Decrease In Staining Intensity Research Articles

Deletion of Dual Specificity Phosphatase 1 Does Not Predispose Mice to Increased Spontaneous Osteoarthritis.

BackgroundOsteoarthritis (OA) is a degenerative joint disease with poorly understood etiology and pathobiology. Mitogen activated protein kinases (MAPKs) including ERK and p38 play important roles in the mediation of downstream pathways involved in cartilage degenerative processes. Dual specificity phosphatase 1 (DUSP1) dephosphorylates the threonine/serine and tyrosine sites on ERK and p38, causing deactivation of downstream signalling. In this study we examined the role of DUSP1 in spontaneous OA development at 21 months of age using a genetically modified mouse model deficient in Dusp1 (DUSP1 knockout mouse).ResultsUtilizing histochemical stains of paraffin embedded knee joint sections in DUSP1 knockout and wild type female and male mice, we showed similar structural progression of cartilage degeneration associated with OA at 21 months of age. A semi-quantitative cartilage degeneration scoring system also demonstrated similar scores in the various aspects of the knee joint articular cartilage in DUSP1 knockout and control mice. Examination of overall articular cartilage thickness in the knee joint demonstrated similar results between DUSP1 knockout and wild type mice. Immunostaining for cartilage neoepitopes DIPEN, TEGE and C1,2C was similar in the cartilage lesion sites and chondrocyte pericellular matrix of both experimental groups. Likewise, immunostaining for phosphoERK and MMP13 showed similar intensity and localization between groups. SOX9 immunostaining demonstrated a decreased number of positive cells in DUSP1 knockout mice, with correspondingly decreased staining intensity. Analysis of animal walking patterns (gait) did not show a discernable difference between groups.ConclusionLoss of DUSP1 does not cause changes in cartilage degeneration and gait in a mouse model of spontaneous OA at 21 months of age. Altered staining was observed in SOX9 immunostaining which may prove promising for future studies examining the role of DUSPs in cartilage and OA, as well as models of post-traumatic OA.

The role of PTEN tumor suppressor pathway staining in carcinoma in situ of the bladder

ObjectivesThe PI3k/Akt pathway has been associated with the development and progression of bladder tumors, with most studies focused on papillary or muscle-invasive tumors. We sought to characterize the expression patterns of the PI3K/Akt pathway in a large cohort of high-risk preinvasive carcinoma in situ (CIS) tumors of the bladder. Our goal was to understand whether PI3K/Akt pathway alterations associated with CIS resemble early- or late-stage bladder cancers. Material and methodsWe evaluated tissue specimens from 97 patients with CIS of the bladder, of which 14 had a concomitant papillary tumor. All patients were treated with intravesical bacillus Calmette-Guerin. All specimens were evaluated for PTEN, p-AKT, and p-S6 immunoreactivity. Markers were evaluated for percentage and intensity of staining and were scored using a 0 to 3+grading system. ResultsPTEN staining was noted as least intense in 67% of tumor specimens and 22% of normal urothelium. P-Akt and p-S6 had intense staining in 77% and 90% of tumor specimens vs. 44% and 68% in normal tissue, respectively. Low-intensity staining for PTEN at 12 months correlated with higher recurrence risk (P = 0.026). ConclusionWe describe a large cohort of CIS bladder tumors with decreased staining intensity of PTEN and increased staining intensity of p-AKT and p-S6, similar to high-grade and high-stage papillary tumors. Low-intensity staining of PTEN at 12 months was associated with an increased risk of recurrence.

Calcium signaling and cell volume regulation are altered in Sjögren's Syndrome

Objective. Sjögren's Syndrome (SS) is a chronic autoimmune disease, leading to deficient secretion from salivary and lacrimal glands. Saliva production is normally increased by cholinergic innervation, giving rise to intracellular calcium signaling and water transport through water channels (aquaporins, AQPs). The aim of this study was to investigate possible pathophysiological changes in cell volume regulation, AQP expression and localization, and intracellular calcium signaling in glandular cells from SS patients compared to controls. Materials and methods. A total of 35 SS patients and 41 non-SS controls were included. Real time qPCR was combined with immunohistochemistry to analyze the mRNA expression and cellular distribution of AQP1, 3 and 5. Cell volume regulation and intracellular calcium signaling were examined in fresh acinar cells. Results. We show for the first time a reduced mRNA expression of AQP1 and 5 in SS compared to controls, accompanied by a decrease in staining intensity of AQP1, 3 and 5 in areas adjacent to local lymphocytic infiltration. Furthermore, we observed that the SS cells' capacity for volume regulation was abnormal. Similarly, the calcium response after parasympathetic agonist (carbachol) stimulation was markedly decreased in SS cells. Conclusions. It is concluded that mRNA expression of AQP1 and 5, protein distribution of AQP1, 3 and 5, glandular cell volume regulation and intracellular calcium signaling are all altered in SS, pointing to possible pathophysiological mechanisms in SS.

