Avidin-biotin Peroxidase Complex Research Articles

Nuclear Localization of STAT5A Modified with O-Linked N-Acetylglucosamine and Early Involution in the Mammary Gland of Hirosaki Hairless Rat

Hirosaki hairless rat (HHR) is a mutant strain spontaneously derived from Sprague-Dawley rats (SDR), and its inheritance is autosomal recessive. In addition to hair loss, female HHRs show involution of the mammary gland at an early stage of lactation. In the present study we investigated the mammary gland development in HHR. Morphological examinations revealed that HHR mammary glands are underdeveloped in virgins and exhibit distended alveoli on day 1 of lactation (L1), followed by involution. Milk secretion was observed on L1 in HHR. Whey acidic protein and other proteins were increased in milk of HHR and heterozygous rats on SDS-polyacrylamide gel electrophoresis. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed apoptosis induction in HHRs at an early stage of lactation. By Western blotting, signal transducer and activator of transcription (STAT) 5A levels in cytoplasmic and nuclear fractions of the mammary glands were not different between HHR and SDR on L1 and L7. Nuclear localization of STAT5A in HHR and SDR was confirmed by immunohistochemistry. Tyr-phosphorylated STAT5A was not detected in HHR but was detected in SDR nuclear fractions. Several proteins modified with O-linked N-acetylglucosamine (O-GlcNAc) were detected in HHR nuclear extract on L1, although not in SDR or heterozygous rats by Western blotting. When HHR nuclear extract was applied to wheat germ agglutinin-agarose, a part of STAT5A was recovered in bound fractions. STAT5A of SDR or heterozygous rat nuclei were not bound to the lectin. Electrophoretic mobility shift assay revealed that STAT5A modified with O-GlcNAc is bound to the STAT5-responsive element. These results indicate that the mammary glands of HHR showed terminal differentiation for a short period, followed immediately by involution. In HHR, STAT5A is modified with O-GlcNAc but is not Tyr-phosphorylated. This type of glycosylation is suggested to be involved in the transient activation of STAT5A in HHR.

Unfavourable outcome of Hashimoto encephalopathy due to status epilepticus

Sirs: A 27-year-old woman with a history of Hashimoto’s thyroiditis presented with psychotic symptoms and myoclonus. Four months earlier, her relatives had noticed myoclonic jerks in the four limbs and depression. During the following weeks, the patient also manifested visual hallucinations and generalized tonic-clonic seizures. In addition, progressive cognitive deterioration and slowed psychomotor activity became evident. One month later, she experienced two episodes of convulsive status epilepticus lasting over 50 minutes that resolved after intravenous lorazepam and phenobarbital had been given. On admission to our department, neurological examination revealed disorientation, fine finger tremor and spontaneous and stimulus-induced myoclonus at the four limbs. Neuropsychological evaluation showed Mini-Mental Status Examination’s score of 23/30, verbal memory deficit, frontal dysfunction and impaired attention. The patient was taking L-thyroxine, 100 μg/day, valproate, 750 mg/day and phenobarbital, 100 mg/day. Laboratory analysis demonstrated high titres of thyroid microsomal antibodies (1781 IU/ml; normal < 60) antithyreoglobulin antibodies (127.5 IU/ml; normal < 28.7) and TSH (40.9 μU/ml; normal < 4.2 μU/ml). FT3 and FT4 were within the normal limits. Antinuclear and antiDNA antibodies were normal. Serological test for syphilis and HIV were negative. CSF examination showed slightly elevated protein levels (55 mg/dl, normal < 40) and two oligoclonal bands. 14-3-3 protein was absent and there was no evidence of bacterial or viral infection. EEG showed diffuse slowing of background activity and bifrontal theta activity. Photic stimulation induced massive myoclonus in the four limbs (Figure). Brain MRI and 99Tc SPECT were unremarkable. Genetic study excluded Unverricht-Lundborg and Lafora disease, and MERRF. As diagnosis of Hashimoto encephalopathy (HE) was made, intravenous treatment with methylprednisolone 1 g followed by prednisolone 40 mg/day was started. No significant improvement was observed and, 6 days later, the patient manifested progressively increasing massive myoclonus, culminating in convulsive status epilepticus. Intravenous benzodiazepines failed to interrupt status epilepticus and pentothal anesthesia was initiated. Ictal activity was never fully suppressed at EEG prolonged monitoring. The patient died after a prolonged comatose state from cardiac failure two weeks later. Autopsy was performed. Microscopic examination revealed focal infiltration of thyroid by lymphocytes. Fixed brain tissue was paraffin-processed, and 10 μm sections were obtained. Gross examination revealed few hemorrhagic areas in the pons. Immunohistochemical staining by using the avidin-biotinperoxidase complex method for assessment of astrocytes (glial fibrillary acid protein), microglia (HLA-DR) and T cells (CD3, CD4, CD8, CD19, CD45), did not show lymphocytic infiltrates. PrPSc staining was negative and no evidence of PAS+ inclusion bodies was found in the cerebral parenchyma. The clinical picture of our patient suggested the diagnosis of diffuse (type II) HE [1, 2]. HE is defined by the association of encephalopathy with the presence of antithyroid antibodies [1, 2]. Steroid treatment is usually effective but refractory cases have been reported [2]. Accordingly, clinical LETTER TO THE EDITORS

