HE-stained Sections Research Articles

Visualization of features in digital slides of HE-stained hepatic histological sections

Background In histological diagnosis of hepatocellular nodule, 2 cm or less in diameter, histological atypism of the early hepatocellular carcinoma is so slight that it is very difficult to differentiate from benign nodules. The nuclear density (the number of nuclei per unit area) and the morphological features such as a roundness factor of nuclear contours are important features. We developed a function to automatically estimate those features in digital slides of HE-stained hepatic histological sections. Methods and results Digital slides obtained using NanoZoomer were divided into 0.3 × 0.3 mm block images. Nuclei and their contours were extracted using the energy function.1 Then, the nuclear density and the roundness factor were calculated using extracted nuclei. Twenty block images sampled from three digital slides were used to evaluate the accuracy. RMS errors of the nuclear density and the roundness factor were 9.5% and 0.48%, respectively. Calculated feature values of all blocks were converted to appropriate colors to visualize the features distribution. Resultant color images help the user easily notice the areas having feature values different from others. This research was partially supported by KAKENHI (24500514).

Confounding Factors and Diagnostic Accuracy of Imprint Cytology

Background: Eradication of malignant tumors at the primary site with oncological safe margin is a critical requirement for obtaining better survival rate and less recurrence. Touch imprint cytology (TIC) has proven itself as a quick, simple, inexpensive, highly accurate and reliable intraoperative technique to assess surgical margins in squamous cell carcinomas of the oral cavity. However, it is still unclear how the mode of excision, i.e. by scalpel (SC) and electrocautery (EC), or the method of staining, i.e. Papanicolaou (PAP) and cytohaem, affect the diagnostic accuracy of TIC. Objective: To study the influence of confounding factors like mode of excision (EC/SC) and staining (PAP/cytohaem) on the diagnostic accuracy of intraoperative TIC technique for assessing surgical margins in oral squamous cell carcinoma in comparison to paraffin-embedded HE-stained sections. Materials and Methods: Thirty patients underwent surgical treatment for primary oral squamous cell carcinoma. Three hundred and forty-eight touch imprint slides were prepared from 174 margins of 30 resected tumor specimens. Two adjacent tissues from the margin to be evaluated were imprinted to observe differences between surfaces excised by EC and SC. The set of imprint from each margin tissue was stained with PAP and cytohaem. The TIC results of 180 EC-excised margins and 168 SC-excised margins were compared. Results of 174 imprints stained with RAPID-PAP were compared to their counterpart comprising of 174 cytohaem-stained imprints. The slides were diagnosed as positive, negative or suspicious for tumor. Finally, TIC results were checked against their respective histopathological sections. Results: No statistically significant difference was found between the results of imprints from EC/SC-excised margins (Z = 0.44, p = 0.70) or the imprints stained with PAP/cytohaem (Z = 0.44, p = 0.70). Conclusion: Confounding factors like mode of excision and staining procedure do not significantly influence the results of imprint cytology.

Erythromycin induced neuroprotection during prolonged deep hypothermic circulatory arrest in an acute porcine model

Methods Piglets were treated with erythromycin (25 mg/kg, iv) (n = 8) or vehicle (n = 6) and subjected to 75 minutes of DHCA at 18°C, 12 hours after pretreatment. Three served as normal controls. After gradual rewarming, treatment animals were sacrificed and brains were perfusion-fixed and cryopreserved. Motor cortex was dissected from the left hemisphere and paraffin embedded for histologic staining with hematoxylin and eosin (HE). To assess neuronal damage, HE-stained paraffin sections (10 μm) were examined by light microscopic examination at x400 magnification. Layer V of the motor cortex was counted. Neuronal injury was recorded when there was evidence of eosinophilic cytoplasm, cytoplasmic vacuolation, cell body shrinkage or nuclear pyknosis. Neuronal injury was scored on a scale of 0-5.

