Therapeutic effect of DNA vaccine encoding the 60-kDa-heat shock protein from Paracoccidoides brasiliensis on experimental paracoccidioidomycosis in mice

Paracoccidioidomycosis (PCM) is a systemic mycosis autochthonous to Latin America and endemic to Brazil, which has the majority of the PCM cases. PCM is acquired through the inhalation of propagules of fungi from genus Paracoccidioides spp. and mainly affects the lungs. We have previously shown that P. brasiliensis-infected mice treated with single-dose of recombinant 60-kDa-heat shock protein from P. brasiliensis (rPbHsp60) had a worsening infection in comparison to animals only infected. In this study, we investigate whether the treatment of infected mice with PB_HSP60 gene cloned into a plasmid (pVAX1-PB_HSP60) would result in efficient immune response and better control of the disease. The harmful impact of single-dose therapy with protein was not seen with plasmid preparations. Most importantly, three doses of pVAX1-PB_HSP60 and protein induced a beneficial effect in experimental PCM with a reduction in fungal load and lung injury when compared with infected mice treated with pVAX1 or PBS. The increase of the cytokines IFN-γ, TNF, and IL-17 and the decrease of IL-10 observed after treatment with three doses of pVAX1-PB_HSP60 appears to be responsible for the control of infection. These results open perspectives of the therapeutic use of Hsp60 in PCM.     

Unraveling the susceptibility of paracoccidioidomycosis: Insights towards the pathogen-immune interplay and immunogenetics

Paracoccidioidomycosis (PCM) is a life-threatening systemic mycosis caused by Paracoccidioides spp. This disease comprises three clinical forms: symptomatic acute and chronic forms (PCM disease) and PCM infection, a latent form without clinical symptoms. PCM disease differs markedly according to severity, clinical manifestations, and host immune response. Fungal virulence factors and adhesion molecules are determinants for entry, latency, immune escape and invasion, and dissemination in the host. Neutrophils and macrophages play a paramount role in first-line defense against the fungus through the recognition of antigens by pattern recognition receptors (PRRs), activating their microbicidal machinery. Furthermore, the clinical outcome of the PCM is strongly associated with the variability of cytokines and immunoglobulins produced by T and B cells. While the mechanisms that mediate susceptibility or resistance to infection are dictated by the immune system, some genetic factors may alter gene expression and its final products and, hence, modulate how the organism responds to infection and injury. This review outlines the main findings relative to this topic, addressing the complexity of the immune response triggered by Paracoccidioides spp. infection from preclinical investigations to studies in humans. Here, we focus on mechanisms of fungal pathogenesis, the patterns of innate and adaptive immunity, and the genetic and molecular basis related to immune response and susceptibility to the development of the PCM and its clinical forms. Immunogenetic features such as HLA system, cytokines/cytokines receptors genes and other immune-related genes, and miRNAs are likewise discussed. Finally, we point out the occurrence of PCM in patients with primary immunodeficiencies and call attention to the research gaps and challenges faced by the PCM field.     

宫颈鳞癌患者外周血单个核细胞Th1/Th2平衡状态的研究

目的探讨宫颈癌患者外周血辅助T细胞1(Th1)和辅助T细胞2(Th2)型细胞因子的表达状况。方法 30例宫颈癌患者采集外周血,采用流式细胞分析法,检测患者外周血CD4+细胞胞内细胞因子干扰素γ(IFN-γ)和白介素4(IL-4)的表达。应用酶联免疫吸附方法(ELISA)检测培养上清IFN-γ、IL-4的水平,并与10例健康对照组进行比较。结果宫颈癌患者IFN-γ染色阳性(Th1)的细胞比例及培养上清IFN-γ浓度明显低于健康对照组,IL-4染色阳性(Th2)细胞比例及培养上清IL-4浓度在两组间差异无统计学意义。结论宫颈癌患者存在Th1/Th2细胞功能失衡,呈Th2优势应答模式,机体免疫抑制,导致肿瘤发生,同时为宫颈鳞癌的生物治疗提供靶点。     

