Apoptotic Body Counts Research Articles

Markedly increased duodenal villous surface apoptosis in patients taking pre-exposure prophylaxis (PrEP) against human immunodeficiency virus.

Pre-exposure prophylaxis (PrEP) consists of combination antiretroviral therapy and is increasingly utilized to prevent human immunodeficiency virus (HIV) in high-risk populations. Two index cases noted during routine care showed markedly increased duodenal villous surface apoptosis in patients on PrEP. We sought to examine the prevalence of this finding and identify any clinicopathologic correlations. Gastrointestinal biopsy specimens from 23 male patients aged 18-40 years taking PrEP and 23 control patients were reviewed. Patients with HIV, inflammatory gastrointestinal diseases, and celiac disease were excluded. Apoptoses were counted on surface epithelium and deep crypts. The highest apoptotic body count per tissue fragment was recorded. Clusters were defined as groups of ≥5 apoptoses. Apoptotic counts between patients taking PrEP and controls were compared using t-tests. In PrEP patients, the median age was 35 years (range 25-40) and 83% (19/23) were white. The control patients were demographically similar (median age: 32 years [range 23-40]; 70% [16/23] white). Duodenal apoptosis in villous surface epithelium was increased in PrEP patients, with 14/23 (60.9%) patients having ≥10 surface apoptoses compared to 2/23 (8.7%) controls (P = 2.1 × 10-3 ) and 14/23 (61%) having clusters compared to 3/23 (13%) controls (P = 2.0 × 10-3 ). There was no significant association between increased surface apoptosis or clusters and clinical symptoms or duration of PrEP use. Markedly increased villous surface apoptosis, particularly in clusters, is often seen in the duodenum of patients taking PrEP. Although the mechanism and significance are unknown, knowledge of this peculiar finding may prevent unnecessary additional testing.

A practical histological approach to the diagnosis of autoimmune hepatitis: experience of an Italian tertiary referral center.

Liver biopsy is crucial for the diagnosis of autoimmune hepatitis (AIH), and new reproducible histological criteria would be highly desirable, especially in acute-on-chronic cases. The aims of the present study were (i) to evaluate the AIH histopathological criteria as a function of the time and modality of AIH onset, and (ii) to validate the count of apoptotic bodies in the portal tracts as a histopathological criterion for AIH diagnosis. Sixty-five patients were retrospectively enrolled: 20 underwent biopsy for the first diagnosis and 45 had a previous histological AIH diagnosis. Biopsies were revised, and all histological variables were collected, including the lymphocytic apoptotic bodies in the portal tracts. Clinical and serological data were revised as well. First-diagnosis patients showed a higher grade of inflammation (p = 0.001), but also worse portal fibrosis (p = 0.001). The apoptotic body count was higher in first-diagnosis patients than in follow-up patients (p = 0.002), and it was strongly correlated to inflammation. Using the apoptotic body count among the simplified AIH score variables, the first-biopsy patients in the “definite” category rose from 42 to 68%. Our results confirm the histopathological criteria proposed by the literature and introduce the count of portal apoptotic bodies for the diagnosis of active AIH, especially in first biopsies without other classic features, as well as in AIH diagnostic score, albeit future studies are required to find a definite cutoff.

Evaluation of crypt apoptotic bodies and apoptotic indices in pediatric celiac disease by routine staining and H2AX immunostaining.

Celiac disease (CD) is an immune-mediated disorder with premature apoptosis occurring along the entire crypt-villous axis. H2AX is the end product of the intrinsic apoptotic pathway. This is the first study to assess apoptotic body counts (ABC) by H&E and apoptotic indices (AI) by immunohistochemistry (IHC) in pediatric CD. The aim of the current study was to evaluate ABC in pediatric patients with CD prior to and following institution of a gluten free diet (GFD). Sixty-three pediatric endoscopic duodenal samples were assessed and divided into three groups. A total of 21 samples from treatment naïve CD patients, 21 from the same patients after instituting a GFD, and 21 from non-celiac patients as a control group. Histopathological evaluation of ABC by H&E, and immunohistochemistry assessment of apoptotic indices (AI) by H2AX antibody were performed. The mean maximum ABC and AI were significantly higher in treatment naïve CD than in GFD and control samples. These values were also significantly higher in treatment naïve Marsh 3C (flat) than in Marsh 1, 2, 3A, and 3B (non-flat) CD cases. GFD samples with persistent flat lesions had significantly higher ABC and AI than GFD non-flat cases. ROC analysis of the mean maximum ABC and AI of treatment naïve CD cases had a statistically significant predictive potential for persistent villous atrophy at a cut-off level ⩾6.61 (P = 0.008) and ⩾105.4 (P = 0.003), respectively. Histopathological evaluation of crypt apoptotic bodies could provide predictive potential for continued villous atrophy following GFD.

