Semi-quantitative Real-time PCR Research Articles

Abstract 676: Tissue inhibitor of metalloproteinase-2 (TIMP2) deficiency enhances tumor burden via increasing HIF-2á expression

Abstract The tissue inhibitor of metalloproteinase family of proteins (TIMPs 1-4) function as natural MMP inhibitors, and have been shown to play a role in maintenance and remodeling of the ECM as well as other cellular processes including proliferation, apoptosis and angiogenesis. A number of studies have shown that the down-regulation or silencing of TIMP2 accelerates tumor development, however, the mechanism is not well understood. High HIF-2á levels in non-small cell lung cancer (NSCLC) correlate with decreased overall survival, while inhibition of HIFs targeted genes VEGF or VEGFR2 are associated with improved clinical outcome. Similarly, TIMP2 mRNA levels were found to be low in NSCLC compared to the corresponding non-neoplastic surrounding lung (p<0.05). The current study was undertaken to examine the molecular mechanisms associated with tumor hypoxia and TIMP2 expression in TIMP2 deficient mice (T2D-/-) and wild type (WT) littermates. Mice were given (1×106)/50ìl Lewis lung carcinoma cells transfected with luciferase (LL/2-Luc-M38, Caliper) via intratracheal installation. Tumor progression was monitored by IVIS imaging 2 weeks after administration and continued until week 4 when mice were sacrificed and lungs harvested for further analysis. The IVIS analysis shows higher tumor burden in T2D-/- compared to WT littermates, suggesting that TIMP2 deficiency augments tumor development in these mice (p<0.05). H&E analysis of lung tissue sections revealed a significant increase in the number of tumor nodules in lungs of T2D mice compared to controls (p = 0.003). In an effort to investigate the relationship between TIMP2 and HIF-2á expression; we determined the expression levels of HIF-2á in healthy non-tumor bearing T2D mice compared to WT littermates. Using semi-quantitative real-time PCR, we confirmed a strong basal up regulation of HIF-2á (but not HIF-1á) mRNA in T2D mice lungs compared to WT (p<0.002). In tumor bearing mice, this increase was even higher in the lungs of T2D mice compared to WT (p<0.007). Since VEGF is the direct downstream target of HIF-2á, we determined the expression of VEGF in these mice. As expected, we found a similar trend of VEGF expression in T2D mice. The basal level of VEGF expression was increased in the lungs of healthy T2D mice compared to WT (p<0.006) and even higher in tumors of T2D mice compared WT (p<0.002). Given that the HIF-2á−VEGF signaling pathway plays a major role in neovascularization and cancer progression, microvessel density was assessed in tumor tissue by calculating the mean number of CD31+ vessels. Accordingly, we found a higher number of CD31+ vessels in T2D mice compared to WT (p<0.0001). Collectively, these findings support our previous work on demonstrating TIMP2 as an inhibitor of tumor angiogenesis and also suggest that TIMP2 regulates hypoxic mediators within the tumor microenvironment, therefore offering TIMP2 as a novel bio-therapeutic molecule for lung cancer therapy. Citation Format: Sarvesh Kumar, Sandra M. Jensen, Ananda Chowdhury, Beiyang Wei, William G. Stetler-Stevenson. Tissue inhibitor of metalloproteinase-2 (TIMP2) deficiency enhances tumor burden via increasing HIF-2á expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 676.

Moraxella catarrhalis Might Be More Common than Expected in Acute Otitis Media in Young Finnish Children.

According to studies based on bacterial cultures of middle ear fluids, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis have been the most common pathogens in acute otitis media. However, bacterial culture can be affected by reduced viability or suboptimal growth of bacteria. PCR detects bacterial DNA from samples with greater sensitivity than culture. In the present study, we analyzed the middle ear pathogens with both conventional culture and semiquantitative real-time PCR in 90 middle ear fluid samples obtained from children aged 5 to 42 months during acute otitis media episodes. Samples were tested for the presence of S. pneumoniae, H. influenzae, M. catarrhalis, Alloiococcus otitidis, Staphylococcus aureus, and Pseudomonas aeruginosa One or more bacterial pathogens were detected in 42 (47%) samples with culture and in 69 (77%) samples with PCR. According to PCR analysis, M. catarrhalis results were positive in 42 (47%) samples, H. influenzae in 30 (33%), S. pneumoniae in 27 (30%), A. otitidis in 6 (6.7%), S. aureus in 5 (5.6%), and P. aeruginosa in 1 (1.1%). Multibacterial etiology was seen in 34 (38%) samples, and M. catarrhalis was detected in most (85%) of those cases. Fifteen signals for M. catarrhalis were strong, suggesting a highly probable etiological role of the pathogen. In conclusion, even though M. catarrhalis is often a part of mixed flora in acute otitis media, a considerable proportion of cases may be primarily attributable to this pathogen.

