Indirect Immunoperoxidase Technique Research Articles

Upregulation of redox-regulating protein, thioredoxin, in endomyocardial biopsy samples of patients with myocarditis and cardiomyopathies.

An important role of redox regulation in myocardial diseases and heart failure has been postulated. Thioredoxin (TRX) is a redox-regulating protein. Recent studies indicated a possible association between plasma TRX concentrations and the severity of heart failure. Accordingly, we investigated the myocardial expression of TRX in patients with myocarditis and cardiomyopathies. Four cases of hypertrophic cardiomyopathy (HCM), 10 of dilated cardiomyopathy (DCM), 6 of myocarditis, and 5 of controls were studied. Right and left ventricular endomyocardial biopsy samples were obtained at the diagnostic cardiac catheterization. The samples were processed for immunohistological staining for TRX, which was done by the indirect immunoperoxidase technique. 8-hydoxy-2'-deoxyguanosine (8-OHdG), one of the major DNA base-modified products, was also detected for an established marker for oxidative stress. TRX immunoreactivity was none or trivial in control specimens. Positive TRX staining was found in 6 cases; 3 in active myocarditis and 3 in DCM. The positive staining was found in infiltrating cells and damaged myocytes in the perinecrotic lesions. Damaged myocytes were also positive for 8-OHdG All the 3 cases of DCM positive for TRX stain showed severe left ventricular hypertrophy on electrocardiogram and highly elevated left ventricular end-diastolic pressure (> 24 mmHg), suggesting the overload of oxidative stress by hemodynamic impairment. Myocardial TRX was upregulated in myocarditis and cardiomyopathies with active necrotic stage associated with DNA damage, which may reflect the oxidative stress overload in hemodynamically uncontrolled status.

Immunohistochemical staining of IGF-I, IGF-binding proteins-1 and -3, and transforming growth factor beta-3 in the umbilical cords of preeclamptic patients.

To detect the immunoreactivity of insulin-like growth factor-I, insulin-like growth factor-binding proteins-1 and -3 and transforming growth factor beta-3 in the umbilical cords of normal and preeclamptic patients. Umbilical cords were obtained from 15 normal and 15 preeclamptic patients. Immunoreactivities were determined using either indirect immunofluorescence or immunoperoxidase techniques on formalin-fixed, paraffin-embedded sections. Staining intensity was graded by a semiquantitative scoring method. The results were compared by Mann-Whitney U-test. The umbilical cords were thinner and the vessels were hypoplastic in the preeclamptic group. Moderate staining intensity for insulin-like growth factor-I, insulin-like growth factor binding protein-1 and -3 and transforming growth factor-beta 3 was observed in normal patients. The preeclamptic group had mild and strong intensities for insulin-like growth factor-I and insulin-like growth factor binding protein-1, respectively, and intensity for insulin-like growth factor binding protein-3 did not change, but diffuse and increased intensity was observed for transforming growth factor-beta 3. Changes in the intensity of insulin-like growth factor-I and its major binding protein and the transformation of growth factor-beta 3 may play a role in the pathogenesis of preeclampsia by altering the structure and responsiveness of the umbilical cord.

Strategies for vascular targeting in tumors.

Strategies for vascular targeting in tumors.

Hereditary renal amyloidosis caused by a new variant lysozyme W64R in a French family

The number of proteins with mutations resulting in amyloidosis has continued to increase. Five proteins--transthyretin, fibrinogen alpha-A chain, apolipoprotein AI, lysozyme, apolipoprotein AII, cystatin C and gelsolin--can be associated with hereditary amyloidosis involving the kidney. A French family with a history of autosomal dominant hereditary amyloidosis with early sicca syndrome and nephropathy leading to renal failure after the fifth to the seventh decade was studied. Several tissue specimens obtained from the proband and his relatives were examined. Immunohistochemistry was performed on paraffin embedded sections using the indirect immunoperoxidase technique. We searched for mutations in the five exons and flanking introns of the lysozyme gene. Amyloid deposits from the bowel, labial salivary gland and kidney were intensively stained by anti-lysozyme antibody. Sequence analysis of lysozyme exon 2 from the affected individuals revealed a nucleotide substitution predicting a substitution of the amino acid at position 64 in the mature protein from tryptophane, an aromatic residue to the cationic residue arginine (W64R). We report a novel mutation (W64R) of the lysozyme that is associated with hereditary amyloidosis and prominent nephropathy. Since the treatment of hereditary amyloidosis greatly varies with the nature of the amyloid protein, thorough characterization of the latter is crucial for the management of the disease.

