[Growth hormone of dried blood spot for the diagnosis of growth hormone deficiency]. / Hormona de crecimiento en sangre de papel filtro para el diagnóstico de deficiencia de hormona de crecimiento.

INTRODUCTION: The diagnosis of growth hormone deficiency (GHD) is difficult to determine, and could be associated with severe complications, especially in the neonatal period. The stimulation test of growth hormone (GH) secretion is considered the gold standard for diagnosis, but it has methodological complications and is associated with adverse effects. Neonates present physiological increased secretion of GH, representing a diagnostic window. OBJECTIVE: To evaluate if the dried blood spot on filter paper obtained in the neonatal period, as part of a neonatal screening for con genital hypothyroidism and phenylketonuria, allows differentiating patients with GHD from those who do not have it. PATIENTS AND METHOD: Study of cases and controls by measuring the GH concen tration in dried blood spot on filter paper obtained in the neonatal period, comparing controls with GHD with cases with discarded deficiency. The sample was extracted from the filter paper, obtaining two 0.125 inch discs per each patient from the center of the blood spot on the paper, for a highly sen sitive ELISA assay for human GH based on the use of polyclonal antibodies against 22 kDa recom binant human GH. RESULTS: Seven cases of GHD and ten controls were obtained. The median GH concentration of the dried blood spot in the cases is 2.0 ng/ml (Interquartile range 3.6 ng/ml) and 2.05 ng/ml (Interquartile range 2.0 ng/ml) in the controls, Mann-Whitney U test 30.5 (p = 0.68). The two cases with multiple pituitary-hormone deficiency (MPHD) present concentrations lower than 1 ng/ml. CONCLUSION: The dried blood spot sample did not differentiate GHD patients from control cases, although MPHD cases present much lower concentrations compared to isolated growth hor mone deficiency (IGHD).

Pollen derived low molecular compounds enhance the human allergen specific immune response in vivo.

BACKGROUND: Besides allergens, pollen release bioactive, low molecular weight compounds that modulate and stimulate allergic reactions. Clinical relevance of these substances has not been investigated to date. OBJECTIVE: To elucidate the effect of a non-allergenic, low molecular weight factors from aqueous birch pollen extracts (Bet-APE < 3 kDa) on the human allergic immune response in vivo. METHODS: Birch and grass pollen allergic individuals underwent skin prick testing with allergen alone, allergen plus Bet-APE < 3 kDa, or allergen plus pre-identified candidate substances from low molecular pollen fraction. Nasal allergen challenges were performed in non-atopic and pollen allergic individuals using a 3 day repeated threshold challenge battery. Subjects were either exposed to allergen alone or to allergen plus Bet-APE< 3 kDa. Local cytokine levels, nasal secretion weights, nasal congestion and symptom scores were determined. RESULTS: Skin prick test reactions to pollen elicited larger weals when allergens were tested together with the low molecular weight compounds from pollen. Similar results were obtained with candidate pollen-associated lipid mediators. In nasal lining fluids of allergic patients challenged with allergen plus Bet-APE < 3 kDa, IL-8 and IgE was significantly increased as compared to allergen-only challenged patients. These patients also produced increased amounts of total nasal secretion and reported more severe rhinorrhea than the allergen-only challenged group. CONCLUSIONS: Low molecular compounds from pollen enhance the allergen specific immune response in the skin and nose. They are therefore of potential clinical relevance in allergic patients.

The putative serine protease inhibitor Api m 6 from Apis mellifera venom: recombinant and structural evaluation.

BACKGROUND: Immunoglobulin (Ig) E-mediated reactions to honeybee venom can cause severe anaphylaxis, sometimes with fatal consequences. Detailed knowledge of the allergic potential of all venom components is necessary to ensure proper diagnosis and treatment of allergy and to gain a better understanding of the allergological mechanisms of insect venoms. OBJECTIVE: Our objective was to undertake an immunochemical and structural evaluation of the putative low-molecular-weight serine protease inhibitor Api m 6, a component of honeybee venom. METHODS: We recombinantly produced Api m 6 as a soluble protein in Escherichia coli and in Spodoptera frugiperda (Sf9) insect cells.We also assessed specific IgE reactivity of venom-sensitized patients with 2 prokaryotically produced Api m 6 variants using enzyme-linked immunosorbent assay. Moreover, we built a structural model ofApi m 6 and compared it with other protease inhibitor structures to gain insights into the function of Api m 6. RESULTS: In a population of 31 honeybee venom-allergic patients, 26% showed specific IgE reactivity with prokaryotically produced Api m 6, showing it to be a minor but relevant allergen. Molecular modeling of Api m 6 revealed a typical fold of canonical protease inhibitors, supporting the putative function of this venom allergen. Although Api m 6 has a highly variant surface charge, its epitope distribution appears to be similar to that of related proteins. CONCLUSION: Api m 6 is a honeybee venom component with IgE-sensitizing potential in a fraction of venom-allergic patients. Recombinant Api m 6 can help elucidate individual component-resolved reactivity profiles and increase our understanding of immune responses to low-molecular-weight allergens

Api m 10, a genuine A. mellifera venom allergen, is clinically relevant but underrepresented in therapeutic extracts.

