ELISPOT-menetelmän testaaminen primaaristen immuunivajavuustilojen diagnosointiin

Primaarisilla immuunivajavuustiloilla tarkoitetaan ryhmää sairauksia, jotka johtuvat immuunijärjestelmän solujen sisäisestä häiriöstä. Primaarisessa immuunivajavuustilassa kyseessä on synnynnäinen puutos immuunijärjestelmässä geneettisen häiriön pohjalta. ELISPOT (Enzyme linked immunospot) -menetelmässä T-lymfosyyttejä stimuloidaan spesifisillä antigeeneilla. Stimuloidut solut alkavat tuottaa sytokiinia ja yksittäisten sytokiinia tuottavien solujen määrä voidaan mitata. Tutkimukseni tarkoituksena oli primaaristen immuunivajavuustilojen diagnostiikkaan soveltuvan ELISPOT-menetelmän alustava pystyttäminen ja testaaminen. Tavoitteena oli tutkia, vaikuttaako solujen pakastaminen ELISPOTin antamiin tuloksiin ja voisiko ELISPOT-menetelmässä käyttää pakastettuja soluja alustavien viitearvojen määrittämiseen. Tutkittiin myös yhden ja kolmen vuorokauden inkubaatioajan vaikutusta solujen sytokiinintuotantoon sekä optimaalisia mitogeenikonsentraatioita. Tuoreiden ja pakastettujen näytteiden vertailu tehtiin kolmella henkilökunnan näytteellä. Henkilöistä kerättyjen ja eristettyjen tuoreiden mononukleaarisolujen antamia tuloksia verrattiin samoista henkilöistä aikaisemmin kerättyjen ja pakastettujen solujen antamiin tuloksiin. Viitearvojen alustava määrittäminen tehtiin pakastetuista potilasnäytteistä. Mitogeenikonsentraatioiden titraaminen ja inkubaatioaikojen vertailu tehtiin henkilökunnan tuoreista näytteistä. Tulokset osoittivat, että tuoreiden ja pakastettujen solujen sytokiinintuotanto eroaa toisistaan ja pakastettuja soluja ei voi käyttää viitearvojen määrittämiseen tässä menetelmässä. Inkubaatioaikavertailu osoitti, että yhden vuorokauden inkubaatioaika antoi riittävästi spotteja, mutta kolmen vuorokauden inkubaatioaika lisäsi solujen sytokiinintuotantoa niin paljon, että tuloksia oli vaikea tulkita. Mitogeenikonsentraatioita titraamalla saatiin selville Con A-mitogeenin optimaalinen ja suboptimaalinen pitoisuus ELISPOT-menetelmää varten. PWM:lla ja PHA:lla titrauksia tulee vielä jatkaa optimaalisen konsentraation selvittämiseksi. Näille mitogeeneille käytetty solumäärä 200 000 solua/kuoppa oli liian suuri ja spotteja tuli liikaa, jotta niitä olisi voitu lukea. Mitogeenien konsentraatioiden titraamista kannattaa tutkia lisää. Lisäksi tulevaisuudessa kannattaa alustavien viitearvojen määrittäminen tehdä tuoreista soluista. Solumääriä kannattaa tulevaisuudessa myös titrata, jotta ELISPOT-menetelmälle parhaiten soveltuva solumäärä löytyisi.

Ejaculate Characteristics Depend on Social Environment in the Horse (Equus caballus).

Sperm competition theory predicts semen characteristics to be affected by the social environment. We used the polygamous horse (Equus caballus) to experimentally study within-subject plasticity in response to different social environments. Stallions were sequentially exposed, over a period of 8 weeks each, to other stallions and then singly to mares, or vice versa (in adjacent boxes separated by grills). Ejaculates were collected to determine semen characteristics. Highest sperm numbers were found in stallions that were first exposed to other stallions and then to mares, while lowest sperm numbers were observed in stallions that had been exposed to mares but not yet to other stallions. One of three sperm velocity measures (curvilinear velocity) was consistently elevated in stallions that were first exposed to stallions and then to mares. Sperm number after exposure to mares and curvilinear sperm velocity after exposure to stallions were both positively correlated to average blood testosterone levels during the corresponding period of exposure. We conclude that ejaculate characteristics are plastic traits affected by the social environment in horses.

