Mentored Training to Increase Diversity among Faculty in the Biomedical Sciences: The NHLBI Summer Institute Programs to Increase Diversity (SIPID) and the Programs to Increase Diversity among Individuals Engaged in Health-related Research (PRIDE).

OBJECTIVE: To report baseline characteristics of junior-level faculty participants in the Summer Institute Programs to Increase Diversity (SIPID) and the Programs to Increase Diversity among individuals engaged in Health-Related Research (PRIDE), which aim to facilitate participants' career development as independent investigators in heart, lung, blood, and sleep research. DESIGN AND SETTING: Junior faculty from groups underrepresented in the biomedical-research workforce attended two, 2-3 week, annual summer research-education programs at one of six sites. Programs provided didactic and/or laboratory courses, workshops to develop research, writing and career-development skills, as well as a mentoring component, with regular contact maintained via phone, email and webinar conferences. Between summer institutes, trainees participated in a short mid-year meeting and an annual scientific meeting. Participants were surveyed during and after SIPID/PRIDE to evaluate program components. PARTICIPANTS: Junior faculty from underrepresented populations across the United States and Puerto Rico participated in one of three SIPID (2007-2010) or six PRIDE programs (2011-2014). RESULTS: Of 204 SIPID/PRIDE participants, 68% were female; 67% African American and 27% Hispanic/Latino; at enrollment, 75% were assistant professors and 15% instructors, with most (96%) on non-tenure track. Fifty-eight percent had research doctorates (PhD, ScD) and 42% had medical (MD, DO) degrees. Mentees' feedback about the program indicated skills development (eg, manuscript and grant writing), access to networking, and mentoring were the most beneficial elements of SIPID and PRIDE programs. Grant awards shifted from primarily mentored research mechanisms to primarily independent investigator awards after training. CONCLUSIONS: Mentees reported their career development benefited from SIPID and PRIDE participation.

A Perspective on Promoting Diversity in the Biomedical Research Workforce: The National Heart, Lung, and Blood Institute's PRIDE Program.

Aspiring junior investigators from groups underrepresented in the biomedical sciences face various challenges as they pursue research independence. However, the biomedical research enterprise needs their participation to effectively address critical research issues such as health disparities and health inequities. In this article, we share a research education and mentoring initiative that seeks to address this challenge: Programs to Increase Diversity among Individuals Engaged in Health Related Research (PRIDE), funded by the National Heart, Lung, and Blood Institute (NHLBI). This longitudinal research-education and mentoring program occurs through summer institute programs located at US-based academic institutions. Recruited participants are exposed to didactic and lab-based research-skill enhancement experiences, with year-round mentoring over the course of two years. Mentor-mentee matching is based on shared research interests to promote congruence and to enhance skill acquisition. Program descriptions and sample narratives of participants' perceptions of PRIDE's impact on their career progress are showcased. Additionally, we highlight the overall program design and structure of four of seven funded summer institutes that focus on cardiovascular disease, related conditions, and health disparities. Mentees' testimonials about the value of the PRIDE mentoring approach in facilitating career development are also noted. Meeting the clinical and research needs of an increasingly diverse US population is an issue of national concern. The PRIDE initiative, which focuses on increasing research preparedness and professional development of groups underrepresented in the biomedical research workforce, with an emphasis on mentoring as the critical approach, provides a robust model that is impacting the careers of future investigators.

The role of indoleamine 2, 3 dioxygenase in regulating host immunity to leishmania infection.

Pathogen persistence in immune-competent hosts represents an immunological paradox. Increasing evidence suggests that some pathogens, such as, Leishmania major (L. major) have evolved strategies and mechanisms that actively suppress host adaptive immunity. If this notion is correct conventional vaccination therapies may be ineffective in enhancing host immunity, unless natural processes that suppress host immunity are also targeted therapeutically. The key problem is that the basis of pathogen persistence in immune-competent individuals is unknown, despite decades of intense research. This fact, coupled with poor health care and a dearth of effective treatments means that these diseases will remain a scourge on humans unless a better understanding of why the immune system tolerates such infections emerges from research. Indoleamine 2,3-dioxygenase (IDO) has been shown to act as a molecular switch regulating host responses, and IDO inhibitor drugs shown to possess potential in enhancing host immunity to established leishmania infections. It is hoped that this review will help stimulate and help generate critical new knowledge pertaining to the IDO mechanism and how to exploit it to suppress T cell mediated immunity, thus offer an innovative approach to studying the basis of chronic leishmania infection in mice.

