A clinically applicable method to preserve urine and bladder washing cells for flow cytometric monitoring of bladder cancer patients

We describe a method to fix exfoliated bladder cells that is suitable for followup of bladder cancer patients by deoxyribonucleic acid flow cytometry. After fixation with room temperature methanol plus acetic acid (20:1, volume:volume) urine and bladder washing samples from these patients can be stored at room temperature for 3 to 7 days and then assessed reliably for the presence of aneuploidy and the percentage of hyperdiploid cells. For those with active transitional cell carcinoma diagnostic accuracy comparing fresh to fixed specimens was improved from 58 to 92% with urine and from 50 to 100% with washing samples. For patients with a history of transitional cell carcinoma who currently are free of disease the false positive rate remains unchanged after fixation. The procedure described is suitable for use in the outpatient clinic and should permit shipping of samples without refrigeration to a central flow cytometry facility for analysis.     

Flow cytometric analysis of testicular tissue: Detection of injury and recovery

Summary DNA flow cytometry was used to examine the effects of five chemotherapeutic agents, vinblastine, cisplatinum, 5-fluorouracil, cyclophosphamide and Adriamycin, on mouse testes. Alterations in the haploid (1C), diploid (2C) and tetraploid (4C) cell compartments were assessed at 11, 29 and 56 days after giving mice a single intraperitoneal injection of each agent. Analysis of the histograms proved to be a rapid and reproducible method of monitoring injury in this animal model. Because this procedure can detect subtle testicular injuries, it is predicted that DNA histograms obtained from fine needle aspirates will gain increasing acceptance in the management of the oligo- and azoospermic patient.     

Role of bacterial adherence and the mucus barrier on bacterial translocation: effects of protein malnutrition and endotoxin in rats.

OBJECTIVE: The purpose of the study was to investigate the potential relations between mucosal bacterial adherence, intestinal mucus and mucin content, and bacterial translocation. SUMMARY BACKGROUND DATA: The attachment of bacteria to mucosal surfaces is the initial event in the pathogenesis of most bacterial infections that originate at mucosal surfaces, such as the gut. The intestinal mucus layer appears to function as a defensive barrier limiting micro-organisms present in the intestinal lumen from colonizing enterocytes. Consequently, studies focusing on the biology of bacterial adherence to the intestinal mucosa likely are to be important in clarifying the pathogenesis of gut origin sepsis. METHODS: To explore the relations between intestinal bacterial adherence, mucus bacterial binding, and bacterial translocation, two models were used. One (protein malnutrition) in which profound alterations in intestinal morphology occurs in the absence of significant translocation and one (endotoxin challenge) in which bacterial translocation occurs and intestinal morphology is relatively normal. RESULTS: Protein malnutrition was not associated with bacterial translocation and measurement of enteroadherent, mucosally associated bacterial population levels documented that the total number of gram-negative enteric bacilli adherent to the ileum and cecum was less in the protein-malnourished rats than in the normally nourished animals (p < 0.01). Furthermore, there was an inverse relation between the duration of protein malnutrition and bacterial adherence to the intestinal mucosa (r = 0.62, p < 0.002). In contrast, after endotoxin challenge, the level of enteroadherent bacteria was increased and bacterial translocation was observed. The binding of Escherichia coli to immobilized ileal mucus in vitro was decreased significantly in protein-malnourished rats, whereas E. coli binding to insoluble ileal mucus was increased in the rats receiving endotoxin. CONCLUSIONS: This study indicates that the adherence of bacteria to the intestinal mucosal surface is an important factor in bacterial translocation, that intestinal mucus modulates bacterial adherence, and that increased levels of mucosally associated bacteria are associated with a loss intestinal barrier function to bacteria.     

Hemorrhagic shock-induced bacterial translocation is reduced by xanthine oxidase inhibition or inactivation

Experiments were performed to determine whether bacterial translocation (BT) after hemorrhagic shock is due to a reperfusion injury mediated by xanthine oxidase-derived oxidants. Rats were subjected to 30 minutes of shock (30 mm Hg) followed by reinfusion of shed blood. Twenty-four hours after hemorrhage and reinfusion, the mesenteric lymph node, liver, and spleen were harvested from each animal for bacterial culture, and the ileum and cecum were examined histologically. Sham-shocked (control) rats were instrumented, but blood was not withdrawn. The incidence of BT was higher in the shocked rats (61%) than in the sham-shocked animals (7%) (p less than 0.01). Allopurinol (50 mg/kg, administered orally), a competitive inhibitor of xanthine oxidase, reduced the incidence of shock-induced BT to 14% (p = 0.02). Similarly, rats fed a tungsten-supplemented molybdenum-free diet, which inactivates xanthine oxidase, reduced shock-induced BT to 10% (p = 0.02). The histologic damage cause by hemorrhagic shock was prevented by blocking xanthine oxidase activity. Thus hemorrhagic shock-induced bacterial translocation from the gut appears to be mediated by oxidants generated by activation of the xanthine oxidase system.     

