Cryopreservation of Collared Peccary (Pecari tajacu Linnaeus, 1758) Somatic Cells is Improved by Sucrose and High Concentrations of Fetal Bovine Serum.

BACKGROUND: The formation of somatic cell banks is affected by, amongst other factors, the cryoprotectant solution used. The selection of an effective solution, therefore, is a primary parameter. OBJECTIVE: We optimized the cryoprotectant used for collared peccary somatic cell cryopreservation. MATERIALS AND METHODS: We categorized cells into different groups based on their cryopreservation and evaluated the morphology, viability, proliferative activity, metabolism, and oxidative stress. One group was cryopreserved in 10% DMSO with 10% fetal bovine serum (DMSO-10FBS), and another with 50% FBS (DMSO-50FBS). The cryopreservation of both groups included the presence of 0.2 M sucrose (DMSO-SUC-10FBS and DMSO-SUC-50FBS). Non-cryopreserved cells and cells cryopreserved with 10% DMSO (DMSO) supplemented with 0.2 M sucrose (DMSO-SUC) were used as controls. RESULTS: There was no difference observed in morphology or viability among the groups. Proliferative activity was reduced in DMSO-10FBS when compared to controls. Although cryopreservation reduced metabolism, no difference was observed among solutions. A lower level of reactive oxygen species was observed in cells of DMSO-SUC-50FBS when compared to other cryoprotectants. Only cells of DMSO-SUC-50FBS had mitochondrial potential similar to non-cryopreserved cells. CONCLUSION: 10% DMSO supplemented with 50% FBS and 0.2 M SUC was observed to be the most efficient cryoprotectant for preserving collared peccary somatic cells.

Caracterização histomorfológica do sistema tegumentar auricular de cateto - Pecari tajacu Linnaeus, 1758) / Histomorphological characterization of collared peccary (Pecari tajacu Linnaeus, 1758) ear integumentary system

A criopreservação de tecido somático derivado da pele de catetos consiste numa alternativa para a conservação da biodiversidade por meio da associação com a transferência nuclear. Nesse contexto, a manipulação de tecidos da pele é uma etapa crucial para o sucesso dessa biotécnica. Portanto, o objetivo do presente estudo, foi caracterizar o sistema tegumentar auricular periférico de catetos, visando aprimorar a conservação tecidual. Para tanto, fragmentos auriculares de oito animais foram avaliados quanto às camadas teciduais, aos componentes, à atividade proliferativa e à viabilidade metabólica, usando-se as colorações hematoxilina-eosina e tricrômico de Gomori, quantificação de AgNORs e microscopia eletrônica de transmissão. Assim, tamanhos de 104,2µm e 222,6µm foram observados para epiderme e derme, com uma proporção volumétrica de 36,6% e 58,7%, respectivamente. Além disso, na epiderme, foram evidenciadas as camadas basal (22,5µm), intermediárias (53,5µm) e córnea (28,2µm), com valores médios de 65,3 células epidermais, 43,4 melanócitos e 14,8 halos perinucleares. Já a derme apresentou 127 fibroblastos, com 2,5 AgNORs/nucléolo. Adicionalmente, a atividade metabólica foi de 0,243. Em conclusão, o sistema tegumentar auricular periférico de catetos possui algumas marcantes variações em relação a outros mamíferos, quanto ao número de camadas e espessura da epiderme, quantidade de células epidermais, melanócitos e parâmetros proliferativos.(AU)

The cryopreservation of somatic tissue derived from skin of collared peccaries is an alternative for biodiversity conservation through association with nuclear transfer. In this context, tissue manipulation of skin is a critical step for the success of this biotechnique. Therefore, the aim was to characterize the peripheral ear integumentary system derived from collared peccaries, directing to improve tissue conservation. Thus, ear fragments of eight animals were evaluated for tissue layers, components, proliferative activity and metabolic viability, using hematoxylin-eosin and Gomori Trichrome, AgNORs quantification and transmission electronic microscopy. Hence, sizes of 104.2 µm and 222.6 µm were observed in the epidermis and dermis, with a volumetric ratio of 36.6% and 58.7%, respectively. Moreover, basal layer (22.5 µm), intermediate (53.5 µm) and cornea (28.2 µm), with mean values of 65.3 epidermal cells, 43.4 melanocytes and 14.8 perinuclear halos were evidenced in the epidermal. Already the dermis has 127 fibroblasts with 2.5 AgNORs/nucleolus. Additionally, the metabolic activity was 0.243. In conclusion, the peripheral ear integumentary system derived from collared peccaries possessed some important variations compared to other mammals, as the number of layers and thickness of the epidermis, number of epidermal cells, melanocytes and proliferative parameters.(AU)

Pneumatic dilation versus laparoscopic Heller myotomy for the treatment of achalasia: variables related to a good response.

