Armido Rubino

Enterotoxicity and Cytotoxicity of Vibrio parahaemolyticus Thermostable Direct Hemolysin in In Vitro Systems

ABSTRACT Vibrio parahaemolyticus is a marine bacterium known to be a common cause of seafood gastroenteritis worldwide. The thermostable direct hemolysin (TDH) has been proposed to be a major virulence factor of V. parahaemolyticus. TDH causes intestinal fluid secretion as well as cytotoxicity in a variety of cell types. In this study, we investigated the interplay between the hemolysins enterotoxic and cytotoxic effects by using both human and rat cell monolayers. As revealed by microspectrofluorimetry, the toxin causes a dose-dependent increase in intracellular free calcium in both Caco-2 and IEC-6 cells. This effect was reversible only when low toxin concentrations were tested. The TDH-activated ion influx pathway is not selective for calcium but admits ions such sodium and manganese as well. Furthermore, in the same range of concentration, the hemolysin triggers a calcium-dependent chloride secretion. At high concentrations, TDH induces a dose-dependent but calcium-independent cell death as assessed by functional, biochemical, and morphological assays.

Etiology and risk factors of severe and protracted diarrhea

Severe and protracted diarrhea (SPD) is the most severe form of diarrhea in infancy and has also been defined as intractable diarrhea. Its etiology is poorly defined. We have retrospectively evaluated the etiology, the outcome, and the risk factors of 38 children, admitted with protracted diarrhea and need for total parenteral nutrition (TPN) from 1977 to 1993. Children with anatomic abnormalities and/or primary immunodeficiency were excluded. There was an inverse relationship between the number of patients and the age of diarrheal onset (mean age, 2.9 ± 3.5 months). Etiology of SPD was an enteric infection in 18 cases (eight Salmonella, three Staphylococcus, five rotavirus, one adenovirus, one Cryptosporidium), multiple alimentary intolerance (eight cases), familial microvillous atrophy (two), autoimmune enteropathy (two), celiac disease, lymphangectasia, eosinophilic enteropathy, intestinal pseudoobstruction, and intestinal neurodysplasia (1 case each). Etiology was not detected in three cases. Overall, 12 children died, five are presently being treated, and 21 had full remission. Comparative evaluation of risk factors between children with SPD and a control population of children with diarrhea but without the need for TPN showed that low birth weight, no breast feeding, history of fatal diarrhea in a relative, and early onset of diarrhea had a significantly higher incidence in the former. Social background was similar in the two populations. We conclude that a specific etiology can be identified in the majority of cases of SPD. The etiologic spectrum of SPD is broad, but an enteric infection is the most common cause of SPD. The severity of this condition is related, at least in part, to established risk factors.

The management of portal hypertension in cystic fibrosis

We have found that with proper selection and preoperative preparation, a major portosystemic shunt can be done with considerable safety in a majority of cystic fibrosis patients and thus provide them with significant palliation and improved quality of life.

Development of Dipeptide Transport in the Intestinal Mucosa of Rabbits

Summary: The uptake of 0.5 mM glycyl-L-proline (Gly-Pro), a substrate which has affinity for the dipeptide specific carrier system and is not hydrolyzed in the brush border, was studied in vitro in jejunum and ileum of rabbits from the 25th day of gestational age into adulthood. For comparison, uptake of 0.5 mM glycine was studied too. Results show that influx of Gly-Pro is present from the earliest age studied and show a steep prenatal increase and peaks at birth, with values, in the newborn jejunum, of 45.2 ± 3.3 μmoles/ghr (mean ± S.E.). Thereafter, the uptake slowly declines toward the adult level of 3.8 ± 0.5 μmoles/ghr. A similar pattern was present in the ileum. On the contrary, free glycine uptake shows no statistically significant change during the whole period studied.The characteristics of the Gly-Pro influx process have been studied in the period of its peak activity (1-to 6-day-old rabbits) in both jejunum and ileum. The process is not inhibited by a 40-fold excess of glycine, proline, phenylalanine, leucine, lysine, glutamic acid, while it is strongly inhibited by a 10-fold excess of the dipeptides leucyl-leucine, alanyl-proline, glycyl-leucine and lysylleucine. A large excess of Gly-Pro has no inhibitory effect on the uptake of free glycine. The kinetic constants for Gly-Pro uptake are: Vmax = 126.7 ± 26.4 μmoles/ghr and Kt = 0.98 ± 0.26 mM in the jejunum and Vmax = 59.6 ± 9.2 and Kt = 0.70 ± 0.10 in the ileum. The removal of Na from the incubation and preincubation medium approximately halves the Vmax in both segments, leaving unchanged the affinity constants.These results indicate that a very efficient dipeptide influx process occurs in the newborn rabbit. This process displays many characteristics in common with that described in the adult: it is not shared by amino acids free in the lumen; has a broad specificity for dipeptides and shows the same pattern of Na dependence. However, its maximal velocity is in the newborn much higher than in the adult.Therefore, (1) the developmental patterns of the transport systems for Gly-Pro and for free glycine are different; this provides ontogenetic evidence for the distinction between dipeptide and free amino acid carrier systems; and (2) dipeptide uptake plays a predominant role in the physiology of the protein absorptive-digestive processes of the youngster.Speculation: Dipeptide uptake is likely to play a major role in the final stages of protein absorption in the youngster. Thus, wherever proteic oral supplementation is indicated in the newborn or young infant, the administration of free amino acids does not appear to be physiologically appropriate.

