Removal of sulfamethizole from aqueous solution using advanced oxidation processes: effects of pH and salinity.

This study investigates the removal of sulfamethizole (SFZ) in ozone (O3), O3/Na2S2O8 (sodium persulfate), UV/Na2S2O8, UV/O3, and UV/O3/Na2S2O8 systems. The effects of pH and salinity on SFZ mineralization were evaluated. The mineralization of SFZ followed pseudo-first-order kinetics. At pH 5, the rate constants of SFZ mineralization in O3, O3/Na2S2O8, UV/Na2S2O8, UV/O3, and UV/O3/Na2S2O8 systems were 0.576, 0.924, 0.702, 1.26, and 5.21 h-1, respectively. The SFZ mineralization rate followed the order pH 5 > pH 7 > pH 9 in all tested advanced oxidation processes. Salinity increased the rate of SFZ mineralization in O3 and O3/Na2S2O8 systems and decelerated it in UV/Na2S2O8, UV/O3, and UV/O3/Na2S2O8 systems. UV/O3/Na2S2O8 was the best system for mineralizing SFZ, and sulfate radicals were the predominant species in UV/O3/Na2S2O8.

Review paper: Implications of the "cancer stem cell" hypothesis on murine models of colon cancer and colitis-associated cancer.

The use of murine models to investigate human diseases has been an invaluable tool. In the areas of inflammation and oncogenesis, such models have provided unique insights into pathogenesis and mechanisms to evaluate potential therapy. As such, one facet of these disease processes links inflammation and cancer. Inflammation is associated with at least 15% of the world's malignancies. One example of this relationship is documented in the association between colitis and colorectal cancer. To date, the precise molecular events linking inflammation and cancer remain unclear. A new paradigm that may bridge these processes includes the cancer stem cell hypothesis. In this review, murine models of colitis, colon cancer, and colitis-associated cancer are discussed in reference to the potential of this paradigm to clarify the relationship of these devastating diseases.

CDX2 does not suppress tumorigenicity in the human gastric cancer cell line MKN45.

CDX2 is a Drosophila caudal-related homeobox transcription factor that is expressed specifically in the intestine. In mice, ectopic expression of CDX2 in the gastric mucosa gives rise to intestinal metaplasia and in one model, gastric carcinoma. In humans, increased CDX2 expression is associated with gastric intestinal metaplasia and tubular adenocarcinomas. These patterns of expression have shown that CDX2 is important for the initiation of intestinal metaplasia in the gastric mucosa, but the role of CDX2 in established gastric cancer remains unclear. We sought to determine whether CDX2 contributes to tumorigenic potential in established gastric cancer. The CDX2 gene in MKN45 gastric carcinoma cells was disrupted using targeted homologous recombination. The resulting CDX2-/- cells are essentially identical to their parental cells, with the exception of CDX2 ablation. We found no significant differences in the proliferation of CDX2-/- cells compared to CDX2+/+ cells, in vitro or in vivo. Molecular analyses show that loss of CDX2 predominantly altered the expression of genes involved in intestinal glandular differentiation and adhesion. However, there were no microscopic differences in tumor differentiation. We conclude that disruption of CDX2 in MKN45 cells does not significantly affect their tumorigenic potential.

Alloantigenicity of human endothelial cells. IV. Derivation, characterization, and utilization of gonadal vein endothelia to control endothelial alloantigenicity during lymphocyte-endothelial interactions.

Most previous studies to evaluate endothelial cell-T lymphocyte interactions have used human peripheral blood as a source of T lymphocytes and human umbilical vein endothelial cells as a source of endothelia. Implicit in this experimental system are allogeneic lymphocyte-endothelial interactions, which are largely ignored. To overcome this problem, we isolated gonadal vein endothelial cells (GVEC) along with matched splenic macrophages and T lymphocytes from cadaveric donors, thus providing a completely autologous series of cells for experimentation. First, GVEC were analyzed for morphology, surface phenotype, and cytokine mRNA expression, and found to be indistinguishable from human umbilical vein endothelial cells. Using this system, we observed that irradiated GVEC were able to promote a 2- to 3-fold increase in the proliferation of matched autologous splenic T cells after PHA stimulation. This indicates that the costimulator activity of endothelial cells reported by others is an intrinsic property of endothelial cells, and is not a consequence of endothelial alloantigens. We also used this system to assess the relative abilities of GVEC and macrophages obtained from the same donor to stimulate the proliferation of purified allogeneic CD3+ PBL. We found the following hierarchy of alloantigenicity in this experimental system: splenic macrophages > IFN-gamma-treated GVEC >> untreated GVEC = TNF alpha-treated GVEC. These studies demonstrate that allogeneic macrophages are intrinsically more antigenic than endothelial cells derived from the same donor. Furthermore, they illustrate the utility of this experimental system to obtain data regarding lymphocyte-endothelial interactions that are otherwise unobtainable.