Calretinin and microtubule-associated protein-2 (MAP-2) immunohistochemistry in the diagnosis of Hirschsprung's disease

Background/PurposeIdentifying ganglion cells by rectal suction biopsy is a basic diagnostic tool for the diagnosis of Hirschsprung's disease (HD). However, the difficult interpretation of conventionally processed slides often necessitates ancillary staining methods. The aim of this study was to evaluate the usefulness of calretinin and microtubule-associated protein-2 (MAP-2) immunohistochemistry in the diagnosis of HD. MethodsWe analyzed 52 rectal suction biopsy specimens (37 from 15 HD patients and 15 from 7 non-HD patients) for ganglion cells with calretinin and MAP-2 immunohistochemistry. We also analyzed full-thickness, frozen biopsy samples obtained from 15 HD patients who underwent surgery utilizing calretinin and MAP-2 immunohistochemistry. ResultsBoth calretinin and MAP-2 positively stained ganglion cells in the submucosal plexus of the ganglionic bowel but not aganglionic bowel. Calretinin usually stained ganglion cell cytoplasm and nuclei more intensely than MAP-2, which only stained cytoplasm. No nerve fiber staining in the submucosal layer was observed for either antibody. In 21.1% (11/52) of samples, calretinin and MAP-2 staining found ganglion cells which were reported not to have ganglion cells in the original surgical pathology reports. Immunohistochemical staining for calretinin using paraffin-embedded tissue sections after cryostat sections clearly demonstrated decreased staining intensity compared to MAP-2. ConclusionCalretinin and MAP-2 are useful diagnostic markers for diagnosing HD in rectal suction biopsies. These complementary methods could ameliorate the diagnostic difficulties associated with HD.

The renoprotective effect of cGMP phosphodiesterase inhibitor and nitroprusside in a rat model of cyclosporin A-induced nephrotoxicity

The cyclosporin A (CsA) nephrotoxicity limits its usefulness as an immunosuppression. We studied the administration of both nitroprusside and phosphodiesterase-5 inhibitor (udenafil) in order to determine whether these agents could ameliorate the renal injury in CsA nephrotoxicity. 30 8-week-old SD rats were divided into 5 groups: the control (1), SQ with 15 mg/kg CsA (Group 2), CsA along with 5 mg/kg IP nitroprusside (Group 3), CsA with 10 mg/kg PO udenafil (Group 4), and CsA with udenafil and nitroprusside (Group 5). Group showed an increase in creatinine compared o the control group. Group 5 showed a decrease in creatinine compared to Group 2. In TUNEL, Group 2 increased apoptosis in proximal tubules compared to control. Group 5 showed a decrease in apoptosis compared to Groups 2, 3, and 4. In IHC, the eNOS in Group 2 was stronger than in the controls. Groups 3, 4, and 5 showed decreased staining intensity compared to Group 2. In IHC, the VEGF in Groups 2, 3, and 4 increased compared to the controls. The eNOS protein expression was increased in both Groups 3 and 5 compared to the controls. The VEGF protein expression was increased in Groups 3 and 5 compared to Group 2. The eNOS mRNA was decreased in Group 2 compared to the control group and tended to increase in Groups 3, 4, and 5 compared to Group 2. The VEGF mRNA was increased in Group 2 and tended to increase more in Groups 3 and 5. The udenafil and nitroprusside ameliorated renal injury in rat model of CsA nephrotoxicity. The mechanism appears to be associated with decreasing tubular apoptosis by decreasing eNOS and increasing VEGF.

