Preferential induction of a Th1 immune response and inhibition of specific IgE antibody formation by plasmid DNA immunization.

We compared the antigen-specific antibody isotypes and lymphokine secretion by CD4+ T cells in BALB/c mice immunized intradermally with either Escherichia coli beta-galactosidase (beta-gal) or plasmid DNA (pDNA) encoding beta-gal in a cytomegalovirus-based expression vector (pCMV-LacZ). pCMV-LacZ induced mainly IgG2a, whereas beta-gal in saline or alum induced IgG1 and IgE beta-gal-specific antibodies. In addition, splenic CD4+ T helper (Th) cells isolated from pDNA-immunized mice secreted interferon-gamma but not interleukin (IL)-4 and IL-5, whereas Th cells from beta-gal-injected mice secreted IL-4 and IL-5 but not interferon-gamma after in vitro stimulation with antigen. Together these data demonstrate that pDNA immunization induced a T helper type 1 (Th1) response, whereas protein immunization induced a T helper type 2 (Th2) response to the same antigen. Interestingly, priming of mice with pCMV-LacZ prevented IgE antibody formation to a subsequent i.p. beta-gal in alum injection. This effect was antigen-specific, because priming with pCMV-LacZ did not inhibit IgE anti-ovalbumin antibody formation. Most importantly, intradermal immunization with pCMV-LacZ (but not pCMV-OVA) of beta-gal in alum-primed mice caused a 66-75% reduction of the IgE anti-beta-gal titer in 6 weeks. Also, pCMV-LacZ induced specific IgG2a antibody titers and interferon-gamma secretion by Th cells in the beta-gal in alum-primed mice. The data demonstrate that gene immunization induces a Th1 response that dominates over an ongoing protein-induced Th2 response in an antigen-specific manner. This suggests that immunization with pDNA encoding for allergens may provide a novel type of immunotherapy for allergic diseases.

Human immunodeficiency virus type 1 infection despite prior immunization with a recombinant envelope vaccine regimen.

With efforts underway to develop a preventive human immunodeficiency virus type 1 (HIV-1) vaccine, it remains unclear which immune responses are sufficient to protect against infection and whether prior HIV-1 immunity can alter the subsequent course of HIV-1 infection. We investigated these issues in the context of a volunteer who received six HIV-1LAI envelope immunizations and 10 weeks thereafter acquired HIV-1 infection through a high-risk sexual exposure. In contrast to nonvaccinated acutely infected individuals, anamnestic HIV-1-specific B- and T-cell responses appeared within 3 weeks in this individual, and neutralizing antibody preceded CD8+ cytotoxic responses. Despite an asymptomatic course and an initial low level of detectable infectious virus, a progressive CD4+ cell decline and dysfunction occurred within 2 years. Although vaccination elicited immunity to HIV-1 envelope, which was recalled upon HIV-1 exposure, it was insufficient to prevent infection and subsequent immunodeficiency.

Male fetal progenitor cells persist in maternal blood for as long as 27 years postpartum.

Rare nucleated fetal cells circulate within maternal blood. Noninvasive prenatal diagnosis by isolation and genetic analysis of these cells is currently being undertaken. We sought to determine if genetic evidence existed for persistent circulation of fetal cells from prior pregnancies. Venous blood samples were obtained from 32 pregnant women and 8 nonpregnant women who had given birth to males 6 months to 27 years earlier. Mononuclear cells were sorted by flow cytometry using antibodies to CD antigens 3, 4, 5, 19, 23, 34, and 38. DNA within sorted cells, amplified by PCR for Y chromosome sequences, was considered predictive of a male fetus or evidence of persistent male fetal cells. In the 32 pregnancies, male DNA was detected in 13 of 19 women carrying a male fetus. In 4 of 13 pregnancies with female fetuses, male DNA was also detected. All of the 4 women had prior pregnancies; 2 of the 4 had prior males and the other 2 had terminations of pregnancy. In 6 of the 8 nonpregnant women, male DNA was detected in CD34+CD38+ cells, even in a woman who had her last son 27 years prior to blood sampling. Our data demonstrate the continued maternal circulation of fetal CD34+ or CD34+CD38+ cells from a prior pregnancy. The prolonged persistence of fetal progenitor cells may represent a human analogue of the microchimerism described in the mouse and may have significance in development of tolerance of the fetus. Pregnancy may thus establish a long-term, low-grade chimeric state in the human female.

