Placental concentrations of xenoestrogenic organochlorine pesticides and polychlorinated biphenyls and assessment of their xenoestrogenicity in the PA-MAMI mother-child cohort.

BACKGROUND: Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCB), they have contributed to the exposure of women to persistent organic pollutants (POPs). These compounds can cross the placental barrier and interfere with the hormonal system of newborns. AIM: To determine concentrations of OCPs and PCBs and their xenoestrogenic activity in placentas of women from the PA-MAMI cohort of Panama. METHODS: Thirty-nine placenta samples from women in the Azuero peninsula (Panama) were analyzed. Five OCPs [p-p'-dichlorodiphenyldichloroethylene (p-p'-DDE), beta-hexachlorohexane (ß-HCH), γ-hexachlorohexane (lindane), hexachlorobenzene (HCB) and mirex] and three PCB congeners (PCB-138, PCB-153 and PCB-180) were quantified in placenta extracts. The xenoestrogenic activity of extracts was assessed with the E-Screen bioassay to estimate the total effective xenoestrogen burden (TEXB). RESULTS: All placental samples were positive for at least three POP residues and >70% for at least six. The frequencies of quantified OCPs ranged from 100% for p,p'-DDE and HCB to 30.8% for ß-HCH. The highest median concentration was for lindane (380.0 pg/g placenta), followed by p,p'-DDE (280.0 pg/g placenta), and HCB (90.0 pg/g placenta). Exposure to p,p'-DDE was associated with greater meat consumption, suggesting that animal fat is a major source of exposure to DDT metabolites. The frequency of detected PCBs ranged between 70 and 90%; the highest median concentration was for PCB 138 (17.0 pg/g placenta), followed by PCB 153 (16.0 pg/g placenta). All placentas were positive in the estrogenicity bioassay with a median TEXB-α of 0.91 pM Eeq/g of placenta. Exposure to lindane was positively associated with the xenoestrogenicity of TEXB- α, whereas this association was negative in the case of exposure to PCB 153. CONCLUSIONS: To our best knowledge, this study contributes the first evidence on the presence of POPs and xenoestrogenic burden in placentas from Latin-American women. Given concerns about the consequences of prenatal exposure to these compounds on children's health, preventive measures are highly recommended to eliminate or minimize the risk of OCP exposure during pregnancy.

Clinical standards for drug-susceptible TB in children and adolescents.

BACKGROUND: These clinical standards aim to provide guidance for diagnosis, treatment, and management of drug-susceptible TB in children and adolescents.METHODS: Fifty-two global experts in paediatric TB participated in a Delphi consensus process. After eight rounds of revisions, 51/52 (98%) participants endorsed the final document.RESULTS: Eight standards were identified: Standard 1, Age and developmental stage are critical considerations in the assessment and management of TB; Standard 2, Children and adolescents with symptoms and signs of TB disease should undergo prompt evaluation, and diagnosis and treatment initiation should not depend on microbiological confirmation; Standard 3, Treatment initiation is particularly urgent in children and adolescents with presumptive TB meningitis and disseminated (miliary) TB; Standard 4, Children and adolescents should be treated with an appropriate weight-based regimen; Standard 5, Treating TB infection (TBI) is important to prevent disease; Standard 6, Children and adolescents should receive home-based/community-based treatment support whenever possible; Standard 7, Children, adolescents, and their families should be provided age-appropriate support to optimise engagement in care and clinical outcomes; and Standard 8, Case reporting and contact tracing should be conducted for each child and adolescent.CONCLUSION: These consensus-based clinical standards, which should be adapted to local contexts, will improve the care of children and adolescents affected by TB.

Theoretical and experimental study of acoustic waves generated by thermocavitation and its application in the generation of liquid jets.