Adipose tissue microbiota in humans: an open issue.

A specific 'adipose tissue' microbiota has been recently identified in mice and hypothesized in humans. The purpose of this study was to verify the presence of microbiota of human whole adipose tissue and isolated adipocytes by combining culture-dependent and independent methods. Standard microbiological cultural techniques and 16S ribosomal RNA (16S rRNA) gene sequencing (Illumina technology) on DNA and RNA were employed to study (a) whole abdominal subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) from 14 obese and five normal-weight subjects and (b) mature adipocytes isolated from SAT and VAT after collagenase digestion or mechanical separation. To optimize the 16S rRNA gene detection, we used different DNA extraction methods (lysis with proteinase K, proteinase K+lysozyme and microbeads) and amplification procedures (semi-quantitative standard PCR and real-time quantitative PCR). Microbiological cultures were negative in all analyzed samples. In enzymatically isolated adipocytes, 90% of the sequenced bacterial DNA belonged to Clostridium histolyticum, the bacterium from which the collagenase enzyme was isolated. Bacterial 16S rRNA gene was not detected from DNA and RNA of whole SAT and VAT, as well as of mechanically isolated mature adipocytes, even after blocking with a specific primer the nonspecific amplification of human mitochondrial 12S rRNA. Our results do not support the presence of a human adipose tissue microbiota. In addition, they emphasized the technical problems encountered when applying metagenomic studies to human tissues with very low or absent bacterial load.

Viral shedding from diverse body fluids in a patient with severe fever with thrombocytopenia syndrome

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is a tick-borne disease characterized by high fever, thrombocytopenia, leukopenia, and multiple organ failure and is caused by a novel bunyavirus. Human-to-human transmission has been reported previously, but the mode of transmission has not been clarified thoroughly. Study designWe identified a case of a 73-year-old woman with SFTS and performed a semi-quantitative real-time reverse transcription PCR (real-time RT-PCR) assay on her blood, tracheal aspirate, gastric aspirate and urine to detect SFTS virus (SFTSV). ResultsDuring 7-day hospitalization, all the serum samples showed positive Ct values lower than 35 in both the S and M segments, suggesting the presence of the SFTSV RNA. After initiation of plasma exchange, serum SFTSV load markedly decreased, but still remained positive. The SFTS viral RNA was also detected in other body fluids, including tracheal aspirate and gastric aspirate. ConclusionThese results suggest that droplet transmission can occur through close contact with infected patients.

Modified gold nanoparticles for intracellular delivery of anti-liver cancer siRNA

To overcome the rapid enzymatic degradation and low transfection efficiency of siRNA, the delivery carriers for siRNA is a therapeutic demand to increase its stability. Gold nanoparticles (AuNPs) modified by branched polyethyleneimine (bPEI) were developed as an efficient and safe intracellular delivery carriers for siRNA. The current study implied that siRNA designed against an oncogene c-Myc could be delivered by a modified AuNPs complex without significant cytotoxicity. The comparative semi-quantitative and quantitative real time PCR were used to measure the c-Myc gene expression after transfection with naked siRNA and siRNA/bPEI/AuNPs, but AuNPs interfered with PCR. However, the c-Myc protein translation was successfully detected in the transfected HuH7 cells with naked siRNA and siRNA/bPEI/AuNPs and it was found to be inhibited by siRNA/bPEI/AuNPs more than naked siRNA. The results validate the successful silencing of c-Myc gene. Accordingly, it may confirm the promising and effective delivery of siRNA by bPEI/AuNPs. The complex enhances the cellular uptake of siRNA without significant cytotoxicity and confirms that bPEI modified AuNPs could be used as a good candidate for safe cellular delivery of siRNA.

Molecular Diagnosis of Urinary Tract Infections by Semi-Quantitative Detection of Uropathogens in a Routine Clinical Hospital Setting.

BackgroundThe objective of our study was the development of a semi-quantitative real-time PCR to detect uropathogens. Two multiplex PCR reactions were designed to detect Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Proteus mirabilis, Enterococcus faecalis, and Pseudomonas aeruginosa. 16S based PCR was performed in parallel to detect Gram-positive and Gram-negative bacteria. Firstly to identify non-targeted agents of infection in the same urine specimen, and secondly to quantify background flora. The method was evaluated in comparison with standard bacterial culture, and a commercial PCR kit for detection of uropathogens.FindingsAnalysis with a known panel of 116 clinical isolates yielded a PCR specificity of 100%. Analysis of urine specimens from 211 patients revealed a high correlation of PCR Cq values with both culture positivity and quantity. Concordance between PCR and culture was 98% when both methods yielded results. PCR was found to be more sensitive than culture. With a cut-off Cq value of 33, the negative predictive value of PCR was 94%. The 16S PCR confirmed most results. One specimen was positive by 16S PCR suggesting another cause of infection not detected by the specific PCR assays.ConclusionWe conclude that it is feasible to detect and identify uropathogens by multiplex real-time PCR assay.