Fecal Microbiota Transplantation and Hydrocortisone Ameliorate Intestinal Barrier Dysfunction and Improve Survival in a Rat Model of Cecal Ligation and Puncture-Induced Sepsis.

Sepsis is a life-threatening syndrome which can progress to multiple organ dysfunction with high mortality. Intestinal barrier failure exerts a central role in the pathophysiological sequence of events that lead from sepsis to multiple organ dysfunction. The present study investigated the role of hydrocortisone (HC) administration and fecal microbiota transplantation (FMT) in several parameters of the gut barrier integrity, immune activation, and survival, in a model of polymicrobial sepsis in rats. Forty adults male Wistar rats were randomly divided into four groups: sham (group I), cecal ligation and puncture (CLP) (group II), CLP + HC (2.8 mg/kg, intraperitoneally single dose at 6 h) (group III), and CLP + FMT at 6 h (group IV). At 24 h post-CLP, ileal tissues were harvested for histological and immunohistochemical analyses while endotoxin, IL-6, and IL-10 levels in systemic circulation were determined. In a second experiment the same groups were observed for 7 days for mortality, with daily administration of hydrocortisone (group III) and FMT (group IV) in surviving rats. HC administration and FMT significantly reduced mortality of septic rats by 50%. These interventions totally reversed intestinal mucosal atrophy by increasing villous density and mucosal thickness (μm, mean ± SD: Group I: 620 ± 35, Group II: 411 ± 52, Group III: 622 ± 19, Group IV: 617 ± 44). HC and FMT reduced the apoptotic body count in intestinal crypts whereas these increased the mitotic/apoptotic index. Activated caspase-3 expression in intestinal crypts was significantly reduced by HC or FMT (activated caspase-3 (+) enterocytes/10 crypts, mean ± SD: Group I: 1.6 ± 0.5, Group II: 5.8 ± 2.4, Group III: 3.6 ± 0.9, Group IV: 2.3 ± 0.6). Both treatments increased Paneth cell count and decreased intraepithelial CD3(+) T lymphocytes and inflammatory infiltration of lamina propria to control levels. In the sham group almost the total of intestinal epithelial cells expressed occludin (92 ± 8%) and claudin-1 (98 ± 4%) and CLP reduced this expression to 34 ± 12% for occludin and 35 ± 7% for claudin-1. Administration of HC significantly increased occludin (51 ± 17%) and claudin-1 (77 ± 9%) expression. FMT exerted also a significant restoring effect in tight junction by increasing occludin (56 ± 15%) and claudin-1 (84 ± 7%) expression. The beneficial effects of these treatments on gut barrier function led to significant reduction of systemic endotoxemia (EU/mL, mean ± SD: Group I: 0.93 ± 0.36, Group II: 2.14 ± 1.74, Group III: 1.48 ± 0.53, Group IV: 1.61 ± 0.58), while FMT additionally decreased IL-6 and IL-10 levels. Fecal microbiota transplantation and stress dose hydrocortisone administration in septic rats induce a multifactorial improvement of the gut mechanical and immunological barriers, preventing endotoxemia and leading to improved survival.

An association between crypt apoptotic bodies and mucosal flattening in celiac disease patients exposed to dietary gluten

BackgroundCeliac disease (CD) is characterized histologically by inflammation and villous atrophy. Villous atrophy is thought to result from a disruption of epithelial cellular proliferation and death. Epithelial cells in intestinal mucosa normally proliferate in the crypts and migrate towards the lumen, eventually dying. Apoptotic bodies in crypts are usually abnormal and are associated with certain disease states. The presence of crypt apoptosis in celiac disease has not been thoroughly examined by routine histologic assessment of crypt apoptotic body count (ABC).MethodsWe quantified the ABC in duodenal biopsies from celiac patients before and after initiation of a gluten-free diet (GFD). We examined twenty-three duodenal biopsies from adult patients with celiac disease at diagnosis and following GFD and determined the maximum ABC in 10 consecutive crypts. Fourteen biopsies from heartburn patients served as controls.ResultsMean duration between paired biopsies was 2.9 (0.5–8.5) years. Mean maximum ABC in active celiac disease was 5.44 per crypt and decreased to 2.60 with GFD (p = <.0001). The mean maximum ABC in controls was 1.79, lower than both active celiac disease and GFD (p = <.0001 and p = .019 respectively). Flat lesions with total villous atrophy (mean: 6.44) showed a higher ABC compared to non-flat lesions (mean: 4.87); p = .04.ConclusionsCrypt ABC is markedly elevated in active celiac disease and decreases significantly with GFD, however it does not achieve normalcy. Total villous atrophy is associated with a higher ABC than all other lesions. Crypt apoptosis is likely a significant contributor to villous atrophy in celiac disease and can be appreciated by routine histologic examination.