Expression of inductive nitric oxide synthase and vascular endothelial growth factor in colonic carcinoma, and their effects on tumor angiogenesis

To investigate the expression of inductive nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF), and their relations with clinicopathological parameters in colonic carcinoma. Immunohistochemistry (streptomycin avidin-biotin peroxidase complex, SP) was used to detect the expression of iNOS, VEGF, collagen IV, FVIII Ag in colonic adenocarcinoma, and micro vessel density (MVD) was counted. The positive rates of iNOS and VEGF in colonic carcinoma were 76% and 80% respectively. The MVD in iNOS-positive carcinoma and iNOS-negative carcinoma were 37.0+/- 5.7 and 30.5+/- 4.9. The MVD in VEGF-positive carcinoma and VEGF-negative carcinoma were 39.5+/- 8.5 and 30.7+/- 4.4. The expressions of iNOS and VEGF were positively correlated with MVD. The expression of VEGF was correlated with tumor invasion, differentiation and lymph no de metastasis, but not with patients age,sex and histologic type. The expression of iNOS and VEGF may play a role in the angiogenesis, metastasis and invasion of colonic carcinoma.

VEGF significance in peritoneal recurrence from gastric cancer

In gastric cancer, the management of peritoneal dissemination in the Peritoneal cavity is extremely important; however, peritoneal dissemination in the final stage of gastric cancer remains untreatable. Peritoneal dissemination involves several steps, including tumor-cell attachment, invasion, and growth in the peritoneum. Many cytokines, growth factors, matrix metalloproteinases (MMPs), and angiogenic factors play important roles in these steps. So far, few studies have investigated the correlation, if any, between peritoneal dissemination and the angiogenic factor, vascular endothelial growth factor (VEGF). Immunohistochemical staining, using the avidin-biotin peroxidase complex method, was performed on slides of surgical specimens from 40 patients with stage II gastric cancer with serosal invasion, who underwent surgery at our hospital between 1990 and 2000. Anti-human VEGF rabbit polyclonal IgG was used as the primary antibody. VEGF expression was classified in one of four categories depending on the percentage of tumor-cell staining (P). VEGF expression was also classified in one of three categories depending on the staining intensity (I). The VEGF expression score was calculated as P x I. There were ten patients with peritoneal recurrence. Of these, seven had macroscopic type-4 scirrhous-type gastric carcinoma. In the immunohistochemical study, the VEGF score of patients with peritoneal recurrence was 9.40 +/- 2.46; on the other hand, that of patients without peritoneal recurrence was 3.47 +/- 2.36. The VEGF score of patients with peritoneal recurrence was significantly higher than that of patients without peritoneal recurrence. In patients with macroscopic type 4, the VEGF score of those with peritoneal recurrence was 9.14 +/- 2.19, while on the other hand, that of the patients without peritoneal recurrence was 3.80 +/- 3.03. The VEGF score of these patients with peritoneal recurrence was significantly higher than that of those without peritoneal recurrence. The survival rate in the VEGF low-expression group was significantly higher than that in the VEGF high-expression group. Multivariate analysis showed that the VEGF score was a significant parameter of peritoneal recurrence. These results suggested that VEGF was correlated with peritoneal metastasis from gastric cancer, and that VEGF was a useful indicator of peritoneal recurrence.