Enteric cryptosporidiosis in pediatric HIV infection

BACKGROUND Enteric cryptosporidiosis is a frequent problem in adults with human immunodeficiency virus (HIV) infection, but little is known of its features in children. The aim of this study was to investigate the incidence and the clinical features of cryptosporidiosis in HIV-infected children. METHODS Thirty-five children with symptomatic HIV infection were screened every 2 months, and in case of diarrhea, for the presence of Cryptosporidium. Intestinal function tests were performed, and the fecal osmotic gap was measured in children with cryptosporidiosis. RESULTS Seventy episodes of diarrhea occurred in 16 children in a median period of 17 months. Cryptosporidium was detected in five cases, all with full-blown acquired immunodeficiency syndrome. Cryptosporidiosis was significantly more protracted than any other form of diarrhea and was associated with dehydration and severe weight loss. Intestinal function was not modified during cryptosporidiosis. Osmotic gap values were consistent with secretory rather than osmotic diarrhea. In four cases, recovery was observed without specific treatment. CONCLUSIONS Enteric cryptosporidiosis is a severe problem in advanced stages of HIV infection. It does not induce intestinal malabsorption. It induces diarrhea of secretory type. Recovery may be observed independently of therapy.

In vivo and in vitro effects of human growth hormone on rat intestinal ion transport.

ABSTRACT: It has been reported that: 1) ovine growth hormone stimulates intestinal water, sodium, and chloride absorption and 2) specific growth hormone receptors are present in the rat intestine. Aims of this work were to investigate the effects of acute administration of hGH on water and ion transport in the rat ileum in vivo and in vitro. In vivo, the absorption rates of water, sodium, chloride, and potassium were determined in the rat perfused ileum, during a basal period and after i.v. administration of 6 μg/kg recombinant DNA-derived hGH. In vitro, electrical parameters were measured before and after the hormone addition to the mucosal or the serosal side of rat ileal mucosa mounted in Ussing chambers. In vivo, growth hormone induced a rapid increase in the absorption rates of water, sodium, chloride, and potassium. In vitro, the serosal, but not the mucosal, addition of growth hormone induced a rapid decrease of transepithelial potential difference and of short-circuit current. The effect was time- and dose-dependent, saturable, but not reversible in the short time. The electrical effect was abolished in the absence of chloride, indicating that it was related, at least in part, to inhibition of basal active chloride secretion. Growth hormone also reduced the short-circuit current increase induced by the secretagogues Escherichia coli heat-stable enterotoxin, theophylline, and calcium ionophore A23187. These results indicate that hGH has a rapid absorptive effect that is related, at least in part, to a direct intestinal antisecretory mechanism. It also reduces active intestinal secretion induced by various secretagogues.

Impaired intestinal function in symptomatic HIV infection.

A longitudinal (10-22 month) evaluation of intestinal symptoms and function was performed in five children with symptomatic human immunodeficiency virus (HIV) infection. All received cotrimoxazole, ketoconazole, and immunoglobulins. A search for enteric pathogens and intestinal function tests were repeatedly performed in all patients. Mild episodes of diarrhea were observed in two children. One had cows milk protein intolerance. Giardia lamblia was found in an asymptomatic carrier. Evidence for impaired intestinal function was found in all patients. These consisted of positive D-xylose and iron oral loads, increased steatorrhea, increased fecal excretion of alpha 1-antitrypsin, abnormal intestinal permeability, and increased food antibody levels. Our results suggest that severe diarrhea may be uncommon in children with HIV infection receiving antimicrobial prophylaxis, but that the intestinal function is frequently, and often markedly, impaired.