BACKGROUND: Generalized systemic reactions to stinging hymenoptera venom constitute a potentially fatal condition in venom-allergic individuals. Hence, the identification and characterization of all allergens is imperative for improvement of diagnosis and design of effective immunotherapeutic approaches. Our aim was the immunochemical characterization of the carbohydrate-rich protein Api m 10, an Apis mellifera venom component and putative allergen, with focus on the relevance of glycosylation. Furthermore, the presence of Api m 10 in honeybee venom (HBV) and licensed venom immunotherapy preparations was addressed. METHODS: Api m 10 was produced as soluble, aglycosylated protein in Escherichia coli and as differentially glycosylated protein providing a varying degree of fucosylation in insect cells. IgE reactivity and basophil activation of allergic patients were analyzed. For detection of Api m 10 in different venom preparations, a monoclonal human IgE antibody was generated. RESULTS: Both, the aglycosylated and the glycosylated variant of Api m 10 devoid of cross-reactive carbohydrate determinants (CCD), exhibited IgE reactivity with approximately 50% of HBV-sensitized patients. A corresponding reactivity could be documented for the activation of basophils. Although the detection of the native protein in crude HBV suggested content comparable to other relevant allergens, three therapeutical HBV extracts lacked detectable amounts of this component. CONCLUSION: Api m 10 is a genuine allergen of A. mellifera venom with IgE sensitizing potential in a significant fraction of allergic patients independent of CCD reactivity. Thus, Api m 10 could become a key element for component-resolved diagnostic tests and improved immunotherapeutic approaches in hymenoptera venom allergy.

The Chilean socio-ethno-genomic cline.

Studies of the current Chilean population performed using classical genetic markers have established that the Chilean population originated primarily from the admixture of European people, particularly Spaniards, and Amerindians. A socioeconomic-ethno-genetic cline was established soon after the conquest. Spaniards born in Spain or Chile occupied the highest Socioeconomic Strata, while Amerindians belonged to the lowest. The intermediate strata consisted of people with different degrees of ethnic admixture; the larger the European admixture, the higher the Socioeconomic Level. The present study of molecular genomic markers sought to calculate the percentage of Amerindian admixture and revealed a finer distribution of this cline, as well as differences between two Amerindian groups: Aymara and Mapuche. The use of two socioeconomic classifications - Class and Socioeconomic Level - reveals important differences. Furthermore, Self-reported Ethnicity (self-assignment to an ethnic group) and Self-reported Ancestry (self-recognition of Amerindian ancestors) show variations and differing relationships between socioeconomic classifications and genomic Amerindian Admixture. These data constitute a valuable input for the formulation of public healthcare policy and show that the notions of Ethnicity, Socioeconomic Strata and Class should always be a consideration in policy development.

Stratum corneum lipids, skin barrier function and filaggrin mutations in patients with atopic eczema.

BACKGROUND: Prior to the discovery of filaggrin (FLG) mutations, evidence for an impaired skin barrier in atopic dermatitis (AD) has been documented, and changes in ceramide profile, altered skin pH and increased trans-epidermal water loss (TEWL) in patients with AD have been reported. Until now, no studies have analysed stratum corneum (SC) lipids combined with skin barrier parameters in subjects of known FLG genotype. METHODS: A cohort of 49 German individuals genotyped for the most common FLG mutations (R501X, 2282del4) had SC samples taken for lipid analysis by high-performance thin layer chromatography. In addition, TEWL, erythema, skin hydration and pH were measured. In 27 of the 49 individuals, a 24-h irritation patch test with sodium lauryl sulphate was performed. For the analysis, both the AD group and the control group were stratified by FLG mutation status (FLGmut/FLGwt). RESULTS: In the FLGmut AD group, significantly lower levels of ceramide 4 and significantly higher levels of ceramide 7 were observed when compared to both healthy control groups. However, ceramide 7 levels also significantly differed between FLGwt AD and FLGwt controls, as did ceramide 1 levels. No significant differences were observed for ceramide 2, 3, 5 and 6. FLGmut individuals had significantly higher skin pH values than individuals not carrying FLG mutations. Patients with AD with FLG mutations had significantly higher erythema compared to patients with AD without FLG mutations. CONCLUSION: Our results confirm previous observations of altered ceramide levels in AD, which however appear to show no clear relationship with FLG mutations.