Abstract 2119: Acetylated secretory APE1/Ref-1 induces apoptotic cell death in orthotopic xenografts of triple-negative breast cancer

Abstract The anticancer properties of acetylated secretory apurinic/apyrimidinic endonuclease-1 (Ape1/Ref-1) was suggested in triple-negative breast cancer (TNBC) cells; Posttranslational modification, hyperacetylation in MDA-MB-231 cells caused extracellular secretion of acetylated-APE1/Ref-1 (Ac-APE1/Ref-1) and initiated apoptotic cell death by auto-, paracrine binding to the receptor for advanced glycation end products (RAGE). In the present study, we observed potential therapeutic efficacy of Ac-APE1/Ref-1 in preclinical orthotopic models of TNBC in response to hyperacetylation. The extracellular Ac-APE1/Ref-1 was confirmed by proximity ligation assay in hyperacetylated tumor tissue, showing direct binding of Ac-APE1/Ref-1 and RAGE. Treatment of orthotopic TNBC xenografts with acetylating agents induced a strong growth inhibition in the tumor development as observed in computed tomography: it caused an increase of RAGE expression and activation of caspase-3 and PARP. The tumors also exhibited markedly higher count of apoptotic bodies and reduced proliferation index and neovascularization compared with control tumors. However, the Ac-APE1/Ref-1-stimulated apoptotic cell death was remarkably retarded in RAGE-knockout tumor even in hyperacetylation compared with RAGE-overexpressed one. The functional role of secreted Ac-APE1/Ref-1 in hyperacetylated TNBC was confirmed in vivo, demonstrating its relevance to the anticancer agent. Our findings suggest that Ac-APE1/Ref-1 protein possesses potent chemotherapeutic efficacy against TNBC, resistant to standard chemotherapeutic agents, warranting further evaluation as an anticancer agent Citation Format: Yu Ran Lee, Hee Kyoung Joo, Eun Ok Lee, Myoung Soo Park, Byeong Hwa Jeon, Sunga Choi. Acetylated secretory APE1/Ref-1 induces apoptotic cell death in orthotopic xenografts of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2119. doi:10.1158/1538-7445.AM2017-2119

Crypt apoptotic body counts in normal ileal biopsies overlap with graft-versus-host disease and acute cellular rejection of small bowel allografts

Crypt apoptosis in intestinal epithelium is an important diagnostic feature of graft-versus-host disease (GVHD) and acute cellular rejection (ACR) of intestinal transplants (ITx). In ITx pathology, 2 or fewer apoptotic bodies in 10 consecutive crypts are considered normal, whereas 6 or more is consistent with mild ACR. The presence of 3 to 5 apoptotic bodies is problematic and is often classified as indeterminate for ACR. The minimum diagnostic threshold for GVHD is controversial but also depends on the apoptotic body count (ABC). We investigated how many crypt apoptotic bodies could be identified in histologically normal ileal biopsies from healthy subjects (native intestines, no bone marrow transplant) who underwent screening colonoscopy and had ileal biopsy to confirm complete colonoscopy. We recorded the number of biopsy pieces per specimen and the maximum ABC in 10 consecutive crypts. Twenty-six of 40 patients (65%) had an ABC of 3 or more in 10 crypts, thus only 35% were "normal." Four (10%) had an ABC of ≥6 (positive for ACR). Twenty-two (55%) had 3-5 (indefinite for ACR). Depending on the criteria, up to 60% of the cases could be diagnosed as positive for GVHD.

