Antigen Labeling Research Articles

Regenerative capacity of normal and irradiated liver following partial hepatectomy in rats

Purpose: To investigate the regenerative capacity and proliferation related to cell cycle modulators in irradiated livers after partial hepatectomy (PH) in rats.Methods and materials: Two experimental groups were given a single dose of either 4-Gy or 8-Gy photon radiation to the whole liver following PH. The control group underwent only PH, without irradiation. The liver specimens were analysed for apoptosis, proliferation and cell cycle related genes between 0.5 and 12 days.Results: Mean change in weight of the remnant liver in the 8-Gy group was significantly lower than in the control and 4-Gy groups. The apex of proliferating cell nuclear antigen labelling and bromodeoxyuridine incorporation index in two irradiated groups were also apparently lower than that in control group. After PH, transforming growth factor beta-1 (TGFβ1), and the type II receptor of TGFβ (TGFβR-II), anti-tumour protein 53(p53) and anti-tumour protein21(p21) protein expression in the irradiated livers was higher than in unirradiated ones. Significant apoptosis was noted in 8-Gy group. However, the maximal value of hepatocyte growth factor (HGF) mRNA and protein expression in the irradiated group was suppressed and restoration of liver function was delayed.Conclusion: Whole liver lower dose irradiation can attenuate regenerative capacity following partial hepatectomy in rats.

Occurrence and Tissue Distribution of Porcine Circovirus Type 2 Identified by Immunohistochemistry in Danish Finishing Pigs at Slaughter

Infection with porcine circovirus type 2 (PCV2) may be subclinical or lead to the development of porcine circovirus disease (PCVD), which includes the entities of post-weaning multisystemic wasting syndrome (PMWS) and the porcine respiratory disease complex (PRDC). PCV2 infection and PMWS occur in the early post-weaning period and are also recognized in finishing pigs of 12-19 weeks of age. The aim of the present study was to assess the role of PCV2 infection in disease of finishing pigs. Accordingly, the occurrence and tissue distribution of PCV2 was examined in Danish finishing pigs at the time of slaughter. Multiple lymph nodes and the spleen, lungs and kidneys from 136 pigs with PRDC (case group) and 36 pigs without lung lesions (control group) were examined by immunolabelling for the presence of PCV2. Additionally, follicular dendritic cells (FDC) were identified immunohistochemically. One or more tissues of 61% of the pigs were positive for PCV2 antigen. Up to 78% of the pigs had mild lymphoid depletion, indistinct lymphoid follicles and/or histiocytic infiltration of the lymph nodes, but these lesions were not associated with PCV2. No association was found between the presence of lung or kidney lesions and detection of PCV2. Three distinct patterns of cellular PCV2 antigen labelling were recognized: (1) labelling of cells with stellate morphology and reticular distribution, (2) labelling of isolated non-epithelial, cells, and (3) epithelial labelling. The reticular pattern was most common and localized to the centres of lymphoid follicles, corresponding to the morphology and distribution of FDCs. This observation may be interpreted to suggest that PCV2 may interact with FDCs to cause depletion of B lymphocytes. Alternatively, the FDCs may be a reservoir of infective PCV2 in subclinically infected animals or represent a simple storage site for PCV2 antigen in pigs that have recovered from infection.

Quantum Dot Labeling and Imaging of Glial Fibrillary Acidic Protein Intermediate Filaments and Gliosis in the Rat Neural Retina and Dissociated Astrocytes

The use of antibody and peptide functionalized semiconductor quantum dots holds considerable potential for specific labeling of target antigens and high resolution optical imaging of biological preparations. Despite this potential, their use in neuroscience is not yet widespread; a number of technical and methodological challenges must still be overcome in order to produce reliable and reproducible labeling protocols. We have optimized and used anti-GFAP functionalized quantum dots for specific labeling of intermediate filaments in astrocyte and Müller glial cells in sections of intact rat neural sensory retina and dissociated primary spinal cord astrocytes. These techniques produced stable and robust imaging of retinal astrocytes and Müller cells with minimal non-specific background labeling and intense fluorescence resulting in a high signal to noise ratio. This resulted in clear and efficient labeling of normal levels of GFAP in the retina and the ability to differentiate it from pathologically high levels of GFAP associated with reactive gliosis in a laser induced injury model. Labeling and imaging of dissociated astrocytes demonstrated the presence of what appeared to be highly complex organizations of fine intermediate filaments that spanned between cells to form intricate networks of filamentous intercellular bridges. The presence of these structures in situ and in vivo as well as any potential functions remain to be determined, but their identification should be greatly facilitated by quantum dot labeling protocols.