Zinc: mechanisms of host defense

Zinc deficiency in humans decreases the activity of serum thymulin (a thymic hormone), which is required for maturation of T-helper cells. T-helper 1 (Th(1)) cytokines are decreased but T-helper 2 (Th(2)) cytokines are not affected by zinc deficiency in humans. This shift of Th(1) to Th(2) function results in cell-mediated immune dysfunction. Because IL-2 production (Th(1) cytokine) is decreased, this leads to decreased activities of natural-killer cell and T cytolytic cells, which are involved in killing viruses, bacteria, and tumor cells. In humans, zinc deficiency may decrease the generation of new CD4+ T cells from the thymus. In cell culture studies (HUT-78, a Th(0) human malignant lymphoblastoid cell line), as a result of zinc deficiency, nuclear factor-kappaB (NF-kappaB) activation, phosphorylation of IkappaB, and binding of NF-kappaB to DNA are decreased and this results in decreased Th(1) cytokine production. In another study, zinc supplementation to humans decreased the gene expression and production of pro-inflammatory cytokines and decreased oxidative stress markers. In HL-60 cells (a human pro-myelocytic leukemia cell line), zinc deficiency increased the levels of TNF-alpha, IL-1beta, and IL-8 cytokines and mRNA. In these cells, zinc induced A20, a zinc finger protein that inhibited NF-kappaB activation via tumor necrosis factor receptor associated factor pathway, and this decreased gene expression of pro-inflammatory cytokines and oxidative stress markers. We conclude that zinc has an important role in cell-mediated immune functions and also functions as antiinflammatory and antioxidant agent.     

Functional memory CD8+ T cells can be generated in vivo without evident T help

Synthetic cytotoxic T cell (CTL) epitope peptides provide an effective and safe means of vaccination against cancers and viruses, as these peptides can induce specific CD8+ effector T cells in vivo. However, the effector CD8+ T cells induced by the minimal CTL epitope peptides do not last past about 3 weeks after the induction and no functional memory CD8+ T cells are generated. It is held that simultaneous induction of CD4+ T cells by incorporating peptides containing T-helper epitopes in the vaccine at the time of primary vaccination are necessary for the induction of long-lived functional memory CD8+ T cells. We now report that, surprisingly, incorporation of medium length (>20 AA) peptides devoid of detectable T-helper epitopes in a minimal CTL epitope-based vaccine can also induce long-lasting functional tumour antigen specific memory CD8+ T cells that are capable of promoting protection against tumour challenge. This observation may have implications for the formulation of therapeutic anti-cancer and anti-virus peptide vaccines where a strong induction of CD4 T help would be undesirable.     

Measuring Dosage: A Key Factor When Assessing the Relationship Between Prenatal Case Management and Birth Outcomes

To assess whether a measure of prenatal case management (PCM) dosage is more sensitive than a dichotomous PCM exposure measure when evaluating the effect of PCM on low birthweight (LBW) and preterm birth (PTB). We constructed a retrospective cohort study (N = 16,657) of Iowa Medicaid-insured women who had a singleton live birth from October 2005 to December 2006; 28 % of women received PCM. A PCM dosage measure was created to capture duration of enrollment, total time with a case manager, and intervention breadth. Propensity score (PS)-adjusted odds ratios (ORs), and 95 % confidence intervals (95 % CIs) were calculated to assess the risk of each outcome by PCM dosage and the dichotomous PCM exposure measure. PS-adjusted ORs of PTB were 0.88 (95 % CI 0.70–1.11), 0.58 (95 % CI 0.47–0.72), and 1.43 (95 % CI 1.23–1.67) for high, medium, and low PCM dosage, respectively. For LBW, the PS-adjusted ORs were 0.76 (95 % CI 0.57–1.00), 0.64 (95 % CI 0.50–0.82), and 1.36 (95 % CI 1.14–1.63), for high, medium, and low PCM dosage, respectively. The PCM dichotomous participation measure was not significantly associated with LBW (OR = 0.95, 95 % CI 0.82–1.09) or PTB (0.97, 95 % CI 0.87–1.10). The reference group in each analysis is No PCM. PCM was associated with a reduced risk of adverse pregnancy outcomes for Medicaid-insured women in Iowa. PCM dosage appeared to be a more sensitive measure than the dichotomous measure of PCM participation.     