Maternal plasma human immunodeficiency virus type 1 RNA level: a determinant and projected threshold for mother-to-child transmission.

To prevent mother-to-child human immunodeficiency virus type 1 (HIV-1) transmission, it is important to identify its determinants. Because HIV-1 RNA levels can be reduced by antiviral therapy, we examined the role of maternal plasma HIV-1 RNA level in mother-to-child transmission. We used quantitative competitive PCR to measure HIV-RNA in 30 infected pregnant women and then followed their infants prospectively; 27% of the women transmitted HIV-1 to their infants and maternal plasma HIV-1 RNA level correlated strikingly with transmission. Eight of the 10 women with the highest HIV-1 RNA levels at delivery (190,400-1,664,100 copies per ml of plasma) transmitted, while none of the 20 women with lower levels (500-155,800 copies per ml) did (P = 0.0002). Statistical analysis of the distribution of HIV-1 RNA loads in these 30 women projected a threshold for mother-to-child transmission in a larger population; the probability of a woman with a viral RNA level of < or = 100,000 copies per ml not transmitting is predicted to be 97%. Examination of serial HIV-1 RNA levels during pregnancy showed that viral load was stable in women who did not initiate or change antiviral therapy. These data identify maternal plasma HIV-1-RNA level as a major determinant of mother-to-child transmission and suggest that quantitation of HIV-1 RNA may predict the risk of transmission.

Systemic immunization with papillomavirus L1 protein completely prevents the development of viral mucosal papillomas.

Infection of mucosal epithelium by papillomaviruses is responsible for the induction of genital and oral warts and plays a critical role in the development of human cervical and oropharyngeal cancer. We have employed a canine model to develop a systemic vaccine that completely protects against experimentally induced oral mucosal papillomas. The major capsid protein, L1, of canine oral papillomavirus (COPV) was expressed in Sf9 insect cells in native conformation. L1 protein, which self-assembled into virus-like particles, was purified on CsCl gradients and injected intradermally into the foot pad of beagles. Vaccinated animals developed circulating antibodies against COPV and became completely resistant to experimental challenge with COPV. Successful immunization was strictly dependent upon native L1 protein conformation and L1 type. Partial protection was achieved with as little as 0.125 ng of L1 protein, and adjuvants appeared useful for prolonging the host immune response. Serum immunoglobulins passively transferred from COPV L1-immunized beagles to naive beagles conferred protection from experimental infection with COPV. Our results indicate the feasibility of developing a human vaccine to prevent mucosal papillomas, which can progress to malignancy.

Induction of antigen-specific cytolytic T cells in situ in human melanoma by immunization with synthetic peptide-pulsed autologous antigen presenting cells.

Human melanoma cells can process the MAGE-1 gene product and present the processed nonapeptide EADPTGHSY on their major histocompatibility complex class I molecules, HLA-A1, as a determinant for cytolytic T lymphocytes (CTLs). Considering that autologous antigen presenting cells (APCs) pulsed with the synthetic nonapeptide might, therefore, be immunogenic, melanoma patients whose tumor cells express the MAGE-1 gene and who are HLA-A1+ were immunized with a vaccine made of cultured autologous APCs pulsed with the synthetic nonapeptide. Analyses of the nature of the in vivo host immune response to the vaccine revealed that the peptide-pulsed APCs are capable of inducing autologous melanoma-reactive and the nonapeptide-specific CTLs in situ at the immunization site and at distant metastatic disease sites.

Reproduction and sera embryotoxicity after immunization of monkeys with the laminin peptides YIGSR, RGD, and IKVAV.

