Therapeutic tools for familial ALS.

Familial ALS (FALS) accounts for 10 to 15% of ALS cases. In more than 70% of FALS patients, a causal gene is identified and animal models have been developed for a subset of them, mainly for the most frequently mutated genes. Therapeutic tools to treat those patients are dominated by gene-specific therapy and the most advanced approaches target the SOD1 gene mutations. Either by direct delivery of antisense oligonucleotides (ASO) or using viral vectors such as adenoviruses (AAV) to deliver ASOs, gene specific therapies have shown promising results in animal models. The recent use of subpial injections of AAV9+anti SOD1 ASO now shows that the disease is completely prevented or stopped in the animal, depending on the moment of injection, e.g., before or after disease onset. However, the use of viral vectors in humans seems to be limited at least by their immunogenicity. Antibody-based therapies are also efficient to treat animal models, but to a lesser extent. Most of the experiments targeted the SOD1 protein in its misfolded conformation. This approach seems better tolerated than the AAV one, an important limit being the choice of the epitope. Unexpectedly, some advances in treating the C9ORF72 animal model have been obtained using a modulation of microbiota, and this strategy has the great advantage to have an easy route of administration and a good safety profile. The landscape of experimental FALS treatment is rapidly evolving and results are promising. This is an important unmet need for ALS patients and several human phase I, II and III trials are ongoing.

Development of quantitative RNA biomarkers for detecting dioxin-like and estrogenic pollutants in Costa Rican native fish species.

Monitoring the environmental impact on native species is crucial for the correct management of tropical ecosystems. The Costa Rican fish Parachromis dovii (Cichlidae) and Poecilia gillii (Poecillidae) were used as sentinel species for freshwater bodies under considerable pressure by intensive agriculture Cichlidae development. Suitable qRT-PCR probes for the quantification of hepatic mRNA levels of two stress-related genes--vitellogen in (estrogenic effects) and cytochrome P4501A(CYP1A, dioxin-like compounds)--for both species were designed and validated in experimental treatments with model effectors (17beta-estradiol and beta-naphtoflavone, respectively), demonstrating their usefulness as markers of exposure to these two kinds of pollutants. Analysis of fish transplanted across pesticide contaminated sites near Palo Verde National Park, Pacific Coast of Costa Rica did show significant changes on hepatic Cyp1A in both species. In P. dovii, Cyp1A levels were enhanced in Barbubal and in the impacted Cabuyo sites in the rainy season whereas in P. gillii fish Cyp1A transcripts were down-regulated differently across rainy and dry seasons. Vitellogen in mRNA levels in P. gillii varied between males and females with males showing always low values which indicated no estrogenic effects. Within females, vitellogenin levels varied over 100,000 fold depending on their maturation stage, further demonstrating the ability of the method to monitor changes (natural or induced) in the reproductive system of the fish.

Pesticide residue analyses and biomarker responses of native Costa Rican fish of the Poeciliidae and Cichlidae families to assess environmental impacts of pesticides in Palo Verde National Park.

Pesticide chemical residues in water samples and biomarker responses in transplanted fish were used to monitor environmental hazards of pesticides in Palo Verde National Park (Costa Rica). The Costarican fish, Parachromis dovii (Ciclhidae) and Poecilia gillii (Poecillidae), were selected as sentinel species. Contaminant analyses detected up to 15 different pesticide residues in water with hexachlobenzene (2261 ng l(-1)), phorate (473 ng l(-1)), epoxiconazole (314) and bromacil (117 ng l(-1)) being the compounds found in higher concentrations. Biomarker responses evidenced impacts on cholinesterase activities in transplanted fish at Barbudal site probably due to the presence of organophosphate insecticides such as phorate. High enzyme activities of glutathione S-transferase and catalase and elevated levels of lipid peroxides were also observed at a site impacted by rice fields (Cabuyo); those effects could be associated with the presence of hexachloro benzene and triazole fungicides. In general, P. dovii biomarkers were affected to a greater extent than those of P. gillii in fish transplanted to sites associated with agriculture, which suggests the former species is a good candidate for future surveys.

