Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.

Factors associated with the emergence of K65R in patients with HIV-1 infection treated with combination antiretroviral therapy containing tenofovir

BACKGROUND: The human immunodeficiency virus type 1 reverse-transcriptase mutation K65R is a single-point mutation that has become more frequent after increased use of tenofovir disoproxil fumarate (TDF). We aimed to identify predictors for the emergence of K65R, using clinical data and genotypic resistance tests from the Swiss HIV Cohort Study. METHODS: A total of 222 patients with genotypic resistance tests performed while receiving treatment with TDF-containing regimens were stratified by detectability of K65R (K65R group, 42 patients; undetected K65R group, 180 patients). Patient characteristics at start of that treatment were analyzed. RESULTS: In an adjusted logistic regression, TDF treatment with nonnucleoside reverse-transcriptase inhibitors and/or didanosine was associated with the emergence of K65R, whereas the presence of any of the thymidine analogue mutations D67N, K70R, T215F, or K219E/Q was protective. The previously undescribed mutational pattern K65R/G190S/Y181C was observed in 6 of 21 patients treated with efavirenz and TDF. Salvage therapy after TDF treatment was started for 36 patients with K65R and for 118 patients from the wild-type group. Proportions of patients attaining human immunodeficiency virus type 1 loads <50 copies/mL after 24 weeks of continuous treatment were similar for the K65R group (44.1%; 95% confidence interval, 27.2%-62.1%) and the wild-type group (51.9%; 95% confidence interval, 42.0%-61.6%). CONCLUSIONS: In settings where thymidine analogue mutations are less likely to be present, such as at start of first-line therapy or after extended treatment interruptions, combinations of TDF with other K65R-inducing components or with efavirenz or nevirapine may carry an enhanced risk of the emergence of K65R. The finding of a distinct mutational pattern selected by treatment with TDF and efavirenz suggests a potential fitness interaction between K65R and nonnucleoside reverse-transcriptase inhibitor-induced mutations.

PTH and 1.25 vitamin D response to a low-calcium diet is associated with bone mineral density in renal stone formers

BACKGROUND: Renal calcium stones and hypercalciuria are associated with a reduced bone mineral density (BMD). Therefore, the effect of changes in calcium homeostasis is of interest for both stones and bones. We hypothesized that the response of calciuria, parathyroid hormone (PTH) and 1.25 vitamin D to changes in dietary calcium might be related to BMD. METHODS: A single-centre prospective interventional study of 94 hyper- and non-hypercalciuric calcium stone formers consecutively retrieved from our stone clinic. The patients were investigated on a free-choice diet, a low-calcium diet, while fasting and after an oral calcium load. Patient groups were defined according to lumbar BMD (z-score) obtained by dual X-ray absorptiometry (group 1: z-score <-0.5, n = 30; group 2: z-score -0.5-0.5, n = 36; group 3: z-score >0.5, n = 28). The effect of the dietary interventions on calciuria, 1.25 vitamin D and PTH in relation to BMD was measured. RESULTS: An inverse relationship between BMD and calciuria was observed on all four calcium intakes (P = 0.009). On a free-choice diet, 1.25 vitamin D and PTH levels were identical in the three patient groups. However, the relative responses of 1.25 vitamin D and PTH to the low-calcium diet were opposite in the three groups with the highest increase of 1.25 vitamin D in group 1 and the lowest in group 3, whereas PTH increase was most pronounced in group 3 and least in group 1. CONCLUSION: Calcium stone formers with a low lumbar BMD exhibit a blunted response of PTH release and an apparently overshooting production of 1.25 vitamin D following a low-calcium diet.

Elevated level of endothelin-1 in cerebrospinal fluid and lack of nitric oxide in basilar arterial plasma associated with cerebral vasospasm after subarachnoid haemorrhage in rabbits