Differential effects of gallium nitrate on T lymphocyte and endothelial cell activation.

The immunosuppressive agents used clinically to prevent allograft rejection exert their effects by interfering with antigen-dependent T cell activation, endothelial cell function, or both. Gallium nitrate (GN) is immunosuppressive both in vitro and in vivo, and has potential for clinical use in transplant recipients. Therefore, we analyzed the influence of GN on gonadal vein endothelial cell (GVEC) and T cell activation. GVEC were stimulated with IFN gamma or TNF alpha in the presence or absence of GN, and tested for changes in levels of MHC class I, MHC class II, vascular cell adhesion molecule-1, and intercellular adhesion molecule-1 expression. GN did not interfere with the baseline or cytokine-enhanced expression of these molecules. Rather, it increased the expression of intercellular adhesion molecule-1 on GVEC, and this effect was further augmented in the presence of IFN gamma. In contrast, GN inhibited T cell proliferation stimulated by allogeneic GVEC or allogeneic monocytes in a dose-dependent manner. In transwell experiments, GN blocked the induction of MHC class II expression on isolated GVEC caused by alloactivated T cells, but not by recombinant IFN gamma. This suggests that GN can interfere indirectly with inflammatory responses of endothelial cells by interfering with local T cell activation and lymphokine production. Once lymphokines are produced, GN does not interfere with their effects on endothelial cells. GN is thought to act through transferrin receptors, but GVEC, unlike T cells, do not increase their expression of transferrin receptors, after stimulation with cytokines. This may explain their relative lack of sensitivity to GN. In general, GN appears to stimulate endothelial cells but suppress T cells. This paradoxic effect suggests that therapy with GN may enhance T cell-independent inflammatory responses, such as cellular infiltration and repair of tissue damage, while suppressing T cell-dependent responses, such as T cell-mediated tissue destruction and allograft rejection.

Prevention of acute murine cardiac allograft rejection: anti-CD4 or anti-vascular cell adhesion molecule one monoclonal antibodies block acute rejection but permit persistent graft-reactive alloimmunity and chronic tissue remodelling.

We treated C57BL/6 mouse recipients of DBA/2 cardiac allografts with anti-CD4 monoclonal antibodies (mAb) or anti-vascular cell adhesion molecule 1 mAb to promote long-term allograft survival and subjected both the recipient animals and the long-surviving allografts to a battery of histologic and immunologic tests. The results were similar regardless of the mAb used for antirejection therapy. At all tested times after transplantation, the allografts displayed histologic evidence of ongoing microvascular endothelial activation and interstitial leukocytic infiltration. Reverse transcription polymerase chain reaction analyses revealed continuous intragraft expression of messenger RNA for interleukin 1, interleukin 2, interleukin 4, interleukin 6, tumor necrosis factor, interferon gamma, and transforming growth factor beta. All grafts had histologic evidence of ongoing vascular and parenchymal tissue remodeling, including interstitial fibrosis and vascular neointimal hyperplasia. The graft recipients retained limiting dilution analysis--detectable, donor-reactive cytolytic T lymphocyte, and helper T lymphocyte in their spleens and produced high liters of donor-reactive alloantibodies. Variable amounts of allogeneic microchimerism were detectable in some, but not all of the long-surviving graft recipients. In general, these observations indicate that (1) a similar immune status is achieved in long-surviving allografts and their recipients when either anti-CD4 mAb or anti-vascular cell adhesion molecule-1 mAb was used for antirejection therapy, in spite of the major differences in lineage and distribution of cells targeted by these two mAbs, (2) this immune status is characterized by continuous, long-term inflammatory and immune processes very similar to those observed during acute allograft rejection, and (3) in spite of these processes the allografts continue to function, although they invariably develop a chronic rejection-like histopathologic condition that may ultimately limit graft function. In this regard, the recipients of long-surviving allografts do not seem to be tolerant of their graft alloantigens.

