Binding studies on isolated porcine small intestinal mucosa and in vitro toxicity studies reveal lack of effect of C. perfringens beta-toxin on the porcine intestinal epithelium

Beta-toxin (CPB) is the essential virulence factor of C. perfringens type C causing necrotizing enteritis (NE) in different hosts. Using a pig infection model, we showed that CPB targets small intestinal endothelial cells. Its effect on the porcine intestinal epithelium, however, could not be adequately investigated by this approach. Using porcine neonatal jejunal explants and cryosections, we performed in situ binding studies with CPB. We confirmed binding of CPB to endothelial but could not detect binding to epithelial cells. In contrast, the intact epithelial layer inhibited CPB penetration into deeper intestinal layers. CPB failed to induce cytopathic effects in cultured polarized porcine intestinal epithelial cells (IPEC-J2) and primary jejunal epithelial cells. C. perfringens type C culture supernatants were toxic for cell cultures. This, however, was not inhibited by CPB neutralization. Our results show that, in the porcine small intestine, CPB primarily targets endothelial cells and does not bind to epithelial cells. An intact intestinal epithelial layer prevents CPB diffusion into underlying tissue and CPB alone does not cause direct damage to intestinal epithelial cells. Additional factors might be involved in the early epithelial damage which is needed for CPB diffusion towards its endothelial targets in the small intestine.

Time-dependent supraimplant mucosa changes: short communication

PURPOSE The aim of this short communication was to analyze time-dependent changes of the supraimplant mucosa architecture in the esthetic zone. MATERIALS AND METHODS Five patients underwent single-tooth replacement with implant crowns in the anterior maxilla. The supraimplant soft tissue was conditioned with fixed provisional crowns. Quadrantlike digital impressions were taken with an intraoral optical scanning device at three time points: t0, immediately after removal of the provisional (baseline); t1, after 5 minutes; and t2, after 10 minutes. To analyze time-dependent mucosal changes, the corresponding digital files were superimposed for each patient, and baseline (t0) scans were compared with t1 and t2 scans, respectively. Wilcoxon rank sum tests were used for statistical calculations with a strict level of significance at P < .01. RESULTS Mean values for supraimplant soft tissue changes were statistically significantly different after 5 minutes (5.5%; standard deviation ± 0.3%) in comparison to the results after 10 minutes (21.7%; standard deviation ± 1.8%). The direction of mucosa shrinkage showed a trend toward palatal sites. CONCLUSION Based on the findings of this analysis, changes in supraimplant mucosa architecture seem to be affected only slightly during the first 5 minutes after removal of soft tissue support.

ADAPTATION OF INTESTINAL MUSCLE IN THE RAT AFTER INTESTINAL BYPASS

After intestinal bypass, the mucosa of the in-continuity segment (ICS) of intestine undergoes adaptive hyperplasia which results in increased absorptive function per length of intestine. In the present study, 70% of the small intestine was bypassed in rats to determine if intestinal muscle also adapts after bypass. To determine the effect of bypass on intestinal transit, a poorly absorbed marker substance was introduced into the orad portion of the ICS or bypassed loop (BL). Significantly less marker (P < 0.05) was passed from the ICS into the colon in 50 minutes in fed rats at 14 days compared to at 3 days after bypass. In 150 minutes there was more marker in the colon of fed rats at 3 and 14 days but not at 35 days after bypass than in control. In the BL, transit was slowed significantly in fed rats at 3 and 35 days and in fasted rats at 3 days but not 35 days after bypass compared to control. The circular muscle from the BL and the distal but not proximal portion of the ICS developed significantly more carbachol-stimulated force in vitro at 35 but not 3 days after bypass compared to unoperated but not sham-operated controls. At 35 days after bypass, the muscle layers had a greater muscle wet weight and protein content compared to both unoperated and sham-operated control in both the proximal and distal portions of the ICS. Similarly, there was more muscle in histological sections of the BL and distal portion of the ICS at 35 days after bypass compared to either control. Nonetheless, at 35 days after bypass actomyosin content as a fraction of muscle weight or total protein content was not different from control. The results support the hypothesis that there was a functional adaptation, i.e. slowed transit in fed rats that allowed more time for absorption. Feeding caused slowed transit in the BL as well as the ICS. Other results suggest that an increased amount of functional muscle formed in the distal portion of the ICS after bypass. ^