BACKGROUND: The role of endothelin-1 (ET-1) and nitric oxide (NO) as two important mediators in the development of cerebral vasospasm (CVS) after subarachnoid haemorrhage (SAH) is controversial. The objective of this study was to determine whether local levels of ET-1 and NO in cerebral arterial plasma and/or in cerebrospinal fluid (CSF) are associated with the occurrence of CVS after SAH. METHODS: CVS was induced using the one-haemorrhage rabbit model and confirmed by digital subtraction angiography of the rabbits' basilar artery on day 5. Prior to sacrifice, local CSF and basilar arterial plasma samples were obtained by a transclival approach to the basilar artery. Systemic arterial plasma samples were obtained. ET-1 levels were determined by immunometric technique (pg/ml +/- SEM) and total nitrate/nitrite level spectrophotometrically (micromol/l +/- SEM). FINDINGS: Angiographic CVS was documented after SAH induction (n = 12, P < 0.05). The ET-1 level in CSF was significantly elevated by 27.3% to 0.84 +/- 0.08 pg/ml in SAH animals (n = 7) in comparison to controls (0.66 +/- 0.04 pg/ml, n = 7, P < 0.05). There was no significant difference in ET-1 levels in systemic and basilar arterial plasma samples of SAH animals compared to controls. A significant lack of local NO metabolites was documented in basilar arterial plasma after SAH (36.8 +/- 3.1 micromol/l, n = 6) compared to controls (61.8 +/- 6.2 micromol/l, n = 6, P < 0.01). CONCLUSION: This study demonstrates that an elevated ET-1 level in CSF and local lack of NO in the basilar arterial plasma samples are associated with CVS after experimental SAH.

LQTS-associated mutation A257G in α1-syntrophin interacts with the intragenic variant P74L to modify its biophysical phenotype.

The SNTA1-encoded α1-syntrophin (SNTA1) missense mutation, p.A257G, causes long QT syndrome (LQTS) by pathogenic accentuation of Nav1.5's sodium current (I Na). Subsequently, we found p.A257G in combination with the SNTA1 polymorphism, p.P74L in 4 victims of sudden infant death syndrome (SIDS) as well as in 3 adult controls. We hypothesized that p.P74L-SNTA1 could functionally modify the pathogenic phenotype of p.A257G-SNTA1, thus explaining its occurrence in non-LQTS populations. The SNTA1 variants p.P74L, p.A257G, and the combination variant p.P74L/p.A257G were engineered using PCR-based overlap-extension and were co-expressed heterologously with SCN5A in HEK293 cells. I Na was recorded using the whole-cell method. Compared to wild-type (WT), the significant increase in peak I Na and window current found with p.A257G was reversed by the intragenic variant p.P74L (p.P74L/p.A257G). These results report for the first time the intragenic rescue of an LQT-associated SNTA1 mutation when found in combination with the SNTA1 polymorphism p.P74L, suggesting an ever-increasing picture of complexity in terms of genetic risk stratification for arrhythmia.

Higher CNS Penetration-Effectiveness of Long-term Combination Antiretroviral Therapy Is Associated With Better HIV-1 Viral Suppression in Cerebrospinal Fluid

Objectives: To determine HIV-1 RNA in cerebrospinal fluid (CSF) of successfully treated patients and to evaluate if combination antiretroviral treatments with higher central nervous system penetration-effectiveness (CPE) achieve better CSF viral suppression. Methods: Viral loads (VLs) and drug concentrations of lopinavir, atazanavir, and efavirenz were measured in plasma and CSF. The CPE was calculated using 2 different methods. Results: The authors analyzed 87 CSF samples of 60 patients. In 4 CSF samples, HIV-1 RNA was detectable with 43–82 copies per milliliter. Median CPE in patients with detectable CSF VL was significantly lower compared with individuals with undetectable VL: CPE of 1.0 (range, 1.0–1.5) versus 2.3 (range, 1.0–3.5) using the method of 2008 (P = 0.011) and CPE of 6 (range, 6–8) versus 8 (range, 5–12) using the method of 2010 (P = 0.022). The extrapolated CSF trough levels for atazanavir (n = 12) were clearly above the 50% inhibitory concentration (IC50) in only 25% of samples; both patients on atazanavir/ritonavir with detectable CSF HIV-1 RNA had trough levels in the range of the presumed IC50. The extrapolated CSF trough level for lopinavir (n = 42) and efavirenz (n = 18) were above the IC50 in 98% and 78%, respectively, of samples, including the patients with detectable CSF HIV-1 RNA. Conclusions: This study suggests that treatment regimens with high intracerebral efficacy reflected by a high CPE score are essential to achieve CSF HIV-1 RNA suppression. The CPE score including all drug components was a better predictor for treatment failure in the CSF than the sole concentrations of protease inhibitor or nonnucleoside reverse transcriptase inhibitor in plasma or CSF.