Leishmania major attenuates host immunity by stimulating local indoleamine 2,3-dioxygenase expression.

Inflammation stimulates immunity but can create immune privilege in some settings. Here, we show that cutaneous Leishmania major infection stimulated expression of the immune regulatory enzyme indoleamine 2,3 dioxygenase (IDO) in local lymph nodes. Induced IDO attenuated the T cell stimulatory functions of dendritic cells and suppressed local T cell responses to exogenous and nominal parasite antigens. IDO ablation reduced local inflammation and parasite burdens, as did pharmacologic inhibition of IDO in mice with established infections. IDO ablation also enhanced local expression of proinflammatory cytokines and induced some CD4(+) T cells to express interleukin (IL) 17. These findings showed that IDO induced by L. major infection attenuated innate and adaptive immune responses. Thus, IDO acts as a molecular switch regulating host responses, and IDO inhibitor drugs are a potential new approach to enhance host immunity to established leishmania infections.

APC dysfunction is correlated with defective suppression of T cell proliferation in human type 1 diabetes.

It is widely believed that CD4(+)CD25(+) regulatory T cells (Treg) are defective in type 1 diabetes (T1D) and other autoimmune diseases. However, this conclusion is based on the suboptimal in vitro suppression results from very small numbers of subjects. Furthermore, the cells responsible for the suboptimal suppression have not been defined. Therefore, we carried out extensive in vitro suppression assays using both autologous and heterologous donors of Tregs, effector T cells and antigen-presenting cells (APC) from both T1D patients and normal controls. Our in vitro suppression data indicated that a significantly higher proportion (40.0%) of T1D patients have "very low suppression" activity (defined as<25%) by autologous Treg compared to controls (6.3%) (p=0.002). Meta-analysis of the published results confirmed this observation with 45.7% low suppressors in T1D and 7.8% in controls (p=0.00002). Interestingly, suppression assays using heterologous Tregs, effector T cells and APC suggest that the source of APC is correlated with the suppression activity. The frequencies of CD4(+)CD25(+) and CD4(+)CD25(hi) T cells were found to increase with age in normal controls but not in T1D patients, resulting in significantly higher frequencies of CD4(+)CD25(+) (p=0.001) and CD4(+)CD25(hi) (p=0.009) T cells in young T1D subjects than age-matched controls but slightly lower CD4(+)CD25(+) (p=0.003) and CD4(+)CD25(hi) (p=0.08) T cells in old T1D subjects than age-matched controls.

Type 1 diabetes patients have significantly lower frequency of plasmacytoid dendritic cells in the peripheral blood.

Dendritic cells uniquely orchestrate the delicate balance between T cell immunity and regulation and an imbalance favoring immunogenic rather than tolerogenic DC is believed to contribute to the development of autoimmune diseases such as type 1 diabetes (T1D). In this study, we determined the frequencies of three blood DC subsets (pDC, mDC1 and mDC2) in 72 T1D patients and 75 normal controls using the Miltenyi blood DC enumeration kit. The frequency of blood pDC was found to be negatively correlated with subject age in both normal controls and T1D patients (p=0.0007), while the frequency of mDC1 and mDC2 do not change significantly with subject age. More importantly, the mean frequency of pDC in blood was, after adjusting for age, significantly lower in T1D (mean=0.127%) than controls (mean=0.188%) (p<6.0 x 10(-5)), whereas no difference was observed for mDC1 and mDC2 between T1D and controls. Furthermore, T1D patients have a lower proportion of pDC and higher proportion of mDC1 among the total blood DC population than normal controls. These results indicate that the frequency of blood pDC and the pDC/mDC1 ratio are negatively associated with T1D.

Cell signaling pathways involved in drug-mediated fetal hemoglobin induction: Strategies to treat sickle cell disease.