Development of therapeutic and prophylactic vaccine against Tuberculosis using monkey and transgenic mice models.

PURPOSE: BCG is not efficacious against M. tuberculosis (TB) in adult. Therefore, novel TB vaccines were established by using three kinds of animal models (cynomolgus monkey model which is the best animal model of human TB, IL-2R knock out SCID mice as a human immune model, and granulysin transgenic mouse). METHODS AND RESULTS: DNA vaccine expressing TB Hsp65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. The BCG prime followed by Hsp65+IL-12/HVJ vaccine boost showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). In contrast, 33% of monkeys were alive in BCG alone group. Furthermore, the prolongation of survival period of the monkey was observed by the combination of BCG and DNA vaccine even when the boost was performed after long-term period (4month) from prime. This combination also improved the erythrocyte sedimentation rate (ESR), increased the body weight, and augmented the proliferation of PBL and IL-12 production at higher levels than BCG alone or saline. Furthermore, this vaccine exerted therapeutic efficacy in IL-2R knock out SCID-PBL/hu mice, which were transplanted with human T cells. Granulysin is an important defensive molecule expressed by human T cells and NK cells and has a cytolytic activity against microbes including Mycobacterium tuberculosis (TB) and tumors. Expression of 15kD (15K) granulysin protein and mRNA in CD8 positive T cells in the patients infected with drug sensitive (TB) or multi-drug resistant (MDR-TB) M. tuberculosis were lower than that in the healthy volunteers, suggesting that granulysin treatment might improve the tuberculous disease in human. Therefore, we established two kinds of granulysin transgenic mice (15K granulysin transgenic mice and 9K granulysin transgenic mice). It was demonstrated that 15K granulysin transgenic mice as well as 9K granulysin transgenic mice exerted in vivo anti-TB effect, including the decrease of the number of TB and augmentation of the CTL activity. These are the first findings which demonstrate in vivo effects of 15K granulysin and 9K granulysin against TB infection. Moreover, DNA vaccine expressing 15K granulysin showed a therapeutic activity against TB in mice. CONCLUSION: These data indicate that monkey, IL-2R gene-knock out SCID-PBL/hu and granulysin transgenic mice models provide useful tools for the development of novel vaccines (HVJ-Envelope/Hsp65 DNA + IL-12 DNA vaccine and granulysin vaccine) against TB.

Novel therapeutic vaccine: granulysin and new DNA vaccine against Tuberculosis.

PURPOSE: Multi-drug resistant (MDR) Mycobacterium Tuberculosis (M.TB) is a big problem in the world. We have developed novel TB therapeutic vaccines. METHODS AND RESULTS: DNA vaccine expressing mycobacterial heat shock protein 65 and IL-12 was delivered by the hemagglutinating virus of Japan (HVJ)-envelope. M. TB, MDR-TB or extremenly drug resistant (XDR-TB) was injected i.v. into DBA/1 mice, and treated with the vaccine three times. This HVJ-E/Hsp65DNA+IL-12DNA vaccine provided strong therapeutic efficacy against MDR-TB and XDR-TB (prolongation of survival time and the decrease in the number of TB) in mice. Therapeutic effect of this vaccine on TB infection was also demonstrated in chronic TB infection murine model using aerosol infection intratracheally. On the other hand, granulysin protein produced from CTL has lethal activity against TB. Granulysin protein vaccine also exerted strong therapeutic effect. Furthermore, we extended our studies to monkey model, which is currently the best animal model of human TB. Hsp65DNA+IL-12 DNA vaccine exerted strong therapeutic efficacy (100% survival and augmentation of immune responses) in the TB-infected monkeys. In contrast, the survival of the saline control group was 60% at 16 week post-challenge. HVJ-Envelope/HSP65 DNA+IL-12 DNA vaccine increased the body weight of TB-infected monkeys, improved the erythrocyte sedimentation rate, and augmentated the immune responses (proliferation of PBL and IL-2 production). The enhancement of IL-2 production from monkeys treated with this vaccine was correlated with the therapeutic efficacy of the vaccine. CONCLUSION: These data indicate that novel vaccines might be useful against TB including XDR-TB and MDR-TB for human therapeutic clinical trials.

