Meta-analyses qualify metzincins and related genes as acute rejection markers in renal transplant patients

Definition of acute renal allograft rejection (AR) markers remains clinically relevant. Features of T-cell-mediated AR are tubulointerstitial and vascular inflammation associated with excessive extracellular matrix (ECM) remodeling, regulated by metzincins, including matrix metalloproteases (MMP). Our study focused on expression of metzincins (METS), and metzincins and related genes (MARGS) in renal allograft biopsies using four independent microarray data sets. Our own cases included normal histology (N, n = 20), borderline changes (BL, n = 4), AR (n = 10) and AR + IF/TA (n = 7). MARGS enriched in all data sets were further examined on mRNA and/or protein level in additional patients. METS and MARGS differentiated AR from BL, AR + IF/TA and N in a principal component analysis. Their expression changes correlated to Banff t- and i-scores. Two AR classifiers, based on METS (including MMP7, TIMP1), or on MARGS were established in our own and validated in the three additional data sets. Thirteen MARGS were significantly enriched in AR patients of all data sets comprising MMP7, -9, TIMP1, -2, thrombospondin2 (THBS2) and fibrillin1. RT-PCR using microdissected glomeruli/tubuli confirmed MMP7, -9 and THBS2 microarray results; immunohistochemistry showed augmentation of MMP2, -9 and TIMP1 in AR. TIMP1 and THBS2 were enriched in AR patient serum. Therefore, differentially expressed METS and MARGS especially TIMP1, MMP7/-9 represent potential molecular AR markers.

Cardiovascular changes after PMMA vertebroplasty in sheep: the effect of bone marrow removal using pulsed jet-lavage

Clinically, the displacement of intravertebral fat into the circulation during vertebroplasty is reported to lead to problems in elderly patients and can represent a serious complication, especially when multiple levels have to be treated. An in vitro study has shown the feasibility of removing intravertebral fat by pulsed jet-lavage prior to vertebroplasty, potentially reducing the embolization of bone marrow fat from the vertebral bodies and alleviating the cardiovascular changes elicited by pulmonary fat embolism. In this in vivo study, percutaneous vertebroplasty using polymethylmethacrylate (PMMA) was performed in three lumbar vertebrae of 11 sheep. In six sheep (lavage group), pulsed jet-lavage was performed prior to injection of PMMA compared to the control group of five sheep receiving only PMMA vertebroplasty. Invasive recording of blood pressures was performed continuously until 60 min after the last injection. Cardiac output and arterial blood gas parameters were measured at selected time points. Post mortem, the injected cement volume was measured using CT and lung biopsies were processed for assessment of intravascular fat. Pulsed jet-lavage was feasible in the in vivo setting. In the control group, the injection of PMMA resulted in pulmonary fat embolism and a sudden and significant increase in mean pulmonary arterial pressure. Pulsed jet-lavage prevented any cardiovascular changes and significantly reduced the severity of bone marrow fat embolization. Even though significantly more cement had been injected into the lavaged vertebral bodies, significantly fewer intravascular fat emboli were identified in the lung tissue. Pulsed jet-lavage prevented the cardiovascular complications after PMMA vertebroplasty in sheep and alleviated the severity of pulmonary fat embolism.

INI1 (BAF 47) immunohistochemistry is an essential diagnostic tool for children with hepatic tumors and low alpha fetoprotein

Malignant rhabdoid tumor (MRT) of the liver is a rare malignancy with grave prognosis. This entity should be considered in the differential diagnosis of any aggressive liver tumor with low levels of alpha fetoprotein. We report 2 cases of hepatic MRT presenting in infancy. In these 2 cases, we show that loss of INI1 facilitates making the correct diagnosis of primary hepatic MRT utilizing BAF 47 (INI1 gene product) immunostains. Difficulty encountered in making this rare diagnosis, including the need for repeated biopsies, can be avoided if MRT is considered in the differential diagnosis early on and BAF 47 immunohistochemistry is ordered.

