Scriptaid and 5-aza-2'deoxycytidine enhanced expression of pluripotent genes and in vitro developmental competence in interspecies black-footed cat cloned embryos.

Somatic cell nuclear transfer offers the possibility of preserving endangered species including the black-footed cat, which is threatened with extinction. The effectiveness and efficiency of somatic cell nuclear transfer (SCNT) depends on a variety of factors, but 'inappropriate epigenetic reprogramming of the transplanted nucleus is the primary cause of the developmental failure of cloned embryos. Abnormal epigenetic events such as DNA methylation and histone modifications during SCNT perturb the expression of imprinted and pluripotent-related genes that, consequently, may result in foetal and neonatal abnormalities. We have demonstrated that pregnancies can be established after transfer of black-footed cat cloned embryos into domestic cat recipients, but none of the implanted embryos developed to term and the foetal failure has been associated to aberrant reprogramming in cloned embryos. There is growing evidence that modifying the epigenetic pattern of the chromatin template of both donor cells and reconstructed embryos with a combination of inhibitors of histone deacetylases and DNA methyltransferases results in enhanced gene reactivation and improved in vitro and in vivo developmental competence. Epigenetic modifications of the chromatin template of black-footed cat donor cells and reconstructed embryos with epigenetic-modifying compounds enhanced in vitro development, and regulated the expression of pluripotent genes, but these epigenetic modifications did not improve in vivo developmental competence.

Congenital infections with Trypanosoma cruzi or Toxoplasma gondii are associated with decreased serum concentrations of interferon-γ and interleukin-18 but increased concentrations of interleukin-10.

Little is known about the immune responses of newborns with congenital Chagas disease (CCD) or congenital toxoplasmosis (CT) but they probably differ to those seen in adults with Chagas disease or toxoplasmosis, leading to differences in pathology. The concentrations of interleukin-18 (IL-18), interferon-γ (IFN-γ) and interleukin 10 (IL-10) in the sera of infants with CCD or CT were determined and compared with those in the sera of uninfected controls (born to mothers who were seropositive or seronegative for Trypanosoma cruzi). The infants with CCD or CT were found to have lower IL-18 and IFN-γ concentrations but higher IL-10 concentrations than the uninfected controls. The IL-18 and IFN-γ concentrations were also significantly lower in the infants with CCD than in those with CT. Although the infants with symptomatic CT had significantly higher serum concentrations of IL-18 than those with asymptomatic infection with Toxoplasma, the infants with symptomatic CCD had similar serum concentrations of IL-18 to the infants with asymptomatic Tr. cruzi infection. Taken together, these results indicate that IL-10 contributes to the suppression of pro-inflammatory immune responses and therefore, perhaps, to clinically overt CCD and CT.

Derivation of cat embryonic stem-like cells from in vitro-produced blastocysts on homologous and heterologous feeder cells.

The domestic cat is a focal mammalian species that is used as a model for developing assisted reproductive technologies for preserving endangered cats and for studying human diseases. The generation of stable characterized cat embryonic stem cells (ESC) lines to use as donor nuclei may help to improve the efficiency of interspecies somatic cell nuclear transfer for preserving endangered cats and allow the creation of knockout cell lines to generate knockout cats for studying function of specific genes related to human diseases. It will also enable the possibility of producing gametes in vitro from ESC of endangered cats. In the present study, we report the generation of cat embryonic stem-like (cESL) cells from blastocysts derived entirely in vitro. We generated 32 cESL cell lines from 331 in vitro derived blastocysts from which inner cell masses were isolated by immunosurgery or by a mechanical method. Inhibition of cat dermal fibroblast (CDF) proliferation after exposure to mitomycin-C was both dose and time dependent, where doses of 30 to 40 microg/mL for 5 h were most efficient. These dosages were higher than that required to inhibit cell proliferation of mouse fetal fibroblasts (MFF; 10 microg/mL for 2.5 h). Mitomycin-C did not significantly increase necrosis of cells from either species, and had an anti-proliferative effect at concentrations below cytotoxicity. A clear species-specific relationship between feeder layers and derivation of cESL cell lines was observed, where higher numbers of cESL cell lines were generated on homologous cat feeder layers (n = 26) than from those derived on heterologous mouse feeder layers (n = 6). Three cESL cell lines generated from immunosurgery and cultured on CDF maintained self-renewal and were morphologically undifferentiated for nine and twelve passages (69-102 days). These lines showed a tightly packed dome shaped morphology, exhibited alkaline phosphatase activity and immuno-expression of the pluripotent marker OCT-4 and surface marker SSEA-1. Primary colonies at P0 to P3 and cat blastocysts expressed transcription factors OCT-4, NANOG and SOX-2 and the proto-oncogene C-MYC. However, expression was at levels significantly lower than in vitro produced blastocysts. During culture, cESL colonies spontaneously differentiated into fibroblasts, cardiomyocytes, and embryoid bodies. Development of techniques to prevent differentiation of cESL cells will be essential for maintaining defined cell lines.

