In vitro study of alcohol dehydrogenase and acetaldehyde dehydrogenase encapsulated into human erythrocytes by an electroporation procedure.

The optimal conditions for electroporated/resealed loading of alcohol dehydrogenase (ADH) and/or acetaldehyde dehydrogenase (ALDH) into human erythrocytes were established prior to the study, with the following characteristics: 300 V, 1 ms pulse time, eight pulses every 15 min and 1 h resealing at 37 degreesC. High encapsulation yield and carrier cell recoveries were achieved. Cell volumes increase while hemoglobin contents decrease; in consequence a decrease in cell hemoglobin concentration was observed. A lower hypotonic resistance of loading erythrocytes (throughout osmotic fragility curves) and unaltered oxygen transport capability (as given by oxygen equilibrium curves) were observed. The stability against time (up to 168 h-7 days) of encapsulated individual enzymes, either ADH- or ALDH-red blood cells (RBCs), was studied at 4 degreesC and 37 degreesC, in comparison with that of free enzyme solutions. Both enzymes were released from carrier RBCs to the incubation medium. The stability of carrier RBCs was studied under similar conditions. Non-significant variations in hematological parameters were observed. However, the hemoglobin derivative forms showed modifications. The continuous degradation of ethanol by ADH-RBCs and coencapsulated ADH- and ALDH-RBCs, as a function of time (up to 70 h) suggests the use of these carrier RBCs as agents for complete metabolization of ethanol. The mentioned properties bare the possibility of using ADH and ALDH as carrier systems in in vivo situations.

Efficacy comparison of oral rehydration solutions containing either 90 or 75 millimoles of sodium per liter.

In a randomized trial, 62 infants 2 to 35 months of age with dehydration due to acute watery diarrhea were allocated to one of two groups: group A received solution A (World Health Organization-recommended oral rehydration solution), which contained (mmol/L): Na+ 90, K+ 20, Cl- 80, citrate3- 10, and glucose 110; group B received solution B (Pedialyte RS; Abbott Laboratories, North Chicago), which contained (in mmol/L): Na+ 75, K+ 20, Cl- 65, citrate3- 10, and glucose 139. Oral therapy was given until clinical signs of hydration status were normal. During the 48-hour trial, the following laboratory data were collected: blood gases, serum electrolytes, glucose, urea, and creatinine values and sodium and potassium concentrations in stool and urine; serial weights and clinical signs were also reported. Six of the 62 infants, three in each group, required intravenous fluids because of high stool output. Results of clinical outcome and normalization of altered serum electrolyte values were similar in both groups. During the 48-hour trial, eight patients in group A and four in group B had mild, asymptomatic hypernatremia. Pedialyte RS was found to be a safe glucose/electrolyte solution for oral rehydration therapy.

Outbreak of neonatal listeriosis associated with mineral oil.

In June, 1989, an outbreak of nosocomial listeriosis occurred in Costa Rica. Listeria monocytogenes was isolated from 9 ill infants 4 to 8 days old who were born after the delivery of an infant with early onset listeriosis. One nosocomial infection was fatal, 2 required mechanical ventilation and 1 resulted in hemiparesis. A higher proportion of cases than other infants born during the outbreak were delivered by cesarean section (55% vs. 24%, P = 0.04). Compared with the mothers of 36 random controls, case mothers were more often primiparous (odds ratio, 6.2, P = 0.03) or received general anesthesia before delivery (odds ratio, 4.4, P = 0.09). All infants were bathed with mineral oil from a multidose container. Culture of the oil by cold enrichment grew L. monocytogenes 4b with the same electrophoretic enzyme type as the outbreak strain. We hypothesize that aspiration of contaminated oil may have resulted in systemic listeriosis. General anesthesia may have increased the risk of aspiration. Lung tissue from the infant who died showed lipid-laden macrophages consistent with oil aspiration and had evidence of L. monocytogenes DNA detected by polymerase chain reaction. This is the first nosocomial outbreak of listeriosis in which a common source suggested epidemiologically was microbiologically confirmed. The high attack rate (greater than 200 times the United States rate of perinatal listeriosis) emphasizes the susceptibility of healthy neonates to L. monocytogenes. The results of our study led to the discontinuation of the use of mineral oil for bathing neonates in Costa Rica.

