Infant morbidity in an Indian slum birth cohort.

OBJECTIVE: To establish incidence rates, clinic referrals, hospitalisations, mortality rates and baseline determinants of morbidity among infants in an Indian slum. DESIGN: A community-based birth cohort with twice-weekly surveillance. SETTING: Vellore, South India. SUBJECTS: 452 newborns recruited over 18 months, followed through infancy. MAIN OUTCOME MEASURES: Incidence rates of gastrointestinal illness, respiratory illness, undifferentiated fever, other infections and non-infectious morbidity; rates of community-based diagnoses, clinic visits and hospitalisation; and rate ratios of baseline factors for morbidity. RESULTS: Infants experienced 12 episodes (95% confidence interval (CI) 11 to 13) of illness, spending about one fifth of their infancy with an illness. Respiratory and gastrointestinal symptoms were most common with incidence rates (95% CI) of 7.4 (6.9 to 7.9) and 3.6 (3.3 to 3.9) episodes per child-year. Factors independently associated with a higher incidence of respiratory and gastrointestinal illness were age (3-5 months), male sex, cold/wet season and household involved in beedi work. The rate (95% CI) of hospitalisation, mainly for respiratory and gastrointestinal illness, was 0.28 (0.22 to 0.35) per child-year. CONCLUSIONS: The morbidity burden due to respiratory and gastrointestinal illness is high in a South Indian urban slum, with children ill for approximately one fifth of infancy, mainly with respiratory and gastrointestinal illnesses. The risk factors identified were younger age, male sex, cold/wet season and household involvement in beedi work.

Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes.

A new approach for delivering vaccine antigens is the use of inexpensive, plentiful, plant-based oral vaccines. Norwalk virus capsid protein (NVCP), assembled into virus-like particles, was used as a test antigen, to determine whether immune responses could be generated in volunteers who ingested transgenic potatoes. Twenty-four healthy adult volunteers received 2 or 3 doses of transgenic potato (n=20) or 3 doses of wild-type potato (n=4). Each dose consisted of 150 g of raw, peeled, diced potato that contained 215-751 microgram of NVCP. Nineteen (95%) of 20 volunteers who ingested transgenic potatoes developed significant increases in the numbers of specific IgA antibody-secreting cells. Four (20%) of 20 volunteers developed specific serum IgG, and 6 (30%) of 20 volunteers developed specific stool IgA. Overall, 19 of 20 volunteers developed an immune response of some kind, although the level of serum antibody increases was modest.

Subunit rotavirus vaccine administered parenterally to rabbits induces active protective immunity.

Virus-like particles (VLPs) are being evaluated as a candidate rotavirus vaccine. The immunogenicity and protective efficacy of different formulations of VLPs administered parenterally to rabbits were tested. Two doses of VLPs (2/6-, G3 2/6/7-, or P[2], G3 2/4/6/7-VLPs) or SA11 simian rotavirus in Freund's adjuvants, QS-21 (saponin adjuvant), or aluminum phosphate (AlP) were administered. Serological and mucosal immune responses were evaluated in all vaccinated and control rabbits before and after oral challenge with 10(3) 50% infective doses of live P[14], G3 ALA lapine rotavirus. All VLP- and SA11-vaccinated rabbits developed high levels of rotavirus-specific serum and intestinal immunoglobulin G (IgG) antibodies but not intestinal IgA antibodies. SA11 and 2/4/6/7-VLPs afforded similar but much higher mean levels of protection than 2/6/7- or 2/6-VLPs in QS-21. The presence of neutralizing antibodies to VP4 correlated (P < 0.001, r = 0.55; Pearson's correlation coefficient) with enhanced protection rates, suggesting that these antibodies are important for protection. Although the inclusion of VP4 resulted in higher mean protection levels, high levels of protection (87 to 100%) from infection were observed in individual rabbits immunized with 2/6/7- or 2/6-VLPs in Freund's adjuvants. Therefore, neither VP7 nor VP4 was absolutely required to achieve protection from infection in the rabbit model when Freund's adjuvant was used. Our results show that VLPs are immunogenic when administered parenterally to rabbits and that Freund's adjuvant is a better adjuvant than QS-21. The use of the rabbit model may help further our understanding of the critical rotavirus proteins needed to induce active protection. VLPs are a promising candidate for a parenterally administered subunit rotavirus vaccine.

