C.M. Gomes

Influence of vitrification on mouse metaphase II oocyte spindle dynamics and chromatin alignment

OBJECTIVE To evaluate influences of vitrification and warming of metaphase II (MII) mouse oocytes on survival, spindle dynamics, spindle morphology, and chromatin alignment on metaphase plates. DESIGN Experimental animal study. SETTING University animal laboratory. ANIMAL(S) Eight-week-old B6D2F1 mice. INTERVENTION(S) Denuded MII oocytes were used fresh (control), exposed to vitrification/warming solutions (Sol Expos), or vitrified and warmed (Vitr). MAIN OUTCOME MEASURE(S) Oocyte recovery and survival after warming and the influence of solution exposure and cryopreservation on spindle dynamics and chromatin alignment. RESULT(S) Cryopreservation of two or 10 oocytes per straw resulted in recovery (100% +/- 0% and 95% +/- 4%, respectively; mean +/- SE) and survival (95% +/- 2% and 98% +/- 2%, respectively). Immediately after warming (Vitr), significantly fewer oocytes assessed with immunocytochemistry contained spindles, compared with control and Sol Expos. When oocytes were placed into a 37 degrees C environment for 2 hours after exposure or warming, the ability to recognize spindles by immunocytochemistry was not significantly different between groups. Using live-cell time-lapse imaging with LC-Polscope, similar time-dependent spindle formation dynamics were observed. At 2 hours after collection or treatment, spindle morphology and length were not significantly different between the groups, nor was the incidence of aberrant alignment of chromatin on metaphase plates. CONCLUSION(S) Immediately after warming of vitrified MII oocytes, beta-tubulin is depolymerized and chromatin remains condensed on the metaphase plate. Within a 2-hour period, beta-tubulin repolymerizes, forming morphologically normal metaphase spindles with properly aligned chromatin.

A prospective study on the dynamics of the clinical and immunological evolution of human Leishmania (L.) infantum chagasi infection in the Brazilian Amazon region

This prospective study was carried out from October 2003 to December 2005 and involved a cohort of 946 individuals of both genders, aged 1-89 years, from an endemic area for American visceral leishmaniasis (AVL), in Pará State, Brazil. The aim of the study was to analyze the dynamics of the clinical and immunological evolution of human Leishmania (L.) infantum chagasi infection represented by the following clinical-immunological profiles: asymptomatic infection (AI); symptomatic infection (SI=AVL); subclinical oligosymptomatic infection (SOI); subclinical resistant infection (SRI); and indeterminate initial infection (III). Infection diagnosis was determined by the indirect fluorescent antibody test and leishmanin skin test. In total, 231 cases of infection were diagnosed: the AI profile was the most frequent (73.2%), followed by SRI (12.1%), III (9.9%), SI (2.6%) and SOI (2.2%). The major conclusion regarding evolution dynamics was that the III profile plays a pivotal role from which the cases evolve to either the resistant, SRI and AI, or susceptible, SOI and SI, profiles; only one of the 23 III cases evolved to SI, while most evolved to either SRI (nine cases) or SOI (five cases) and eight cases remained as III.

A cross-sectional study on the clinical and immunological spectrum of human Leishmania (L.) infantum chagasi infection in the Brazilian Amazon region

The objectives of this study were to identify individuals with symptomatic and/or asymptomatic infection due to Leishmania (L.) infantum chagasi; to study the two types of infection, both clinically and immunologically, and to determine the prevalence rate of infection at the beginning of the study. This was a cross-sectional study with a cohort of 946 individuals, of both genders, from the age of 1 year, living in the municipality of Barcarena, PA, Brazil, an area endemic for American visceral leishmaniasis (AVL). The leishmanin skin test (LST) and the indirect fluorescent test (IFAT), were used for the diagnosis of infection. One hundred and twenty cases of infection were diagnosed, with a prevalence rate of 12.6%; eight cases showed high seroreactivity (1280-10240, IgG) in IFAT and no LST reaction; four of these cases were typical AVL and four had subclinical oligosymptomatic infection. Using two immunological methods with a clinical examination of the infected individuals enabled the identification of five clinical-immunological profiles which may promote a better understanding of the interaction between L. (L.) i. chagasi and the human immune response: asymptomatic infection (AI) 73.4%; subclinical resistant infection (SRI) 15%; subclinical oligosymptomatic infection (SOI) 3%; symptomatic infection (AVL) 3% and indeterminate initial infection (III) 5%.

Insulin‐like Growth Factor‐I Induces Phosphorylation in Leishmania {Leishmania) mexicana Promastigotes and Amastigotes

Protein phosphorylation controls major steps of proliferation and differentiation in eukaryotic cells. However there are few studies done in protozoa particularly when being triggered by external stimuli. In this paper we have examined the tyrosine‐ and serine/threonine‐phosphorylated proteins in both promastigote and amastigote‐like forms of Leishmania (Leishmania) mexicana stimulated with insulin‐like growth factor (IGF)‐I. Stimulation with IGF‐I induces major tyrosine phosphorylation of a 185‐kDa protein in promastigotes and 60‐ and 40‐kDa proteins in amastigotes. Analysis of total phosphorylation revealed additional sets of phosphorylated proteins: a 110‐kDa protein band in promastigotes and two other proteins of 120 and 95 kDa in the amastigote‐like forms. To further analyze the IGF‐I‐mediated response we compared it with the phosphorylation pattern obtained with a known inducer of protein kinase C, phorbol myristate acetate. This analysis showed overlapping phosphorylation of most of the proteins but mainly of the 185‐ and 110‐kDa proteins in the promastigotes and the 95‐. 60‐ and 40‐kDa proteins in the amastigote‐like forms. We thus conclude that there are phosphorylation‐dependem pathways in Leishmania parasites induced by IGF‐I that are stage‐specific.