Evidence for a conserved CCAP-signaling pathway controlling ecdysis in a hemimetabolous insect, Rhodnius prolixus

A vital feature in the success of Ecdysozoa is their ability to shed their exoskeleton (a process called ecdysis) such that they can grow or change their morphology. In holometabolous insects, these behaviors are orchestrated by the sequential actions of neuropeptides, one of which is crustacean cardioactive peptide (CCAP). Little is known about the control of ecdysis in hemimetabolous insects. Here, we report that CCAP is essential for successful ecdysis in the hemimetabolous insect, Rhodnius prolixus; the vector of Chagas disease. The first indication of CCAP's involvement in ecdysis was the observation of decreased staining intensity of CCAP-containing neurons immediately following ecdysis, indicative of the release of CCAP. The critical importance of the CCAP signaling pathway was further demonstrated by knockdown (as determined by qPCR and immunohistochemistry) of the CCAP and CCAPR transcripts utilizing dsRNA. This technique reduced the staining intensity of CCAP-containing neurons, and knocked down the transcript levels by up to 92%, with lethal consequences to the insect. Insects with these transcripts knocked down had very high mortality (up to 84%), typically at the expected time of the ecdysis sequence, or had ecdysis extremely delayed. This is the first report of the susceptibility of R. prolixus to dsRNA knockdown of neuropeptide and receptor transcripts, and the data clearly demonstrates the conserved nature of the CCAP signaling pathway in ecdysis between holometabolous and hemimetabolous insects.

Abstract P1-07-05: Evaluation of tissue processing factors affecting HER2 IHC staining intensity in breast cancer cell lines

Abstract Background: In breast cancer the HER2 status as established by IHC (e.g. HercepTest™) determines eligibility for HER2 targeted therapy. Pre-analytical procedures including tissue processing create variation in HER2 intensity seen in breast cancer tissues. Therefore, there is a need for a greater understanding of the effects of the individual tissue processing steps on HER2 staining intensity. Control cell lines (CCL) are commonly used as staining run controls in immunohistochemistry (IHC) in both pharmacodiagnostic, routine and in clinical research. As a surrogate for cancer tissue, these immortalized cancer cells can serve as a model system for investigating the effect of process variables on staining intensity. This study was designed to investigate if variations in cell/tissue processing (fixation, embedding and sectioning) affect the IHC staining intensity in a HER2 formalin- fixed, paraffin-embedded (FFPE) control cell lines. To introduce a more objective scoring we have evaluated the application of digital image analysis combined with design of experiment (DOE) statistics. Methods: The following tissue processing parameters were investigated: embedding medium (HistoGel™ or agarose), fixation time (18 or 48 hours in neutral buffered formalin), density of cells (25% or 50%) and thickness of cut sections (2 μm, 4 μm or 6 μm). Factors were set up in a 2k factorial experimental design; all slides were stained with Dako HercepTest™. All slides were scanned, analyzed and compared on two different image analysis systems: ACIS®III (Dako) and Scanscope CS (Aperio). The DOE statistical analysis revealed the contribution of individual factors as well as any additive effect of a combination of factors. Analysis was based on the level of intensity as well as histoscores. Results: In a 24 DOE experiment, several of the parameters described above were associated with decreased staining intensity: increased fixation time (p < 0.001), decreased section thickness (p < 0.001) and decreased cell density (p < 0.001). In a separate 23 experiment, it was shown that fixation media in combination with cell density was associated with changes in staining intensity (p values from 0.001 to 0.022). Three different algorithms and two different slide scanners were tested and results were consistent regardless of the image analysis system or the algorithm used. Conclusions: This study identified several tissue processing factors which impact IHC staining intensity in a HER2 expressing CCL. The discovery of these effects has advanced the understanding of processing methodology and the influence of pre-analytical factors on HER2 IHC staining intensity. The application of a DOE setup with quantification by image analysis can be a useful tool for the systematic evaluation of several variables compared with the standard “one factor at a time” design. A multifactor effect between fixation media and cell density was detected which would not have been possible in a “one factor at a time” design. Implementing the knowledge achieved from this study provides a methodology for standardizing tissue processing and thereby improving HER2 testing in breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P1-07-05.

