Showing papers in "Journal of Histochemistry and Cytochemistry in 2014"
TL;DR: This review summarizes the current knowledge of mast cell function in both normal and pathological conditions with regards to their regulation, phenotype and role.
Abstract: Since first described by Paul Ehrlich in 1878, mast cells have been mostly viewed as effectors of allergy. It has been only in the past two decades that mast cells have gained recognition for their involvement in other physiological and pathological processes. Mast cells have a widespread distribution and are found predominantly at the interface between the host and the external environment. Mast cell maturation, phenotype and function are a direct consequence of the local microenvironment and have a marked influence on their ability to specifically recognize and respond to various stimuli through the release of an array of biologically active mediators. These features enable mast cells to act as both first responders in harmful situations as well as to respond to changes in their environment by communicating with a variety of other cells implicated in physiological and immunological responses. Therefore, the critical role of mast cells in both innate and adaptive immunity, including immune tolerance, has gained increased prominence. Conversely, mast cell dysfunction has pointed to these cells as the main offenders in several chronic allergic/inflammatory disorders, cancer and autoimmune diseases. This review summarizes the current knowledge of mast cell function in both normal and pathological conditions with regards to their regulation, phenotype and role.
432 citations
TL;DR: This study illustrates the inability of Picrosirius red staining to differentiate collagen types, since the absorbed amount of polarized light by this dye strictly depends on the orientation of the collagen bundles.
Abstract: Specific staining of the extracellular matrix components is especially helpful in studying tissue remodeling, particularly in the case of connective tissue pathologies. As developed by Junqueira and colleagues in 1979, specific staining by Picrosirius red is one of the most important stains to study collagen networks in different tissues. Under polarized light, collagen bundles appear green, red or yellow, and are easily differentiated from the black background, thus allowing for quantitative morphometric analysis. As Junqueira and colleagues point out, many studies use color staining to differentiate collagen bundles and to specify collagen types, yet other studies report that polarized colors only reflect fiber thickness and packing. Using a simple histological example, our study illustrates the inability of Picrosirius red staining to differentiate collagen types, since the absorbed amount of polarized light by this dye strictly depends on the orientation of the collagen bundles.
357 citations
TL;DR: Essential controls required for validation of immunohistochemical findings are summarized and represents a standard of practice for the use of Immunohistochemistry in research and diagnostic investigations and can be cited by authors as support for the validity of interpretations of the immunohists reported in publications.
Abstract: Immunohistochemistry is widely used in biomedical research to localize specific epitopes of molecules in cells and tissues. The validity of interpretations based on immunohistochemistry requires appropriate positive and negative controls that are often not reported in publications. This omission may lead to incorrect interpretations and irreproducible results in the literature and contribute to wasted time, effort, and resources as well as erosion of confidence in scientific investigation by the general public, legislative bodies and funding agencies. The present article summarizes essential controls required for validation of immunohistochemical findings and represents a standard of practice for the use of immunohistochemistry in research and diagnostic investigations. Adherence to the guidelines described in the present article can be cited by authors as support for the validity of interpretations of the immunohistochemistry reported in their publications.
182 citations
TL;DR: This review summarizes the current knowledge of HDACs in tumorigenesis and tumor progression as well as their contribution to the hallmarks of cancer and discusses the potential role of histone deacetylase inhibitors as emerging epigenetic drugs to cure cancer.
Abstract: In the current era of genomic medicine, diseases are identified as manifestations of anomalous patterns of gene expression. Cancer is the principal example among such maladies. Although remarkable progress has been achieved in the understanding of the molecular mechanisms involved in the genesis and progression of cancer, its epigenetic regulation, particularly histone deacetylation, demands further studies. Histone deacetylases (HDACs) are one of the key players in the gene expression regulation network in cancer because of their repressive role on tumor suppressor genes. Higher expression and function of deacetylases disrupt the finely tuned acetylation homeostasis in both histone and non-histone target proteins. This brings about alterations in the genes implicated in the regulation of cell proliferation, differentiation, apoptosis and other cellular processes. Moreover, the reversible nature of epigenetic modulation by HDACs makes them attractive targets for cancer remedy. This review summarizes the current knowledge of HDACs in tumorigenesis and tumor progression as well as their contribution to the hallmarks of cancer. The present report also describes briefly various assays to detect histone deacetylase activity and discusses the potential role of histone deacetylase inhibitors as emerging epigenetic drugs to cure cancer.
110 citations
TL;DR: Together, these studies provide a set of guidelines for diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue and these characterization techniques are transferable to similar questions in other tissue types, as demonstrated on frozen human liver tissue and paraff in different fixatives.
