Microscale three-dimensional polymeric platforms for in vitro cell culture systems.

This paper describes fabrication schemes to create multidimensional polymeric platforms to study cell function. A key feature of these constructs is the replication of in vivo geometry and dimensional size scales that will aid in the understanding of fundamental cell-environment interactions. Advantages of these microtextured membranes include the high degree of reproducibility, optical clarity, and the ability to create multiple features on the micron and sub-micron size scale. We have demonstrated the creation of controlled microscale features on hydrogels as well as biodegradable materials such as poly(lactic-glycolic acid). These microtopographies selectively degrade under physiological conditions. Because of the flexibility of substrate material and the ease of creating micron size structures, this technique can be applied to a multitude of physiological and biological systems.

Fiber knob modifications overcome low, heterogeneous expression of the coxsackievirus-adenovirus receptor that limits adenovirus gene transfer and oncolysis for human rhabdomyosarcoma cells.

Exploiting the lytic life cycle of viruses has gained recent attention as an anticancer strategy (oncolysis). To explore the utility of adenovirus (Ad)-mediated oncolysis for rhabdomyosarcoma (RMS), we tested RMS cell lines for Ad gene transduction and infection. RMS cells were variably transduced by Ad. Compared with control cells, RMS cells were less sensitive or even resistant to oncolysis by wild-type virus. RMS cells expressed the Ad internalization receptors, alpha(v) integrins, but had low or undetectable expression of the major attachment receptor, coxsackievirus-Ad receptor (CAR). Mutant Ads with ablated CAR binding exhibited only 5-20% of transgene expression in RMS cells seen with a wild-type vector, suggesting that residual or heterogeneous CAR expression mediated the little transduction that was detectable. Immunohistochemical analysis of archived clinical specimens showed little detectable CAR expression in five embryonal and eight alveolar RMS tumors. Stable transduction of the cDNA for CAR enabled both efficient Ad gene transfer and oncolysis for otherwise resistant RMS cells, suggesting that poor CAR expression is the limiting feature. Gene transfer to RMS cells was increased >2 logs using Ads engineered with modified fiber knobs containing either an integrin-binding RGD peptide or a polylysine peptide in the exposed HI loop. The RGD modification enabled increased oncolysis for RMS cells by a conditionally replicative Ad, Ad delta24RGD, harboring a retinoblastoma-binding mutation in the E1A gene. Thus, the development of replication-competent vectors targeted to cell surface receptors other than CAR is critical to advance the use of Ad for treating RMS.

Nucleophosmin/B23 is a target of CDK2/cyclin E in centrosome duplication.

In animal cells, duplication of centrosomes and DNA is coordinated. Since CDK2/cyclin E triggers initiation of both events, activation of CDK2/cyclin E is thought to link these two events. We identified nucleophosmin (NPM/B23) as a substrate of CDK2/cyclin E in centrosome duplication. NPM/B23 associates specifically with unduplicated centrosomes, and NPM/B23 dissociates from centrosomes by CDK2/cyclin E-mediated phosphorylation. An anti-NPM/B23 antibody, which blocks this phosphorylation, suppresses the initiation of centrosome duplication in vivo. Moreover, expression of a nonphosphorylatable mutant NPM/ B23 in cells effectively blocks centrosome duplication. Thus, NPM/B23 is a target of CDK2/cyclin E in the initiation of centrosome duplication.

Novel diagnostic tests to detect Helicobacter pylori infection: A pediatric perspective.

Because of the widespread problem of Helicobacter pylori infections, there is an increased need for rapid, reliable and inexpensive diagnostic tests. Five recently developed tests that offer potential advantages because they are less invasive or permit easier acquisition of samples than available tests are assessed. The tests assessed are whole blood, saliva and urine assays that measure systemic antibody response to H pylori, stool tests that measure H pylori antigens and string tests that recover H pylori organisms.

Transjugular intrahepatic portosystemic shunts (TIPS) in children.

