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Tumor-associated carcinoembryonic antigen (CEA) is a natural target for vaccines against colorectal cancers. Our previous experience with a DNA vaccine with scFv6.C4, a CEA surrogate, showed a CEA-specific immune response with 40% of tumor-free mice after challenge with B16F10-CEA and 47% with MC38-CEA cells. These percentages increased to 63% after using FrC as an adjuvant. To further enhance the vaccine efficacy, we tested GM-CSF and IFNγ as adjuvants. C57BL/6J-CEA2682 mice were immunized 4 times with uP-PS/scFv6.C4, uP-PS/scFv6.C4 + uP-IFNγ, or uP-PS/scFv6.C4 + uP-GMCSF. After one week, the mice were challenged with MC38-CEA, and tumor growth was monitored over 100 days. Immunization with scFv6.C4 and scFv6.C4 + GM-CSF resulted in a gradual increase in the anti-CEA antibody titer, while scFv6.C4 + IFNγ immunization led to a rapid and sustained increase in the titer. The addition of IFNγ also induced higher CD4 + and CD8 + responses. When challenged, almost 80% of the scFv6.C4 + IFNγ-vaccinated mice did not develop tumors, while the others had a significant tumor growth delay. The probability of being tumor-free was 2700% higher using scFv6.C4 + IFNγ than scFv6.C4. The addition of GM-CSF had no additional effect on tumor protection. DNA immunization with scFv6.C4 + IFNγ, but not GM-CSF, increased the antitumor effect via readily sustained specific humoral and cytotoxic responses to CEA.

OBJECTIVE The aim of this study was to investigate which factors contribute to the incidence of immature oocytes (germinal vesicle -GV- and metaphase I -MI-) and how they impact the intracytoplasmic sperm injection (ICSI) outcomes of sibling mature oocytes. METHODS Data from 3,920 cycles performed from June/2010 to August/2016 in a private university-affiliated IVF center were evaluated for the influence of controlled ovarian stimulation protocol (COS) on immature oocytes incidence and its effects on ICSI outcomes. RESULTS MI (p=0.004) and GV (p=0.029) number were negatively correlated with gonadotropin dose. Patients stimulated by rFSH had increased GV/oocyte rate in both GnRH agonists (p

To study the implications of sperm DNA fragmentation (SDF) in intracytoplasmic sperm injection cycles for non-male factor infertility.Prospective cohort study.Private university-affiliated IVF center.Data from 475 cycles performed from June 2016 to June 2017.Cycles were divided according to SDF rate into two groups:30% SDF (n = 433) and ≥30% SDF (n = 42). Laboratory and clinical outcomes were compared between groups by generalized linear models adjusted for potential confounders.Embryo quality and miscarriage rates.Fertilization rate was similar between groups (≥30% SDF, 85.28% ± 1.06% vs.30% SDF, 90.68% ± 3.61%). Significantly lower rates of normal cleavage speed (≥30% SDF, 61.12% ± 4.21% vs.30% SDF, 72.53% ± 1.24%), high-quality embryos at day 3 (≥30% SDF, 23.07% ± 5.56% vs.30% SDF, 36.41% ± 1.53%), blastocyst formation (≥30% SDF, 39.09% ± 2.73% vs.30% SDF, 58.83% ± 7.59%), blastocyst quality (≥30% SDF, 11.97% ± 1.22% vs.30% SDF, 30.09% ± 2.39%), and implantation (33.24% ± 1.66% vs.30% SDF, 46.40% ± 4.61%) were observed in cycles with higher SDF, despite similar pregnancy rates (≥30% SDF, 30.40% vs.30% SDF, 32.40%). A 2.5-fold miscarriage rate was observed in cycles with an SDF above the established cutoff (≥30% SDF, 42.8% vs.30% SDF, 16.8%).Higher SDF is correlated with poor embryo development, lower implantation rate, and higher miscarriage rate in non-male factor infertility intracytoplasmic sperm injection cycles. Since defects in sperm may be hidden, the SDF test can bring additional information to the sperm quality evaluation of men with unknown infertility history.