Dopaminergic Receptors and Tyrosine Hydroxylase Expression in Peripheral Blood Mononuclear Cells: A Distinct Pattern in Central Obesity.

BackgroundDopamine (DA) may be involved in central obesity (CO), an inflammatory condition, through its role in the central nervous system and in periphery, where it may affect immune cell function through five different DA receptors (DR). Whether dopaminergic pathways in peripheral immune cells are implicated in the inflammatory condition linked to CO is however unknown.MethodsIn a cohort of blood donors with and without CO, categorized by waist circumference (WC) (CO: WC ≥0.80 m in women and ≥0.94 m in men), we studied the expression of DR and tyrosine hydroxylase (TH), the rate-limiting enzyme in the synthesis of DA, in peripheral blood mononuclear cells (PBMCs) and their relation with anthropometric and metabolic/endocrine and inflammatory parameters. DR D1-5 and TH expression was assessed by semi quantitative real-time PCR. As inflammatory markers we investigated the immunophenotype of monocyte subsets by flow cytometry, staining for CD14, CD16, CD11b and CD36.ResultsCO individuals showed higher plasma levels of leptin and higher inflammatory pattern of monocytes compared with non-CO. PBMC expression of DR D2, DR D4 and DR D5 as well as of TH were lower in CO in comparison with non-CO. DR D2, and DR D5 expression correlated with lower WC and weight, and with lower inflammatory pattern of monocytes, and TH expression correlated with lower WC. DR D4 expression correlated with lower plasma levels of glycosylated hemoglobin, and DR D2 expression correlated with lower CO.ConclusionsResults show that CO is associated with peripheral inflammation and downregulation of dopaminergic pathways in PBMCs, possibly suggesting DR expressed on immune cells as pharmacological targets in obesity for better metabolic outcome.

Constitutive Overexpression of the Plasma Membrane Na+/H+ Antiporter for Conferring Salinity Tolerance in Rice

At the cellular level, the Salt Overly Sensitive (SOS) signaling pathway comprising SOS3, SOS2, and SOS1 has been proposed to mediate cellular signaling under salt stress to maintain ion (Na+) homeostasis. In this regulatory pathway, both OsSOS1 encoding plasma membrane and OsNHX1 encoding vacuolar Na+/H+ antiporters are regulated by SOS3?SOS2 protein kinase complex. In the present study, the rice variety BRRI dhan28 - which is popular with farmers and high yielding, but salt sensitive, was transformed with the OsSOS1 gene isolated from salt tolerant Pokkali rice and driven by the constitutive promoter, CaMV35S. The construct was transformed through a tissue culture-independent Agrobacteriummediated in planta transformation method that circumvents the problems associated with tissue culture-based indica rice transformation methods. Integration of the foreign genes (OsSOS1) into the genome of transgenic plants was confirmed by gene-specific PCR and Southern blot analysis. The level of transgene expression (SOS1) was also quantified by semi-quantitative RT PCR and real time PCR. Genetic segregation ratio for T1 progenies was calculated and found to follow the Mendelian law of inheritance in case of positive transformants. The transformants were shown to be salt tolerant compared to wild type in molecular analysis as well as physiological screening. Future work will involve transformation of both the OsSOS1 and OsNHX1 genes together; with the expectation for enhancing the tolerance level compared to currently available transgenic rice.Plant Tissue Cult. & Biotech. 25(2): 257-272, 2015 (December)

Silencing of hERG1 Gene Inhibits Proliferation and Invasion, and Induces Apoptosis in Human Osteosarcoma Cells by Targeting the NF-κB Pathway.