Protection and humoral immune responses against Bordetella pertussis infection in mice immunized with acellular or cellular pertussis immunogens

In the present study, protection against Bordetella pertussis infection and humoral immunological responses in mice has been assessed upon immunization with custom-made acellular pertussis vaccines (ACVs) and whole-cell pertussis vaccine (WCV). Mice were immunized, next intranasally infected with B. pertussis and during 14 days the number of bacteria in the trachea and lungs and the level of serum antibodies were determined. ACV contained five immunogens, filamentous hemagglutinin, pertactin, fimbriae serotypes 2 and 3, and chemically detoxified pertussis toxin (PMC-5), or three immunogens, filamentous hemagglutinin, pertactin, and genetically detoxified (BC-3) or chemically detoxified pertussis toxin (SKB-3). Immunization with a high or low dose of ACV or WCV resulted in significant protection against B. pertussis, with differences in the degree of protection between the vaccines. The lowest protection was found with a low dose of SKB-3 and WCV. The pattern of cytokine production by spleen cells of immunized, non-infected, mice indicated that T-helper 1 cells are activated by vaccination with WCV, and T-helper 1 and T-helper 2 cells are involved in the immune response upon vaccination with ACVs. Each vaccine stimulated the production of IgG, but not IgA, antibodies. In mice immunized with ACV, elimination of B. pertussis from trachea and lungs correlated significantly with the titre of IgG1, but not IgG2a, antibodies.     

Zinc deficiency and lymphocyte subpopulations. A study by flow cytometry

Zinc deficiency is well known to alter immunity. We report the case of a 18-yr-old female with relapsing Crohn's disease who experienced acrodermatitis enteropathica due to zinc deficiency during total parenteral nutrition (TPN). Blood lymphocytes have been studied by flow cytometry: before zinc treatment an important decrease of T-helper lymphocytes with high level of OKM-5+ lymphocytes had been observed. Zinc-supplemented diet induced within a few days, a rise of T-helper lymphocytes and a proportional reduction of OKM5+ cells. Increased values of high metabolism surface marker (OKT-9) were also observed, as well as cytoplasmic modifications. The authors suggest that lymphocyte surface markers could be useful to monitor TPN in patients at high risk for zinc deficiency.     

ADAPTATION IN PROTEIN CALORIE MALNUTRITION (PCM)

Endocrine adaptation to starvation and low protein hypocaloric diets is discussed as a basis for understanding the clinical expression of PCM. Infections common in PCM increase acute phase reactants, which may contribute to the derangement of homeostasis in PCM.     

Major lymphocyte populations and T-cell expression of ICAM-1 and L-selectin adhesion molecules in Kuwaitis with asthma and rhinitis

The authors evaluate major immunologic features of asthma and allergies in a Kuwaiti population. They analyzed peripheral venous blood from 17 asthmatic and 17 healthy long-term residents of Kuwait by using two-color flow cytometry for major lymphocyte subpopulations; they also evaluated 10 healthy individuals who had recently arrived in Kuwait. Relative to healthy subjects, asthmatics exhibited increased percentages of T+ NK cells (p < .01), T-helper cells (p < .05), T-cytotoxic and NK cells for both total numbers (p < .01-.001) and percentages (p < .05-.01), and increased percentages of T cells expressing CD54 (ICAM-1; p < .001) and CD62 (L-selectin; p < .01). However, B cells were present at significantly lower levels in asthmatics, both in total numbers (p < .05) and percentages (p < .01). In comparison with healthy individuals who had recently arrived in Kuwait, healthy long-term residents exhibited elevated numbers of pan-T cells (p < .01) and T-helper cells (p < .05). These results help establish immunological parameters for asthma and allergies in Kuwaiti populations.     

Surveying Hard-to-Reach Programs: Identifying the Population of Medicaid Prenatal Case Management Programs

Objectives Community-based prenatal case management (PCM) is a means to improve birth outcomes for medically or socially high-risk pregnant women. To conduct national surveys of PCM programs, a useful sampling frame of existing programs is needed. However, as a relatively small optional Medicaid reimbursed program, PCM programs are hard to reach. Methodological approaches are needed to address issues arising when attempting to access and survey hard-to-reach participants, including programs. Methods State Medicaid offices were contacted to determine whether they reimbursed for PCM, and lists of Medicaid providers were obtained from those states. Most providers on the lists were contacted to confirm that they provide PCM and to verify the program director contact information. Findings Multiple attempts, using different modes of communication, were required to identify states reimbursing for PCM through Medicaid (n = 33). Of providers on the lists obtained from 29 of the 33 states, 34% of those listed provided PCM, suggesting over coverage rather than omissions. Provider contact information was outdated, duplicative, or not specific to PCM. The final count was 1,184 PCM programs in 29 states. Conclusion Identifying hard-to-reach programs requires persistence and creativity, as well as a rigorous approach to generating a census of programs.     