Monkeys with excellent reproductive histories were immunized with the laminin peptides YIGSR, RGD, IKVAV, and YD, a control sequence with no known biological function. Sera from the YIGSR-immunized monkey became toxic, causing neural tube defects in whole rat embryo cultures, and this monkey experienced fetal loss after immunization. Sera from the RGD-immunized monkey also became embryotoxic in culture after immunization, but this monkey appeared to become infertile as she failed to initiate a pregnancy for at least 2 years after immunization. In contrast, embryos cultured on sera from the IKVAV- or YD-immunized monkeys were predominantly normal and both monkeys completed successful pregnancies. Antibody levels to the respective peptides or to laminin were not predictive of embryotoxicity, but antibody binding to homogenized yolk sacs as well as to yolk sacs of cultured embryos was associated with sera embryotoxicity and reproductive outcomes in vivo. These observations suggested that the laminin sequences YIGSR and RGD may play a role in immune-mediated reproductive failure by reacting directly with embryonic tissue and could provide a basis for identifying individuals at risk for both spontaneous abortion and infertility.

DNA-mediated immunization to the hepatitis B surface antigen in mice: aspects of the humoral response mimic hepatitis B viral infection in humans.

Intramuscular injection of plasmid DNA expression vectors encoding the three envelope proteins of the hepatitis B virus (HBV) induced humoral responses in C57BL/6 mice specific to several antigenic determinants of the viral envelope. The first antibodies appeared within 1-2 weeks after injection of DNA and included antibodies of the IgM isotype. Over the next few weeks, an IgM to IgG class switch occurred, indicating helper T-lymphocyte activity. Peak IgG titers were reached by 4-8 weeks after a single DNA injection and were maintained for at least 6 months without further DNA injections. The antibodies to the envelope proteins reacted with group- and subtype-specific antigenic determinants of the HBV surface antigen (HBsAg). Expression vectors encoding the major (S) and middle (preS2 plus S) envelope proteins induced antibodies specific to the S protein and preS2 domain, and preS2 antibodies were prominent at early time points. In general, the expression vectors induced humoral responses in mice that mimic those observed in humans during the course of natural HBV infection.

Priming of tumor-specific T cells in the draining lymph nodes after immunization with interleukin 2-secreting tumor cells: three consecutive stages may be required for successful tumor vaccination.

Although both CD4+ and CD8+ T cells are clearly required to generate long-lasting anti-tumor immunity induced by s.c. vaccination with interleukin 2 (IL-2)-transfected, irradiated M-3 clone murine melanoma cells, some controversy continues about the site and mode of T-cell activation in this system. Macrophages, granulocytes, and natural killer cells infiltrate the vaccination site early after injection into either syngeneic euthymic DBA/2 mice or athymic nude mice and eliminate the inoculum within 48 hr. We could not find T cells at the vaccination site, which argues against the concept that T-cell priming by the IL-2-secreting cancer cells occurs directly at that location. However, reverse transcription-PCR revealed transcripts indicative of T-cell activation and expansion in the draining lymph nodes of mice immunized with the IL-2-secreting vaccine but not in mice vaccinated with untransfected, irradiated M-3 cells. We therefore propose that the antigen-presenting cells, which invade the vaccination site, process tumor-derived antigens and, subsequently, initiate priming of tumor-specific T lymphocytes in lymphoid organs. These findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2-secreting tumor cells: (i) tumor-antigen uptake and processing at the site of injection by antigen-presenting cells, (ii) migration of antigen-presenting cells into the regional draining lymph nodes, where T-cell priming occurs, and (iii) circulation of activated T cells that either perform or initiate effector mechanisms leading to tumor cell destruction.

Evidence suggesting that the odortypes of pregnant women are a compound of maternal and fetal odortypes.

Odortypes--namely, body odors that distinguish one individual from another on the basis of genetic polymorphism at the major histocompatibility complex and other loci--are a fundamental element in the social life and reproductive behavior of the mouse, including familial imprinting, mate choice, and control of early pregnancy. Odortypes are strongly represented in urine. During mouse pregnancy, an outcrossed mother's urine acquires fetal major histocompatibility complex odortypes of paternal origin, an observation that we took as the focus of a search for odortypes in humans, using a fully automated computer-programmed olfactometer in which trained rats are known to distinguish precisely the odortypes of another species. Five women provided urine samples before and after birth, which in each case appropriately trained rats were found to distinguish in the olfactometer. Whether this olfactory distinction of mothers' urine before and after birth reflects in part the odortype and hence genotype of the fetus, and not just the state of pregnancy per se, was tested in a second study in which each mother's postpartum urine was mixed either with urine from her own infant or with urine of a different, same-aged infant. Responses of trained rats were more positive with respect to the former (congruous) mixtures than to the latter (incongruous) mixtures, implying that, as in the mouse, human fetal odortypes of paternal genomic origin are represented in the odortype of the mother, doubtless by circulatory transfer of the pertinent odorants.