Background Regulatory bodies recommend inconsistent ejaculatory abstinence lengths before semen analysis. The literature exploring the effect of ejaculatory abstinence length on the outcomes of intracytoplasmic sperm injection is scarce. Objective To study the influence of ejaculatory abstinence length on semen quality and intracytoplasmic sperm injection outcomes. Materials and methods This prospective cohort study included 818 patients undergoing conventional semen analysis from October 2015 to October 2016, in a private university-affiliated IVF centre. Generalized linear models adjusted for potential confounders were used to investigate the associations between ejaculatory abstinence length and seminal parameters and intracytoplasmic sperm injection outcomes. Results Increasing ejaculatory abstinence length was positively correlated with semen volume, sperm concentration, total sperm count, total motile sperm count and sperm DNA fragmentation index. Significant inverse correlations were observed between ejaculatory abstinence length and fertilization rate, blastocyst formation rate, implantation rate and pregnancy rate. A discriminant analysis showed a mean ejaculatory abstinence length in the positive pregnancy group of 3.14 ± 1.64 days and 4.83 ± 3.66 days in the negative pregnancy group. A cut-off point was established halfway between ejaculatory abstinence length averages, at 4 days. The ejaculatory abstinence ≤4 days group showed significant lower semen volume, sperm concentration, total sperm count and total motile sperm count compared to ejaculatory abstinence > 4 days group. The ejaculatory abstinence ≤ 4 days group showed significant lower sperm DNA fragmentation index, and higher rates of fertilization, high-quality embryos on day 3, blastocyst development, implantation and pregnancy compared to ejaculatory abstinence > 4 days group. The implantation rate was significantly higher and the pregnancy rate tended to be higher with one day of ejaculatory abstinence, compared to 2-4 days of ejaculatory abstinence. Conclusions Ejaculatory abstinence periods of >4 days have a detrimental effect on sperm DNA and intracytoplasmic sperm injection outcomes. One day of ejaculatory abstinence significantly improves implantation rate and tends to increase pregnancy rate, compared to 2, 3 and 4 days of ejaculatory abstinence.

CD8+ T lymphocytes play an important role in controlling infections by intracellular pathogens. Chemokines and their receptors are crucial for the migration of CD8+ T-lymphocytes, which are the main IFNγ producers and cytotoxic effectors cells. Although the participation of chemokine ligands and receptors has been largely explored in viral infection, much less is known in infection by Trypanosoma cruzi, the causative agent of Chagas disease. After T. cruzi infection, CXCR3 chemokine receptor is highly expressed on the surface of CD8+ T-lymphocytes. Here, we hypothesized that CXCR3 is a key molecule for migration of parasite-specific CD8+ T-cells towards infected tissues, where they may play their effector activities. Using a model of induction of resistance to highly susceptible A/Sn mice using an ASP2-carrying DNA/adenovirus prime-boost strategy, we showed that CXCR3 expression was upregulated on CD8+ T-cells, which selectively migrated towards its ligands CXCL9 and CXCL10. Anti-CXCR3 administration reversed the vaccine-induced resistance to T. cruzi infection in a way associated with hampered cytotoxic activity and increased proapoptotic markers on the H2KK-restricted TEWETGQI-specific CD8+ T-cells. Furthermore, CXCR3 receptor critically guided TEWETGQI-specific effector CD8+ T-cells to the infected heart tissue that express CXCL9 and CXCL10. Overall, our study pointed CXCR3 and its ligands as key molecules to drive T. cruzi-specific effector CD8+ T-cells into the infected heart tissue. The unveiling of the process driving cell migration and colonization of infected tissues by pathogen-specific effector T-cells is a crucial requirement to the development of vaccine strategies., Author summary Chemokine receptors and cell adhesion molecules are essential for T lymphocytes migration into infected tissues. Previously, our group demonstrated that CXCR3 receptor was highly expressed on specific CD8+ T-cells surface after immunization and infection by T. cruzi. Also, recirculation of specific CD8+ T-cells was more important than proliferation to control the infection by T cruzi. As CD8+ T lymphocytes are responsible for controlling T. cruzi infection by releasing IFN-γ or by direct cytotoxicity against infected target cells, our aim was to analyze the role of the chemokine receptor CXCR3 in the migration of specific CD8+ T-cells towards infected tissues. Our results revealed that intervention on CXCR3 by administration of a blocking anti-CXCR3 antibody decreased CD8+ T-cell migration, hampering the access of parasite-specific effector cell into the heart tissue of mice infected by T. cruzi. Therefore, to induce the appropriate migration footmarks is crucial for drive the pathogen-specific effector to sites of infection and, therefore, to clarify this requirement is a crucial strategy for vaccine development.