A study of primary central nervous system lymphoma in northern India

BACKGROUND Primary central nervous system lymphoma (PCNSL) is rare and accounts for 1 to 2% of all lymphomas. There are conflicting reports about the rise in incidence of PCNSL cases in the last two decades; this has largely been attributed to an increase in incidence of AIDS and other immunosuppressive states in some studies. This study was undertaken to view the trend of PCNSL at the Postgraduate Institute of Medical Education and Research, Chandigarh, which is a referral hospital in northern India. METHODS The PCNSL cases from our surgical material of 15 years (1985–1999) were reviewed. Slides were examined independently by three histopathologists. Immunophenotyping was done on paraffin-embedded tissue using indirect immunoperoxidase technique. RESULTS Out of a total of 3,325 intracranial tumors diagnosed during this period (1985–1999), there were 40 cases (1.2%) of PCNSL; gliomas accounted for 1,531 cases (46.04%). The age ranged from 24–75 years with the sex ratio (M:F) being 2:1. HIV serology, available in 14 cases, was negative in all. The parietal lobe was the most common site of involvement. Diffuse large cell lymphoma was the most common morphological type. Immunohistochemistry could be done in 31 cases; 28 cases were found to be B-cell type whereas 3 cases were T-cell type. No statistically significant increase was seen on comparing the number of cases at 5-year intervals. CONCLUSION This study revealed that there has been no significant increase in PCNSL cases over the last 15 years.

Clearing mechanisms of Vibrio vulnificus biotype I in the black tiger shrimp Penaeus monodon.

Vibrio species' infections are a common sequelae to environmental stress or other disease processes in shrimp, but the mechanism by which the shrimp eliminate the bacteria is poorly understood. In this study, the penetration, fate and the clearing of V. vulnificus were investigated in Penaeus monodon. A bacterial disease isolate from a shrimp farm was identified as V. vulnificus biotype I. Polyclonal antiserum was raised in rabbits against the bacterium and the specificity was verified by ELISA and immunoblot against a range of Vibrio spp. and other gram-negative bacteria. The bacteria were then administered to P. monodon juveniles by injection, immersion and oral intubation. An indirect immunoperoxidase technique was employed in a time course study to follow the bacteria and bacterial antigens in the tissue of the shrimp. Bacteria were cleared by a common route, regardless of the method of administration. Observations in immersion challenge were similar to a combination of those for oral and injection challenges. With immersion, bacteria entered the shrimp through damaged cuticle or via insertion points of cuticular setae. Shortly after entry, whole bacterial cells were observed in the haemolymph and connective tissue. They were either phagocytosed by haemocytes, or broken down outside host cells. Haemocytes containing bacterial cells or antigens (HCB) were observed in the connective tissue and haemolymph. HCB accumulated around the hepatopancreas, midgut, midgut-caecum, gills, heart and lymphoid organ. Free bacterial antigens also accumulated in the heart and lymphoid organ. Bacteria entering through the mouth by oral intubation or immersion were broken down so that only soluble or very fine particles entered the hepatopancreas. Bacterial antigens passed through the hepatopancreas into the haemolymph. Antigens were initially observed in the haemolymph sinuses and subsequently accumulated in the heart and lymphoid organ. Bacterial antigens were released from the shrimp, initially through the gills and subsequently through hepatopancreatic B-cells, branchial podocytes and sub-cuticular podocytes.