Passive immunity to bovine rotavirus in newborn calves fed colostrum supplements from cows immunized with recombinant SA11 rotavirus core-like particle (CLP) or virus-like particle (VLP) vaccines.

Heterotypic passive immunity to IND (P/5/G6) bovine rotavirus (BRV) was evaluated. Three groups of calves (n = 5 per group) were fed 1% pooled colostrum supplements (birth to 7 days of age) from BRV seropositive cows vaccinated with recombinant SA11(P/2/G3) rotavirus-like particles (VLPs), recombinant SA11 rotavirus core-like particles (CLPs), or inactivated SA11 rotavirus (SA11). Control calves (n = 5 per group) received either pooled colostrum from unvaccinated (BRV field exposure seropositive) control cows, or no colostrum. IgG1 antibody titers to IND BRV for the pooled colostrum were: 1,048,576 (VLP); 1,048,576 (CLP); 262,144 (SA11); and 16,384 (control colostrum). Elevated titers of BRV neutralizing (VN) antibodies were present in VLP colostrum (98,000), and SA11 colostrum (25,000), but not in CLP colostrum (1400), compared to colostrum from nonvaccinates (2081). Calves were orally inoculated with virulent IND BRV at 2 days of age and challenged at post-inoculation day (PID) 21. Calves were monitored daily for diarrhea and faecal BRV shedding through PID 10 and post-challenge day (PCD) 10. After colostrum feeding, the IgG1 antibody titers were highest in serum and faeces of calves fed VLP and CLP colostrum, but VN and IgA antibodies were highest in calves fed VLP colostrum. After BRV inoculation, calves fed colostrum from vaccinated cows had significantly fewer days of BRV-associated diarrhea and BRV shedding than control calves. All calves fed VLP colostrum were protected from diarrhea after BRV inoculation; two calves shed BRV. In the CLP colostrum group, one calf developed BRV-associated diarrhea and all calves shed virus. In the SA11 colostrum group, three calves developed BRV-associated diarrhea and four calves shed virus. BRV-associated diarrhea and shedding occurred in 9 of 10 control calves. Active IgM antibody responses occurred in faeces and/or serum of most calves after BRV inoculation. However, the highest active antibody responses (IgM and IgG1 in serum, and IgM, IgG1 or IgA in faeces) after BRV inoculation were in calves fed control or no colostrum, in association with clinical diarrhea in most of these calves. After challenge at PID 21, BRV-associated diarrhea and shedding were of short duration or absent, in all groups. These results demonstrate the efficacy of colostrum from VLP vaccinated cows to provide heterologous, passive protection against BRV diarrhea and shedding in calves. In comparison, calves fed CLP or SA11 colostrum were only partially protected against BRV diarrhea or shedding.

Pharmacokinetics and cardiovascular effects of ma-huang (Ephedra sinica) in normotensive adults.

The purpose of this study was to evaluate heart rate and blood pressure responses to a commercially available source of ma-haung, a natural source of the sympathomimetic substance, ephedrine, and to evaluate the pharmacokinetic properties of the product in normotensive, healthy adults. On day 1, twelve study participants were monitored with an ambulatory blood pressure device between hours 7 and 20. On day 2, they ingested four capsules of powdered ma-huang at hours 8 and 17 while again wearing the monitor between hours 7 and 20. Serial plasma samples were obtained and concentrations of ephedrine were analyzed by high-performance liquid chromatography. Pharmacokinetic parameters of ephedrine were determined from plasma concentration-time profiles. The ephedrine alkaloid content of each capsule was also determined by high-performance liquid chromatography. Six participants experienced a statistically significant increase in heart rate, but the effects on blood pressure were variable. The half-life, volume of distribution, clearance, and maximum concentration in plasma of ephedrine in the ma-huang product were similar to values previously reported for a 20 mg, immediate-release ephedrine tablet. Values for the absorption rate were considerably lower and time to reach maximum concentration was longer for the capsules, compared with the standard tablet. Variability in alkaloid content of ephedrine was low and yielded a mean dose of ephedrine at 19.4 mg; pseudoephedrine at 4.9 mg; and methylephedrine at 1.2 mg for a four-capsule dose. In summary, ma-haung had variable effects on blood pressure and increased heart rate in healthy, normotensive adults. Pharmacokinetic parameters for ephedrine were in agreement with those previously reported; however, the absorption rate was much slower after ingestion of ma-huang.

