Presence, characterization, and genotype profiles of Mycobacterium avium subspecies paratuberculosis from unpasteurized individual and pooled milk, commercial pasteurized milk, and milk products in India by culture, PCR, and PCR-REA methods.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease in ruminants, a chronic enteritis evocative of human inflammatory bowel disease. In industrialized countries MAP has been cultured from pasteurized milk, compounding the increasing concern that MAP may be zoonotic. The purpose of this study was to evaluate commercially available unpasteurized and pasteurized milk and its products for the presence of viable MAP or MAP DNA from an area of northern India with a population of 150 million people. METHODS: We studied 43 samples (16 unpasteurized, 27 pasteurized) purchased in Mathura, Agra, or New Delhi, for the presence of MAP by culture or by PCR for IS900 MAP DNA. Positives results were confirmed as MAP by restriction endonuclease analysis and/or DNA sequencing. RESULTS: Colonies appeared in 1.5-20 months post-inoculation. Of the unpasteurized samples, 44% (7/16) were MAP culture-positive and 6% (1/16) were positive for IS900 MAP DNA. Of the pasteurized samples, 67% (18/27) were MAP culture-positive and 33% (9/27) were IS900-positive. Subsequently, 100% (25/25) of the cultured colonies were IS900 and IS1311 MAP DNA-positive. CONCLUSIONS: This is the first report from a developing country of MAP cultured from both pasteurized and unpasteurized milk and milk products. Thus we corroborate the presence of viable MAP in the food chain reported from industrialized countries. With the increasing concern that MAP may be zoonotic, these findings have major implications for healthcare in India. The decreased sensitivity in detecting MAP DNA by PCR directly from milk should be ascribed to our employing only one set of PCR primers.

Identification of overlapping AP-2/NF-kappa B-responsive elements on the rat cholecystokinin gene promoter.

In this study we evaluate both proximal and more distal transcriptional regulation of the 5' flanking region of the rat cholecystokinin gene in transfected GH3 (rat pituitary tumor) cells. Transcriptional activity was measured on the intact (-400 to +73) 5' flanking region of cholecystokinin (CCK), as well as with DNA constructs, which were deleted in both the conventional 5' to 3', as well as an unconventional 3' to 5' direction. Our in vivo studies indicate complex phorbol ester and forskolin interactions in the 10-base pair region between -130 and -140. We conclude, there are at least two transcriptional factors involved in regulation of the rat CCK transcription in this region. In vitro studies utilizing heterologous nuclear (HeLa) extract, as well as purified transcription factors AP-2 and NF-kappa B, identify overlapped AP-2- and NF-kappa B-responsive elements within the 17-base pair sequence between -149 and -134 of the distal 5' flanking region. In this region complex transcriptional regulation occurs, which indicates inhibition of AP-2 CCK promoter complexing by NF-kappa B. Six-point mutations introduced into this sequence prevent AP-2 and NF-kappa B binding to CCK promoter, as well as its transcriptional activation by phorbol ester and forskolin in GH3 cells.

Eukaryotic gene transfer with liposomes: effect of differences in lipid structure.

Liposome mediated gene transfer has a great potential in gene therapy. In this review we discuss the physical and chemical properties of cationic liposomes that affect their abilities to mediate gene transfer into eukaryotic cells. The specific focus is on functional domains of cationic lipids. We address polar head variations, counterions, linker bonds, acyl chain variations, as well as composition of liposomes. We additionally discuss different functional groups of lipids affecting lipid bilayer packing, lipid association with DNA, fusion with the cellular membranes and the release of transferred DNA from endosomes into the cytoplasm.

Liposome mediated gene transfer into GH3 cells, and rat brain, liver and gut: comparison of different polar or aliphatic domains.