Proliferative activity in the cerebellar external granular layer evaluated by bromodeoxyuridine labeling

We have optimised an indirect immunoperoxidase technique demonstrating bromodeoxyuridine (BrdU) incorporation into dividing cells for cerebellar tissue sections of four-day-old rats injected with this marker. This permits confident identification of granule-cell precursors engaged in DNA synthesis in the external granular layer of the developing cerebellum. Preservation of BrdU immunoreactivity is attained using methanol/acetic acid fixation and different pretreatments before immunostaining, while unlabeled nuclei can be recognized clearly after Feulgen or hematoxylin counterstaining. We established conditions to ensure satisfactory BrdU uptake without affecting cell-cycle progression during the postlabeling time period. The dose of BrdU employed provides saturation S-phase labeling from at least 1 h after BrdU delivery. Various kinetic parameters and phase durations have been determined in experiments involving a single injection or cumulative labeling sequences, and the cycle time was calculated based on two models of generative behavior: steady-state and exponential growth. The working hypothesis of steadystate kinetics can be adopted successfully if the existence of neuroblasts with different proliferation rates is taken into account.

Neural cell adhesion molecule and neurothelin expression in human ameloblastoma

Purpose: The objective of the present study was to determine whether neural cell adhesion molecules (NCAM; CD56) and neurothelin (CD147) are expressed by adontogenic cells in the ameloblastoma. Materials and Methods: Frozen sections from ameloblastoma specimens (n = 7) were stained with monoclonal antibody-recognizing CD56 and CD147 molecules using indirect immunoperoxidase and indirect immunofluorescent techniques. Results: CD56 and CD147 molecules were strongly expressed by the peripheral columnar cells of the tumor nests. Neurothelin reactivity was also present in the stellate reticulum of the nests and in some stromal components. Conclusions: The presence of these antigens in ameloblastoma supports the classic view about the neural crest origin of cells giving use to this tumor. © 2001 Ameriacn Association of Oral and Maxillofacial Surgeons

Cadherins and integrins in renal cell carcinoma: an immunohistochemical study.

The aim of this study was to determine the expression of cadherins and integrins in renal cell carcinoma (RCC) and their relationship with tumor morphology and TNM status. Cadherin and integrin expression was investigated using an indirect immunoperoxidase technique, applying antibodies to E-, N-, P- and VE-cadherin and to alpha1, alpha2, alpha3, alpha4, alpha5, alpha6, and alpha(v) integrin subunits. Correlation of semiquantitatively scored adhesion molecule levels with histopathological parameters (cytology, growth pattern, nuclear grade) and TNM status was performed for 24 RCCs (17 clear cell, 3 granular, 3 spindle cell and 1 chromophobe cell type according to the WHO classification). E-cadherin and N-cadherin were present in most cases (88% and 67%, respectively) and were usually coexpressed. T3 RCCs displayed higher E-cadherin and N-cadherin levels than T1/T2 tumors regardless of tumor grade, suggesting that impairment of their function might exist without actual loss from tumor cells. P-cadherin was found focally in two RCCs only, while VE-cadherin was present on stromal vessel endothelium in five tumors, showing no differences with regard to cell type, growth pattern, tumor grade or TNM status. All integrins were present in the studied RCCs (ranging from 12% for alpha5 to 79% for alpha3), including those that are normally absent from adult kidney tissue (alpha4 and alpha5). Tumors of higher grade showed increased alpha(v) and decreased alpha6 levels, while RCCs with metastases less often showed diffuse alpha3 presence and never expressed alpha5 integrin. Our results suggest that the level of expression of N-cadherin and some integrins (most notably alpha3, alpha6 and alpha5) is associated with the capacity of RCC for local and distant spread, regardless of tumor grade.

