Long term persistence and risk factors for anorectal symptoms following low anterior resection for rectal cancer.

BACKGROUND: Rectal cancer is commonly treated by chemoradiation therapy, followed by the low anterior resection anal sphincter-preserving surgery, with a temporary protecting ileostomy. After reversal of the stoma a condition known as low anterior resection syndrome (LARS) can occur characterized by a combination of symptoms such as urgent bowel movements, lack of control over bowel movements, and difficulty fully emptying the bowels. These symptoms have a significant negative impact on the quality of life for individuals who have survived the cancer. Currently, there is limited available data regarding the presence, risk factors, and effects of treatment for these symptoms during long-term follow-up. AIMS: To evaluate long term outcomes of low anterior resection surgery and its correlation to baseline anorectal manometry (ARM) parameters and physiotherapy with anorectal biofeedback (BF) treatment. METHODS: One hundred fifteen patients (74 males, age 63 ± 11) who underwent low anterior resection surgery for rectal cancer were included in the study. Following surgery, patients were managed by surgical and oncologic team, with more symptomatic LARS patients referred for further evaluation and treatment by gastroenterologists. At follow up, patients were contacted and offered participation in a long term follow up by answering symptom severity and quality of life (QOL) questionnaires. RESULTS: 80 (70%) patients agreed to participate in the long term follow up study (median 4 years from stoma reversal, range 1-8). Mean time from surgery to stoma closure was 6 ± 4 months. At long term follow up, mean LARS score was 30 (SD 11), with 55 (69%) patients classified as major LARS (score > 30). Presence of major LARS was associated with longer time from surgery to stoma reversal (6.8 vs. 4.8 months; p = 0.03) and with adjuvant chemotherapy (38% vs. 8%; p = 0.01). Patients initially referred for ARM and BF were more likely to suffer from major LARS at long term follow up (64% vs. 16%, p < 0.001). In the subgroup of patients who underwent perioperative ARM (n = 36), higher maximal squeeze pressure, higher maximal incremental squeeze pressure and higher rectal pressure on push were all associated with better long-term outcomes of QOL parameters (p < 0.05 for all). 21(54%) of patients referred to ARM were treated with BF, but long term outcomes for these patients were not different from those who did not perform BF. CONCLUSIONS: A significant number of patients continue to experience severe symptoms and a decline in their quality of life even 4 years after undergoing low anterior resection surgery. Prolonged time until stoma reversal and adjuvant chemotherapy emerged as the primary risk factors for a negative prognosis. It is important to note that referring patients for anorectal physiology testing alone tended to predict poorer long-term outcomes, indicating the presence of selection bias. However, certain measurable manometric parameters could potentially aid in identifying patients who are at a higher risk of experiencing unfavorable functional outcomes. There is a critical need to enhance current treatment options for this patient group.

AHNAK KO mice are protected from diet-induced obesity but are glucose intolerant.

AHNAK is a 700 KD phosphoprotein primarily involved in calcium signaling in various cell types and regulating cytoskeletal organization and cell membrane architecture. AHNAK expression has also been associated with obesity. To investigate the role of AHNAK in regulating metabolic homeostasis, we studied whole body AHNAK knockout mice (KO) on either regular chow or high-fat diet (HFD). KO mice had a leaner phenotype and were resistant to high-fat diet-induced obesity (DIO), as reflected by a reduction in adipose tissue mass in conjunction with higher lean mass compared to wild-type controls (WT). However, KO mice exhibited higher fasting glucose levels, impaired glucose tolerance, and diminished serum insulin levels on either diet. Concomitantly, KO mice on HFD displayed defects in insulin signaling, as evident from reduced Akt phosphorylation and decreased cellular glucose transporter (Glut4) levels. Glucose intolerance and insulin resistance were also associated with changes in expression of genes regulating fat, glucose, and energy metabolism in adipose tissue and liver. Taken together, these data demonstrate that (a) AHNAK is involved in glucose homeostasis and weight balance (b) under normal feeding KO mice are insulin sensitive yet insulin deficient; and (c) AHNAK deletion protects against HFD-induced obesity, but not against HFD-induced insulin resistance and glucose intolerance in vivo.

PPARγ gene expression is autoregulated in primary adipocytes: ligand, sumoylation, and isoform specificity.