The transjugular intrahepatic portosystemic shunt procedure is an accepted treatment for adults with complications of portal hypertension. We performed a retrospective review of all pediatric TIPS placements performed at the University of California, San Francisco between 1990 and 1996. Twelve procedures were attempted in nine children, with a mean age (+/- SD) of 9.4 +/- 3.9 years (range, 5 to 15 years) and a mean weight of 31 +/- 18 kg (range, 16 to 70 kg). The indications for TIPS placement were portal hypertension complicated by chronic variceal hemorrhage not controlled with sclerotherapy (n = 7) and hypersplenism with thrombocytopenia (n = 2). TIPS placement was successfully completed initially in seven of nine (78%) patients. Unfavorable vascular anatomy was the cause of failure in two cases. The seven patients who underwent successful TIPS placement were followed up for an average of 136 days (range, 1 to 800 days); two still have patent shunts, three underwent liver transplantation, one had a splenorenal shunt after stenosis, and one died of underlying liver disease. Variceal bleeding was controlled in four of five patients who successfully underwent TIPS placement. Shunt occlusion occurred in four patients; patency was restored by transjugular shunt revision in three, and a splenorenal shunt was performed in one.

Donor selection limits use of living-related liver transplantation.

The purpose of this investigation was to assess the applicability of living-related liver transplantation in an established regional transplant program by determining the frequency of acceptable living donors from an unselected population of pediatric transplant candidates and identify specific factors limiting application of this technique. During the period May 1992 to May 1994, all children accepted as transplant candidates at the University of California-San Francisco were evaluated for potential living-related liver transplantation. Indications for transplantation and patient demographics represented the spectrum anticipated at a regional center. Donor evaluation was performed using a three-phase evaluation process we have previously reported. Retrospective analysis identified 75 potential donors for 38 pediatric candidates (age range, 17 days to 14.5 years; mean, 5.1 years). Twenty-three percent of potential donors declined evaluation. Of the 75 potential donors, only 10 (13%) were found to be acceptable for donation. The leading causes for donor declination were significant medical history (23%), ABO blood group incompatibility (23%), and psychosocial history (20%). Of the 38 recipient candidates, 9 (23%) were offered living-donor transplantation. Five patients have received living-donor transplantation, and 4 patients await the procedure when medical indications exist. Seventy-seven percent of recipient candidates received or are awaiting cadaveric transplantation. These results suggest that current donor criteria markedly limit the application of living-related liver transplantation.

Primary antral gastritis in young American children. Low prevalence of Helicobacter pylori infections.

Although Helicobacter pylori (H. pylori) is considered by many to be the major cause of primary antral gastritis (PAG), several important questions concerning its pathogenetic role remain unanswered. The most basic unresolved issue relates to the low prevalence of H. pylori in children in developed countries. If H. pylori is the cause of PAG, the prevalence of PAG should also be low, but previous studies have not provided data on this issue. To answer this question, we prospectively studied 408 children who underwent esophagogastroduodenoscopy and biopsy from January 1, 1988, to December 31, 1990, for symptoms consistent with peptic disease or immunocompromise. Although the prevalence of PAG was similar (about 20%) in the four age groups of patients studied (< 5, 5-9, 10-14, and 15-20 years), the prevalence of H. pylori infections was significantly greater in the oldest age group (67%, P < 0.0001). Only four of 39 children < 10 years old with PAG had evidence of H. pylori. H. pylori is an uncommon finding in our population of young American children with PAG, indicating that it does not play an important role in the pathogenesis of this disorder in this age group.

Is rice based oral rehydration therapy effective in young infants?