Achalasia is a motor disorder characterized by esophageal aperistalsis and failure of lower esophageal sphincter relaxation. The cardinal symptoms are dysphagia, food regurgitation and weight loss. The most effective treatments are pneumatic dilation (PD) of the cardia and Heller esophageal myotomy with partial fundoplication. There is still controversy regarding which treatments should be initially done. The aims of this study were to evaluate clinical response and the variables related to good results in both treatments. Ninety-two patients with achalasia diagnosed by esophageal manometry were randomized to receive either PD or laparoscopic Heller myotomy with partial fundoplication. After the procedure, patients were followed up clinically and submitted to esophageal manometry and pH monitoring. Three months after treatment, 73% of the patients from PD group and 84% of the surgery group had good results (P = 0.19). After 2 years of follow-up, 54% of the PD group and 60% of the surgery group (P = not significant) were symptom free. Variables related to a good response to PD were a 50% drop in lower esophageal sphincter pressure (LESP) or a LESP <10 mmHg after treatment. Patients over 40 years old with LESP ≤32 mmHg before treatment and a drop in LESP >50% after treatment significantly achieved better responses after surgical treatment when compared with PD. The reflux rate was significantly higher in the PD group (27.7%) compared with the surgery group (4.7%), P = 0.003. We concluded that surgical treatment and PD for achalasia are equally effective even after 2 years of follow-up. The choice of treatment for achalasia should be based on the following parameters: treatment availability, rate of good results, complication rates, variables related to good responses and also the patient's wish.

Hyperthermia induced after recirculation triggers chronic neurodegeneration in the penumbra zone of focal ischemia in the rat brain.

Chronic neurodegenerative processes have been identified in the rat forebrain after prolonged survival following hyperthermia (HT) initiated a few hours after transient global ischemia. Since transient global ischemia and ischemic penumbra share pathophysiological similarities, this study addressed the effects of HT induced after recirculation of focal brain ischemia on infarct size during long survival times. Adult male Wistar rats underwent intra-luminal occlusion of the left middle cerebral artery for 60 min followed by HT (39.0-39.5 degrees C) or normothermia. Control procedures included none and sham surgery with and without HT, and middle cerebral artery occlusion alone. Part I: 6-h HT induced at recirculation. Part II: 2-h HT induced at 2-, 6-, or 24-h recirculation. Part III: 2-h HT initiated at recirculation or 6-h HT initiated at 2-, 6- or 24-h recirculation. Survival periods were 7 days, 2 or 6 months. The effects of post-ischemic HT on cortex and striatum were evaluated histopathologically by measuring the area of remaining tissue in the infarcted hemisphere at -0.30 mm from bregma. Six-hour HT initiated from 6-h recirculation caused a significant decrease in the remaining cortical tissue between 7-day (N = 8) and 2-month (N = 8) survivals (98.46 +/- 1.14 to 73.62 +/- 8.99%, respectively). When induced from 24-h recirculation, 6-h HT caused a significant reduction of the remaining cortical tissue between 2- (N = 8) and 6-month (N = 9) survivals (94.97 +/- 5.02 vs 63.26 +/- 11.97%, respectively). These data indicate that post-ischemic HT triggers chronic neurodegenerative processes in ischemic penumbra, suggesting that similar fever-triggered effects may annul the benefit of early recirculation in stroke patients over the long-term.

Hyperthermia induced after recirculation triggers chronic neurodegeneration in the penumbra zone of focal ischemia in the rat brain

Chronic neurodegenerative processes have been identified in the rat forebrain after prolonged survival following hyperthermia (HT) initiated a few hours after transient global ischemia. Since transient global ischemia and ischemic penumbra share pathophysiological similarities, this study addressed the effects of HT induced after recirculation of focal brain ischemia on infarct size during long survival times. Adult male Wistar rats underwent intra-luminal occlusion of the left middle cerebral artery for 60 min followed by HT (39.0-39.5°C) or normothermia. Control procedures included none and sham surgery with and without HT, and middle cerebral artery occlusion alone. Part I: 6-h HT induced at recirculation. Part II: 2-h HT induced at 2-, 6-, or 24-h recirculation. Part III: 2-h HT initiated at recirculation or 6-h HT initiated at 2-, 6- or 24-h recirculation. Survival periods were 7 days, 2 or 6 months. The effects of post-ischemic HT on cortex and striatum were evaluated histopathologically by measuring the area of remaining tissue in the infarcted hemisphere at -0.30 mm from bregma. Six-hour HT initiated from 6-h recirculation caused a significant decrease in the remaining cortical tissue between 7-day (N = 8) and 2-month (N = 8) survivals (98.46 ± 1.14 to 73.62 ± 8.99 percent, respectively). When induced from 24-h recirculation, 6-h HT caused a significant reduction of the remaining cortical tissue between 2- (N = 8) and 6-month (N = 9) survivals (94.97 ± 5.02 vs 63.26 ± 11.97 percent, respectively). These data indicate that post-ischemic HT triggers chronic neurodegenerative processes in ischemic penumbra, suggesting that similar fever-triggered effects may annul the benefit of early recirculation in stroke patients over the long-term.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays.