Galectin‐3 expression in papillary microcarcinoma of the thyroid

Galectin-3 is a beta-galactoside binding protein, recently recognized as a promising molecular marker of thyroid malignancy. As reported in several studies, galectin-3 is highly expressed in papillary thyroid carcinoma, but its expression has not been investigated in papillary microcarcinoma, which is a variant of papillary thyroid carcinoma. Using a monoclonal antibody to galectin-3 and the avidin-biotin-peroxidase complex (ABC) immunohistochemical technique, we analysed galectin-3 expression in 63 cases of papillary microcarcinoma. The results showed immunohistochemical reactivity for galectin-3 in 51 (80.9%) cases. Intensity of staining varied from strong or moderate to weak. Galectin-3 localization was mostly cytoplasmic, but also membranous or nuclear in some cells. Immunohistochemical expression of galectin-3 was not found in 12 (19.1%) cases. Most galectin-3 negative microcarcinomas (10/12) were of the non-classical type, i.e. without papillary architecture. Neither the frequency nor the intensity of a positive reaction was related to tumour size. Galectin-3 gene is expressed at the protein level in most papillary microcarcinomas, although with slightly lower frequency than that reported for clinically evident papillary thyroid carcinoma. The presence of galectin-3 in clinically silent microcarcinomas may indicate that galectin-3 is not related to growth or aggressiveness of papillary thyroid microcarcinomas but rather plays some other role in thyroid tumour biology.

Intracellular localization of NS3 and C proteins in chronic hepatitis C

The cellular localization of hepatitis C virus (HCV) proteins in chronic hepatitis C remains a debatable issue. Aim of the studies included cellular and subcellular localization of two HCV proteins, NS3 and C protein, in liver biopsies from 20 children with chronic hepatitis C employing commercially available monoclonal antibodies and a semiquantitative technique of the proteins expression. In the studies advantage was taken of (Strept)avidin-biotin peroxidase complex and ImmunoMax technique. The cytoplasmic localization of both proteins dominated over nuclear localization. The total amount of NS3 protein exceeded that of C protein. At the ultrastructural level, we observed both proteins in endoplasmic reticulum membranes and mitochondria, but small amount of both proteins was seen also in cell nuclei. The amount of either protein did not correlate with liver histology in our patients. No correlation could be disclosed between content of both proteins and HCV RNA levels in serum. Content of NS3 demonstrated negative correlation with activity of alanine transaminase. In conclusion, the new aspect in present studies involved localization of C and NS3 proteins at the levels of light and electron microscopy in children. For the first time also intramitochondrial localization of NS3 protein was demonstrated.

Multiple hepatic masses in a 38-year-old male 10 years after renal transplantation

The patient is a 38-year-old male, diagnosed with IgA nephropathy at age 24, who underwent renal allograft transplantation 10 years ago. He presented with complaints of fever and non-productive cough. Clinical examination was notable for a low grade temperature (99.5 F), bibasilar crackles and a distended abdomen with overlying varices. Laboratory studies showed anaemia of chronic disease and normal liver function tests, except for a mildly depressed serum albumin level. Serum creatinine was elevated from his baseline of 2.5mg/dl to 4.2mg/dl. Additional past medical history includes former heavy use of alcohol, with none following his transplant. A right sided pulmonary infiltrate was detected on chest X-ray. Following antibiotic therapy for presumed pneumonia, his clinical symptoms resolved and his creatinine reverted to baseline level. An out-patient computed tomography (CT) scan was performed to evaluate the abdominal findings. Massive liver nodules in the lobes were present, replacing the hepatic parenchyma. The largest measured 13.5 cm in the left lobe, with a separate 12.5 cm mass in the right lobe (Figure 1). Radiologically, the lesions were perplexing, with a broad differential diagnosis including abscesses, haemangiomas, benign cystic lesions, metastases or hepatocellular carcinoma. Percutaneous core needle biopsy yielded a moderately cellular spindle cell neoplasm with minimal cellular pleomorphism and no mitotic activity (Figure 2). The spindle cells contained elongated, blunt-ended nuclei with finely stippled chromatin. Immunohistochemistry by the standard avidin–biotin–peroxidase complex method demonstrated myogenic differentiation of the tumour cells, which were immunoreactive to smooth muscle actin. In situ hybridization for EBV virus was strongly positive in >90% of the spindle cell nuclei (Figure 3). These findings are indicative of an EBV-associated myogenic tumour. A reduction of the immunosuppressive agents was recommended to the patient, who declined for fear of impending rejection. Currently he is being followed, with slow expansion in the size of the liver nodules. Six months following diagnosis of the liver lesions, a palpable 4 cm nodule in the right gluteal region developed. Additional smaller nodules in the same region were noted by imaging studies. Percutaneous needle core biopsy illustrated a histologically identical EBV-associated myogenic tumour.