Rice based oral rehydration therapy (ORT) solutions have been shown to be superior to glucose oral rehydration salts (World Health Organisation (WHO) ORS) in reducing stool volume and duration of diarrhoea in children and adults. Rice based ORT has been used only sparingly in young infants, however, because of theoretical concerns about digestibility. A randomised controlled trial of rice based ORT (50 g rice and electrolytes identical to WHO ORS) and WHO ORS was carried out in 52 male infants less than 6 months old with moderately severe acute diarrhoea to evaluate efficacy and digestibility. Nineteen (70%) of 27 children who received rice based ORT and 18 (72%) of 25 children who received WHO ORS were treated successfully. The mean (SD) diarrhoeal stool output for the first 24 hours of treatment was significantly lower in the infants receiving the rice based ORT than in those receiving WHO ORS (101.0 (60.5) v 137.1 (74.6) g/kg). The stool output was also significantly less in the rice based ORT group in the second 24 hours. Infants in the rice based ORT group drank significantly less rehydration solution than infants in the WHO ORS group (mean (SD) 165.4 (77.4) v 217.9 (86.1) during the first 24 hours of treatment. There was no difference in the duration of diarrhoea between the groups. The volume of breast and formula feeding was similar in the two groups. No difference was seen in the frequency of finding reducing substances or acid pH in the stools of either group of children. The results suggest that rice based ORT is as effective as WHO ORS in infants with moderately severe diarrhoea and that rice based ORT is as well tolerated as WHO ORS in infants.

Nutrient absorption and weight gain in persistent diarrhea: comparison of a traditional rice-lentil/yogurt/milk diet with soy formula.

We prospectively studied clinical outcome and nutrition absorption in male children (6-36 months of age) with persistent diarrhea (PD) and severe protein-energy malnutrition (mean z score for age, -4.2 +/- 0.8). Fifty-one children were randomly allocated to receive a combination of khitchri, a rice-lentil mixture, yogurt, and half-strength buffalo milk (group A; n = 26) or full-strength soy formula (group B; n = 25) for 14 days. The initial caloric intake (p < 0.02) and mean weight gain (p < 0.02) were greater in the group B patients. The mean stool volume and frequency were comparable between the two groups. Seven children in group A were considered clinical failures by criteria of weight loss and PD, compared to three in group B. The coefficients of absorption (COA) for protein were similar between the two groups, but group B patients had higher values of COA for energy and fat (p < 0.05) in the second week of nutritional rehabilitation. The majority (63%) of the children improved on the khichri-yogurt-milk diet but the nutritional outcome was not as good as with the soy formula. These data indicate that a traditional diet can be successfully used in the dietary management of PD and severe malnutrition but that a more optimal composition, yielding a higher success rate, should be sought.

Is anemia an accurate predictor of vitamin A status in Pakistani children?

Serum retinol and hemoglobin levels were determined in 532 children aged 6-60 months living in urban slums of Karachi, Pakistan. Overall 67% (358 of 532) of children had hemoglobin levels of less than 11 g/dl, the World Health Organization definition of anemia for this age group. Estimations for red blood cell indices (hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and red blood cell count) were done on a sample of 391 children to classify anemia morphologically. A similar percentage (69.8% [273 of 391 children]) of anemia was found in this group. The anemia was predominantly microcytic and hypochromic. Normocytic normochromic anemia was present in only 16.5%. Serum retinol levels were significantly correlated with hemoglobin (P < 0.002), hematocrit (P < 0.01), and red blood cell (P < 0.001) levels. However, anemia was found to be a poor predictor (positive predictive value [PPV] = 2.5%) for the presence of vitamin A deficiency (retinol < 10 micrograms/dl). The PPV increased to 54% if 20 micrograms/dl, which is an indicator of marginal vitamin A status, was used as the cutoff point. The sensitivity of the hemoglobin test was found to be 75% in correctly identifying vitamin A deficiency when retinol levels of 10 micrograms/dl and 20 micrograms/dl were both used as the cutoff points for deficiency. The specificity for the hemoglobin test varied from 33% to 40% when levels of 10 micrograms/dl and 20 micrograms/dl were used as the cutoff points for vitamin A deficiency.(ABSTRACT TRUNCATED AT 250 WORDS)

Vitamin A status of children in the urban slums of Karachi, Pakistan, assessed by clinical, dietary, and biochemical methods.