Inhibitory effect of bread crust antioxidant pronyl-lysine on two different categories of colonic premalignant lesions induced by 1,2-dimethylhydrazine

Colorectal malignancies continue to be one of the most frequent and life-threatening diseases throughout the world. Pronyl-lysine, a product obtained from bread crust, is a potent free radical scavenging antioxidant exerting chemopreventive activity by reducing oxidative stress. This study was conducted to investigate the effects of pronyl-lysine supplementation on the formation of colonic precancerous lesions, circulatory lipid peroxidation, and enzymic antioxidant status in 1,2-dimethylhydrazine-induced colon carcinogenesis. Male Wistar rats were randomized into seven groups; group 1 was control rats, group 2 received pronyl-lysine (2 mg/kg body weight orally) everyday, rats in groups 3-7 were administered subcutaneous 1,2-dimethylhydrazine (20 mg/kg body weight) once a week for 15 consecutive weeks. In addition, group 4 (pre-initiation), 5 (initiation), 6 (post-initiation), and 7 (entire period) received pronyl-lysine (2 mg/kg body weight orally) everyday. At the end of 34 weeks, indicative markers of lipid peroxidation and changes in antioxidant defense system were measured in circulation. The results showed that 1,2-dimethylhydrazine significantly increased total aberrant crypt foci formation, total number of dysplastic foci, beta-catenin accumulated crypts and proliferating cell nuclear antigen labeling index in the colon, and enhanced lipid peroxidation markers and decreased enzymic antioxidant activities in the plasma and erythrocyte lysate as compared with untreated controls. Pronyl-lysine supplementation significantly reversed the changes as compared with the rats treated with 1,2-dimethylhydrazine alone. The effect of pronyl-lysine was more pronounced when supplemented throughout the study period (group 7). These findings suggest that pronyl-lysine suppresses 1,2-dimethylhydrazine-induced colon carcinogenesis effectively.

Immunohistochemical Characterization of Canine Prostatic Intraepithelial Neoplasia

The development of prostate cancer is believed to be a multistep process, progressing sequentially from normal epithelium, to prostatic intraepithelial neoplasia (PIN) and, finally, to invasive neoplasia. Malignant stem cells within the basal cell layer of the prostatic epithelium are believed to play an important role in the failure of androgen-ablation therapy that occurs in the most advanced form of prostate cancer. The aim of the present study was to immunohistochemically characterize the lesions of canine PIN. Prostatic tissue from five dogs with PIN was compared with normal prostate tissue from nine further dogs. There was an increase in the number of basal epithelial cells in lesions consistent with PIN as defined by expression of the nuclear protein p63. These lesions had elevated expression of proliferating cell nuclear antigen (PCNA) and heterogeneous labelling for the nuclear androgen receptor (AR). These findings suggest that the basal cells present in PIN may play a role in canine prostate carcinogenesis and that the proliferation of these cells occurs despite the heterogeneous expression of the AR.

Immunohistochemical Expression of Vascular Endothelial Growth Factor and Vascular Endothelial Growth Factor Receptor in Canine Cutaneous Fibrosarcomas

The expression of five markers associated with tumour angiogenesis, proliferation and apoptosis was studied in 24 canine cutaneous fibrosarcomas. Tumours were assigned histological grades and were immunohistochemically evaluated for the expression of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor-2 (VEGFR-2). Additionally, intra-tumour microvessel density (iMVD) was assessed by immunohistochemical labelling for expression of von Willebrand factor (vWf) and tumour proliferation index (PI) was measured following labelling of Ki-67 antigen. Finally, tumour apoptotic index (AI) was determined by application of the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP end-labelling method (TUNEL). VEGF and VEGFR-2 expression were detected in 22/24 (92%) and 24/24 (100%) of fibrosarcomas, respectively. There was correlation between VEGF and VEGFR-2 expression (r = 0.51) and between histological grade and PI (r = 0.82). A significant difference in PI between tumours of different histological grade was found (P < 0.05). The median PI in grade 2 and 3 tumours (30.6 and 54.7, respectively) was significantly higher than in grade 1 tumours (6.4). Therefore, only PI correlates significantly with the histological grade of canine cutaneous fibrosarcomas. The potential for autocrine activity for VEGF exists in canine cutaneous fibrosarcomas, as VEGF and VEGFR-2 expression was found in most tumours.