Effects of anthocyanin extracted from black soybean seed coat on spermatogenesis in a rat varicocele-induced model

Varicocele is the most common cause of primary male infertility and is associated with oxidative stress. The aim of the present study was to investigate the effects of anthocyanin on a rat model of varicocele. Twenty-four male rats were divided into four experimental groups: a normal control group, a varicocele-induced control group and two varicocele-induced groups treated with either 40 or 80mgkg(-1), p.o., anthocyanin for 4 weeks. Varicocele was induced by the partial obstruction of the left renal vein. After 8 weeks, the testes and epididymides from rats in all groups were removed, weighed and subjected to histological examination and semen analysis. Apoptosis in the testes was determined by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) and oxidative stress was assessed by measuring 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels. Although no significant differences in sperm counts were observed among the groups, anthocyanin treatment of the varicocele-induced groups resulted in significantly increased testes weight, sperm motility and spermatogenic cell density (P<0.05). Anthocyanin treatment also significantly decreased apoptotic body count and 8-OHdG concentrations (P<0.05). We suggest that the antioxidant effect of anthocyanin prevented the damage caused by varicocele-induced reactive oxygen species.

The Changes of Testis and the Effects of Anthocyanin on Spermatogenesis in Rat Induced Varicocele

Purpose: Varicocele is known as a main cause of primary male infertility and it supposed to be associated with oxidative stress. Anthocyanin is known as a natural plant pigment and novel antioxidant. This study was designed to investigate the effects of anthocyanin on a rat model of varicocele. Materials and Methods: Twenty four male rats, induced varicocele by partial obstruction of left renal vein, were divided into four experimental groups: the group induced varicocele for four weeks without anthocyanin, the group received anthocyanin (80 mg/kg) right after varicocele induction, group induced varicocele for eight weeks without anthocyanin, and the group received anthocyanin (80 mg/kg) after four weeks observation following varicocele induction. After anthocyanin treatment, testes from the rats in all groups were removed, weighed, and subjected to histological examination. Apoptosis in the testes was measured by the TUNEL assay. And the oxidative stress was evaluated by measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG). Results: Induction of varicocele led to decreasing left testis weight, decreasing spermatogenic cell density significantly (p <0.05). Also it led to increasing apoptotic body counts and increasing concentration of 8-OHdG significantly (p<0.05). However administration of anthocyanin right after varicocele induction prevent this change meaningfully (p<0.05). In group received anthocyanin after four weeks observation following varicocele induction, interestingly, there was no significant difference in testis weight, spermatogenic cell density, apoptotic body count and concentration of 8-OHdG compared to group induced varicocele for eight weeks without anthocyanin administration. Conclusions: These results suggest that anthocyanin is effective in decreasing the oxidative stress of testis in rat induced varicocele and may be effective in making a healthy sperm in patient of varicocele in early stage. However in patient under way in advanced stage, it is supposed that the anthocyanin cannot help having a protective effect from oxidative stress narrowly unless the condition of oxidative stress by varicocele is corrected. Further studies are needed to better understand the mechanisms and actions of anthocyanin and varicocele, and these studies may lead

Ginsenoside Rh2 induces Bcl‐2 family proteins‐mediated apoptosis in vitro and in xenografts in vivo models

The cancer chemoprevention effects of ginseng saponins have been demonstrated against a variety of experimental tumors; however, their molecular mechanisms in vitro and in in vivo models are not well studied. This study was undertaken to gain insights into the molecular mechanisms of ginsenoside Rh2 (Rh2)-induced cell death in human breast cancer cell lines as well as in in vivo xenografts. Rh2 treatment significantly inhibited viability of both MCF-7 and MDA-MB-231 human breast cells in a concentration-dependent manner, which correlated with mitochondria-mediated apoptosis. Rh2-induced apoptosis was accompanied by the down-regulation of antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1. It also caused induction of the proapoptotic members Bak, Bax, and Bim leading to mitochondrial translocation of Bax and activation of caspases. Moreover, Rh2-induced apoptosis was partially, yet significantly protected by transient transfection of MCF-7 cells with Bax- and Bak-targeted siRNAs. Oral gavage of 5 mg Rh2/kg of mouse (three times a week) significantly caused apoptosis of MDA-MB-231 xenografts. An increase in Bax and Bak and a decrease in Bcl-2 and Bcl-xL transcript levels, in accordance with their protein expression, were observed in tumor tissue. Tumors from Rh2-treated mice exhibited a markedly higher count of apoptotic bodies and reduced proliferation index compared with control tumors. Our data suggest that Rh2 used in traditional oriental medicine for the treatment of various ailments, may be an attractive agent for the treatment and/or prevention of human breast cancers.