IN VITRO INDUCTION OF XENOIMMUNE RESPONSES TO LIPOSOMES CONTAINING HUMAN COLON TUMOR ANTIGENS

Liposomes prepared with human LS174T colon tumor cell membranes induce specific primary and secondary xenogeneic immune responses in BALB/c splenocytes in vitro. The multilamellar vesicular liposomes were prepared by adding sonicated membrane fragments in 8 mM CaCl(,2) to a dried lipid film. Cytoxic splenocytes generated in vivo exhibited specificity for the LS174T cell; liposomes elicited higher levels of cytotoxicity than did membranes (P < 0.01). Secondary blastogenic responses elicited in in vivo-primed spleen cells by liposomes also produced a significantly greater (P < 0.005) response than membranes. Subsequently, in vitro induction of primary blastogenic and cytotoxic responses by liposomes were accomplished and revealed similar kinetics to that of whole LS174T cell immunogens. Specificity of the in vitro-primed spleen cells was clearly demonstrated (P < 0.01) on a variety of human tumor cells using both the primed lymphocyte and cell-mediated cytotoxicity assays. The results of competitive inhibition tests with autologous lymphoblasts demonstrated that 30% of the cytotoxic activity was directed against lymphocyte antigens.^ The adjuvant effect of liposomes was shown to be mediated primarily by tumor antigens exposed on the outer surface of liposomes. Trypsinization of the liposomes which eliminated 96% of the surface protein reduced the ability of liposomes to induce cytotoxic splenocytes. The generation of cytolytic activity with liposomes of increasing protein concentration showed that while 10 (mu)g protein was threshold, 100 (mu)g protein induced maximal responses. In addition, membrane fluidity studies revealed that rigid liposomes were significantly (P < 0.05) more efficacious than fluid liposomes in inducing cytotoxicity.^ The induction of the primary response required the presence of nonadherent splenocytes bearing the Thy-1, Lyt-1, and Lyt-2 surface markers. The role of a Lyt-123 subpopulation was suggested by the inability of both the Lyt-1 and Lyt-2 depleted populations to completely restore the cytolytic levels to normal. In addition, the interaction of I-A('+) spleen adherent cells with liposomes for at least 8 hours was required to generate maximal cytotoxic activity. The phenotype of the cytotoxic effector was Thy-1('+), Lyt-2('+), and I-A('d-).^ Incorporation of tumor antigens into liposomes has thus enabled primary immunization in vitro to human colon cancer antigens and may afford an adaptable means to evaluate and to select specific immune responses, as well as to identify colon tumor-specific determinants.^

Role of cytochrome P450 in DNA damage produced by treatment of colon cells with 1,2-dimethylhydrazine

The study of colon cancer has taken advantage of the development of a model in animals in which tumors in the colon are easily induced by chemical treatment. When 1,2-dimethylhydrazine (DMH) is injected into rats tumor growth is observed in colon in preference to other tissues. This observation led us to investigate the Cytochrome P450 system in colon and its participation in the particular “colon sensitivity” to DMH. It has been established that the Cytochrome P450 system participates in the metabolism of DMH and the methyl carbonium product of Cytochrome P450 activation of DMH is responsible for DNA damage which is considered an initial step to carcinogenesis. The Cytochrome P450 system is a reasonable place to search for an explanation of this organotropic effect of DMH and we feel that the knowledge obtained from this study can take us closer to understanding the development of colonic malignancy. In our study we used a human colon cell line (LS174T) treated with DMH. The Cytochrome P450 system in the cells was manipulated with inducers of different isoforms of Cytochrome P450. The effect of DMH on colon cells was measured by determination of O-6-methylguanine which is a DNA adduct derived from the metabolism of this chemical and is associated with development of tumors. Our results support the hypothesis that Cytochrome P450 plays an important role in the damage to cellular DNA by DMH. This damage is increased after induction of Cytochromes P450 1A1 and 2E1. The effect of inhibition of the methyltransferase and glutathione systems on protection against DMH damage in colon demonstrated the importance of the protective role of the former and the lack of effective protection of the latter system. ^