A randomized controlled clinical trial to evaluate a new xenograft for alveolar socket preservation

The aim of this clinical trial was to compare the effect of Bio-Oss(®) and a new bovine xenograft (Osseus(®) ) in alveolar sockets after a 24-week healing period. A total of 20 adult volunteers ages 30-60 were subjected to single tooth extraction. A tooth extraction was performed at the baseline. All sites were randomly allocated to two test groups (TG1: grafted using a new bovine xenograft, Osseus(®) , and TG2: grafted using commercially available bovine xenograft-Bio-Oss(®) ). Six months later, a sample of the grafted area was obtained and implants were inserted in the same site. Histological sections were examined focusing on the presence of fibrous connective tissue (CT), and newly formed bone in direct contact with the graft. The HE-stained sections were subjected to histomorphometrical evaluation using Image Pro-Plus(®) software (Release 7.0). The definitive crown was placed 3 months later. Upon completion of the study, no patients were removed from the study and all inserted implants (10 in each group) were eventually integrated. After 6 months, in the TG1, the mean value of new bone formation was 33.7 (± 7.1), for CT was 32.3 (± 8.9) and for the remaining biomaterial was 10.7 (± 16.2). In the TG2, the mean value of new bone formation was 19.3 (± 22.6), of the CT was 49.9 (± 14.1) and of the remaining biomaterial was 22.6 (± 7.9). No statistically significant difference was observed between TG1 and TG2 after 6 months (P > 0.05), and both biomaterials afforded a more favorable implant position.

Cytotoxic Effects of Polyhexanide on Cellular Repopulation and Calcification of Decellularized Equine Carotids in vitro and in vivo

Disinfection of biological implants is indispensable for clinical safety. Here, decellularized equine carotid arteries (dECAs) were disinfected by polyhexanide (PHX), an effective, well-tolerated and nontoxic wound disinfectant and evaluated as vascular grafts for their repopulation and local biocompatibility in vivo. dECAs were terminally disinfected by a combination of 0.1% PHX and 70% ethanol (dECA_PHX-ET) or exclusively ethanol (dECA-ET) and subsequently implanted as arteriovenous shunts in sheep for 14 weeks. Repopulation was determined by immunohistochemistry for endothelial- (ECs) or smooth muscle cells (SMCs) using antibodies against CD31 and smooth muscle actin. Histological evaluation was performed on HE-stained sections. Cytotoxicity of dECAs was measured directly by seeding the scaffolds with L-929 fibroblasts, which were visualized by calcein staining. Indirect cytotoxicity was determined by WST-8 viability assay by incubation of L-929 with dECA extracts. dECA_PHX-ET completely lacked repopulation with ECs and SMCs, showed leukocyte infiltration, strong calcification and poor neovascularization indicating insufficient biocompatibility and inflammatory graft degeneration. PHX-treatment reduced cell viability to 33.2 ± 12.6% and disturbed cell growth at direct contact. In contrast, dECA_ET had no direct cytotoxic effect and only slightly influenced cell viability (82.9 ± 12.5%), showed a substantial repopulation by ECs and SMCs including neovascularization, and were only slightly calcified. The disinfectant polyhexanide seems to exert severe cytotoxic effects when used for the processing of decellularized matrices and may result in degenerative graft deterioration. In contrast, dECAs exclusively disinfected with ethanol were well integrated. Thus, ethanol seems to be a more suitable tool for graft processing than polyhexanide.

Biocompatibility of Intracanal Medications Based on Calcium Hydroxide

Objective. The aim of this study was to evaluate the rat subcutaneous tissue reaction to calcium hydroxide-based intracanal medicaments, UltraCal XS (calcium hydroxide, barium sulphate, aqueous matrix), Hydropast (calcium hydroxide, barium sulphate, and propyleneglycol), and Calen (Calcium hydroxide, zinc oxide, colophony, and polyethyleneglycol), used as a control. Methods. Forty-eight rats (Rattus Norvegicus Holtzman) were distributed in three groups: Calen, UltraCal XS, and Hydropast. Polyethylene tubes filled with one of the medicaments were implanted in the dorsal subcutaneous. After 7 and 30 days, the implants were removed and the specimens were fixed and embedded in paraffin. Morphological and quantitative analyses were carried out in the HE-stained sections. The numerical density of inflammatory cells in the capsule was evaluated and statistical analyses were performed (P ≤ 0.05). Results. At 7 days, all materials induced an inflammatory reaction in the subcutaneous tissue adjacent to the implants. In all groups, a significant reduction in the number of inflammatory cells and giant cells was verified in the period of 30 days. Conclusion. These results indicate that the calcium hydroxide-based medicaments evaluated present biocompatibility similar to Calen.