BACKGROUND: Cationic liposomes may be used in gene transfer. However, different liposome configurations have varying efficiency in different tissues. AIMS: To compare multiple lipids during gene transfer into the intestinal mucosa, liver and central nervous system in the adult rat. We evaluate different lipid aliphatic and polar head domains. MATERIALS AND METHODS: Nine cationic or neutral phospholipids, combined with a cationic cholesterol derivative, have been compared to Lipofectin. Transfection was into GH3 cells and the adult rat brain, liver or intestinal mucosa. Results Optimum DNA:lipid ratio was lowest (1:2) in the intestinal mucosa and highest in GH3 cells (1:40). Lipofectin ", was most effective in brain and GH3 cells but had no activity in intestinal mucosa. Saturated cationic lipids transfect differently in GH3 cells and GI mucosa than in liver and brain. However, with saturated neutral phospholipids, GH3 cells, intestinal mucosa and liver transfect similarly. DOTAP the longest unsaturated cationic lipid (18:1) was most effective in the intestine, whereas DMEPC the shortest saturated neutral lipid (14:0) was optimal in the liver. CONCLUSIONS: In this study we propose a rational approach, based on systematic variations of lipids, to optimize liposome mediated gene transfer into the ventricular system of the brain, the liver and gastro-intestinal tract in the adult rat. Additionally, we demonstrate the feasibility of gene transfer into the mucosal cells of the gastro-intestinal tract as well as throughout the ventricular system of the rat brain. This requires liposomes which contain a cationic cholesterol derivative.

Esophageal anatomy and function in laparoscopic gastric restrictive bariatric surgery: implications for patient selection.

BACKGROUND: The purpose of this study was to assess factors of clinical importance in morbidly obese patients having a laparoscopically adjustable gastric band (LAP-BAND) implanted in order to achieve weight loss. METHODS: Preoperative evaluation of hiatus hernia and esophageal (dys)motility were compared with the need for reoperation. Results are presented for the first 50 consecutive patients entered. RESULTS: Nine of the first 50 patients required reoperation (18%). Five (10%) were for LAP-BAND slippage on the stomach. Of these five, reoperation was required in four of 12 (33%) with hiatus hernia (P = 0.0093); three of nine (33%) with a motility disorder (P = 0.025); and three of six (50%) with both hiatus hernia and a motility disorder (P = 0.0076). CONCLUSIONS: We identify two factors, hiatus hernia and esophageal dysmotility, which are associated, both independently as well as in combination, with reoperation for LAP-BAND slippage. Both patients and their physicians should consider these data when considering the LAP-BAND as possible therapy for morbid obesity.

Differential cholecystokinin brain/gut transcription initiation in the rat: evidence for brain-specific start sites.

Brain/gut cholecystokinin (CCK) has well-established translational and post-translational differences. By contrast, CCK transcription regulation is reported to be the same in brain and gut. Accordingly, the rat CCK gene has been evaluated for differential brain/gut transcription initiation. Using the 5'-flanking region of the rat CCK gene, DNase I protection assays were performed. We evaluated reporter constructs deleted of the conventional transcription start site in cell culture. Finally, brain and gut mRNA was evaluated using both a reverse transcription serial primer polymerase chain reaction assay as well as rapid amplification of cDNA ends (RACE) analysis. TFIID protein protects against DNase I digestion between -177 and -196 bp. In tissue culture, spontaneous 5'-flanking region transcription initiation occurs until deletion proceeds upstream of -140 bp. RACE analysis performed on mRNA from the rat brain identifies heretofore undescribed transcription initiation at -43 bp 5' as well as at +1,212 (in exon II). These alternative transcription initiation sites are utilized in brain, but not gut. The rat CCK gene has alternative 5'-flanking region transcription initiation sites. These alternative sites are utilized only in the brain. These data may provide insights into how brain and gut respond to their differing physiological demands.

Metabolism of alcohol by human gastric cells: relation to first-pass metabolism.

BACKGROUND & AIMS: The bioavailability of orally administered alcohol is incomplete, indicating first-pass metabolism. There is debate regarding the site of first-pass metabolism and specifically whether the stomach has the metabolic capacity to account for first-pass metabolism. The aim of this study was to assess ethanol metabolism by human gastric mucosa cells in primary culture. METHODS: Cells were incubated with [1-14C]ethanol, and the quantity of ethanol oxidized was measured by the production of [1-14C]acetate. RESULTS: Gastric cells cultured from men produced 7.3 +/- 3.5 mumol acetate.10(6) cells-1.h-1, which was more than that generated in cells from women (3.2 +/- 0.6; P < 0.05). Acetate production was inhibited by 4-methylpyrazole (a class I alcohol dehydrogenase [ADH] inhibitor) and by m-nitrobenzaldehyde (a selective substrate for class IV ADH isoenzyme) but not by sodium azide (a catalase inhibitor). Cimetidine (a gastric ADH inhibitor) reduced acetate production by as much as 59%, whereas ranitidine had no significant effect. CONCLUSIONS: Human gastric cells metabolize sufficient alcohol to account for the bulk of first-pass metabolism. At least two isozymes of gastric ADH contribute to this metabolism. Cimetidine, but not ranitidine, inhibits gastric alcohol metabolism in keeping with its inhibition of in vivo first-pass metabolism.