Borrelia in granuloma annulare, morphea and lichen sclerosus: a PCR-based study and review of the literature

Morphea, granuloma annulare (GA) and lichen sclerosus et atrophicans (LSA) have also been suggested to be linked to Borrelia infection. Previous studies based on serologic data or detection of Borrelia by immunohistochemistry and polymerase chain reaction (PCR) reported contradictory results. Thus, we examined skin biopsies of morphea, GA and LSA by PCR to assess the prevalence of Borrelia DNA in an endemic area and to compare our results with data in the literature.

Peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) promotes skeletal muscle lipid refueling in vivo by activating de novo lipogenesis and the pentose phosphate pathway

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through peroxisome proliferator-activated receptor coactivator 1 (PGC-1 ). PGC-1 enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains unresolved, however. Here, we investigated a possible role of elevated PGC-1 levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1 -mediated de novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1 and human muscle biopsies pre- and post-exercise. We demonstrate that PGC-1 enhances lipogenesis in skeletal muscle through liver X receptor -dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1 and the liver X receptor-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1 coordinates lipogenesis, intramyocellular lipid accumulation, and substrate oxidation in exercised skeletal muscle in vivo.

Statin therapy and the expression of genes that regulate calcium homeostasis and membrane repair in skeletal muscle

In skeletal muscle of patients with clinically diagnosed statin-associated myopathy, discrete signs of structural damage predominantly localize to the T-tubular region and are suggestive of a calcium leak. The impact of statins on skeletal muscle of non-myopathic patients is not known. We analyzed the expression of selected genes implicated in the molecular regulation of calcium and membrane repair, in lipid homeostasis, myocyte remodeling and mitochondrial function. Microscopic and gene expression analyses were performed using validated TaqMan custom arrays on skeletal muscle biopsies of 72 age-matched subjects who were receiving statin therapy (n = 38), who had discontinued therapy due to statin-associated myopathy (n = 14), and who had never undergone statin treatment (n = 20). In skeletal muscle, obtained from statin-treated, non-myopathic patients, statins caused extensive changes in the expression of genes of the calcium regulatory and the membrane repair machinery, whereas the expression of genes responsible for mitochondrial function or myocyte remodeling was unaffected. Discontinuation of treatment due to myopathic symptoms led to a normalization of gene expression levels, the genes encoding the ryanodine receptor 3, calpain 3, and dystrophin being the most notable exceptions. Hence, even in clinically asymptomatic (non-myopathic) patients, statin therapy leads to an upregulation in the expression of genes that are concerned with skeletal muscle regulation and membrane repair.

An in vitro triple cell co-culture model with primary cells mimicking the human alveolar epithelial barrier

A triple cell co-culture model was recently established by the authors, consisting of either A549 or 16HBE14o- epithelial cells, human blood monocyte-derived macrophages and dendritic cells, which offers the possibility to study the interaction of xenobiotics with those cells. The 16HBE14o- containing co-culture model mimics the airway epithelial barrier, whereas the A549 co-cultures mimic the alveolar type II-like epithelial barrier. The goal of the present work was to establish a new triple cell co-culture model composed of primary alveolar type I-like cells isolated from human lung biopsies (hAEpC) representing a more realistic alveolar epithelial barrier wall, since type I epithelial cells cover >93% of the alveolar surface. Monocultures of A549 and 16HBE14o- were morphologically and functionally compared with the hAEpC using laser scanning microscopy, as well as transmission electron microscopy, and by determining the epithelial integrity. The triple cell co-cultures were characterized using the same methods. It could be shown that the epithelial integrity of hAEpC (mean ± SD, 1180 ± 188 Ω cm(2)) was higher than in A549 (172 ± 59 Ω cm(2)) but similar to 16HBE14o- cells (1469 ± 156 Ω cm(2)). The triple cell co-culture model with hAEpC (1113 ± 30 Ω cm(2)) showed the highest integrity compared to the ones with A549 (93 ± 14 Ω cm(2)) and 16HBE14o- (558 ± 267 Ω cm(2)). The tight junction protein zonula occludens-1 in hAEpC and 16HBE14o- were more regularly expressed but not in A549. The epithelial alveolar model with hAEpC combined with two immune cells (i.e. macrophages and dendritic cells) will offer a novel and more realistic cell co-culture system to study possible cell interactions of inhaled xenobiotics and their toxic potential on the human alveolar type I epithelial wall.