Objective: This study aimed to look into the use of serum metabolites as potential biomarkers of response to controlled ovarian stimulation (COS) in patients undergoing intracytoplasmic sperm injection (ICSI) cycles. Methods: This case-control study analyzed serum samples from 30 patients aged 25 retrieved oocytes (HR, n=10). The metabolic profiles of the serum samples were compared between the groups through Principal Component Analysis (PCA). Receiver Operating Characteristic (ROC) curves were built to assess the power of the model at predicting response to COS. Results: PCA clearly distinguished between PR, NR and HR, and 10 ions were chosen as potential biomarkers of response to COS. These ions were more specific for PR than for NR. The ROC curve considering PR and NR had an area under the curve of 99.6% (95% CI: 88.9 - 100%). Conclusion: The preliminary evidence discussed in this study suggests that serum metabolites may be used as predictive molecular markers of ovarian response to controlled stimulation. The integration of clinical and “omics” findings may allow the migration toward an era of personalized treatment in reproductive medicine.

Objective To study which factors affect perinatal outcomes in intracytoplasmic sperm injection (ICSI) cycles. Methods Data was obtained from 402 live births born to 307 patients undergoing ICSI cycles in a private university-affiliated IVF center between Jan/2014 and Dec/2015. The influences of the cycles' characteristics on the number of gestational weeks to livebirth (GW), baby birth weight (BW), and baby birth length (BL) were evaluated by linear regression models, adjusted for maternal age and body mass index, number of transferred embryos, number of gestational sacs, and number of born infants. In a subsequent analysis, GW, BW and baby sex were utilized for cycle classification into the groups Appropriate for gestational age (AGA n=256) and Small for gestational age (SGA n=146), which were compared by general linear models adjusted for the same confounder variables. Results The number of follicles (β=-0.069 p=0.018) and retrieved oocytes (β=-0.087 p=0.049) were negatively correlated with BL. The endometrial thickness was positively correlated with GW (β=0.198 p=0.003) and BW (β=28.351 p=0.044). When each baby was classified into AGA and SGA groups, it was observed that SGA babies were derived from cycles with higher estradiol levels at hCG day (SGA: 3897.01±550.35 vs. AGA: 2324.78±101.86 p=0.006) and higher number of retrieved oocytes (SGA: 16.70±1.78 vs. AGA: 12.92±0.42 p=0.042). The endometrial thickness was significantly lower in the SGA group (SGA: 10.2±0.23 vs. AGA: 11.68±0.17 vs. p=0.029). Conclusion Higher ovarian response to stimulation and suboptimal endometrial development are associated with adverse perinatal outcomes in ICSI cycles.