Presence of Gastric Autoantibodies Impairs Gastric Secretory Function in Patients with Helicobacter pylori-positive Duodenal Ulcer

Background: Although the association between Helicobacter pylori infection and gastric autoimmunity is now well established, to date little is known about the significance of anticanalicular autoantibodies in patients with duodenal ulcer (DU). We therefore investigated the prevalence of serum antiparietal cell autoreactivity in DU patients as well as the relationship between these autoantibodies, gastric histopathology and gastric secretory function in this setting. Methods: Forty-one consecutive patients with H. pylori-positive DU were initially recruited. In all patients, basal (BAO) and pentagastrin stimulated acid output (PAO), fasting and meal-induced serum gastrin levels, as well as serum pepsinogen I concentrations, were measured. Antral and body gastritis was evaluated according to the Sydney system. Serum anticanalicular autoreactivity was determined by the indirect immunoperoxidase technique. Results: Serum anticanalicular autoantibodies were found in 7 out of 34 patients (20%). The presence of these antibodies was associated with a significantly higher grade of body gastritis (activity: 1.9 versus 0.9) as well as with significantly higher fasting and meal stimulated gastrin levels (mean fasting gastrin, 76.4 (15.2) pg/ml versus 59.3 (20.5) pg/ml). In addition, PAO values were significantly lower in patients with gastric autoantibodies than in those without this autoreactivity (mean 0.35 (0.16) mmol kg-1 h-1 versus 0.49 (0.16) mmol kg-1 h-1). In contrast, no significant differences were found between patients with and without anticanalicular autoantibodies as regards fasting serum pepsinogen I concentrations. Conclusions: Serum anticanalicular autoantibodies can be detected in 20% of patients with DU and are associated with a more severe pattern of body gastritis, higher gastrin levels and decreased peak acid secretion values. Their presence could account for the normal or reduced acid output which can be seen in a subset of DU patients.

Expression of β1 integrins in human dental pulp in vivo: a comparative immunohistochemical study on healthy and chronic marginal periodontitis samples

The objective of this study was to determine the tissue distribution of beta 1 integrin chains in sound human dental pulps and to compare the findings with connective tissue compartments of other organs and to pulp tissue in teeth extracted due to periodontal disease. Freshly frozen pulp tissue samples from teeth extracted for orthodontic reasons were examined and compared to samples from teeth extracted due to chronic (marginal) periodontitis. beta 1 integrin chains were determined using an indirect-immunoperoxidase technique. Seven monoclonal antibodies recognizing alpha 1, alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and beta 1 chains of Very Late Activation Antigen (VLA) integrins were used for this purpose. VLA-1, VLA-2, VLA-3 and VLA-5 were expressed by vascular endothelium and vascular smooth muscle in varying intensities in both groups. VLA-6 reactivity was observed in the basal surfaces of arterial, venous and capillary endothelia. Our results indicate that there was no significant difference in the expression of VLA integrins in sound pulp tissue when compared to the samples from chronic (marginal) periodontitis and the connective tissue compartments of other viscera. The present findings suggest that human dental pulp tissue is not different from other connective tissue compartments in the body with respect to VLA integrin expression, and chronic marginal periodontitis does not affect pulp tissue to a histopathologically detectable extent.

Inhibin expression in normal and pre-eclamptic placental tissue.

Serum inhibin levels increase during normal pregnancy, but are significantly higher in patients with pre-eclampsia. The aim of this study was to demonstrate possible increased expression of inhibin within the placentas of women with pre-eclampsia compared with non-pre-eclamptic controls. Cellular expression of inhibin alpha and beta A subunits was studied using immunohistochemistry on formalin-fixed, paraffin-embedded placental sections from cases of pre-eclampsia (n = 23) and gestational age-matched non-pre-eclamptic controls (n = 16). Immunohistochemistry was performed using monoclonal antibodies against inhibin alpha and beta A subunits by the indirect immunoperoxidase technique. Intensity of staining was graded by a semiquantitative scoring method. Differences in distribution and intensity of staining between control and pre-eclamptic placentas were analyzed using a nonparametric Mann-Whitney U test. Staining for both inhibin alpha and beta A was predominantly confined to the cytoplasm of syncytiotrophoblast, with weak expression within intermediate trophoblast. The intensity of staining for inhibin alpha was significantly greater in the syncytiotrophoblast of pre-eclamptic patients (mean staining intensity controls = 0.97, disease = 1.87; p < 0.001). Inhibin beta A staining was generally stronger than for the alpha subunit, and was also significantly increased in pre-eclamptic patients compared with controls (mean controls = 1.72, disease 2.19; p < 0.05). This is the first evidence for increased placental inhibin presence in pre-eclampsia, suggesting increased inhibin production within the placenta, a finding that could account for increased serum inhibin levels in pre-eclampsia.