Beneficial effects of splenectomy on massive hepatectomy model in rats

Possible spleno-hepatic relationships during hepatectomy remain unclear. The purpose of this study was to investigate the impact of splenectomy during massive hepatectomy in rats. Rats were divided into the following two groups: 90% hepatectomy (Hx group), hepatectomy with splenectomy (Hx+Sp group). The following parameters were evaluated; survival rate, biochemical parameters, quantitative RT-PCR for hemeoxygenase-1 (HO-1) and tumor necrosing factor alpha (TNFalpha), immunohistochemical staining for HO-1, proliferating cell nuclear antigen labeling index and liver weights. The survival rate after massive hepatectomy significantly improved in Hx+Sp group as well as serum biochemical parameters, compared with Hx group (P < 0.05). HO-1 positive hepatocytes and its mRNA expression significantly increased and TNFalpha mRNA expression significantly decreased in Hx+Sp group compared with Hx group (P < 0.05). Moreover, liver regeneration was significantly accelerated at 48 and 72 h after hepatectomy in Hx+Sp group. Splenectomy had beneficial effects on massive hepatectomy by ameliorating liver injuries and promoting preferable liver regeneration.

Regeneration of Graft Livers and Limited Contribution of Extrahepatic Cells After Partial Liver Transplantation in Humans

Background Liver regeneration is still not fully understood. Partial liver transplantation (LT) can provide the opportunity to investigate the mechanisms of liver regeneration, including the contribution of extrahepatic cells to liver regeneration. Methods Of 61 patients transplanted with partial liver graft between August 1997 and October 2006, 56 patients were studied, including 49 adults and 7 children. Sequential computed tomography volumetric analysis was performed for volume measurement, while proliferating cell nuclear antigen (PCNA) labeling index was investigated for liver cell proliferation in nonprotocol liver biopsy specimens. In addition, 15 male recipients who had female liver grafts were investigated in order to detect Y chromosomes as extrahepatic cells in nonprotocol liver biopsy specimens. Results Graft volume per standard liver volume was markedly increased after adult-to-adult living-donor (LD) LT. In pediatric transplants, there was no volume increase over time. PCNA labeling index was vigorous in adult-to-adult LDLT in the early period after LDLT. No Y chromosome was evident in hepatocytes from female-donor male-recipient grafts during or after liver regeneration. However, in the cases of failing grafts of this type, many Y-chromosome-positive cells were observed in the graft liver. The character of those cells was CD34(−), CK9(−), hepatocyte-specific antigen(−), and CD68(+/−). Conclusion In adult-to-adult LDLT, vigorous liver regeneration occurs in the graft liver, demonstrated by not only volumetric but cell kinetic analysis. Involvement of extrahepatic cells in normal liver regeneration seems limited.

Two-Stage Portal Vein Ligation Facilitates Liver Regeneration in Rats

Background/Aims: Recent reports have demonstrated that some patients are unable to undergo scheduled liver resection after preoperative portal vein embolization due to insufficient hypertrophy of the future remnant liver. The present study examined whether two-stage portal vein ligation (PVL) increases hypertrophy of the future remnant liver compared to conventional PVL in rats. Methods: Rats were divided into 3 groups: group A, ligation of left primary branch; group B, ligation of right and left primary branches; group C, ligation of the left primary branch, followed by 2-stage PVL 7 days postoperatively. To evaluate liver regeneration, the proliferating cell nuclear antigen labeling index (LI), mitotic index (MI) in the caudate lobe and weight ratio of caudate lobe to body weight were measured. Results: The weight ratio of caudate lobe to body weight was significantly higher in group C than in groups A or B 14 days postoperatively. In groups A and B, LI and MI in the caudate lobe peaked 2 days postoperatively, then decreased to preoperative levels by 7–8 days postoperatively, but remained significantly elevated until 10–14 days postoperatively in group C. Conclusion: Two-stage PVL increases hypertrophy of the future remnant liver compared to conventional PVL in rats.