Objective To describe the cases of preimplantation genetic testing for monogenic diseases (PGT-M) in fertile couples who had undergone intracytoplasmic sperm injection (ICSI) cycles in a Brazilian in vitro fertilisation (IVF) centre and determine whether these cases were different from those reported from the European Society of Human Reproduction and Embryology (ESHRE). Methods This retrospective collection included data obtained from ICSI-PGT-M cycles between 2011 and 2016. The disease indication, number of biopsied embryos, biopsy stage, diagnosed and affected embryos, and cycles with embryo to transfer as well as implantation, pregnancy and miscarriage rates were analysed and compared to cycles without genetic diagnosis (PGT) and with ESHRE PGD Consortium collection XIV-XV. Results From 5,070 cycles performed, 72 had indications for PGT-M. The most common time for biopsy was cleavage-stage; 93% of the embryos had a diagnostic result, 59.4% of which were genetically transferable, resulting in 68% of the cycles with transferred embryos, a 22.1% implantation rate, and a 28.6% pregnancy rate. No differences in clinical outcomes of cycles with PGT-M or without PGT were observed. The day of biopsy and diagnostic success as well as implantation, pregnancy and miscarriage rates were similar to ESHRE collection. Conclusions Although the proportion of cases with PGT-M was low, its efficacy was similar to what was reported in the European collection and represents a viable alternative for families at risk of transmitting a genetic disorder to their offspring. The main difference between our and ESHRE collection were the disease indications, which reflected the admixed, multi-ethnic Brazilian population.

The carcinoembryonic antigen (CEA) is the main tumor-associated antigen of colorectal cancers. Previously, we developed a DNA vaccine using scFv6.C4, a CEA surrogate, against CEA-expressing tumors; 40% of the vaccinated mice were tumor-free after tumor challenge. In order to enhance vaccine efficacy, fragment C of Tetanus Toxin (FrC) was tested as adjuvant. C57BL/6J-CEA2682 mice were electroporated intramuscularly 4 times with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4, challenged by s.c. injection of 1 × 105 MC38-CEA cells, and tumor growth was monitored over 100 days. The humoral and cellular immune responses were assessed by ELISA, immunocytochemistry, in-vitro lymphocyte proliferation, and CTL cytotoxicity assays. Immunization with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4 induced similar anti-CEA antibody titers. However, immunocytochemistry analysis showed stronger staining with uP-PS/scFv6.C4-FrC-immunized mice sera. When challenged with MC38-CEA cells, 63% of the FrC-vaccinated mice did not develop tumors, half of the rest had a significant tumor growth delay, and the probability of being free of tumors was on average 40% higher than that of scFv6.C4-immunized mice. Addition of the adjuvant led to higher CD4+ and CD8+ proliferative responses and strong CD8+ CTL response against MC38-CEA cells. DNA immunization with scFv6.C4 and FrC increased antitumor effect via induction of high and specific humoral and cellular immune responses to CEA.

The aim of this study was to investigate which factors contribute to the incidence of biochemical pregnancy (BP) in intracytoplasmic sperm injection (ICSI) cycles. This cohort study included cycles performed from June 2010 to September 2016 in a private, university-affiliated IVF centre. Cycles were split into four groups, depending on the pregnancy outcomes: Clinical Pregnancy (CP, n = 903), Biochemical Pregnancy (BP, n = 55), Miscarriage (MI, n = 142) and Negative Pregnancy (NP, n = 2034). The effects of ovarian stimulation, laboratory data and seminal parameters on pregnancy outcomes were evaluated using adjusted general linear models. Discriminant analyses were conducted to construct a model for pregnancy prediction and to establish cut-offs for BP. The total sperm count (p = 0.035), total and progressive sperm motility (p = 0.001 and p = 0.023, respectively), total motile sperm count (TMSC, p = 0.029) and the endometrial thickness (p 0.001) were lower among BP group cycles. Lower rates of high-quality cleavage-stage embryos were observed in the BP group compared to CP and MI groups (p 0.001). In discriminant analyses, cut-offs for BP prediction were established for the following factors: endometrial thickness11 mm, sperm motility55.5% and total dose of follicle-stimulating hormone (FSH)2400 IU. The incidence of biochemical pregnancy was four times higher when the aforementioned factors did not meet the defined cut-offs. The combination of suboptimal endometrial development and poor seminal and embryo quality contribute to an increased incidence of biochemical pregnancy in ICSI cycles.