Reviewing the role of the dendritic Langerhans cells in the immunopathogenesis of American cutaneous leishmaniasis

The role of dendritic Langerhans cells (LCs) in the immunopathogenesis of American cutaneous leishmaniasis (ACL) was reviewed in the light of more recent clinical and immunological features of ACL, caused by the principal human pathogenic leishmanial parasites found in Brazil: Leishmania (Viannia) braziliensis and L. (L.) amazonensis. The report shows a species-specific correlation between the LC density and the CD4+ and CD8+ T-cell profiles in the cellular infiltrate of skin lesions of ACL patients, providing the conclusion that LCs might be influencing the dichotomy of interaction between L. (V.) braziliensis and L. (L.) amazonensis with the human T-cell immune response. While L. (V.) braziliensis shows a clear tendency to direct infection to the hypersensitivity pole of the ACL clinical-immunological spectrum marked by a strong Th1-type immune response, L. (L.) amazonensis shows the opposite, directing infection to the hyposensitivity pole associated with a marked Th2-type immune response. These are probably the main immunological mechanisms of LCs regarding the immune response dichotomy that modulates infection outcome by these Leishmania parasites.

A case of oocyte and embryo vitrification resulting in clinical pregnancy

OBJECTIVE To examine a case of clinical pregnancy following oocyte and day 3 embryo vitrification. DESIGN Case report. SETTING Huntington Centro de Medicina Reprodutiva, a private infertility clinic, in São Paolo, Brazil. PATIENT(S) A 31-year-old woman, gravida 1, para 1, with polycystic ovary syndrome. INTERVENTION(S) In vitro fertilization with oocyte and embryo vitrification. MAIN OUTCOME MEASURE(S) Clinical pregnancy defined as fetal cardiac activity on ultrasound examination. RESULT(S) Transvaginal ultrasound examination, at 7 6/7 weeks, revealed a single live intrauterine pregnancy with positive cardiac activity. CONCLUSION(S) Although further research is needed, this case suggests that repeat vitrification and warming of oocytes and embryos not only are possible but can result in pregnancy.

Leishmania sp. identification by PCR associated with sequencing of target SSU rDNA in paraffin-embedded skin samples stored for more than 30 years.

Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocol was applied to identify Leishmania strains in 33 paraffin-embedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.

Oocyte meiotic-stage-specific differences in spindle depolymerization in response to temperature changes monitored with polarized field microscopy and immunocytochemistry

OBJECTIVE To compare the polymerization status of mouse oocyte spindles exposed to various temperatures at various stages of meiosis. DESIGN Experimental animal study. SETTING University animal laboratory. ANIMAL(S) CF1 mice. INTERVENTION(S) Immature oocytes matured to metaphase I (MI), telophase I (TI), and metaphase II (MII) were incubated at 37 °C (control), room temperature (RT), or 4 °C for 0, 10, 30, and 60 minutes. Spindle analysis subsequently was performed using polarized field microscopy and immunocytochemistry. Spindles of TI and MII oocytes that underwent vitrification and warming were analyzed also by immunocytochemistry. MAIN OUTCOME MEASURE(S) Detection of polymerized meiotic spindles. RESULT(S) At RT, and after 60 minutes at 4 °C, a significant time-dependent decrease in the percentage of polymerized meiotic spindles was observed in MI and MII oocytes, but not in TI oocytes. The polymerization of TI spindles at 4 °C was similar to that of TI spindles at 4 °C that underwent vitrification and warming. CONCLUSION(S) Significant differences in the microtubule dynamics of MI, TI, and MII oocytes incubated at different temperatures were observed. In particular, meiotic spindles in TI oocytes exhibited less depolymerization than did metaphase spindles.

Administration of a pharmacophysiologic dose of recombinant human chorionic gonadotropin at menses promotes corpus luteum rescue.

material removed at the second vacuum aspiration. This is the second reported case of fallopian tube incarceration, but the first in which the complication was suspected at the time of the procedure and the injury was repaired shortly after the incarceration. In the first case the patient underwent hysteroscopy and laparoscopy 5 years after the surgical termination [3]. When uterine perforation is suspected based on an abnormal finding in the retrieved material, although rare, tubal incarceration must be considered—especially because it can be relatively asymptomatic. Prompt diagnosis may lead to preservation of the incarcerated tube.

Leishmania amazonensis downregulates macrophage iNOS expression via Histone Deacetylase 1 (HDAC1): a novel parasite evasion mechanism

The induced expression of nitric oxide synthase (iNOS) controls the intracellular growth of Leishmania in infected macrophages. Histones deacetylases (HDACs) negatively regulate gene expression through the formation of complexes containing transcription factors such as NF‐κB p50/50. Herein, we demonstrated the occupancy of p50/p50_HDAC1 to iNOS promoter associated with reduced levels of H3K9Ac. Remarkably, we found increased levels of HDAC1 in L. amazonensis‐infected macrophages. HDAC1 upregulation was not found in L. major‐infected macrophages. The parasite intracellular load was reduced in HDAC1 knocked‐down macrophages, which presented increased nitric oxide levels. HDAC1 silencing led to the occupancy of CBP/p300 to iNOS promoter and the rise of H3K9Ac modification. Importantly, the immunostaining of skin samples from hiporeactive cutaneous leishmaniasis patients infected with L. amazonensis, revealed high levels of HDAC1. In brief, L. amazonensis induces HDAC1 in infected macrophages, which contribute to parasite survival and is associated to hiporeactive stage found in L. amazonensis infected patients.