Recently, the human ether à go-go (eag) related gene 1 (hERG1) channel, a member of the voltage-dependent potassium channel (Kv) family, was determined to have a critical role in cancer cell proliferation, invasion, tumorigenesis and apoptosis. However, the expression levels and functions of hERG1 in osteosarcoma cells remain poorly characterized. In this study, hERG1 transcript and protein levels in osteosarcoma cells and tissues were measured using semi-quantitative real time PCR (RT-PCR), Western blot, and immunohistochemistry. The effects of hERG1 knockdown on osteosarcoma cell proliferation, apoptosis and invasion were examined using CCK-8, colony formation, flow cytometry, caspase-3 activity, wound healing and transwell based assays. Furthermore, semi-quantitative RT-PCR, Western blot and a luciferase reporter assay were used to assess the effects of hERG1 inhibition on the nuclear factor-κB (NF-κB) pathway. In addition, the effect of NF-κB p65-siRNA and NF-κB p65 expression on the survival of osteosarcoma cells was investigated. Through this work, a relationship for hERG1 with the NF-κB pathway was identified. Osteosarcoma cells and tissues were found to express high levels of hERG1. Knockdown of hERG1 significantly suppressed cellular proliferation and invasion, and induced apoptosis, while inhibition of hERG1 significantly decreased activation of NF-κB. Overall, hERG1 may stimulate nuclear translocation of p65, thus regulating the NF-κB pathway through the activation of the hERG1/beta1 integrin complex and PI3K/AKT signaling. Taken together, these results demonstrate that hERG1 is necessary for regulation of osteosarcoma cellular proliferation, apoptosis and migration. Furthermore, this regulation by hERG1 is, at least in part, through mediation of the NF-κB pathway.

Updates in the pathophysiological mechanisms of Parkinson's disease: Emerging role of bone marrow mesenchymal stem cells.

To explore the approaches exerted by mesenchymal stem cells (MSCs) to improve Parkinson's disease (PD) pathophysiology. MSCs were harvested from bone marrow of femoral bones of male rats, grown and propagated in culture. Twenty four ovariectomized animals were classified into 3 groups: Group (1) was control, Groups (2) and (3) were subcutaneously administered with rotenone for 14 d after one month of ovariectomy for induction of PD. Then, Group (2) was left untreated, while Group (3) was treated with single intravenous dose of bone marrow derived MSCs (BM-MSCs). SRY gene was assessed by PCR in brain tissue of the female rats. Serum transforming growth factor beta-1 (TGF-β1), monocyte chemoattractant protein-1 (MCP-1) and brain derived neurotrophic factor (BDNF) levels were assayed by ELISA. Brain dopamine DA level was assayed fluorometrically, while brain tyrosine hydroxylase (TH) and nestin gene expression were detected by semi-quantitative real time PCR. Brain survivin expression was determined by immunohistochemical procedure. Histopathological investigation of brain tissues was also done. BM-MSCs were able to home at the injured brains and elicited significant decrease in serum TGF-β1 (489.7 ± 13.0 vs 691.2 ± 8.0, P < 0.05) and MCP-1 (89.6 ± 2.0 vs 112.1 ± 1.9, P < 0.05) levels associated with significant increase in serum BDNF (3663 ± 17.8 vs 2905 ± 72.9, P < 0.05) and brain DA (874 ± 15.0 vs 599 ± 9.8, P < 0.05) levels as well as brain TH (1.18 ± 0.004 vs 0.54 ± 0.009, P < 0.05) and nestin (1.29 ± 0.005 vs 0.67 ± 0.006, P < 0.05) genes expression levels. In addition to, producing insignificant increase in the number of positive cells for survivin (293.2 ± 15.9 vs 271.5 ± 15.9, P > 0.05) expression. Finally, the brain sections showed intact histological structure of the striatum as a result of treatment with BM-MSCs. The current study sheds light on the therapeutic potential of BM-MSCs against PD pathophysiology via multi-mechanistic actions.

Epigenetic regulation of APC in the molecular pathogenesis of gallbladder cancer.

Background & objectives:Loss of function of adenomatous polyposis coli (APC) has been reported in cancer. The two promoters of APC, 1A and 1B also have roles in cancer. But, the epigenetic role of APC promoters is not yet clear in gallbladder cancer (GBC) and gallstone diseases (GSD). We undertook this study to determine the epigenetic role of APC in GBC and GSD.Methods:Methylation-specific (MS)-PCR was used to analyze the methylation of APC gene. The expression of APC gene was studied by semi-quantitative PCR, real-time PCR and immunohistochemistry (IHC) in GBC, GSD and adjacent normal tissues.Results:Of the two promoters, APC 1A promoter was found methylated in 96 per cent GBC (P=0.0155) and 80 per cent GSD (P=0.015). Exon 1 was downregulated in grade II (P=0.002) and grade III (P=0.0001) of GBC, while exon 2 was normally expressed. Scoring analysis of IHC revealed 0 or negativity in 34.48 per cent (P=0.057) and 1+ in 24.14 per cent (P=0.005) GBC cases suggesting loss of APC expression.Interpretation & conclusions:The present findings indicate epigenetic silencing of APC in advanced GBC. The methylation pattern, followed by expression analysis of APC may be suggested for diagnostic, prognostic and therapeutic purposes in GBC in future.