Calcium oxalate: crystallographic analysis in solid aggregates in urinary sediments.

A relationship between crystallographic structure and morphological form of calcium oxalates in urinary sediments is established. The common tetragonal bipyramids have been confirmed as weddellite from their electron diffraction patterns. Other solid forms, such as needles, biconcave disks, and dumbbell forms, that can appear in hyperoxalurias, of both metabolic and alimentary origin, have been identified as whewellite. Micrographs reveal fibrous structure on those whewellite polycrystalline aggregates.

CTG repeats at the myotonic protein kinase gene in a healthy Chilean population sample.

OBJECTIVES: To study the variability at the myotonic dystrophy protein kinase (DMPK) gene in a Chilean sample of healthy people. DM1 is an autosomal dominant disorder caused by an expansion of a (CTG) repeat at the 3'-UTR of the gene DMPK. Healthy individuals have alleles under 35 repeats and diseased individuals have over 50. METHODS: Genotyping the number of (CTG) repeats at this gene in a sample of healthy Chilean people. RESULTS: Allele frequencies were significantly different from those of other populations. The most frequent allele was with five repeats. The frequency of larger alleles (>18 CTG repeats) was 11%, close to the European frequency (12%) and higher than the Japanese (8%) and Aboriginal Pehuenche samples (8%). CONCLUSIONS: Allelic frequencies in the Chilean sample studied were intermediate between those of the two ancestral populations (European and Pehuenche).

Allele frequencies for 12 autosomal short tandem repeat loci in two Bolivian populations.

Two hundred and sixty unrelated subjects who asked for paternity testing at two Bolivian Laboratories in La Paz and Santa Cruz were studied. The loci D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820, TH01, TPOX, and CSF1PO were typed from blood samples, amplifying DNA by polymerase chain reactions and electrophoresis. Allele frequencies were estimated by simple counting and the unbiased heterozygosity was calculated. Hardy-Weinberg equilibrium was studied and gene frequencies were compared between the two samples. All loci conformed to the Hardy-Weinberg law and allele frequencies were similar in samples from the two cities. The Bolivian gene frequencies estimated were significantly different from those described for Chile and the United States Hispanic-Americans for most of the loci.

Assessing the health benefits of urban air pollution reductions associated with climate change mitigation (2000-2020): Santiago, São Paulo, México City, and New York City.

To investigate the potential local health benefits of adopting greenhouse gas (GHG) mitigation policies, we develop scenarios of GHG mitigation for México City, México; Santiago, Chile; São Paulo, Brazil; and New York, New York, USA using air pollution health impact factors appropriate to each city. We estimate that the adoption of readily available technologies to lessen fossil fuel emissions over the next two decades in these four cities alone will reduce particulate matter and ozone and avoid approximately 64,000 (95% confidence interval [CI] 18,000-116,000) premature deaths (including infant deaths), 65,000 (95% CI 22,000-108,000) chronic bronchitis cases, and 46 million (95% CI 35-58 million) person-days of work loss or other restricted activity. These findings illustrate that GHG mitigation can provide considerable local air pollution-related public health benefits to countries that choose to abate GHG emissions by reducing fossil fuel combustion.

Experimental study of the protective effect of glutathione against cisplatin-induced nephrotoxicity.

Glutathione (GSH) is the most important intracellular thiol-compound which participates in the detoxification mechanisms of the cell. Its high affinity to react with platinum complexes would give rise to lower or non-toxic metabolites and prevent cisplatin nephrotoxicity. In order to determine if GSH can protect against cisplatin-induced renal toxicity, 120 female Wistar rats received LD-100 or LD-50 of cisplatin with or without GSH, at two different dose levels and by two different routes. Biochemical and histological changes as survival was observed in each group. The administration of GSH did not modify cisplatin LD-100. When cisplatin LD-50 was used, a significant improvement in the survival rate was observed in the group which received GSH as chemoprotector (100% vs 40%). The average values of urea and creatinine were significantly lower in the group treated with GSH (115 vs 370 mg/dl and 1.07 vs 4.02 mg/dl respectively). The degree of the tissue injury was also lower in the GSH group. The administration of GSH prior to cisplatin reduces its nephrotoxicity in this animal model. Further clinical trials are necessary to verify this protective effect when cisplatin is used as a cyclic administration and at different dose levels.

Glutathione administration against cisplatin-induced nephrotoxicity does not modify its cytotoxic activity.