Abstract: Tissue autofluorescence frequently hampers visualization of immunofluorescent markers in formalin-fixed paraffin-embedded respiratory tissues. We assessed nine treatments reported to have efficacy in reducing autofluorescence in other tissue types. The three most efficacious were Eriochrome black T, Sudan black B and sodium borohydride, as measured using white light laser confocal Λ2 (multi-lambda) analysis. We also assessed the impact of steam antigen retrieval and serum application on human tracheal tissue autofluorescence. Functionally fitting this Λ2 data to 2-dimensional Gaussian surfaces revealed that steam antigen retrieval and serum application contribute minimally to autofluorescence and that the three treatments are disparately efficacious. Together, these studies provide a set of guidelines for diminishing autofluorescence in formalin-fixed paraffin-embedded human respiratory tissue. Additionally, these characterization techniques are transferable to similar questions in other tissue types, as ...
92 citations
TL;DR: The intriguing possibility that loss of bone mass during aging may be manipulated by controlling the levels and activity of intracellular VEGF in bone marrow mesenchymal stem cells is raised.
Abstract: Vascular endothelial growth factor A (VEGF), a key factor in angiogenesis, plays an essential role in skeletal development and postnatal homeostasis. VEGF serves as a survival factor for chondrocytes and couples the resorption of cartilage with bone formation during endochondral ossification. Recently, it has also been found to regulate the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells. Surprisingly, this regulatory function of VEGF is not based on paracrine signaling involving cell surface receptor activation. Instead, the mechanism appears to utilize intracellular VEGF, which is functionally linked to the nuclear envelope protein lamin A. Lamin A and VEGF control osteoblast and adipocyte differentiation by regulating the levels of the osteoblast and adipocyte transcription factors Runx2 and PPARγ, respectively. These data raise the intriguing possibility that loss of bone mass during aging may be manipulated by controlling the levels and activity of intracellular VEGF in bone marrow mesenchymal stem cells.
77 citations
TL;DR: The data suggests that early dietary manipulation of intestinal microbiota may be a novel mechanism to delay T1D onset in genetically pre-disposed individuals.
Abstract: Infant formula and breastfeeding are environmental factors that influence the incidence of Type 1 Diabetes (T1D) as well as the acidity of newborn diets. To determine if altering the intestinal microbiome is one mechanism through which an acidic liquid plays a role in T1D, we placed non-obese diabetic (NOD)/ShiLtJt mice on neutral (N) or acidified H2O and monitored the impact on microbial composition and diabetes incidence. NOD-N mice showed an increased development of diabetes, while exhibiting a decrease in Firmicutes and an increase in Bacteroidetes, Actinobacteria, and Proteobacteria from as early as 2 weeks of age. NOD-N mice had a decrease in the levels of Foxp3 expression in CD4(+)Foxp3(+) cells, as well as decreased CD4(+)IL17(+) cells, and a lower ratio of IL17/IFNγ CD4+ T-cells. Our data clearly indicates that a change in the acidity of liquids consumed dramatically alters the intestinal microbiome, the presence of protective Th17 and Treg cells, and the incidence of diabetes. This data suggests that early dietary manipulation of intestinal microbiota may be a novel mechanism to delay T1D onset in genetically pre-disposed individuals.
74 citations
TL;DR: It is suggested that the expression of SOX2 and OCT4 in tumor cells indicates resistance to radiotherapy and that these two factors were important predictors of poor survival in patients with LACSCC.
Abstract: Radiotherapy (RT) as a preoperative or postoperative adjuvant or primary treatment is the most common management modality for locally advanced cervical cancer. Radioresistance of tumor cells remains a major therapeutic problem. Consequently, we aimed to explore if the stem cell biomarkers SOX2 and OCT4 protein could be used to predict radioresistance in patients with locally advanced cervical squamous cell carcinoma (LACSCC). These 132 patients were divided into two groups (radiation-resistant and radiation-sensitive groups) according to progress-free survival (PFS). Using pretreatment paraffin-embedded tissues, we evaluated SOX2 and OCT4 expression using immunohistochemical staining. The percentage of overexpression of SOX2 and OCT4 in the radiation-resistant group was much higher than that in the radiation-sensitive group (p<0.001 and p <0.001, respectively). The patients with high expression of SOX2 and OCT4 showed a shorter PFS than those with low expression. Our study suggests that the expression of SOX2 and OCT4 in tumor cells indicates resistance to radiotherapy and that these two factors were important predictors of poor survival in patients with LACSCC (hazard ratio [95% CI], 2.294 [1.013, 5.195] and 2.300 [1.050, 5.037], respectively; p=0.046 and p=0.037, respectively).
73 citations
TL;DR: This work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.