Identificación de características forenses avanzadas a partir del ADN: etnogeografía, patología delictiva y morfoanatomía

Resumen: El uso de la molécula de ADN ha revolucionado el mundo de la Genética forense, convirtiéndose en una herramienta poderosa para resolver un gran número de casos judiciales. El ADN permite identificar a una persona determinada con una probabilidad de error ínfima. Utilizando el análisis del ADN los ámbitos clásicos de actuación han sido: la resolución de grandes delitos, las pruebas de paternidad o de parentesco en general y por último la identificación de restos humanos que estuviesen muy dañados. Sin embargo, los investigadores policiales buscan obtener más información a partir de las moléculas de ADN extraídas de muestras biológicas (sangre, saliva, semen, células epiteliales, etc.). En este trabajo abordaremos los conocimientos que en la actualidad se tienen de algunas de las características del aspecto externo de los individuos a partir del análisis del ADN de las muestras, su interés desde el punto de vista jurídico y sus limitaciones. En concreto nos centraremos en cuatro grandes áreas de estudio: el análisis del origen geográfico de los ancestros, la genética del comportamiento, la genética médica y las predicciones de características externas tales como el color de los ojos, del cabello, de la piel, la estatura y la edad. Palabras clave: Genética forense, ADN. Origen étnico-geográfico. Patología delictiva. Características externas visibles.

A análise seminal deve ser requisitada para homens com histórico de fertilidade prévia?

OBJETIVOS: determinar se a história prévia de fertilidade pode predizer o atual status de fertilidade de um paciente masculino examinado por infertilidade do casal. MÉTODOS: estudo retrospectivo envolvendo análises seminais de 183 pacientes consecutivos subférteis avaliados entre setembro de 2002 e março de 2004. Foram excluídos do estudo os pacientes que haviam se submetido a radioterapia, quimioterapia, orquiectomia ou vasectomia. Os valores médios de todas as análises foram usados em pacientes com múltiplas análises de sêmen. Pacientes com concentração espermática superior a 20x10(6) espermatozóides/mL, motilidade superior a 50% e espermatozóides com morfologia estrita superior a 14% foram considerados normais. Os pacientes foram divididos em dois grupos, segundo o status de fertilidade: infertilidade primária (118 pacientes) e infertilidade secundária (65 pacientes). Os dados foram analisados pelos testes estatísticos chi2 e teste t de Student. RESULTADOS: não houve diferença na idade média entre os pacientes com infertilidade primária, 37,3±6,3, e infertilidade secundária, 38,1±5,9; p=0,08. No grupo de pacientes com infertilidade primária, 51,9% (61 pacientes) tiveram uma concentração espermática normal, 70,3% (83 pacientes) tiveram a motilidade espermática normal e 26,37% (31 pacientes), por sua vez, morfologia normal. No grupo de pacientes com infertilidade secundária, 53,8% (35 pacientes) tiveram concentração espermática normal, 75,4% (49 pacientes) tiveram motilidade espermática normal e 32,3% (21 pacientes), morfologia normal. Nenhuma diferença significativa foi detectada na concentração espermática (21,3x10(6)/mL versus 23,1x10(6)/mL; p=0,07), motilidade (45,2 versus 48,1%; p=0,08) e morfologia (6,1 versus 6,4%; p=0,09) entre os grupos de pacientes com infertilidade primária e secundária. CONCLUSÕES: a análise seminal deve ser solicitada mesmo em casos de fertilidade masculina prévia. Os médicos não devem presumir que um paciente possui uma análise seminal normal, baseados no fato de este possuir história de estabelecimento de uma gravidez no passado.

Criopreservação de sêmen humano: comparação entre métodos de congelação e tipos de envase