Cell saver autologous transfusion: metabolic consequences of washing blood with normal saline.

OBJECTIVE: To evaluate acid-base and electrolyte changes in high volume cell saver autologous blood transfusion when normal saline (0.9% NaCl) is used as the wash solution. DESIGN: Open-label study. MATERIALS AND METHODS: Nine anesthetized and anticoagulated mongrel dogs underwent 15 cycles of cell saver autologous blood transfusion. Eight percent of the circulating blood volume (125 mL) was withdrawn, washed with normal saline, and retransfused for each cycle. MEASUREMENTS AND MAIN RESULTS: Analyses of acid-base, electrolyte, and hematologic parameters were performed on both systemic and the washed blood. The washed blood had increased levels of sodium and chloride. There were decreased levels in pH, Pco2, total CO2 (bicarbonate), lactic acid, potassium, total and ionized calcium, magnesium, inorganic phosphorus, total protein, and albumin. Systemically, in the animals, by the end of the study, there were significant increases in the levels of chloride, inorganic phosphorus, hemoglobin, and hematocrit and significant decreases in the levels of pH, total CO2, total and ionized calcium, magnesium, total protein, and albumin. CONCLUSIONS: Acid-base, electrolyte, and hematologic changes occur when normal saline is used as the wash solution in high volume cell saver autologous blood transfusion. The washed blood with its elevation of sodium and chloride appears to reflect the constituents of the wash solution, normal saline. The depletion in the washed blood of PCO2, total CO2, potassium, total calcium, ionized calcium, magnesium, phosphorus, total protein, and albumin we feel is because of the absence of these electrolytes in the wash solution and their physical removal during salvaged blood separation and washing. The systemic acid-base and electrolyte changes primarily reflect the electrolyte pattern of the reinfused washed blood except for inorganic phosphorus. Inorganic phosphorus was maintained systemically, despite its wash out in the cell salvage process. This paradoxical finding may be caused by intracellular to extracellular inorganic phosphorus flux caused by the progressive systemic metabolic acidosis.

On the etiology of Crohn disease.

Crohn disease (CD) is a chronic, panenteric intestinal inflammatory disease. Its etiology is unknown. Analogous to the tuberculoid and lepromatous forms of leprosy, CD may have two clinical manifestations. One is aggressive and fistulizing (perforating), and the other is contained, indolent, and obstructive (nonperforating) [Gi]-berts, E. C. A. M., Greenstein, A. J., Katsel, P., Harpaz, N. & Greenstein, R. J. (1994) Proc. Natl. Acad. Sci. USA 91, 12721-127241. The etiology, if infections, may be due to Mycobacterium paratuberculosis. We employed reverse transcription PCR using M. paratuberculosis subspecies-specific primers (IS 900) on total RNA from 12 ileal mucosal specimens (CD, n = 8; controls, n = 4, 2 with ulcerative colitis and 2 with colonic cancer). As a negative control, we used Myobacterium avium DNA, originally cultured from the drinking water of a major city in the United States. cDNA sequence analysis shows that all eight cases of Crohn's disease and both samples from the patients with ulcerative colitis contained M. paratuberculosis RNA. Additionally, the M. avium control has the DNA sequence of M. paratuberculosis. We demonstrate the DNA sequence of M. paratuberculosis from mucosal specimens from humans with CD. The potable water supply may be a reservoir of infection. Although M. paratuberculosis signal in CD has been previously reported, a cause and effect relationship has not been established. In part, this is due to conflicting data from studies with empirical antimycobacterial therapy. We conclude that clinical trials with anti-M. paratuberculosis therapy are indicated in patients with CD who have been stratified into the aggressive (perforating) and contained (nonperforating) forms.

Is there clinical, epidemiological and molecular evidence for two forms of Crohn's disease?

Crohn's disease is an idiopathic chronic panenteric intestinal inflammatory disease. Data concerning the pathogenesis of, and the immune responses occurring in, Crohn's disease are often conflicting. Current therapy is empirical and either non-specifically immunosuppressive or surgically ablative in nature. Although controversial, Crohn's disease may be thought of as having two different presentations, an aggressive fistulizing form and an indolent obstructive form. This is analogous to the tuberculoid and lepromatous manifestations of leprosy. If correct, this subclassification may provide key insights into the pathogenesis and differing host immune responses in Crohn's disease and also allow the development of more rational therapies.

