levels were divided into quartiles. Patients in 1st, 2nd, and 3rd quartile were classified as low CRP (n = 408) and patients in the 4th quartile as high CRP (n = 136). Univariate analysis showed that the proportion of patients with SCR was higher in the high CRP group (10.3% vs 3.4%, P = 0.0067). Multivariate analysis showed that independent predictors of high CRP were body mass index (odds ratio [OR] 1.072 and 95% confidence interval [CI] 1.027-1.119), a positive urine culture at the day of the biopsy (OR 2.760 and 95% CI 1.205-6.323), and the presence of SCR at 1-year surveillance biopsy (OR 7.260 and 95% CI 3.530-14.935). In summary, we describe that subclinical acute rejection constitutes an independent predictor of systemic inflammation as measured by CRP.

Development of Kidney Transplant Fibrosis Is Inversely Associated With Plasma Marine Fatty Acid Level.

OBJECTIVE(S): We assessed associations between plasma levels of polyunsaturated fatty acids (PUFAs) and degree of inflammation and interstitial fibrosis in transplanted kidneys. DESIGN: The design of the study was single center cohort study. SUBJECTS: A study population of 156 patients who received a kidney transplant at Oslo University Hospital during 2010. MAIN OUTCOME MEASURE: Kidney transplant biopsies were obtained at 2 months and 1 year after transplantation. Degree of inflammation and interstitial fibrosis in the cortex of transplanted kidneys were estimated semi-quantitatively. Plasma phospholipid fatty acids levels were measured in a stable phase 2 months posttransplant. We used multivariate linear regression to assess associations between plasma levels of PUFAs and degree of inflammation and interstitial fibrosis at 2 months and 1 year postoperatively and change in degree of interstitial fibrosis during the first year after transplantation, adjusting for inflammation and fibrosis risk factors. RESULTS: Higher plasma marine n-3 PUFA levels were associated with less development of interstitial fibrosis in the kidney transplant (unstandardized ß-coefficient -1.12, standardized ß-coefficient -0.18, P = .03) during the first year after transplantation. Plasma levels of alpha linoleic acid, linoleic acid, and arachidonic acid were not associated with development of interstitial fibrosis. No associations were found between plasma levels of PUFAs and inflammation inside fibrotic areas or outside fibrotic areas in the kidney transplant at neither 2 months nor 1 year postoperatively. Linolenic acid levels in plasma were positively associated with change in renal function during the first year after transplantation. CONCLUSION: The inverse association between plasma marine n-3 PUFA levels and development of interstitial fibrosis during the first year after kidney transplantation suggests that marine fatty acid consumption might halt progression of fibrosis.

Tacrolimus and mycophenolate regimen and subclinical tubulo-interstitial inflammation in low immunological risk renal transplants.

The aim was to evaluate the relationship between maintenance immunosuppression, subclinical tubulo-interstitial inflammation and interstitial fibrosis/tubular atrophy (IF/TA) in surveillance biopsies performed in low immunological risk renal transplants at two transplant centers. The Barcelona cohort consisted of 109 early and 66 late biopsies in patients receiving high tacrolimus (TAC-C0 target at 1-year 6-10 ng/ml) and reduced MMF dose (500 mg bid at 1-year). The Oslo cohort consisted of 262 early and 237 late biopsies performed in patients treated with low TAC-C0 (target 3-7 ng/ml) and standard MMF dose (750 mg bid). Subclinical inflammation, adjusted for confounders, was associated with low TAC-C0 in the early (OR: 0.75, 95% CI: 0.61-0.92; P = 0.006) and late biopsies (OR: 0.69, 95% CI: 0.50-0.95; P = 0.023) from Barcelona. In the Oslo cohort, it was associated with low MMF in early biopsies (OR: 0.90, 95% CI: 0.83-0.98; P = 0.0101) and with low TAC-C0 in late biopsies (OR: 0.77, 95% CI: 0.61-0.97; P = 0.0286). MMF dose was significantly reduced in Oslo between early and late biopsies. IF/TA was not associated with TAC-C0 or MMF dose in the multivariate analysis. Our data suggest that in TAC- and MMF-based regimens, TAC-C0 levels are associated with subclinical inflammation in patients receiving reduced MMF dose.

Inflammation in Early Kidney Allograft Surveillance Biopsies With and Without Associated Tubulointerstitial Chronic Damage as a Predictor of Fibrosis Progression and Development of De Novo Donor Specific Antibodies.