Check samples for laboratory self-assessment in DNA flow cytometry. The National Cancer Institute's Flow Cytometry Network experience

The National Cancer Institute's Flow Cytometry Network (NCI-FCN) is attempting to facilitate the transfer of flow cytometry (FCM) of exfoliated bladder cells from the research laboratory to the clinical laboratory. Demonstrating interinstitutional consistency in FCM analysis of replicate specimens simulating clinical barbotage specimens, fixed to allow easy transportation and storage at room temperature was one specific objective. Simulated barbotage specimens were prepared by mixing cultured aneuploid bladder carcinoma cells with normal or mitogen-stimulated peripheral blood mononuclear cells in different ratios. The samples were fixed in 10% formalin for 30 minutes, stored in buffer, and enucleated with pepsin, pH 1.5, before staining with propidium iodide for FCM DNA analysis. Preservation in ethanol or other common DNA cytochemical reagents was found to be unsatisfactory. In contrast, the formalin-fixed samples showed excellent preservation of quantitative DNA fluorescence and coefficient of variation of histogram peaks for over 2 weeks. Exchange of eight fixed specimens among five network laboratories that analyzed them as "unknowns" showed good overall agreement on histogram data and interpretation, although some noteworthy interlaboratory differences were found. This technique could be used for self-assessment surveys of clinical laboratory performance in DNA FCM of bladder barbotage specimens.     

Adoptive transfer of T lymphocytes to T-cell-depleted mice inhibits Escherichia coli translocation from the gastrointestinal tract.

Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal (GI) tract to extraintestinal sites, such as the mesenteric lymph node (MLN), spleen, liver, kidneys, and blood. Previously, we reported that depletion of CD4+ and/or CD8+ T cells promotes bacterial translocation from the GI tract to the MLN. In the present study, CD4+ and/or CD8+ T cells, harvested from donor mice, were adoptively transferred to mice previously depleted of T cells by thymectomy plus intraperitoneal injection of rat anti-mouse T-cell monoclonal antibodies. The adoptively transferred CD4+ and/or CD8+ T cells inhibited the translocation of Escherichia coli from the GI tract. Migration of the adoptively transferred T cells to the spleens and MLNs of the recipient mice was determined by utilizing Thy 1.1+ donor cells adoptively transferred into mice whose cells express the Thy 1.2 marker. These results provide further evidence of the importance of T cells in the host immune defense against bacterial translocation from the GI tract.     

T lymphocytes in host defense against bacterial translocation from the gastrointestinal tract.

Flow cytofluorometric analyses of lymphocytes harvested from the mesenteric lymph node (MLN), mucosal epithelium, and lamina propria of C57BL/6 mice demonstrate that expression of alpha/beta or gamma/delta T-cell receptors (TCR) and CD4 or CD8 molecules by T lymphocytes in the intestinal immune system varies depending upon their anatomic location. The MLN contained equivalent numbers of CD4+ and CD8+ T cells, the vast majority of which were alpha/beta TCR positive (alpha/beta TCR+). The lamina propria T cells were predominantly CD4+ and alpha/beta TCR+, while the intestinal intraepithelial lymphocytes consisted of equivalent numbers of alpha/beta and gamma/delta T cells, the majority of which were CD8+. There were no significant changes in these T-cell phenotypic profiles when the mice were antibiotic decontaminated or monoassociated with Escherichia coli. Mice were depleted of CD4+ T cells and/or CD8+ T cells in vivo by intraperitoneal injections of monoclonal antibody GK 1.5 (rat anti-mouse CD4) and/or monoclonal antibody 2.43 (rat anti-mouse CD8). T-cell depletion was confirmed in the MLN, lamina propria, and the intestinal epithelium by flow cytometry. E. coli C25 translocation from the gastrointestinal (GI) tract to the MLN was significantly increased in mice depleted of CD4+ T cells, CD8+ T cells, or both. T-cell-deficient athymic beige/nude mice also exhibited greater levels of E. coli C25 translocation to the MLN than beige/het euthymic littermates. Salmonella typhimurium translocation also was increased following CD4+ and CD8+ T-cell depletion in mice monoassociated with S. typhimurium. Depletion of CD4+ and/or CD8+ T cells also increased the translocation to the MLN of certain indigenous GI flora bacteria. These results confirm that T-cell-mediated immunity is involved in the host defense against bacterial translocation from the GI tract.     

