Increase In Staining Intensity Research Articles

Ultrastructural Findings and Tumor Necrosis Factor-alpha and Intercellular Adhesion Molecule-1 Expression in Psoriasis Patients Before and After Oral Cyclosporin A Therapy

Elevated levels of proinflammatory cytokines, including tumor necrosis factor-alpha, are found in skin lesions and plasma of patients with psoriasis. Clinical improvement of psoriasis with cyclosporin A treatment is accompanied by downmodulation of proinflammatory epidermal cytokines. In this study to determine the effects of cyclosporin A on the ultrastructural changes and tumor necrosis factor-alpha and intercellular adhesion molecule-1 expression in psoriasis, biopsy specimens before and after cyclosporin A treatment were evaluated ultrastructurally and immunohistochemically. Ten patients were given 3–7.5 mg/kg oral cyclosporin A for 6 months. Before and after treatment full thickness of 4-mm punch biopsies were obtained from patients and from 6 healthy volunteers. Samples were processed for electron microscopic and immunohistochemical evaluation. The treatment was well tolerated with complete clinical improvement. The ultrastructural changes such as reduction of tonofilaments, dilatation of intercellular space, and interruption in lamina densa were recovered by cyclosporin A treatment. The increased staining intensity of tumor necrosis factor-alpha and intercellular adhesion molecule-1 on epidermal keratinocytes and endothelial cells was reduced after cyclosporin A therapy. Cyclosporin A treatment results in total normalization of the electron microscopic picture of psoriasis and its beneficial effect depends on the direct inhibition of tumor necrosis factor-alpha and consequently intercellular adhesion molecule-1.

Elevated Akt activation and its impact on clinicopathological features of renal cell carcinoma.

Akt has been implicated in the pathogenesis and progression of numerous human malignant tumors because Akt regulates many key effector molecules involved in controlling the balance of survival and apoptosis. Elevated Akt activation has been demonstrated in various types of malignant tumors. In the current study we evaluated Akt activation in renal cell carcinoma and investigated its association with pathological features and clinical outcome. Akt activation was evaluated by immunohistochemistry using antiphospho-specific Akt antibody, which recognizes only activated Akt, in 48 patients with renal cell carcinoma. High immunostaining tumors were defined as tumors with increased staining intensity compared with adjacent nonneoplastic kidney tissue and low immunostaining tumors were defined as tumors with equivalent or decreased staining intensity. The association of Akt activation status with clinicopathological features was analyzed. Of 48 patients examined 18 (37.5%) demonstrated high antiphospho-specific Akt immunostaining compared with adjacent nonneoplastic kidney tissue, while 30 (62.5%) demonstrated low immunostaining. Elevated immunostaining was significantly associated with tumor grade (p = 0.0354) and metastatic disease (p = 0.0044), while it was not associated with tumor stage or histological subtype. In addition, high antiphospho-specific Akt immunostaining was significantly associated with a poor cancer specific survival rate on univariate analysis (p = 0.0109) but not on multivariate analysis. Elevated Akt activation could be a common finding, especially in high grade tumor and metastatic disease. It could have an important role in the pathogenesis and progression of renal cell carcinoma.

Sequence of protein expression of bone sialoprotein and osteopontin at the developing interface between repair cementum and dentin in human deciduous teeth