Reactive nitrogen species modulate the effects of rhein, an active component of senna laxatives, on human epithelium in vitro.

Background Senna laxatives are used worldwide. However, their misuse can lead to chronic mucosal inflammation with the accumulation of pigment-laden leukocytes and may cause colon cells to undergo apoptosis. This study explores the mechanisms by which rhein, an active component of senna, acts on a human intestinal cell line to induce ion secretion, apoptosis, and indirect chemotaxis of polymorphonuclear leukocytes. Methods Human colonic adenocarcinoma (CaCo-2) monolayer cells, in the presence or in the absence of rhein, were used to monitor the production of reactive nitrogen species using the Griess reaction. Modified Ussing chambers were used to study electrolyte secretion. The capacity to recruit human polymorphonuclear leukocytes was evaluated using masked well chemotaxis chambers. Rhein-induced apoptosis was investigated by counting apoptotic nuclei stained with Hoechst 33258 dye. Results Rhein caused a dose-dependent increase in short-circuit current that was abolished in chloride-free bathing buffer or by preincubating with 100 &mgr;mol/L NG-nitro-L-arginine (L-NAME) methyl ester. The concentration that maximally stimulated intestinal secretion, 50 &mgr;mol/L rhein, induced nitrate production. Supernatants obtained from CaCo-2 cultures after incubation with 50 &mgr;mol/L rhein stimulated a time-dependent polymorphonuclear leukocytes chemotaxis that was significantly decreased with 100 &mgr;mol/L L-NAME, whereas rhein per se was not active. Neutralizing antibodies anti–interleukin-8 (IL-8) and anti-ENA78 also inhibited chemotaxis. Overnight rhein incubation produced an increased number of apoptotic cells in the culture supernatant that was significantly decreased by preincubation with 100 &mgr;mol/L L-NAME. Light-degraded rhein had no effects on CaCo-2 monolayers. Conclusions The integrity of rhein is crucial to generating nitric oxide, which mediates, with different time courses, ion secretion, chemotaxis, and apoptosis of human-derived cells.

Reference values of the steatocrit and its modifications in diarrheal diseases

Conflicting results have been reported on the use of the steatocrit to measure fecal fat excretion. Aiming to assess the reliability of this method and its usefulness in the diagnosis of intestinal enteropathies, we measured the steatocrit in 747 healthy children and 442 children with diarrhea grouped according to diagnosis. The steatocrit was found to correlate strictly (r = 0.93) with the chemical measurement of fecal fat. Reference values and ranges were established. The maximal steatocrit was observed in neonates; afterwards, it progressively decreased to an undetectable level in children older than 2 years of age. A steatocrit abnormally high for age was found in 20% of patients with acute diarrhea and in 53% of those with chronic diarrhea. All celiac patients with a gluten-containing diet showed a marked increase of steatocrit. We conclude that the steatocrit is a reliable and easy-to-perform test, which quickly provides valuable information in the diagnostic workup of the child with diarrhea.

Characteristics and Mechanism of Action of a Heat-Stable Enterotoxin Produced by Klebsiella pneumoniae from Infants with Secretory Diarrhea

ABSTRACT: Escherichia coli heat-stable enterotoxins (ST) are classified into STa and STb according to their physicochemical and biologic characteristics. STa induces diarrhea, activating the guanylate cyclase-cGMP system. ST-like enterotoxins can be produced by bacteria other than E. coli, including Klebsiella pneumoniae. A Klebsiella ST has previously been shown to share some chemical and immunologic characteristics with E. coli ST. Aiming to define better the nature of Klebsiella ST, we have screened 237 children with diarrhea and 179 controls for ST-producing Klebsiella, using the SMA. We detected 26 Klebsiella strains from patients, two of which were positive in the SMA, and 36 from controls, all negative for ST. A partial purification was performed using an acetone precipitation followed by ultrafiltration and gel filtration techniques. Klebsiella toxin was heat-stable, methanol-soluble, sensitive to mercaptoethanol, active at acid pH values, but not at pH >8. The time course of Klebsiella toxin in the SMA resembled that of E. coli STa. Klebsiella ST caused reduced Na absorption and net Cl secretion in rabbit ileal mucosa mounted in Ussing chambers. It was found to increase the cGMP but not the cAMP concentration. Finally, Klebsiella ST did not react with anti-£. coli STa MAb in a competitive ELISA. We conclude that K. pneumoniae may induce diarrhea through the production of an STa similar but not identical to E. coli STa.