beta-catenin interacts with and inhibits NF-kappaB in colon cancer

The β-catenin pathway plays an important role in the progression of colon cancer as well as many other cancer types. Almost all colorectal tumors show an upregulation of β-catenin activity either through mutations in the β-catenin regulator APC or through mutations in β-catenin itself. Upregulation of β-catenin leads to the transcription of many target genes involved in tumorigenesis. NF-κB is a transcription factor which activates many target genes, including both anti-apoptotic and pro-apoptotic molecules. Recently, it has been shown that GSK-3β, a negative regulator of β-catenin, is involved in the activation of NF-κB. However, the mechanism of this regulation of NF-κB by GSK-3β is unclear. As GSK-3β inhibits β-catenin we hypothesized that β-catenin may be responsible for the regulation of NF-κB by GSK-3β; i.e. β-catenin may inhibit NF-κB activity. In this study we show that β-catenin physically interacts with NF-κB leading to the inhibition of NF-κB transcriptional and DNA-binding activities. We also show that in colon cancer cells with high β-catenin expression there is a suppressed NF-κB activity and depletion of β-catenin increases NF-κB activity. Similarly, in colon cancer cells that have a low level of β-catenin NF-κB activity is high and introduction of β-catenin reduces NF-κB activity. Importantly, we show that this suppression of NF-κB by β-catenin leads to a reduction of NF-κB target gene Fas expression. Also Fas-mediated apoptosis is reduced in β-catenin overexpressing cells, which can be reversed upon depletion of β-catenin. Introduction of the NF-κB subunit p65 can restore Fas expression indicating that the effect of β-catenin on Fas is through NF-κB. Furthermore, β-catenin expression was found to inversely correlate with Fas expression in human colon and breast primary tumor tissues. As Fas downregulation is important for tumors to evade immune surveillance, β-catenin inhibition of NF-κB and Fas downregulation likely plays and important role for colon cancer progression. Additionally, we found that phosphoinositide 3-kinase plays a role in the regulation of β-catenin inhibition of NF-κB through the disruption of the β-catenin/NF-κB complex. This study provides a link between two important signal transduction pathways as well as another mechanism of β-catenin oncogenesis. ^

Salivary cortisol, psychological stress and depressive symptoms among patients undergoing colon cancer screenings

As the second leading cause of cancer-related deaths in the United States, colon cancer has a high cure rate if detected early by a colonoscopy (U.S. Cancer Statistics Working Group, 2007). However, more than 41 million at-risk Americans are not properly receiving colonoscopy screenings according to the recommendations of the Center for Disease Control. This study provides insight into the physiological and psychological benefits of the colonoscopy procedure over and above cancer detection and prevention. Thirty-six patients receiving colonoscopic screening at the University of Connecticut Health Center participated in this study. A questionnaire battery that assessed perceived stress, depressive symptoms, colon cancer related worry, and social support, and optional saliva sampling was completed 2 weeks prior to and post colonoscopy. It was hypothesized that salivary cortisol concentrations, perceived stress, and self-reported depressive symptoms would show significant decreases from pre to post colonoscopy, and that these variables would all be positively correlated with one another. Results showed significant, positive correlations between depressive symptoms and both salivary cortisol (r (34)= .348, p< .05) and perceived stress (r (34)= .635, p< .01). Morning salivary cortisol levels decreased significantly from pre to post colonoscopy to levels below the population mean (t (16)=-3.711, p<. 01). No such differences were observed in either perceived stress or depressive symptoms. These results indicate that by decreasing cortisol concentrations to levels below that of the population mean, the colonoscopy provided physiological health benefits to patients beyond cancer screening. From a health psychology standpoint, this may encourage some of the 41 million Americans not receiving proper colon cancer screenings to adopt this potentially life-saving health behavior.