Comparison of acute and chronic protein-energy malnutrition on host antitumor immune mechanisms

Protein-calorie malnutrition (PCM) is prevalent in cancer patients. However, the effect of PCM on anti-tumor immunity is unclear and critically important in an era of improving results with adoptive immunotherapy. This study examined the effect of short- and long-term PCM on tumor-specific and natural immune effector mechanisms in a murine neuroblastoma (C1300 NRB) model. A/J mice received an isocaloric 2.5% or 24% casein diet for 3 or 8 weeks before inoculation with tumor. Three weeks later lymphocytes from tumor-bearing mice were harvested for determination of cytotoxic T lymphocyte (CTL) generation and natural killer (NK) cell cytotoxicity. Both 3 and 8 weeks of PCM significantly reduced mean total body weight by 25% (p less than 0.001) and 41% (p less than 0.001), respectively, compared with regularly nourished mice. Short-term PCM did not inhibit CTL or NK cytotoxicity, whereas long-term PCM significantly diminished CTL generation (p less than 0.001) but preserved NK cytotoxic function. These results indicate that CTL development against autologous tumor, in contrast to basal NK function, is dependent on host nutritional status. Mean tumor growth, determined by tumor-weight to carcass-weight ratio, was unchanged for both short- and long-term protein-energy deprived groups compared with results in regularly nourished mice. These findings suggest that NK function is the predominant effector mechanism inhibiting C1300 NRB growth and that NK tumoricidal capacity is preserved during PCM.     

Evaluation of the phase contrast microscopy method for the detection of fibrous and other elongated mineral particulates by comparison with a STEM technique

The USPHS/NIOSH Membrane Filter Method is used to monitor for asbestos in occupational and mining atmospheres, and employs the phase-contrast optical microscope (PCM) that under optimum conditions has a resolution of approximately 0.25 micron. While amphibole cleavage fragments are usually visible by PCM, asbestos fibers (such as amosite and chrysotile) have finer widths that may render them invisible by PCM. In this study, personal air-monitoring filters containing chrysotile, amosite and amphibole cleavage fragments from various sources have been analyzed by PCM in accordance with the USPHS/NIOSH Method and scanning transmission electron microscopy (STEM) to assess the effectiveness of the PCM technique. Each STEM specimen was prepared using a direct-transfer technique to ensure that particle size distribution and concentration were not altered. STEM results for chrysotile samples are highly variable, with 9% to 81% of regulatory particles having widths smaller than 0.25 micron--the resolution of the optical microscope. Amosite samples have 27% to 38% of regulatory particles with widths below microscope resolution, indicating that routine particle counts by PCM on these samples would underestimate true fiber content by approximately one-third. All amphibole cleavage fragment samples had regulatory particles that would be observed by PCM. Multiplication factors have been suggested for application to routine counts by PCM to more accurately assess true particle content for mineral particulates on personal air-monitoring filters.     

VA cooperative study on alcoholic hepatitis. II: Prognostic significance of protein-calorie malnutrition

Three hundred and fifty-two patients with alcoholic hepatitis were evaluated for protein-calorie malnutrition (PCM). In order to facilitate data analysis of nutritional status, a PCM score was calculated for each patient using eight nutritional parameters. The PCM score correlated significantly with mortality, clinical severity of the liver disease, and biochemical liver dysfunction. When 30 day changes in the PCM scores were compared with 30 day caloric intake (expressed as percent basal energy expenditure (BEE], a marginally significant correlation was observed (p = 0.05). However, those patients who showed improvement in their PCM score over 30 days of hospitalization also improved their 6-mo and 1-yr survival. These data indicate that nutrition, as determined by the PCM score, has prognostic significance. Additional studies are needed to establish the beneficial role for vigorous protein-calorie nutritional therapy in the management of alcoholic hepatitis.     