Age-Related Changes in the myenteric Plexus of the Gastric Fundus in Albino Rats

Introduction It is well known that during ovarian follicular development, the majority of follicles undergo atresia at various stages of their development. Recent studies have reported that the degenerative changes associated with atresia appear initially in the granulosa cell layer, and follicular atresia occurs by apoptosis, which is an essential physiological process. The present study aimed to examine granulosa cell apoptosis using different, histological, immunohistochemical, and biochemical methods. Materials and methods Fifteen adult female albino rats were used in this study. The rats were sacrificed and bilateral oophorectomy was carried out; the ovaries of rats were processed and paraffin sections were prepared for staining with H&E and also to study immunohistochemistry for apoptotic proteins such as caspase-3 and antiapoptotic proteins such as B-cell lymphoma-2 (Bcl-2). Some ovarian specimens were processed by electrophoresis for DNA separation. Results Apoptosis in granulosa cell showed morphological characteristics, including nuclei with marginated chromatin, a single condensed pyknotic nucleus, multiple nuclear fragments, and apoptotic bodies containing variable amounts of chromatin. Moreover, apoptotic changes were detected by immunohistochemical staining in the form of increased staining intensity to caspase-3 (apoptotic protein) and decreased staining intensity to Bcl-2 (antiapoptotic protein), and also increased fragmentation of DNA, which was indicated by a ladder step appearance that was detected using DNA electrophoresis.

Effectiveness of Topical Infliximab in a Mouse Model of Experimental Dry Eye

To investigate the efficacy of a topical anti-tumor necrosis factor-α agent, infliximab, in a mouse model of experimental dry eye (EDE). EDE was induced in C57BL/6 mice, with or without topical treatment consisting of balanced salt solution or 0.001%, 0.01%, or 0.1% infliximab solutions. Tear volume and corneal smoothness were measured on days 5 and 10 after treatment. Levels of interleukin (IL)-1β, IL-6, IL-17, and interferon γ (IFN-γ) were measured in the conjunctiva using a multiplex immunobead assay 10 days after treatment. Periodic acid-Schiff staining, immunohistochemistry, and flow cytometry were also performed 10 days after treatment. Mice treated with 0.01% or 0.1% infliximab showed a significant improvement in tear volume and corneal smoothness compared with controls. The 0.01% and 0.1% infliximab-treated groups showed decreased levels of conjunctival IL-1β, IL-6, IL-17, and interferon γ and a decreased staining intensity of tumor necrosis factor-α. The density of conjunctival goblet cells was higher, whereas the number of CD4*CXCR3* T cells was lower, in the 0.01% and 0.1% infliximab-treated groups compared with the EDE and balanced salt solution control groups. However, there was no significant difference in all parameters between the 0.001% infliximab-treated group and control group. : Topical application of infliximab can improve tear production and ocular surface irregularity, decrease inflammatory cytokines and cells on the ocular surface, and increase conjunctival goblet cell density. These results suggest that topical infliximab eye drops at a concentration of 0.01% and 0.1% may be useful for the treatment of dry eye disease.

Role and predictive strength of transglutaminase type 2 expression in premalignant lesions of the cervix

The demonstration that type 2 transglutaminase (TG2) can incorporate polyamine into the E7 oncoprotein of human papillomavirus (HPV) type 18 has led to the hypothesis that TG2 can have a role in the host cellular response to HPV infection. The aim of this study was to investigate whether HPV-related pathology, in infected human cervical epithelium, was associated with modulation of TG2 expression. Normal controls and HPV-infected cervical biopsies were analyzed for the expression of TG2, and the findings were compared with lesion grade. The correlation between TG2 expression and p16, a marker for HPV-induced dysplasia, and the retinoblastoma protein (Rb), a target of the E7 protein of HPV, was also investigated. Results obtained showed that TG2 was absent in normal squamous mucosa, whereas it was present in 100% CIN I lesions. Low-grade lesions showed significantly higher TG2 expression than high-grade lesions (P<0.0001). In 94% of CIN I more than 50% of the cells were positive for TG2, with a strong staining intensity (+3), whereas a decreased staining intensity and a low number of positive cells were found in CIN II/III. In CIN I cases, both nuclear and cytoplasmic staining were found in cells exhibiting classical morphological features of HPV infection. In addition, during progression from low-grade squamous intraepithelial lesions to severe dysplasia, TG2 expression was inversely correlated with p16 (Pearson: −0.930), whereas a positive correlation was observed between the expression of TG2 and pRb (Pearson: 0.997). TG2 is expressed in HPV infection as an early phenomenon, not restricted to high-risk genotypes. TG2 upregulation is probably part of host cell reaction against HPV-induced tissue modification. It may act as a cellular antioxidant defense factor, playing an important role in counteracting oxidative damage in neoplastic disease.