Being a key-factor in glucose homeostasis, PPARγ transcriptional activity (TA) is of high importance. However, its mediation by ligands and post-translational modifications in insulin target tissues are unclear. We investigated effects of rosiglitazone (Rg) and sumoylation on PPARγ-TA by overexpressing expression vectors and promoter-reporters for PPARγ1 and PPARγ2 in primary rat adipocytes. Wild type (WT) PPARγ1 and PPARγ2 dose-dependently repressed transcription from their promoters to a maximum of 40-50%. PPARγ2 mutants defective in either MAP-kinase phosphorylation (S112A) or the ligand-binding domain (LBD; P495L, L496A/E499A) exhibited decreased repression of PPARγ2 promoter. Rg enhanced repression by S112A, but not by LBD-defective mutants. Sumoylation-defective PPARγ1 mutants K77R and K365R repressed PPARγ2 promoter activity similar to WT, while Rg enhanced repression by K77R but not by K365R. Sumoylation-defective PPARγ2 mutants K107R and K395R exhibited impaired TA and impaired responsiveness to Rg. GLUT4 promoter, previously shown by us to be repressed by WT-PPARγ1 and WT-PPARγ2, was similarly repressed by both sumoylation-defective PPARγ1 mutants, while both sumoylation-defective PPARγ2 mutants exerted reduced repression. Surprisingly, Rg alleviated only WT-PPARγ2-induced repression, while augmenting that induced by WT-PPARγ1 and all sumoylation-defective mutants. Promoter and chromatin immunoprecipitation analyses revealed that PPARγ2 autorepression involves its direct binding to its promoter. In concert with effects at promoter level, Rg decreased endogenous level of PPARγ2 mRNA, while increasing that of GLUT4. We suggest a hypothetical model for PPARγ gene regulation in primary adipocytes that is isoform-specific and Rg/sumoylation-dependent. These findings are important due to the role of PPARγ and Rg in insulin sensitivity.

CYP2E1 impairs GLUT4 gene expression and function: NRF2 as a possible mediator.

Impaired GLUT4 function/expression in insulin target tissues is well-documented in diabetes and obesity. Cytochrome P450 isoform 2E1 (CYP2E1) induces oxidative stress, leading to impaired insulin action. CYP2E1 knockout mice are protected against high fat diet-induced insulin resistance and obesity; however the molecular mechanisms are still unclear. We examined whether CYP2E1 impairs GLUT4 gene expression and function in adipose and muscle cells. CYP2E1 overexpression in skeletal muscle-derived L6 cells inhibited insulin-stimulated Glut4 translocation and 2-deoxyglucose uptake, with the latter inhibition being blocked by vitamin E. CYP2E1 overexpression in L6 and primary rat adipose (PRA) cells suppressed GLUT4 gene expression at promoter and mRNA levels, whereas CYP2E1 silencing had opposite effects. In PRA, CYP2E1-induced suppression of GLUT4 expression was blocked by chlormethiazole (CYP2E1-specific inhibitor) and the antioxidants vitamin E and N-acetyl-l-cysteine. CYP2E1 effect was mediated by the transcription factor NF-E2-related factor 2 (NRF2), as evident from its complete reversal by a coexpressed dominant-negative, but not wild-type NRF2. GLUT4 transcription was suppressed by NRF2 overexpression, and enhanced by NRF2 silencing. Promoter and ChIP analysis showed a direct and specific binding of NRF2 to a 58-326 GLUT4 promoter region that was required to maintain CYP2E1 suppression; this binding was enhanced by CYP2E1 overexpression. We suggest a mechanism for CYP2E1 action that involves: a) suppression of GLUT4 gene expression that is mediated by NRF2; b) impairment of insulin-stimulated Glut4 translocation and function. CYP2E1 and NRF2 are introduced as negative regulators of GLUT4 expression and function in insulin-sensitive cells.

Adult rat liver cells transdifferentiated with lentiviral IPF1 vectors reverse diabetes in mice: an ex vivo gene therapy approach.