Specific [3H]-spiroperidol binding sites in human prefrontal cortex: potential site multiplicity and overall serotonin-like selectivity.

[3H]-Spiroperidol specifically binds at sites in human prefrontal cortex. The binding of this ligand is apparently anomalous at 37 degrees C, with a substantial loss of specific binding occurring between 5 and 40 min incubation. However, at 21 degrees C, this loss of binding is not observed even at 60 min. At 21 degrees C, [3H]-spiroperidol binding in human prefrontal cortex is apparently occurring at multiple sites or multiple affinity states of single classes of sites, or at a combination of both. The overall selectivity is predominantly serotonergic, rather than dopaminergic.

Cations decrease specific [3H]-spiroperidol binding in human prefrontal cortex.

Ligand binding at many physiologically relevant receptors is regulated by divalent cations. To determine whether [3H]-spiroperidol binding sites in prefrontal cortex might be physiologically relevant receptors, we examined the effect of ions on the binding of this ligand in postmortem human prefrontal cortex. Our results indicate that several cations decreased [3H]-spiroperidol binding in a dose-dependent fashion. Of these, Cd++ and Zn++ were the most able to decrease [3H]-spiroperidol binding with IC50 of 5.5 +/- 2.4 X 10(-6)M and 5.6 +/- 1.1 X 10(-5)M respectively. These findings indicate that [3H]-spiroperidol may bind at physiologically relevant receptors in human prefrontal cortex.

The percentage of CD31+ T cells decreases after open but not laparoscopic surgery.

BACKGROUND: Efficient killing of tumor cells depends on T cells that migrate from the circulation to the peripheral tissues; these cells express CD31. This study was undertaken to determine the impact of open (OS) and laparoscopic (LS) colorectal surgery on the percentage of circulating CD3+CD31+ cells. METHODS: Peripheral blood was collected from 27 OS and 24 LS colon cancer patients preoperatively (preOP) and on postoperative days 1 (POD1) and 3 (POD3). CD31+ T cells were assessed by flow cytometry using monoclonal antibodies. RESULTS: In the OS group, the percentage of CD3+CD31+ cells was significantly lower in POD1 and POD3 samples compared to the preOP results. LS surgery did not result in a significant change in the percentage of these T cells. A significant correlation was found between the decrease in the percentage of CD3+CD31+ cells and the length of incision in OS patients. CONCLUSIONS: The percentage of CD3+CD31+ cells decreases following OS but not LS and may be related to incision length. This may compromise T cell function in the peripheral tissues in the postoperative period.

Lymphocyte proliferation in mice after a full laparotomy is the same whether performed in a sealed carbon dioxide chamber or in room air.

PURPOSE: Our laboratory has demonstrated that significantly more cell-mediated immunosuppression occurs after full laparotomy than after either anesthesia control or carbon dioxide (CO2) pneumoperitoneum. We further demonstrated that the postoperative immunosuppression is related to the length of the incision. Other investigators believe that the immunosuppression observed after laparotomy is caused by peritoneal exposure to small amounts of lipopolysaccharide found in circulating air. They believe that the better-preserved immune function associated with laparoscopic surgery results from the avoidance of air contamination of the peritoneal cavity. To investigate this hypothesis, we determined and compared postoperative lymphocyte proliferation rates after (a) laparotomy in room air, (b) laparotomy in a CO2 chamber, (c) CO2 insufflation in a murine model, and (d) anesthesia alone. METHODS: Female C3H/He mice (n = 21) were divided randomly into four groups: (a) anesthesia control, (b) air laparotomy, (c) CO2 laparotomy, and (d) CO2 insufflation. The control mice underwent no procedure. The group 2 animals underwent a full midline incision (xiphoid to pubis) and exposure to room air for 20 min and then were clipped closed. The group 3 mice underwent a full midline incision in a sealed CO2 chamber for 20 min, and the group 4 mice insufflation with CO2 gas at 4 to 6 mm Hg for 20 min. Splenocytes were harvested from all the animals on day 2 after the interventions. Lymphocyte proliferation then was assessed using the nonradioactive colorimetric MTS/PMS system 72 h after concanavalin-A stimulation. RESULTS: There was no significant difference in lymphocyte proliferation between the air and CO2 laparotomy groups. Lymphocyte proliferation in the anesthesia control and CO2 insufflation groups was significantly higher than in both the air laparotomy (p<0.05) and CO2 laparotomy (p<0.05) groups (p values by Tukey-Kramer test). There was no significant difference between the anesthesia control and CO2 pneumoperitoneum groups. CONCLUSIONS: Our results suggest that full laparotomy performed in a sealed CO2 chamber compared to room air laparotomy resulted in similar suppression of lymphocyte proliferation. Furthermore, no significant suppression of lymphocyte proliferation was observed in the CO2 pneumoperitoneum group. These results, with regard to lymphocyte proliferation rates, refute the hypothesis that postoperative immunosuppression is related to air exposure and support the alternative hypothesis that immunosuppression is related to incision length.