Molecular evidence for two forms of Crohn disease.

Recent epidemiological evidence suggests that there are two forms of Crohn disease (CD): perforating and nonperforating. We hypothesized that, just as with tuberculoid and lepromatous leprosy, differences in the two forms of CD would be both identified and determined by differences in the host immune response. Resected intestinal tissue from control patients as well as perforating and nonperforating CD patients was evaluated for mRNA levels. We employed 32P PCR amplification with published or custom-designed primers of a housekeeping gene (beta-actin); a human T-cell marker (CD3-delta); and the cytokines tumor necrosis factor alpha, transforming growth factor beta, granulocyte/macrophage colony-stimulating factor, interleukin (IL) 1 beta, IL-1ra, and IL-6. Differences were identified with IL-1 beta (control = 162 +/- 57 vs. perforating = 464 +/- 154 vs. nonperforating = 12,582 +/- 4733; P < or = 0.02) and IL-1ra (control = 1337 +/- 622 vs. perforating = 2194 +/- 775 vs. nonperforating = 9715 +/- 2988; P < or = 0.02). These data corroborate the epidemiological observation that there are two forms of CD. Nonperforating CD, the more benign form, is associated with increased IL-1 beta and IL-1ra mRNA expression. We conclude that it is the host immune response that determines which form of CD becomes manifest in any given individual and discuss the investigative, diagnostic, and therapeutic implications of these observations.

Obesity surgery: dietary and psychosocial expectations and reality.

Psychosocial and dietary habits have been compared in patients considering (Pre n = 33) or who had (Post n = 32) surgery for morbid obesity. Failure of conventional diets was attributed to lack of self-discipline (Pre 92% [22/24] vs. Post 87% [20/23], NS). Consumption of fast foods fell (Pre 70% [23/33] vs. Post 16% [5/32], p < 0.0001). Preoperative patients had unrealistic social expectations. They exaggerated the prospect of improved friendship (Pre 67% [22/32] vs. Post 34% [11/21], p < 0.05), erroneously anticipated better sex (Pre 78% [25/32] vs. Post 50% [15/30], p < or = 0.05), predicted better acceptance at work (Pre 85% [23/27] vs. Post 50% [15/30], p < or = 0.05), and misanticipated improved relationship with their partner (Pre 77% [20/26] vs. Post [47% 8/17], p < or = 0.05). Two factors predicted becoming employed following surgery: age (became employed [n = 5] 28 +/- 2 years vs. remained unemployed [n = 12] 44 +/- 4 years, p < 0.05) and percentage of excess weight lost (became employed 76 +/- 11 vs. remained unemployed 51 +/- 7, p < 0.05). The free support group was "useful" (17/17), yet only 5% attended regularly. Patients considering obesity surgery had specific unrealistic psychosocial expectations. They infrequently availed themselves of postoperative professional help. We identify the features associated with gaining employment.

Age, as well as cell turnover kinetics, regulates brain/gut repair.

The prototypical brain/gut peptide cholecystokinin (CCK) has been used to assess brain and gut repair kinetics following cytotoxic injury in the rat. Studies addressed the effect of repetitive injury as well as aging. Injury was induced by one of the two alkylating agents, one active in the brain, the other systematically. Consistently the responses differ between brain and intestine. Total RNA falls (as predicted) in the intestine (control 1.5 +/- 1.4 versus cytotoxic 0.21 +/- 0.06 tRNA mg/organ, P < or = 0.0001), but rises (unexpectedly) in the brain (control 0.79 +/- 0.04 versus cytotoxic 1.02 +/- 0.03 tRNA mg/organ, P < or = 0.001). CCK mRNA concentration falls in the brain (predicted) (control 27 +/- 1 versus cytotoxic 11 +/- 1 pg CCK mRNA/micrograms tRNA, P < or = 0.001), but rises in the intestine (unexpectedly) (control 0.18 +/- 0.02 versus cytotoxic 0.3 +/- 0.04 pg CCK mRNA/micrograms tRNA, P < or = 0.001). CCK peptides do not change in the brain (control 39 +/- 4 versus cytotoxic 34 +/- 4 nmol/g, P < or = NS), but rise (unexpectedly) in the intestine (control 43 +/- 4 versus cytotoxic 250 +/- 27 nmol/g, P < or = 0.001). We ascribe these observations to differing brain/gut cell turnover kinetics. These data indicate that a rebound phenomenon occurs during gut recovery from cytotoxic injury. We additionally show a differential age-related response to cytotoxic injury. Younger animals tolerated the injury better than old ones (mortality: young 27% (3/11) versus old 66% (8/12), P < or = 0.001). Additionally, intestinal recovery is more rapid in younger animals. These data suggest that with increasing age, chemotherapeutic dosages may need to be modulated. It is additionally possible that clinically applicable algorithms may be developed using our animal model.