AIMS/HYPOTHESIS: We examined a clinical model of ex vivo transdifferentiation of primary adult hepatocytes to insulin-secreting cells for the treatment of type 1 diabetes. MATERIALS AND METHODS: Isolated rat hepatocytes were transduced in primary culture with a human lentivirus containing pancreatic duodenal homeobox 1 (PDX1, now known as insulin promoter factor 1, homeodomain transcription factor [IPF1]). Insulin expression and secretion of the newly engineered cells were assessed in vitro by RT-PCR, in situ hybridisation, immunostaining and radioimmunoassay. PDX1-transduced hepatocytes were further studied in vivo by injecting them under the renal capsule of diabetic SCID mice. RESULTS: Isolated rat hepatocytes were efficiently transduced with the lentiviral vector, as assessed by green fluorescent reporter gene expression. The transduced cells exhibited insulin at both mRNA (RT-PCR, in situ hybridisation) and protein levels (immunostaining and radioimmunoassay). Moreover, insulin secretion by the engineered cells was dependent on glucose and sulfonylurea. Other beta cell genes, including those encoding solute carrier family 2 (facilitated glucose transporter), member 2 (Slc2a2), glucokinase (Gck), ATP-binding cassette, sub-family C (CFTR/MRP), member 8 (Abcc8), the potassium inwardly-rectifying channel, subfamily J, member 11 (Kcnj11) and proprotein convertase subtilisin/kexin type 1 (Pcsk1) were also expressed. The PDX1-transduced hepatocytes expressed several pancreatic transcription factors related to early pancreatic endocrine development (endogenous Pdx1, neurogenic differentiation factor 1 [Neurod1], and NK6 transcription factor related, locus 1 [Nkx6-1]) as well as the late-stage pancreatic transcription factors (paired box gene 4 [Pax4], paired box gene 6 [Pax6], and v-maf musculoaponeurotic fibrosarcoma oncogene homolog A [Mafa]). Transplantation of 3 x 10(6) transdifferentiated liver cells under the renal capsule of seven streptozotocin-induced diabetic SCID mice resulted in significant reduction of non-fasting blood glucose levels from 30.7 +/- 1.3 to 8.7 +/- 3.7 mmol/l (mean +/- SEM, p = 0.01), in 6 to 8 weeks. Removal of the graft resulted in severe hyperglycaemia. CONCLUSIONS/INTERPRETATION: Ex vivo lentiviral-mediated PDX1 expression in isolated adult liver cells represents a potential model for type 1 diabetes mellitus therapy.

L-Glutamic acid gamma-monohydroxamate. A potentiator of vanadium-evoked glucose metabolism in vitro and in vivo.

We report that the vanadium ligand L-Glu(gamma)HXM potentiates the capacity of free vanadium ions to activate glucose uptake and glucose metabolism in rat adipocytes in vitro (by 4-5-fold) and to lower blood glucose levels in hyperglycemic rats in vivo (by 5-7-fold). A molar ratio of two L-Glu(gamma)HXM molecules to one vanadium ion was most effective. Unlike other vanadium ligands that potentiate the insulinomimetic actions of vanadium, L-Glu(gamma)HXM partially activated lipogenesis in rat adipocytes in the absence of exogenous vanadium. This effect was not manifested by D-Glu(gamma)HXM. At 10-20 microM L-Glu(gamma)HXM, lipogenesis was activated 9-21%. This effect was approximately 9-fold higher (140 +/- 15% of maximal insulin response) in adipocytes derived from rats that had been treated with vanadium for several days. Titration of vanadium(IV) with L-Glu(gamma)HXM led to a rapid decrease in the absorbance of vanadium(IV) at 765 nm, and (51)V NMR spectroscopy revealed that the chemical shift of vanadium(IV) at -490 ppm disappeared with the appearance of a signal characteristic to vanadium(V) (-530 ppm) upon adding one equivalent of L-Glu(gamma)HXM. In summary, L-Glu(gamma)HXM is highly active in potentiating vanadium-activated glucose metabolism in vitro and in vivo and facilitating glucose metabolism in rat adipocytes in the absence of exogenous vanadium probably through conversion of trace intracellular vanadium into an active insulinomimetic compound. We propose that the active species is either a 1:1 or 2:1 L-Glu(gamma)HXM vanadium complex in which the endogenous vanadium(IV) has been altered to vanadium(V). Finally we demonstrate that L-Glu(gamma)HXM- and L-Glu(gamma)HXM.vanadium-evoked lipogenesis is arrested by wortmannin and that activation of glucose uptake in rat adipocytes is because of enhanced translocation of GLUT4 from low density microsomes to the plasma membrane.