Diarrhea associated with rotaviruses, enterotoxigenic Escherichia coli, Campylobacter, and other agents in Costa Rican children, 1976-1981.

Rotaviruses, enterotoxigenic Escherichia coli, Salmonella, Shigella, and parasites were investigated in outpatient diarrheic children, and in hospitalized diarrheic and non-diarrheic children, between January 1976 and June 1979. In outpatient cases studied within 4 days of onset of symptoms, rotaviruses were the most common agents (45.3%); E. coli heat-stable enterotoxin ranked second (13.4%); Shigella was third (8.1%); Salmonella was fourth (7.3%). In 63.2%, one or more enteric agents were detected. In hospitalized non-diarrheic children, asymptomatic shedding of pathogens was rarely observed. A later survey of outpatient diarrheic children revealed Campylobacter fetus jejuni in 8%. In 5.5 years of observation rotaviruses were endemic with excess frequency in the dry and cooler months of December and January. The excess occurrence of bacterial pathogens did not coincide with that of rotaviruses.

Mouse erythrocytes as carriers for coencapsulated alcohol and aldehyde dehydrogenase obtained by electroporation in vivo survival rate in circulation, organ distribution and ethanol degradation.

Alcohol dehydrogenase and aldehyde dehydrogenase (ADH and ALDH) have been coencapsulated into mouse erythrocytes by an electroporation technique. The optimal conditions were achieved as follows: 420 V, four pulses of 1 ms every 15 min. at 37 degrees C, completed by resealing: 1 h at 37 degrees C. An encapsulation yield ranging from 11-12% was obtained for ADH+ALDH-loaded erythrocytes. Carrier cell recovery was 52%. Electroporated-RBCs observed under Scanning electron microscopy exhibited a tendency toward invaginated sphero-stomatocytes. These invaginations were not found in electroporated/resealed RBCs. The intravenous administration of 51Cr-RBCs manifested a bimodal pharmacokinetic profile: an initial phase (t1/2alpha) with a rapid decrease of plasma 51Cr-RBCs followed by a slow and prolonged elimination phase (t1/2beta). The values corresponding to in vivo survival rate during the elimination phase indicated that the survival rate of 51Cr-electroporated loaded-RBCs was slightly lower (t1/2beta, 4.5 days) than 51Cr-native RBCs (t1/2beta, 5.3 days). The mean clearance values from blood of electroporated 51Cr-RBCs (unloaded and loaded) were higher (0.51 %51Cr/day and 0.54 %51Cr/day, respectively) than the obtained for native 51Cr-RBCs (0.18 %51Cr/day). The target organs for electroporated RBCs proved to be the same as for native RBCs. However, electroporated RBCs showed highest accumulation in liver, spleen and lung, since they were promptly recognized by the reticuloendothelium system. Mice induced to the state of acute ethanol intoxication and treated with ADH+ALDH-RBCs clearly showed a lower level of ethanol concentration in plasma (less than 43% ethanol) than the intoxicated mice treated with native RBCs. En consequence the clearance values of ethanol from blood in intoxicated mice treated with ADH+ALDH-RBCs (0.39 ml/min) were higher than the treated with native RBCs (0.20 ml/min). The results obtained suggest that ADH+ALDH-loaded erythrocytes could be used as a potential carrier system for in vivo removal of high levels of ethanol from blood caused by excessive alcohol consumption.

In vitro and in vivo study of glutamate dehydrogenase encapsulated into mouse erythrocytes by a hypotonic dialysis procedure.

Glutamate dehydrogenase (GDH) has been encapsulated into mouse erythrocytes by a hypotonic dialysis/isotonic resealing method. Although a low GDH entrapment yield was achieved (3.8%), this percentage appeared sufficient enough to metabolize high quantities of ammonia. Carrier cell recovery yield was 56%. Due to the decrease in cell volume and haemoglobin content, constant mean cell haemoglobin concentration (MCHC) values were obtained. The osmotic fragility curves (OFC) indicated that dialyzed/resealed-RBCs are more resistant to hypotonic haemolysis than native-RBCs. The successful in vitro ammonia degradation by GDH-RBCs was reflected in its total disappearance from the incubation medium at around 48 h. In contrast, initial ammonia levels were not affected during the incubation in the presence of native-RBCs and remained constant. Two different methods were used for the preparation of hyperammonaemic mice model. Since the intraperitoneal (i.p.) administration of ammonium acetate produced high ammonia levels that lasted only a few minutes, the i.p. administration of urease was chosen, given that it generated elevated ammonia levels for longer periods of time. Hyperammonaemic mice quickly removed high levels of circulating ammonia in the presence of GDH-RBCs, whereas in the presence of native-RBCs ammonia was slowly metabolized. These results suggest that loaded GDH-erythrocytes can be used as a potential carrier systems for the in vivo removal of high levels of ammonia from blood.