[Acute toxoplasmosis: clinical and laboratory data in eleven patients]. / Toxoplasmosis aguda: datos clínicos y de laboratorio en 11 pacientes.

INTRODUCTION: The clinical and laboratory data of immunocompetent patients with acute toxoplasmosis (AT) are described. PATIENTS AND METHODS: We performed a retrospective study of patients with AT attended between 1996 and 2004. Diagnostic criteria consisted of compatible clinical findings (generalized and cervical lymphadenopathies) and specific serology against Toxoplasma gondii (high IgG and IgM and/or reactive IgA). IgG and IgM determinations were performed by ELFA and IgA determinations by ELISA. IgM-CMV, heterophil antibodies, hemogram, hepatic chemistry were also determined and funduscopic examination was performed. RESULTS: Eleven immunocompetent patients with AT were evaluated. The mean age was 8.8 years (95 % CI: 3.6-12.9). The patients were evaluated between the first and the third month after symptom onset. Of the 11 patients, hard elastic lymphadenopathies were found in 10, single cervical lymphadenectomy in three and generalized lymphadenectomy in seven. One patient showed no symptoms. In one patient, nodal histology showed the Piringer-Kuchinka triad. None of the patients showed alterations in the hemogram, hepatic chemistry or funduscopic examination. The mean IgG value was 4.143 UI/ml (95 % CI: 2.570 and 5.717). IgM was reactive in nine of the 11 patients (81.8 %) and IgA in seven out of 10 patients (70 %). In all patients, at least one of these two immunoglobulins was reactive. In all patients, clinical outcome was favorable without parasiticide treatment. CONCLUSION: Except for one asymptomatic patient, all the patients had generalized lymphadenopathies and only 27.2 % showed cervical lymphadenopathies. A negative IgM or IgA result does not rule out a diagnosis of AT. Parasiticide treatment is unnecessary in this entity. Acute toxoplasmosis should be considered early in children with lymphadenopathies to avoid invasive procedures.

Toxoplasmosis aguda: datos clínicos y de laboratorio en 11 pacientes / Acute toxoplasmosis: clinical and laboratory data in eleven patients

Introducción Se describen la clínica y el laboratorio de pacientes inmunocompetentes con toxoplasmosis aguda (TA). Pacientes y métodos Se trata de un estudio retrospectivo de pacientes con TA asistidos entre 1996 y 2004. Criterios diagnósticos: clínica compatible (adenopatías generalizadas o cervicales) y serología específica contra Toxoplasma gondii (IgG elevada e IgM y/o IgA reactiva). La determinación de IgG e IgM se realizó por ELFA, las IgA por ELISA. Se realizó IgM-CMV, anticuerpos heterófilos, hemograma, hepatograma y fundoscopia. Resultados Se evaluaron 11 pacientes inmunocompetentes con TA, edad media: 8,8 años (intervalo de confianza del 95 % [IC 95 %]: 3,6-12,9). Concurrieron entre 1 y 3 meses del inicio de los síntomas. En 10/11 se hallaron adenopatías elásticas, cervicales únicas en tres y generalizadas en siete. Un paciente fue asintomático. La histología del ganglio de un paciente presentó la tríada de Piringer-Kuchinka. Ninguno presentó alteraciones del hemograma, hepatograma y del fondo de ojo. La media de la IgG: 4,143 U/ml (IC 95 %: 2,570-5,717). La IgM se detectó en 9/11 (81,8 %), la IgA en 7/10 (70 %). Todos los pacientes presentaron al menos una de estas dos inmunoglobulinas reactivas. La evolución clínica fue favorable sin tratamiento parasiticida. Conclusión Excepto un paciente asintomático, todos presentaron adenopatías generalizadas y sólo el 27,2 % cervicales. Un resultado negativo de IgM o IgA no descarta el diagnóstico. El tratamiento parasiticida es innecesario en esta patología. Es importante pensar tempranamente en esta etiología en niños con adenopatías, esto ahorraría procedimientos invasivos