Increased incidence of colorectal adenomas in follow-up evaluation of patients with newly diagnosed hyperplastic polyps.

BACKGROUND: Although adenomatous polyps have been established clearly as precursor lesions for most cases of colorectal cancer, the role, if any, of hyperplastic polyps remains uncertain. The aim of the current study was to determine whether a patient with an index finding of hyperplastic polyp on colonoscopy is at increased risk for adenomatous polyps. METHODS: We conducted a retrospective cohort study using the records of a single surgeon's colonoscopic experience over a 20-year period (June 1973 to December 1994). Patients found to have hyperplastic lesions on index colonoscopy were compared with those who had "clean" index colonoscopies. The two groups were compared for the subsequent diagnosis of adenomatous polyps on follow-up colonoscopies. Those with cancer or adenomas at index colonoscopy or in their history were excluded. We used Cox proportional hazard modeling with subsequent adenoma or cancer diagnosis at follow-up colonoscopy as the outcome, controlling for age and gender. RESULTS: We identified 42 patients for whom hyperplastic polyps were the only colorectal neoplasms found on the index examination, in contrast to 362 control patients who had a "clean" index examination. In this cohort study, patients found to have only hyperplastic polyps on initial examination had a rate of subsequent adenoma diagnoses (42%) twice that of patients with a clean initial colonoscopy (21%). Mean follow-up time was 4.3 years. The relative rate ratio was 2.0 (95% confidence interval, 1.2-3.4). CONCLUSIONS: This study suggests that patients found to have hyperplastic polyps on initial colonoscopic examination may have twice the risk of adenomas on follow-up colonoscopy, as compared with those who have clean initial examinations. If this finding is borne out in larger prospective studies, surveillance strategies may need to be modified accordingly.

Increased platelet-derived growth factor (PDGF) release after laparotomy stimulates systemic tumor growth in mice.

BACKGROUND: Our laboratory has demonstrated that tumors grow larger and are more easily established following laparotomy than after carbon dioxide (CO2) pneumoperitoneum or anesthesia alone. We have also shown that tumor cells incubated with serum from laparotomized mice proliferated significantly faster in vitro than those incubated with plasma from mice that underwent laparoscopy or anesthesia alone. We hypothesized that differing levels of a plasma-soluble growth factor(s) postoperatively causes tumors to proliferate faster after laparotomy. This study's purpose was to isolate and characterize the plasma growth factor(s) responsible for the increased growth of systemic tumors after laparotomy. METHODS: Female Balb/C mice (n = 100) were randomized to two groups: anesthesia control (AC) or midline sham laparotomy (4 cm) (Open). Plasma was collected on Postoperative day 4. For the tumor proliferation assay, C-26 colon cancer cells were incubated in media with either 10% AC or Open "raw" plasma (not passed through column), or AC or Open plasma that had been passed through the column. For elution of heparin-binding proteins, plasma from each group was passed through a heparin-sepharose column. Elution of bound proteins was accomplished with a 0.1-2 M NaCl gradient. Each fraction was examined for protein content. For the anti-platelet-derived growth factor (PDGF) neutralizing antibody study, C-26 cells were incubated with one of four plasma preparations: AC or Open plasma alone, or AC or Open plasma incubated with anti-PDGF antibody. For both studies, tumor proliferation was determined after 2 days with an MTS/PMS assay. Results from each group were compared and differences determined using analysis of variance (ANOVA) and Tukey-Kramer tests. RESULTS: On heparin chromatography, a single elution peak consistent with PDGF was present in both AC and Open plasma and was 1.5 times greater in the Open plasma. The first tumor proliferation assay showed that tumor cells incubated with Open plasma proliferated 2.5 times faster than those with AC plasma (p < 0.0001). Passage of AC plasma through the column did not alter its mitogenic activity, but Open plasma thus treated demonstrated significantly decreased mitogenic activity. The second tumor proliferation assay showed that anti-PDGF antibody had no effect on the mitogenic activity of the AC plasma but decreased the mitogenic activity of the Open plasma to the AC plasma level. CONCLUSIONS: Laparotomy is associated with higher levels of a heparin-binding plasma factor, consistent with PDGF. The enhanced mitogenic activity of the OP plasma was neutralized with anti-PDGF antibody. Increased plasma levels of PDGF after laparotomy may be responsible for accelerated tumor growth following laparotomy in mice.