Abstract: Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest—in this case CB2—but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.
72 citations
TL;DR: The status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles and potential substrates.
Abstract: Cell adhesion to extracellular matrix is a complex process involving protrusive activity driven by the actin cytoskeleton, engagement of specific receptors, followed by signaling and cytoskeletal organization. Thereafter, contractile and endocytic/recycling activities may facilitate migration and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles and potential substrates.
71 citations
TL;DR: IUGR was shown to impair the mucosal immunity of the SI in neonatal piglets, and the ileum was the major site of impairment.
Abstract: Intrauterine growth restriction (IUGR) is a very common problem in both piglet and human neonate populations. We hypothesized that IUGR neonates have impaired intestinal mucosal immunity from birth. Using neonatal piglets as IUGR models, immune organ weights, the weight and length of the small intestine (SI), intestinal morphology, intraepithelial immune cell numbers, levels of cytokines and immunoglobulins, and the relative gene expression of cytokines in the SI were investigated. IUGR neonatal piglets were observed to have lower absolute immune organ weight and SI length, decreased relative weights of the thymus, spleen, mesenteric lymph node, and thinner but longer SIs. Damaged and jagged villi, shorter microvilli, presence of autophagosomes, swelled mitochondria, and decreased villus surface areas were also found in the SIs of IUGR neonatal piglets. We also found a smaller number of epithelial goblet cells and lymphocytes in the SIs of IUGR neonates. In addition, we detected reduced levels of the cytokines TNF-α and IFN-γ and decreased gene expression of cytokines in IUGR neonates. In conclusion, IUGR was shown to impair the mucosal immunity of the SI in neonatal piglets, and the ileum was the major site of impairment.
TL;DR: It is indicated that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis, which may help to develop a novel therapeutic strategy against osteoclineogenesis-related diseases.
Abstract: Previous studies have implicated autophagy in osteoclast differentiation. The aim of this study was to investigate the potential role of p62, a characterized adaptor protein for autophagy, in RANKL-induced osteoclastogenesis. Real-time quantitative PCR and western blot analyses were used to evaluate the expression levels of autophagy-related markers during RANKL-induced osteoclastogenesis in mouse macrophage-like RAW264.7 cells. Meanwhile, the potential relationship between p62/LC3 localization and F-actin ring formation was tested using double-labeling immunofluorescence. Then, the expression of p62 in RAW264.7 cells was knocked down using small-interfering RNA (siRNA), followed by detecting its influence on RANKL-induced autophagy activation, osteoclast differentiation, and F-actin ring formation. The data showed that several key autophagy-related markers including p62 were significantly altered during RANKL-induced osteoclast differentiation. In addition, the expression and localization of p62 showed negative correlation with LC3 accumulation and F-actin ring formation, as demonstrated by western blot and immunofluorescence analyses, respectively. Importantly, the knockdown of p62 obviously attenuated RANKL-induced expression of autophagy- and osteoclastogenesis-related genes, formation of TRAP-positive multinuclear cells, accumulation of LC3, as well as formation of F-actin ring. Our study indicates that p62 may play essential roles in RANKL-induced autophagy and osteoclastogenesis, which may help to develop a novel therapeutic strategy against osteoclastogenesis-related diseases.
TL;DR: TRPV1 expression was found to be regulated at the level of individual blood vessels, where some vessels expressed, while others did not express TRPV 1 in the same tissue sections, and this tightly regulated expression and function suggests a physiological role for vascular TRV1.
Abstract: Transient receptor potential (TRP) cation channels are emerging in vascular biology. In particular, the expression of the capsaicin receptor (TRPV1) was reported in vascular smooth muscle cells. This study characterized the arteriolar TRPV1 function and expression in the rat. TRPV1 mRNA was expressed in various vascular beds. Six commercially available antibodies were tested for TRPV1 specificity. Two of them were specific (immunostaining was abolished by blocking peptides) for neuronal TRPV1 and one recognized vascular TRPV1. TRPV1 was expressed in blood vessels in the skeletal muscle, mesenteric and skin tissues, as well as in the aorta and carotid arteries. TRPV1 expression was found to be regulated at the level of individual blood vessels, where some vessels expressed, while others did not express TRPV1 in the same tissue sections. Capsaicin (a TRPV1 agonist) evoked constrictions in skeletal muscle arteries and in the carotid artery, but had no effect on the femoral and mesenteric arteries or the aorta. In blood vessels, TRPV1 expression was detected in most of the large arteries, but there were striking differences at level of the small arteries. TRPV1 activity was suppressed in some isolated arteries. This tightly regulated expression and function suggests a physiological role for vascular TRPV1.