Intestinal cytokine response after gut ischemia: role of gut barrier failure

OBJECTIVE: To investigate the effect of intestinal ischemia with and without a reperfusion injury on intestinal cytokine production and gut permeability. SUMMARY BACKGROUND DATA: In humans and in animal models, the gut has been implicated as a cytokine-producing organ after ischemia/reperfusion (I/R)-type injuries. Because of the limitations of in vivo models, it has been difficult to demonstrate directly that the gut releases cytokines after an I/R injury or whether there is a relation between the magnitude of the ischemic process and the cytokine response. METHODS: Ileal mucosal membranes from rats subjected to sham or 45 or 75 min of superior mesenteric occlusion (SMAO) or 45 minutes of SMAO and 30 minutes of reperfusion (SMAO 45/30) were mounted in the Ussing chamber system. Levels of tumor necrosis factor-alpha and interleukin-6 were serially measured in the mucosal and serosal reservoirs of the Ussing system, as was mucosal permeability as reflected by the passage of bacteria or phenol red across the ileal membrane. In a second group of experiments, Escherichia coli C25 was added to the mucosal reservoir to determine if the cytokine response would be increased. RESULTS: Mucosal and serosal levels of tumor necrosis factor-alpha were equally increased after SMAO, with the highest levels in the 75-minute SMAO group. The highest levels of interleukin-6 were found in rats subjected to 75 minutes of SMAO or SMAO 45/30; the serosal levels of interleukin-6 were four to sixfold higher than the mucosal levels. The addition of E. coli C25 resulted in a significant increase in the amount of interleukin-6 or tumor necrosis factor-alpha recovered from the mucosal reservoir. Increased ileal membrane permeability was observed only in rats subjected to 75 minutes of SMAO or SMAO 45/30. CONCLUSION: These results directly document that the levels of tumor necrosis factor-alpha and interleukin-6 released from the gut increase after an ischemic or I/R injury, such as SMAO, and that there is a relation between the magnitude of the gut ischemic or I/R insult and the cytokine response.     

Priming of neutrophil [Ca2+]i signaling and oxidative burst by human fracture fluids

BACKGROUND: Patients with major fracture/soft-tissue injuries are at risk for adult respiratory distress syndrome after secondary infection. Fracture fluids (FF) are rich in neutrophil (PMN) -specific chemokines such as interleukin-8. PMN respond to both interleukin-8 and bacterial stimuli with calcium ([Ca2+]i) fluxes, which can initiate respiratory burst (RB). We hypothesize that small amounts of FF entering the circulation could exaggerate PMN [Ca2+]i and RB responses, potentially increasing the risk of adult respiratory distress syndrome. METHODS: FF were obtained from 10 patients at open fixation of the femur 2 to 5 days postinjury. Volunteer PMN were isolated and loaded with fura dye. PMN were preincubated either in 30% autologous plasma (AP)/70% buffer, or in 5% FF/25% AP/70% buffer. Cells were resuspended in buffer with 1,2,3-dihydrorhodamine and stimulated with low-dose n-formyl-methionyl-leucyl-phenylalanine (fMLP). [Ca2+]i was assayed by fura fluorescence at 505 nm after excitation at 340/380 nm. RB was assessed by 1,2,3-dihydrorhodamine fluorescence at 530 nm after 488 nm excitation. RESULTS: PMN basal [Ca2+]i was higher after FF incubation than AP incubation (94+/-12 vs. 61+/-9 nmol/L, p = 0.0002). Peak [Ca2+]i response to fMLP was 475+/-47 nmol/L after FF but only 356+/-22 nmol/L after AP (p = 0.01). Two hundred seconds after fMLP, [Ca2+]i remained higher after FF (172+/-17 vs. 145+/-9 nmol/L, p = 0.04). Basal RB was slightly higher after FF than AP (13.4+/-0.3 vs. 11.3+/-0.3 units, p = 0.051) as was the maximal rate of extracellular oxidant release (1.10+/-0.17 vs. 0.76+/-0.16 units/s, p = 0.004) and total oxidant production (42.5+/-0.8 vs. 31.7+/-0.8 units, p = 0.006). CONCLUSION: Small amounts of FF in plasma can exaggerate PMN [Ca2+]i flux and RB responses to subsequent bacterial stimuli. These findings are consistent with the hypothesis that release of FF into the circulation primes PMN and, thus, may predispose to adult respiratory distress syndrome. Such PMN priming events might have important implications for both the operative and medical management of patients with major fractures.