Alterations of antioxidant biomarkers and type I collagen deposition in the parotid gland of streptozotocin-induced diabetic rats

Background and objectiveAcarbose is a competitive inhibitor of intestinal alpha-glycosidases that slows the breakdown of sucrose and starch, thereby reducing glucose and fructose absorption. The aim of this study was to evaluate the effect of acarbose treatment on antioxidant parameters and deposition of type I collagen in the parotid glands of diabetic rats. MethodsDiabetes mellitus was induced by intravenous injection of streptozotocin, and rats were divided into four groups: non-diabetic (NDM), diabetic (DM), diabetic treated with 25mg/kg acarbose (DMA) and non-diabetic treated with acarbose (NDMA). Changes in enzymatic antioxidant systems, such as the activity of SOD and GPx enzymes, were evaluated, and the specific staining pattern of the type I collagen fibres was investigated in the rat parotid glands. ResultsThe DM group presented high levels of SOD and GPx enzymes, which were reduced by acarbose treatment. Tissue damage, which was indicated by an increased MDA concentration in the parotid glands of rats in the DM group, was also reversed in the DMA group. Moreover, type I collagen fibres from DM rats were more intensely stained than those of NDM rats. Acarbose treatment was effective in decreasing collagen deposition, which was shown by a decrease in staining intensity of approximately 25%. ConclusionsThese results suggest that the diabetic state influences the type I collagen concentration in the parotid glands of rats. In addition, acarbose treatment was helpful in preventing the deposition of such fibres, as well the increase in oxidative stress induced by hyperglycemia.

Histochemical Evaluation of Human Prostatic Tissues withCratylia mollisSeed Lectin

Lectins, proteins which selectively recognize carbohydrates, have been used in histochemistry for the evaluation of changes in glycosylation in processes of cellular differentiation and/or dedifferentiation. Cratylia mollis seed lectins (Cramoll 1,4 and Cramoll 3), conjugated to horseradish peroxidase, were used as histochemical probes in human prostate tissues: normal (NP), hyperplasia (BPH), and prostate carcinoma (PCa). The staining pattern of Con-A and Cramoll 1,4 in BPH was more intense than in NP. These lectins also showed staining differences between BPH and PCa; the latter showing decreased staining intensity with an increased degree of malignancy. PNA and Cramoll 3 stained epithelial cells similarly in all diagnoses although they did present intense staining of PCa glands lumen. Corpora amylacea were not differentially recognized by any of the lectins. Cramoll 1,4 and Cramoll 3 seed lectins present themselves as candidates for histochemical probes for prostate pathologies when compared to commercial lectins such as Con-A and PNA.

DHPR α1S subunit controls skeletal muscle mass and morphogenesis

The alpha1S subunit has a dual function in skeletal muscle: it forms the L-type Ca(2+) channel in T-tubules and is the voltage sensor of excitation-contraction coupling at the level of triads. It has been proposed that L-type Ca(2+) channels might also be voltage-gated sensors linked to transcriptional activity controlling differentiation. By using the U7-exon skipping strategy, we have achieved long-lasting downregulation of alpha1S in adult skeletal muscle. Treated muscles underwent massive atrophy while still displaying significant amounts of alpha1S in the tubular system and being not paralysed. This atrophy implicated the autophagy pathway, which was triggered by neuronal nitric oxide synthase redistribution, activation of FoxO3A, upregulation of autophagy-related genes and autophagosome formation. Subcellular investigations showed that this atrophy was correlated with the disappearance of a minor fraction of alpha1S located throughout the sarcolemma. Our results reveal for the first time that this sarcolemmal fraction could have a role in a signalling pathway determining muscle anabolic or catabolic state and might act as a molecular sensor of muscle activity.

Brain-Specific Deletion of Extracellular Signal-Regulated Kinase 2 Mitogen-Activated Protein Kinase Leads to Aberrant Cortical Collagen Deposition

