Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos

Abstract This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Resumo Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P 0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P 0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P 0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P 0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Use of melatonin as an inhibitor of apoptotic process for cryopreservation of zebrafish (Danio rerio) embryos / Uso da melatonina como inibidor do processo apoptotico para a criopreservação de embriões de zebrafish (Danio rerio)

This study investigated the use of melatonin to arrest the effects of apoptosis in vitrified zebrafish (D. rerio) embryos. Dechorionated embryos at 22-24 somite-stage were divided (n = 60/treatment) into a non-vitrified (Control Group, 0 M melatonin) and vitrified treatments with 0 M (T1), 1 µM (T2) and 1 mM of melatonin (T3). For vitrified treatments, a solution methanol/propylene glycol based was used and the embryos stored in -196 °C for a week. After thaw, survival rate, scanning electron microscopy, expression of anti (bcl-2) and pro-apoptotic (bax/caspase-3) genes, reactive oxygen species (ROS) formation and DNA fragmentation analyses were performed. No live embryos were obtained from vitrified treatments, observing a rapid degeneration immediately after thawing, with the vitelline layer rupture and leakage of its content, followed by breakdown of epithelial cells and melanisation of the tissue. Regarding the apoptotic process, T3 had the highest relative gene expression, for the three genes (P < 0.05) furthermore, T2 had similar expression of pro-apoptotic genes to CG (P < 0.05). ROS formation revealed that CG presented lower percentage of embryo surface area affected (3.80 ± 0.40%) (P < 0.05), in contrast, no differences were found among the other groups. T1 was most significantly (P < 0.05) damaged by DNA fragmentation. The vitrified groups with melatonin had similar damage levels of CG (P > 0.05). The inclusion of 1 µM of melatonin in the vitrifying solution, countered the effects of apoptotic process in post-thaw embryos, suggesting its utility in cryopreserving fish embryos.

Este estudo investigou o uso da melatonina para conter os efeitos da apoptose em embriões vitrificados de zebrafish (D. rerio). Embriões descorionados no estágio de 22-24 somitos foram divididos (n = 60 / tratamento) em tratamento não vitrificado (Grupo Controle, melatonina 0 M) e tratamentos vitrificados com 0 M (T1), 1 µM (T2) e 1 mM de melatonina (T3). Para os tratamentos vitrificados, utilizou-se uma solução à base de metanol/propilenoglicol e os embriões foram armazenados em -196 °C por uma semana. Após o descongelamento, foram realizadas análises de taxa de sobrevivência, microscopia eletrônica de varredura, expressão dos genes anti (bcl-2) e pró-apoptóticos (bax/caspase-3), formação de espécies reativas de oxigênio (EROS) e análises de fragmentação de DNA. Não foram obtidos embriões vivos a partir dos tratamentos vitrificados, observando uma rápida degeneração imediatamente após o descongelamento, com ruptura da camada vitelina e vazamento de seu conteúdo, seguida de quebra das células epiteliais e melanização do tecido. Em relação ao processo apoptótico. T3 apresentou expressão gênica relativa alta para os três genes (P <0,05), além disso, T2 apresentou expressão semelhante as dos genes pró-apoptóticos de GC (P <0,05). A formação de EROS revelou que GC apresentou menor percentual de área de superfície embrionária afetada (3,80 ± 0,40%) (P <0,05), ao contrário, não foram encontradas diferenças entre os outros grupos. T1 foi mais significativamente (P <0,05) danificado pela fragmentação do DNA. Os grupos vitrificados com melatonina apresentaram níveis de dano semelhantes ao do GC (P> 0,05). A inclusão de 1 µM de melatonina na solução de vitrificação, contrariou os efeitos do processo apoptótico em embriões pós-descongelamento, sugerindo sua utilidade na criopreservação de embriões de peixes.

Penicillin-Induced Stevens–Johnson Syndrome: A Case Report.

Stevens–Johnson syndrome (SJS) is one of the manifestations of severe form of cutaneous adverse drug reactions (CADRs). Penicillin group of antibiotics is well-known to cause the CADRs. Few cases of amoxicillin and only one case of dicloxacillininduced SJS have been reported. To the best of our literature search, we have not come across a single case of amoxicillin– dicloxacillin-induced SJS. Here, we report a case of amoxicillin–dicloxacillin-induced SJS in a 28-year-old female patient. The rationality of amoxicillin–dicloxacillin fixed drug combination is doubtful. Hence, prescribing rational drug therapy and promptly reporting the adverse drug reactions is essential so that noncompliance to treatment with resultant therapeutic failure and augmented antimicrobial resistance can be avoided.

Cutaneous Drug Eruption Caused by Amoxicillin: A Case Report.

Amoxicillin is a commonly used antimicrobial agent to combat various infections. Penicillin group of drugs are known to cause cutaneous drug eruptions especially in pediatric population. Most of the time, these eruptions are mild in nature, however, sometimes they represent the early manifestation of rare, severe drug-induced cutaneous reactions, such as Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). Here, we report a case of maculopapular, erythematous rash induced by amoxicillin in an 18-year-old male patient. The case is being reported to emphasize the need for efficient pharmacovigilance in order to motivate adverse drug reaction reporting so as to gather more and more data regarding adverse drug reactions.