Biocompatibility of an experimental MTA sealer implanted in the rat subcutaneous: Quantitative and immunohistochemical evaluation

The tissue reaction promoted by an experimental mineral trioxide aggregate sealer (MTAS) in the rat subcutaneous was evaluated by morphological and morphometric analyses. In the animals from each group (n = 20), polyethylene tubes filled with MTAS, Portland cement (PC) or MTA were implanted in the dorsal subcutaneous. In the control group, empty tubes were implanted. After 7, 14, 30, and 60 days, the specimens were fixed and embedded in paraffin. In the HE-stained sections, the numerical density of inflammatory cells (IC) in the capsule was evaluated and statistical analyses performed (p ≤ 0.05). The expression of osteopontin (OPN) was evaluated by immunohistochemistry. The von Kossa method for detection of calcified structures was also performed. A moderate inflammatory process in the capsule was seen in all groups, at 7 and 14 days. At 60 days, significant reduction in the number of IC was verified in comparison to initial periods; however, significant differences were not verified among the groups. OPN immunolabeling was observed in the fibroblasts cytoplasm of the capsule next to the implants. Structures von Kossa-positive were observed in the capsule adjacent to all materials implanted at 7, 14, and 30 days. The results strongly indicate that MTAS presents biocompatibility similarly to MTA and PC.

Histopathology of incidental findings in cynomolgus monkeys ( macaca fascicularis ) used in toxicity studies.

The purpose of our publication is to widely communicate pictures of spontaneous findings occurring in cynomolgus monkeys. Focal lymphoplasmacytic infiltration is commonly seen in the general organs. The frequency and severity of these lesions may be influenced by the administration of drugs with an effect on the immune system. Lymphoplasmacytic infiltration in the lamina propria of the stomach is also frequently seen in cynomolgus monkeys, and it is caused mainly by a Helicobacter pylori infection. Various degrees of brown pigments are observed in various organs, and it is possible to distinguish the material of the pigments by its morphological features and site. A focal/segmental glomerular lesion is occasionally seen in a section of the kidney, and the minimal lesion has no influence on the urinalysis. We showed the common glomerular lesions in HE-stained sections, as well as in PAM- or PAS-stained sections, for understanding the details. Young and pubertal monkeys are usually used in toxicity studies; therefore, understanding various maturation stages of the genital system is important. In particular, the female genital system needs to be understood in the morphology, because their cyclic changes are different from other laboratory animals. Thus, we present the normal features of the cyclic changes of the female genital organs. Furthermore, we provide more information on spontaneous findings in cynomolgus monkeys for exact diagnoses in toxicity studies.

Lymph vessel density in seminomatous testicular cancer assessed with the specific lymphatic endothelium cell markers D2-40 and LYVE-1: Correlation with pathologic parameters and clinical outcome