Experimental periodontal regeneration studies have revealed the weak binding of repair cementum to the root surface, whereas attachment of cementum to dentin preconditioned by odontoclasts appears to be superior. The aim of this study has been, therefore, to analyze the structural and partial biochemical nature of the interface that develops between resorbed dentin and repair cementum by using human deciduous teeth as a model. Aldehyde-fixed and decalcified tooth samples were embedded in acrylic or epoxy resins and sectioned for light and transmission electron microscopy. Antibodies against bone sialoprotein (BSP) and osteopontin (OPN), two noncollagenous proteins accumulating at hard tissue interfaces in bone and teeth, were used for protein A-gold immunocytochemistry. Light microscopy revealed a gradually increasing staining intensity of the external dentin matrix starting after the withdrawal of the odontoclast. Labeling for both BSP and OPN was first detected among the exposed collagen fibrils and in the intratubular dentin matrix when odontoclasts had withdrawn but mesenchymal cells were present. Subsequently, collagen fibrils of the repair cementum were deposited concomitantly with the appearance of labeling for BSP and OPN over the intratubular, intertubular, and peritubular dentin matrix. Labeled mineralization foci indicated the advancing mineralization front, and the collagenous repair matrix became integrated in an electron-dense organic material that showed labeling for BSP and OPN. Thus, no distinct planar interfacial matrix layer lies between the resorbed dentin and the repair cementum. The results suggest that odontoclasts precondition the dentin matrix such that the repair cementum becomes firmly attached.

B7-H4 Is Highly Expressed in Ductal and Lobular Breast Cancer

This study was designed to investigate the expression of B7-H4 protein, a member of the B7 family that is involved in the regulation of antigen-specific immune responses, in normal breast and in primary and metastatic breast carcinomas. Archival formalin-fixed tissue blocks from breast cancers and normal somatic tissues were evaluated for B7-H4 expression by immunohistochemistry with manual and automated image analysis. The proportion of B7-H4-positive cells and the intensity of B7-H4 staining were compared with histologic type, grade, stage, hormone receptor status, and HER-2/neu status. B7-H4 was detected in 165 of 173 (95.4%) primary breast cancers and in 240 of 246 (97.6%) metastatic breast cancers. B7-H4 staining intensity was greater in invasive ductal carcinomas [24.61 relative units (RU)] and in invasive lobular carcinomas (15.23 RU) than in normal breast epithelium (4.30 RU, P = 0.0003). Increased staining intensity was associated with negative progesterone receptor status (P = 0.014) and history of neoadjuvant chemotherapy (P = 0.004), and the proportion of B7-H4-positive cells was associated with negative progesterone receptor (P = 0.001) and negative HER-2/neu (P = 0.024) status. However, there was no statistically significant relationship between the proportion of B7-H4-positive cells or staining intensity and grade, stage, or other clinicopathologic variables. Low levels of B7-H4 expression were also detected in epithelial cells of the female genital tract, lung, pancreas, and kidney, but B7-H4 was generally absent in most other normal somatic tissues. The nearly ubiquitous expression of B7-H4 in breast cancer, independent of tumor grade or stage, suggests a critical role for this protein in breast cancer biology.

Development of a Tissue Array for Primary Melanoma with Long-Term Follow-Up: Discovering Melanoma Cell Adhesion Molecule as an Important Prognostic Marker

Refining current prognostic capability is essential for improving the management of melanoma. This study was undertaken to develop a tumor array for the rapid assessment of novel prognostic markers in a series of specimens from melanoma patients with 7- to 10-year follow-up. A melanoma database of 120 patients with archival specimens was created after histopathological review of original specimens. A tissue array was developed allowing 480 biopsy samples from the 120 primary melanoma specimens to be embedded into a single paraffin block. This was sectioned and stained for the adhesion marker melanoma cell adhesion molecule (MCAM); after further review, 76 of the 120 specimens were suitable for further analysis. The slides were assessed by two independent observers without previous knowledge of the clinical outcome for staining positivity and stain intensity. Assessment of association between MCAM and clinicopathological features was carried out using chi-squared analysis, and univariate and Cox multivariate analyses were performed on the data. There was a high correlation between MCAM intensity and both Clark's level and Breslow thickness (Spearman correlation p < 0.001 for both). The data revealed that MACM was a highly specific prognostic marker for survival in univariate analysis (chi2 = 18, p < 0.0001). Subgroup analysis by stratification of the staining intensity revealed a sequentially worsening survival with increasing staining intensity (chi2 = 22.33, p < 0.0001). Multivariate analysis of survival showed MCAM to be an independent prognostic marker more accurate than all other clinicopathological parameters (p < 0.0001), including the Breslow depth. Further analysis within only intermediate-thickness tumors showed MCAM intensity added further refinement to outcome prediction (chi2 = 22.33, p < 0.0001). The tissue array provided a rapid method of analyzing up to 480 specimens within a single paraffin block. This will benefit many areas of plastic surgery research. The identification of adhesion markers revealed a valuable prognostic marker for predicting outcome and a potential target for therapeutic manipulation.