Antibody Elution Method for Multiple Immunohistochemistry on Primary Antibodies Raised in the Same Species and of the Same Subtype

Double or multiple antigen labeling in IHC classically relies on the existence of primary antibodies raised in different species or of different IgG isotypes to ensure the specific labeling with the secondary detection systems. However, suitable pairs of primary antibodies are not always available or the best choice (e.g., as diagnostic tools). During the last few years, several methods have been proposed to overcome this, but none of them offers the flexibility needed for reliable double or multiple enzymatic or fluorescent IHC. We present here a procedure that elutes the antibodies after a first round of immunolabeling, which, in combination with precipitation-based detection systems, allows multiple IHC rounds even for primary antibodies raised in the same species and IgG isotype. Compared with other proposed methods, this procedure ensures a reliable enzymatic or fluorescent staining without cross-reactivity and without loss of tissue antigenicity, thus offering a flexible tool for colocalization studies and pathological diagnosis. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Effects of Bicyclol on Liver Regeneration After Partial Hepatectomy in Rats

Bicyclol is a synthetic antihepatitis drug with antioxidative property. The present study was performed to investigate the effect of bicyclol on liver regeneration after partial hepatectomy in rats. Bicyclol (300 mg/kg) was given to rats subjected to 70% hepatectomy three times before operation. At 6, 24, and 48 h after resection, samples were collected for the measurement of serum alanine aminotransferase (ALT), total bilirubin (TBil), hepatic glycogen, malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH). Moreover, liver regeneration rate, proliferating cell nuclear antigen (PCNA) labeling, proliferation index, and histopathological examination were evaluated at 48 h after hepatectomy. As a result, bicyclol significantly increased regeneration rate, mitotic index (MI), PCNA labeling index, and proliferation index in PH rats. Additionally, bicyclol remarkably inhibited the elevation of serum ALT and TBil levels, alleviated the formation of liver MDA, restored impaired antioxidant SOD and GSH, increased hepatic glycogen content, and also attenuated hepatic vacuolar degeneration. These results suggested that bicyclol had a beneficial effect on liver regenerative capacity of the remnant liver tissue after hepatectomy, probably due to its antioxidative property.

Biphasic effect of short-term pregnancy hormone treatment on N-methyl-N-nitrosourea-induced mammary carcinogenesis in young and old rats

This study examined the development of N-methyl-N-nitrosourea (MNU)-induced mammary carcinomas in young and old female Lewis rats following short-term treatment with estrogen and progesterone to mimic pregnancy. Rats exposed at 4 weeks of age to MNU were treated at 6 weeks of age (early MNU/young E/P treatment) or at 24 weeks of age (early MNU/old E/P treatment) with a 21-day slow-release pellet containing 0.5 mg 17β-estradiol and 32.5 mg progesterone (E/P). Other rats were exposed to MNU at 22 and again at 23 weeks of age, and were treated with E/P at 24 weeks of age (late MNU/old E/P treatment). All experimental groups were compared with respective MNU-exposed age-matched E/P-untreated rats. Overt mammary carcinomas (≥1 cm in diameter) that were positive for hormone receptors were reduced in young E/P-treated rats, while hormone receptor-negative overt mammary carcinomas increased in old E/P-treated rats. The rate of development of small-sized mammary carcinoma (<1 cm in diameter) was similar in early MNU/young E/P-treated and late MNU/old E/P-treated groups, but higher in early MNU/old E/P-treated rats compared with respective E/P-untreated rats. At the termination of the experiment, normal mammary gland architecture had not been influenced by E/P treatment, although E/P treatment of older rats caused an increase in proliferating cell nuclear antigen (PCNA) labeling of the mammary tissue. Thus, the impact of short-term E/P treatment on MNU-induced rat mammary carcinogenesis is age-dependent and shows biphasic effects; the development of hormone-dependent overt mammary carcinomas was reduced in young rats but the development of hormone-independent overt mammary carcinomas increased in older rats. The enhanced outgrowth of hormone-independent overt mammary carcinomas by E/P treatment in old age is due to accelerated cell proliferation at the promotion/progression phase of mammary carcinogenesis. Age at short-term E/P treatment is crucial for breast cancer control.