OBJETIVOS: comparar duas diferentes técnicas de congelação e dois tipos de envase do sêmen humano durante processo de criopreservação. MÉTODOS: estudo experimental, no qual foi analisada a criopreservação de 18 amostras de sêmen de 18 voluntários. Após a adição de meio crioprotetor, "Test-yolk buffer" , as amostras de sêmen foram envasadas em palhetas com capacidade de 0,25 mL ou em criotubos de 2 mL e submetidas à criopreservação por dois métodos, um lento e outro rápido, totalizando quatro tratamentos distintos: RP (congelação pelo método rápido e envasado em palheta), RT (rápido-criotubo), LP (lento-palheta) e LT (lento-criotubo). As amostras, após 24 horas, foram descongeladas em temperatura ambiente e mantidas a 37ºC. Os dados coletados foram analisados através do teste t de Student, com p<0,05, utilizando o programa de computador SPSS for Windows® versão 11.0.0. RESULTADOS: houve redução da motilidade espermática após o processo de criopreservação. A taxa de motilidade inicial foi 58,1% e as motilidades após os diferentes métodos de criopreservação foram: 19,2% (RP), 27% (RT), 21,1% (LP) e 30,3% (LT). Houve redução significativa na morfologia normal. A taxa de morfologia normal inicial foi 14,2% e as morfologias após os diferentes métodos de criopreservação foram: 12,8% (RP), 12,6% (RT), 12,6% (LP) e 12,4% (LT). CONCLUSÕES: o método de criopreservação lento com envase em criotubo esteve associado à melhor motilidade espermática após o descongelamento. Não houve diferença entre os métodos quando avaliada a morfologia espermática.

Sperm morphological and morphometric evaluation in captive collared peccaries (Pecari tajacu)

The aim of this study was to compare different staining methods for the evaluation of sperm morphology by light microscopy and also to describe the morphometry of the entire sperm in collared peccaries (Pecari tajacu). Semen from 10 males was obtained by electroejaculation and evaluated for sperm motility, vigor, and concentration. Semen smears were prepared through three different staining methods: Bengal rose, brome-phenol blue, and eosin-nigrosin. Smears were evaluated under light microscopy and sperm morphologic alterations were determined in percentage. In addition, sperm morphometric analysis was conducted by light microscopy coupled to image analyzer software. The smears stained with Bengal Rose provide the best results for the visualization of the sperm tail, midpiece, and head. The use of eosin-nigrosin stain did not allow an adequate impregnation, and some sperm presented a few contrasts with the background. A higher incidence of bent coiled tails was verified in the use of brome-phenol blue staining (P<0.05). Through morphometric evaluation, it was observed that the tail occupies the greatest proportion (89%) of the sperm which presents a discretely elongated head. According to the results, the use of the Bengal Rose stain is recommended for the morphologic evaluation of the collared peccary sperm.

The seasonal and ovarian status effects on in vitro production of domestic cat embryos between Equator and Tropic of Capricorn

From the Tropic of Capricorn to Equator, the seasonality of domestic cat is known to be absent, i.e., these animals are considered non-seasonal breeders at these regions. We hypothesized that this particularity might have some influence on in vitro embryo production. The aim of this experiment was to determine the percentage of cleavage and morulae and blastocyst formation produced from oocytes recovered from queen ovaries of three distinct status - follicular, luteal or inactive - during two different reproductive seasons experienced by cats in southeast of Brazil (22°53'09" S and 48°26'42" W) - non breeding season (NBS), comprehending January to March; and breeding season (BS), August to October. Thirty queens were neutered. Ovaries were classified according to their status and were sliced in PBS for cumulus oocyte complex (COC) releasing. Grade I COC were washed three times in H-MEM supplemented with BSA, glutamine, sodium pyruvate, cysteine, streptomycin and penicillin. Oocytes were incubated in groups of 20-30 in 400µL of DMEM supplemented with FSH, LH, estradiol, IGF-I and basic fibroblast growth factor under mineral oil for 30 or 36 hours at 38°C in humidified environment of 5% de O2, 5% CO2 and 90% N2. COC were fertilized in Ham's F-10 medium supplemented with BSA, cysteine, pyruvate and streptomycin/penicillin (culture medium) with fresh semen selected through swim up technique. Eighteen hours later, the presumptive zygotes were denuded, the percentage of cleavage was determined and every 10 zygotes were transferred to 100mL drops of culture medium for culture during three days. After 72 hours of culture the percentage of morulae formation was evaluated and these structures were transferred to drops of the same culture medium. At the eighth day of culture blastocyst formation was analyzed. During NBS, from a total of 272 (inactive), 162 (luteal) and 134 (follicular) fertilized oocytes, the percentage of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 24.63, 16.54 and 8.09 respectively; for those derived from luteal ovaries, the percentage was 21.6, 12.96 and 8.64, and for those from follicular ovaries, they were 24.62, 16.41 and 8.21. Considering BS, from a total of 102 (inactive), 198 (luteal) and 86 (follicular) fertilized oocytes, the relative frequency (%) of cleaved zygotes, morulae and blastocysts derived from inactive ovaries were 64.7, 41.17 and 23.53 respectively; for those derived from luteal ovaries, the percentage was 64.14, 40.41 and 23.73, and for those from follicular ovaries, they were 63.95, 39.54 and 24.41. The results of this experiment demonstrate that no statistically significant difference (P<0.05) was verified in the frequency of cleaved embryos and morulae and blastocyst formation when comparing the three ovarian conditions in the same season. However the breeding season presented better results considering cleavage and morulae and blastocyst formation.