Evaluation of the frequency of putative prostate cancer stem cells in primary and metastatic prostate cancer

Tumour cells with a stem cell-like phenotype have recently been identified in prostate tumors and it has been suggested that this population may be responsible for the diversity of cell types within tumors and also for the initiation of metastases. These cells carry a number of defined markers: they are cd133 and cd44+ve and express high levels of alpha2beta1 integrin. In this study we have, for the first time, assessed matched primary and bone marrow biopsies from prostate cancer patients for the distribution of cells carrying these and a number of other putative stem cell markers.

Uromodulin is expressed in renal primary cilia and UMOD mutations result in decreased ciliary uromodulin expression

Uromodulin (UMOD) mutations are responsible for three autosomal dominant tubulo-interstitial nephropathies including medullary cystic kidney disease type 2 (MCKD2), familial juvenile hyperuricemic nephropathy and glomerulocystic kidney disease. Symptoms include renal salt wasting, hyperuricemia, gout, hypertension and end-stage renal disease. MCKD is part of the 'nephronophthisis-MCKD complex', a group of cystic kidney diseases. Both disorders have an indistinguishable histology and renal cysts are observed in either. For most genes mutated in cystic kidney disease, their proteins are expressed in the primary cilia/basal body complex. We identified seven novel UMOD mutations and were interested if UMOD protein was expressed in the primary renal cilia of human renal biopsies and if mutant UMOD would show a different expression pattern compared with that seen in control individuals. We demonstrate that UMOD is expressed in the primary cilia of renal tubules, using immunofluorescent studies in human kidney biopsy samples. The number of UMOD-positive primary cilia in UMOD patients is significantly decreased when compared with control samples. Additional immunofluorescence studies confirm ciliary expression of UMOD in cell culture. Ciliary expression of UMOD is also confirmed by electron microscopy. UMOD localization at the mitotic spindle poles and colocalization with other ciliary proteins such as nephrocystin-1 and kinesin family member 3A is demonstrated. Our data add UMOD to the group of proteins expressed in primary cilia, where mutations of the gene lead to cystic kidney disease.

Polyunsaturated fatty acid-enriched diets used for the treatment of canine chronic enteropathies decrease the abundance of selected genes of cholesterol homeostasis

Lipids are important for cell function and survival, but abnormal concentrations may lead to various diseases. Cholesterol homeostasis is greatly dependent on the active transport by membrane proteins, whose activities coordinate lipid status with cellular function. Intestinal Niemann-Pick C1-Like 1 protein (NPC1L1) and scavenger receptor B1 (SR-B1) participate in the uptake of extracellular cholesterol, whereas ATP binding cassette A1 (ABCA1) mediates the efflux of excessive intracellular cholesterol. Caveolin-1 binds cholesterol and fatty acids (FA) and participates in cholesterol trafficking. Sterol response element binding protein-2 (SREBP-2) is a sensor that regulates intracellular cholesterol synthesis. Given that cholesterol is a constituent of chylomicrons, whose synthesis is enhanced with an increased FA supply, we tested the hypothesis that feeding polyunsaturated FA (PUFA)-enriched diets in treatment of canine chronic enteropathies alters the mRNA expression of genes involved in cholesterol homeostasis. Using quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we compared the mRNA abundance of NPC1L1, SR-B1, ABCA1, caveolin-1, and SREBP-2 in duodenal mucosal biopsies of dogs with food-responsive diarrhea (FRD; n=14) and inflammatory bowel disease (IBD; n=7) before and after treatment with cholesterol-free PUFA-enriched diets and in healthy controls (n=14). The abundance of caveolin-1, ABCA1, and SREBP-2 were altered by PUFA-enriched diets (P<0.05), whereas that of NPC1L1 and SR-B1 mRNA remained unchanged. The gene expression of caveolin-1, ABCA1, and SREBP-2 was down-regulated (P<0.05) by PUFA-enriched diets in IBD dogs only. Our results suggest that feeding PUFA-enriched diets may alter cholesterol homeostasis in duodenal mucosal cells of dogs suffering from IBD.