Loss of Raf-1 kinase inhibitor protein (RKIP) is strongly associated with high-grade tumor budding and correlates with an aggressive phenotype in pancreatic ductal adenocarcinoma (PDAC)

BACKGROUND Raf-1 kinase inhibitor protein (RKIP) has emerged as a significant metastatic suppressor in a variety of human cancers and is known to inhibit Ras/Raf/MEK/ERK signaling. By suppressing the activation of the NFkB/SNAIL circuit, RKIP can regulate the induction of epithelial-mesenchymal transition (EMT). The aim of this study was to evaluate RKIP expression and to determine its association with clinicopathological features, including EMT in form of tumor budding in pancreatic ductal adenocarcinoma (PDAC). METHODS Staining for RKIP was performed on a multipunch Tissue Microarray (TMA) of 114 well-characterized PDACs with clinico-pathological, follow-up and adjuvant therapy information. RKIP-expression was assessed separately in the main tumor body and in the tumor buds. Another 3 TMAs containing normal pancreatic tissue, precursor lesions (Pancreatic Intraepithelial Neoplasia, PanINs) and matched lymph node metastases were stained in parallel. Cut-off values were calculated by receiver operating characteristic (ROC) curve analysis. RESULTS We found a significant progressive loss of RKIP expression between normal pancreatic ductal epithelia (average: 74%), precursor lesions (PanINs; average: 37%), PDAC (average 20%) and lymph node metastases (average 8%, p<0.0001). RKIP expression was significantly lower in tumor buds (average: 6%) compared to the main tumor body (average 20%; p<0.005). RKIP loss in the tumor body was marginally associated with advanced T-stage (p=0.0599) as well as high-grade peritumoral (p=0.0048) and intratumoral budding (p=0.0373). RKIP loss in the buds showed a clear association with advanced T stage (p=0.0089). CONCLUSIONS The progressive loss of RKIP seems to play a major role in the neoplastic transformation of pancreas, correlates with aggressive features in PDAC and is associated with the presence of EMT in form of tumor budding.

Congenital Hepatic Fibrosis in the Franches-Montagnes Horse Is Associated with the Polycystic Kidney and Hepatic Disease 1 (PKHD1) Gene

Congenital hepatic fibrosis has been described as a lethal disease with monogenic autosomal recessive inheritance in the Swiss Franches-Montagnes horse breed. We performed a genome-wide association study with 5 cases and 12 controls and detected an association on chromosome 20. Subsequent homozygosity mapping defined a critical interval of 952 kb harboring 10 annotated genes and loci including the polycystic kidney and hepatic disease 1 (autosomal recessive) gene (PKHD1). PKHD1 represents an excellent functional candidate as variants in this gene were identified in human patients with autosomal recessive polycystic kidney and hepatic disease (ARPKD) as well as several mouse and rat mutants. Whereas most pathogenic PKHD1 variants lead to polycystic defects in kidney and liver, a small subset of the human ARPKD patients have only liver symptoms, similar to our horses with congenital hepatic fibrosis. The PKHD1 gene is one of the largest genes in the genome with multiple alternative transcripts that have not yet been fully characterized. We sequenced the genomes of an affected foal and 46 control horses to establish a comprehensive list of variants in the critical interval. We identified two missense variants in the PKHD1 gene which were strongly, but not perfectly associated with congenital hepatic fibrosis. We speculate that reduced penetrance and/or potential epistatic interactions with hypothetical modifier genes may explain the imperfect association of the detected PKHD1 variants. Our data thus indicate that horses with congenital hepatic fibrosis represent an interesting large animal model for the liver-restricted subtype of human ARPKD.

Induction of the autophagy-associated gene MAP1S via PU.1 supports APL differentiation

The PU.1 transcription factor is essential for myeloid development. We investigated if the microtubule-associated protein 1S (MAP1S) is a novel PU.1 target with a link to autophagy, a cellular recycling pathway. Comparable to PU.1, MAP1S expression was significantly repressed in primary AML blasts as compared to mature neutrophils. Accordingly, MAP1S expression was induced during neutrophil differentiation of CD34(+) progenitor and APL cells. Moreover, PU.1 bound to the MAP1S promoter and induced MAP1S expression during APL differentiation. Inhibiting MAP1S resulted in aberrant neutrophil differentiation and autophagy. Taken together, our findings implicate the PU.1-regulated MAP1S gene in neutrophil differentiation and autophagy control.

Exercise-associated glucose metabolism in individuals with type 1 diabetes mellitus.

PURPOSE OF REVIEW The primary focus of this review is threefold: first, to summarize available knowledge on exercise-associated glucose metabolism in individuals with type 1 diabetes mellitus (T1DM); second, to elucidate physiological mechanisms predisposing to glycemic variations in patients in T1DM; and third, to describe novel approaches derived from physiological perceptions applicable to stabilize exercise-related glycemia in individuals with T1DM. RECENT FINDINGS Recent studies corroborate the concept that despite partial differences in counter-regulatory mechanisms individuals with T1DM do not fundamentally differ in their glucose response to exercise when compared with healthy individuals if studies are performed under standardized conditions with insulin and glucose levels held close to physiological ranges. Novel approaches derived from a better understanding of exercise-associated glucose metabolism (e.g., the concept of intermittent high-intensity exercise) may provide alternative ways to master the challenges imposed by exercise to individuals with T1DM. SUMMARY Exercise still imposes high demands on patients with T1DM and increases risks for hypoglycemia and hyperglycemia. Deeper insight into the associated metabolic pathways has revealed novel options to stabilize exercise-associated glucose levels in these patients.