ObjectivesTo evaluate the role of lymph vessel density (LVD) and lymphangiogenesis in seminomatous testicular cancer (STC) by using the lymphatic endothelial cell (LEC) markers LYVE-1 and D2-40. Methods and materialsParaffin embedded tumor specimens from 40 patients with STC were stained by specific D2-40 and Lyve-1 antibodies. LVD was measured in different representative and standardized areas. Fluorescence double immunostaining for Lyve-1 and Ki-67 was performed and results were correlated with clinicopathologic data. The median follow-up period was 55 (range 10–135) months. ResultsMean intratumoral LVD (D2-40: 1.30 ± 1.99; Lyve-1: 1.82 ± 2.34) was significantly lower than peritumoral LVD (D2-40: 4.94 ± 2.58; Lyve-1: 4.62 ± 2.73) and LVD in nontumoral areas (D2-40: 4.81 ± 3.79; Lyve-1: 4.22 ± 3.19). There was no significant difference between LVD measures when using D2-40 or LYVE-1. Detection rates of lymphatic vascular invasion (LVI) were significantly higher than in conventional HE-stained sections (77.5% vs. 52.5%). No proliferating lymphatic vessels were found. ConclusionsWe found that LVD is decreased within tumor areas of STC. Despite a higher peritumoral LVD, no signs of proliferating endothelial cells were observed, suggesting a lack of lymphangiogenesis in STC. Detection of LVI can be optimized by specific D2-40 or LYVE-1 staining.

Elemental Bioimaging in Kidney by LA–ICP–MS As a Tool to Study Nephrotoxicity and Renal Protective Strategies in Cisplatin Therapies

A laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)-based methodology is presented for Pt, Cu, and Zn bioimaging on whole kidney 3 μm sagittal sections from rats treated with pharmacological doses of cisplatin, which were sacrificed once renal damage had taken place. Pt turned out to accumulate in the kidney cortex and corticomedullary junction, corresponding to areas where the proximal tubule S3 segments (the most sensitive cells to cisplatin nephrotoxicity) are located. This demonstrates the connection between platinum accumulation and renal damage proved by histological examination of HE-stained sections and evaluation of serum and urine biochemical parameters. Cu and Zn distribution maps revealed a significant displacement in cells by Pt, as compared to control tissues. A dramatic decrease in the Pt accumulation in the cortex was observed when cilastatin was coadministered with cisplatin, which can be related to its nephroprotective effect. Excellent imaging reproducibility, sensitivity (LOD 50 fg), and resolution (down to 8 μm) were achieved, demonstrating that LA-ICP-MS can be applied as a microscopic metal detector at cellular level in certain tissues. A simple and quick approach for the estimation of Pt tissue levels was proposed, based on tissue spiking.

Abstract 407: Detection of phospho-Stat3-mediated immunosuppressive microenvironment in benign lymph nodes of patients with early stage non-small cell lung cancer

Abstract Objectives: Surgical resection offers the most promising chance for cure in patients who present with early-stage disease, although the majority will experience recurrence due to microscopic disease at the time of surgery. In this study, we sought to identify markers of metastatic potential within benign regional lymph nodes of patients with completely resected non-small cell lung cancer (NSCLC) with a focus on VEGFR-1, myeloid cells and pStat3. Methods: Paraffin-embedded benign pulmonary lymph node specimens from 46 lung cancer patients with stage I-III resected disease were stained for CD68 (AbD Serotec, Raleigh, NC), CD163 (AbD Serotec, Raleigh, NC), VEGFR-1 (Santa Cruz, Santa Cruz, CA), phospho-Stat3 (Cell Signaling, Danvers, MA), MMP-9 (Cell Signaling, Danvers, MA) and VEGF-A (Abcam, Cambridge, MA) by immunohistochemistry or immunofluorescence. Anthracosis in the lymph nodes was evaluated using HE-stained sections. The sections were analyzed with Image Pro Plus or evaluated by two independent observers who were not aware of the patient information (double-blinded). Results: In benign lymph nodes from resected lung cancer patients, CD68 was highly expressed, while VEGFR-1 demonstrated low expression. Furthermore, these cells were associated with anthracotic deposition. Upon additional evaluation, we found that the infiltrating cells with anthracotic pigment display CD68+ CD163+, suggestive of an M2 macrophage phenotype, which are immunosuppressive and cancer promoting. These M2 macrophages expressed MMP-9 and VEGF-A, and in at least 40% of the patient lymph node tissue, pStat3 was elevated in these anthracotic macrophages. The infiltration of CD68+ macrophages was closely correlated with the intensity of anthracosis (P<0.0001, One-way ANOVA). In some lymph nodes, the infiltration of M2 macrophages was dramatically increased. We also observed positive correlations between overall pStat3 level with M2 macrophage infiltration and anthracosis intensity (P<0.05 and P<0.01, respectively, One-way ANOVA). In addition, pStat3 levels in anthracotic cells increased with the intensity of anthracosis in the lymph nodes. Conclusion: Our results indicate the presence of M2 macrophages and anthracosis in benign lymph nodes of patients with stage I-III NSCLC. The infiltration of M2 macrophages in benign pulmonary lymph nodes might serve as a predictive factor for micrometastasis and patient prognosis. Correlations between M2 macrophage in lymph nodes with smoking status, patient stage and survival will also be evaluated. Supported by NIH R01 CA115815 (hy) and NIH K12 CA001727 (klr; skp). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 407. doi:10.1158/1538-7445.AM2011-407