Degenerative joint disease in cattle and buffaloes in the Amazon region: a retrospective study

A retrospective study of the epidemiological and clinic-pathological aspects of cattle and buffaloes with degenerative joint disease (DJD) was conducted in the state of Pará, Brazil. From 1999 to 2014, eleven cattle and 24 buffaloes were evaluated. All the treated animals with suspected DJD underwent a clinical examination of the musculoskeletal system. In seven cattle and eight buffaloes with clinical signs of the disease postmortem examination was performed. The common clinical signs observed in both species were chronic lameness, stiff gait, postural changes, audible crackles in the affected limb, prolonged recumbency, difficulty in getting up and progressive weight loss. The lesions observed at necropsy were: irregular articular surfaces, erosion of the articular cartilage and the underlying bone tissue, and proliferation of the periarticular bone tissue with formation of osteophytes. The most affected joints in cattle and buffaloes wereof the hind limb. In buffaloes, the main predisposing factor to the onset of DJD was phosphorus deficiency. In cattle, defects of the anatomical conformation of the hind limbs, chronic trauma due to the activities performed, such as semen collection, and advanced age possibly contributed to the emergence of the disease.

Maintenance of Brazilian Biodiversity by germplasm bank

Abstract: Currently the importance of using alternative strategies for biodiversity conservation is emphasized and since the establishment of germplasm bank is an alternative to the conservation of endangered species. This is a technique of great importance for the maintenance of Brazilian fauna. Since the early70'sthere was a growing concern about the need to preserve essential genetic resources for food and agriculture, mainly for conservation of genetic material from farm animals. Thus was created the Brasilia Zoo, in July 2010, the first Germplasm Bank of Wild Animals in Latin America, as an alternative strategy for the conservation of threatened or endangered species, using both gametes and somatic cells and stem cells. Then we argue to create new banks or research networks among different regions with aimed to tissue preservation.

Vitrification of bovine preantral follicles with dimethylsulfoxide and sucrose plus α-tocopherol

Abstract: The objective of this study was to evaluate the vitrification of bovine preantral follicles with dimethylsulfoxide (D) and sucrose (S) plus α-tocopherol 5mmol/L (T5) or 10mmol/L (T10) and, evaluate the thawed with minimal essential medium (m) with or without sucrose (s). Ovaries of cows were collected from slaughterhouse for the experiment I (n=66) and II (n=51). In the laboratory ovarian fragments were randomly assigned either to fresh control and 8 vitrification treatments (Controle and Dm; Dms, DSm; DSms; DST5m; DST5ms; DST10m; DST10ms). Ovarian fragments were placed in vitrification solution (5 min) and immersed in liquid nitrogen (-196°C), after a week, the fragments were thawed and analyzed. In the experiments I, preantral follicles were morphologically observed for histological evaluation, (normal; degenerated and developing of stage). In the experiment II, preantral follicles were mechanically isolated from ovarian tissue and examined with trypan blue, where dead and live corresponded to stained or non-stained. The treatments DSm, DSms and DST10m were effective in preserving the morphology in situ. However, the viability of isolated preantral follicles after vitrification remained high only in treatment DST10m. Thus, DST10m preserves survival rates and morphological integrity during vitrification of bovine preantral follicles.