Natural Variation in Maize Aphid Resistance Is Associated with 2,4-Dihydroxy-7-Methoxy-1,4-Benzoxazin-3-One Glucoside Methyltransferase Activity

Plants differ greatly in their susceptibility to insect herbivory, suggesting both local adaptation and resistance tradeoffs. We used maize (Zea mays) recombinant inbred lines to map a quantitative trait locus (QTL) for the maize leaf aphid (Rhopalosiphum maidis) susceptibility to maize Chromosome 1. Phytochemical analysis revealed that the same locus was also associated with high levels of 2-hydroxy-4,7-dimethoxy-1,4-benzoxazin-3-one glucoside (HDMBOA-Glc) and low levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside (DIMBOA-Glc). In vitro enzyme assays with candidate genes from the region of the QTL identified three O-methyltransferases (Bx10a-c) that convert DIMBOA-Glc to HDMBOA-Glc. Variation in HDMBOA-Glc production was attributed to a natural CACTA family transposon insertion that inactivates Bx10c in maize lines with low HDMBOA-Glc accumulation. When tested with a population of 26 diverse maize inbred lines, R. maidis produced more progeny on those with high HDMBOA-Glc and low DIMBOA-Glc. Although HDMBOA-Glc was more toxic to R. maidis than DIMBOA-Glc in vitro, BX10c activity and the resulting decline of DIMBOA-Glc upon methylation to HDMBOA-Glc were associated with reduced callose deposition as an aphid defense response in vivo. Thus, a natural transposon insertion appears to mediate an ecologically relevant trade-off between the direct toxicity and defense-inducing properties of maize benzoxazinoids.

Heterogeneity analysis of Metastasis Associated in Colon Cancer 1 (MACC1) for survival prognosis of colorectal cancer patients: a retrospective cohort study.

BACKGROUND Metastasis of colorectal cancer (CRC) is directly linked to patient survival. We previously identified the novel gene Metastasis Associated in Colon Cancer 1 (MACC1) in CRC and demonstrated its importance as metastasis inducer and prognostic biomarker. Here, we investigate the geographic expression pattern of MACC1 in colorectal adenocarcinoma and tumor buds in correlation with clinicopathological and molecular features for improvement of survival prognosis. METHODS We performed geographic MACC1 expression analysis in tumor center, invasive front and tumor buds on whole tissue sections of 187 well-characterized CRCs by immunohistochemistry. MACC1 expression in each geographic zone was analyzed with Mismatch repair (MMR)-status, BRAF/KRAS-mutations and CpG-island methylation. RESULTS MACC1 was significantly overexpressed in tumor tissue as compared to normal mucosa (p < 0.001). Within colorectal adenocarcinomas, a significant increase of MACC1 from tumor center to front (p = 0.0012) was detected. MACC1 was highly overexpressed in 55% tumor budding cells. Independent of geographic location, MACC1 predicted advanced pT and pN-stages, high grade tumor budding, venous and lymphatic invasion (p < 0.05). High MACC1 expression at the invasive front was decisive for prediction of metastasis (p = 0.0223) and poor survival (p = 0.0217). The geographic pattern of MACC1 did not correlate with MMR-status, BRAF/KRAS-mutations or CpG-island methylation. CONCLUSION MACC1 is differentially expressed in CRC. At the invasive front, MACC1 expression predicts best aggressive clinicopathological features, tumor budding, metastasis formation and poor survival outcome.

Ectodysplasin-1 deficiency in a German Holstein bull associated with loss of respiratory mucous glands and chronic rhinotracheitis

A 2-year-old German Holstein bull was identified as a carrier of a mutation within the X-chromosomal ED1 gene, which encodes a TNF-related signalling molecule mainly involved in ectodermal development. The clinicopathological appearance was associated with hypotrichosis, hypodontia, and a reduced number of eccrine glands, in addition to chronic rhinotracheitis and partial squamous metaplasia. Furthermore, for the first time in an ED1-deficient animal, a complete lack of respiratory mucous glands was observed. This suggests that the ED1 gene plays a role in the development of mucous glands, the absence of which resembles a feature of X-linked anhidrotic ectodermal dysplasia (ED1) in human patients.