For the present study we asked whether the endometrial fluid lipidomic may be a useful approach to predict endometrial receptivity in freeze-all cycles. For this case-control study, endometrial fluid samples were collected from 41 patients undergoing freeze-all cycles. Samples were split depending on the pregnancy outcome: positive group (n = 24) and negative group (n = 17). Data were acquired by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were applied. A list of potential biomarker ion ratios was obtained and the values were used to build a receiver operating characteristic (ROC) curve to predict pregnancy success. The lipid categories were attributed by LIPID MAPS database. Ion ratios were established according to their correlations and used for the analysis. The PCA showed a tendency of separation between the studied groups, whereas the PLS-DA was able to clearly distinguish them. Fifteen ratios (13 hyper-represented in the negative and two hyper-represented in the positive group) were selected according to their importance for model prediction. These ratios were used to build the ROC curve, which presented an area under curve of 84.0% (95%CI: 69.2-97.4%; p = 0.009). These findings suggest that lipidomic profiling of endometrial fluid may be a valuable tool for identifying the time interval comprising the window of implantation.

To investigate the effect of perivitelline space (PVS) abnormalities on the outcomes of intracytoplasmic sperm injection (ICSI) cycles in which the entire cohort was affected.Data from 9752 oocytes obtained from 1151 ICSI cycles performed from June/2010 to August/2016 in a private university-affiliated IVF centre. Cycles were divided into four groups according to the presence or absence of PVS abnormalities: PVS-L group (cycles with the entire oocyte cohort affected by large PVS, n = 265), PVS-G group (cycles with the entire oocyte cohort affected by PVS granularity, n = 280), PVS-L + PVS-G group (cycles with the entire oocyte cohort affected by PVS-L and PVS-G, n = 204), and control group (cycles with the entire oocyte cohort free of PVS abnormalities, n = 402). The effect of PVS abnormalities on ICSI outcomes was assessed by GLM adjusted for potential confounders.Groups with PVS abnormalities presented substantially higher FSH/follicle (p 0.001) and FSH/oocyte (p 0.001) ratios, and lower numbers of follicles (p 0.001), oocytes (p 0.001) and embryos (p = 0.002) compared to the control group. PVS-L + PVS-G implantation (p = 0.044) and pregnancy (p = 0.004) rates were significantly lower than in cycles with isolated PVS abnormalities and controls.Cycles in which the entire oocyte cohort is affected by both large PVS and PVS granularity have compromised implantation and pregnancy rates.

Human sperm morphology has been described as an essential parameter for the diagnosis of male infertility and a prognostic indicator of natural or assisted pregnancies. Nevertheless, standard morphological assessment remains a subjective analysis and its impact on intracytoplasmic sperm injection (ICSI) is also of limited value. The objective of this prospective cohort study was to investigate whether motile sperm organelle morphology examination (MSOME) can improve semen analysis by better defining male infertility and providing a better prognosis for ICSI up to a year later. Data were obtained from 483 patients undergoing conventional semen analysis from June 2015 to June 2017 in a private university-affiliated in vitro fertilization (IVF) center. The correlation of MSOME with seminal parameters was evaluated. One hundred and thirty patients underwent ICSI up to a year later, and the correlation between MSOME and ICSI outcomes was established. Except for volume, all seminal parameters were positively correlated with MSOME I+II. MSOME was also distinct between World Health Organization (WHO) classification groups, with normozoospermic and oligoasthenoteratozoospermic presenting the higher and the lower proportion of MSOME I+II, respectively. MSOME I+II was prognostic for fertilization rate, high-quality cleavage-stage embryos rate, and blastocyst rate. The normality cutoff value based on blastocyst rate was MSOME I+II≥ 5.5%. MSOME could be a useful tool for the diagnosis of infertility severity as it is correlated with sperm morphology, motility, and concentration. Men who had higher MSOME I+II had better ICSI outcomes. The future use of MSOME as a routine method for semen analysis may be a reliable form of assessing male infertility.

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells.