Expression of epidermal growth factor receptor, ERBB2 and KIT in adult soft tissue sarcomas

Little is known about the expression of receptor tyrosine kinases in adult soft tissue sarcomas (STS). In the current study, the authors analyzed the expression of epidermal growth factor receptor (EGFR), ERBB2, and KIT in 281 patients with STS who were treated in a single institution. Verification of the presence of an association with prognosis was performed. The current study included 281 adult patients with STS of the extremity and trunk who were diagnosed and treated in the National Cancer Center, Tokyo. Expression was assessed using immunohistochemical stains for EGFR, ERBB2, and KIT on formalin-fixed, paraffin-embedded tissue sections by standard avidin-biotin peroxidase complex technique and EGFR detection system. Positive staining of EGFR was observed in 168 of 281 (60%) patients. Positive staining was common in pleomorphic malignant fibrous histiocytomas (89%), myxofibrosarcomas (89%), synovial sarcomas (76%), malignant peripheral nerve sheath tumors (89%), and leiomyosarcomas (73%). It was less common in well differentiated liposarcomas (38%), fibrosarcomas (36%), and myxoid liposarcomas (6%). In contrast, positive staining of ERBB2 and KIT was very limited. Increased levels of EGFR were significantly associated with a decreased probability of overall survival (P = 0.01), although by univariate analysis; probability of overall survival at 5 years was 64% in patients with increased levels of EGFR and 79% in patients without such overexpression. The overexpression of EGFR was significantly associated with histologic grade (P < 0.001). Moreover, stratified log-rank test revealed that there is an interrelation between EGFR overexpression and histologic grade. EGFR overexpression was found to be a negative prognostic factor of adult STS, which is strongly associated with histologic grade. STS patients with EGFR overexpression may benefit from treatment with currently available biospecific inhibitors for EGFR.

Localization and changes of intraneural inflammatory cytokines and inducible-nitric oxide induced by mechanical compression

Study design Investigation of intraneural inflammation induced by mechanical compression. Objectives In order to investigate the mechanism of neuropathy, this study used a median nerve compression model in dogs. Immunohistochemistry was used to examine the localization and changes of inflammatory cytokines and nitric oxide (NO). Summary of background data The manifestation of pain at sites of inflammation has a close relationship with the release of mediators from macrophages such as interleulin-1 (IL-1) and tumor necrosis factor-α (TNF-α), as well as with NO. However, the mediators involved in inflammation of nerve due to mechanical compression remain almost unknown. Methods In this study, the median nerve of dogs was compressed with a clip for three weeks to observe the changes caused by compression. Immunohistochemistry was done by the avidin–biotin-peroxidase complex method to observe the changes of T cells (CD45) and macrophages (Mac-1) after compression. Antibodies against IL-1β, TNF-α, and inducible nitric oxide synthesis (i-NOS) were used to examine the localization and changes of these mediators caused by nerve compression. Results In control animals, resident T cells were detected, but there were no macrophages. IL-1β was positive in the Schwann cells and vascular endothelial cells. However, no cells showed TNF-α or i-NOS positively. After nerve compression, numerous T cells and macrophages appeared among the demyelinized nerve fibers. The macrophages were positive for IL-1β, TNF-α and i-NOS. Conclusion Inflammatory cytokines and NO may be involved in intraneural inflammatory changes arising from mechanical compression. Such mediators may be of importance in the manifestation of neuropathy.