Karyotype of cryopreserved bone marrow cells

The analysis of chromosomal abnormalities is important for the study of hematological neoplastic disorders since it facilitates classification of the disease. The ability to perform chromosome analysis of cryopreserved malignant marrow or peripheral blast cells is important for retrospective studies. In the present study, we compared the karyotype of fresh bone marrow cells (20 metaphases) to that of cells stored with a simplified cryopreservation method, evaluated the effect of the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) as an in vitro mitotic index stimulator, and compared the cell viability and chromosome morphology of fresh and cryopreserved cells whenever possible (sufficient metaphases for analysis). Twenty-five bone marrow samples from 24 patients with hematological disorders such as acute myeloid leukemia, acute lymphoblastic leukemia, myelodysplastic syndrome, chronic myeloid leukemia, megaloblastic anemia and lymphoma (8, 3, 3, 8, 1, and 1 patients, respectively) were selected at diagnosis, at relapse or during routine follow-up and one sample was obtained from a bone marrow donor after informed consent. Average cell viability before and after freezing was 98.8 and 78.5%, respectively (P < 0.05). Cytogenetic analysis was successful in 76% of fresh cell cultures, as opposed to 52% of cryopreserved samples (P < 0.05). GM-CSF had no proliferative effect before or after freezing. The morphological aspects of the chromosomes in fresh and cryopreserved cells were subjectively the same. The present study shows that cytogenetic analysis of cryopreserved bone marrow cells can be a reliable alternative when fresh cell analysis cannot be done, notwithstanding the reduced viability and lower percent of successful analysis that are associated with freezing.

Production of a cloned calf from a fetal fibroblast cell line

The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5%) were successfully fused and 24 (28.9%) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8%) of which were pregnant on day 35 and 3 (16.7%) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).

Minor sperm abnormalities in young male post-pubertal patients with juvenile dermatomyositis

The objective of the present study was to identify sperm abnormalities in young male patients with juvenile dermatomyositis (JDM). In 2005, 18 male JDM patients, diagnosed according to the criteria of Bohan and Peter, were followed at the Pediatric Rheumatology Unit and Rheumatology Division, of our Institution. Of the 18 males, 11 were pre-pubertal and 7 were post-pubertal. Two of 7 post-pubertal JDM male patients were excluded: one for orchidopexy for cryptorchidism and the other for testicular ectopia in the left testis. The remaining 5 post-pubertal JDM patients were prospectively evaluated on the basis of two semen analyses, according to the World Health Organization (WHO), urologic evaluation, testicular Doppler ultrasound hormone profile. The data of the JDM patients were compared with those of 5 age-matched healthy controls. The median age 18, was similar in JDM patients and controls. All JDM patients had teratozoospermia (abnormal sperm morphology), as did 4 (80%) of the controls. One of JDM patients had previous oligoasthenoteratozoospermia treated with intravenous cyclophosphamide with normalization of the number and concentration of the sperm after 5 years. All sperm parameters (sperm concentration, total sperm count and total motile sperm count by WHO, and sperm morphology by Kruger strict criteria), testicular volumes by Prader orchidometer and ultrasound, and hormones were similar in JDM patients compared with controls. The frequency of anti-sperm antibodies was similar in both groups. All JDM patients had minor sperm abnormalities in the head, midpiece, and/or tail of spermatozoids. Serial semen analyses in larger study populations are necessary to identify the extent and duration of sperm abnormalities in male patients with idiopathic inflammatory myopathies.

Lyophilized allografts without pre-treatment with glutaraldehyde are more suitable than cryopreserved allografts for pulmonary artery reconstruction

Various methods are available for preservation of vascular grafts for pulmonary artery (PA) replacement. Lyophilization and cryopreservation reduce antigenicity and prevent thrombosis and calcification in vascular grafts, so both methods can be used to obtain vascular bioprostheses. We evaluated the hemodynamic, gasometric, imaging, and macroscopic and microscopic findings produced by PA reconstruction with lyophilized (LyoPA) grafts and cryopreserved (CryoPA) grafts in dogs. Eighteen healthy crossbred adult dogs of both sexes weighing between 18 and 20 kg were used and divided into three groups of six: group I, PA section and reanastomosis; group II, PA resection and reconstruction with LyoPA allograft; group III, PA resection and reconstruction with CryoPA allograft. Dogs were evaluated 4 weeks after surgery, and the status of the graft and vascular anastomosis were examined macroscopically and microscopically. No clinical, radiologic, or blood-gas abnormalities were observed during the study. The mean pulmonary artery pressure (MPAP) in group III increased significantly at the end of the study compared with baseline (P=0.02) and final [P=0.007, two-way repeat-measures analysis of variance (RM ANOVA)] values. Pulmonary vascular resistance of groups II and III increased immediately after reperfusion and also at the end of the study compared to baseline. The increase shown by group III vs group I was significant only if compared with after surgery and study end (P=0.016 and P=0.005, respectively, two-way RM ANOVA). Microscopically, permeability was reduced by ≤75% in group III. In conclusion, substitution of PAs with LyoPA grafts is technically feasible and clinically promising.