TL;DR: Some endoneurial fibroblast-like cells in normal peripheral nerves could have a different proliferative and/or regenerative potential than others, and these EFLCs may play a role in the initial phase of nerve repair.
Abstract: Endoneurial fibroblast-like cells (EFLCs) are one of the cell populations present in the peripheral nervous system. The role and immunophenotypic characteristics of EFLCs are not well known and led us to perform a histological and cytological study of EFLCs in normal human and rat peripheral nerves. We found that all EFLCs express CD34, neural/glial antigen 2 (NG2), and prolyl-4-hydrolase-beta. In addition, half of the EFLCs in normal peripheral nerves express platelet-derived growth factor receptor-β (PDGFR-β) and some also express the intermediate filament nestin in vivo (at a lower level than Schwann cells, which express high levels of nestin). Using cell cultures of purified EFLCs, we characterized subpopulations of EFLCs expressing PDGFR-β alone or PDGFR-β and nestin. Experimental nerve lesions in rat resulted in an increase in nestin-positive EFLCs, which returned to normal levels after 8 days. This suggests that some EFLCs could have a different proliferative and/or regenerative potential than others, and these EFLCs may play a role in the initial phase of nerve repair. These "activated" EFLCs share some immunophenotypic similarities with pericytes and Interstitial cells of Cajal, which have progenitor cell potentials. This raises the questions as to whether a proportion of EFLCs have a possible role as endoneurial progenitor cells.
TL;DR: The results suggest that the expression of GRs in oligodendrocytes and their progenitor cells mediates several functions in vivo, including differentiation and myelination, as a major target of glucocorticoids and their cofactors.
Abstract: The expression of glucocorticoid receptors (GRs) was investigated immunohistochemically in two different lineages of oligodendrocytes, using carbonic anhydrase (CA) II and neuron glial antigen (NG) 2 as markers of mature oligodendrocytes and oligodendrocyte progenitors, respectively. We focused on the gray matter regions, including CA1, CA3 and the dentate gyrus of the hippocampus, the primary somatosensory cortex barrel field and the basolateral amygdala, and the white matter regions, including the corpus callosum, external capsule and fimbria of the hippocampus. More than 80% of CAII-immunoreactive (IR) cells and more than 95% of NG2-IR cells expressed GRs in various regions of the brain. In contrast, neither CAII-IR cells nor NG2-IR cells expressed mineralocorticoid receptors (MRs) in the same regions. The intensity of GR expression was drastically reduced in CA II-IR cells and NG2-IR cells in the same regions in adrenalectomized mice. Finally, steroid receptor co-activator (SRC)-1 and p300, both of which are cofactors for GR, were expressed in the gray and white matter regions in NG2-IR cells, but not in CAII-IR cells. These results suggest that the expression of GRs in oligodendrocytes and their progenitor cells mediates several functions in vivo, including differentiation and myelination, as a major target of glucocorticoids and their cofactors.
TL;DR: It is found that the elution is dependent upon the antibody affinity and is reduced by species-specific crosslinking via a dimeric or Fab fragments of a secondary antibody, and 2-ME/SDS buffer is the method of choice to perform multiple rounds of immunostaining on a single routine section.
Abstract: Inconsistent results obtained with published methods for the elution of antibodies from tissue sections prompted the assessment of both old and new methods in combination with monoclonal rabbit antibodies of known, increased affinity (above 1×10(-9) KD). We tested an acidic (pH 2) glycine buffer, a 6 M urea hot buffer and a 2-Mercaptoethanol, SDS buffer (2-ME/SDS). Some antibodies were not removed by the glycine pH 2 or 6 M urea hot buffers, indicating that antibodies survive much harsher conditions than previously believed. We found that the elution is dependent upon the antibody affinity and is reduced by species-specific crosslinking via a dimeric or Fab fragments of a secondary antibody. The high affinity bond of exogenous streptavidin with the endogenous biotin can be removed by 6 M urea but not by the other buffers. 2-ME/SDS buffer is superior to glycine pH 2 and 6 M urea hot elution buffers for all antibodies because of its irreversible effect on the structure of the antibodies. It also has a mild retrieving effect on some antigens present on routinely treated sections and no detrimental effect on the immunoreactivity of the tissue. Therefore, 2-ME/SDS buffer is the method of choice to perform multiple rounds of immunostaining on a single routine section.
TL;DR: H&E staining is a more reliable indicator of acute bleeding events induced by LPS in this model within a short time span, and is compared to Perl’s Prussian blue in this paper.