Nerve growth factor and Neurotrophin-3 modulate the rabies infection of adult sensory neurons in primary cultures

With the aim of determining if the proportion of rabies virus (RV)-infected adult neurons from dorsal root ganglion are affected by in vitro treatment with different neurotrophins, experiments using Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF) or Neurotrophin-3 (NT-3) as supplements for cells in culture were performed. Cultures treated with three different concentrations of each of the neurotrophins mentioned were infected with Challenge Virus Standard RV strain. An indirect immunoperoxidase technique was performed for the detection and counting of infected cells. NGF (2 ng ml −1 and 10 ng ml −1) and NT-3 (1 ng ml −1 and 5 ng ml −1) induced a significant reduction of infected neurons. None of the cultures treated with BDNF showed changes in the percentage of infected neurons. Likewise, the proportion of infected non-neuronal cells (Schwann cells and fibroblasts) was not altered by the treatment with neurotrophins. In addition, morphometric analysis of total and virus-immunoreactive neurons in culture were carried out, the neurotrophin treatment induced variations in the profile of neurons preferentially infected, since cell diameters in the infected cell population are different in the presence of NGF and NT-3. Data presented here could indicate a putative participation of neurotrophin receptor or biochemical modifications induced by neurotrophin treatment that affect the infection. The primary culture of dorsal root ganglion cells from adult mice is a very useful model for studying the basic phenomena of the RV-neuron interaction.

Expression of prolactin receptors in rat reproductive tissues during periovulatory prolactin imbalance.

Indirect immunoperoxidase technique in combination with cytophotometry showed that prolactin imbalance in the periovulatory period is characterized by considerable changes in the expression of prolactin receptors in uterine glands and endometrial stroma (but not in the myometrium) and in the pituitary gland and hypothalamus, while receptor compartmentalization pattern remains unchanged.

Modulation of ICAM-1 tissue expression in patients with liver transplantation (LT) and acute rejection (AR) after glucocorticoid treatment.

Acute rejection (AR) is a frequent complication following liver transplantation (LT). ICAM-1 may be involved in its pathogenesis. High doses of glucocorticoids are the standard treatment in these patients. The aim of this study was to describe corticoid effects on ICAM-1 tissue expression in liver biopsies of patients with LT and AR. The study included liver biopsies performed before and after treatment in 12 patients with LT and proven AR. In 10 patients AR was reversible and in 2, was steroid resistant. For immunohistochemistry, an indirect immunoperoxidase technique was used. Each histology section was semiquantitatively evaluated as follows: 0: < 10% staining, 1: 10-25%, 2: 25-50%, 3: > 50%. The control group comprised nine patients with LT and normal liver biopsies. In pre-treatment liver biopsy samples, ICAM-1 was markedly expressed on sinusoidal cells (2.41 +/- 0.66), and there was also expression on periportal (0.66 +/- 0.65) and perivenular hepatocytes (0.83 +/- 0.57). By contrast, in the liver tissue from the control group, sinusoidal ICAM-1 reactivity was significantly lower (0.88 +/- 0.33; P < 0.05), and hepatocytes showed no reliable ICAM-1 expression. After steroid treatment the intensity of ICAM-1 decreased significantly in sinusoids (1.5 +/- 0.67; P < 0.05) and in perivenular hepatocytes (0.25 +/- 0.86; P < 0.05). Additionally, we also observed a decreased ICAM-1 reactivity in portal hepatocytes (0.25 +/- 0.62), but these differences did not reach statistical significance. Remarkably, after treatment, hepatocytes did not show ICAM-1 reactivity in resolved AR, but in corticoid-resistant patients AR did not change or increase. In conclusion, in patients with LT and AR, ICAM-1 was expressed in hepatocytes and with more intensity in sinusoid cells. Additionally, a down-regulation of the ICAM-1 tissue expression after corticoid treatment may exist, although in corticoid-resistant AR no modulation on ICAM-1 tissue expression was observed.