264 A NEW BULKING AGENT TO TREAT VESOURETERAL REFLUX: AN EXPERIMENTAL STUDY

You have accessJournal of UrologyPediatrics: Basic Research1 Apr 2011264 A NEW BULKING AGENT TO TREAT VESOURETERAL REFLUX: AN EXPERIMENTAL STUDY Salvador Lima, Artur Rangel, Lamartine Aguiar, Francisco Sampaio, Luiz Cardoso, and Henrique Gomes Salvador LimaSalvador Lima Recife, Brazil More articles by this author , Artur RangelArtur Rangel Recife, Brazil More articles by this author , Lamartine AguiarLamartine Aguiar Recife, Brazil More articles by this author , Francisco SampaioFrancisco Sampaio Rio de Janeiro, Brazil More articles by this author , Luiz CardosoLuiz Cardoso Rio de Janeiro, Brazil More articles by this author , and Henrique GomesHenrique Gomes Rio de Janeiro, Brazil More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.355AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The use of bulking agents in Urology became very popular especially after the approval of new agents suchs as Deflux. We present the experimental results of a new agent a cellulosic exopolysaccharide (CE), synthesized from sugar molasses by Zooglea sp [Paterson-Beedle. Carbohydr Polymers. 2000;42:375–383] with specific characteristics that can represent a new option in the treatment of VUR. METHODS Ten adult rabbits had the new agent injected into the bladder (submucosa) using a small abdominal incision. Three points in each half of the bladder were injected with 0.2ml of Deflux and the same amount of CE.The animals were sacrificed after 3 and 90 days, Collagen and elastic fibers were evidenced using the Sirius red and Weigert's resorsin fuchsin staining methods, respectively. Smooth muscle cells were identified by immunolabeling with smooth muscle alpha-actin. The density of blood vessels was assessed in oulined areas of injected material or normal bladder wall using the ImageJ software and HE-stained sections, and was expresed as as mean±SD number of blood vessels per mm2. RESULTS In 3-day samples, injected CE and Deflux were located mostly under the lamina propria and were structurally homogeneous and free from inflammatory cells or blood vessels. However, in 3-month samples, with the exception of a few small areas, CE was organized as short bundles, although the material stained weakly for collagen and was negative for elastic fibers, in contrast to the adjacent tissue. CE areas were populated by fibroblast-like cells, which were negative for smooth muscle immunolabeling. Newly formed blood vessels were also present in these areas, with a density 45.0±8.4, whereas in normal bladders the value was 35.2±2.9. A few small inflammatory infiltrates were observed in the CE areas, especially around blood vessels. Deflux areas in 3-month samples were fragmented but still homogeneous and free from cells or blood vessels. CONCLUSIONS The results obtained thus far indicate that this biomaterial exhibits little immunogenicity and integrates better in the host tissue as compared with Deflux. Therefore, CE should be an efficient material when incorporation is desired, for example as a suppportive scaffold in reconstructive surgery. CE may still be an effective bulking agent if its metabolizatin in the longer term does not involve reduction in volume. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e106 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Salvador Lima Recife, Brazil More articles by this author Artur Rangel Recife, Brazil More articles by this author Lamartine Aguiar Recife, Brazil More articles by this author Francisco Sampaio Rio de Janeiro, Brazil More articles by this author Luiz Cardoso Rio de Janeiro, Brazil More articles by this author Henrique Gomes Rio de Janeiro, Brazil More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