Anthocyanin Extracted from Black Soybean Reduces Prostate Weight and Promotes Apoptosis in the Prostatic Hyperplasia-Induced Rat Model

Anthocyanin is a natural plant pigment and potent antioxidant. This study was designed to investigate the effects of anthocyanin extracted from black soybeans on a rat model of benign prostatic hyperplasia (BPH), a disease associated with the geriatric population. Thirty male rats were divided into five experimental groups: a control group, a BPH-induced group, and three BPH-induced groups that received oral doses of anthocyanin (40, 80, and 160 mg/kg). Prostate hyperplasia was induced by the administration of testosterone propionate for 4 weeks. Following BPH induction, the anthocyanin-treated groups received the compound for 4 weeks. After anthocyanin treatment, the prostates from the rats in all groups were removed, weighed, and subjected to histological examination. Apoptosis in the prostates was measured by the TUNEL assay. The mean prostate weight for the control animals was 674.17 ± 28.24 mg, whereas the BPH-induced rats had a mean prostate weight of 1098.33 ± 131.31 mg. The mean prostate weights for the rats receiving 40, 80, and 160 mg/kg anthocyanin were 323.00 ± 22.41, 324.00 ± 26.80, and 617.50 ± 31.08 mg, respectively. The average prostate weight in the BPH-induced group was significantly higher than in the control group (p < 0.05), whereas the prostate weights in the anthocyanin-administered groups were significantly lower than in the BPH-induced group (p < 0.05). Injected testosterone led to prostatic hyperplasia as observed histologically, but anthocyanin administration helped to prevent this change. Apoptotic body counts were significantly higher in groups receiving anthocyanin than in the BPH-induced group (p < 0.05). These results suggest that anthocyanin may be effective in decreasing the volume and suppressing the proliferation of the prostate. Further studies are needed to better understand the mechanisms and actions of anthocyanin, and these studies may lead to the clinical application of anthocyanin in treating BPH.

CD4+CD25+ T Cells Play a Complex Role in the Pediatric Combined Liver-Intestinal Graft Acceptance

CD4+CD25+ T Cells Play a Complex Role in the Pediatric Combined Liver-Intestinal Graft Acceptance

Abstract 5395: Phenethyl isothiocyanate sensitizes androgen-independent human prostate cancer cells to Docetaxel-induced apoptosis in vitro and in vivo

Abstract Phenethyl isothiocyanate (PEITC), a constituent of edible cruciferous vegetables such as watercress, not only affords significant protection against chemically-induced cancer in experimental animals but also suppresses growth of cancer cells in culture and in vivo. The present study demonstrates, for the first time, that PEITC is highly effective in sensitizing PC-3 and DU145 androgen-independent human prostate cancer cells to Docetaxel-induced apoptosis in vitro as well as in vivo. Pharmacologic concentrations of PEITC (1 and 2 µmol/L) synergistically increased cytotoxicity of Docetaxel in PC-3 and DU145 cells as judged by trypan blue dye exclusion assay and Sulforhodamine B assay. The PEITC-mediated sensitization of growth inhibition by Docetaxel was associated with increased apoptosis (judged by DAPI assay and analysis of cytoplasmic histone-associated DNA fragmentation), suppression of anti-apoptotic proteins Bcl-2 and XIAP, and induction of multidomain proapoptotic proteins Bax and Bak. Moreover, the HCT-116 human colon cancer cells lacking XIAP protein were significantly more sensitive to growth inhibition and apoptosis induction by the PEITC-Docetaxel combination compared with parental HCT-116 cells. The PEITC-Docetaxel combination was markedly more efficacious against PC-3 xenografts in vivo compared with PEITC or Docetaxel alone. For example, the average tumor volume on last day of measurement (day 38) in mice treated with PEITC-Docetaxel combination (564 ± 114 mm3) was significantly lower compared with control mice [gavaged with 0.1 mL phosphate-buffered saline (PBS) five times per week and 0.1 mL PBS on day 15, 22 and 29 after tumor cell implantation; n= 6; 1071 ± 148 mm3; P= 0.019 by two-tailed Student's t-test], PEITC alone treated mice (6 μmol PEITC in 0.1 mL PBS by garage five times per week; n= 7; 1043 ± 71 mm3; P= 0.004), and Docetaxel alone treated mice (5 mg Docetaxel/kg body weight by intraperitoneal injection on day 15, 22 and 29 after tumor cell implantation; n= 6; 931 ± 126 mm3; P= 0.053). Consistent with cellular data, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay revealed significantly higher count of apoptotic bodies in tumor sections from mice treated with the PEITC-Docetaxel combination compared with PEITC or Docetaxel alone treatment groups. Moreover, the PEITC and/or Docetaxel-mediated changes in the levels of apoptosis regulating proteins in the tumor were generally consistent with the alterations observed in cultured cells. The results of the present study offer obligatory impetus to test PEITC-Docetaxel combination for the treatment of androgen-independent prostate cancer in a clinical setting. This investigation was supported by the National Cancer Institute grant 2 RO1 CA101753-06. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5395.