Introduction The clinical and laboratory data of immunocompetent patients with acute toxoplasmosis (AT) are described. Patients and methods We performed a retrospective study of patients with AT attended between 1996 and 2004. Diagnostic criteria consisted of compatible clinical findings (generalized and cervical lymphadenopathies) and specific serology against Toxoplasma gondii (high IgG and IgM and/or reactive IgA). IgG and IgM determinations were performed by ELFA and IgA determinations by ELISA. IgM-CMV, heterophil antibodies, hemogram, hepatic chemistry were also determined and funduscopic examination was performed. Results Eleven immunocompetent patients with AT were evaluated. The mean age was 8.8 years (95 % CI: 3.6-12.9). The patients were evaluated between the first and the third month after symptom onset. Of the 11 patients, hard elastic lymphadenopathies were found in 10, single cervical lymphadenectomy in three and generalized lymphadenectomy in seven. One patient showed no symptoms. In one patient, nodal histology showed the Piringer-Kuchinka triad. None of the patients showed alterations in the hemogram, hepatic chemistry or funduscopic examination. The mean IgG value was 4.143 UI/ml (95 % CI: 2.570 and 5.717). IgM was reactive in nine of the 11 patients (81.8 %) and IgA in seven out of 10 patients (70 %). In all patients, at least one of these two immunoglobulins was reactive. In all patients, clinical outcome was favorable without parasiticide treatment. Conclusion Except for one asymptomatic patient, all the patients had generalized lymphadenopathies and only 27.2 % showed cervical lymphadenopathies. A negative IgM or IgA result does not rule out a diagnosis of AT. Parasiticide treatment is unnecessary in this entity. Acute toxoplasmosis should be considered early in children with lymphadenopathies to avoid invasive procedures

[Congenital Chagas disease: experience in the Hospital de Niños, Ricardo Gutiérrez, Buenos Aires, Argentina]. / Enfermedad de Chagas congenita: experiencia del Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina.

Epidemiological, clinical, diagnostic, and therapeutic data from children who were born to mothers infected with T. cruzi who came to our hospital are presented. In addition, we exhibit the preliminary results of a technique that detects the anti F2/3 antibodies: these would be able to confirm the cure earlier than conventional serology. We also show the results of PCR diagnosis. Most of the mothers (76,1%) resided in Argentina, the rest were from Bolivia and Paraguay The median average age at diagnosis of the patients was 8,5 months (range 15 days-10 years). Out of 168 children, 64,98% were asymptomatic at diagnosis. The diagnosis criteria were: T. cruzi observation by microhematocrit technique in patients less than 7 month old. Two reactive serological tests in patients older than 8 months. A nifurtimox dose used in these patients was 10-13 mg/kg/d during 60 days. Although 31% presented side effects, none of them had to be dropped from the treatment. Cure criteria was conventional serology negativization. Of the patient population, we cured 87,2% of them, 98% of those under 3 years, and 100% of those who received treatment before age 8 months. We compared the time of negativization between conventional serology and anti F2/3 in 21 children. The latter were very useful to demonstrate (p>0,001) the success of the treatment, in those that started treatment after 8 months of age. PCR testing of a group of all patients, showed a diagnostic sensibility of 80,3% and a specificity of 97,8%.

Enfermedad de Chagas congenita: experiencia del Hospital de Niños Ricardo Gutiérrez, Buenos Aires, Argentina / Congenital Chagas disease: experience in the Hospital de Niños, Ricardo Gutiérrez, Buenos Aires, Argentina

Epidemiological, clinical, diagnostic, and therapeutic data from children who were born to mothers infected with T. cruzi who came to our hospital are presented. In addition, we exhibit the preliminary results of a technique that detects the anti F2/3 antibodies: these would be able to confirm the cure earlier than conventional serology. We also show the results of PCR diagnosis. Most of the mothers (76,1%) resided in Argentina, the rest were from Bolivia and Paraguay The median average age at diagnosis of the patients was 8,5 months (range 15 days-10 years). Out of 168 children, 64,98% were asymptomatic at diagnosis. The diagnosis criteria were: T. cruzi observation by microhematocrit technique in patients less than 7 month old. Two reactive serological tests in patients older than 8 months. A nifurtimox dose used in these patients was 10-13 mg/kg/d during 60 days. Although 31% presented side effects, none of them had to be dropped from the treatment. Cure criteria was conventional serology negativization. Of the patient population, we cured 87,2% of them, 98% of those under 3 years, and 100% of those who received treatment before age 8 months. We compared the time of negativization between conventional serology and anti F2/3 in 21 children. The latter were very useful to demonstrate (p>0,001) the success of the treatment, in those that started treatment after 8 months of age. PCR testing of a group of all patients, showed a diagnostic sensibility of 80,3% and a specificity of 97,8%.