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

A serosurvey for hantavirus infection in wild rodents from the states of Rio de Janeiro and Pernambuco, Brazil.

Sera from 269 rodents obtained during the routine surveillance operations in plague areas of Rio de Janeiro and Pernambuco states, Brazil were tested by ELISA for specific IgG antibodies against a recombinant nucleocapsid (N) protein of Araraquara hantavirus. ELISA-positive sera were submitted to reverse transcriptase-polymerase chain reaction (RT-PCR) for amplification of the virus genome and later sequencing for identification of the viral variant. The samples from the state of Pernambuco were antibody negative, and although four from Rio de Janeiro were ELISA-positive, they failed to yield viral cDNA by RT-PCR. This is the first report of the presence of antibodies to a hantavirus among rodents from Rio de Janeiro and suggests the possibility of human cases of hantavirus pulmonary syndrome (HPS) in that state, although no case has yet been reported.

Expression of a hantavirus N protein and its efficacy as antigen in immune assays

Hantavirus cardiopulmonary syndrome (HCPS) has been recognized as an important public heath problem. Five hantaviruses associated with HCPS are currently known in Brazil: Juquitiba, Araraquara, Laguna Negra-like, Castelo dos Sonhos, and Anajatuba viruses. The laboratory diagnosis of HCPS is routinely carried out by the detection of anti-hantavirus IgM and/or IgG antibodies. The present study describes the expression of the N protein of a hantavirus detected in the blood sample of an HCPS patient. The entire S segment of the virus was amplified and found to be 1858 nucleotides long, with an open reading frame of 1287 nucleotides that encodes a protein of 429 amino acids. The nucleotide sequence described here showed a high identity with the N protein gene of Araraquara virus. The entire N protein was expressed using the vector pET200D and the Escherichia coli BL21 strain. The expression of the recombinant protein was confirmed by the detection of a 52-kDa protein by Western blot using a pool of human sera obtained from HCPS patients, and by specific IgG detection in five serum samples of HCPS patients tested by ELISA. These results suggest that the recombinant N protein could be used as an antigen for the serological screening of hantavirus infection.

Midazolam in neonatal seizures with no response to phenobarbital.

The outcome of 45 neonates with EEG-confirmed seizures (ESz) was analyzed with regard to treatment. ESz persisted in 17 of 32 neonates receiving phenobarbital/phenytoin (13 had a poor outcome, 4 died). In contrast, ESz were rapidly controlled in 13 of 13 nonresponders to phenobarbital/phenytoin treated with midazolam (4 had poor outcome, 2 died). Nonresponders to phenobarbital/phenytoin had a significantly worse outcome than responders did. Midazolam effectively controlled ESz in nonresponders to phenobarbital/phenytoin and correlated with significantly improved long-term neurodevelopment.

Assessing the relative quality of anesthesiology and critical care medicine Internet mailing lists.

UNLABELLED: We studied the relative quality of a subset of anesthesiology and critical care medicine Internet mailing lists regarding the publishing capacity of their members to compare them with the major journals and conferences regarding these specialties. Using systematic searches on MEDLINE and according to the Science Citation Index 1995, we investigated the impact factor of mailing list subscribers, of the first authors of the selected articles, and of the first authors of published abstracts from conferences. We studied six mailing lists, seven journals, and four conferences. Journals and conferences showed a higher percentage of published authors and higher average impact factor among their first authors than the mailing lists did per subscriber. However, when only the subset of publishing authors from the three media was considered, no significant differences were found. We conclude that qualified authors may be found among the subscribers of Internet medical mailing lists on anesthesiology and critical care medicine. These professional discussion groups could complement peer-reviewed publications and conferences in professional information exchange and continuing medical education. IMPLICATIONS: Internet publishing is not governed by rules that assure certain basic quality standards. Methods for assessing these standards are needed. We compared discussion groups with medical journals and conferences on anesthesiology and critical care medicine by calculating the impact factor of their members and first authors, respectively. Our study shows that qualified authors may be found in all three media.

Can examination of WWW usage statistics and other indirect quality indicators distinguish the relative quality of medical web sites?