Glutathione (GSH) is an intracellular thiol compound which has been shown to protect against cisplatin-induced nephrotoxicity, in animal models and clinical trials. In order to determine whether GSH interferes with cisplatin activity, the lymphoma L5178Y was implanted in 50 DBA/2 mice, and then they were treated with cisplatin with or without previous GSH. Two similar experiments were carried out with three different groups: Group 1: Control group without cisplatin; Group 2: Treatment with cisplatin without GSH, and Group 3: GSH administration prior to cisplatin. Tumor area and survival have been considered as parameters to measure the activity of cisplatin. The average Values of tumor areas in the mice pretreated with GSH were not significantly different from those corresponding to the group treated with cisplatin alone. Sixty days survival was 55% and 73% in the groups pretreated with GSH and with cisplatin alone respectively, the difference was not statistically significant. In conclusion, GSH administration prior to cisplatin does not modify its cytotoxic activity.

Stable carbon and nitrogen isotope analysis of TNT: two-dimensional source identification.

Data from a combination of laboratory and fieldwork is presented to initiate testing of stable carbon and nitrogen isotope ratios to trace sources of TNT in contaminated soil and groundwater. Evaluation of these extraction methods resulted in 99.9 and 99.8% recovery of TNT with Soxhlet and solid-phase extraction (SPE), respectively. As a result of the high extraction efficiency, isotope fractionation did not occur, thus providing an accurate stable isotope value on TNT from laboratory and field samples. Subsequent experiments evaluated the stability of isotope signatures through incubations lasting up to four weeks with a 70% decline in the TNT concentration. During these experiments, no significant variation in stable carbon and nitrogen isotope ratios was measured. Five different sources of TNT, compared for stable carbon and nitrogen isotope ratios, showed a range of 4.2 and 15%, respectively. This large range in the isotope ratios suggests excellent potential to trace sources in a complex environment. Finally, a site was surveyed for concentrations and isotope values of TNT extracted from groundwaters. Values from this site were substantially different relative to the variation measured on standards and in laboratory incubation experiments. The data set indicates good potential to use stable isotopes to determine TNT sources and fate in the environment.

Frequency of the hypervariable DNA loci D18S849, D3S1744, D12S1090 and D1S80 in a mixed ancestry population of Chilean blood donors.

Blood donors (N = 150) at San José Hospital (Santiago, Chile) were typed for one VNTR locus (D1S80) and three STR loci (D18S849, D3S1744, D12S1090). A questionnaire was used to determine the socioeconomic level of the donors, because it is known that some genetic markers (e.g., the ABO and Rh groups) are differentially distributed between different socioeconomic strata. This methodology revealed that two of the three socioeconomic strata distinguishable in Santiago were present in our sample of blood donors, with stratum II representing the middle strata and stratum III the low strata. Allele frequency was determined for each locus and socioeconomic stratum, and it was found that the allele distributions of each locus in socioeconomic strata II and III were statistically similar. All loci conformed to the Hardy-Weinberg law and there was no evidence for association between the alleles of the four loci, allelic frequencies being similar to those found in North American Hispanic populations. The results support the view that the analysis of these loci may have useful applications in population genetics as well as in identity tests.

Effect of the fine fraction of particulate matter versus the coarse mass and other pollutants on daily mortality in Santiago, Chile.

Daily counts of non-accidental deaths in Santiago, Chile, from 1988 to 1996 were regressed on six air pollutants--fine particles (PM2.5), coarse particles (PM10-2.5), CO, SO2, NO2, and O3. Controlling for seasonal and meteorological conditions was done using three different models--a generalized linear model, a generalized additive model, and a generalized additive model on previously filtered data. Single- and two-pollutant models were tested for lags of 1-5 days and the average of the previous 2-5 days. The increase in mortality associated with the mean levels of air pollution varied from 4 to 11%, depending on the pollutants and the way season of the year was considered. The results were not sensitive to the modeling approaches, but different effects for warmer and colder months were found. Fine particles were more important than coarse particles in the whole year and in winter, but not in summer. NO2 and CO were also significantly associated with daily mortality, as was O3 in the warmer months. No consistent effect was observed for SO2. Given particle composition in Santiago, these results suggest that combustion-generated pollutants, especially from motor vehicles, may be associated with increased mortality. Temperature was closely associated with mortality. High temperatures led to deaths on the same day, while low temperatures lead to deaths from 1 to 4 days later.

Paternity analysis when the putative father is missing: first case in Chile.

Genetic marker analysis is a powerful tool for solving paternity-related problems when the putative father is missing. This report describes the first time this approach was employed in Chile to solve such a problem. In the case presented, the alleged father was missing as a result of the political detentions that took place in Chile during 1973. It was not possible to obtain any biological sample from him because he was missing. Thus, the case was resolved by means of genetic marker analysis of the alleged father's close relatives.