Numerical simulations using the Finite-Difference Time-Domain method were used to study the propagation of an acoustic wave within a truncated ellipsoidal cavity. Based in our simulations, a fluidic device was designed and fabricated using a 3D printer in order to focus an acoustic wave more efficiently and expel a liquid jet. The device consists of an ellipsoidal shaped chamber filled with a highly absorbent solution at the operating wavelength (1064 nm) in order to create a vapor bubble using a continuous wavelength laser. The bubble rapidly expands and collapses emitting an acoustic wave that propagates inside the cavity, which was measured by using a needle hydrophone. The bubble collapse, and source of the acoustic wave, occurs in one focus of the cavity and the acoustic wave is focused on the other one, expelling a liquid jet to the exterior. The physical mechanism of the liquid jet generation is momentum transfer from the acoustic wave, which is strongly focused due to the geometry of the cavity. This mechanism is different to the methods that uses pulsed lasers for the same purpose. The maximum speed of the generated liquid microjets was approximately 20 m/s. One potential application of this fluidic device can be found for inkjet printing, coating and, maybe the most attractive, for drug delivery.

17α-Ethynylestradiol alters the peritoneal immune response of gilthead seabream.

17α-Ethynylestradiol (EE2), a synthetic estrogen used in most oral contraceptives pills and hormone replacement therapies, is found in many water bodies, where it can modulate the fish immune response. EE2 acts as an endocrine disruptor in gilthead seabream, Sparus aurata L., a marine teleost fish of great economic value in Mediterranean aquaculture, as it induces hepatic vitellogenin gene (vtg) expression. Moreover, EE2 also alters the capacity of gilthead seabream to appropriately respond to infection although it does not behave as an immunosuppressor. Nevertheless, these previous studies have mainly focused on the head kidney leukocytes and no information exists on peritoneal leukocytes, including mast cells. In the present work, juvenile gilthead seabream fish were fed a pellet diet supplemented with EE2 for 76 days and intraperitoneally injected with hemocyanin plus imject alum adjuvant at the end of EE2 treatment and 92 days later, and the peritoneal immune response was analyzed. EE2 supplementation induced vtg expression but returned to basal levels by 3 months post-treatment. Interestingly, gilthead seabream peritoneal leukocytes express the genes encoding for the nuclear estrogen receptor α and the G protein-coupled estrogen receptor 1 and the dietary intake of EE2 induced these expression. Moreover, EE2 induced an inflammatory response in the peritoneal cavity in unvaccinated fish, which was largely maintained for several months after the cessation of the treatment. However, the impact of EE2 in vaccinated fish was rather minor and transient. Taken together, the study provides fresh information about endocrine immune disruption, focusing on peritoneal leukocytes.

Histamine and mast cell activator compound 48/80 are safe but inefficient systemic adjuvants for gilthead seabream vaccination.

Histamine has a key role in the regulation of inflammatory and innate immune responses in vertebrates. Gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost of great commercial value, was the first fish species shown to possess histamine-containing mast cells (MCs) at mucosal tissues. MCs are highly abundant in the peritoneal exudate of gilthead seabream and compound 48/80 (Co 48/80), often used to promote MC activation and histamine release, is able to promote histamine release from gilthead seabream MCs in vitro and in vivo. The aim of the present study was to analyze the effect of histamine and Co 48/80 on the immune responses of gilthead seabream. For this purpose, histamine and Co 48/80 were intraperitoneally injected alone or combined with 109 heat-killed Vibrio anguillarum cells and their effects on head kidney and peritoneal exudate were analyzed. The results indicated that although histamine and Co 48/80 were both able to alter the percentage of peritoneal exudate and head kidney immune cell types, only Co 48/80 increased reactive oxygen species production by peritoneal leukocytes. In addition, histamine, but not Co 48/80, was able to slightly impair the humoral adaptive immune response, i.e. production of specific IgM to V. anguillarum. Notably, both histamine and Co 48/80 reduced the expression of the gene encoding histamine receptor H2 in peritoneal exudate leukocytes. These results show for the first time in fish that although systemic administration of histamine and Co 48/80 is safe, neither compound can be regarded as an efficient adjuvant for gilthead seabream vaccination.

Thyroid hormone stimulates the proliferation of Sertoli cells and single type A spermatogonia in adult zebrafish (Danio rerio) testis.