Bromodeoxyuridine Incorporation in Differentiated Thyroid Cancer

Objective: Bromodeoxyuridine (BUdR), a thymidine analog, is incorporated into the DNA in the synthetic (S) phase of the cell cycle. BUdR uptake quantifies DNA synthesis and helps predict the prognosis of various cancers. We, therefore, studied BUdR uptake in differentiated thyroid cancers to determine whether it correlates with other clinical prognostic factors in patients with differentiated thyroid cancer. Materials and Methods: Fresh thyroid cancer tissue from 45 patients was incubated in 10 μm BUdR in Dulbecco's modified Eagle medium with 10% fetal calf serum for 1 hour at 37℃, fixed in 70% ethanol and embedded in paraffin. Five tm sections were deparaffinized, denatured, and incubated for 1 hour in 1:3000 purified monoclonal antibodies. The immunohistochemical staining was performed using the avidin-biotin peroxidase complex technique. The extent of BUdR incorporation was measured using the labeling index (% of total cells labeled with BUdR). Results: BUdR labeling index was lower in good risk cancers confined to the thyroid (DeGroot Class Ⅰ) or regional nodes (DeGroot Class Ⅱ) (0.65%±0.23%, N=26) than in poor risk cancers with local invasion (DeGroot Class Ⅲ) or distant metastases (DeGroot Class Ⅳ) (1.13%±0.67%, N=19) (P=0.002 using the t-test). BUdR labeling index was similarly lower in cancers from low risk patients with AGES score ＜4 (0.66%±0.27%, N=29) than those from high risk patients with AGES score ≥4 (1.l9%±0.69%, N=16) (P=0.001 using the t-test). BUdR labeling index correlated with patient age (r=0.313, p=0.00l), and tumor size (r=0.479, p=0.00l). Six patients had tumor BUdR labeling indexes greater than 1.5%. All had DeGroot Class III or IV cancers and had AGES scores greater than 4. Conclusions: We concluded that BUdR labeling index correlated with tumor size, patient age, and risk classification of DeGroot Class or AGES score in patients with differentiated thyroid cancer, and it may become a useful factor in predicting thyroid cancer behavior.

Decrease of the surface fraction of surfactant proteins containing Clara cells and type II pneumocytes in a rat asthma model

In asthma surfactant proteins (SP) might differ in distribution and composition and thus play a role in pathophysiology of this disease. Therefore, the well-established animal model of ovalbumin sensitized and challenged rats were used to study the distribution of surfactant proteins in Clara cells and type II pneumocytes. Serial sections of paraffin embedded lung tissue were sequentially immunostained by the avidin–biotin–peroxidase complex (ABC) technique. Antisera against SP-A, SP-B and Clara cell specific protein (CC10) were used. We determined stereologically the surface fraction of immunolabelled cells and semiquantitatively the percentage of test fields containing labelled alveolar macrophages. In allergen sensitized and provocated rat lungs: (1) the surface fraction of SP-A and SP-B positive Clara cells was significantly reduced, (2) the surface fraction of Clara cells stained with CC10 was coincided with controls, (3) the surface fraction of SP-A and not of SP-B possitive type II pneumocytes decreased significantly, (4) a significantly higher percentage of test fields with SP-A labelled alveolar macrophages was evaluated. Thus, in this animal model of asthma the inflammatory process after allergen challenge is accompanied by alterations in the distribution patterns of SP in Clara cells and type II pneumocytes.