BACKGROUND: Interstitial fibrosis and tubular atrophy (IFTA) associated with interstitial inflammation in nonscarred areas (IFTA+i) is associated with poorer graft outcome than inflammation without IFTA or IFTA without inflammation. METHODS: We evaluated if histological categories at week 6 could predict the development of interstitial fibrosis and de novo donor specific anti-HLA antibodies (dnDSA) at 1 year. Biopsies were classified according to Banff criteria as normal (i+t≤1 and ci+ct≤1), inflammation (i+t≥2 and ci+ct≤1), IFTA (i+t≤1 and ci+ct≥2) or IFTA+i (i+t≥2 and ci+ct≥2). RESULTS: We analyzed 598 standard immunological risk recipients. The histological diagnosis at 6 weeks was: normal (n = 206), inflammation (n = 29), IFTA (n = 255), and IFTA+i (n = 108). Moderate/severe interstitial fibrosis (ci≥2) at 1 year was observed in 4.2% of patients with prior (6 weeks) normal histology, in 3.4% with inflammation, in 13.8% with IFTA, and in 24.5% with IFTA+i (P = 0.0001). Fifty-three recipients (8.9%) had dnDSA at 1 year. Independent predictors of development of dnDSA at 1 year were: HLA-DR mismatches (odds ratio [OR], 1.95; 95% confidence interval [95% CI], 1.09-3.49), the presence of inflammation (OR, 5.49; 95% CI, 1.67-18.03) or IFTA+i (OR, 4.09; 95% CI, 1.67-10.0) in the 6-week surveillance biopsy. CONCLUSIONS: Early subclinical inflammation in surveillance biopsies with or without tubulointerstitial chronic lesions is associated with an increased risk of dnDSA development.

Total inflammation in early protocol kidney graft biopsies does not predict progression of fibrosis at one year post-transplant.

INTRODUCTION: There is an uncertainty whether total inflammation in early protocol kidney graft biopsies is associated with fibrosis progression. We investigated whether total inflammation, both in fibrotic and non-fibrotic areas, at week 6 would predict fibrosis progression at one yr post-transplant. METHODS: We included 156 single adult ABO compatible kidney recipients with adequate week 6 and one yr transplant protocol biopsies (312 biopsies). Biopsies were scored according to the current Banff criteria. In addition, fibrosis and inflammation in fibrotic and non-fibrotic areas were scored in a 10-grade semi-quantitative eyeballing system from 0% to 100%. RESULTS: Fibrosis increased significantly from week 6 to one yr both by the 10-grade scoring system from 0.69 ± 1.07 to 1.45 ± 1.86, (mean ± SD), p < 0.001 and by Banff interstitial fibrosis (ci) scoring 0.81 ± 0.65 to 1.13 ± 0.87, p < 0.001. The 10-grade scoring system detected a larger proportion of fibrosis progressors than the Banff scoring 40.4% vs. 35.5%, p < 0.001. No significant positive association was found between inflammation at week 6 and progression of fibrosis in either of the scoring systems. CONCLUSIONS: Total inflammation in kidney transplant biopsies at week 6 did not predict progression of fibrosis at one yr post-transplant.

Ichthyosis prematurity syndrome caused by a novel missense mutation in FATP4 gene-a case report from India.

Ichthyosis prematurity syndrome (IPS) is reported mainly from Scandinavia where most of the cases are homozygous or compound heterozygous for the nonsense mutation c.504C>A (p.Cys168*) in exon3 indicating a common ancestor for this mutation. The occurrence of IPS in an Indian patient suggests that it is more widespread than previously reported.

Autologous cell sources in therapeutic vasculogenesis: In vitro and in vivo comparison of endothelial colony-forming cells from peripheral blood and endothelial cells isolated from adipose tissue.

BACKGROUND AIMS: Autologous endothelial cells are promising alternative angiogenic cell sources in trials of therapeutic vasculogenesis, in the treatment of vascular diseases and in the field of tissue engineering. A population of endothelial cells (ECs) with long-term proliferative capability, endothelial colony-forming cells (ECFCs), can be isolated from human peripheral blood. ECFCs are considered an endothelial precursor population. They can be expanded in cell factories in sufficient numbers for clinical applications, but because the number of isolated primary ECs is low, the culture period required may be long. Another EC population that is easily available in the autologous setting and may be expanded in vitro through several population doublings are ECs from adipose tissue (AT-ECs). METHODS: Through extensive comparisons using whole-genome microarray analysis, morphology, phenotype and functional assays, we wanted to evaluate the potential of these EC populations for use in clinical neovascularization. RESULTS: Global gene expression profiling of ECFCs, AT-ECs and the classical EC population, human umbilical vein ECs, showed that the EC populations clustered as unique populations, but very close to each other. By cell surface phenotype and vasculogenic potential in vitro and in vivo, we also found the ECFCs to be extremely similar to AT-ECs. CONCLUSIONS: These properties, together with easy access in the autologous setting, suggest that both AT-ECs and ECFCs may be useful in trials of therapeutic neovascularization. However, AT-ECs may be a more practical alternative for obtaining large quantities of autologous ECs.

Effects of restoring normoglycemia in type 1 diabetes on inflammatory profile and renal extracellular matrix structure after simultaneous pancreas and kidney transplantation.