Abstract 218: Ginsenoside Rh2 induced bcl2 family proteins mediated apoptosis in vitro and xenograft in vivo

Abstract Natural products have received increasing attention in recent years for the discovery of novel cancer preventive and therapeutic agents. The root of Panax ginseng has been used for thousands of years in Korean medicine practice for treatment of diverse ailments. More recent studies have indicated that ginsenoside saponins, a constituents of Panax ginseng can induce retardation of growth and cell death of cancer in culture and in vivo. In this study, we used two human breast cancer cells, MCF7 (wild type p53, ER responsive), MDA-MB-231 (mutated p53, ER-independent) and xenograft nude mouse model to investigate further insights into the mechanism of Ginsenoside Rh2-induced cell death. It was observed that Rh2 treatment significantly decreases viability at 40 to 60 μM concentrations as judged by trypan blue dye exclusion assay in both two cell lines. Rh2 treatment also increased in a concentration- and time-dependent cell death in cytoplasmic histone-associated DNA fragmentation assay. Bcl-2 family proteins have emerged as critical regulators of apoptosis by functioning either as promoters (e.g., Bax and Bak) or inhibitors (e.g., Bcl-2 and Bcl-xL) of the cell death process. Rh2-induced apoptosis was correlated with induction of Bax and Bak and a decrease in Bcl-xL and Bcl-2 protein levels in both two cell lines. Transient transfection of MCF7 cells with Bak- and Bax-targeted siRNAs conferred partial yet significant protection against ginsenoside Rh2-induced apoptosis. The increased expression level of Bax was associated with initiation of intrinsic apoptotic pathway. The pathway initiated translocation of Bax from cytosol following release of mitochondrial cytochrome c dependent on ginsenoside Rh2 treatment time. To test whether Rh2-mediated inhibition of MDA-MB-231 xenograft growth in vivo at the 2mg dose was due to increased apoptosis, tumor tissues from control and Rh2-treated mice were examined for histologic evidence of apoptosis. The tumors from Rh2-treated mice exhibited markedly higher count of apoptotic bodies and reduced proliferation index. The tumor section also exhibited the changed protein level compared with control one in specific for Bax or Bcl-2 immunohistochemistry. It was consistent in RT-PCR using appropriate Bcl2- family gene primers. In conclusion, our data suggests Rh2-mediated suppression of MDA-MB-231 xenograft growth in vivo and MF7 cells in vitro highly correlates with increased apoptosis. The results of the present study show that the ability of Rh2 to inhibit cell growth or cause apoptotic cell death in two human breast cancer cells is not influenced by either p53 functional status or ER responsiveness. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 218.

Honokiol, a Constituent of Oriental Medicinal Herb Magnolia officinalis, Inhibits Growth of PC-3 Xenografts In vivo in Association with Apoptosis Induction

This study was undertaken to determine the efficacy of honokiol, a constituent of oriental medicinal herb Magnolia officinalis, against human prostate cancer cells in culture and in vivo. Honokiol-mediated apoptosis was assessed by analysis of cytoplasmic histone-associated DNA fragmentation. Knockdown of Bax and Bak proteins was achieved by transient transfection using siRNA. Honokiol was administered by oral gavage to male nude mice s.c. implanted with PC-3 cells. Tumor sections from control and honokiol-treated mice were examined for apoptotic bodies (terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling assay), proliferation index (proliferating cell nuclear antigen staining), and neovascularization (CD31 staining). Levels of Bcl-2 family proteins in cell lysates and tumor supernatants were determined by immunoblotting. Exposure of human prostate cancer cells (PC-3, LNCaP, and C4-2) to honokiol resulted in apoptotic DNA fragmentation in a concentration- and time-dependent manner irrespective of their androgen responsiveness or p53 status. Honokiol-induced apoptosis correlated with induction of Bax, Bak, and Bad and a decrease in Bcl-xL and Mcl-1 protein levels. Transient transfection of PC-3 cells with Bak- and Bax-targeted siRNAs and Bcl-xL plasmid conferred partial yet significant protection against honokiol-induced apoptosis. Oral gavage of 2 mg honokiol/mouse (thrice a week) significantly retarded growth of PC-3 xenografts without causing weight loss. Tumors from honokiol-treated mice exhibited markedly higher count of apoptotic bodies and reduced proliferation index and neovascularization compared with control tumors. Our data suggest that honokiol, which is used in traditional oriental medicine for the treatment of various ailments, may be an attractive agent for treatment and/or prevention of human prostate cancers.