Distribution of luteinizing hormone-releasing hormone in the upper brainstem and diencephalon of the cat: An immunocytochemical study

The distribution of luteinizing hormone-releasing hormone (LH-RH)-immunostained cell bodies and fibres was studied in the brainstem and diencephalon of the cat using an indirect immunoperoxidase technique. The brainstem and the thalamus were devoid of immunostained cell bodies, whereas in the hypothalamus immunopositive perikarya were observed in the supraoptic nucleus, the anterior hypothalamus, the preoptic region and in the arcuate nucleus. Our findings also showed that the hypothalamus is richer in immunostained fibres, and that in this region such fibres are more widely distributed than in the thalamus and upper brainstem. No immunopositive fibres were observed in the lower brainstem. Our results point to a more widespread distribution of LH-RH-immunostained perikarya in the cat hypothalamus than that previously reported in the cat; a similar distribution to that found in the rat, and a more restricted distribution than in primates. Additionally, our study shows a more widespread distribution of immunostained fibres in the cat brainstem and diencephalon than that previously described for other mammals. In this context, our results describe for the first time in the mammals central nervous system fibres containing LH-RH located in the stria medullaris of the thalamus, the supramammillary decussation, the laterodorsal and lateroposterior thalamic nuclei, the nucleus reuniens, the supraoptic nucleus, and the optic chiasm. Thus, our findings reveal that LH-RH-immunostained structures are widely distributed in the upper brainstem and in the diencephalon of the cat, suggesting that the peptide may be involved in several physiological functions.

Expression of the E-cadherin/catenin (alpha-, beta-, and gamma-) complex correlates with the macroscopic appearance of early gastric cancer.

E-cadherin and its associated cytoplasmic proteins, alpha-, beta-, and gamma-catenins, play an essential role in the control of epithelial differentiation. We have previously shown that loss or down-regulation of E-cadherin/catenin correlates with poor survival in advanced gastric adenocarcinoma. The aim of this study was to assess the expression of E-cadherin and catenins in early gastric cancers (EGCs). Immunohistochemical staining for E-cadherin and alpha-, beta-, and gamma-catenins was performed on 41 paraffin-embedded gastrectomy specimens of EGC using an indirect immunoperoxidase technique. The pattern of expression and cellular localization of the E-cadherin/catenin complex in tumour cells were correlated with the macroscopic appearance of the tumour according to the Japanese Endoscopic Society classification. The tumours were classified as follows: three type I (protruding) and 38 type II (superficial), of which ten were type IIa (elevated), one was type IIb (flat), and 27 were type IIc (depressed). E-cadherin and alpha-, beta-, and gamma-catenins were expressed at the cell-cell junctions in normal mucosa. Forty out of 41 tumours showed abnormal expression (loss of membranous immunoreactivity and/or nuclear staining) of at least one component of the E-cadherin catenin complex. Loss of E-cadherin immunoreactivity was more frequently seen in type IIb (1/1, 100%) and type IIc (27/27, 100%) than in type I (1/3, 33%) and type IIa (1/10, 10%) (p<0.01). Abnormal expression of E-cadherin and alpha-catenin was more frequently seen in diffuse-type than in intestinal type tumours (p<0.05). Abnormal immunoreactivity of beta- and gamma-catenin, including nuclear localization, was observed in 34% and 7.3% of tumours, respectively, but there was no significant correlation with tumour type or endoscopic appearance. In conclusion, abnormal expression of the E-cadherin/catenin complex occurs in EGC and seems to correlate with macroscopic appearances.