Intestinal current measurement for diagnostic classification of patients with questionable cystic fibrosis: validation and reference data

In questionable cystic fibrosis (CF), mild or monosymptomatic phenotypes frequently cause diagnostic difficulties despite detailed algorithms. CF transmembrane conductance regulator (CFTR)-mediated ion transport can be studied ex vivo in rectal biopsies by intestinal current measurement (ICM).

Multimodal target correction by local bone registration: a PET/CT evaluation

PET/CT guidance for percutaneous interventions allows biopsy of suspicious metabolically active bone lesions even when no morphological correlation is delineable in the CT images. Clinical use of PET/CT guidance with conventional step-by-step technique is time consuming and complicated especially in cases in which the target lesion is not shown in the CT image. Our recently developed multimodal instrument guidance system (IGS) for PET/CT improved this situation. Nevertheless, bone biopsies even with IGS have a trade-off between precision and intervention duration which is proportional to patient and personnel exposure to radiation. As image acquisition and reconstruction of PET may take up to 10 minutes, preferably only one time consuming combined PET/CT acquisition should be needed during an intervention. In case of required additional control images in order to check for possible patient movements/deformations, or to verify the final needle position in the target, only fast CT acquisitions should be performed. However, for precise instrument guidance accounting for patient movement and/or deformation without having a control PET image, it is essential to be able to transfer the position of the target as identified in the original PET/CT to a changed situation as shown in the control CT.

Can molecular markers stratify the diagnostic value of high-grade prostatic intraepithelial neoplasia?

The diagnostic performance of isolated high-grade prostatic intraepithelial neoplasia in prostatic biopsies has recently been questioned, and molecular analysis of high-grade prostatic intraepithelial neoplasia has been proposed for improved prediction of prostate cancer. Here, we retrospectively studied the value of isolated high-grade prostatic intraepithelial neoplasia and the immunohistochemical markers ?-methylacyl coenzyme A racemase, Bcl-2, annexin II, and Ki-67 for better risk stratification of high-grade prostatic intraepithelial neoplasia in our local Swiss population. From an initial 165 diagnoses of isolated high-grade prostatic intraepithelial neoplasia, we refuted 61 (37%) after consensus expert review. We used 30 reviewed high-grade prostatic intraepithelial neoplasia cases with simultaneous biopsy prostate cancer as positive controls. Rebiopsies were performed in 66 patients with isolated high-grade prostatic intraepithelial neoplasia, and the median time interval between initial and repeat biopsy was 3 months. Twenty (30%) of the rebiopsies were positive for prostate cancer, and 10 (15%) showed persistent isolated high-grade prostatic intraepithelial neoplasia. Another 2 (3%) of the 66 patients were diagnosed with prostate cancer in a second rebiopsy. Mean prostate-specific antigen serum levels did not significantly differ between the 22 patients with prostate cancer and the 44 without prostate cancer in rebiopsies, and the 30 positive control patients, respectively (median values, 8.1, 7.7, and 8.8 ng/mL). None of the immunohistochemical markers, including ?-methylacyl coenzyme A racemase, Bcl-2, annexin II, and Ki-67, revealed a statistically significant association with the risk of prostate cancer in repeat biopsies. Taken together, the 33% risk of being diagnosed with prostate cancer after a diagnosis of high-grade prostatic intraepithelial neoplasia justifies rebiopsy, at least in our not systematically prostate-specific antigen-screened population. There is not enough evidence that immunohistochemical markers can reproducibly stratify the risk of prostate cancer after a diagnosis of isolated high-grade prostatic intraepithelial neoplasia.