Diallyl Trisulfide Suppresses Growth of PC-3 Human Prostate Cancer Xenograft In vivo in Association with Bax and Bak Induction

The present study was undertaken to determine the effect of garlic constituent diallyl trisulfide (DATS) on growth of PC-3 human prostate cancer xenograft in vivo. DATS was given orally (6 micromoL, thrice weekly) to male athymic mice s.c. implanted with PC-3 cells. Tumor sections from control and DATS-treated mice were examined for apoptotic bodies by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Protein levels of apoptosis and cell cycle regulating proteins in tumor tissues of control and DATS-treated mice were determined by immunoblotting. The effect of DATS treatment on in vivo angiogenesis was determined by immunohistochemical analysis of CD31 in tumors. Oral gavage of DATS significantly retarded growth of PC-3 xenografts in athymic mice without causing weight loss. For instance, 20 days after starting therapy, the average tumor volume in control mice was approximately 3-fold higher compared with DATS-treated mice. Tumors from DATS-treated mice exhibited a markedly higher count of apoptotic bodies compared with control tumors. Consistent with the results in cultured PC-3 cells, the DATS-mediated suppression of PC-3 xenograft growth correlated with induction of proapoptotic proteins Bax and Bak. Although DATS treatment inhibited migration of cultured PC-3 cells in association with down-regulation of vascular endothelial growth factor receptor-2 protein, formation of new blood vessels was comparable in tumors of control and DATS-treated mice as judged by CD31 immunostaining. The present study indicates that DATS administration inhibits growth of PC-3 xenografts in vivo in association with induction of Bax and Bak.

Gastric Graft-Versus-Host Disease Revisited

Accurate diagnosis of gastrointestinal graft-versus-host disease (GvHD) is important, as it contributes significantly to postallogeneic stem cell transplant (SCT) morbidity and mortality. To test the hypothesis that proton pump inhibitor (PPI) therapy may interfere with histologic evaluation of gastric GvHD by inducing apoptosis, we evaluated epithelial apoptotic body counts in antral and fundic biopsies from SCT recipients and control patients, both taking and not taking PPIs at the time of endoscopic biopsy. Hematoxylin and eosin-stained slides of gastric biopsies from 130 patients (75 allogeneic SCT with GvHD on clinical and histologic grounds, and a comparison group of 55 age- and sex-matched nontransplant patients with histologically normal gastric biopsies) were reviewed. The groups were further stratified into patients taking (PPI+) and not taking PPIs (PPI-) at the time of biopsy. Apoptotic bodies (AB)/10 (400 x) high power fields (HPF) were quantified for each case. Mean apoptotic body counts were then calculated for each case group. Seventy antral cases (31 control and 39 transplant) were also evaluated via gastrin immunohistochemistry, and the mean number of gastrin positive cells/400 x HPF calculated. In the PPI- groups, apoptosis was increased in biopsies from transplant patients, compared with controls, both in antral and fundic mucosa. In PPI+ patients, there was significantly more apoptosis in the gastric body in transplant patients than in controls. However, comparing antral biopsies from control and transplant PPI+ patients, there was no significant difference in AB quantitation. More apoptosis was seen in antral biopsies from PPI+ control patients when compared with PPI- control patients (P = 0.009). Mean numbers of gastrin positive cells/400 x HPF were increased in both control and transplant patients taking PPIs (85 and 58, respectively) compared with samples from those patients not taking PPIs (48 and 51, respectively). PPI therapy is associated with increased apoptosis in antral biopsies and may interfere with the evaluation of GvHD in biopsies from this site. A similar increase in apoptosis was not seen in fundic biopsies; biopsy of the gastric fundus rather than antrum may be preferable for the diagnosis of upper gastrointestinal GvHD.