Leukocyte adhesion molecules and x-ray energy dispersive spectroscopy in Sturge-Weber disease.

We examined the light microscopic and ultrastructural features associated with Sturge-Weber disease, including x-ray energy dispersive spectroscopy to evaluate the chemical composition of the mineralized deposits and immunofluorescence microscopy with leukocyte adhesion molecules to examine the blood vessel proliferation further. Two patients (a 17-year-old girl and a 9-month-old boy) with Sturge-Weber disease comprise this series. Mineralized deposits stained strongly positive with von Kossa and negative with Prussian blue. Transmission electron microscopy of tissue removed during a functional hemispherectomy procedure in both cases indicated that most concretions were adjacent to or in the basal lamina of parenchymal vessels; no deposits were observed in leptomeningeal vessels. Energy dispersive spectroscopy of the deposits showed emission peaks corresponding predominantly to calcium, with lesser amounts of phosphorus. Fluorescent monoclonal antibodies to leukocyte adhesion molecules (endothelial cell, vascular cell, and intercellular: ELAM-1, VCAM-1, and ICAM-1) demonstrated strong positive staining of the meningeal vessels with all three antibodies. Cortical vessels were positive only for ICAM-1. Findings based on routine staining and energy dispersive spectroscopy indicate that the mineralized deposits detected in Sturge-Weber disease are composed primarily of calcium phosphate and are located primarily in and adjacent to the vascular basal lamina. There is an aberrant expression of ELAM-1 and VCAM-1 in the meningeal vascular proliferation similar to what is observed with other vascular malformations and tumors. Parenchymal vessel changes may be secondary to the meningeal vascular proliferation.

Isotype-specific antibody responses to rotavirus and virus proteins in cows inoculated with subunit vaccines composed of recombinant SA11 rotavirus core-like particles (CLP) or virus-like particles (VLP).

The isotype antibody responses to bovine IND P5, G6 and simian SA11 P2, G3 rotavirus and SA11 rotavirus proteins (VP4, VP6 and VP7) in serum, colostrum and milk were analysed by ELISA in three groups of vaccinated cows and nonvaccinated controls. Pregnant cows were vaccinated intramuscularly and intramammarily with recombinant baculovirus-expressed SA11 rotavirus VLP (triple-layered virus-like particles containing rotavirus VP2, VP4, VP6 and VP7); CLP (double-layered core-like particles containing rotavirus VP2 and VP6); or inactivated SA11 rotavirus, respectively. Rotavirus antigen titers were highest (30-200-fold) in ELISA in the VLP vaccine compared to the inactivated SA11 vaccine. The IgG1, IgG2 and IgM geometric mean antibody titers (GMT) to rotavirus (titers to bovine rotavirus vs SA11 rotavirus did not differ significantly for any isotype or group) and the IgG2 GMT to VP6 in serum at calving in the vaccinated groups were significantly (P < 0.05) higher than in the control group. In colostrum, IgG1 and IgA rotavirus antibody titers were significantly elevated for VLP (IgG1 GMT 832225; IgA GMT 16384), CLP (IgG1 GMT 660561; IgA GMT 10321) and SA11 (IgG1 GMT 131072; IgA GMT 1448) vaccinated cows compared to control cows (IgG1 GMT 11585; IgA GMT 45). The IgG1 and IgA GMT to rotavirus were significantly elevated (6-100-fold) in milk of VLP and CLP vaccinated cows compared to SA11 vaccinated or control cows. The isotype antibody responses to VP6 in serum, colostrum and milk paralleled the responses to rotavirus, but titers were approximately 2-10-fold lower. Only cows vaccinated with VLP had significantly enhanced serum, colostral and milk antibody titers to rotavirus VP4 and VP7. These results demonstrate that rotavirus antibody titers in serum, colostrum and milk are significantly enhanced by use of non-infectious VLP, CLP and inactivated SA11 rotavirus vaccines, but the VLP or CLP vaccines induced the highest antibody responses, corresponding to their higher rotavirus antigen titers measured by ELISA.

Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.

Alternatives to cell culture systems for production of recombinant proteins could make very safe vaccines at a lower cost. We have used genetically engineered plants for expression of candidate vaccine antigens with the goal of using the edible plant organs for economical delivery of oral vaccines. Transgenic tobacco and potato plants were created that express the capsid protein of Norwalk virus, a calicivirus that causes epidemic acute gastroenteritis in humans. The capsid protein could be extracted from tobacco leaves in the form of 38-nm Norwalk virus-like particles. Recombinant Norwalk virus-like particle (rNV) was previously recovered when the same gene was expressed in recombinant baculovirus-infected insect cells. The capsid protein expressed in tobacco leaves and potato tubers cosedimented in sucrose gradients with insect cell-derived rNV and appeared identical to insect cell-derived rNV on immunoblots of SDS/polyacrylamide gels. The plant-expressed rNV was orally immunogenic in mice. Extracts of tobacco leaf expressing rNV were given to CD1 mice by gavage, and the treated mice developed both serum IgG and secretory IgA specific for rNV. Furthermore, when potato tubers expressing rNV were fed directly to mice, they developed serum IgG specific for rNV. These results indicate the potential usefulness of plants for production and delivery of edible vaccines. This is an appropriate technology for developing countries where vaccines are urgently needed.

Protoplasmic astrocytoma. A clinicopathologic study of 16 tumors.

UNLABELLED: Protoplasmic astrocytomas, composed of process-poor astrocytic cells and a microcystic background, are rare variants of low grade astrocytoma. Three hundred eight low grade astrocytomas over a 23-year period were reviewed. Sixteen (5.2%) were classified as pure protoplasmic astrocytoma. Patients (12 males, 4 females) ranged in age from 2.5 to 41 years (mean, 20.7 years). All patients presented with seizures and three had headaches. Duration of symptoms ranged from 7 months to 28 years (mean, 6.6 years). Nine tumors (56%) were left-sided and seven right-sided (44%). Seven (44%) occurred in the temporal lobe, six (37%) in the frontal lobe, two (13%) in the parietal lobe, and one (6%) in the thalamus. Surgery consisted of partial lobectomy with total tumor resection in nine and biopsy alone in seven. Five patients received adjuvant therapy with no apparent effect on survival. At frozen section, protoplasmic astrocytoma was most confused with fibrillary low grade astrocytoma (n = 6). Follow-up revealed 10 patients with no evidence of disease 2 to 108 months postoperatively (mean, 41 months), 5 patients were alive with disease 10 to 84 months postoperatively (mean, 56 months) and 1 patient died with disease at 36 months. Of patients with total tumor resection, eight had no evidence of disease and one died with disease. IN CONCLUSION: (1) protoplasmic astrocytomas in this study were more frequently observed in males at a younger mean age than fibrillary low grade astrocytomas as reported in literature; (2) temporal and frontal lobes were the most likely site of origin; and (3) complete excision may be beneficial, whereas adjuvant therapy appeared to have no effect on outcome.

Structure and function of the polypeptides in simian virus 40. I. Existence of subviral deoxynucleoprotein complexes.

Velocity sedimentation analysis of simian virus 40 degraded in alkaline buffers, pH 10.5, yields two components: soluble protein containing the largest polypeptides, VP1 and VP2, of the virion, and a deoxynucleoprotein complex (DNP-I) containing the viral deoxyribonucleic acid (DNA) and the small polypeptides, VP4, 5, and 6, and all or part of VP3. Dissociation of DNP-I by equilibrium centrifugation in CsCl yields a complex (DNP-II) of the viral DNA and residual, tightly bound polypeptide; VP4, 5, and 6, but not VP3, are recovered after separation from DNP-II. Treatment of the virus with beta-mercaptoethanol and iodination experiments suggest that VP1 and VP2 might exist as compact structures cross-linked with disulfide bonds, perhaps forming the capsid. VP4, 5, and 6 form a relatively stable complex with the viral DNA and are supposed to be the internal proteins. The location of VP3 is not well defined; at least a portion of it is tightly bound to the viral DNA.