Effects of cysteine on the pharmacokinetic parameters of omeprazole in rats with protein-calorie malnutrition: partial restoration of some parameters to control levels by oral cysteine supplementation

BACKGROUND: It has been reported that omeprazole is mainly metabolized via the hepatic cytochrome (CYP) 1A1/2, 3A1/2, and 2D1, and the expressions and mRNA levels of CYP1A2, 2C11, and 3A1/2 decreased in protein-calorie malnutrition (PCM) rats compared with controls. Interestingly, the decreased CYP1A2, 2C11, and 3A1/2 in PCM rats returned fully or partially to control levels by oral cysteine supplementation (PCMC rats). Hence, it could be expected that some pharmacokinetic parameters of omeprazole might change in PCM rats and partially restore to control levels in PCMC rats. The purpose of this study is to investigate the pharmacokinetic changes of omeprazole in PCM rats and restoration of the parameters in PCMC rats to control levels. METHODS: Omeprazole was administered intravenously (20 mg/kg) and orally (40 mg/kg) to control, PCM, and PCMC rats. RESULTS: The following pharmacokinetic parameters were changed in PCM rats and partially returned to control levels in PCMC rats: the area under the plasma concentration-time curve (AUC; 387, 762, and 539 microg min/mL for control, PCM, and PCMC rats, respectively, after intravenous [IV] administration, and the corresponding values after oral administration: 115, 304, and 201 microg min/mL), total body clearance (51.7, 25.5, and 37.1 mL/min/kg, respectively), nonrenal clearance (51.5, 25.4, and 36.1 mL/min/kg, respectively), and in vitro intrinsic clearance (0.158, 0.118, and 0.138 mL/min/mg protein). CONCLUSIONS: PCM was associated with significant changes in some omeprazole pharmacokinetics and the pharmacokinetic parameters restored to control levels by oral cysteine.     

Maitake D-Fraction enhances antitumor effects and reduces immunosuppression by mitomycin-C in tumor-bearing mice

OBJECTIVE: D-Fraction, a polysaccharide extracted from maitake mushrooms (Grifola frondosa), has been reported to exhibit an antitumor effect through activation of immunocompetent cells, including macrophages and T cells, with modulation of the balance between T-helper 1 and 2 cells. We examined whether D-Fraction could decrease the effective dosage of the chemotherapeutic agent, mitomycin-C (MMC), necessary to control carcinoma in mice. METHODS AND RESULTS: We determined that 0.25 mg.kg-1.d-1 was the optimal dosage of MMC because consecutive administration for 17 d resulted in antitumor effects and a survival ratio of 100% in mice bearing mammary cancer cells (MM-46). Although the dosage of MMC was lower than the effective level, spleen weight and total number of nuclear cells in the mouse spleen decreased, indicating that MMC showed immunosuppressive activity. In contrast, the combination of D-Fraction and MMC recovered the decreases in the dose response induced by MMC and inhibited tumor cell growth more than MMC alone. These effects were achieved through increased immunocompetent cell proliferation. We evaluated the expression of CD28 on splenic CD8+ T cells and the amount of interleukin-12 produced by whole spleen cells including macrophages after administering D-Fraction. The results showed enhancement of the T-helper 1 dominant response. CONCLUSION: These results suggest that D-Fraction can decrease the effective dosage in tumor-bearing mice by increasing the proliferation, differentiation, and activation of immunocompetent cells and thus provide a potential clinical benefit for patients with cancer.     

Lymphocyte subpopulation before and after operation in patients with benign vs malignant breast disease.

Multiple parameters of immune function were measured serially before and one and five weeks following operation in 14 patients with fibrocystic disease of the breast (Group A) and in 20 patients with stage 1-2 infiltrating duct carcinoma (Group B). These parameters included the following: WBC, total number and percentage of lymphocytes, numbers of B cells, T cells, T-active, T-helper and T-suppressor cells and the ratio between the latter as well as spontaneous suppressor or helper activity and the graft-versus-host reaction. Prior to operation no statistically significant difference was found between the two groups except for the number of T-helper cells, which was higher in Group B (p less than 0.05), and the spontaneous suppressor activity, which was higher in Group B (p less than 0.05). The finding of such a high percentage (80%) of negative graft-versus-host reactions five weeks after operation together with the high suppressor activity may indicate the presence of tumor micrometastases. The burden of surgery and general anesthesia was stronger in Group B, with a pronounced difference found between the groups (p = 0.0005), but the interaction between the influence of time (surgery and anesthesia) and the groups was not as great (p = 0.4864) and was found to be different for each group.     