[Evaluation of asthmatic children presenting at the emergency room at Barzilai Hospital].

Bronchial asthma in the pediatric age group has become prevalent recently. Many children who suffer from asthma arrive at the emergency room (ER) with exacerbations which did not respond to medical treatment at home. Between July and December 1997, 136 children 8 months to 14 years of age (61% below 3 years), were studied in our pediatric ER. Investigation included physical examination and pulse oximetry, which were used as guidelines for scoring the children on arrival and post-treatment. Spirometry was done in those who could cooperate. For each patient a detailed questionnaire about medical and sociodemographic factors was filled. Primary pediatricians used mainly beta-agonist and corticosteroid inhalators, while pediatric pulmonologists used mainly inhaled steroids. There was no relationship between severity of attack on arrival at the ER, mode of treatment and speed of recovery in the ER. More children treated by a general pediatrician more were admitted to hospital. Low parental education and paternal smoking were risk factors for recurrent hospital admissions. Our results indicate that parents must be educated to stop smoking, especially those with asthmatic children, and primary pediatricians should be updated with regard to proper treatment and follow-up of asthma.

[Sudden death from asthma in childhood, is it preventable?].

Sudden death from asthma is rare but occurs in the young age group. We recently faced this rare situation when 3 asthmatic children were dead on arrival at the local emergency room. All 3 had been treated with beta-2 agonist inhalation on a regular basis, without anti-inflammatory treatment, 2 of the children died while inhaling the beta-2 agonist. It is important that there be clear guidelines and full education about the management of asthma, during and between exacerbations, to prevent such deaths.

[Pediatric flexible bronchoscopy].

Between 1993-1996, 200 pediatric flexible bronchoscopies were performed. Indications were: chronic cough (158 children), persistent pulmonary infiltrates (89), recurrent stridor (28), suspected tracheobronchial foreign body (20), suspected tuberculosis (17) and hemoptysis (3). Some children had more than 1 indication. 124 patients were boys (mean 4.18 +/- 2.86 years; range 1 month-15 years) and 76 were girls (mean 4.39 +/- 2.7 years; range 4 months-15 years). The procedure included direct vision recorded by video-camera and bronchoalveolar lavage; the lavage fluid was sent for culture, Gram and Ziehl-Nielsen strains and for cytology. There were a few minor side effects: mild stridor which resolved within a few hours (10 children) and transient fever (3). This simple, flexible instrument was effective and helpful in the diagnosis and treatment of children with respiratory symptoms in a secondary hospital facility.

[Tuberculosis in early childhood].

The incidence of tuberculosis (TB) has been increasing worldwide. While the incidence in the young is lower than in adults, it mirrors that of the adult population. Infants do not exhibit typical symptoms, and may present with only a low-grade fever and a mild cough which resolves within a few days. Often physical examination is within normal limits. Over the past year we diagnosed TB, confirmed by culture and histology, in 6 children under 2 years of age. We wish to increase awareness among pediatricians of the clinical forms of this disease in children.

Modulation of the activity of glucose transporters (GLUT) in the aged/obese rat adipocyte: suppressed function, but enhanced intrinsic activity of GLUT.

To study the contribution of glucose transporters (GLUT) to insulin resistance in aging, GLUT intrinsic activity was assessed in a cell-free system. Adipocytes were isolated from 18-month-old rats and young controls and incubated either with or without 7 nM insulin. Plasma membrane (PM) and low density microsomal fractions were prepared from the cells, and GLUT levels were assessed in these fractions before and after reconstitution into liposomes. Glucose transport rates were measured in intact cells and liposomes. Functional and intrinsic activities of GLUT were assessed from the ratio between these transport rates and GLUT levels in the respective fractions. Basal 3-O-methylglucose transport rates were unaffected by aging, which is consistent with unchanged levels of GLUT in PM. Insulin-stimulated glucose transport was 60% lower in aging, as was the extent of GLUT recruitment to PM. The effect of insulin stimulation of GLUT functional activity by 6-fold at PM was attenuated by 40% in aging. Conversely, the basal intrinsic activity of GLUT was significantly enhanced in aging (by 280% and 230% in PM and density microsomal liposomes, respectively) and was further stimulated by insulin by about 160% in PM, compared to only about 117% stimulation in controls. In conclusion, our data show that insulin stimulates the intrinsic activity of GLUT in rat adipocytes, and this activity is further enhanced in aging. Impaired glucose uptake in aging can be attributed to depleted GLUT4 levels and impaired function of GLUT at the cell surface. The discrepancy observed between impaired function and enhanced intrinsic activity of GLUT suggests the presence of additional factors that modulate the full functional expression of GLUT at the cell surface.