Abstract: Cerebral microbleeds are microscopic hemorrhages with deposits of blood products in the brain, which can be visualized with MRI and are implicated in cerebrovascular diseases. Hematoxylin and eosin (HE Prussian blue-positive stains, by comparison, showed no significant increase with LPS treatment and did not correlate with either H&E-positive stains or surface microhemorrhages. H&E staining is thus a more reliable indicator of acute bleeding events induced by LPS in this model within a short time span.
TL;DR: Pretreatment microRNA profiling, especially miR-224 expression, might serve as an ancillary tool for the better assessment of expected survival rates for patients under sorafenib treatment.
Abstract: Sorafenib represents the first effective targeted therapy for advanced stage hepatocellular carcinoma (HCC); however, adequate patient stratification regarding sorafenib-responsiveness is still missing. Our aim was to analyse the association between the pretreatment microRNA profile of HCC and patient survival under sorafenib treatment. Total RNA was extracted from diagnostic fine-needle aspiration biopsy (FNAB) cytological smears of 20 advanced stage HCC patients collected between June 2008 and July 2012. All patients underwent sorafenib administration after FNA. Clinicopathological and survival data were recorded. Fourteen frequently deregulated miRNAs in HCC (miR-17-5p, miR-18a, miR-21, miR-34a, miR-122, miR-195, miR-210, miR-214, miR-221, miR-222, miR-223, miR-224, miR-140, miR-328) were tested by qRT-PCR. NormFinder software was used to select proper miR (mir-140) as a reference. Satisfactory amount of total RNA was obtained from all the considered samples (mean 10.8 ± 9.3 µg, range 0.2-32.2 µg). Among the analysed miRNAs, high miR-214 expression was associated with smaller tumor size (p=0.019), whereas high miR-17-5p expression correlated with better Eastern Cooperative Oncology Group performance status (p=0.003). The survival analysis revealed that high miR-224 expression was associated with increased progression-free and overall survival (PFS p=0.029; OS p=0.012). Pretreatment microRNA profiling, especially miR-224 expression, might serve as an ancillary tool for the better assessment of expected survival rates for patients under sorafenib treatment.
TL;DR: Investigation of the localization of AQP1 and AQP4 in normal rat spinal cord compared with the expression of marker proteins for astrocytes, neurons, and endothelial cells revealed characteristic localization and expression patterns that could provide insights toward gaining an understanding of the role of AQPs in the spinal cord.
Abstract: Aquaporin (AQP) is a water-selective channel protein. In the brain, AQPs play critical roles in the production of cerebrospinal fluid and in edema formation. In contrast, the expression and role of AQPs in spinal cord are unclear. We aimed to investigate the localization of AQP1 and AQP4 in normal rat spinal cord compared with the expression of marker proteins for astrocytes, neurons, and endothelial cells. Immunohistochemistry demonstrated that AQP1 and AQP4 are expressed along all levels of the spinal cord from the cervical to lumbar levels. AQP1 immunolabeling was observed in the dorsal horns in the gray matter, whereas the labeling was weak and mainly seen close to glia limitans in the white matter. AQP1 was co-labeled with marker proteins for unmyelinated neuronal fibers (peripherin) and endothelial cells (RECA-1) of blood vessels that had penetrated through the glia limitans. In contrast, AQP1 did not colocalize with GFAP, an astrocyte marker, at any level of the spinal cord. AQP4 was exclusively localized at the astrocytes, but AQP4 expression in spinal cord exhibited a less polarized and more spatial distribution than that of brain astrocytes. The observed characteristic localization and expression patterns of AQP1 and AQP4 could provide insights toward gaining an understanding of the role of AQPs in the spinal cord.
TL;DR: This RFP-MT1 transgenic model provides a unique tool for studying the distribution of the MT1 receptor in the brain of mice, its cell-specific expression and its function in vivo.
Abstract: The pineal hormone melatonin activates two G-protein coupled receptors (MT1 and MT2) to regulate in part biological functions. The MT1 and MT2 melatonin receptors are heterogeneously distributed in the mammalian brain including humans. In the mouse, only a few reports have assessed the expression of the MT1 melatonin receptor expression using 2-iodomelatonin binding, in situ hybridization and/or polymerase chain reaction (PCR). Here, we described a transgenic mouse in which red fluorescence protein (RFP) is expressed under the control of the endogenous MT1 promoter, by inserting RFP cDNA at the start codon of MTNR1a gene within a bacterial artificial chromosome (BAC) and expressing this construct as a transgene. The expression of RFP in the brain of this mouse was examined either directly under a fluorescent microscope or immunohistochemically using an antibody against RFP (RFP-MT1). RFP-MT1 expression was observed in many brain regions including the subcommissural organ, parts of the ependyma lining the lateral and third ventricles, the aqueduct, the hippocampus, the cerebellum, the pars tuberalis, the habenula and the habenula commissure. This RFP-MT1 transgenic model provides a unique tool for studying the distribution of the MT1 receptor in the brain of mice, its cell-specific expression and its function in vivo.