EGFR is involved in control of gastric cell proliferation through activation of MAPK and Src signalling pathways in early-weaned rats

Early weaning (EW) increases proliferation of the gastric epithelium in parallel with higher expression of transforming growth factor alpha and its receptor epidermal growth factor receptor (EGFR). The primary objective of the present study was to examine involvement of EGFR signalling in regulating mucosal cell proliferation during the early weaning period. Fifteen-day-old rats were split into two groups: suckling (control) and EW, in which pups were separated from the dam. Animals were killed daily until the 18th day, 3 days after onset of treatment. To investigate the role of EGFR in proliferation control, EW pups were injected with AG1478, an EGFR inhibitor; signalling molecules, proliferative indices and cell cycle-related proteins were evaluated. EW increased ERK1/2 and Src phosphorylation at 17 days, but p-Akt levels were unchanged. Moreover, at 17 days, AG1478 administration impaired ERK phosphorylation, whereas p-Src and p-Akt were not altered. AG1478 treatment reduced mitotic and DNA synthesis indices, which were determined on HE-stained and BrdU-labelled sections. Finally, AG1478 injection decreased p21 levels in the gastric mucosa at 17 days, while no changes were detected in p27, cyclin E, CDK2, cyclin D1 and CDK4 concentrations. EGFR is part of the mechanism that regulates cell proliferation in rat gastric mucosa during early weaning. We suggest that such responses might depend on activation of MAPK and/or Src signalling pathways and regulation of p21 levels.

Diagnostic Algorithm to Differentiate Lymphoma From Inflammation in Feline Small Intestinal Biopsy Samples

Differentiating between inflammatory bowel disease (IBD) and small intestinal lymphoma in cats is often difficult, especially when only endoscopic biopsy specimens are available for evaluation. However, a correct diagnosis is imperative for proper treatment and prognosis. A retrospective study was performed using surgical and endoscopic intestinal biopsy specimens from 63 cats with a history of chronic diarrhea or vomiting or weight loss. A diagnosis of lymphoma or inflammation was based on microscopic examination of hematoxylin and eosin (HE)-stained sections alone, HE-stained sections plus results of immunohistochemical labeling (IHC) for CD3e and CD79a, and HE staining, immunophenotyping, and polymerase chain reaction (PCR) results for B and/or T cell clonality. In addition, various histomorphologic parameters were evaluated for significant differences between lymphoma and IBD using Fisher's exact test. The sensitivity and specificity of each parameter in the diagnosis of lymphoma were also determined. Results of Bayesian statistical analysis demonstrated that combining histologic evaluation of small intestinal biopsy specimens with immunophenotyping and analysis of clonality of lymphoid infiltrates results in more accurate differentiation of neoplastic versus inflammatory lymphocytes. Important histologic features that differentiated intestinal lymphoma from IBD included lymphoid infiltration of the intestinal wall beyond the mucosa, epitheliotropism (especially intraepithelial nests and plaques), heterogeneity, and nuclear size of lymphocytes. Based on the results of this study, a stepwise diagnostic algorithm that first uses histologic assessment, followed by immunophenotyping and then PCR to determine clonality of the lymphocytes, was developed to more accurately differentiate between intestinal lymphoma and IBD.

New Findings Concerning Eosinophilic Substance Deposition in Mouse Nasal Septum

An eosinophilic substance (ES) is usually observed in the mouse nasal septum. In contrast to textbooks and one report describing ES as amyloid, a previous study by the authors revealed that ES is not amyloid but consists of collagen and an amorphous material. Furthermore, it was suggested that the amorphous material was produced by clear HE-stained nasal gland epithelial cells present at the dorsal portion directly above the vomeronasal organ. In this histological examination, ES deposition showed sex difference (more intense in males than in females). ES increased with age but not in seniles, suggesting that the increase has a limit. In the detailed examination using subserial HE-stained nasal sections, it was revealed that the clear HE-stained nasal glands continued to the vomeronasal glands, which communicated with the lumen of the vomeronasal organ, and the vomeronasal gland epithelial cells contained strongly periodic acid-Schiff (PAS) positive granules, similar to the clear HE-stained nasal gland epithelial cells. ES also deposited in the interstitium of the vomeronasal glands. The results suggested a possibility that ES deposition may be related to vomeronasal organ.