Regression of flow-induced pulmonary arterial vasculopathy after flow correction in piglets

Chronic thromboembolic pulmonary hypertension is due to partial obstruction of the pulmonary arterial bed and may resolve after pulmonary thromboendarterectomy. Persistent pulmonary hypertension, the main complication after pulmonary thromboendarterectomy, may reflect vessel alterations induced by high flow in unobstructed lung territories. The aim of this study was to determine whether correcting high flow led to reversal of the vasculopathy in piglets. The effects of high pulmonary blood flow were investigated 5 weeks after creation of an aortopulmonary shunt (n = 10), and reversibility of vessel disease was evaluated at 1 week (n = 10) and 5 weeks after shunt closure (n = 10), compared to sham-operated animals (n = 10). Hemodynamic variables, pulmonary artery reactivity, and morphometry were recorded. We also investigated the endothelin, angiopoietin, and nitric oxide synthase pathways. High flow increased medial thickness in distal pulmonary arteries (55.6% +/- 1.2% vs 35.9% +/- 0.8%; P < .0001) owing to an increase of smooth muscle cell proliferation (proliferating cell nuclear antigen labeling). The endothelium-dependent relaxation was altered (P < .05). This phenomenon was associated to an overexpression of endothelin-1, endothelin-A, angiopoietin 1, angiopoietin 2, and Tie-2 (P < .05). After 1 week of shunt closure, all overexpressed genes returned to control values, the proliferation of smooth muscle cells stopped, and smooth muscle cell apoptosis increased (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling), preceding the normalization of the wall thickness hypertrophy and the pulmonary artery vasoreactivity observed at 5 weeks after shunt closure. These results demonstrate that endothelin-1 and angiopoietin pathways are involved in vasculopathy development and may be important therapeutic targets for preventing persistent pulmonary hypertension after pulmonary thromboendarterectomy.

Suppression of Right Ventricular Hypertrophy After Extensive Pulmonary Resection in Rats by Granulocyte Colony-Stimulating Factor

The objective of the present study was to investigate the effects of granulocyte colony-stimulating factor (G-CSF) on right ventricular hypertrophy following extensive pulmonary resection in rats. Adult rats were divided into four groups: (1) Group S (right thoracotomy only); (2) Group L (right three lobectomy); (3) Group LG10 (Group L+G-CSF [10microg/kg/d]); and (4) Group LG100 (Group L+G-CSF [100microg/kg/d]). At postoperative day 21, weight ratio of the right ventricular to the left ventricle plus septum (RV/LV+S, indicator of right ventricular hypertrophy) were measured, and a histopathological study was conducted to determine percentage wall thickness of peripheral pulmonary arteries and proliferating cell nuclear antigen labeling index (indicator of oxidative DNA damage) of right ventricles. Mean RV/LV+S for Group S was 0.27+/-0.02, significantly smaller than that for the lobectomy groups (Group L, LG10, LG100; 0.47+/-0.05, 0.35+/-0.02, 0.38+/-0.05). G-CSF significantly suppressed right ventricular hypertrophy. Mean medial wall thickness of peripheral pulmonary arteries for Group S was 13.6% +/- 4.9%, significantly smaller than that for Group L (22.9% +/- 9.6%). Compared with Group L, G-CSF reduced medial wall thickness (LG10, 17.6% +/- 9.5%; LG100, 18.0% +/- 11.2%). Incidence of proliferating cell nuclear antigen positive nuclei for Group S was 1.07% +/- 0.49%, significantly smaller than that for Group L (13.77% +/- 5.87%). G-CSF significantly reduced the incidence of proliferating cell nuclear antigen positive nuclei (LG10, 4.04% +/- 2.14%; LG100, 3.18% +/- 1.66%). G-CSF administration not only reduce medial wall thickness of peripheral pulmonary arteries but also directly protect cardiomyocytes of the right ventricle, thus suppressing right ventricular hypertrophy. These results suggest that low-dose G-CSF administration can prevent right heart failure following extensive pulmonary resection.