Mechanical retraction in laparoscopic surgery: intraoperative cholangiography.

The utility of mechanical retraction in laparoscopic surgery is presented. The steps required for its utilization during intraoperative laparoscopic cholangiography are outlined.

Postoperative hypertension: a multicenter, prospective, randomized comparison between intravenous nicardipine and sodium nitroprusside.

OBJECTIVE: To compare the efficacy and safety of iv nicardipine with sodium nitroprusside in the treatment of postoperative hypertension after both cardiac and noncardiac surgery. DESIGN: Multicenter, prospective, randomized, open-label study. SETTING: Six tertiary referral medical centers (recovery rooms and surgical ICUs). PATIENTS: A total of 139 patients with postoperative hypertension: i.v. nicardipine (n = 71), sodium nitroprusside (n = 68). INTERVENTION: Administration of i.v. nicardipine or sodium nitroprusside. MEASUREMENTS: Vital signs (BP, heart rate), hemodynamic variables, medication dosage, total number of dose changes, and time to achieve BP control were recorded. MAIN RESULTS: Both medications were equally effective in reducing BP in both the cardiac and noncardiac surgical groups. Under the conditions of the study, i.v. nicardipine controlled hypertension more rapidly than sodium nitroprusside (i.v. nicardipine 14.0 +/- 1.0 mins and sodium nitroprusside 30.4 +/- 3.5 mins, p = .0029). The total number of dose changes required to achieve therapeutic BP response was significantly less in the i.v. nicardipine-treated patients (i.v. nicardipine 1.5 +/- 0.2 vs. sodium nitroprusside 5.1 +/- 1.4, p < .05). Adverse effects were observed with both drugs (i.v. nicardipine 7% [5/71] and sodium nitroprusside 18% [12/68] [NS]). CONCLUSIONS: Intravenous nicardipine is as effective as sodium nitroprusside in the therapy of postoperative hypertension. Specific advantages have been identified. The use of i.v. nicardipine should be considered in the therapy of postoperative hypertension.

Mechanical retraction in laparoscopic surgery: cholecystectomy.

Mechanical retraction in laparoscopic surgery results in stable exposure, dependable field of vision, and more efficient use of personnel. The steps required to use mechanical retraction in laparoscopic cholecystectomy are outlined in this report.

Cholecystokinin upregulation during intestinal repair.

To determine whether cholecystokinin (CCK), a small intestinal hormone, may have autocrine or paracrine functions, gene regulation in the rat stomach and duodenum has been evaluated following cytotoxic injury. We quantified total RNA, CCK messenger RNA (mRNA), total protein, small and large forms of CCK peptides and gastrin. The stomach and the intestine respond differently. Following cytotoxic injury duodenal total RNA falls (1.5 +/- 0.1 vs 0.18 +/- 0.04 mg/g P less than or equal to 0.0001), and CCK mRNA content is depleted (260 +/- 23 vs 41 +/- 8 pg CCK mRNA/duodenum P less than or equal to 0.0001), yet there is a paradoxical increase in CCK mRNA concentration (176 +/- 20 vs 303 +/- 38 pg CCK mRNA/mg total RNA P less than or equal to 0.01). Increases occurred in both molecular species of CCK peptides evaluated: CCK8 (8 +/- 7 vs 26 +/- 2 pmole/g P less than or equal to 0.0001), large forms of CCK (42 +/- 4 vs 250 +/- 27 pmole/g P less than or equal to 0.0001). By contrast, in the stomach, only decreases were observed. These data identify sites of anatomical and biosynthetic upregulation during gastrointestinal repair. Changes are dependent upon the length of the period of recovery, differ between stomach and duodenum, and may be age related. Intestinal CCK may have para- and or autocrine roles in addition to its hormone function.

The brain-gut axis: a model system to study gene regulation.

The data summarized show unequivocal differences in gene regulation between the brain and the intestine in the rat. It is possible that the differences may occur partly as a reflection of cell turnover kinetics. The mechanisms accounting for these differences should provide fertile ground for investigation. I conclude that the brain-gut axis provides a model for studying differential gene regulation in animals.