BACKGROUND: The Internet offers a great amount of health related websites, but concern has been raised about their reliability. Several subjective evaluation criteria and websites rating systems have been proposed as a help for the Internet users to distinguish among web resources with different quality, but their efficacy has not been proven. OBJECTIVES: To evaluate the agreement of a subset of Internet rating systems editorial boards regarding their evaluations of a sample of pediatric websites. To evaluate certain websites characteristics as possible quality indicators for pediatric websites. METHODS: Comparative survey of the Results of systematic evaluations of the contents and formal aspects of a sample of pediatric websites, with the number of daily visits to those websites, the time since their last update, the impact factor of their authors or editors, and the number of websites linked to them. RESULTS: 363 websites were compiled from eight rating systems. Only 25 were indexed and evaluated by at least two rating systems. This subset included more updated and more linked websites. There was no correlation among the Results of the evaluation of these 25 websites by the rating systems. The number of inbound links to the websites significantly correlated with their updating frequency (p<.001), with the number of daily visits (p=.005), and with the Results of their evaluation by the largest rating system, HealthAtoZ (p<.001). The websites updating frequency also significantly correlated with the RESULTS of the websites evaluation by HealthAtoZ, both about their contents (p=.001) and their total values (p<.05). The number of daily visits significantly correlated (p<.05) with the Results of the evaluations by Medical Matrix. CONCLUSIONS: Some websites characteristics as the number of daily visits, their updating frequency and, overall, the number of websites linked to them, correlate with their evaluation by some of the largest rating systems on the Internet, what means that certain indexes obtained from the usage analysis of pediatric websites could be used as quality indicators. On the other hand, the citation analysis on the Web by the quantification of inbound links to medical websites could be an objective and feasible tool in rating great amounts of websites.

Comparative analysis of pediatric mailing lists on the Internet.

OBJECTIVE: To analyze quantitative aspects and the relative quality of various pediatric discussion groups on the Internet and to contrast them with qualitative aspects of a selected number of pediatric journals. MATERIAL AND METHODS: An extensive number of mailing lists on the Internet of interest to pediatricians was compiled. Twelve of them concerned with pediatric specialties were selected. Six representative journals of pediatric specialties were also analyzed and compared with the corresponding mailing lists. From the list of subscribers we studied the potential quality of each mailing list. The postings sent by each member to the on-line discussions were also analyzed. As an estimate of the standing as author of each list member as well as of each first author of the selected journals, we calculated several indexes of quality using the 1995 Medline database and the impact factors of the biomedical journals reported by the 1994 Science Citation Index. RESULTS: The most popular lists were NICU-NET and PICU, both having more than 1100 subscribers. PEDPATH and PEDIHEART had the highest percentage of subscribers who were published authors, and their papers also yielded the highest impact factors. The most active lists were NICU-NET and PICU. The most participative ones were CHILD-NEURO and PED-LUNG. CHILD-NEURO had the highest percentage of authors among the participants. PEDPATH and CHILD-NEURO had the authors with the highest impact factors among the people who participated in the discussions. These latter two lists also showed the highest impact factor per posting. Those which had the highest yield (highest activity with highest quality per posting) were CHILD-NEURO and PEDIHEART. The average impact factor per first author of the analyzed journals was always higher than the average impact factor per participant of the lists. CONCLUSIONS: The electronic-mail discussion groups on the Internet are new nonacademic forums in which knowledge and experience in pediatrics can be shared. They cannot replace but they complement other more academic sources such as medical journals.

Nocardiosis after corticosteroid therapy for malignant thymoma.

BACKGROUND: Nocardia asteroides is an opportunistic infection caused by an aerobic actinomycete, which, in the immunocompromised host, can be associated with severe invasive disease with a predilection for the brain. METHODS AND RESULTS: The authors describe a 62-year-old man with a malignant thymoma that was clinically responsive to oral prednisone. N. asteroides sepsis subsequently developed, leading to his death. CONCLUSIONS: Nocardiosis should be considered a potential pathogen in this immunocompromised setting.

Brachial artery stenosis secondary to ergotism and responsive to nifedipine.

Ergotism is an uncommon drug reaction that may lead to severe ischemic vasoconstriction, which is usually unilateral and more commonly involves the lower extremities. Successful therapeutic measures have included invasive vascular surgery and the use of various available intravenous and oral vasodilators. Reported here is the case of a 56-year-old woman with Marfan's syndrome and chronic migraine headaches who presented with upper extremity pulselessness that responded promptly to oral nifedipine (Procardia). This relatively inexpensive agent with potent peripheral arterial vasodilative properties appears to be the agent of choice in severe ergotamine poisoning.