Identification of a Novel Calcium (Ca^(2+))-Activated Chloride Channel Accessory Gene in Xenopus laevis

Calcium (Ca^(2+))-activated chloride channel accessories (CLCAs) are putative anion channel-related proteins with diverse physiological functions. Exploring CLCA diversity is important for prediction of gene structure and function. In an effort to identify novel CLCA genes in Xenopus laevis, we successfully cloned and characterized a Xenopus laevis cDNA predicted to encode the xCLCA3 gene. Cloning of xCLCA3 was achieved by computational analysis, rapid amplification of cDNA ends (RACE), and a tissue distribution analysis by semi-quantitative reverse transcription (RT) PCR or real-time PCR. We obtained a 2958 bp xCLCA3 cDNA sequence with an open reading frame encoding 943 amino acids. According to the primary structure analysis, xCLCA3 contains a predicted signal sequence, multiple sites of N-linked (N-) glycosylation, N-myristoylation, PKA, PKC, and casein kinase II phosphorylation sites, five putative hydrophobic segments, and the HExxH metalloprotease motif. Additionally, the transmembrane prediction server yielded a preserved N-terminal CLCA domain and a von Willebrand factor type A domain with one transmembrane domain in the C-terminal region. Expression analysis showed that xCLCA3 is expressed in a number of tissues, with strong expression in the brain, colon, small intestine, lung, kidney, and spleen, and poor expression in the heart and liver. These results suggest that xCLCA3 may be a candidate CLCA family member as well as a metalloprotease, rather than just an ion channel accessory protein.

Analysis of persistent changes to γ-aminobutyric acid receptor gene expression in Plutella xylostella subjected to sublethal amounts of spinosad.

A multi-generational approach was used to investigate the persistent effects of a sub-lethal dose of spinosad in Plutella xylostella. The susceptibility of various sub-populations of P. xylostella to spinosad and the effects of the insecticide on the gene expression of γ-aminobutyric acid receptor (GABAR) were determined. The results of a leaf dip bioassay showed that the sensitivity of P. xylostella to spinosad decreased across generations. The sub-strains had been previously selected based on a determined LC25 of spinosad. Considering that GABA-gated chloride channels are the primary targets of spinosad, the cDNA of P. xylostella was used to clone GABARα by using reverse transcription-polymerase chain reaction (RT-PCR). The mature peptide cDNA was 1477-bp long and contained a 1449-bp open reading frame encoding a protein of 483 amino acids. The resulting amino acid sequence was used to generate a neighbor-joining dendrogram, and homology search was conducted using NCBI BLAST. The protein had high similarity with the known GABAR sequence from P. xylostella. Subsequent semi-quantitative RT-PCR and real-time PCR analyses indicated that the GABAR transcript levels in the spinosad-resistant strain (RR, 145.82-fold) and in Sub1 strain (selected with LC25 spinosad for one generation) were the highest, followed by those in the spinosad-susceptible strain, the Sub10 strain (selected for ten generations), and the Sub5 strain (selected for five generations). This multi-generational study found significant correlations between spinosad susceptibility and GABAR gene expression, providing insights into the long-term effects of sub-lethal insecticide exposure and its potential to lead to the development of insecticide-resistant insect populations.

The Chemokine Receptor Profile As Distinctive Criterion Between Normal B-Cell Subsets and As Potential Discriminative Marker to Identify the Cell of Origin in Patients with Chronic Lymphocytic Leukemia and Richter Syndrome