The possible involvement of immune complexes in pulmonary granuloma formation in experimental hypersensitivity pneumonitis

Hypersensitivity pneumonitis (HP) is a disease caused by inhaled organic dust. The alveolitis and granuloma are characteristic features of HP. The precise mechanism of granuloma formation is still unclear. The roles of immune complexes and primed T cell in relation to granuloma formation were studied. Local lymph node cells were obtained from C57BL/6 mice immunized by injection of ovalbumin (OA) or keyhole limpet haemocyanin (KLH) emulsified in complete Freund's adjuvant into footpads. Thereafter enriched T cells were obtained by passage through nylon wool columns. Antigens (OA, KLH) were chemically coupled to Sepharose 4B beads (OA-beads, KLH-beads). The immune complex was made by the incubation of the antigen-coupled beads and the immunized mouse serum at 37 degrees C for 2 hours. The recipient mouse irradiated by 6.5 Gy was transferred with 3 X 10(7) T cells and challenged with intratracheal instillation of 1 X 10(4) antigen-coupled beads or 1 X 10(4) immune complex-coupled beads. The recipient mice were sacrificed day 3 after cell transfer and challenge. The radius of pulmonary granuloma was measured by microscopic examination of the HE-stained section. The radius of granuloma increased in the recipient mouse which received the primed T cell and was challenged with immune complex-coupled beads compared with any other combination of cell transfer and antigen challenge. This result suggests that not only T cell but also immune complexes play an important role in granuloma formation in HP.

Histological development of human testicular cords from 70 to 90 days of gestation

The development of the testicular cord structure was investigated in 4 human fetuses between 70 and 90 days of gestation, in which the testicular cords are differentiating into the seminiferous tubules. Histological examinations were performed using stains with haematoxylin-eosin (HE), Masson's trichrome (MT), periodic acid schiff (PAS), anti-proliferating cell nuclear antigen (PCNA) monoclonal antibodies, and TUNEL methods. It was found that the testicular cords structures were indefinitely observed in HE-stained sections of four fetuses. However, the basement membranes of the testicular cord were clearly stained with MT, showing the tubular structure. Furthermore, cells in the testicular cords were positive with PAS, but the interstitial tissues outside the testicular cords were negative. PCNA-positive cells were detected not only inside but also outside the testicular cords, however, TUNEL positive cells are not detected throughout all testicular tissues.

Cytokeratin 8/18 is a Useful Immunohistochemical Marker for Hepatocellular Proliferative Lesions in Mice

In order to clarify whether cytokeratin (CK) 8/18 is a useful immunohistochemical marker for hepatocellular proliferative lesions in mice, partially hepatectomized male ICR mice were given 0.6% piperonyl butoxide (PBO) for 8 (Experiment I) or 25 weeks (Experiment II) after N-diethylnitrosamine (DEN) initiation treatment, and the livers were subjected to histological examinations on hematoxylin and eosin (HE) stained sections, CK8/18 immunohistochemistry and gamma-glutamyl transpeptidase (GGT) histochemistry. In Experiment I, the multiplicity of hepatocellular foci in paraffin-embedded sections which were observed in HE-stained sections and positive for CK8/18 was 10.17 and 18.50, respectively, while that of hepatocellular foci in frozen sections which were observed in HE-stained sections and positive/negative for GGT was 6.17 and 8.17, respectively. In Experiment II, the total multiplicity of hepatocellular foci in paraffin-embedded sections which were observed in HE-stained sections and positive/negative for CK8/18 was 4.47 and 23.17, respectively, while that of hepatocellular foci in frozen sections which were observed in HE-stained sections and positive/negative for GGT was 2.50 and 3.50, respectively. Most of the hepatocellular adenomas and carcinomas observed in HE-stained sections were positive for CK8/18, but some of the adenomas were negative for CK8/18. These findings indicate that more hepatocellular proliferative lesions can be detected in CK8/18 immunohistochemistry in addition to those observed in HE-stained sections, and suggest that CK8/18 may become a useful immunohistochemical marker for detecting hepatocellular proliferative lesions in mice.