The mitogen-activated protein kinases extracellular signal-regulated kinase (ERK)1 and 2 are essential intracellular mediators of numerous transmembrane signals. To investigate neural-specific functions of ERK2 in the brain, we used a Cre/lox strategy using Nestin:Cre to drive recombination in neural precursor cells. Nestin:Cre;ERK2(fl/fl) conditional knockout (cKO) mice have architecturally normal brains and no gross behavioral deficits. However, all cKO mice developed early-onset (postnatal day 35 to 40) frontal cortical astrogliosis, without evidence of neuronal degeneration. Frontoparietal cortical gray matter, but not underlying white matter, was found to contain abundant pericapillary and parenchymal reticulin fibrils, which were shown by immunohistochemistry to contain fibrillar collagens, including type I collagen. ERK1 general KO mice showed neither fibrils nor astrogliosis, indicating a specific role for ERK2 in the regulation of brain collagen. Collagen fibrils were also observed to a lesser extent in GFAP:Cre;ERK2(fl/fl) mice but not in CamKII-Cre;ERK2(fl/fl) mice (pyramidal neuron specific), consistent with a possible astroglial origin. Primary astroglial cultures from cKO mice expressed elevated fibrillar collagen levels, providing further evidence that the phenotype may be cell autonomous for astroglia. Unlike most other tissues, brain and spinal cord parenchyma do not normally contain fibrillar collagens, except in disease states. Determining mechanisms of ERK2-mediated collagen regulation may enable targeted suppression of glial scar formation in diverse neurological disorders.

Joint Extraintestinal Manifestations in Ulcerative Colitis

An extraintestinal manifestation (EIM) very often occurs in ulcerative colitis (UC) patients. EIM modifies the natural course of UC and decreases the quality of life in these patients. The aim of this study was to analyze clinical and laboratory findings in UC patients with joint EIM. 319 UC patients were examined. Among them were 131 (41.1%) patients with distal UC, 102 (32.0%) suffered from left-sided UC and 86 (26.9%) had pancolitis. 95 (29.8%) UC patients had joint EIM. Arthritis correlated with extensive forms of UC and was more often determined in patients with left-sided UC and pancolitis. Arthralgia was a prevalent symptom of joint EIM in patients with distal UC. Colon microbiocenosis and the mucosal barrier in UC patients were analyzed. The cytokine status with privileged cytokine profile changes was investigated. In all UC patients, dysbiosis with a decreasing quantity of bifidobacteria, lactobacilli and зscherichia coli was found, but an increase of facultative flora was also found. At the same time, an association of facultative flora in UC patients with arthritis was observed. In these patients, Staphylococcus, Klebsiella and Proteus were found more often in stool cultures. These associations correlated with a modification of the colonocytes’ cell receptor maturity of mucus, a condition with a decreased staining intensity by lectins. A cytokine imbalance with an increase of proinflammatory and a decrease of anti-inflammatory cytokines was found in all UC patients. The privileged cytokine profile changes in UC patients with joint EIM were analyzed. Maximal increases of IL-1 and TNF with decreases of IL-10 in plasma in patients with joint EIM were observed.

Glomerular Protein Levels of Matrix Metalloproteinase-1 and Tissue Inhibitor of Metalloproteinase-1 Are Lower in Diabetic Subjects

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulate extracellular matrix turnover throughout the body, including in renal glomeruli. We investigated protein levels of multiple MMPs (MMP-1, MMP-2, MMP-3, and MMP-9) and TIMP-1 in glomeruli and investigated whether disease phenotypes were associated with levels of these proteins. Renal cortex was collected from 100 adult autopsy subjects arrayed across 17 tissue microarrays. Immunohistochemical staining intensity for each MMP and TIMP-1 was determined using quantitative color deconvolution techniques. We observed significantly decreased glomerular MMP-1 and TIMP-1 staining in subjects with diabetes, hypertension, and an estimated glomerular filtration rate <30 ml/min/1.73 m(2) in univariate analyses. MMP-1 staining, but not TIMP-1 staining, was inversely correlated with increased glomerular fibrosis (r = -0.40). In multivariable analysis, diabetes was robustly associated with decreased staining intensity. This study indicates that in human subjects, the long-term sequelae of diseases such as diabetes that cause chronic renal failure result in decreased TIMP-1 and MMP-1 proteins in renal glomeruli. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Histochemical changes in the liver of freshwater fish, Rasbora daniconius, exposed to paper mill effluent

This investigation deals with the effect of paper mill effluent, on the histochemical components of the liver of Rasbora daniconius . The Paper mill effluent was collected directly from the 45 Km away from Aurangabad (MS) India. Histochemical studies on protein, lipid and glycogen contents of liver Rasbora daniconius showed a progressive decrease in staining intensity to Mercury bromophenol blue (Hg-BPB), Sudan black B and Bets’s Carmine at 30 days exposure when treated at sublethal concentrations (1.9% and 0.95%) of paper mill effluent. The magnitudes of these changes were dose dependent. This study, therefore, concludes that paper mill effluent creates metabolic crisis and impairment in fish liver. The depletion in level of protein, lipid and glycogen points towards exhaustion of cell- energy to meet high demand of fish in stressful condition.