Exercise-induced angiogenesis correlates with the up-regulated expression of neuronal nitric oxide synthase (nNOS) in human skeletal muscle

The contribution of neuronal nitric oxide synthase (nNOS) to angiogenesis in human skeletal muscle after endurance exercise is controversially discussed. We therefore ascertained whether the expression of nNOS is associated with the capillary density in biopsies of the vastus lateralis (VL) muscle that had been derived from 10 sedentary male subjects before and after moderate training (four 30-min weekly jogging sessions for 6 months, with a heart-rate corresponding to 75% VO(2)max). In these biopsies, nNOS was predominantly expressed as alpha-isoform with exon-mu and to a lesser extent without exon-mu, as determined by RT-PCR. The mRNA levels of nNOS were quantified by real-time PCR and related to the capillary-to-fibre ratio and the numerical density of capillaries specified by light microscopy. If the VL biopsies of all subjects were co-analysed, mRNA levels of nNOS were non-significantly elevated after training (+34%; P > 0.05). However, only five of the ten subjects exhibited significant (P ≤ 0.05) elevations in the capillary-to-fibre ratio (+25%) and the numerical density of capillaries (+21%) and were thus undergoing angiogenesis. If the VL biopsies of these five subjects alone were evaluated, the mRNA levels of nNOS were significantly up-regulated (+128%; P ≤ 0.05) and correlated positively (r = 0.8; P ≤ 0.01) to angiogenesis. Accordingly, nNOS protein expression in VL biopsies quantified by immunoblotting was significantly increased (+82%; P ≤ 0.05) only in those subjects that underwent angiogenesis. In conclusion, the expression of nNOS at mRNA and protein levels was statistically linked to capillarity after exercise suggesting that nNOS is involved in the angiogenic response to training in human skeletal muscle.

Different molecular and structural adaptations with eccentric and conventional strength training in elderly men and women

Reprogramming of gene expression contributes to structural and functional adaptation of muscle tissue in response to altered use. The aim of this study was to investigate mechanisms for observed improvements in leg extension strength, gain in relative thigh muscle mass and loss of body and thigh fat content in response to eccentric and conventional strength training in elderly men (n = 14) and women (n = 14; average age of the men and women: 80.1 ± 3.7 years) by means of structural and molecular analyses. Biopsies were collected from m. vastus lateralis in the resting state before and after 12 weeks of training with two weekly resistance exercise sessions (RET) or eccentric ergometer sessions (EET). Gene expression was analyzed using custom-designed low-density PCR arrays. Muscle ultrastructure was evaluated using EM morphometry. Gain in thigh muscle mass was paralleled by an increase in muscle fiber cross-sectional area (hypertrophy) with RET but not with EET, where muscle growth is likely occurring by the addition of sarcomeres in series or by hyperplasia. The expression of transcripts encoding factors involved in muscle growth, repair and remodeling (e.g., IGF-1, HGF, MYOG, MYH3) was increased to a larger extent after EET than RET. MicroRNA 1 expression was decreased independent of the training modality, and was paralleled by an increased expression of IGF-1 representing a potential target. IGF-1 is a potent promoter of muscle growth, and its regulation by microRNA 1 may have contributed to the gain of muscle mass observed in our subjects. EET depressed genes encoding mitochondrial and metabolic transcripts. The changes of several metabolic and mitochondrial transcripts correlated significantly with changes in mitochondrial volume density. Intramyocellular lipid content was decreased after EET concomitantly with total body fat. Changes in intramyocellular lipid content correlated with changes in body fat content with both RET and EET. In the elderly, RET and EET lead to distinct molecular and structural adaptations which might contribute to the observed small quantitative differences in functional tests and body composition parameters. EET seems to be particularly convenient for the elderly with regard to improvements in body composition and strength but at the expense of reducing muscular oxidative capacity.