Fluorescence of 2-aminopurine reveals rapid conformational changes in the RB69 DNA polymerase-primer/template complexes upon binding and incorporation of matched deoxynucleoside triphosphates.

We have used 2-aminopurine (2AP) as a fluorescent probe in the template strand of a 13/20mer primer/template (D) to detect deoxynucleoside triphosphates (N)-dependent conformational changes exhibited by RB69 DNA polymerase (ED) complexes. The rates and amplitudes of fluorescence quenching depend hyperbolically on the [dTTP] when a dideoxy-primer/template (ddP/T) with 2AP as the templating base (n position) is used. No detectable fluorescence changes occur when a ddP/T with 2AP positioned 5' to the templating base (n + 1 position) is used. With a deoxy-primer/template (dP/T) with 2AP in the n position, a rapid fluorescence quenching occurs within 2 ms, followed by a second, slower fluorescence quenching with a rate constant similar to base incorporation as determined by chemical quench. With a dP/T having 2AP in the n + 1 position, there is a [dNTP]-dependent fluorescence enhancement that occurs at a rate comparable to dNMP incorporation. Collectively, the results favor a minimal kinetic scheme in which population of two distinct biochemical states of the ternary EDN complex precedes the nucleotidyl transfer reaction. Observed differences between dP/T and ddP/T ternary complexes indicate that the 3' hydroxyl group of the primer plays a critical role in determining the rate constants of transitions that lead to strong deoxynucleoside triphosphate binding prior to chemistry.

Influence of a medium-impact aquaerobic program on health-related quality of life and fitness level in healthy adult females.

AIM: The aim of the study was to examine the influence of a medium-impact aquaerobic program on the health-related quality of life (HRQoL) and health-related fitness (HRF) level of middle-aged healthy female subjects. METHODS: Twenty apparently healthy women (mean age: 43.1 [standard deviation: 9.7] years) participated in the study. Criteria for inclusion were absence of diagnosed illnesses, as well and signs and symptoms of disease as evaluated by the Physical Activity Readiness Questionnaire. Participants carried out a medium-impact aquaerobic exercise program consisting of 2 weekly sessions of 60 min during 8 months. Before and after the exercise program, HRQoL was assessed by the 36-item short form health survey (SF-36) questionnaire, and HRF was measured using a simplified version of the AFISAL-INEFC HRF test battery. RESULTS: Following the exercise program, an increase in all domains of HRQoL, except general health and role-emotional, was observed. Total body mass and body fat percentage decreased, and estimated aerobic power increased. CONCLUSION: Completion of a medium-impact aquaerobic program (2 weekly sessions of 60 min during 8 months) improves HRQoL in most domains, particularly bodily pain and vitality, and shows to be among the most effective programs for improving perceived quality of life. Moreover, this exercise program proved to have a positive influence on the body composition and functional capacity of the subjects, being effective in reducing fat body mass and improving cardiorespiratory function.

Antimicrobial Resistance Genes in Pigeons from Public Parks in Costa Rica.