TL;DR: The results show the sum of p-mTOR/p-AKT and p-MAPK/EGFR is a potential predictive marker of survival in NSCLC patients and offers the capacity to understand factors that predict outcome better than current approaches of evaluating biomarkers individually or in pairs.
Abstract: Activation of numerous pathways has been documented in non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR) has emerged as a common therapeutic target. The mitogen-activated protein kinase (MAPK) and AKT signaling pathways are downstream of EGFR and deregulated via genetic and epigenetic mechanisms in many human cancers. We evaluated selected markers in the EGFR pathway with reference to outcome. Tissues from 220 cases of NSCLC patients presented in a tissue microarray were assayed with immunohistochemistry for phosphorylated AKT, phosphorylated MAPK, phosphorylated mTOR, and EGFR and then quantified by automated image analysis. Individually, the biomarkers did not predict. Combined as ratios, p-mTOR/p-AKT, and p-MAPK/EGFR function as prognostic markers of survival (p=0.008 and p=0.029, respectively), however, no significance was found after adjustment (p=0.221, p=0.103). The sum of these ratios demonstrates a stronger correlation with survival (p<0.001) and remained statistically significant after adjustment (p=0.026). The algebraic combination of biomarkers offer the capacity to understand factors that predict outcome better than current approaches of evaluating biomarkers individually or in pairs. Our results show the sum of p-mTOR/p-AKT and p-MAPK/EGFR is a potential predictive marker of survival in NSCLC patients.
TL;DR: The generation and characterization of a novel recombinant HA-binding probe obtained by fusion of a modified TSG-6 Link module with mutationally inactivated heparin-binding sequence and the Fc portion of human IgG1 is described for tissue HA detection in histological samples.
Abstract: Tumor necrosis factor-Stimulated Gene 6 protein (TSG-6) is a hyaluronan (HA)-binding glycoprotein containing an HA-binding Link module. Because of its well-defined structure, HA binding properties and small size, TSG-6 is an excellent candidate as an alternative to animal-derived HA-binding protein (HABP) for the detection of HA. The present work describes the generation and characterization of a novel recombinant HA-binding probe obtained by fusion of a modified TSG-6 Link module with mutationally inactivated heparin-binding sequence and the Fc portion of human IgG1 (TSG-6-ΔHep-Fc) for tissue HA detection in histological samples. Direct binding assays indicated strong binding of TSG-6-ΔHep-Fc to HA, with little residual binding to heparin. Histolocalization of HA in formalin-fixed, paraffin-embedded tissue sections using biotin-TSG-6-ΔHep-Fc resulted in hyaluronidase-sensitive staining patterns similar to those obtained with biotin-HABP, but with improved sensitivity. HA was detected in many human tissues, and was most abundant in soft connective tissues such as the skin dermis and the stroma of various glands. Digital image analysis revealed a linear correlation between biotin-HABP and biotin-TSG-6-ΔHep-Fc staining intensity in a subset of normal and malignant human tissues. These results demonstrate that TSG-6-ΔHep-Fc is a sensitive and specific probe for the detection of HA by histological methods.
TL;DR: GDH activity in mice is highest in the liver with NAD+ as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM.
Abstract: Glutamate dehydrogenase (GDH) catalyses the reversible conversion of glutamate into α-ketoglutarate with the concomitant reduction of NAD(P)(+) to NAD(P)H or vice versa. GDH activity is subject to complex allosteric regulation including substrate inhibition. To determine GDH kinetics in situ, we assessed the effects of various glutamate concentrations in combination with either the coenzyme NAD(+) or NADP(+) on GDH activity in mouse liver cryostat sections using metabolic mapping. NAD(+)-dependent GDH V(max) was 2.5-fold higher than NADP(+)-dependent V(max), whereas the K(m) was similar, 1.92 mM versus 1.66 mM, when NAD(+) or NADP(+) was used, respectively. With either coenzyme, V(max) was determined at 10 mM glutamate and substrate inhibition was observed at higher glutamate concentrations with a K(i) of 12.2 and 3.95 for NAD(+) and NADP(+) used as coenzyme, respectively. NAD(+)- and NADP(+)-dependent GDH activities were examined in various mouse tissues. GDH activity was highest in liver and much lower in other tissues. In all tissues, the highest activity was found when NAD(+) was used as a coenzyme. In conclusion, GDH activity in mice is highest in the liver with NAD(+) as a coenzyme and highest GDH activity was determined at a glutamate concentration of 10 mM.