AIMS: Patients with type 1 diabetes and end-stage renal disease with simultaneous pancreas and kidney (SPK) or kidney transplants alone (KA) were recruited 9-12 years post transplantation. We investigated differences between these groups with regard to inflammatory parameters and long-term structural changes in kidneys. METHODS: Blood samples were analyzed by ELISA and multiplex for chemokines, cytokines, growth factors, cell adhesion molecules and matrix metalloproteinases. Kidney graft biopsies were analyzed by electron microscopy for glomerular basement membrane thickness. Heparan- and chondroitin sulfate disaccharide structures were determined by size exclusion chromatography mass-spectrometry. RESULTS: The SPK and the KA group had average glycated hemoglobin A1c (HbA1c) of 5.8% (40 mmol/mol) and 8.6% (70 mmol/mol) respectively. SPK recipients also had 16.2% lower body mass index (BMI) and 46.4% lower triglyceride levels compared with KA recipients, compatible with an improved metabolic profile in the SPK group. Plasminogen activator inhibitor (PAI-1), C-reactive protein (CRP) and vascular endothelial growth factor (VEGF) were lower in the SPK group. In kidney graft biopsies of the KA-patients an 81.2% increase in average glomerular basement membrane thickness was observed, accompanied by alterations in heparan sulfate proteoglycan structure. In addition to a decrease in 6-O-sulfated disaccharides, an increase in non-N-sulfated disaccharides with a corresponding slight decrease in N-sulfation was found in kidney biopsies from hyperglycemic patients. CONCLUSIONS: Patients with end stage renal disease subjected to KA transplantation showed impaired inflammatory profile, increased thickness of basement membranes and distinct changes in heparan sulfate structures compared with SPK recipients.

Tendon to bone tunnel healing--a study on the time-dependent changes in biomechanics, bone remodeling, and histology in a rat model.

Tendons and ligaments attach to bone through a transitional connective tissue with complex biomechanical properties. This unique tissue is not regenerated during healing, and surgical reattachment therefore often fails. The present study was designed to evaluate tendon healing in a bone tunnel and to evaluate the utilized rat model. Wistar rats (n = 61) were operated with the Achilles tendon through a bone tunnel in the distal tibia. Healing was evaluated at 2, 3, 4, and 12 weeks by biomechanical testing, bone mineral density and histology. After 2 weeks median (interquartile range) pull-out force was 2.2 N (1.9). The pull-out force increased chronologically, by 12 weeks fivefold to 11.2 N (11.4). Energy absorption, stiffness, and bone mineral density increased similarly. The histological analyses showed inflammation at early stages with increasing callus by time. Our data showed a slow healing response the first 4 weeks followed by an accelerated healing period, favoring that most of the gain in mechanical strength occurred later than 4 weeks postoperatively. These findings support the concern of a vulnerable tendon bone tunnel interface in the early stages of healing.

Quantitative proteomics at different depths in human articular cartilage reveals unique patterns of protein distribution.

The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease.

In patients with type 1 diabetes simultaneous pancreas and kidney transplantation preserves long-term kidney graft ultrastructure and function better than transplantation of kidney alone.

AIMS/HYPOTHESIS: In patients with type 1 diabetes and end-stage renal disease (ESRD) we aimed to determine whether long-term normoglycaemia, as achieved by successful simultaneous pancreas and kidney (SPK) transplantation, would preserve kidney graft structure and function better than live donor kidney (LDK) transplantation alone. METHODS: Estimated GFR (eGFR) was calculated in SPK (n = 25) and LDK (n = 17) recipients in a stable phase 3 months after transplantation and annually during follow-up. Kidney graft biopsies were obtained at follow-up for measurement of glomerular volume (light microscopy), glomerular basement membrane (GBM) and podocyte foot process widths and mesangial volume fraction (electron microscopy). RESULTS: SPK and LDK recipients were similar in age and diabetes duration at engraftment. Donor age was higher in the LDK group. Median follow-up time was 10.1 years. Mean HbA1c levels during follow-up were 5.5 ± 0.4% (37 ± 5 mmol/mol) and 8.3 ± 1.5% (68 ± 16 mmol/mol) in the SPK and LDK group, respectively (p < 0.001). Compared with SPK recipients, LDK recipients had wider GBM (369 ± 109 nm vs 281 ± 57 nm; p = 0.008) and increased mesangial volume fraction (median 0.23 [range 0.13-0.59] vs 0.16 [0.10-0.41]; p = 0.007) at follow-up. Absolute eGFR change from baseline was -11 ± 21 and -23 ± 15 ml min(-1) 1.73 m(-2) (p = 0.060), whereas eGFR slope was -1.1 (95% CI -1.7, -0.5) and -2.6 (95% CI -3.1, -2.1) ml min(-1) 1.73 m(-2) per year in the SPK and LDK group, respectively (p = 0.001). CONCLUSIONS/INTERPRETATION: In patients with type 1 diabetes and long-term normoglycaemia after successful SPK transplantation, kidney graft ultrastructure and function were better preserved compared with LDK transplantation alone.