Lepromatous leprosy of histoid type.

Histoid leprosy is a variant of lepromatous leprosy, which develops as a result of resistance to dapsone monotherapy. Here we report two cases of lepromatous leprosy of histoid type, one with typical and another with atypical presentations.

Distribution and in vitro antimicrobial susceptibility of Acinetobacter species on the skin of healthy humans.

BACKGROUND: Acinetobacter spp. are ubiquitous in the environment and have emerged as important nosocomial pathogens. The distribution of Acinetobacter spp. In some temperate European countries has been reported. However, similar data from a tropical country such as India are not available. METHODS: Six body sites (antecubital fossa, axilla, forehead with hairline, neck, outer surface of nose and toe webs) from men and women volunteers were sampled with saline-soaked cotton swabs enriched in Baumann's enrichment medium. The isolates were identified to the genus level by chromosomal DNA transformation assay and to the species level by a 16-test biochemical system. The minimum inhibitory concentration for 39 antibiotics was determined by the two-fold agar dilution method. RESULTS: Seven genospecies of Acinetobacter were found at 6 body sites on healthy human skin. Acinetobacter lwoffii was the most dominant comprising 40% of the total number of isolates, followed by A. junii (35%) and A. haemolyticus (16.5%). The antecubital fossa had the highest colonization frequency (48.5%). The overall positivity rate of samples was higher from women (26.3%) compared to men volunteers (25%). Only two Acinetobacter genospecies 1-3 isolates were isolated while no A. radioresistens were isolated. Susceptibility testing revealed no major differences among the 7 Acinetobacter spp. tested. Fluoroquinolones were the most active, while low-to-intermediate resistance was exhibited towards beta-lactams and aminoglycosides. Acinetobacter spp. isolated from the skin showed susceptibility to commonly used antibiotics. CONCLUSION: Seven Acinetobacter genospecies were isolated from 6 different body sites from the skin of healthy human volunteers. Acinetobacter lwoffii was the dominant isolate. The rate of skin carriage was higher in men than in women and the maximally colonized site was the antecubital fossa. All the genospecies displayed susceptibility to most of the commonly used antimicrobials.

Prevalence of rheumatic diseases in a rural population in western India: a WHO-ILAR COPCORD Study.

BACKGROUND: COPCORD (Community oriented program from control of rheumatic diseases) is a global initiative of the WHO/International League of Associations from Rheumatology (ILAR). The prevalence data from the first Indian COPCORD survey (Stage 1), carried out in village Bhigwan (Dist. Pune), in 1996, is presented. AIM: To study the rural prevalence of rheumatic-musculoskeletal symptoms/diseases (RMSD). METHODS: A cross-sectional survey of the village (non-randomised selection) was completed in five weeks, using validated questionnaires, served by 21 trained volunteers. 746 patients (18.2%, 95% CI: 17-1-19-4) were identified (Phase 1) from 4092 adults (response 89%), and systematically evaluated (Phase 2 and 3) by a medical team, including a rheumatologist; limited investigations were carried out and diagnosis confirmed during a planned 12 week initial follow-up. Standard clinical criteria were used for the diagnosis; point prevalence estimates (prev)/confidence interval (CI) are shown in parenthesis. RESULTS: There was a dominant distribution of 'pain at all sites' (articular/soft tissues) in the females; painful neck (9.5%), back (17.3%), and calf (8.5%) appeared significant when compared to the Bhigwan males and the Indonesian and the Chinese rural COPCORD results. 55% RMSD were due to soft tissue rheumatism (5.5%) and an ill-defined/unclassifiable symptom-related-diagnosis (7.1%). Osteoarthritis (5.8%) and inflammatory arthritis (IA) were seen in 29% and 10% patients respectively. 240 patients (5.9%) with chronic knee pains did not show any clinical evidence of OA. The prev of rheumatoid arthritis (0.5%, 95% CI: 0.3-0.7), as classified by the American College of Rheumatology, was the highest ever reported from an Asian rural COPCORD study. Though unclassifiable IA (0.9%, 95% CI: 0.6-1.1) was seen, well defined reactive arthritis, TB, leprosy and connective tissue disorders were not observed. Gout was diagnosed in five patients (0.12%). CONCLUSIONS AND DISCUSSION: The Bhigwan COPCORD survey demonstrates a significant rural spectrum of RMSD. It provides a reasonable speculation about the Indian rheumatological burden. Further, an eight year prospective study is in progress to identify new cases and risk factors, and educate people (Stages 2 and 3).

Transferrin receptor expression by blast cells in acute lymphoblastic leukemia correlates with white cell count & immunophenotype.