Chemokine receptors are G-protein-coupled cell surface receptors, which dissociate upon activation by their ligands and cause downstream signaling. Several studies have revealed the crucial contribution of chemokine receptors and their ligands in normal B-cell differentiation and development of hematopoietic malignancies. The Richter syndrome (RS) represents the clinico-pathologic transformation of chronic lymphocytic leukaemia (CLL) to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma (DLBCL). Due to the lack of knowledge on the chemokine receptor, we aimed to investigate their expression profile in patients with CLL and Richter syndrome. Therefore, we investigated the mRNA expression levels of 18 known chemokine receptors (CCR1-CCR9, CXCR1-CXCR7, XCR1, CX3CR1) by using semi-quantitative real-time PCR on seven samples of paired (CLL and transformed DLBCL) RS samples, additionally four CLL samples -all of them subsequently transformed into DLBCL-, and eight transformed DLBCL samples originating from CLL. Additionally, 30 samples of de-novo DLBCL, including 10 germinal center B-cell (GCB) lymphomas, 12 non-germinal center B-cell lymphomas (non-GCB), and 8 unclassified DLBCL were included. Four samples of naïve B-cells (CD5 neg), CD5+ naïve B-cells and CD27+ memory B-cells (n=12) served as non-neoplastic controls.No differences in the chemokine receptor profile were detected between CD5+ and negative naïve B-cells. When comparing CD27+ memory B-cells to naïve B-cells a significant lower expression level was found for CCR7 (7-fold), CXCR4 (4-fold), and CXCR5 (1.5 fold). CCR7 (5-fold) and CXCR4 (5-fold) were also lower expressed in CD27+ memory B-cells compared to CD5+ naïve B-cells.Five out of 18 chemokine receptors were differentially expressed comparing the distinct normal B-cell subsets with RS samples. Comparing CLL samples and RS samples to CD5+ naïve B-cells, CXCR4 (12-fold for CLLs and 10-fold for RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for RS samples) were lower expressed, whereas CXCR3 (10-fold for CLLs and 8.5-fold for the transformed samples) was higher expressed and CCR5 de-novo expressed. Compared to naïve B-cells, the same chemokine receptors were deregulated: CXCR4 (10-fold for CLLs and 8.5-fold for the RS samples) and CXCR5 (2-fold for CLLs and 2.4-fold for the transformed samples) were lower expressed, CXCR3 (45-fold for CLLs and 30-fold for the transformed samples) was higher expressed and CCR5 was de-novo expressed. Comparing CLL samples and transformed RS samples to CD27+ memory B-cells, CCR5 (5.1-fold for CLLs and 4.3-fold for the RS samples) and CCR7 (8.7-fold for CLLs and 10-fold for the transformed samples) were higher expressed in both malignancies.Only one chemokine receptor was found to be differentially expressed in our seven paired RS samples: CCR6 showed a trend of a higher expression (1.4-fold) in CLL components.Considering RS and GCB DLBCL, CCR1, CCR5, and CXCR6 were found to be significantly down-regulated in RS (at least 4-fold), in contrast to CCR7 and CXCR4, which showed higher expression levels in RS (6-fold). CCR1 and CCR5 were lower expressed comparing RS and non-GCB DLBCL (25-fold and 8-fold), whereas CCR7 again, together with CXCR7, was higher expressed (3- fold and 6-fold respectively).Our data indicate a difference in the chemokine receptor profile within normal B-cell subsets. These differences are also reflected in the different expression profile of low and high aggressive component of CLL/RS compared to the distinct B cell subtypes. Hence, in future these multiple deregulated CC and CXC receptors might serve as a further hint in identifying the cell of origin of different B-cell malignancies. DisclosuresNo relevant conflicts of interest to declare.

Pseudosyndactyly - an inflammatory and fibrotic wound healing disorder in recessive dystrophic epidermolysis bullosa.

A genetic blistering skin disease, recessive dystrophic epidermolysis bullosa (RDEB), is marked by severe wound healing defects and finger contractures. The purpose of this investigation was to elucidate the mechanisms of impaired wound healing and pseudosyndactyly occurring in RDEB patients by studying the role of known inflammation and fibrosis markers in RDEB pseudosyndactyly tissue. We studied the expression of the fibrosis and/or inflammation markers tenascin-C, α-smooth muscle actin, transforming growth factor-β1, interleukin-1β, and interleukin-6 in scarring and nonscarring tissue from healthy donors and RDEB patients by semiquantitative real time-PCR and, where applicable, by immunoblots. Furthermore, the distribution pattern of α-smooth muscle actin and tenascin-C were assessed by immunofluorescence microscopy. Based on mRNA and protein analysis, we found upregulation of tenascin-C, interleukin-1β, and interleukin-6 - but not of transforming growth factor-β1 - in recessive dystrophic epidermolysis bullosa scar samples taken from pseudosyndactyly hands. Unexpectedly, α-smooth muscle actin was not upregulated. Our results confirm inflammation and fibrosis in recessive dystrophic epidermolysis bullosa, especially in scars, suggesting major roles for these processes in pseudosyndactyly. Our data therefore suggests the potential use of antiinflammatory and antifibrotic drugs in the prevention of pseudosyndactyly.

Characterization and expression analysis of serpins in the Chinese mitten crab Eriocheir sinensis

Serpins are a family of serine protease inhibitors that regulate physiological functions and are found widely in animals, plants and microorganisms. However, there are few reports on the role of serpins in the Chinese mitten crab, Eriocheir sinensis. We cloned two serpin genes from E. sinensis, named Esserpin-2 and Esserpin-3. Quantitative and semi-quantitative real-time PCR results showed that Esserpin-3 mRNA transcripts were detected in all the examined E. sinensis tissues, but were most highly expressed in immune tissues, including the gills, hepatopancreas and intestines. Lower levels of Esserpin-3 expression were detected in the accessory gland, testis and stomach, while the muscle and heart showed minimal expression. Esserpin-3 was differentially expressed throughout testis developmental stages. Esserpin-3 protein was localized in the tube wall and lumen of the accessory gland. In the testes, Esserpin-3 was detected in spermatogonia, spermatocytes and spermatids. As Esserpin-3 was also detected in the sperm contained within the seminal vesicles, it is suggested to be an intrinsic sperm protein. During the acrosome reaction, Esserpin-3 expression reduced gradually and completely disappeared after the reaction. Together, our results indicate that Esserpin-3 is an intrinsic sperm protein involved in regulation of sperm maturation and the acrosome reaction in E. sinensis.