Selective DNA release from DQAsome/DNA complexes at mitochondria-like membranes.

The number of diseases found to be associated with defects of the mitochondrial genome has grown significantly over the past decade (Wallace 1999). Despite major advances in understanding mtDNA defects at the genetic and biochemical level, there is no satisfactory treatment available for the vast majority of patients and the exploration of gene therapeutic approaches is highly warranted. However, mitochondrial gene therapy still appears only theoretical and speculative. Any possibility for gene replacement depends on the use of a yet unavailable mitochondria-specific transfection vector. Mitochondria-specific vectors must posses two properties: they have to transport DNA to the side of mitochondria; they must not release DNA during endocytosis. Amphiphile compounds with delocalized cationic charge centers such as rhodamine 123 and the bolaamphiphile dequalinium have long been known to accumulate in mitochondria. Sufficient lipophilicity combined with delocalization of the positive charge to reduce the free energy change when moving from an aqueous to a hydrophobic environment are believed to be prerequisite for mitochondrial accumulation in response to the mitochondrial membrane potential. We have recently succeeded in preparing cationic vesicles made of dequalinium that we termed DQAsomes (Weissig et al. 1998a). We have shown that DQAsomes bind and protect DNA against DNase activity (Lasch et al. 1999). Based on the intrinsic property of dequalinium to preferentially accumulate in mitochondria in response to the electrochemical gradient at the mitochondrial membrane, we believe that DQAsomes can serve as a vector to deliver DNA to mitochondria in living cells. As a first step in the development of mitochondria-specific DNA delivery systems, we report here that DQAsome/DNA complexes selectively release DNA at cardiolipin-rich liposomes mimicking both the inner and the outer mitochondrial membrane. We demonstrate that DNA remains tightly associated with DQAsomes in the presence of an excess of anionic lipids other than cardiolipin.

[Rotavirus in children with severe malnutrition]. / Rotavirus en niños con desnutrición severa.

The study comprised 19 children with severe protein-calorie malnutrition admitted to Hospital Nacional de Niños. With one exception, all of the patients complained of diarrhea at the examination. Samples of stools were collected for bacteriologic, parasitologic and virologic studies. Rotavirus was shown in 4 cases and adenovirus in another 4. Enteropathogenic Escherichia coli, Shigella and Salmonella were isolated in 9 different children. It was possible to demonstrate at least one potentially pathogenic agent in 84% of the total number of cases. In malnourished infants under one year of age, the index of positiveness was 100%. It is set forth that infection is the most important factor in the etiology of diarrhea of the malnourished infant.

[Meningococcal meningitis in Costa Rica, 1970--1973. Epidemiological study]. / Meningitis meningocócica en Costa Rica, 1970--1973 Estudio epidemiológico.

A retrospective hospital chart review of meningococcal meningitis cases in Costa Rica revealed a large number of cases confirmed in the laboratory by Gram-stained smear and/or culture of spinal fluid. A large percentage of the isolates studied were serogroup C and all of these were sulfonamide resistant. The age-specific attack rates were high in the preschool children with the highest attack rate in children 3 to 5 months of age. Case fatality ratios were 14% overall, with the low fatality rates in individuals 10 to 29 years of age. The outbreak of serogroup C meningitis in 1970--1971 was relatively brief, country-wide, and not predominantly a urban outbreak. The few secondary cases observed occurred within the first 2 weeks of the primary case.

[Disseminated histoplasmosis in children]. / Histoplasmosis diseminada en niños.

Clinical features and treatment evaluation of 38 children with disseminated histoplasmosis were studied. Thirty-six cases presented the severe form and two of them showed the mild chronic type. A higher incidence of this disease was found in infants (65%) and in severe malnutrition (68%) and lethality in the overall group was 61%. Patients died either because they did not receive specific treatment or were given amphotericin for less than 15 days. These figures stress the importance of this treatment in disseminated histoplasmosis.