Antimicrobial resistance is known to be an emerging problem, but the extent of the issue remains incomplete. The aim of this study was to determine the presence or absence of nine resistance genes (blaTEM , catI, mecA, qnrS, sulI, sulII, tet(A), tet(Q), vanA) in the faeces of 141 pigeons from four urban parks in Alajuela, Guadalupe, Tres Ríos and San José in Costa Rica. The genes were identified by real-time PCR directly from enema samples. About 30% of the samples were positive for genes catI and sulI; between 13% and 17% were positive for qnrS, sulII, tet(A) and tet(Q); and 4% were positive for blaTEM . The mecA and vanA genes were not detected. The average of antimicrobial resistance genes detected per pigeon was 2. Eight different patterns of resistance were identified, without differences in the sampling areas, being the most common pattern 2 (sulII positive samples). During rainy season, the genes more frequently found were sulI and tet(A). In conclusion, the urban inhabiting pigeons tested are currently carrying antimicrobial resistance genes, potentially acting as reservoirs of resistant bacteria and vectors to humans. To the authors' knowledge, this is the first study carried out on direct detection of resistance genes in the digestive metagenomes of pigeons.

Polymerization and structure of nucleotide-free actin filaments.

Two factors have limited studies of the properties of nucleotide-free actin (NFA). First, actin lacking bound nucleotide denatures rapidly without stabilizing agents such as sucrose; and second, without denaturants such as urea, it is difficult to remove all of the bound nucleotide. We used apyrase, EDTA and Dowex-1 to prepare actin that is stable in sucrose and approximately 99 % free of bound nucleotide. In high concentrations of sucrose where NFA is stable, it polymerizes more favorably with a lag phase shorter than ATP-actin and a critical concentration close to zero. NFA filaments are stable, but depolymerize at low sucrose concentrations due to denaturation of subunits when they dissociate from filament ends. By electron microscopy of negatively stained specimens, NFA forms long filaments with a persistence length 1.5 times greater than ADP-actin filaments. Three-dimensional helical reconstructions of NFA and ADP-actin filaments at 2.5 nm resolution reveal similar intersubunit contacts along the two long-pitch helical strands but statistically significant less mass density between the two strands of NFA filaments. When compared with ADP-actin filaments, the major difference peak of NFA filaments is near, but does not coincide with, the vacated nucleotide binding site. The empty nucleotide binding site in these NFA filaments is not accessible to free nucleotide in the solution. The affinity of NFA filaments for rhodamine phalloidin is lower than that of native actin filaments, due to a lower association rate. This work confirms that bound nucleotide is not essential for actin polymerization, so the main functions of the nucleotide are to stabilize monomers, modulate the mechanical and dynamic properties of filaments through ATP hydrolysis and phosphate release, and to provide an internal timer for the age of the filament.

Adenosquamous carcinoma of the duodenum. An immunohistochemical study.

A rare adenosquamous carcinoma of the duodenum occurred in an 84-year-old woman. Histologically the neoplasm showed evidence of glandular and squamous differentiation, and both components showed malignant characteristics. Intermediate elements sharing squamous and glandular features, were found and were immunoreactive for carcinoembryonic antigen, epithelial membrane antigen and cytokeratins of both low and high molecular weight. A review of the literature and discussion about the histogenesis is included.

Evaluation of Mycobacterium leprae antigens in the monitoring of a dapsone-based chemotherapy of previously untreated lepromatous patients in Cebu, Philippines.

Thirty-five previously untreated lepromatous patients receiving dapsone-based therapy were monitored throughout their 5-year period of treatment by serology and by pathology. Sequentially collected sera were used to evaluate the usefulness of four Mycobacterium leprae antigens as used in ELISA to monitor the progress of their therapy. ELISA results were compared with each other and with bacterial load over the treatment period and with duration of treatment. The ELISAs, based on the measurement of IgM antibody reactivity to the two neoglycoproteins (NDO and NTO) representing the phenolic glycolipid antigen of M. leprae, were found to be the most effective in monitoring treatment. A whole M. leprae based ELISA was less efficient in monitoring treatment because it failed to measure antibodies in 8 out of 35 patients and because it provided consistently lower values than either NTO or NDO. The ELISA-inhibition test based on the detection of antibodies to a species-specific epitope on the 36 K antigen of M. leprae was less suitable because of persistent reactivity during therapy, consequently resulting in no significant correlation with ELISA reactivities to NTO or NDO.