Diazoxide preconditioning of endothelial progenitor cells from streptozotocin-induced type 1 diabetic rats improves their ability to repair diabetic cardiomyopathy.

Type 1 diabetes mellitus (DM) is a strong risk factor for the development of diabetic cardiomyopathy (DCM) which is the leading cause of morbidity and mortality in the type 1 diabetic patients. Stem cells may act as a therapeutic agent for the repair of DCM. However, deteriorated functional abilities and survival of stem cells derived from type 1 diabetic subjects need to be overcome for obtaining potential outcome of the stem cell therapy. Diazoxide (DZ) a highly selective mitochondrial ATP-sensitive K(+) channel opener has been previously shown to improve the ability of mesenchymal stem cells for the repair of heart failure. In the present study, we evaluated the effects of DZ preconditioning in improving the ability of streptozotocin-induced type 1 diabetes affected bone marrow-derived endothelial progenitor cells (DM-EPCs) for the repair of DCM in the type 1 diabetic rats. DM-EPCs were characterized by immunocytochemistry, flow cytometry, and reverse transcriptase PCR for endothelial cell-specific markers like vWF, VE cadherin, VEGFR2, PECAM, CD34, and eNOS. In vitro studies included preconditioning of DM-EPCs with 200 μM DZ for 30 min followed by exposure to either 200 μM H2O2 for 2 h (for oxidative stress induction) or 30 mM glucose media (for induction of hyperglycemic stress) for 48 h. Non-preconditioned EPCs with and without exposure to H2O2 and 30 mM high glucose served as controls. These cells were then evaluated for survival (by MTT and XTT cell viability assays), senescence, paracrine potential (by ELISA for VEGF), and alteration in gene expression [VEGF, stromal derived factor-1α (SDF-1α), HGF, bFGF, Bcl2, and Caspase-3]. DZ preconditioned DM-EPCs demonstrated significantly increased survival and VEGF release while reduced cell injury and senescence. Furthermore, DZ preconditioned DM-EPCs exhibited up-regulated expression of prosurvival genes (VEGF, SDF-1α, HGF, bFGF, and Bcl2) on exposure to H2O2, and VEGF and Bcl2 on exposure to hyperglycemia while down regulation of Caspase-3 gene. Eight weeks after type 1 diabetes induction, DZ preconditioned, and non-preconditioned DM-EPCs were transplanted into left ventricle of diabetic rats (at a dose of 2 × 10(6) DM-EPCs/70 μl serum free medium). After 4 weeks, DZ preconditioned DM-EPCs transplantation improved cardiac function as assessed by Millar's apparatus. There was decrease in collagen content estimated by Masson's trichrome and sirius red staining. Furthermore, reduced cell injury was observed as evidenced by decreased expression of Caspase-3 and increased expression of prosurvival genes Bcl2, VEGF, and bFGF by semi-quantitative real-time PCR. In conclusion, the present study demonstrated that DZ preconditioning enhanced EPCs survival under oxidative and hyperglycemic stress and their ability to treat DCM.

The role of CNS TLR2 activation in mediating innate versus adaptive neuroinflammation

Toll-like receptor 2 (TLR2) is expressed on immune cells in the periphery and the CNS and mediates both innate and adaptive immune responses. Recent studies have implicated TLR2 in systemic pathogenesis of adaptive immunity in experimental autoimmune encephalomyelitis (EAE). In addition, TLR2 is expressed on oligodendrocyte progenitor cells and its activation inhibits their differentiation and myelination. We investigated the roles of CNS TLR2 activation in mediating neuro-inflammatory responses in intact versus EAE animals. We examined the effects of intra-cerebro-ventricular (ICV) injection of Zymosan, a TLR2 agonist, on naive versus EAE animals. The neuro-inflammatory response was characterized by immune-fluorescent staining for IBA-1+ microglia/macrophages and CD3+ T cells, and by semi-quantitative real time PCR for TLR2 and immune cytokines. The nature of the immune cells isolated from EAE brain tissue was assessed by their proliferative response to the PLP peptide autoantigen. Survival and clinical scores were monitored; demyelination and axonal loss were quantified by Gold-Black and Bielschowsky stains. Our findings showed that Zymosan injection in naïve mice induced a massive neuro-inflammatory response without any clinical manifestations. In EAE mice, ICV Zymosan induced a severe acute toxic response with 80% mortality. Surviving animals returned to pre-injection clinical score, and their course of disease was not altered as compared to control EAE group. Demyelination and axonal loss were not affected by ICV Zymosan injection. Quantification of immune response in the brain by real time PCR, immunofluorescent stains and proliferative response to PLP peptide suggested that TLR2 activation induces innate but not adaptive immune response. We conclude that EAE mice are hypersensitive to CNS TLR2 activation with a severe toxic response. This might represent the susceptibility of multiple sclerosis patients to even trivial infections. As CNS TLR2 activation does not alter the clinical and pathological course of EAE, it implies that CNS TLR2 activation affects the innate but not adaptive brain immune responses.