Study of the Impact of Liver Transplantation on the Outcome of Intestinal Grafts in Children

Successful small bowel transplantation remains a challenge due to the septic and immune content of the gut. The possible beneficial role of the liver was assessed in pediatric recipients of isolated intestinal and liver intestinal combined transplantation, receiving the same immunosuppressive therapy. Fifteen children who underwent small bowel transplantation (seven SbTx) or combined liver-small bowel transplantation (eight LSbTx) at a single center between 1994 and 1998 were retrospectively reviewed and compared with fifteen controls (eight normal and seven appendicitis as inflammatory control). Transplant and patient survival, acute rejection episodes were analyzed and compared. Epithelial apoptotic body counts (ABC) and NF-kB (p65), Caspase-3 and Bax intestinal immunostaining from days 0 to 20 after transplantation were assessed. Graft and patient survivals at 5 years were respectively 75% and 75% in LSbTx; 43% and 57% in SbTx (NS). Histological analysis showed higher ABC in LSbTx intestinal mucosa (P = 0.05 on day 5, P < 0.01 thereafter). Immunostaining of biopsies on day 0 after reperfusion showed different expression of NF-kB, Caspase-3 and Bax on endothelial (P < 0.05 for NF-kB and Bax), mononuclear (P < 0.05 for Bax) and epithelial cells in LSbTx and SbTx. Our results suggest a protective role of the liver toward intestinal transplantation even in absence of significative difference, probably due to the small number of children. Early changes in NF-kB immunostaining in the biopsies sampled on day 0, pointed to a possible beneficial effect of the liver in the very early phase following transplantation, perhaps through the differential control of ischemia-reperfusion.

Effect of Oral Glutamine Administration on Bacterial Tanslocation, Endotoxemia, Liver and Ileal Morphology, and Apoptosis in Rats with Obstructive Jaundice

Postoperative complications in patients with obstructive jaundice remain increased when associated with endotoxemia and the inflammatory response due to gut barrier failure. Administration of glutamine has been proposed to maintain the integrity of the gut mucosa and thus reduce bacterial translocation (BT), but the effects of this pretreatment on apoptosis and histologic morphology of various organs affected by BT in obstructive jaundice have not been studied. We therefore studied the effects of oral glutamine supplementation on endotoxemia, BT, liver and terminal ileal morphology, and apoptosis in an experimental model of obstructive jaundice. A total of 60 male Wistar rats were randomly divided into four groups of 15 each: I, controls; II, sham-operated; III, bile duct ligation (BDL); IV, BDL + glutamine (4.5 g/kg/day in drinking water). Ileal samples for histology, DNA and protein content, liver biopsies, mesenteric lymph nodes (MLNs) for culture, and portal and systemic blood samples for endotoxin measurements were obtained 10 days later. Compared to the controls, a significant increase in contaminated MLN and liver samples and increased endotoxemia were noted in group III (p < 0.01) but were significantly reduced in group IV (p < 0.05). Group IV also had a significantly higher number of mitoses per crypt (M/c) (p < 0.05), less apoptotic body counts (ABCs) (p < 0.05), and a higher DNA content than did group III (p < 0.05). Liver biopsies from group III displayed typical changes of large duct obstruction that significantly improved after glutamine treatment, with decreased ductular proliferation. We concluded that supplementation of oral glutamine in the presence of obstructive jaundice ameliorates BT, endotoxemia, and apoptosis and improves the ileal and liver histology.

Increased apoptosis is specific for acute rejection in rat small bowel transplant

<h2>Abstract</h2> Apoptosis has been associated with several events in solid organ transplantation, including ischemia/reperfusion (IR) injury and acute rejection. To determine whether apoptosis-profiles may distinguish these two conditions, we analyzed apoptosis rates in a rat orthotopic small bowel transplant (SBT) model. SBT was performed in Lewis rats with either freshly harvested or preserved (4 h, in UW at 4°C) syngeneic and allogeneic (Brown-Norway) grafts. Bowel samples were collected 2 h after reperfusion and on small bowel transplant postoperative days (POD) 1, 4, and 7. Apoptosis was detected by measuring levels of histone-associated DNA fragments and caspase 3 expression, and by determining apoptotic body counts. All markers measured 2 h after reperfusion increased profoundly in association with preservation. After a significant decrease on POD 1, apoptosis rates rose again between POD 4 and 7 only in allogeneic grafts. This distinct second increase in apoptosis may be an early and specific sign of acute rejection.