Enhanced proliferation of acinar and progenitor cells by prophylactic pilocarpine treatment underlies the observed amelioration of radiation injury to parotid glands

Background Administration of pilocarpine before irradiation can ameliorate radiation-induced hyposalivation. Indirect evidence suggests that this effect may be mediated through induction of a compensatory response. In this study, this hypothesis is tested directly, by assessing the proliferation of progenitor and secretory cells in irradiated and non-irradiated parotid gland tissue. Methods In a rat model, parotid glands were unilaterally irradiated with a single dose of 15 Gy, 60 min after administration of pilocarpine (4.0 mg/kg). Rats were sacrificed for proliferating cell nuclear antigen (PCNA) labelling, assessing the number of proliferating progenitor and secretory cells, before, and 10 h, 1, 3, 7, 10, 20 and 30 days after irradiation. Results A small radiation-induced increase in PCNA expressing cells was observed, both in the acinar (secretory cells) and intercalated duct cell (containing the progenitor cells) compartment. This increment was significantly enhanced in pilocarpine pre-treated glands. In fact, in this group of animals increased proliferation was observed both in the irradiated and the shielded gland. Conclusions Amelioration of early loss of rat salivary gland function after radiation by pilocarpine pre-treatment is, at least in part, due to compensatory mechanisms through increased proliferation of undamaged cells.

Platelets contribute to the reduction of liver fibrosis in mice

Several recent studies have reported that liver cirrhosis (LC) can be ameliorated, but few adequate strategies are available against liver fibrosis. Although LC clinically shows thrombocytopenia and hypersplenism, the correlation with liver fibrosis and platelets remains unclear. The aim of the present study was to investigate the effect of platelets on liver fibrosis in mouse models. To induce liver fibrosis, C57BL6 female mice were injected i.p. with 1 mL/kg carbon tetrachloride (CCl(4)) twice a week for 8 weeks. Thrombocytosis was achieved by giving thrombopoietin or splenectomy in addition to CCl(4) intoxication. At 8 weeks, whole blood and liver specimens were obtained for studies as follows: peripheral platelet counts, histopathological examination, hydroxyproline assay, immunostaining, quantification of mRNA expression, and microarray analysis. Thrombocytosis significantly reduced liver fibrosis and hydroxyproline content of liver tissues compared to mice with CCl(4) administration alone. Platelets suppressed increments in mRNA expression for transforming growth factor-beta, and increased matrix metalloproteinase-9 expression in the liver. Microarray analysis of the liver revealed that platelets upregulated gene expressions involved in cell proliferation compared to expression in mice with CCl(4) intoxication alone. Platelets also increased liver volume, proliferative cell nuclear antigen labeling index, and mitotic index in fibrotic mice. These results clearly show that platelets reduce liver fibrosis and promote liver regeneration, even under cirrhotic conditions. We, therefore, propose that platelets could offer a potent tool in the treatment of liver cirrhosis.

Toll-Like Receptor 3 Is a Negative Regulator of Embryonic Neural Progenitor Cell Proliferation

Toll-like receptors (TLRs) play important roles in innate immunity. Several TLR family members have recently been shown to be expressed by neurons and glial cells in the adult brain, and may mediate responses of these cells to injury and infection. To address the possibility that TLRs play a functional role in development of the nervous system, we analyzed the expression of TLRs during different stages of mouse brain development and assessed the role of TLRs in cell proliferation. TLR3 protein is present in brain cells in early embryonic stages of development, and in cultured neural stem/progenitor cells (NPC). NPC from TLR3-deficient embryos formed greater numbers of neurospheres compared with neurospheres from wild-type embryos. Numbers of proliferating cells, as assessed by phospho histone H3 and proliferating cell nuclear antigen labeling, were also increased in the developing cortex of TLR3-deficient mice compared with wild-type mice in vivo. Treatment of cultured embryonic cortical neurospheres with a TLR3 ligand (polyIC) significantly reduced proliferating (BrdU-labeled) cells and neurosphere formation in wild type but not TLR3(-/-)-derived NPCs. Our findings reveal a novel role for TLR3 in the negative regulation of NPC proliferation in the developing brain.