Progesterone receptors and proliferating cell nuclear antigen expression in equine luteal tissue

Steroid hormones act via specific receptors, and these play an important physiological role in the ovary. The objective of this study was to evaluate the cellular distribution of progesterone receptors and their staining intensity in different equine luteal structures during the breeding season, as well as their relationship to luteal cell composition, cell proliferation pattern and plasma progesterone (P4) concentration. There was an increase in proliferating cell nuclear antigen (PCNA) expression in large luteal cells from the corpus hemorrhagicum (CH) to mid-luteal phase, followed by a decrease toward the late luteal stage. In the CH, the number of large luteal cells was lower than in other structures. Only large luteal cells showed positive staining for P(4) receptors. An increase in staining intensity for P(4) receptors was observed between CH and mid-phase corpus luteum, and CH and late-phase corpus luteum. Synthesis of P(4) started at a very early stage of the luteal structure and was accompanied by an increase in P(4) receptors and PCNA expression, and proliferation of large luteal cells, until mid-luteal phase. These data suggest that large luteal cells might play an important role in the regulation or synthesis of P(4) in equine luteal structures.

Clinicopathologic characteristics of nodular glomerulosclerosis in Chinese patients with type 2 diabetes

Nodular glomerulosclerosis is a distinct entity that is highly specific for diabetic glomerulopathy. However, clinicopathologic characteristics of this nodular lesion are largely undefined in patients with type 2 diabetes. An autopsy study was conducted to investigate the clinical, histopathologic, and histochemical characteristics of 351 consecutive cases with type 2 diabetes. In addition, immunohistochemical staining was performed in a representative subset of 50 cases. Nodular glomerulosclerosis was found in 37.6%. Hypertension, elevated serum creatinine and urea levels, renal failure, myocardial infarction, low body mass index, and large glomerular matrix area were the significant risk factors for nodular glomerulosclerosis. Proteinuria (protein > 0.5 g/24 h), renal insufficiency (serum creatinine > or = 2.0 mg/dL [> or =178 micromol/L]), and renal failure were found in 28.1%, 33.6%, and 7.6% of patients with type 2 diabetes with nodular glomerulosclerosis, respectively. Glomerular matrix fractions were 42.1% +/- 13.3%, 32.3% +/- 15.3%, and 22.7% +/- 8.0% in patients with nodular glomerulosclerosis, non-nodular glomerulopathy (glomerulopathy in the absence of Kimmelstiel-Wilson nodule), and near-normal glomeruli with age-related minimal changes, respectively (analysis of variance, P < 0.001). Immunoreactivity for collagen type IV, fibronectin, and laminin was localized at the periphery of mesangial nodules. Mesangial cells at the periphery of mesangial nodules showed increased staining intensity for alpha-smooth muscle actin (alpha-SMA) and transforming growth factor-beta1 (TGF-beta1). Nodular lesions also showed a marked increase in number of glomerular CD68-positive macrophages. In patients with type 2 diabetes, nodular glomerulosclerosis is related to hypertension, advanced renal disease, and prevalent myocardial infarction. Glomerular macrophage infiltration, expression of alpha-SMA by mesangial cells, and overexpression of TGF-beta1 are the cellular changes associated with abnormal extracellular matrix deposition in nodular glomerulosclerosis.