Summary Objective: To evaluate the effect of male factor infertility on intracytoplasmic sperm injection (ICSI) outcomes compared with a control group presenting isolated tubal factor. Method: This retrospective study included 743 couples undergoing ICSI as a result of isolated male factor and a control group consisting of 179 couples undergoing ICSI as a result of isolated tubal factor, performed in a private university- -affiliated in vitro fertilization center, between January/2010 and December/2016. Patients were divided into two groups according to maternal age: women ≤35 years old and >35 years old. The effects of infertility causes on laboratorial and clinical ICSI outcomes were evaluated using Student's t-test and (2 test. Results: No differences in controlled ovarian stimulation outcomes were observed between male factor cycles and tubal factor cycles in the two age groups. Implantation (male factor 35.5% vs. tubal factor 32.0%, p=0.340), pregnancy (male factor 46.9% vs. tubal factor 40.9%, p=0.184) and miscarriage (male factor 10.3% vs. tubal factor 10.6%, p=0.572) rates were similar between the infertility groups, irrespective of female age. Considering maternal age, the cancelation rate was higher in older women (>35 years old) undergoing ICSI as a result of male factor infertility (17.4% vs. 8.9%, p=0.013). Conclusion: Our results showed that there is no difference in the outcomes of pregnancy between couples with male or tubal factor infertility, which indicates that ICSI surpasses the worse specific outcomes associated with male factor. Resumo Objetivo: Avaliar o efeito do fator masculino de infertilidade em resultados de injeção intracitoplasmática de espermatozoides (ICSI) em comparação com um grupo controle que apresenta o fator tubário isolado. Método: Este estudo retrospectivo incluiu 743 casais submetidos a ICSI por fator masculino e 179 casais por fator tubário, realizada em um centro privado de fertilização in vitro associado à universidade, entre janeiro de 2010 e dezembro de 2016. Os pacientes foram divididos em dois grupos de acordo com a idade materna: mulheres ≤ 35 e > 35 anos de idade. Os efeitos das causas de infertilidade nos resultados laboratoriais e clínicos da ICSI foram avaliados pelos testes T de Student e Qui-quadrado. Resultados: Não foram observadas diferenças nos parâmetros de estimulação ovariana entre os ciclos com fatores masculinos e com fatores tubários. A taxa de implantação (fator masculino 35,5% vs. fator tubário 32,0%, p=0,340), de gravidez (fator masculino 46,9% vs. fator tubário 40,9%, p=0,184) e de aborto (fator masculino 10,3% vs. fator tubário 10,6%, p=0.572) foram semelhantes entre os grupos de infertilidade, independentemente da idade feminina. Considerando a idade materna, a taxa de cancelamento foi maior em mulheres > 35 anos cuja causa de infertilidade era o fator masculino (17,4% vs. 8,9%, p=0,013). Conclusão: Não há diferenças nos resultados de gravidez entre casais com infertilidade dos fatores masculino ou tubário isolados, o que indica que ICSI supera os piores resultados associados ao fator masculino.

Objective To determine the effect of FSH doses on intracytoplasmic sperm injection (ICSI) outcomes according to the age of the patient. Methods Patients undergoing controlled ovarian stimulation (COS) for ICSI cycles in a university-affiliated in vitro fertilization center were split into age groups: ≤35 y.o. (n=1523); >35 and ≤38 y.o. (n=652); >38 and ≤40 y.o. (n=332); and >40 y.o. (n=370). The effect of FSH dose on COS, laboratorial and clinical outomes was determined by linear regression models. Results The FSH dose didn't affect the ovarian response in terms of total number of follicles, retrieved oocytes and mature oocytes within the age groups, but we found that the lower the age, the lower the FSH dose needed per oocyte retrieved. In the group of patients ≤35 y.o., we also found a positive effect of the FSH dose on oocyte yield. Despite that, for patients ≤38 y.o. there was a negative effect of the FSH dose on embryo quality and blastocyst formation rate, and an increase in the cycle's cancelation rate. In patients ≥39 y.o., there were no effects of the FSH doses on the analysed variables. Conclusions Ovarian stimulation with high doses of FSH is not recommended in younger women (≤38 y.o.), once we found a decrease in embryo quality and an increase in cycle's cancelation rate. Mild ovarian stimulation protocols may be more appropriate; however, it may not be applicable for women in advanced age, since a higher FSH dose is needed for oocyte retrieval in these patients.