Immunohistochemical Recognition of the Epidermal Growth Factor Receptor by the h-R3 Antibody in the Skin of Experimental Animals

The h-R3 is a humanized monoclonal antibody (Mab) that binds to the external domain of the human epidermal growth factor receptor (EGF-R). It has being used for the treatment of head and neck tumors. Since it is known that different animal species have different EGF-R amino acid sequences, it raises the handicap that, a priori, the animal species relevant for the pharmacodynamic, pharmacokinetic, and toxicologic studies of this Mab are unknown. To elucidate relevant laboratory animal species, the authors investigated the immunohistochemical recognition by h-R3 Mab of EGF-R in small skin biopsy samples obtained from NMRI nu/nu, C57Bl/6, and OF-1 mice; Sprague-Dawley rats; Hartley guinea pigs; New Zealand rabbits; and Cercopithecus aethiops and Macaca fascicularis monkeys. Additionally, three human skin biopsies were obtained from trauma victims. The immunolocalization of EGF-R in different tissue sections was performed using the avidin-biotin peroxidase complex (ABC) technique. The skin sections from different tissues were tested with the biotinylated h-R3 Mab or with a biotinylated irrelevant Mab, used as negative control. The staining intensity was qualified as:-, no staining; +, weak staining; ++, moderate staining; +++, strong staining. Macaca fascicularis monkey, New Zealand rabbit, and OF-1 mouse skins had a strong staining intensity; Cercopithecus aethiops monkey and Sprague-Dawley rat skins showed a moderate to strong staining intensity; C57Bl/6 mouse skins showed a moderate staining intensity. No staining was observed in the skins from Hartley guinea pigs and MNRI nu/nu mice. The fact that h-R3 Mab recognizes other animal EGF receptors in addition to the human EGF-R makes it possible to perform relevant pharmacodynamic, pharmacokinetic, and toxicologic studies in some laboratory animals.

Melatonin-like immunoreactivity in the pineal gland of the cow: an immunohistochemical study.

With a view to checking the presence of melatonin in the pineal gland of the cow, in the present work we used six adult animals, ranging in age from one to six years, which were sacrificed at dawn. Sections of 6 micro m thickness of Bouin-fixed and paraffin-embedded pineal glands were incubated in an anti-melatonin serum, which was provided by the Institute for Molecular and Cellular Recognition, Gunma University, Maebshi, Japan. After incubation and successive washings in PBS, some of the sections were treated with the avidin-biotin-peroxidase complex (ABC) technique using antisera from Sigma, and developed with the method of Graham and Karnovsky (which employs 3,3'-diaminobenzidine and H2O2 as developer). Other sections were incubated in a goat-anti-rabbit IgG (H+L) bound to fluorochrome Cy5 for immunofluorescence studies. An intense reaction for melatonin was observed in the cytoplasm but not in the nucleus of melatonin secreting pinealocytes located in peripheral and intermediate zones of the pineal gland. Immunoabsorption of the antimelatonin primary antibody with melatonin at a dilution of 10 mM per 0.1 ml of serum prevented the reaction, as happened when any of the antisera used in the procedure were used. Immunoabsorption of anti-melatonin serum with different amounts of bovine albumin (ranging between 1/5 to 1/50) failed to inhibit the immunoreactivity. When a bovine anti-albumin antibody was employed, working with the above methods, no immunoreaction was detected. Our data suggest that the pinealocytes of cows sacrificed at dawn contain immunoreactive melatonin.

Ultrastructural localization of gastrin-releasing peptide (GRP) in the uterine gland of cow.

Gastrin-releasing peptide (GRP) is thought to act mainly as a neurotransmitter and localized almost exclusively to neurons and neuroendocrine cells. Recently, the localization of GRP in mammalian uterus and placenta has been demonstrated. Moreover, the exocrine manner of GRP release was deduced in ewes from the distribution of GRP on the uterine gland cells and its secretion as well as in the circulation. However, these reports have been examined at light-microscopic level. The present study was designed to make clear the localization of GRP in the uterine gland cells of nonpregnant and pregnant cows using an avidin-biotin-peroxidase complex (ABC) method at light-microscopic level and a pre-embedding immunogold with silver enhancement method at electron-microscopic level. The light-microscopic observation showed positive staining for GRP immunoreactivity in the supranuclear region and in the secreted materials of the uterine gland cells. At the electron-microscopic level, the supranuclear secretory granules and the secreted materials on the surface of the cell were labeled with immunogold particles representing GRP immunoreactivity in the uterine gland cells of nonpregnant and pregnant cows. Western blotting analysis showed a larger molecular form of GRP in the endometrial tissues taken from nonpregnant and pregnant cows. The present results revealed the localization of GRP in the uterine gland cells at light- and electron-microscopic levels and suggested the release of GRP from the cell into the lumen of the gland by exocrine manner.