Influence of calcium ions in the flow cytometric analysis of human CD8-positive cells.

The CD8 co-receptor is an important marker used to identify various lymphocyte subsets. A significant decrease in CD8alpha staining intensity was observed in the presence of divalent cation chelators. Peripheral blood mononuclear cells (PBMC) obtained from healthy volunteers were treated with calcium chelators, stained with different anti-human CD8 mAbs, and analyzed by flow cytometry. Calcium chelators caused a dose-dependent decrease in fluorescence intensity, using specific anti-human CD8alpha mAbs. This phenomenon was not due to CD8 internalization and could be reversed by the addition of calcium ions. In contrast, calcium depletion increased staining intensity with one anti-CD8beta mAb. Divalent cation chelators are used as cell anti-clumping agents in MACS or FACS applications. Researchers should be aware that such treatment could lead to the almost complete loss of fluorescence with selected anti-human CD8alpha mAbs. Since CD8 staining is used in conjunction with tetramer staining to identify antigen-specific cytotoxic human T cells, the effect of calcium depletion should be taken into account in experimental design.

Influence of chronic exposure to treated sewage effluent on the distribution of white blood cell populations in rainbow trout (Oncorhynchus mykiss) spleen.

Impairment of immune function in aquatic animals has been proposed as a possible consequence of low-level contamination of surface waters with anthropogenic substances such as through the discharge of wastewater into rivers, lakes, and oceans. The study at hand investigated the effects of chronic (32 weeks) exposure to sewage treatment plant (STP) effluent on the prevalence and distribution of different leucocyte populations in spleen samples of rainbow trout (Oncorhynchus mykiss). To simulate an infection, fish were injected intraperitoneally (ip) with inactivated Aeromonas salmonicida salmonicida, 6 weeks prior to the termination of the experiment. Immunohistological analysis revealed a marked decrease in thrombocyte numbers, an increase of monocytes, altered distribution of B-cells, and higher surface immunoglobulin expression, as well as activation of MHC class II expression in the spleen after exposure to 15% (v/v) effluent. The most prominent finding of the present study, however, was the occurrence of intraplasmatic deposits or inclusions with strong autofluorescence in spleen sections from effluent-exposed trout. In addition to effluent effects, injection of trout with A. salmonicida stimulated infiltration of monocytes, increased staining intensity on thrombocytes, and enhanced MHC class I expression in larger leucocytes surrounding melanomacrophage centres. In general, the current study demonstrates a marked, potentially adverse effect of STP effluent on spleen leucocytes and on the integrity of spleen tissue. The observed response suggests a constant unspecific stimulation of different leucocyte populations and is reminiscent of chronic inflammation.

RETENTION OF REGULATORY ACTIVITY IN CD4+CD25+ T-CELLS ISOLATED FROM RENAL TRANSPLANT RECIPIENTS FOLLOWING EX-VIVO EXPANSION