Evaluation of Mycobacterium leprae antigens in the serological monitoring of a clofazimine-based chemotherapeutic study of dapsone resistant lepromatous leprosy patients in Cebu, Philippines.

Thirty-one dapsone resistant lepromatous leprosy patients receiving clofazimine based therapy were serologically monitored throughout their 5-year period of treatment. Sequentially collected sera were used to examine 4 Mycobacterium leprae antigens to evaluate their usefulness in ELISA's for monitoring the progress of their therapy. The ELISA results were compared with decline in bacterial load over the treatment period and with duration of treatment. In addition the ELISA's were compared with each other. The ELISA's based on the measurement of IgM antibodies to the two neoglycoproteins (NDO and NTO) representing the phenolic glycolipid antigen of M. leprae were found to be the most effective with regard to monitoring treatment. A whole M. leprae based ELISA was less efficient in monitoring treatment because it failed to measure antibodies in 5 out of 31 patients. The ELISA-inhibition test based on the detection of antibodies to a species-specific epitope on the 36 K antigen of M. leprae was less suitable because of persistent reactivity during therapy.

Hazardous pesticides in Central America.

Pesticides are an extensively documented occupational and environmental hazard in Central America. Yet, severe problems persist. Toxic pesticide use in the Region increased during 1985-1999. High exposure levels and ineffectiveness of personal protective equipment evidence the difficulties for risk reduction. Acute poisonings remain a severe problem. Delayed and/or long-lasting health effects include dermatoses, cancer, and genotoxic, neurotoxic, and respiratory effects. The use of hazardous pesticides persists through deficiencies in government-driven assessment and risk management; excessive focus on regional harmonization; short-term economic interests; strong links between industry and governments; aggressive marketing; weak trade unions; and failure of universities to reach decision makers. Regulation based on local data is lacking. An agreement of the Ministries of Health for restricting the most toxic pesticides in Central America has potential for progress. The most effective way to reduce risk is to greatly reduce pesticide use. Actions needed include development of multidisciplinary strategies for local studies on health and environmental impact of pesticides; development of sustainable nonchemical agricultural technologies; evaluation of interventions; extending and sharing of expertise within the Region; strengthening of unions and communities; and redefining the role of industry toward development of safer products, with responsible marketing and reliable information.

Rabdomiólisis por hipocalemia: manifestación atípica del síndrome de Conn / Rhabdomyolysis due to hypokalemia: an atypical manifestation of Conn's syndrome

Resumen La rabdomiólisis es una afección con espectro de manifestación amplio que puede cursar desde una enfermedad leve asintomática hasta complicaciones mortales por desequilibrio hidroelectrolítico, arritmias o lesión renal aguda. Se comunica el caso de una paciente de 35 años de edad, hipertensa, que ingresó por debilidad muscular posterior a un cuadro gastrointestinal. Tenía hipocalemia severa, elevación de creatincinasa, función renal conservada, hipocalcemia y alcalosis metabólica. Su evaluación integral culminó en el diagnóstico de hiperaldosteronismo primario secundario a un adenoma productor de aldosterona que fue removido de manera quirúrgica sin complicaciones. La manifestación del síndrome de Conn con rabdomiólisis por hipocalemia es excepcional porque la mayoría de los casos se diagnostican con normocalemia o hipocalemia leve a partir del protocolo de hipertensión secundaria. Es necesario un alto nivel de sospecha y evaluación integral para llegar al diagnóstico certero.