Calponin expression and myoepithelial cell differentiation in canine, feline and human mammary simple carcinomas

Calponin is a 34-kDa smooth muscle-specific protein that has been shown to be a highly sensitive marker of myoepithelial cells in canine, feline and human mammary tissue and tumours. The expression of calponin was studied in 15 canine, 32 feline and 28 human simple mammary carcinomas using a monoclonal mouse antihuman calponin antibody and the avidin-biotin peroxidase complex (ABC) immunohistochemical technique. Calponin expression was compared with the expression of cytokeratin 14, a marker of normal mammary myoepithelial cells in the three species. Four different types of calponin-positive cells were identified: (1) Type 1: cytokeratin-14-positive pre-existing myoepithelial cells forming a continuous layer with images of focal disruptions; (2) Type 2: cytokeratin-14-positive isolated nests of fusiform, polygonal or round cells without atypia; (3) Type 3: cytokeratin-14-positive atypical cells indistinguishable from non-reactive atypical cells, which should have never been detected in haematoxylin and eosin-stained sections and (4) Type 4: cytokeratin-14-negative stromal fusiform cells around the neoplastic growth or cell nests, identified as myofibroblasts. Calponin-negative and cytokeratin-14-positive atypical neoplastic cells were observed in three canine, 28 feline and two human carcinomas. The latter were indicative of altered expression of high-molecular-weight cytokeratins in luminal epithelial-type simple carcinomas. Our findings show that calponin is a good marker of myoepithelial cell differentiation in feline, human and, particularly, canine simple carcinomas. The high number (six out of 15) of canine tumours with type 3 cells points to the need of both introducing calponin examination in the routine diagnostic schedule and performing further studies on its prognostic significance.

Production and preliminary characterization of monoclonal antibodies raised against Xanthomonas campestris pv. mangiferaeindicae

Bacterial black spot disease of mango is caused by Xanthomonas campestris pv. mangiferaeindicae (Xcm), which consists of two genotypically and phenotypically distinct groups of strains. Monoclonal antibodies (MABs) were produced – 15 against CFBP 1717, a group I strain, and 9 against CFBP 2919 (yellow-pigmented), a group II strain – and were analyzed for their characteristics. On the avidin-biotin peroxidase complex enzyme-linked immunosorbent assay, the dilution limit of the MABs was between 100 and 200 000 and was 10 times higher when measured on the corresponding ascitic fluid. All kinds of isotypes were represented among the MABs. All the Japanese Xcm strains, designated group I by hrp-restriction fragment length polymorphism (RFLP) analysis, reacted equally with MAB 1A7H12G3, which is the most specific for all but one worldwide group I strains, and to only one strain among group II. Also, to various extents, serological heterogeneity inside the two groups was consistently differentiated based on isozyme and RFLP analyses. MAB 1E2E1 against CFBP2919, because of its narrow specificity, and MAB 1A7H12G3 against CFBP1717, because of its broad specificity, will be useful for epidemiological studies or general control of the pathogen.

Apoptosis of thymocytes in experimental African Swine Fever virus infection.

This paper report on the lesions occurred in the thymus in experimental acute African swine fever (ASF). Twenty-one pigs were inoculated with the highly virulent ASF virus (ASFV) isolate Spain-70. Animals were slaughtered from 1 to 7 days post infection (dpi). Three animals with similar features were used as controls. Thymus samples were fixed in 10% buffered formalin solution for histological and immunohistochemical study and in 2.5% glutaraldehyde for ultrastructural examination. For immunohistochemical study, the avidin-biotin-peroxidase complex (ABC) technique was used to demonstrate viral protein 73 and porcine myeloid-histiocyte antigen SWC3 using specific monoclonal antibodies. Cell apoptosis was evaluated by the TUNEL assay. Blood samples were taken daily from all pigs and were used for leukocyte counts. The results of this study show a severe thymocyte apoptosis not related to the direct action of ASFV on these cells, but probably to a quantitative increase in macrophages in the thymus and their activation. A decrease in the percentage of blood lymphocytes was observed at the same time No significant vascular changes were observed in the study. With these results we suggest that ASFV infection of the thymus does not seem to play a critical role in the acute disease. Although severe apoptosis was observed, animals died because of the severe lesions found in the other organs.