O247* Aims: Emerging evidence suggests that donor alloimmune responses mediated via the indirect pathway contribute to ongoing tissue injury in chronic allograft nephropathy. Recently active regulation of the indirect pathway alloimmune response by a sub-population of CD4+ cells co-expressing the IL2 receptor α chain, CD25 has been demonstrated in experimental and clinical studies. We sought to develop a protocol for the expansion and characterization of CD4+CD25+ T-regulatory (Treg) cells ex vivo. Methods: CD4+CD25+ T cells were purified by magnetic bead positive selection from peripheral blood samples of renal transplant recipients. Cells from 9 patients were expanded ex vivo for three, two weekly, cycles in the presence of IL-2, plate-bound anti-CD3 and irradiated syngeneic feeder cells previously incubated with synthetic peptides corresponding to the hypervariable regions of the β chain of the donor HLA-DR molecule(s) to which the transplant recipient was mismatched. Following the first expansion cycle the cell population was further enriched for the Treg phenotype by flow sorting the cells to acquire cells co-expressing high levels of CD25. Results: Retention of the regulatory phenotype was confirmed at time points of up to six weeks. Triple color flow cytometry examining the expression of molecular markers of regulatory activity including the negative co-stimulatory molecule CTLA4, GITR, CD45RO and OX40 demonstrated a progressive increase in staining intensity that paralleled that of CD25 suggesting enrichment of the regulatory phenotype in the CD25hi population. Quantitative PCR confirmed higher expression levels of FoxP3, a key gene in the development of regulatory function in CD4 cells expressing high levels of CD25 compared to ex-vivo expanded cells that had either lost CD25 expression or expressed it only at low levels. CD4CD25hi cells efficiently suppressed the proliferative responses of PBMCs to a combination of anti-CD3 and anti-CD28 confirming retention of a regulatory activity following expansion ex-vivo. Conclusions: The successful expansion of CD4+CD25+ Treg will greatly enhance our ability to further define the cell phenotype and its functional characteristics in vitro and in vivo as a prelude to possible cellular therapy for the management of CAN, the induction of tolerance and/or the minimization of the immunosuppresive burden in renal transplant recipients.

Up-regulation of cytokeratin expression in canine distemper virus-infected canine footpad epidermis.

Cytokeratin expression was assessed in footpad epidermis from dogs using immunohistochemistry. Four groups of dogs were studied: dogs with experimentally induced distemper and with canine distemper virus (CDV) in footpad epidermis (group 1, n = 7); dogs with experimentally induced distemper and without CDV in footpad epidermis (group 2, n = 4); inoculated dogs without distemper and without CDV in the footpad epidermis (group 3, n = 8), and noninoculated dogs without distemper (group 4, n = 2). No increase in thickness of the footpad epidermis was present in any of these groups. Sections of metacarpal or metatarsal pads were stained for cytokeratin (CK)14 (proliferation-associated), CK10 (correlated with early differentiation), and for involucrin (associated with terminal differentiation). CK14 was present in basal keratinocytes of all groups, but staining intensity decreased towards the corneal layer in groups 2-4, but not in group 1. CK10 was present in the spinous and granular layer of all groups, but staining of the granular layer was much stronger in group 1. Involucrin was present in the granular layer of footpads of group 1 and only in the upper part of this layer in groups 2-4. The results demonstrate increased staining intensity and/or wider distribution within the footpad epidermis in group 1 dogs when compared to the other groups. This was interpreted as up-regulation in expression of these proteins. These findings suggest that presence of CDV antigen and mRNA in footpad epidermis was associated with an increase in expression of CK14, CK10 and involucrin. The potential role of this up-regulation in cytokeratin expression in the development of CDV-induced digital hyperkeratosis remains speculative at the moment and requires further studies.

Immunohistochemical localisation of extracellular matrix proteins in the periodontium during cementogenesis in the rat molar

Objective: The development of the periodontium involves the coordinated expression of numerous extracellular matrix (ECM) macromolecules and their receptors (integrins). The aim of this study was to determine the expression of selected hard and soft tissue matrix molecules and the integrin α5β1 in the periodontal tissues, during cementogenesis in the rat molar. Methods: Using immunohistochemical methods, the distribution of the extracellular matrix proteins, fibronectin, tenascin, and bone sialoprotein (BSP), as well as the integrin subunits α5 and β1 were studied in rats aged 3, 5 and 8 weeks. Results: Fibronectin was widely distributed in the gingival epithelium, gingival connective tissue and in the periodontal ligament. Tenascin expression was less marked compared with fibronectin, but was more distinctly associated with cells and peri-cellular areas of the epithelial-connective tissue interface, the gingiva and within the periodontal ligament. The fibronectin-receptor α5β1 integrins were expressed by epithelial cells, periodontal ligament cells and gingival fibroblasts. A notable finding was the increased staining intensity of fibronectin, tenascin and α5β1 integrin in all 5-week old molar sections in the periodontal ligament matrix and cells, apical to the cemento-enamel junction (CEJ) along the alveolar crest (AC) ridge height. Bone sialoprotein was distinctly associated with the hard tissues of the periodontium as acellular cementum and alveolar bone matrix expressed bone sialoprotein throughout all sections, in all age groups. Conclusions: In conclusion, this study has demonstrated the selective distribution of several hard and soft tissue matrix molecules during periodontogenesis. The results highlight the complex nature of interactions of various proteins and molecules during development. The interactions between these molecules and their specific roles in development and regeneration await further investigation.