Improved efficacy of DNA vaccination against prostate carcinoma by boosting with recombinant protein vaccine and by introduction of a novel adjuvant epitope

DNA vaccine represents an attractive approach for cancer treatment by inducing active immune-deprivation of gastrin-releasing peptide (GRP) from tumor cells, the growth of which is dependent on the stimulation of GRP. In this study, we developed a DNA vaccine using a plasmid vector to deliver the immunogen of six copies of the B cell epitope GRP_18–27 (GRP6). In order to increase the potency of this DNA vaccine, multiple strategies have been applied including DNA-prime protein-boost immunization and introduction of a foreign T-helper epitope into DNA vaccine. Mice vaccinated DNA vaccine boosting with HSP65-GRP6 protein induced high titer and relatively high avidity of anti-GRP antibodies as well as inhibition effect on the growth of murine prostate carcinoma, superior to the treatment using DNA alone or BCG priming HSP65-GRP6 protein boosting. Furthermore, the introduction of a novel foreign T-helper epitope into the GRP DNA vaccine showed a markedly stronger humoral immune response against GRP and tumor rejection even than the DNA-prime protein-boost strategy. No further stronger immunogenicity of this foreign T-helper epitope modified DNA vaccine was observed even using the strategy of modified DNA vaccine-priming and HSP65-GRP6 boosting method. The data presented demonstrate that improvement of potency of anti-GRP DNA vaccine with the above two feasible approaches should offer useful methods in the development of new DNA vaccine against growth factors for cancer immunotherapy.     

Potential role of ubiquinone (coenzyme Q10) in pediatric cardiomyopathy

Pediatric cardiomyopathy (PCM) represents a group of rare and heterogeneous disorders that often results in death. While there is a large body of literature on adult cardiomyopathy, all of the information is not necessarily relevant to children with PCM. About 40% of children who present with symptomatic cardiomyopathy are reported to receive a heart transplant or die within the first two years of life. In spite of some of the advances in the management of PCM, the data shows that the time to transplantation or death has not improved during the past 35 years. Coenzyme Q10 is a vitamin-like nutrient that has a fundamental role in mitochondrial function, especially as it relates to the production of energy (ATP) and also as an antioxidant. Based upon the biochemical rationale and a large body of data on patients with adult cardiomyopathy, heart failure, and mitochondrial diseases with heart involvement, a role for coenzyme Q10 therapy in PCM patients is indicated, and preliminary results are promising. Additional studies on the potential usefulness of coenzyme Q10 supplementation as an adjunct to conventional therapy in PCM, particularly in children with dilated cardiomyopathy, are therefore warranted.     

Developing a Measure of Prenatal Case Management Dosage

Recently, federal funding was designated through the Patient Protection and Affordable Care Act giving states the opportunity to expand their prenatal case management programs (PCM) through home visitation. Studies evaluating the effect of PCM on birth outcomes have shown little or no positive results. One suggested reason for these findings is a lack of attention in the assessment of dosage. The objective of this study is to demonstrate the use of measuring PCM dosage when assessing pregnancy outcomes. A birth cohort (N = 4,582) encompassing Medicaid-insured Iowa residents enrolled in PCM who gave birth to a singleton from October 2005 to December 2006 was constructed from linked Iowa birth, Medicaid Claims, and Women's Health Information Systems datasets. Data was used to create a dosage measure capturing the duration of enrollment, amount of time spent with a case manager, and breadth of interventions. Bivariate analysis and logistic regression were used to assess the relationship between PCM dosage and the birth outcomes. Dosage was significantly associated with LBW (X (2) = 31.1, P < 0.001) and PTB (X (2) = 56.2, P < 0.001). After adjustment for potential confounders, the likelihood of LBW and PTB were aOR: 0.47 (95% CI: 0.36-0.63) and aOR: 0.60 (95% CI: 0.44-0.82) for women with medium dosage (compared to low dosage), respectively. For women with high PCM dosage the likelihood of LBW and PTB was aOR 0.40 (95% CI: 0.31-0.51) and aOR = 0.62 (95% CI: 0.48-0.81), respectively. This study showed that PCM dosage was significantly associated with lower odds of an adverse pregnancy outcome occurring.