Thyroid hormones participate in regulating growth and homeostatic processes in vertebrates, including development and adult functioning of the reproductive system. Here we report a new stimulatory role of thyroid hormone on the proliferation of Sertoli cells (SCs) and single, type A undifferentiated spermatogonia (A(und)) in adult zebrafish testes. A role for T3 in zebrafish testis is suggested by in situ hybridization studies, which localized thyroid receptor α (thrα) in SCs and the ß (thrß) mRNA in Sertoli and Leydig cells. Using a primary zebrafish testis tissue culture system, the effect of T3 on steroid release, spermatogenesis, and the expression of selected genes was evaluated. Basal steroid release and Leydig cell gene expression did not change in response to T3. However, in the presence of FSH, T3 potentiated gonadotropin-stimulated androgen release as well as androgen receptor (ar) and 17α-hydroxylase/17,20 lyase (cyp17a1) gene expression. Moreover, T3 alone stimulated the proliferation of both SCs and A(und), potentially resulting in newly formed spermatogonial cysts. Additional tissue culture studies demonstrated that Igf3, a new, gonad-specific member of the IGF family, mediated the stimulatory effect of T3 on the proliferation of A(und) and SCs. Finally, T3 induced changes in connexin 43 mRNA levels in the testis, a known T3-responsive gene. Taken together, our studies suggest that T3 expands the population of SCs and A(und) involving Igf signaling and potentiates gonadotropin-stimulated testicular androgen production as well as androgen sensitivity.

ATP-binding cassette transporter ABCB5 gene is expressed with variability in malignant melanoma.

BACKGROUND: Melanoma is a malignant neoplasm with high metastatic disease risk and elevated mortality. Incidence of melanoma varies according to geographic region and genetic BACKGROUND: Epidemiological studies indicate that acral melanoma (AM) is among the most common melanomas in the Mexican population. While extensive studies have identified genes associated with melanoma, little is known about the genes involved in the pathogenesis of AM. OBJECTIVE: To compare the gene expression patterns between primary melanoma and normal skin. METHODS: We used 10 samples of fresh acral melanomas and normal skin for the study of differential gene expression and 22 samples of melanoma for in situ hybridization. RESULTS: We first identified a gene that was present in a sample of AM and absent in normal skin. DNA sequencing of this differentially expressed gene revealed that it corresponded to ABCB5, a gene recently implicated in the regulation of progenitor cell fusion. Furthermore, we detected ABCB5 expression in other melanoma specimens by RT-PCR. We showed that nine out of ten melanomas were positive for ABCB5 while only one melanoma and normal skin samples were negative. All ABCB5 expressing melanomas had variable gene expression according to in situ hybridization studies, suggesting that the ABCB5 gene may be differentially regulated by individual melanomas. CONCLUSIONS: The ABCB5 gene may be related to the properties of chemoresistance and aggressiveness of melanoma. The high expression found in samples of acral melanoma may provide more insight on the pathogenesis of this common type of melanoma in the Mexican population, frequently associated with poor prognosis.

Role of telomere length in subtelomeric gene expression and its possible relation to cellular senescence.

Transcriptional silencing of subtelomeric genes is associated with telomere length, which is correlated with age. Long and short telomeres in young and old people, respectively, coincide with gene repression and activation in each case. In addition, differential location of genes with respect to telomeres causes telomere position effect. There is very little evidence of the manner in which age-related telomere length affects the expression of specific human subtelomeric genes. We analyzed the relationship between telomere length and gene expression levels in fibroblasts derived from human donors at ages ranging from 0-70 years. We studied three groups of genes located between 100 and 150 kb, 200 and 250 kb, and > 300 kb away from telomeres. We found that the chromatin modifier-encoding genes Eu-HMTase1, ZMYND11, and RASA3 were overexpressed in adults. Our results suggest that short telomere length-related overexpression of chromatin modifiers could underlie transcriptional changes contributing to cellular senescence.

Salmonella and Shigella in freshly squeezed orange juice, fresh oranges, and wiping cloths collected from public markets and street booths in Guadalajara, Mexico: incidence and comparison of analytical routes.