Abstract Rhabdomyolysis is a condition with a broad spectrum of presentation that can range from mild asymptomatic disease to fatal complications due to electrolyte imbalance, arrhythmias and/or acute renal injury. We report the case of a 35-year-old woman, hypertensive, who was admitted for muscle weakness following a gastrointestinal condition. Biochemically with severe hypokalemia, elevated creatinekinase, conserved renal function, hypocalcemia and metabolic alkalosis. Their comprehensive evaluation culminated in the diagnosis of primary hyperaldosteronism secondary to an aldosterone-producing adenoma which was surgically removed without complications. The presentation of Conn's syndrome with hypokalemia rhabdomyolysis is exceptional since most cases are diagnosed with normokalemia or mild hypokalemia from the secondary hypertension protocol. A high level of suspicion and integral evaluation are necessary to arrive at the correct diagnosis.

Environmental hazards of pesticides from pineapple crop production in the Río Jiménez watershed (Caribbean Coast, Costa Rica).

This study aimed to characterize environmental hazards of pesticides from pineapple production in riparian communities along the Jiménez River watershed. To achieve our objectives riparian ecological quality indices on riparian habitat and macroinvertebrate assemblages were combined with toxicity assays, fish biomarkers, physico-chemical water analysis and pesticide environmental hazards. During two consecutive years and two periods (July and October), three reference and four impacted sites were monitored. The ecological quality of benthic macroinvertebrates and of riparian habitats deteriorated from the reference sites downstream to the polluted reaches along the Jiménez River area affected by pineapple plantations. The toxicity of water to Daphnia magna also increased towards downstream reaches. Biomarkers of fish of the species Poecilia gillii and Bryconamericus scleroparius transplanted across the studied sites evidenced a clear anticholinergic effect towards downstream sites as well as increased levels of lipid peroxidation. Different pesticide residues were frequently detected in water samples collected across the Jiménez River watershed with herbicides (ametryn, bromacil, diuron), organophosphorus insecticides (diazinon and ethoprophos) and triazole fungicides being the greatest reaching levels above 1 µg L(-1) in downstream sites. Principal component and environmental hazard analysis of physico-chemical and biological responses established clear relationships among habitat deterioration and the ecological quality of macroinvertebrate communities, high levels of herbicides and poor plant growth, high levels of organophosphorus insecticides in water and anticholinesterase effects on fish, D. magna mortality and deterioration of macroinvertebrate communities. Fungicide and herbicide residue levels were also related with high levels of lipid peroxidation and high activities of glutathione S transferase in fish liver, respectively. These results indicated, thus, that riparian habitat deterioration due to deforestation and land use for agriculture and pesticide contamination are affecting river ecosystems.

Low-level subchronic arsenic exposure from prenatal developmental stages to adult life results in an impaired glucose homeostasis.

We evaluated how low-level (3 ppm) subchronic inorganic arsenic (iAs) exposure from prenatal developmental stages until adult life affects glucose homeostasis. Biochemical parameters of glucose and lipid metabolism, pancreatic insulin and glycosylated haemoglobin were determined in 4-month-old female offspring of adult Wistar rats. Pancreatic histology was also performed. Statistical comparisons between control and iAs-treated groups were performed by unpaired two-tailed Student's t-test. Statistical significance was set at p<0.05. We found that iAs treatment resulted in an impaired glucose tolerance test, suggestive of impaired glucose metabolism. This group was found to have hyperglycaemia and high levels of HOMA-IR, glycosylated haemoglobin, cholesterol and pancreatic insulin compared to control rats. However, plasma insulin, triglycerides and high-density lipoprotein cholesterol were not different from control rats. Moreover, ß-cell damage found in iAs-treated rats consisted of cells with a nucleus with dense chromatin and predominance of eosinophilic cytoplasm, as well as changes in the pancreatic vasculature. The current study provided evidence that subchronic iAs exposure at 3 ppm from prenatal developmental stages to adult life resulted in damage to pancreatic ß cells, affected insulin secretion and demonstrated altered glucose homeostasis, thus supporting a causal association between iAs exposure and diabetes.