TL;DR: This paper presents a method that is easy to use and highly flexible to accommodate both an avidin-biotin detection system as well as a biotin-free polymer detection system.
Abstract: Immunohistochemistry on mouse tissue utilizing mouse monoclonal antibodies presents a challenge. Secondary antibodies directed against the mouse monoclonal primary antibody of interest will also detect endogenous mouse immunoglobulin in the tissue. This can lead to significant spurious staining. Therefore, a “mouse-on-mouse” staining strategy is needed to yield credible data. This paper presents a method that is easy to use and highly flexible to accommodate both an avidin-biotin detection system as well as a biotin-free polymer detection system. The mouse primary antibody is first combined with an Fab fragment of an anti-mouse antibody in a tube and allowed sufficient time to form an antibody complex. Any non-complexed secondary antibody is bound up with mouse serum. The mixture is then applied to the tissue. The flexibility of this method is confirmed with the use of different anti-mouse antibodies followed by a variety of detection reagents. These techniques can be used for immunohistochemistry (IHC), immunofluorescence (IF), as well as staining with multiple primary antibodies. This method has also been adapted to other models, such as using human antibodies on human tissue and using multiple rabbit antibodies in dual immunofluorescence.
TL;DR: PAG is mainly active in mouse kidney, brain and liver, and shows different kinetics depending on which type of PAG is expressed, as well as compared with liver, kidney and brain, which showed 3-fold to 6-fold less PAG activity.
Abstract: Phosphate-activated glutaminase (PAG) converts glutamine to glutamate as part of the glutaminolysis pathway in mitochondria. Two genes, GLS1 and GLS2, which encode for kidney-type PAG and liver-type PAG, respectively, differ in their tissue-specific activities and kinetics. Tissue-specific PAG activity and its kinetics were determined by metabolic mapping using a tetrazolium salt and glutamate dehydrogenase as an auxiliary enzyme in the presence of various glutamine concentrations. In kidney and brain, PAG showed Michaelis-Menten kinetics with a Km of 0.6 mM glutamine and a Vmax of 1.1 µmol/mL/min when using 5 mM glutamine. PAG activity was high in the kidney cortex and inner medulla but low in the outer medulla, papillary tip, glomeruli and the lis of Henle. In brain tissue sections, PAG was active in the grey matter and inactive in myelin-rich regions. Liver PAG showed allosteric regulation with a Km of 11.6 mM glutamine and a Vmax of 0.5 µmol/mL/min when using 20 mM glutamine. The specificity of the me...
TL;DR: It is shown that CFTR is only expressed in neurons and has an early and widespread distribution during development, which suggests that this gene may play previously unsuspected roles in neuronal maturation or function.
Abstract: Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein has recently been shown to be expressed in the human adult central nervous system (CNS). As CFTR expression has also been documented during embryonic development in several organs, such as the respiratory tract, the intestine and the male reproductive system, suggesting a possible role during development we decided to investigate the expression of CFTR in the human developing CNS. In addition, as some, although rare, neurological symptoms have been reported in patients with CF, we compared the expression of normal and mutated CFTR at several fetal stages. Immunohistochemistry was performed on brain and spinal cord samples of foetuses between 13 and 40 weeks of gestation and compared with five patients with cystic fibrosis (CF) of similar ages. We showed in this study that CFTR is only expressed in neurons and has an early and widespread distribution during development. Although we did not observe any cerebral abnormality in patients with CF, we observed a slight delay in the maturation of several brain structures. We also observed different expression and localization of CFTR depending on the brain structure or the cell maturation stage. Our findings, along with a literature review on the neurological phenotypes of patients with CF, suggest that this gene may play previously unsuspected roles in neuronal maturation or function.
TL;DR: The localization of IMPDH2 inside the nucleus of human cells as well as the ultrastructure of R&R inclusions are described, which show that the nuclear pool has an ability to form Rod structures after inhibition by ribavirin.
Abstract: Inosine-5'-monophosphate dehydrogenase catalyzes the critical step in the de novo synthesis of guanosine nucleotides: the oxidation of inosine monophosphate to xanthosine monophosphate. This reaction can be inhibited by specific inhibitors, such as ribavirin or mycophenolic acid, which are widely used in clinical treatment when required to inhibit the proliferation of viruses or cells. However, it was recently found that such an inhibition affects the cells, leading to a redistribution of IMPDH2 and the appearance of IMPDH2 inclusions in the cytoplasm. According to their shape, these inclusions have been termed "Rods and Rings" (R&R). In this work, we focused on the subcellular localization of IMPDH2 protein and the ultrastructure of R&R inclusions. Using microscopy and western blot analysis, we show the presence of nuclear IMPDH2 in human cells. We also show that the nuclear pool has an ability to form Rod structures after inhibition by ribavirin. Concerning the ultrastructure, we observed that R&R inclusions in cellulo correspond to the accumulation of fibrous material that is not surrounded by a biological membrane. The individual fibers are composed of regularly repeating subunits with a length of approximately 11 nm. Together, our findings describe the localization of IMPDH2 inside the nucleus of human cells as well as the ultrastructure of R&R inclusions.