[Intussusception: diagnosis and treatment with saline enema under ultrasound].

Intussusception is the most common abdominal emergency of early childhood. In the past 100 years intussusception has been treated by both surgical and nonsurgical methods. The last few years have brought improved nonsurgical methods of treatment. We present the results of ultrasonic diagnosis, and of treatment by saline enema under ultrasound, in the first 7 infants in our series.

In vivo insulin action in normal and streptozotocin-induced diabetic rats.

Models studying in vivo insulin animal action usually employ single-use anesthetized animals, mainly for technical reasons. We developed a modification of the euglycemic insulin clamp technique and used it to repeatedly assess in vivo insulin effects in awake streptozotocin-induced diabetic rats, and in weight- and/or age-matched controls. Permanent catheters implanted into the left carotid artery and the right jugular vein were used for miniature blood sampling (20 microliters) and recycling. Insulin was infused at 1, 2, 3, 15, and 30 mU/kg.min. Plasma insulin and C-peptide levels and glucose utilization rate were measured at blood glucose levels of 100 mg/dl. Diabetes was associated with diminished elevation of plasma C-peptide and insulin levels after ad lib feeding, 50% decreased (p < 0.005) insulin sensitivity, 31% decreased (p < 0.001) insulin responsiveness, and unchanged insulin clearance rates. Thus, using repeated clamps of the same rat over a prolonged period of time, we demonstrate that diabetes is associated with unchanged clearance but decreased sensitivity and responsiveness to insulin.

Potential role for insulin and cycloheximide in regulating the intrinsic activity of glucose transporters in isolated rat adipocytes.

We examined the hypothesis that insulin stimulation of cellular glucose transport may involve a protein synthesis-dependent regulation of glucose transporter (GTer) activity independent of GTer translocation to the cell surface. Rat adipocytes were isolated, incubated with or without 10 micrograms/ml (36 microM) cycloheximide (CHX) for 60 min, and then with or without 7 nM insulin for 30 min. Glucose transport rates were assessed in intact cells, and both glucose transport rates and GTer levels were assessed in subcellular fractions of membrane vesicles before and after reconstitution into artificial liposomes. GTer functional and intrinsic activities were calculated as the ratio between these transport rates and GTer levels in native and reconstituted membranes, respectively. Insulin increased functional activity by 340% in native plasma membrane (PM) vesicles and intrinsic activity by 60% in reconstituted membranes (from 54 +/- 4 to 86 +/- 4 molecules transported per GTer/sec, P < 0.02). CHX preincubation of cells did not interfere with the insulin effect to stimulate glucose transport rate in either intact cells or in native PMs; it did, however, reduce PM GTer levels by 27-30%, but not affecting those in the intracellular pool. However, CHX additively increased the insulin-stimulated intrinsic activity of PM GTers by 67%. Relative reconstitution efficiencies, assessed by immunoblotting both native and reconstituted membranes against specific antibodies, were similar for GLUT 1 and GLUT 4. Although insulin did not alter this efficiency, CHX slightly decreased it for GLUT 4. Our data suggest that insulin stimulation of glucose transport may involve, as part of its mechanism, modulation of the GTer intrinsic activity. We further hypothesize that CHX effects on increasing this activity state of GTer may involve as yet unknown protein synthesis-dependent regulator(s).

Exofacial epitope-tagged glucose transporter chimeras reveal COOH-terminal sequences governing cellular localization.