Expression of matrix metalloproteinase-26 and tissue inhibitors of metalloproteinases TIMP-3 and -4 in benign endometrium and endometrial cancer☆

Objective Matrix metalloproteinases (MMPs) and their physiological inhibitors, the tissue inhibitors of MMPs (TIMPs), play a key role in tumor cell invasion, angiogenesis, and growth. The aim of this study was to determine the expression and cellular distribution of MMP-26, TIMP-3, and TIMP-4 in endometrial cancers and benign endometrium throughout the menstrual cycle and the correlation with tumor histological subtype, stage, and grade. Methods Immunohistochemical analysis using polyclonal antibodies generated against pro- and active MMP-26, and mono- and polyclonal antibodies specific to TIMP-3 and TIMP-4, respectively, was performed. Results MMP-26, TIMP-3, and TIMP-4 are expressed in endometrial carcinomas ( N = 86) and benign endometrium ( N = 50) from various stages of the menstrual cycle. Semi-quantitative analysis of staining intensity indicated that endometrial carcinomas expressed more MMP-26, TIMP-3, and TIMP-4 compared to benign endometrium from the postmenopausal period, but not from the secretory phase of the menstrual cycle. The highest staining intensity was associated with endometrial epithelial cells, followed by vascular endothelial cells, myometrial smooth muscle cells, and endometrial stromal cells. Increased staining intensity of MMP-26 and TIMP-3 correlated with grade III tumors and MMP-26 and TIMP-4 with the depth of myometrial invasion in tumors histologically characterized as endometrioid adenocarcinoma, clear-cell, and papillary serous carcinoma staged/graded based on FIGO criteria. Conclusion MMP-26 and TIMP-4 are expressed in endometrium and endometrial carcinoma and their elevated expression and correlation with myometrial invasion suggests that MMP-26 and TIMP-4 may play a key role in endometrial tumor progression.

Thyroid Hormone Regulates Hepatic Triglyceride Mobilization and Apolipoprotein B Messenger Ribonucleic Acid Editing in a Murine Model of Congenital Hypothyroidism

Thyroid hormone modulates the expression of numerous genes that in turn regulate lipoprotein metabolism in vivo. We have examined the thyroid hormone-dependent regulation of apolipoprotein B (apoB) RNA editing in a strain of congenitally hypothyroid mice (Pax8(-/-)) that lacks thyroid follicular cells. Neonatal Pax8(-/-) mice demonstrate an approximately 10-fold increase in hepatic triglyceride content associated with a decrease in hepatic apoB RNA editing. Thyroid hormone administration resulted in hepatic triglyceride mobilization in conjunction with an increase in hepatic, but not intestinal, apoB RNA editing and without changing total apoB RNA abundance. ApoB RNA editing is mediated by a multicomponent enzyme complex whose catalytic core contains two proteins, apobec-1 and apobec-1 complementation factor (ACF). Hepatic ACF mRNA and protein abundance decreased in Pax8(-/-) mice, with restoration after thyroid hormone administration, whereas apobec-1 mRNA and protein abundance were unchanged. Immunohistochemical analysis revealed increased staining intensity of ACF within hepatocyte nuclei of treated mice, findings confirmed by Western analysis of isolated nuclei. In vitro RNA editing assays demonstrated that supplementation with recombinant ACF alone restored enzymatic activity of S100 extracts from hypothyroid, Pax8(-/-) mice. These data demonstrate that thyroid hormone modulates murine hepatic lipoprotein metabolism in association with tissue-specific effects on apoB RNA editing mediated through alterations in ACF gene expression.