A survey of the presence of Salmonella and Shigella in freshly squeezed orange juice and related samples was conducted in Guadalajara, Mexico. One hundred samples of freshly squeezed orange juice were collected from 49 street booths and 51 small food service establishments. In addition, 75 fresh orange samples, each consisting of five orange units, and 75 wiping cloths were collected from the same establishments from which juice had been collected. Salmonella was isolated from 14, 20, and 23% of samples of orange juice, orange surfaces, and wiping cloths collected from street vendors, while Shigella was isolated from 6, 17, and 5% of these samples. In general, the frequency of isolation of these pathogens in samples from juice serving establishments at public markets was significantly lower than that found among street vendors (P < 0.05). Salmonella enterica serotypes Agona, Typhimurium, and Anatum were found in orange juice, fresh oranges, and wiping cloth samples, while serotype Mexico was found on fresh oranges and in wiping cloths and serotypes Muenchen and Panama were found only in wiping cloth samples. Regarding Shigella species, Shigella sonnei was found in all three types of sample tested; Shigella dysenteriae was found in juice and orange samples, Shigella boydii in orange and wiping cloth samples, and Shigella flexneri on oranges only. Thirty-one percent and 39% of the juice samples showed aerobic plate counts of > or = 5.0 log CFU/ml and Escherichia coli counts of > 3.0 log CFU/ml, respectively. These high counts may indicate poor sanitation and potential exposure to fecal contamination either in the raw materials or during the orange-crushing and juice-serving process. These data may be useful for a further risk assessment of Salmonella or Shigella in unpasteurized, freshly squeezed juice.

Mutational analysis of K-ras and Ras protein expression in larynx squamous cell carcinoma.

The ras gene family (H, K and N-ras) encodes the Ras protein, a GTPase-activating protein that regulates several signal transduction pathways including cellular proliferation and differentiation. Mutations in codons 12, 13 and 61 of the ras genes constitute one of the most frequent alterations in human cancer. In the Western Hemisphere, a low frequency of mutations in these genes has been observed in head and neck carcinomas; a higher frequency has been found in countries such as India and Taiwan. Increased protein expression is a relatively frequent event in larynx carcinomas. This study was aimed to evaluate the participation of the k-ras gene and Ras expression in 20 Mexican patients with larynx squamous carcinoma, 2 with dysplasia and 4 with normal mucosa. Samples (of 26 patients) were embedded in paraffin and immunohistochemical analysis was performed for the Ras protein, as well as amplification of the k-ras gene exon 1 (108 bp) by laser capture microdissection. Then, DNA extraction, PCR and sequencing were performed looking for possible mutation in codons 12 and 13. All patients with larynx carcinoma were men, median age 62 years. Eighty-five percent of the patients had risk factors such as smoking and/or alcohol consumption, 25% were in clinical stages I and II, and 75% in stages III and IV; 45% of the patients presented tumor recurrence or persistence. In this study, no mutations were found in codons 12 or 13 of the k-ras gene; however, protein expression was observed in 95% of the samples and a higher expression of the protein was associated with tumor recurrence or persistence, although this was not statistically significant. Unexpectedly, well-differentiated carcinomas and dysplasias presented an increase in protein expression. These results suggest that ras may be involved in early stages of larynx carcinogenesis and may be activated by other mechanisms different from mutations, such as epigenetic events.

Fetal-maternal immune interaction: blocking antibody and survival of the fetus.

In the late 1940s it became clear that the homograft reaction was essentially the result of an immune response. Subsequently, Medawar commented on the apparent paradox of the survival of the mammalian fetus in the face of such a potential (cell-mediated) immune response. In an outbred population the fetal-placental unit will be antigenically different to the mother by virtue of its complement of paternal genes and additionally there may be developmental or stage-specific gene products that are immunogenic. Many mechanisms have been proposed to account for the survival of the fetus in the face of a potential immune attack and, while many of these have been investigated in considerable detail, there has been no clear-cut indication that any one plays a predominant role. Either control of immune rejection of the fetus is exercised by an as yet undiscovered mechanism or, more probably, by a combination of some or all of the mechanisms that have been proposed by many workers over the last three decades. Potential controlling processes, which will be reviewed briefly, include: systemic and local modification of maternal responsiveness; altered expression of MHC antigens on extra-embryonic tissues; the placenta as a barrier; and blocking antibody responses. We discuss some of our recent studies in which we have started to look for potential blocking antibodies in a mouse model system. Cells secreting immunoglobulins M and G, characterized in hemolytic plaque assays, have been mapped to areas close to the midgestation mouse embryo, using an immunocryohistological technique. A scaled-down version of hybridoma technology has been used as an analytical probe of the specificity and isotype of immunoglobulin secreted by cells originating either from close to the embryo/fetus or from the para-aortic lymph nodes (PALN). So far monoclonal (IgG1) antibodies with specificity for embryonic cells have been derived together with some monoclonal immunoglobulins with as yet uncharacterized antibody specificity.