The insulin-regulated adipocyte/skeletal muscle glucose transporter (GLUT4) displays a characteristic steady-state intracellular localization under basal conditions, whereas the erythrocyte/brain transporter isoform (GLUT1) distributes mostly to the cell surface. To identify possible structural elements in these transporter proteins that determine their cellular localization, GLUT1/GLUT4 chimera cDNA constructs that contain the hemagglutinin epitope YPYDVPDYA (HA) in their major exofacial loops were engineered. Binding of monoclonal anti-HA antibody to non-permeabilized COS-7 cells expressing HA-tagged transporter chimeras revealed that expression of transporters on the cell surface was strongly influenced by their cytoplasmic COOH-terminal domain. This method also revealed a less marked, but significant effect on cellular localization of amino acid residues between transporter exofacial and middle loops. The subcellular distribution of expressed chimeras was confirmed by immunofluorescence microscopy of permeabilized COS-7 cells. Thus, HA-tagged native GLUT4 was concentrated in the perinuclear region, whereas a chimera containing the COOH-terminal 29 residues of GLUT1 substituted onto GLUT4 distributed to the plasma membrane, as did native GLUT1. Furthermore, a chimera composed of GLUT1 with a GLUT4 COOH-terminal 30-residue substitution exhibited a predominantly intracellular localization. Similar data was obtained in CHO cells stably expressing these chimeras. Taken together, these results define the unique COOH-terminal cytoplasmic sequences of the GLUT1 and GLUT4 glucose transporters as important determinants of cellular localization in COS-7 and CHO cells.

Accessory nipples: any relationship to urinary tract malformation?

One hundred two infants and children age 3 days to 16.5 years, found to have accessory nipples (AN), were enrolled in this study. They were categorized by ethnic origin, sex, positive family history of AN, and number, site, and shape of AN, to determine factors for increased risk of anomalies of the urinary tract. Physical and ultrasound examinations of the abdomen did not reveal evidence of urinary tract malformation in any of the children. The results of this survey support the contentions that AN are not associated with urinary tract malformations, and that no further investigation is required in children with solitary AN.

Insulin resistance and acanthosis nigricans: evidence for a postbinding defect in vivo.

Acanthosis nigricans (AN) with insulin resistance has been traditionally attributed to insulin receptor abnormalities. To further clarify the postbinding defects of in vivo insulin action in this state, we applied the euglycemic insulin clamp technique, combined with the glucose trace infusion method, to 26 subjects: 12 AN patients (eight normoglycemic and four hyperglycemic), eight obese, and eight lean control subjects. The normoglycemic AN group exhibited fasting hyperinsulinemia (666% of control), 160% elevated hepatic glucose production (HGP), 425% increased posthepatic insulin delivery rate, and only slightly reduced (19%) insulin clearance rates, compared with controls. Except for the latter, all these abnormalities were statistically significant (P less than .05), and could not be accounted for by body overweight. AN patients with diabetes mellitus (AN + DM) exhibited a further decreased insulin responsiveness (30%) and clearance (38%), together with a major increase in HGP (320%). All AN patients showed a significant right-shift in the insulin dose-response curve, indicating a decrease in insulin sensitivity. In conclusion, AN is characterized by increased basal rates of HGP, and peripheral insulin resistance, which can be partially attributed to postbinding defects. In AN + DM, a worsening of these abnormalities may be responsible for unmasking the existence of diabetes.

Regulation of glucose transporters in diabetes.

It is now widely accepted that insulin stimulates glucose metabolism in its target tissues via recruitment of transporters from a large intracellular pool to the plasma membrane. Recent studies, however, suggest a two-step model for insulin action, of transporter translocation and transporter activation. Data confirming this hypothesis for the first time are presented. It is shown that insulin significantly enhances the intrinsic activity of glucose transporters in human and rat adipose cells, in physiological as well as in diabetic state. The functional activity of transporters is impaired in the diabetic state, but surprisingly, 'diabetic' transporters exhibit normal or even enhanced intrinsic activity. In both noninsulin-dependent diabetes mellitus and streptozotocin-diabetic rats, insulin resistance is associated with 50% transporter depletion in the intracellular pool, thus leading to a decreased number of transporters appearing in the plasma membrane in response to insulin. It is concluded that impaired glucose transport in diabetes is secondary (1) to intracellular transporter depletion, and (2) to the presence of inhibitory factors interfering with the full expression of glucose transporters at the plasma membrane, thus contributing to postreceptor insulin resistance.

Typhoid fever diagnosed by isolation of S. typhi from gastric aspirate.

The diagnosis of typhoid fever (TF) is usually established by culturing S. typhi from the blood or by serology. In this report we describe three patients in whom the diagnosis of TF was made by the isolation of S. typhi from gastric contents, despite negative blood urine and stool cultures. Culture of gastric contents has not previously been recognized as a diagnostic tool in this disease.