The antiestrogen ICI 182,780 decreases the expression of estrogen receptor-alpha but has no effect on estrogen receptor-beta and androgen receptor in rat efferent ductules

BackgroundThe antiestrogen ICI 182,780 has been used successfully as an alternative experimental model for the study of estrogen action in the rodent adult male reproductive tract. Although ICI 182,780 causes severe alterations in testicular and efferent ductule morphology and function, the effects on the expression of estrogen and androgen receptors in the male have not been shown.MethodsIn the present study, adult male rats were treated with ICI 182,780 for 7 to 150 days, to evaluate the time-response effects of the treatment on the pattern of ERα, ERβ and AR protein expression in the efferent ductules. The receptors were localized using immunohistochemistry.ResultsERα, ERβ and AR have distinct cellular distribution in the testis and efferent ductules. Staining for ERα is nearly opposite of that for ERβ, as ERα shows an increase in staining intensity from proximal to distal efferent ductules, whereas ERβ shows the reverse. Androgen receptor follows that of ERα. ICI 182,780 caused a gradual but dramatic decrease in ERα expression in the testis and efferent ductules, but no change in ERβ and AR expression.ConclusionsThe differential response of ERα and ERβ proteins to ICI 182,780 indicates that these receptors are regulated by different mechanisms in the male reproductive tract.

Expression of a plant-associated human cancer antigen in normal, premalignant and malignant esophageal tissues

To study the relationship between the expression profiles of a plant-associated human cancer antigen and carcinogenesis of esophagus and its significance. We analyzed expression of a plant-associated human cancer antigen in biopsy specimens of normal (n=29), mildly hyperplastic (n=29), mildly (n=30), moderately (n=27) and severely dysplastic (n=29) and malignant esophageal (n=30) tissues by immunohistochemistry. The plant-associated human cancer antigen was mainly confined to the cytoplasm and showed diffuse type of staining. Positive staining was absent or weak in normal (0/30) and mildly hyperplastic tissue samples (2/29), while strong staining was observed in severe dysplasia (23/29) and carcinoma in situ (24/30). There was significant difference of its expression between normal mucosa and severely dysplastic tissues (P<0.001) or carcinoma in situ (P<0.001). Significant difference was also observed between mild dysplasia and severe dysplasia (P<0.001) or carcinoma in situ (P<0.001). An overall trend toward increased staining intensity with increasing grade of dysplasia was found. There was a linear correlation between grade of lesions and staining intensity (r=0.794, P<0.001). Samples from esophageal cancer showed no higher levels of expression than those in severely dysplastic lesions (P>0.05). The abnormal expression of this plant-associated human cancer antigen in esophageal lesions is a frequent and early finding in the normal-dysplasia-carcinoma sequence in esophageal carcinogenesis. It might contribute to the carcinogenesis of esophageal cancer. The abnormal expression of this plant-associated human cancer antigen in esophageal lesion tissues may serve as a potential new biomarker for early identification of esophageal cancer.

Expression of Transforming Growth Factor β1 in Bronchial Biopsies in Asthma and COPD

The role of transforming growth factor β1 (TGF β1) in airway remodeling in asthma and chronic obstructive pulmonary disease (COPD) has not been fully described. To evaluate the possible pathogenetic role of TGF β1 in asthma and COPD, immunohistochemical expression of TGF β1 was described in bronchial biopsies from patients with asthma and COPD compared with healthy individuals. Twelve subjects with asthma, 13 subjects with COPD, and 10 healthy individuals enrolled in the study. Bronchial biopsies were stained with hematoxylin and eosin and anti‐TGF β1 antibody. As a result, immunoreactive TGF β1 was mainly localized in association with connective tissue in all groups. The staining intensity was not statistically different among the groups in bronchial epithelium, whereas it was significantly higher in the group of asthma in the submucosa. Because there is evidence showing a significant increase of staining intensity in the submucosa from asthmatics but not from subjects with COPD, we may conclude that TGF β1 may play a significant role in pathogenesis of asthma but not in COPD.