[Toxocariasis: clinical and laboratory features in 54 patients]. / Toxocariasis: aspectos clínicos y de laboratorio en 54 pacientes.

OBJECTIVE: To describe the clinical and laboratory findings in children with toxocariasis. METHODS: Fifty-four children with reactive serology to Toxocara determined by ELISA were prospectively identified between January 1998 and September 2000. The patients were divided into three groups: asymptomatic children (n 24), those with visceral larva migrans (n 16) and those with ocular larva migrans (n 14). Age, serology titers, and eosinophil count at diagnosis were compared among the groups. The patients received treatment with albendazole 10-15 mg/kg/day for 15 days or thiabendazole 25 mg/kg/day in two series of 7 days. RESULTS: The clinical features were as follows: 24 children (44.4 %) were asymptomatic, pneumonitis was found in 9 (16.7 %), hepatomegaly in 6 (11.1 %), acute posterior uveitis in 5 (9.3 %), strabismus in 5 (9.3 %), leukocoria in 4 (7.4 %), fever in 3 (5.6 %). There was 1 case of keratitis, 1 of cataracts, 1 of myocarditis and 1 case of pneumonia with pleural effusion. Some patients showed more than one clinical feature. Four children experienced loss of vision in the affected eye. No differences in age or serology titers were found among the groups. Eosinophil count was lower in the group with ocular larva migrans than in the other groups (p < 0.001). Children with active disease showed clinical improvement and a 70.4 % decrease in eosinophilic count one year after treatment. Serological titers showed an unpredictable pattern during the follow-up. CONCLUSIONS: Most of the infected children were asymptomatic. In the post-treatment follow-up, clinical improvement and a decrease in eosinophilic count were observed. Further studies are needed to evaluate the efficacy of treatment, especially in asymptomatic children.

Toxocariasis: aspectos clínicos y de laboratorio en 54 pacientes / Toxocariasis: clinical and laboratory features in 54 patients

Objetivo: Describir los hallazgos clínicos y de laboratorio en niños con toxocariasis. Métodos: En forma prospectiva se diagnosticaron 54 niños con serología reactiva por técnica de enzimoinmunoanálisis (ELISA) para Toxocara, entre enero de 1998 y septiembre de 2000. Se dividieron en 3 grupos: asintomáticos, 24; larva migrans visceral, 16; larva migrans ocular, 14. Se compararon la edad, los títulos serológicos y la eosinofilia al diagnóstico entre los grupos. Se indicó como tratamiento 10-15 mg/kg/día de albendazol durante 15 días o 25 mg/kg/día de tiabendazol en 2 series de 7 días. Resultados: Los hallazgos clínicos fueron: asintomáticos, 24 casos (44,4 por ciento); neumonitis, 9 (16,7 por ciento); hepatomegalia, 6 (11,1 por ciento); uveítis posterior aguda, 5 (9,3 por ciento); estrabismo, 5 (9,3 por ciento); leucocoria, 4 (7,4 por ciento); fiebre, 3 (5,6 por ciento). Hubo un caso de queratitis, uno de cataratas, uno de miocarditis y uno de neumonía con derrame. Algunos pacientes presentaron más de un signo clínico. Cuatro pacientes perdieron la visión del ojo lesionado. No se encontraron diferencias en la edad y los títulos serológicos entre los grupos. Se halló menor eosinofilia en los niños con compromiso ocular con relación a los otros grupos (p < 0,001). Se observó mejoría clínica en los niños con infección activa y una disminución del 70,4 por ciento en el recuento de eosinófilos en el seguimiento a un año postratamiento. Los títulos serológicos mostraron un comportamiento errático en el seguimiento. Conclusiones: La mayor parte de los infectados fueron asintomáticos. En el seguimiento postratamiento se evidenció una mejoría clínica y caída en el recuento de eosinófilos. Se plantea la necesidad de ampliar el estudio valorando la eficacia del tratamiento especialmente en aquellos niños asintomáticos (AU)

[Education of the hypertensive patient: are educational booklets useful?]. / L'educazione del paziente iperteso: sono utili gli opuscoli informativi?