Colonoscopy in mice.

INTRODUCTION: Current investigational models of murine colitis and colon cancer necessitate sacrifice of animals in order to obtain colonic tissue. The purpose of this study was to develop a safe method of murine colonoscopy that would allow serial evaluation and mucosal biopsies of the same animal. METHODS: Nine mice (two C3H, two C57/BL6, and five IL-10 deficient) were studied a total of four times each over 4 weeks. Three mice [APC (Min +/-)] were examined three times each. Mice were gavaged with 1 cc of a polyethylene glycol solution on the day prior to colonoscopy. Solid chow was withheld and the mice were maintained on Pedialyte. Mice were anesthetized with ketamine and xylazine. A flexible pediatric cystoscope (2.1-mm diameter) with a single biopsy channel was introduced per anum, and the colon was gently insufflated with air to a mean pressure of less than 5 mmHg. Saline irrigation was used when necessary. A single biopsy was obtained from the rectosigmoid colon during each examination. RESULTS: A total of 46 examinations were carried out. One mouse died after being anesthesized for the fourth examination, and two mice [one IL-10 knockout and one APC (Min+/-)] died one day after the 3rd examination. No other complications were noted. The average length of insertion was 3 cm. Transillumination allowed for localization of the endoscope tip. Biopsies, although quite small, were sufficient for pathologic evaluation and diagnosis. CONCLUSIONS: Murine colonoscopy is a safe and feasible technique. It permits consecutive visual and histopathological examinations, and it allows the investigator to monitor the response of the murine colon to experimental interventions.

Surgical implications of colonoscopy.

Colonoscopy, although increasingly used as a screening tool, has many surgical applications. As a tool for the abdominal surgeon, colonoscopy may be used not only to diagnose neoplasia, but also hemorrhage, and inflammatory and obstructive disorders. Therapeutically, endoscopic polypectomy has impacted the incidence of colorectal cancer, and further visualization techniques have augmented the ability of the endoscopist to discriminate between benign and neoplastic lesions. Colonoscopy remains a necessary implement to facilitate the diagnosis and therapy of those patients with disorders of the colon and rectum.

An in vitro model of T cell activation by autologous cytomegalovirus (CMV)-infected human adult endothelial cells: contribution of CMV-enhanced endothelial ICAM-1.

Cellular immunity is strongly implicated in control of CMV disease; however, many mechanistic details remain unresolved. We previously demonstrated T cell activation responses to CMV-infected allogeneic endothelial cells (EC), suggesting EC as a mediator of CMV response in the transplant recipient. We now test the hypothesis that CMV-specific T cell responses can be directly stimulated by infected EC in an environment free of potentially confounding allogeneic factors. By isolating splenic T cells and gonadal vein endothelial cells (GVEC) from individual cadaveric organ donors, we have developed an in vitro model of T cell interaction with autologous CMV-infected EC. Proliferation assays demonstrated significantly enhanced responses by CMV-seropositive donor-derived T cells cocultured with CMV-infected GVEC, as compared with those elicited by uninfected cells. Similarly, as determined by limiting dilution analysis of IL-2-producing cells, T cell response frequencies to infected GVEC were significantly greater than to uninfected EC. In contrast, responses of CMV-seronegative donor-derived T cells were minimal, regardless of CMV status of stimulator GVEC. Intriguingly, CD4 responses were observed in spite of the fact that CMV-infected EC express no HLA class II. Finally, attenuation of CMV-stimulated T cell proliferation observed in the presence of blocking Ab specific for ICAM-1 suggests a contributing role for CMV-enhanced endothelial ICAM-1 expression in the activation response. These studies demonstrate that EC can stimulate autologous T cell responses to CMV in the absence of accessory APC and suggest potentially novel mechanisms of immune activation.