Hepatic immunohistochemical staining with a monoclonal antibody against HCV-E2 to evaluate antiviral therapy and reinfection of liver grafts in hepatitis C viral infection

Background/Aims: A simple and reproducible hepatic immunohistochemical staining (IHS) for hepatitis C virus (HCV) is not available. We aimed to validate hepatic IHS with monoclonal antibody (Mab) IG222, directed against the HCV-envelope 2 (E2) protein.Methods: A three-step indirect immunoperoxidase method was used for frozen sections and a two-step indirect EnVision technique was used for paraffin-embedded sections.Results: Naturally or in vitro HCV infected primary human hepatocytes were immunoreactive to HCV-E2. In the patient study (n=253), IHS had a sensitivity of 96% and a specificity of 91%. Six patients who showed positivity in the liver with Mab IG222, but remained anti-HCV and HCV-RNA negative, had hepatitis C-like changes in their liver biopsy. In one patient HCV-RNA could be detected in the liver biopsy. We confirmed early graft reinfection in patients transplanted for HCV-related disease (34 patients with serial biopsies). Treatment for acute cellular rejection with steroids was associated with an increase in staining intensity. In nine patients with clearance of HCV-RNA during antiviral therapy, seven achieved negativation of immunoreactivity and two a marked reduction.Conclusions: The IHS with Mab IG222 is an accurate tool for diagnosis and clinical management of chronic hepatitis C.

Expression of the lung resistance-related protein in human and rat hepatocarcinogenesis.

Lung resistance-related protein (LRP) plays an important role in chemoresistance of tumor cells probably by altering nuclear-cytoplasmic transport processes. We analyzed the association between LRP expression and hepatocarcinogenesis in humans and rats by RT-PCR, immunoblotting, and immunohistochemistry. LRP was found in hepatocytes and bile epithelia of normal human and rat liver showing distinct interindividual variations. In human tissues, the LRP expression levels of dysplastic liver nodules, hepatocellular adenomas, and carcinomas were highly variable, including decreased but also distinctly increased staining intensities. Mean expression levels, however, were comparable to the surrounding tissue. Considerable levels of LRP mRNA and protein were also found in human hepatoma cell lines. To study LRP expression from the beginning of hepatocarcinogenesis onward, rats were subjected to a tumor initiation/promotion protocol leading to preneoplastic hepatocytes present as single cells or multicellular clones, followed by adenoma and carcinoma. All of the (pre)neoplastic rat liver lesions expressed, comparable to the surrounding tissue, considerable amounts of LRP. We conclude that LRP might be one mechanism involved in the intrinsically high but variable chemoresistance of normal and (pre)neoplastic hepatocytes.

Iso-butanol saturated water: a simple procedure for increasing staining intensity of resin sections for light and electron microscopy.

The addition of the alcohol iso-butanol (2-methylpropan-1-ol) to water was found to improve the post-staining procedures for semi-thin and ultrathin resin sections, for both light and electron microscopy. Stain penetration was enhanced with samples embedded in both acrylic and epoxy resins and provided structural information not previously seen. These improvements were found with general (non-specific) stains and a fluorescence stain for light microscopy, as well as for a range of heavy metal stains for electron microscopy. The use of this water/solvent medium also gave improved results when used in a variety of immunogold labelling procedures, resulting in a greater specific label density without affecting background gold levels. The use of this solvent/water medium may have wider applications for other types of staining.