Objective: To discuss the requirement from the National Health Surveillance Agency(ANVISA), for assisted reproduction treatment patients to undergo laboratory tests for ZIKV detection, and if the public health authorities and government leaders' recommendations to women simply avoid pregnancy is prudent. Methods: This study was performed in a universityaffiliated in vitro fertilization center in Brazil. We present a critical discussion on the risk of microcephaly due to ZIKV infection and the prevalence of other harmful pathogens to vulnerable pregnant women and infants. We assessed, 954 patients undergoing intracytoplasmic sperm injection cycles(ICSI), between April and November of 2016, concerning the results of ZIKV test, according to different regions in Brazil. Results: Patients undergoing ICSI cycles were split into groups, according to their region of origin: 28(3.0%) were from the North, 27(2.8%) were from the Northeast, 40(4.2%) were from the Midwest, 830(87.2%) were from the Southeast, and 29(3.0%) were from the South. Concerning the diagnosis, 112 samples had a positive or inconclusive result for ZIKV, by chromatography immunoassay. These samples were re-analyzed by ELISA and no result was positive. All positive results were from the Southeast region and none from the Northeast or Midwest regions, which are considered endemic regions. Conclusion: ZIKV test before the onset of assisted reproduction treatments does not rule out the risk of the infection during pregnancy. In addition, although ZIKV infection risk is extremely high, the microcephaly risk due to ZIKV is not higher than the risk of miscarriage and birth defects due to other recognized pathogens. Fertil Med Grp, Sao Paulo, SP, Brazil Ctr Estudos & Pesquisa Reprod Humana Assistida, Inst Sapientiae, Sao Paulo, SP, Brazil Univ Fed Sao Paulo UNIFESP, Dept Cirurgia, Area Reprod Humana, Disciplina Urol, Sao Paulo, Brazil Univ Fed Sao Paulo UNIFESP, Dept Cirurgia, Area Reprod Humana, Disciplina Urol, Sao Paulo, Brazil Web of Science

Carcinoembryonic antigen (CEA) is expressed during embryonic life and in low level during adult life. Consequently, the CEA is recognized by the immune system as a self-antigen and thus CEA-expressing tumors are tolerated. Previously, we constructed a single chain variable fragment using the 6.C4 (scFv6.C4) hybridoma cell line, which gave rise to antibodies able to recognize CEA when C57/Bl6 mice were immunized. Here, the scFv6.C4 ability to prevent the CEA-expressing tumor growth was assessed in CEA-expressing transgenic mice CEA2682. CEA2682 mice immunized with the scFv6.C4 expressing plasmid vector (uP/PS-scFv6.C4) by electroporation gave rise to the CEA-specific AB3 antibody after the third immunization. Sera from immunized mice reacted with CEA-expressing human colorectal cell lines CO112, HCT-8, and LISP-1, as well as with murine melanoma B16F10 cells expressing CEA (B16F10-CEA). Cytotoxic T lymphocytes (CTL) from uP/PS-scFv6.C4 immunized mice lysed B16F10-CEA (56.7%) and B16F10 expressing scFv6.C4 (B16F10-scFv6.C4) (46.7%) cells, against CTL from uP-immunized mice (10%). After the last immunization, 5 × 105 B16F10-CEA cells were injected into the left flank. All mice immunized with the uP empty vector died within 40 days, but uP/PS-scFv6.C4 vaccinated mice (40%) remained free of tumor for more than 100 days. Splenocytes obtained from uP/PS-scFv6.C4 vaccinated mice showed higher T-cell proliferative activity than those from uP vaccinated mice. Collectively, DNA vaccination with the uP-PS/scFv6.C4 plasmid vector was able to give rise to specific humoral and cellular responses, which were sufficient to retard growth and/or eliminate the injected B16F10-CEA cells.