Saffold Virus, a Human Cardiovirus, and Risk of Persistent Islet Autoantibodies in the Longitudinal Birth Cohort Study MIDIA.

The aim of this study was to describe the frequency and distribution of Saffold virus in longitudinal stool samples from children, and test for association with development of persistent autoantibodies predictive of type 1 diabetes. A cohort of Norwegian children carrying the HLA genotype associated with highest risk of type 1 diabetes (“DR4-DQ8/DR3-DQ2”) was followed with monthly stool samples from 3 to 35 months of age. Blood samples were tested for autoantibodies to insulin, glutamic acid decarboxylase65 and Islet Antigen-2. 2077 stool samples from 27 children with ≥2 repeatedly positive islet autoantibodies (cases), and 53 matched controls were analysed for Saffold virus genomic RNA by semi-quantitative real-time reverse transcriptase PCR. Saffold virus was found in 53 of 2077 (2.6%) samples, with similar proportions between cases (2.5%) and controls (2.6%). The probability of being infected by 3 years of age was 28% (95% CI 0.18–0.40). Viral quantities ranged from <1 to almost 105 copies/μl. Estimated odds ratio between islet autoimmunity and infection episodes prior to seroconversion was 1.98 (95% CI: 0.57–6.91, p = 0.29). Saffold virus had no statistically significant association with islet autoimmunity.

Adipocyte progenitor cells initiate monocyte chemoattractant protein-1-mediated macrophage accumulation in visceral adipose tissue.

ObjectiveMacrophages are important producers of obesity-induced MCP-1; however, initial obesity-induced increases in MCP-1 production precede M1 macrophage accumulation in visceral adipose tissue (VAT). The initial cellular source of obesity-induced MCP-1 in vivo is currently unknown. Preliminary reports based on in vitro studies of preadipocyte cell lines and adherent stroma-vascular fraction cells suggest that resident stromal cells express MCP-1. In the past several years, elegant methods of identifying adipocyte progenitor cells (AdPCs) have become available, making it possible to study these cells in vivo. We have previously published that global deletion of transcription factor Inhibitor of Differentiation 3 (Id3) attenuates high fat diet-induced obesity, but it is unclear if Id3 plays a role in diet-induced MCP-1 production. We sought to determine the initial cellular source of MCP-1 and identify molecular regulators mediating MCP-1 production.MethodsId3+/+ and Id3−/− mice were fed either a standard chow or HFD for varying lengths of time. Flow cytometry, semi-quantitative real-time PCR, ELISAs and adoptive transfers were used to assess the importance of AdPCs during diet-induced obesity. Flow cytometry was also performed on a cohort of 14 patients undergoing bariatric surgery.ResultsFlow cytometry identified committed CD45−CD31−Ter119−CD29+CD34+Sca-1+CD24− adipocyte progenitor cells as producers of high levels of MCP-1 in VAT. High-fat diet increased AdPC numbers, an effect dependent on Id3. Loss of Id3 increased p21Cip1 levels and attenuated AdPC proliferation, resulting in reduced MCP-1 and M1 macrophage accumulation in VAT, compared to Id3+/+ littermate controls. AdPC rescue by adoptive transfer of 50,000 Id3+/+ AdPCs into Id3−/− recipient mice increased MCP-1 levels and M1 macrophage number in VAT. Additionally, flow cytometry identified MCP-1-producing CD45−CD31−CD34+CD44+CD90+ AdPCs in human omental and subcutaneous adipose tissue, with a higher percentage in omental adipose. Furthermore, high surface expression of CD44 marked abundant MCP-1 producers, only in visceral adipose tissue.ConclusionsThis study provides the first in vivo evidence, to our knowledge, that committed AdPCs in VAT are the initial source of obesity-induced MCP-1 and identifies the helix-loop-helix transcription factor Id3 as a critical regulator of p21Cip1 expression, AdPC proliferation, MCP-1 expression and M1 macrophage accumulation in VAT. Inhibition of Id3 and AdPC expansion, as well as CD44 expression in human AdPCs, may serve as unique therapeutic targets for the regulation of adipose tissue inflammation.