TL;DR: The current study aimed to compare the efficiency of PEG as an embedding medium for histochemical analyses of primary and secondary metabolites accumulation and found that PEG binds to phenols, such as tannins, flavonoids and lignins, thereby presenting false negatives in T3.
Abstract: Histochemical analyses in plants are commonly performed on hand-made sections of fresh materials. The disadvantages of embedding in historesin, paraffin or paraplast® are the alterations to cellular contents, the high costs and few evident results, depending on the test. Polyethylene-glycol (PEG), as a low cost, hydrophilic medium that maintains most of the cellular features similar to fresh conditions, may be useful for obtaining good histochemical results in thinner and homogeneous sections. The current study aimed to compare the efficiency of PEG as an embedding medium for histochemical analyses of primary and secondary metabolites accumulation. Using hand-made sections of fresh samples (T1) as a comparison, we tested the influence of the use of Karnovsky's solution as a fixative (T2) versus embedding in PEG (T3). The samples herein analyzed comprise leaves, stems, seeds and insect galls of different plant species. Neither the Karnovsky's fixative nor the embedding in PEG altered the histochemical results for starch, lipids, terpenoids, proteins and reducing sugars in T1, T2, and T3. However, PEG binds to phenols, such as tannins, flavonoids and lignins, thereby presenting false negatives in T3.
TL;DR: Results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains, and shows the loss of Gal-6-sulfate in KS of the KSGal 6ST-deficient brains.
Abstract: Keratan sulfate (KS) comprises repeating disaccharides of galactose (Gal) and N-acetylglucosamine (GlcNAc). Residues of Gal and GlcNAc in KS are potentially modified with sulfate at their C-6 positions. The 5D4 monoclonal antibody recognizes KS structures containing Gal and GlcNAc, both 6-sulfated, and has been used most extensively to evaluate KS expression in mammalian brains. We previously showed that GlcNAc6ST1 is an enzyme responsible for the synthesis of the 5D4 epitope in developing brain and in the adult brain, where it is induced after injury. It has been unclear which sulfotransferase is responsible for Gal-6-sulfation within the 5D4 KS epitope in developing brains. We produced mice deficient in KSGal6ST, a Gal-6-sulfotransferase. Western blotting and immunoprecipitation revealed that all 5D4-immunoreactivity to proteins, including phosphacan, were abolished in KSGal6ST-deficient postnatal brains. Likewise, the 5D4 epitope, expressed primarily in the cortical marginal zone and subplate and dorsal thalamus, was eliminated in KSGal6ST-deficient mice. Disaccharide analysis showed the loss of Gal-6-sulfate in KS of the KSGal6ST-deficient brains. Transfection studies revealed that GlcNAc6ST1 and KSGal6ST cooperated in the expression of the 5D4 KS epitope in HeLa cells. These results indicate that KSGal6ST is essential for C-6 sulfation of Gal within KS in early postnatal brains.
TL;DR: The Spearman’s rho value indicated that NRP-1 is positively related to the EMT markers E-cadherin and Slug, which might indicate that N RP- 1 is a partner in the E MT process in EOC tumors.
Abstract: The mechanism by which neuropilin-1 (NRP-1) induces malignancy in Epithelial Ovarian Cancer (EOC) is still unknown. This study is the first to demonstrate the relationship between NRP-1 expression and EMT markers vimentin, N-cadherin, E-cadherin and Slug. We used tissue microarrays containing the three main subtypes of EOC tumors: serous, mucinous cystadenocarcinoma and endometrioid adenocarcinoma and representative cases retrieved from our pathology archives. Immunohistochemistry was performed to detect the expression levels and location of NRP-1 and the aforementioned EMT proteins. NRP-1 was mainly expressed on cancer cells but not in normal ovarian surface epithelium (OSE). The Immunoreactive Scoring (IRS) values revealed that the expression of NRP-1, Slug and E-cadherin in the malignant subtypes of ovarian tissues was significantly higher (5.18 ± 0.64, 4.84 ± 0.7, 4.98 ± 0.68, respectively) than their expression in the normal and benign tissues (1.04 ± 0.29, 0.84 ± 0.68, 1.71 ± 0.66, respectively), wi...