Immunohistochemical study of protein induced by vitamin K absence or antagonist II in hepatocellular carcinoma.

Protein induced by vitamin K absence or antagonist II (PIVKA-II) has been used as a tumor marker of hepatocellular carcinoma (HCC). In this study, we examined the expressions of PIVKA-II in HCC tissues. Expression of PIVKA-II in HCC was evaluated with the avidin-biotin-peroxidase complex method. Paraffin-embedded HCC tissues obtained from 48 patients were subjected to immunohistochemical staining using the anti-PIVKA-II antibody monoclonal antibody (MU-3) (Eisai, Tokyo, Japan). The tissue PIVKA-II positivity rate of 12/23 (52.2%) in HCC with vascular infiltration was significantly higher than the rate of 4/25 (16.0%) in HCC without such infiltration. Further, the tissue PIVKA-II positivity rate of 13/28 (46.4%) in HCC of tumor stage III or IV was significantly higher than the rate of 3/20 (6.0%) in HCC of tumor stage I or II. Overall survival rate tended to be lower in the tissue-PIVKA-II-positive than -negative patients. PIVKA-II may play an important role in progression of HCC and is useful not only as a tumor marker but also as a prognostic factor.

Evaluation of the expression of collagen type I in porous calcium phosphate ceramics implanted in an extra-osseous site

The aim of the present study is to demonstrate the newly formed tissue in calcium phosphate (Ca/P) ceramics after extra-osseous implantation by histological and immunohistochemistry (IHC) methods. Synthesis porous Ca/P ceramics without adding any growth factor and living cell were implanted in the dorsal muscle of dogs for 1 and 2 months. Undecalcified and decalcified sections were stained by hematoxylin and eosin (H&E), and IHC, respectively. The histological results showed the beginning of osteogenesis and angiogenesis after being implanted for 1 month and the obvious new bone formation after being implanted for 2 months. IHC were conducted via the avidin–biotin peroxidase complex (ABC) method and the primary antibody was collagen type I. IHC results indicated that collagen type I was expressed within osteoblast-like cells and newly formed bone-like tissue in Ca/P ceramics after 1 month, and in the mineralized matrix of newly formed bone and osteoblasts, some osteocytes and some lacunae after 2 months. No cartilage and chondrocytes were observed in the histological and IHC-stained sections. Evidence of intramembranous osteogenesis was confirmed.

Immunohistochemical detection of peste des petits ruminants viral antigens in tissues from cases of naturally occurring pneumonia in goats

Peste des petits ruminants (PPR) is an important viral disease of goats and sheep characterized by erosive stomatitis, enteritis, and pneumonia. But the pneumonia is the most frequently recorded lesion in PPR infections. In this study, the presence of PPR viral antigens in goat’s pneumonia was investigated. For this purpose, a total of 1505 goat’s lungs slaughtered in Bitlis and Van slaughterhouses were grossly examined and pneumonia was detected in 74 cases (4.91%). Lesions were more frequently encountered in anteroventral lobes than caudal lobes. Immunohistochemical (IHC) examinations were performed on 42 pneumonic lungs (except the verminous pneumonia). IHC staining of formalin-fixed, paraffin-embedded tissues were carried out by the avidin–biotin–peroxidase complex (ABC) procedure. The presence of PPR viral antigens were detected in 17 (40%) out of 42 pneumonic lungs. Viral antigens were found most frequently in the cytoplasm and rarely in the nucleus, including bronchial glands, bronchial and bronchiolar epithelial cells, type II pneumocytes, syncytial cells, and alveolar macrophages.In conclusion, it was found that there was a close relationship between the pneumonia in goats and the presence of PPR viral antigens. The PPR incidence in pneumonic lungs of goats was detected to be very high in the present study performed in the region of Bitlis and Van.