Post-Conflict Maternal Communication, Children's Understandings, and Child Functioning in Violent and Non-Violent Families

The last two decades have been marked by a growing public awareness of family violence. Research by social scientists has suggested that family violence is widespread (Gelles and Straus, 1988). It is estimated that every year 1.8 to 4 million women are physically abused by their partners (Novello, 1992). In fact, more women are abused by their husbands or boyfriends than are injured in car accidents, muggings, or rapes (Jaffe, Wolfe, and Wilson, 1990). A recent prevalence study by Fantuzzo, Boruch, Beriama, Atkins, and Marcus (1997) found that children were disproportionately present in households where there was a substantial incident of adult female assault. Experts estimate that 3.3 to 10 million children are exposed to marital violence each year (Carlson, 1984; Straus, 1991). Until recently, most researchers did not consider the impact of parental conflict on the children who witness this violence. The early literature in this field primarily focused on the incidence of violence against women and the inadequate response of community agencies (Jaffe et al, 1990). The needs of children were rarely considered. However, researchers have become increasingly aware that children exposed to marital violence are victims of a range of psychological maltreatment (e.g., terrorizing, isolation;Hart, Brassared & Karlson, 1996) and are at serious risk for the development of psychological problems (Fantuzzo, DePaola, Lambert, Martino, Anderson, and Sutton, 1991). Jouriles, Murphy and O'Leary (1989) found that children of battered women were four times more likely to exhibit psychopathology as were children living in non-violent homes. Further, researchers have found associations between childhood exposure to parental violence and the expression of violence in adulthood (Carlson, 1990). Existing research suggests that children who have witnessed marital violence manifest numerous emotional, social, and behavioral problems (Sternberg et al., 1993; Fantuzzo et al., 1991; Jaffe et al, 1990). Studies have found that children of battered women exhibit more internalizing and externalizing behavior problems than non-witnesschildren (Hughes and Fantuzzo, 1994; McCloskey, Figueredo, and Koss, 1995). In addition, children exposed to marital violence have been found to exhibit difficulties with social problem-solving, and have lower levels of social competence than nonwitnesses (Rosenberg, 1987; Moore, Pepler, Weinberg, Hammond, Waddell, & Weiser, 1990). Other reported difficulties include low self esteem (Hughes, 1988), poor school performance (Moore et al., 1990) and problems with aggression (Holden & Ritchie, 1991; Jaffe, Wolfe, Wilson, & Zak, 1986). Further, within the last decade, researchers have found that some children are traumatized by the witnessing experience, showing elevated levels of posttraumatic stress symptoms (Devoe & Graham-Bermann, 1997; Rossman, Bingham, & Emde, 1996; Kilpatrick, Litt, & Williams, 1997). These findings corroborate clinical reports that describe many exposed children as experiencing trauma reactions. It appears that the negative effects of witnessing marital violence are numerous and varied, ranging from mild emotional and behavioral problems to clinically significant levels of posttraumatic stress symptoms. These incidence figures and research findings indicate that children's exposure to violence is a significant problem in our nation today and has serious implications for the future.

Novel particulate BCG-loaded delivery system for mucosal immunization against tuberculosis

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016

Caesarean Birth vs. Vaginal Delivery: Effects upon Maternal Satisfaction, Maternal Confidence and Maternal Perception of the Newborn Behaviour