[Infectious agents in diarrhea of hospitalized children in Costa Rica]. / Agentes infecciosos en la diarrea del niño hospitalizado en Costa Rica.

Shigella, toxigenic Escherichia coli (stable toxin) and rotaviruses were frequently found among 50 children hospitalized with diarrhea studied during one year. These agents were less common among 45 controls without diarrhea, of comparable age and from the same wards as the cases reported. There was a greater frequency of respiratory symptoms in the diarrhea associated with rotaviruses. These were characterized by frequent bowel movements and vomiting and often fever. The bacterial diarrheas showed, in general, a more severe clinical picture than the viral ones. The rotaviruses had a low endemic level during April-October, but their prevalence increased in December and January; in such months these viruses were found in more than 50 per cent of the diarrheal cases.

Comparison of sucrose with glucose in oral therapy of infant diarrhoea.

In a randomised double-blind trial, 51 5--10% dehydrated infants were rehydrated with oral electrolyte solutions containing sucrose or glucose. Most infants in both groups were successfully rehydrated, but the sucrose solution produced a slower correction of electrolyte abnormalities and a higher percentage of patients who needed more than 24 h of therapy. Where there is adequate knowledge of the oral therapy method sucrose can substitute for glucose in many cases; where there is a choice glucose is recommended.

PIP: 51 infants aged 3 to 12 months with dehydration due to acute watery diarrhea were randomly assigned to either the glucose or sucrose oral therapy group on admission to the hospital. Oral rehydration and maintenance without any intravenous fluids was successful in 100% and 92% of patients in the glucose and sucrose groups respectively, as evidenced by the subjects' weight gain, fall in plasma proteins and hematocrit, and disappearance of clinical signs of dehydration. There was a greater improvement in mean HC03 in the glucose group than in the sucrose group. Both groups exhibited decline in mean serum osmolarity. The 2 oral treatment failures in the sucrose group occurred in patients who could not absorb the solutions adequately. This study shows that oral therapy with sucrose is less efficient than oral therapy with glucose. However, in areas where only sucrose is available and where knowledge and means of using oral therapy exist, the oral sucrose solution can be used to treat most infants with diarrhea and 5 to 10% dehydration.

Oral rehydration and maintenance of children with rotavirus and bacterial diarrhoeas.

PIP: The outcome of administering ORT (oral rehydration therapy) to 62 infants admitted to a Costa Rican hospital with acute rotavirus or bacterial diarrheas and with 5-10% dehydration was described. 94% of the infants were successfully treated by administering only ORT. There were no significant differences in the success rates for rotavirus diarrhea patients and for various bacterial diarrhea patients. Success rates were 92% for rotavirus patients, 93% for Escherichia coli patients, 96% for idiopathic diarrhea patients, and 100% for salmonella and shigella patients. Upon admission, the average duration of diarrhea was 2.9 days, vomiting was present 88% of the cases, and all patients exhibited some signs of dehydration. The infants were administered the oral formula recommended by the World Health Organization. Patients received 400 ml of oral solution followed by 200 ml of water. The treatment was repeated until skin tuger was normal. 34% of the infants were rehydrated within 6 hours and 76% within 20 hours. Patients were admitted with a variety of electrolyte abnormalies. 24% had hyponatraemia, 27% had hypokalaemia, and 23% had hypernatraemia. Sodium levels were improved within 24 hours for all patients except for 5 hyponataemia patients. Although rotavirus patients had higher stool glucose concentrations than the other patients, they apparently absorbed enough of the solution to rehydrate successfully. Specific data on changes in weight, plasma protins, hematocrit, blood composition, and stool composition and on therapeutic failures was provided. Investigators concluded that ORT was a safe and effective form of therapy for both rotavirus and bacterial diarrhea and for severe cases of dehydration.^ieng

[Diabetic ketoacidosis]. / Cetoacidosis diabética.

Is the most commun and serious complication of the diabetes mellitus in the childhood and adolescent, the current mortality rate in most tertiary hospitals for children is about 2 percent. Is the metabolic consequence of a deficiency of insulin, it can be precipitating for stress, or inappropriate knowledge of the management and in some cases of subclinical diabetes or it still has not been diagnostic. The majority of instances of diabetic ketoacidosis can be prevented by early recognition of ketosis, and on appropriate ambulatory management.