Intraductal tubular carcinoma, intestinal type, of the pancreas

Presented herein is an unusual case of intraductal tubular carcinoma, intestinal type, of the pancreas. This tumor was characterized by intraductal adenoma with a few malignant foci, and also by entire involvement of the main pancreatic duct and no involvement of its branches. A 67-year-old man was admitted to hospital because of abdominal pain. On endoscopy and endoscopic retrograde cholangiopancreatography, irregular pancreatic duct was seen. No mucus secretion was observed on endoscopy. Because a biopsy showed tubular atypical cells, pancreato-duodenectomy was performed. Grossly, the entire main pancreatic duct had intraductal tumor, sparing its branches. No intraductal mucus was noted. Microscopically, the entire main pancreatic duct had proliferation of tubular adenomatous tumor without secretory mucins. Goblet cells were present in some areas. No pyloric type tubules were recognized. Malignant transformation was present in a few areas. No invasive features were recognized. On mucin histochemistry the tumor cell cytoplasm contained a little or no neutral and acidic mucus, and no secretory mucins were recognized. Immunohistochemically, the tumor cells were positive for cytokeratins (CK), CK 8, 9, 18, 19 and 20, epithelial membrane antigen, CDX2, carbohydrate antigen 19-9, and Ki-67 (labeling 30%), MUC2, MUC5AC and MUC6, and CD10. The tumor cells were negative for C-erbB2, MUC1, trypsin, pancreatic amylase and pancreatic lipase. The tumor cells were negative for p53 protein, but the malignant foci were positive for p53 protein and had high Ki-67 antigen (labeling 60%). The patient was free of disease 4 years after the operation. In summary, presented here is an extremely rare case of intraductal tubular carcinoma, intestinal type, showing focal malignant foci.

Inhibition of COX-2 and activation of peroxisome proliferator-activated receptor γ synergistically inhibits proliferation and induces apoptosis of human pancreatic carcinoma cells

Although inhibition of cyclooxygenase-2 (COX-2) or activation of peroxisome proliferators-activated receptor γ (PPAR-γ) leads to growth inhibition in malignancies, the synergistic anti-tumor effects of combination of COX-2 inhibitor (NS-398) and PPAR-γ agonist (rosiglitazone) on the human pancreatic cancer cells remains unknown. Here, we evaluated the effects of NS-398 and/or rosiglitazone on the cell proliferation and apoptosis in a pancreatic cancer cell line, SW1990. NS-398 and rosiglitazone decreased cell proliferation in a dose- and time-dependent manner. Proliferating cell nuclear antigen (PCNA) labeling index significantly decreased in the cells treated with either NS-398 or rosiglitazone. Both NS-398 and rosiglitazone alone induced apoptotic cell death of SW1990. The combination of NS-398 and rosiglitazone exerted synergistic effects on proliferation inhibition, and apoptosis induction in SW1990 cells, with down-regulation of Bcl-2 and up-regulation of Bax expression. Our results indicate that simultaneous targeting of COX-2 and PPAR-γ inhibits pancreatic cancer development more effectively than targeting each molecule alone.

Dual fluorescence labeling of surface-exposed and internal proteins in erythrocytes infected with the malaria parasite Plasmodium falciparum

There is a need for improved methods for in situ localization of surface proteins on Plasmodium falciparum-infected erythrocytes to help understand how these antigens are trafficked to, and positioned within, the host cell membrane. This protocol for confocal immunofluorescence microscopy combines surface antigen labeling on live cells with subsequent fixation and permeabilization, which enables antibodies to penetrate the cell and label internal antigens. The key steps of the protocol are as follows: indirect labeling of the surface antigen using a fluorescently tagged secondary antibody; fixation and permeabilization; indirect labeling of the internal antigen using a secondary antibody tagged with a spectrally distinct fluorescent dye; and detection of the differentially labeled antigens using a laser scanning confocal microscope. The protocol can be completed in approximately 7 h. Although the protocol is discussed here in the context of malaria parasite-infected cells, it can also be modified to visualize the membrane and intracellular distribution of surface and internal proteins in other eukaryotic cells.