Histologic Influence of Doxazosin and Finasteride in Benign Prostatic Hyperplasia Accompanying Chronic Inflammation

Aim: To evaluate the influence of doxazosin and finasteride on histologic findings in benign prostatic hyperplasia accompanied by prostatitis, and to examine the factors related with prostate carcinogenesis. Materials and Methods: Prostate tissue from 17 cases of prostatic hyperplasia were divided into three groups; group 1: no medication history, group 2: both doxazosin and finasteride for at least 6 months before surgery; group 3: doxazosin for a minimum of 6 months before transurethral resection. Formalin-fixed, paraffin-embedded sections were stained with hematoxylin and eosin stain and immunohistochemistry for COX-2, CD3, CD68, VEGF, and GSTP (glutathione S-transferase pi). Results: CD3 showed a tendency toward decreased staining intensity and extent in group 3 (p = 0.026, p = 0.004, respectively). CD68 showed a different staining extent among the three groups (p = 0.020). For VEGF, the staining extent showed different results between groups 2 and 3 (p = 0.044). There was no correlation between COX-2 and VEGF in group 1. However, a positive correlation between COX-2 and GSTP1 was demonstrated in group 2 (p = 0.000). Conclusions: Doxazosin-treated prostate tissue showed decreased inflammatory reaction. GSTP1 expression was decreased in combined treatment with doxazosin and finasteride. These findings suggest that finasteride may interfere with the anti-inflammatory reaction. Finasteride also decreases angiogenesis. However, the meaning of our observations is limited by small sample size.

Expression of E‐ and N‐cadherin and clinicopathology in hepatocellular carcinoma

Loss or reduced E-cadherin expression and aberrant expression of N-cadherin have been associated with invasiveness of human carcinoma cells and poor prognosis. The role of E- and N-cadherin, however, in hepatocellular carcinoma (HCC) has not yet been elucidated. The aim of the present study was to investigate the expression pattern of E- and N-cadherin in surgically resected HCC specimens according to their relationship with clinicopathological features. The expression patterns of E- and N-cadherin were evaluated on immunohistochemistry in 68 specimens of HCC and adjacent non-tumor tissue. The most different expression pattern between HCC and non-tumor tissue was the decreased staining intensity of E-cadherin (n = 37, 54%) and the dot-like discontinuous staining of N-cadherin (n = 35, 55%). Decreased intensity of E-cadherin and discontinuous staining of N-cadherin in HCC was correlated with advanced stage. The risk factors for expression patterns related to recurrence were loss of E-cadherin expression (odds ratio (OR) = 3.6; 95% confidence interval (CI): 1.1-12.4) and discontinuous staining of N-cadherin (OR = 1.6; 95% CI: 0.8-3.2). In conclusion, discontinuous staining of N-cadherin and loss of E-cadherin expression in HCC predicts a high risk of recurrence after surgical treatment.

Expression and Prognostic Relevance of Annexin A3 in Prostate Cancer

ObjectivesBy differential quantitative protein expression, it has previously been shown that annexin A3 (ANXA3) expression is associated with prostate cancer. However little is known about the role and biology of ANXA3 in the human prostate. The aim of this study was to thoroughly analyze ANXA3 expression patterns and its potential as a prognostic marker in a large set of benign, preneoplastic, and neoplastic prostate tissue samples. MethodsImmunohistochemistry-based ANXA3 protein expression was analyzed for 1589 prostate cancers as well as smaller subsets of benign epithelium and high-grade prostatic intraepithelial neoplasia (PIN) in a tissue microarray format. ResultsAll samples of benign prostatic epithelium and PIN showed ANXA3 protein expression, with PIN lesions showing a decreased staining intensity compared with benign epithelium (p<0.0001). In cancer, ANXA3 protein expression was essentially reduced, resulting in a negative staining rate of 27.2%, which correlated with increasing pT stage and Gleason score (p<0.0001). ANXA3 status in cancer was shown to be an independent adverse prognostic factor and enabled substratification of the large group of intermediate-risk patients (n=969) into high- and low-risk subgroups. ConclusionsANXA3 represents a promising candidate tissue marker, and when combined with the standard prognostic parameters, is suggested to provide a more precise prediction of prognosis in the individual patient, therefore harboring the potential to contribute to future patient management.