Expression of CD146 adhesion molecules (MUC18 or MCAM) in the thymic microenvironment

The thymic microenvironment is thought to play a critical role in T-lymphocyte development, providing signals both via cell surface molecules such as adhesion molecules and soluble molecules. The present investigation is focused on immunoelectron microscopical analysis of distribution patterns of CD146 adhesion molecules (MUC18 or MCAM) in the microenvironment of normal human thymus, using the pre-embedding indirect immunoperoxidase technique. The anti-CD146 monoclonal antibody (mAb) revealed strong membraneous labelling of immature thymocytes at both the light and electron microscopical level. Proliferating thymocytes, most of the epithelial cells, macrophages, endothelial cells and smooth musle cells of small vessels and capillaries showed both membraneous and cytoplasmic labelling with anti-CD146 mAb as was demonstrated by electron microscopy. In contrast, these cells displayed a strong cytoplasmic immunoreactivity at the light microscopical level. The extracellular matrix was also stained with the anti-CD146 mAb. No labelling was observed in interdigitating cells. Interestingly, the CD146 molecule was strongly expressed on apical and lateral membranes of endothelial cells as was demonstrated electron microscopically. This selective CD146 labelling of capillary endothelium mainly localized at the cortico-medullary junction may be manifestations of lymphocyte transmembrane migration and lymphocyte homing. In conclusion, the present study suggests that CD146 is expressed by most elements of the microenvironment of normal human thymus. Therefore, it may be a pan-antigen which is essential for the maintenance of thymic architecture and function.

Endothelial cell adhesion molecules in human dental pulp: A comparative immunohistochemical study on chronic periodontitis

Migration of leukocytes to inflammation sites through vascular endothelium is controlled by the interactions of adhesion molecules expressed on both endothelial cells and leukocytes, most of which are already covered by cluster of differentiation (CD) codes. We examined the expression of a variety of endothelial cell adhesion molecules in human dental pulp vasculature to obtain further evidence on the tissue distribution and function of these molecules by using an indirect immunoperoxidase technique. We obtained the pulp tissue samples from teeth extracted due to orthodontic reasons as controls and compared with those extracted due to chronic periodontitis. In all samples, both CD31 and CD146 were expressed by arterial, venous, and capillary endothelia. There was no significant difference between the staining intensity of normal and inflamed pulp tissues. CD102 expression on the endothelium was significantly stronger in chronic periodontitis pulp samples. CD106, CD62-E, CD62-P, CD105, and CD54 were variably expressed in control and chronic periodontitis groups. Our results indicate that CD102 represents the major endothelial cell adhesion molecule probably involved in the inflammatory reactions in chronic periodontitis.

Computerized histomorphometric assessment of protocol renal transplant biopsy specimens for surrogate markers of chronic rejection.

Chronic transplant rejection has emerged as the commonest cause of long-term renal allograft failure, and early identification of those grafts at risk could allow the targeting of specific therapies aimed at delaying this process. This study explores the usefulness of quantitative immunohistochemistry in defining biopsy-based surrogate markers of allograft damage. A consecutive series of 52 renal transplant recipients immunosuppressed with cyclosporine were studied. Needle core transplant biopsies were performed at 1, 3, and 6 months postoperatively. Immunostaining for collagen III, and smooth muscle actin, tenascin, and infiltrating leukocytes was performed using an indirect immunoperoxidase technique. The interstitial area stained (%) was measured using a semiautomatic image analysis system. The results were related to glomerular filtration rates (GFR) measured at 6, 12, and 24 months after transplantation using rank correlation coefficients. The area fraction of immunostained collagen III correlated with 6-month GFR (r=-0.42, P=0.005) and was predictive of 12-month GFR (r=-0.32, P=0.03). An area fraction of immunostained collagen III of >40% at 6 months was associated with a significantly lower GFR at 24 months, compared with a percentage area of < or =40% (31+/-4 versus 45+/-4 ml/min/1.73 m2, P=0.01). Furthermore, a collagen III of >40% at 6 months identified patients who were at risk of progressive deterioration in graft function. Grafts with poorer long-term function can be predicted using 6-month protocol biopsy specimens immunostained for collagen III. This should prove to be a useful ad interim surrogate marker of allograft damage in studies addressing the effects of new immunosuppressive agents on the development of chronic rejection.