Transferrin receptor (TR) expression by blast cells in 127 cases of acute lymphoblastic leukemia (ALL) at presentation and 19 cases at relapse was examined using three anti-TR monoclonal antibodies to find its correlation with prognostic features such as the total leucocyte count (TLC), the morphology of blast cells and their cytochemical and immunophenotypic properties, as well as age and sex of the patients. Blasts in 62 per cent of thymic (T) ALL cases at presentation showed significant TR expression as compared to only 10.9 per cent in common ALL (CALL) (P < 0.001). This differential expression of TR was also observed among cases with > 50 x 10(9)/l TLC, while in cases with < 50 x 10(9)/ l TLC no such pattern was observed (30% TR positivity in T-ALL vs 20% TR positivity in non-T-ALL). Furthermore, the percentage of TR positive blasts was significantly higher (P < 0.005) in cases with > 50 x 10(9)/l TLC as compared to those with < 50 x 10(9)/l (48.3-54.4% vs 24.9-28.8%). In contrast to CALL cases at presentation, those at relapse showed a very high TR positivity (54-66%), similar to the T-ALL cases (53-84%). This suggests a high proliferative rate of blast cells in ALL at relapse, irrespective of its immunophenotype. There was no correlation of TR expression with blast cell morphology (FAB L1 vs L2), their cytochemical properties and sex of the patients. However, a significantly higher incidence of TR positivity was observed in patients above 10 yr of age compared to those below 10 yr (47% vs 15%; P < 0.001). The incidence of T-ALL was also significantly higher in the former group (56%) compared to the latter (33%) (P < 0.005). Our data suggest that by virtue of its association with features of poor prognosis, e.g., age above 10 yr, expression of thymic markers, high leucocyte count and disease relapse, TR expression by blast cells in ALL could serve as a biological marker of poor prognosis.

Antibody dependent cell mediated cytotoxicity and erythrophagocytosis assays in Rh haemolytic disease of the newborn.

OBJECTIVE: To evaluate the role of antibody dependent cell mediated cytotoxicity (ADCC) and erythrophagocytosis in comparison to IgG subtypes and concentration of anti-D in haemolytic disease of the newborn (HDN). DESIGN, SETTING AND PATIENTS: One hundred Rh (D) immunised women attending an antenatal clinic of Nowrosjee Wadia Maternity Hospital at 32 to 34 weeks of gestation. Results of 90 women having Rh (D) positive infants were correlated with severity based on outcome of pregnancy, cord blood Hb and treatment given to infant. MEASUREMENTS: Immunoradiometric assay (IRMA) was used for anti-D quantitation. In ADCC assay 51Cr release in the culture supernatant was measured, and in the phagocytosis assay lytic activity was measured on spectrophotometer. Cord blood monocytes were used as effector cells in both the assays. RESULTS: Good correlation (P < 0.01) was observed between ADCC vs IRMA and ADCC vs phagocytosis. ADCC% specific lysis was significantly higher and still-birth rate was increased when mothers had IgG1 + IgG3 type of anti-D. Though all variables showed significant correlation (P < 0.01) with severity, ADCC assay was the most predictive. Cord blood haemoglobin showed a significant inverse correlation with ADCC and phagocytosis assays. CONCLUSION: In the absence of a cordocentesis facility severity of Rh HDN could be reliably judged by ADCC assay. If an isotope laboratory is not available, then instead of ADCC, phagocytosis assay may be employed.

Role of antibody dependent cell mediated cytotoxicity in ABO hemolytic disease of the newborn.

Study includes fifty O blood group mothers delivering A or B group infants suffering from jaundice and a control group consisting of thirty one O group mothers of non-jaundiced infants. Lytic ability of maternal IgG anti-A/anti-B was determined by 51Cr ADCC assay in which cord blood monocytes were used as effector cells. In control series mean% specific lysis (SL) was 18 +/- 3.1 for IgG anti-A and 17.9 +/- 3.1 for IgG anti-B. In jaundiced series IgG anti-A was more lytic than IgG anti-B. However, the increase in ADCC lysis was statistically insignificant. Even though > 1:32 titre was more often associated with > 35% SL, in general the immune A/B antibody titre showed poor correlation with ADCC lysis. Majority of the severe ABO-HDN cases had > 35% ADCC lysis.

Air pollution related respiratory morbidity in central and north-eastern Bombay.

A study of 4 comparable communities in central & northeastern Bombay (2 each) among randomly matched 349 subjects in 1988-9, along with ambient sulfur dioxide (SO2), Nitrogen dioxide (NO2) & suspended particulate matter (SPM) air monitoring was carried out. The levels in winter were higher particularly for SO2 in Parel (upto 584 micrograms) in Maravali; Deonar showed lower pollution. There were inter-area differences for housing, income, residential history but age-sex differences were small; these were reduced by matching. Clinical respiratory symptoms were higher in Parel & Maravali (cough 12% and 11.2%, dyspnoea 17% & 13.3% respectively). Cardiac problems are commoner in Parel (11.0%). Smoker had cough more often but not dyspnoea. Maravali had a high prevalence for headache and eye irritation (9.5%). Those using kerosene suffered more than those using gas (22.2% as compared to 9.2%) Lung functions (FVC, FEVI) were lowest in Parel for males and in Maravali for females. Expiratory flow rates were lower at Dadar and then at Maravali. Despite lower SO2 pollution, Maravali residents suffered equally as in Parel. This may be due to added effect of diesel exhausts (NO2, SPM) or other unmeasured chemicals.