Jenny-Marguerite Colon, Hüftbild

Signatur des Originals: S 36/G02907

Léontine Fay, Hüftbild - Jenni-Vertpré, Hüftbild - Jenni-Colon, Hüftbild - Marie Thomase Amélie Dorval, Hüftbild - Alberr, Hüftbild - {[N.N.], Hüftbild

Signatur des Originals: S 36/G04476

High-resolution microarray analysis of chromosome 20q in human colon cancer metastasis model systems

Amplification of human chromosome 20q DNA is the most frequently occurring chromosomal abnormality detected in sporadic colorectal carcinomas and shows significant correlation with liver metastases. Through comprehensive high-resolution microarray comparative genomic hybridization and microarray gene expression profiling, we have characterized chromosome 20q amplicon genes associated with human colorectal cancer metastasis in two in vitro metastasis model systems. The results revealed increasing complexity of the 20q genomic profile from the primary tumor-derived cell lines to the lymph node and liver metastasis derived cell lines. Expression analysis of chromosome 20q revealed a subset of over expressed genes residing within the regions of genomic copy number gain in all the tumor cell lines, suggesting these are Chromosome 20q copy number responsive genes. Bases on their preferential expression levels in the model system cell lines and known biological function, four of the over expressed genes mapping to the common intervals of genomic copy gain were considered the most promising candidate colorectal metastasis-associated genes. Validation of genomic copy number and expression array data was carried out on these genes, with one gene, DNMT3B, standing out as expressed at a relatively higher levels in the metastasis-derived cell lines compared with their primary-derived counterparts in both the models systems analyzed. The data provide evidence for the role of chromosome 20q genes with low copy gain and elevated expression in the clonal evolution of metastatic cells and suggests that such genes may serve as early biomarkers of metastatic potential. The data also support the utility of the combined microarray comparative genomic hybridization and expression array analysis for identifying copy number responsive genes in areas of low DNA copy gain in cancer cells. ^

Oncologic outcomes in diabetic patients with colon cancer

Type II diabetes mellitus is a growing problem worldwide and although its association with increased cardiovascular morbidity and mortality is well known, its role in the development of cancer is now being further elucidated. Recently, there has been increasing evidence that not only are diabetics more susceptible towards development of particular types of cancer, but also have worse oncologic outcomes. This retrospective chart review investigates whether diabetics with colon cancer have a poorer prognosis than their nondiabetic counterparts. Patients with high risk Stage II and Stage III colon cancer who were diagnosed and/or treated at The University of Texas M.D. Anderson Cancer Center from 1/1/2000 till 12/1/2004 were included in our study. We carried out a survival analysis using Kaplan-Meier method and multivariable analysis to assess differences in outcomes of the two population groups. We found that the decreased overall survival in diabetics did not reach statistical significance but this could be due to a lower event rate in our study. Larger studies are required to investigate this further. ^

Adherence to adjuvant chemotherapy and post-treatment surveillance in a large community-based cohort of patients with colon cancer

The objective of this dissertation was to determine the initiation and completion rates of adjuvant chemotherapy, its toxicity and the compliance rates of post-treatment surveillance for elderly patients with colon cancer using the linked Surveillance, Epidemiology, and End Results – Medicare database.^ The first study assessed the initiation and completion rate of 5-fluorouracil-based adjuvant chemotherapy and its relationship with patient characteristics. Of the 12,265 patients diagnosed with stage III colon adenocarcinoma in 1991-2005, 64.4% received adjuvant chemotherapy within 3-months after tumor resection and 40% of them completed the treatment. Age, marital status, and comorbidity score were significant predictors for chemotherapy initiation and completion.^ The second study estimated the incidence rate of toxicity-related endpoints among stage III colon adenocarcinoma patients treated with chemotherapy in 1991-2005. Of the 12,099 patients, 63.9% underwent chemotherapy and had volume depletion disorder (3-month cumulative incidence rate [CIR]=9.1%), agranulocytosis (CIR=3.4%), diarrhea (CIR=2.4%), nausea and vomiting (CIR=2.3%). Cox regression analysis confirmed such association (HR=2.76; 95% CI=2.42-3.15). The risk of ischemic heart diseases was slightly associated with chemotherapy (HR=1.08), but significantly among patients aged <75 with no comorbidity (HR=1.70). ^ The third study determined the adherence rate of follow-up cares among patients diagnosed with stage I-III colon adenocarcinoma in 2000 - June 2002. We identified 7,348 patients with a median follow-up of 59 months. The adherence rate was 83.9% for office visits, 29.4% for CEA tests, and 74.3% for colonoscopy. Overall, 25.2% met the recommended post-treatment care. Younger age at diagnosis, white race, married, advanced stage, fewer comorbidities, and chemotherapy use were significantly associated with guideline adherence.^ In conclusions, not all colon cancer patients received chemotherapy. Receiving chemotherapy was associated with increased risk of developing gastrointestinal, hematological and cardiac toxicities. Patients were more likely to comply with the schedule for office visits and colonoscopy but failed in CEA tests. ^