A good level of knowledge about hypertension can improve patients' compliance to treatment so achieve better therapeutic results. The aim of our study was to evaluate the degree of knowledge about their disease in hypertensives followed in a hospital out-patient unit; and whether an informative booklet could increase this awareness. The patients were presented a questionnaire on the following items: 1. their interest in health news as presented by the mass-media and their judgement on physicians' willingness to provide information about hypertension; 2. the health hazards of being hypertensive; 3. the importance of a family history of hypertension; 4. life style and blood pressure; 5. the reasons for treating hypertension and the length of treatment. Upon completion of the questionnaire, the patients were handed out a booklet in which these same topics were analyzed. At the next follow-up visit, they were invited to answer a set of questions quite similar to the first ones, but presented in a different verbal form. 200 patients completed the first questionnaire; 159, both of them. Basically, they show a high degree of correct knowledge about their disease, giving between 77% and 94% of correct answers to the different questions. After the booklet, for most of the questions the percentage of correct answers remains the same; when it does change, this is usually for the worse. Simply handing out a booklet doesn't help patients to better understand their disease. On the contrary it may have an opposite effect, inducing some degree of confusion.

TAT, F1 + 2, and D-dimer levels in patients after coronary angiography with nonionic or ionic contrast media or after cardiac catheterization.

The prothrombotic effects of nonionic contrast media (NICM) have been evaluated in both biological and clinical studies. The question of whether there is a higher risk of thromboembolism during angiography with NICM than with ionic contrast media (ICM) has not yet been answered, nor has the precise role of the angiographic procedure per se in such complications been determined. The present study was performed to compare in vivo the potential prothrombotic effects during cardiac angiography of an NICM with those of an ICM, to estimate the effects of the procedure per se, and to assess how long these effects might be maintained. We measured blood levels of three markers of activation of blood coagulation: thrombin-antithrombin III (TAT) complexes, prothrombin fragment 1 + 2 (F1 + 2), and the split product of fibrin, D-dimer, before and after coronary angiography in three groups of patients. In group 1, 14 patients underwent coronary angiography with the NICM iopamidol 370. In group 2, 10 patients underwent coronary angiography with the ICM ioxaglate. In group 3, 10 patients were evaluated immediately after cardiac catheterization, before the injection of contrast material, as controls. No statistically significant differences between the three groups were found in TAT, F1 + 2, or D-dimer levels at different times before and after coronary angiography. There was a trend toward a transient increase in TAT levels after coronary angiography with iopamidol, which at first suggested a possible brief activation of hemostasis with this NICM, but a similar trend was also seen in the control group. We hypothesize that not only the type of contrast material, but also the angiographic procedure per se and patient-related factors all play roles in determining a prothrombotic state during coronary angiography.

Hypoglycemic treatments and the fibrinolytic system in maturity-onset diabetes mellitus without thromboembolic manifestations.

The fibrinolytic system was investigated in 115 patients with maturity-onset diabetes mellitus in good metabolic control and without thromboembolic manifestations. The patients were divided into 7 groups according to the hypoglycemic therapy: diet alone, tolbutamide, glibenclamide, phenformin, combination of the last two drugs, insulin, combination of insulin and phenformin. Our results indicate that in maturity-onset diabetes both fibrinolytic activity and inhibitors of fibrinolysis are increased. The enhanced fibrinolytic activity was not affected by different hypoglycemic drugs, whereas the main antiplasmins showed wide variations in the different treatment groups. In particular, a significant reduction of alpha 2M was observed in patients on glibenclamide therapy. In conclusion, our study showed that the high fibrinolytic state seems to be a constant element in diabetes, and that the different behaviors of the fibrinolytic system, reported by various authors in patients taking different hypoglycemic drugs, could be explained by the wide range of plasmin inhibitor levels observed in these conditions.

Hypoglycemic therapy and hemostasis in maturity- onset diabetes mellitus: existence of an early prethrombotic state.

We studied 115 patients with uncomplicated maturity-onset diabetes mellitus, 55 males and 60 females; patients were divided into 7 groups according to the hypoglycemic therapy (diet alone, tolbutamide, glibenclamide, phenformin, combination of the last two drugs, insulin, combination of insulin and phenformin). All patients were tested for a prethrombotic state by the simultaneous determination of the following parameters: ADP-induced platelet aggregation, Factor VIII as antigen and procoagulant activity and the plasma levels of fibrinogen and antithrobin III. Our results show that these parameters were markedly altered in diabetic patients when compared to apparently normal subjects of the same age; on the contrary, differences between groups of diabetic patients were very slight and hardly ever statistically significant. These results suggest that the prethrombotic alterations of hemostatic system are very similar in all groups of diabetic patients; this is true also for patients submitted to diet alone, i.e. with slight and usually recent-onset diabetes mellitus. In conclusion, our study suggests the opportunity, at least from the theoretical point of view, of systematic antiaggregant and/or anticoagulant treatment(s) in maturity-onset diabetes mellitus, whose severe and precocious prethrombotic alterations seem to be independent of the hypoglycemic treatment applied.