In order to test the hypothesis that caesarean birth has negative consequences upon newly mothers’ satisfaction and perceptions, women delivering by caesarean birth (WCB) were compared with women delivering by vaginal birth (WVB). Subjects: 180 newly mothers; 93 WCB and 87 WVB. Instruments: A Socio-Demographic Questionnaire developed for this research, the Childbirth Perceptions Questionnaire and the Mother and Baby Scales. Results: WCB had significantly lower scores in perceptions of baby as alert/responsive and nearly significant lower scores for baby as alert during feeds. WVB showed a significantly higher level of satisfaction with delivery and conduct during labour, as also had significantly lower scores for perceptions of baby as irritable during feeds and for lack of confidence in feeds. After controlling for the kind of anesthesia received in labour, three conclusions must be taken into account: 1) between WCB with regional anaesthesia and WCB with general anaesthesia there is only one significant difference, with the former having higher scores for perception of baby as alert during feeds; 2) between WVB with regional anaesthesia and WVB with no anaesthesia there are only two significant differences, with the former having higher scores for lack of confidence in feeding and having lower scores for global confidence; 3) between WCB with regional anaesthesia and WVB with regional anaesthesia four significant differences emerge, with the former having a lower level of satisfaction with delivery and conduct in labour and having lower scores for perception of baby as alert responsive, and also having higher scores of perception of baby as irritable in feeds and higher scores for lack of confidence in feeding. Data seem compatible with the hypothesis that caesarean birth has some negative consequences upon mothers’ satisfaction and perceptions and, for this reason, psychological surveys should constitute a routine procedure in maternity hospitals, especially when newly mothers pertain to families affected by risks of psychological or social nature.

Murine Model for Preclinical Studies of Var2CSA-Mediated Pathology Associated with Malaria in Pregnancy

Plasmodium falciparum infection during pregnancy leads to abortions, stillbirth, low birth weight, and maternal mortality. Infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) via var2CSA protein exposed on the P. falciparum IE membrane. Plasmodium berghei IE infection in pregnant BALB/c mice is a model for severe placental malaria (PM). Here, we describe a transgenic P. berghei parasite expressing the full-length var2CSA extracellular region (domains DBL1X to DBL6ε) fused to a P. berghei exported protein (EMAP1) and characterize a var2CSA-based mouse model of PM. BALB/c mice were infected at midgestation with different doses of P. berghei-var2CSA (P. berghei-VAR) or P. berghei wild-type IEs. Infection with 10(4) P. berghei-VAR IEs induced a higher incidence of stillbirth and lower fetal weight than P. berghei At doses of 10(5) and 10(6) IEs, P. berghei-VAR-infected mice showed increased maternal mortality during pregnancy and fetal loss, respectively. Parasite loads in infected placentas were similar between parasite lines despite differences in maternal outcomes. Fetal weight loss normalized for parasitemia was higher in P. berghei-VAR-infected mice than in P. berghei-infected mice. In vitro assays showed that higher numbers of P. berghei-VAR IEs than P. berghei IEs adhered to placental tissue. Immunization of mice with P. berghei-VAR elicited IgG antibodies reactive to DBL1-6 recombinant protein, indicating that the topology of immunogenic epitopes is maintained between DBL1-6-EMAP1 on P. berghei-VAR and recombinant DBL1-6 (recDBL1-6). Our data suggested that impairments in pregnancy caused by P. berghei-VAR infection were attributable to var2CSA expression. This model provides a tool for preclinical evaluation of protection against PM induced by approaches that target var2CSA.

Maternal separation followed by isolation-housing differentially affects prepulse inhibition of the acoustic startle response in C57BL/6 mice

Exposure to chronic stress is associated with an increased incidence of neuropsychiatric dysfunction. The current study evaluated two competing hypotheses, the cumulative stress and the match/mismatch hypothesis of neuropsychiatric dysfunction, using two paradigms relating to exposure to “stress”: pre-weaning maternal separation and post-weaning isolation-housing. C57BL/6 offspring were reared under four conditions: typical animal facility rearing (AFR, control), early handling (EH, daily 15 min separation from dam), maternal separation (MS, daily 4 hr separation from dam), and maternal and peer separation (MPS, daily 4 hr separation from dam and from littermates). After weaning, mice were either housed socially (2–3/cage) or in isolation (1/cage) and then tested for prepulse inhibition in adulthood. Isolation-housed MPS subjects displayed greater deficits in prepulse inhibition relative to socially-housed MPS subjects while socially-housed AFR subjects displayed greater deficits in prepulse inhibition relative to isolation-housed AFR subjects. The results indicate that these treatment conditions represent a potentially valuable model for evaluating the match/mismatch hypothesis in regards to neuropsychiatric dysfunction.