Geographic variation in the utilization and survival associated with oxaliplatin chemotherapy in patients with stage III colon cancer

Background: Overall objectives of this dissertation are to examine the geographic variation and socio-demographic disparities (by age, race and gender) in the utilization and survival of newly FDA-approved chemotherapy agents (Oxaliplatin-containing regimens) as well as to determine the cost-effectiveness of Oxaliplatin in a large nationwide and population-based cohort of Medicare patients with resected stage-III colon cancer. Methods: A retrospective cohort of 7,654 Medicare patients was identified from the Surveillance, Epidemiology and End Results – Medicare linked database. Multiple logistic regression was performed to examine the relationship between receipt of Oxaliplatin-containing chemotherapy and geographic regions while adjusting for other patient characteristics. Cox proportional hazard model was used to estimate the effect of Oxaliplatin-containing chemotherapy on the survival variation across regions using 2004-2005 data. Propensity score adjustments were also made to control for potential bias related to non-random allocation of the treatment group. We used Kaplan-Meier sample average estimator to calculate the cost of disease after cancer-specific surgery to death, loss-to follow-up or censorship. Results: Only 51% of the stage-III patients received adjuvant chemotherapy within three to six months of colon-cancer specific surgery. Patients in the rural regions were approximately 30% less likely to receive Oxaliplatin chemotherapy than those residing in a big metro region (OR=0.69, p=0.033). The hazard ratio for patients residing in metro region was comparable to those residing in big metro region (HR: 1.05, 95% CI: 0.49-2.28). Patients who received Oxalipaltin chemotherapy were 33% less likely to die than those received 5-FU only chemotherapy (adjusted HR=0.67, 95% CI: 0.41-1.11). KMSA-adjusted mean payments were almost 2.5 times higher in the Oxaliplatin-containing group compared to 5-FU only group ($45,378 versus $17,856). When compared to no chemotherapy group, ICER of 5-FU based regimen was $12,767 per LYG, and ICER of Oxaliplatin-chemotherapy was $60,863 per LYG. Oxaliplatin was found economically dominated by 5-FU only chemotherapy in this study population. Conclusion: Chemotherapy use varies across geographic regions. We also observed considerable survival differences across geographic regions; the difference remained even after adjusting for socio-demographic characteristics. The cost-effectiveness of Oxaliplatin in Medicare patients may be over-estimated in the clinical trials. Our study found 5-FU only chemotherapy cost-effective in adjuvant settings in patients with stage-III colon cancer.^

Risk factors for surgical site infections among patients undergoing major colon surgery in the United States 2007-2009