We assessed the vitamin A status of 532 children with an age range of 6-60 months who were living in slum areas of Karachi, Pakistan, using three methodologies: clinical eye examination, dietary vitamin A intake, and serum retinol level. No definite clinical signs of xerophthalmia were observed in any of these children. The mean +/- SD vitamin A intake estimated from a food frequency questionnaire for the group with inadequate (low and deficient) serum retinol levels (< 20 micrograms/dl) was 362 +/- 332 retinol equivalents (RE) compared with 431 +/- 332) RE in the group with adequate serum levels (P < 0.005). Deficient serum retinol levels (< 10 micrograms/dl) were present in 12 children (2%); two of these had a healed corneal scar. Low serum retinol levels (10-19 micrograms/dl) were present in 46%, while 51% children had adequate levels (> or = 20 micrograms/dl). The mean +/- SD serum retinol level for the inadequate (< 20 micrograms/dl) and adequate groups were 15.3 +/- 2.8 and 26.6 +/- 6.7 micrograms/dl, respectively. These results suggest that a significant number of children in these communities have low vitamin A levels and thus may constitute an at risk group. These results also suggest that the dietary intake method may be a simple and inexpensive screening tool for assessment of vitamin A status in communities.

Learning by teaching. A resident-taught oral therapy program for acute diarrhea.

An educational program on oral rehydration therapy (ORT) for diarrhea was instituted in our residents' continuity clinics to evaluate the impact that residents teaching parents would have on the knowledge and practices of both groups. Sixty-one residents and 287 parents answered the initial written questionnaire before the teaching program began, and 48 residents and 147 parents completed a second questionnaire at the end of the program. Nineteen residents in two clinics were told to participate frequently in teaching the parents, while 29 residents in three other clinics were given no such instructions. The parents were divided into three groups: 58 received teaching and an instructional handout on the management of diarrhea; 73 received only the instructional handout; and 16 received neither intervention. The 19 "teaching" residents had a significantly improved overall score compared with the "nonteaching" residents (p < .03). No improvement was found in the scores of the 58 parents who received teaching compared with those of the 89 parents who received either a handout or no educational intervention. We conclude that active teaching of ORT may improve the knowledge and practices of residents, but that single teaching encounters, whether or not accompanied by written instructions, may have little impact on parents.

Nutritional management of persistent diarrhea: factors predicting clinical outcome.

In a prospective study we randomized 102 male children (age 6-36 months) with persistent diarrhea to receive a traditional rice-lentil (Khitchri) diet and yogurt (K-Y) or a soy formula (alone for seven days and then in combination with K-Y for seven days). Of 73 children satisfying the study criteria, 66 were successfully treated and there were 7 treatment failures. There was poor concordance between parental accounts of severity of diarrhea or vomiting and that observed after admission. Significant risk factors associated with treatment failure included younger age (p < 0.005) and vomiting at presentation (p < 0.02). The greatest number of risk factors associated with delayed recovery (> 10 days) were identified during an initial evaluation period (the first 8 h after admission). These included greater severity of watery diarrhoea (p < 0.01) and increased ORS intake (p < 0.02). Our data suggest that an initial evaluation period, including objective observations, may identify children with persistent diarrhea who are at greatest need of hospitalization.

Dietary protein sensitivity: is it an important risk factor for persistent diarrhea?

Although the causes of persistent diarrhea are largely unknown, dietary protein sensitivity has been identified as one possible mechanism. Since nutritional therapy appears to be an essential component of treatment for persistent diarrhea, a clearer understanding of the importance of dietary protein allergy is needed to provide optimal dietary recommendations. This paper reviews the current concepts of the pathophysiology, clinical features and criteria for diagnosis of dietary protein sensitivity. Evaluation of the results of several clinical studies indicates that dietary protein sensitivity does occur especially in young infants fed cows' milk but that it is an uncommon event, especially in older infants and children.