Biosynthesis of nitric oxide and citrulline from L-arginine by constitutive nitric oxide synthase present in rabbit corpus cavernosum.

The objective of this study was to determine whether a constitutive isoform of nitric oxide (NO) synthase is present in rabbit corpus cavernosum that could account for the involvement of the L-arginine-NO pathway in neurogenically-elicited relaxation of the corpus cavernosum and, therefore, penile erection. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NO(x) (NO+nitrite [NO2-]+nitrate [NO3-]) by chemiluminescence after reduction of NO(x) to NO by acidic vanadium (III). Equimolar quantities of NO plus citrulline were generated from L-arginine and the formation of both products was time-dependent at 37 degrees C. NO synthase activity was distributed almost entirely to the cytosolic fraction. Enzymatic activity was completely dependent on NADPH, calmodulin, and calcium. Addition of tetrahydrobiopterin increased NO synthase activity by about 30 percent. The NO synthase inhibitor NG-nitro-L-arginine, abolished enzymatic activity. The Km for L-arginine was 17 microM and the Vmax of the reaction was 18 pmol/min/mg protein. These observations indicate that a cytosolic, constitutive isoform of NO synthase, like that found in brain neuronal tissue, is present in rabbit corpus cavernosum.

Nitric oxide synthase from cerebellum catalyzes the formation of equimolar quantities of nitric oxide and citrulline from L-arginine.

This study examined whether constitutive nitric oxide (NO) synthase from rat cerebellum catalyzes the formation of equimolar amounts of NO plus citrulline from L-arginine under various conditions. Citrulline was determined by monitoring the formation of 3H-citrulline from 3H-L-arginine. NO was determined by monitoring the formation of total NOx (NO+nitrite [NO2-] + nitrate [NO3-]) by chemiluminescence after reduction of NOx to NO by acidic vanadium (III). Equal quantities of NO plus citrulline were generated from L-arginine and the formation of both products was linear for about 20 min at 37 degrees C provided L-arginine was present in excess to maintain a zero order reaction rate. Deletion of NADPH, addition of the calmodulin antagonist calmidazolium, or addition of NO synthase inhibitors (NG-methyl-L-arginine, NG-amino-L-arginine) abolished or markedly inhibited the formation of both NO and citrulline. The Km for L-arginine (14 microM; 18 microM) and the Vmax of the reaction (0.74 nmol/min/mg protein; 0.67 nmol/min/mg protein) were the same whether NO or citrulline formation, respectively, was monitored. These observations indicate clearly that NO and citrulline are formed in equimolar quantities from L-arginine by the constitutive isoform of NO synthase from rat cerebellum.

The use of microwave tissue fixation to demonstrate the in vivo phosphorylation of an acidic 80,000 molecular weight protein in the rat neocortex following treatment with soman.

Studies were conducted to determine if soman, a cholinesterase inhibitor, could activate the protein kinase C system in the rat neocortex. Using microwave radiation for rapid tissue fixation, it was demonstrated that treatment with soman increased 32P incorporation into an acidic 80,000 molecular weight, heat-stable protein in vivo. Based on relative molecular weight and isoelectric point this protein appears to be identical to a protein identified as a substrate for protein kinase C. Additionally, a protein of the same molecular weight and isoelectric point could be phosphorylated in tissue slices prepared from the neocortex by cholinergic dependent mechanisms. Also, treatment with soman decreased protein kinase C in the soluble fraction of this brain region; however, no corresponding increase was observed in the particulate fraction. These results suggest that soman can activate protein kinase C in vivo, and demonstrate the utility of using microwave tissue fixation to study protein phosphorylation events in vivo.