Background: Surgical site infections (SSIs) after abdominal surgeries account for approximately 26% of all reported SSIs. The Center for Disease Control and Prevention (CDC) defines 3 types of SSIs: superficial incisional, deep incisional, and organ/space. Preventing SSIs has become a national focus. This dissertation assesses several associations with the individual types of SSI in patients that have undergone colon surgery. ^ Methods: Data for this dissertation was obtained from the American College of Surgeons' National Surgical Quality Improvement Program (NSQIP); major colon surgeries were identified in the database that occurred between the time period of 2007 and 2009. NSQIP data includes more than 50 preoperative and 30 intraoperative factors; 40 collected postoperative occurrences are based on a follow-up period of 30 days from surgery. Initially, four individual logistic regressions were modeled to compare the associations between risk factors and each of the SSI groups: superficial, deep, organ/space and a composite of any single SSI. A second analysis used polytomous regression to assess simultaneously the associations between risk factors and the different types of SSIs, as well as, formally test the different effect estimates of 13 common risk factors for SSIs. The final analysis explored the association between venous thromboembolism (VTEs) and the different types of SSIs and risk factors. ^ Results: A total of 59,365 colon surgeries were included in the study. Overall, 13% of colon cases developed a single type of SSI; 8% of these were superficial SSIs, 1.4% was deep SSIs, and 3.8% were organ/space SSIs. The first article identifies the unique set of risk factors associated with each of the 4 SSI models. Distinct risk factors for superficial SSIs included factors, such as alcohol, chronic obstructive pulmonary disease, dyspnea and diabetes. Organ/space SSIs were uniquely associated with disseminated cancer, preoperative dialysis, preoperative radiation treatment, bleeding disorder and prior surgery. Risk factors that were significant in all models had different effect estimates. The second article assesses 13 common SSI risk factors simultaneously across the 3 different types of SSIs using polytomous regression. Then each risk factor was formally tested for the effect heterogeneity exhibited. If the test was significant the final model would allow for the effect estimations for that risk factor to vary across each type of SSI; if the test was not significant, the effect estimate would remain constant across the types of SSIs using the aggregate SSI value. The third article explored the relationship of venous thromboembolism (VTE) and the individual types of SSIs and risk factors. The overall incidence of VTEs after the 59,365 colon cases was 2.4%. All 3 types of SSIs and several risk factors were independently associated with the development of VTEs. ^ Conclusions: Risk factors associated with each type of SSI were different in patients that have undergone colon surgery. Each model had a unique cluster of risk factors. Several risk factors, including increased BMI, duration of surgery, wound class, and laparoscopic approach, were significant across all 4 models but no statistical inferences can be made about their different effect estimates. These results suggest that aggregating SSIs may misattribute and hide true associations with risk factors. Using polytomous regression to assess multiple risk factors with the multiple types of SSI, this study was able to identify several risk factors that had significant effect heterogeneity across the 3 types of SSI challenging the use of aggregate SSI outcomes. The third article recognizes the strong association between VTEs and the 3 types of SSIs. Clinicians understand the difference between superficial, deep and organ/space SSIs. Our results indicate that they should be considered individually in future studies.^

Mechanisms of multidrug resistance: Differences between natural and acquired adriamycin -resistant human colon carcinoma cells

Mechanisms of multidrug resistance (MDR) were studied in two independent MDR sublines (AdR1.2 and SRA1.2) derived from the established human colon carcinoma cell line LoVo. AdR1.2 was developed by long-term continuous exposure of the cells to adriamycin (AdR) in stepwise increments of concentration, while SRA1.2 was selected by repetitive pulse treatments with AdR at a single concentration. In this dissertation, the hypothesis that the mechanism of drug resistance in SRA1.2 is different than that in AdR1.2 is tested. While SRA1.2 demonstrated similar biological characteristics when compared to the parental LoVo, AdR1.2 exhibited remarkable alterations in biological properties. The resistance phenotype of AdR1.2 was reversible when the cells were grown in the drug-free medium whereas SRA1.2 maintained its resistance for at least 10 months under similar conditions. Km and Vmax of carrier-mediated facilitated diffusion AdR transport were similar among the three lines. However, both resistant sublines exhibited an energy-dependent drug efflux. AdR1.2 appeared to possess an activated efflux pump, and a decreased nucleus-binding of AdR, whereas SRA1.2 showed merely a lower affinity in binding of AdR to the nuclei. Southern blot analysis showed no amplification of the MDR1 gene in either of the two resistant subclones. However, Western blot analysis using the C219 monoclonal antibody against P170 glycoprotein detected a Mr 150-kDa plasma protein (P150) in AdR1.2 but not in SRA1.2 or in the parental LoVo. In vitro phosphorylation studies revealed that P150 was a phosphoprotein; its phosphorylation was Mg$\sp{2+}$-dependent and could be enhanced by verapamil, an agent capable of increasing intracellular AdR accumulation in AdR1.2 cells. The phosphorylation studies also revealed elevated phosphorylation of a Mr 66-kDa plasma membrane protein that was detectable in the AdR1.2 revertant and in AdR1.2 when verapamil was present, suggesting that hyperphosphorylation of the Mr 66-kDa protein may be related to the reversal of MDR. SDS-PAGE of the plasma membrane protein demonstrated overproduction of a Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa, MDR-related protein in both the resistant sublines. The Mr 130-kDa protein was not immunoreactive with C219, but its absence in the AdR1.2 revertant and the parental LoVo suggests that it is an MDR-related plasma membrane protein. In conclusion, the results from this study support the author's hypothesis that the mechanisms responsible for "Acquired" and "Natural" MDR are not identical. ^