Detection of Lymphovascular Invasion in Vulvar Cancer by D2-40 (Podoplanin) as a Predictor for Inguinal Lymph Node Metastases

Background: Lymphatic vessel invasion (LVI) plays a major role in the spread of vulvar cancer and predicts regional lymph node metastasis. D2-40, a monoclonal immunohistochemical marker might be able to increase the detection rate of LVI compared to conventional hematoxylin-eosin (HE) staining. The aim of the study was to evaluate the suitability of D2-40 for the prediction of regional lymph node metastases. Patients and Methods: Immunohistochemical staining with D2-40 was performed on formalin-fixed, paraffin-embedded tissue sections of 32 patients with squamous cell carcinoma of the vulva. Slides were screened for the presence of LVI. Correlation with clinico-pathological features including LVI as retrieved by routine HE-stained sections was assessed. Results: Immunostaining with D2-40 significantly (p = 0.019) increased the frequency of detection of lymphatic invasion compared to conventional HE staining. LVI was correctly identified by D2-40 (D2-40+ LVI) in 65.6% of tumor specimens as compared to 40.6% by routine HE staining (HE+ LVI). D2-40+ LVI significantly (p = 0.026) predicted inguinal lymph node metastases. Conclusions: Immunostaining with D2-40 significantly increased the frequency of detection of lymphatic invasion compared to conventional HE staining in squamous cell carcinomas of the vulva. D2-40+ LVI is a strong predictor for inguinal lymph node metastases.

P12: Histopathological evaluation of adult rat testis after administration of the chemotherapeutic agent Melphalan

Melphalan is an alkylating chemotherapeutic agent currently used in the treatment of various neoplasms. Several lines of evidence suggest that chemotherapeutic regimens that include melphalan administration are related with impairment of testicular function. The aim of the present study was to assess the effect of Melphalan treatment in the histology of rat testis. For that, adult male Wistar rats were dosed weekly with 7.5 mg/kg body weight for a period of three weeks. Animals were sacrificed at different time-points including one (group A; n =15) and 65 (group B; n =15) days following last administration of melphalan. Histopathological indices of testicular injury such as sloughing of seminiferous epithelium, vacuolization of Sertoli and seminiferous tubule cells, retention of step 19 spermatids at IX–XII stages, formation of multinucleated spermatids and seminiferous tubule atrophy were scored on HE-stained sections. Germ cell apoptosis was also assessed semi-quantitatively by use of caspace-3 specific immunohistochemistry. Seven rats (46.6%) of group A but only a single rat of group B (6.67%) had apoptotic germ cells exceeding the normal range. In group A, vacuolization of the seminiferous tubules, vacuolization of the Sertoli cells and sloughing of the seminiferous epithelium was found in 8 (53.3%), 2 (13.3%) and 1(6.67%) rat, respectively. Group B scores for the same lesions were unremarkable. Retented step 19 spermatids were noticed at 7 rats (46.67%) of group A and 4 rats (26.67%) of group B. None of group B animals had notable scores for the presence of multinucleated spermatids and atrophic seminiferous tubules. At group B prominence of multinucleated spermatids and atrophic seminiferous tubules were found in 2 (13.33%) and 6 (40%) rats, respectively. Taken together the results of the present study indicate that a powerful alkylating and mutagenic agent such as melphalan produced significant changes in the spermatogenic compartment of the rat testis. However, the damage was largely reversible, and the spermatogenic process was significantly restored within a two months period following termination of treatment.