Dietary management of persistent diarrhea: comparison of a traditional rice-lentil based diet with soy formula.

Recent studies have indicated that enteral diets can play an important role in the treatment of persistent diarrhea. Khitchri, a local weaning food in Pakistan, is composed of rice and lentils, which have previously been shown to be well tolerated in many children with acute diarrhea. The effectiveness of a khitchri and yogurt (KY) diet, which is inexpensive and widely available in Pakistan, was studied. One hundred two weaned boys (6 to 36 months old) with persistent diarrhea were randomly assigned to receive either soy formula (group A) or the KY diet (group B) for 14 days. Group A also received the KY diet in addition to formula for days 8 through 14. Twenty-nine children did not complete the study because of severe infection (13) or their family's decision to leave the study early (9 in group A and 7 in group B). Sixty-six children successfully completed the study protocol; there were five clinical failures in group A and two in group B. On a comparable caloric intake, there was a significantly lower stool volume (group B: 38 +/- 16 [mean +/- SD] vs group A: 64 +/- 75 g/kg per day, P less than .05) and frequency (B: 4.4 +/- 2.0 vs. A: 6.6 +/- 4.2 stools per day, P less than .005) in children fed KY during the first week of therapy. Group B children also had a significantly greater weight gain than children in group A during the first week (B: 468 +/- 373 g/wk vs A: 68 +/- 286 g/wk, P less than .005).(ABSTRACT TRUNCATED AT 250 WORDS)

Stiff upper lips.

PIP: In an interview with Family Planning World, Helen Axby of the Marie Stopes Clinics explains that the organization's growth at a time when the number of abortions in Great Britain has begun to decline is due to successful management and marketing. Although the British government provides from abortion services, the Marie Stopes Clinics performs over 25,000 abortions a year. According to Axby, the main reason for this is that government restrictions have created a cumbersome process where women often wait 3-4 weeks to receive an abortion. At the Stopes clinics, women receive prompt treatment in a comfortable setting. A consultation is scheduled within a day or two following the initial phone call, and the operation is scheduled at the woman's convenience. Axby explains that the organization conducts extensive advertisement in the Yellow Pages, local newspapers, and women's magazines. Though the clinic operates for profit, its services are far less expensive than what it would cost to obtain them at other private offices. In 1990, the organization grossed about 6.2 million pounds (profits were around 1 million pounds). The profits are used to fund overseas projects. Because of the decline in the number of abortions in Britain, Axby expects the organization to earn less this year. However, Marie Stopes clinics plans to fight to increase its market share of family planning services. Its fastest growing service is vasectomy. Relying on the noninvasive bipolar diathermy technique, the Stopes Clinics have made vasectomy a more simple and comfortable experience for men. Other services include employee screening (for pap smears, breast checks, etc.). Finally, Axby says that the organization might also increase its market share by acquiring its competitors.^ieng

Dietary risk factors associated with acute and persistent diarrhea in children in Karachi, Pakistan.

Feeding practices may have an important impact on diarrheal diseases in developing countries. This study evaluated feeding practices in three groups of male children aged 6-36 mo: 100 with persistent diarrhea (PD), 79 with acute diarrhea (AD), and 86 in a comparison group (CG). The children came from comparably poor socioeconomic settings in Karachi, Pakistan, except that the literacy rates were higher in mothers of the CG (P = 0.0001). Although greater than 95% of all infants were breast-fed, delayed initiation of breast-feeding was more common in the diarrhea groups. Children with diarrhea were also more likely to receive supplemental milk (PD = 92%, AD = 87%) than were children in the CG (69%, P less than 0.05). Feedings were not withheld during diarrhea but changes were made in the nature of foods given. These results indicate that several feeding practices may be important risk factors for diarrhea in Pakistan.