Glioblastoma (GBM) is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs) were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 μM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells.

The large-scale production of cardiomyocytes is a key step in the development of cell therapy and tissue engineering to treat cardiovascular diseases, particularly those caused by ischemia. the main objective of this study was to establish a procedure for the efficient production of cardiomyocytes by reprogramming mesenchymal stem cells from adipose tissue. First, lentiviral vectors expressing neoR and GFP under the control of promoters expressed specifically during cardiomyogenesis were constructed to monitor cell reprogramming into precardiomyocytes and to select cells for amplification and characterization. Cellular reprogramming was performed using 5'-azacytidine followed by electroporation with plasmid pOKS2a, which expressed Oct4, Sox2, and Klf4. Under these conditions, GFP expression began only after transfection with pOKS2a, and less than 0.015% of cells were GFP(+). These GFP(+) cells were selected for G418 resistance to find molecular markers of cardiomyocytes by RT-PCR and immunocytochemistry. Both genetic and protein markers of cardiomyocytes were present in the selected cells, with some variations among them. Cell doubling time did not change after selection. Together, these results indicate that enrichment with vectors expressing GFP and neoR under cardiomyocyte-specific promoters can produce large numbers of cardiomyocyte precursors (CMPs), which can then be differentiated terminally for cell therapy and tissue engineering. Universidade Federal de São Paulo, Gene Therapy Invest Ctr, Dept Biophys, BR-04044010 São Paulo, Brazil Universidade Federal de São Paulo, Dept Surg, BR-04044010 São Paulo, Brazil Universidade Federal de São Paulo, Gene Therapy Invest Ctr, Dept Biophys, BR-04044010 São Paulo, Brazil Universidade Federal de São Paulo, Dept Surg, BR-04044010 São Paulo, Brazil Web of Science

Citrus canker is one of the most important agricultural citrus diseases worldwide. It is caused by Xanthomonas citri subsp. citri (Xcc) bacterium that infects leaves and the fruits produce a cysteine peptidase (CPXaC), which makes it a potential target for the development of effective and rapid detection methods for citrus canker. We report here the studies on the development of piezoelectric immunoassay for CPXaC using a polyclonal antibody against CPXaC (anti-CPXaC). Three different strategies for covalent immobilization of anti-CPXaC on gold surfaces were evaluated by monitoring the frequency (Δf) and energy dissipation (ΔD) variation in real time when 64.5×10−8 mol L−1 CPXaC was added. Anti-CPXaC immobilized with 11-mercaptoundecanoic acid (MUA) showed the best relation between the frequency and dissipation factor variation, and strong values for the kinetic and equilibrium binding constant were obtained. The immunosensor showed a detection limit of 13.0 nmol L−1 with excellent specificity, showing no response for different proteins that include another cysteine peptidase that is used as a target to detect Xylella fastidiosa bacterium, responsible for another important citrus disease. These results provide good perspectives for the use of CPXaC as a new biomarker for citrus canker.

Glioblastoma (GBM) is the most common and lethal primary intracranial tumor. Because GBM is highly invasive and diffusely infiltrates the brain, a complete resection of the tumor is unfeasible. The gene encoding thymidine kinase from herpes simplex virus-1 (HSV-Tk) is the most commonly used suicide gene in preclinical and clinical trials against GBM. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. Aiming at using this strategy in clinical trials, here we evaluated the abundantly present human adipose-tissue-derived mesenchymal stem cells (AT-MSC) to treat GBM in mouse model.AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal proteins and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 μM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection and treated with GCV drastically reduced tumors to smaller than 0.5 mm2. View Large Image | Download PowerPoint SlideImmunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, indicating specific migration of AT-MSC-HSV-Tk to and interaction with tumours. View Large Image | Download PowerPoint SlideIn conclusion, the AT-MSCs are good carriers of the suicide HSV-Tk gene for the treatment of GBM, and the abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.