The developmental regulation of globin gene expression has shaped research efforts to establish therapeutic modalities for individuals affected with sickle cell disease and ß-thalassemia. Fetal hemoglobin has been shown to block sickle hemoglobin S polymerization to improve symptoms of sickle cell disease; moreover, fetal hemoglobin functions to replace inadequate hemoglobin A synthesis in ß-thalassemia thus serving as an effective therapeutic target. In the perinatal period, fetal hemoglobin is synthesized at high levels followed by a decline to adult levels by one year of age. It is known that naturally occurring mutations in the γ-globin gene promoters and distant cis-acting transcription factors produce persistent fetal hemoglobin synthesis after birth to ameliorate clinical symptoms. Major repressor proteins that silence γ-globin during development have been targeted for gene therapy in ß-hemoglobinopathies patients. In parallel effort, several classes of pharmacological agents that induce fetal hemoglobin expression through molecular and cell signaling mechanisms have been identified. Herein, we reviewed the progress made in the discovery of signaling molecules targeted by pharmacologic agents that enhance γ-globin expression and have the potential for future drug development to treat the ß-hemoglobinopathies.

Novel Inducers of Fetal Globin Identified through High Throughput Screening (HTS) Are Active In Vivo in Anemic Baboons and Transgenic Mice.

High-level fetal (γ) globin expression ameliorates clinical severity of the beta (ß) hemoglobinopathies, and safe, orally-bioavailable γ-globin inducing agents would benefit many patients. We adapted a LCR-γ-globin promoter-GFP reporter assay to a high-throughput robotic system to evaluate five diverse chemical libraries for this activity. Multiple structurally- and functionally-diverse compounds were identified which activate the γ-globin gene promoter at nanomolar concentrations, including some therapeutics approved for other conditions. Three candidates with established safety profiles were further evaluated in erythroid progenitors, anemic baboons and transgenic mice, with significant induction of γ-globin expression observed in vivo. A lead candidate, Benserazide, emerged which demonstrated > 20-fold induction of γ-globin mRNA expression in anemic baboons and increased F-cell proportions by 3.5-fold in transgenic mice. Benserazide has been used chronically to inhibit amino acid decarboxylase to enhance plasma levels of L-dopa. These studies confirm the utility of high-throughput screening and identify previously unrecognized fetal globin inducing candidates which can be developed expediently for treatment of hemoglobinopathies.

Dendritic cells: a specialized complex system of antigen presenting cells.

The dendritic cell (DC) network is a specialized system for presenting antigen to naive or quiescent T cells, and consequently plays a central role in the induction of T cell and B cell immunity in vivo. Despite considerable achievements in the last ten years, in our understanding of how DC induce and regulate immune responses, much remains to be learned about this complex system of cells. The history and current status of DC termed "directors of the immune system orchestra" is reviewed. The present understanding of DC cell biology, function and use, taking into account their complexity is discussed.

Viability and infectivity of Cryptosporidium parvum oocysts detected in river water in Hokkaido, Japan.

The viability and infectivity of Cryptosporidium parvum (C. parvum) oocysts, detected in water samples collected from river water in Hokkaido, were investigated using Severe Combined Immunodeficient (SCID) mice. The water samples collected from September 27 through October 10, 2001 by filtration using Cuno cartridge filters were purified and concentrated by the discontinuous centrifugal flotation method. From 1.2 x 10 (5) liters of the raw river water, approximately 2 x 10(4) oocysts were obtained and designated as Hokkaido river water 1 isolate (HRW-1). Oocyst identification was carried out using microscopic and immunological methods. Six 8-week-old female SCID mice were each inoculated orally with 1 x 10 (3) oocysts. Infection was successfully induced, resulting in fecal oocyst shedding. Oocysts were then maintained by sub-inoculation into SCID mice every 3 months. Infectivity was evaluated by making comparisons with two known C. parvum stocks, HNJ-1 and TK-1, which were bovine genotypes detected in fecal samples from a cryptosporidiosis patient and young cattle raised in Tokachi, Hokkaido respectively. The oocyst genotypes were determined from a small subunit ribosomal RNA (SSU-rRNA) gene by polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) analysis. No significant differences were observed in the average number of oocysts per gram of feces (OPG) in any of the isolates. Our data indicates that the C. parvum oocysts detected in the sampled river water were of C. parvum genotype 2. Moreover, our data on the continued isolation, detection and identification of the C. parvum isolates is consistent with the available epidemiological data for the Tokachi area.

A comparison of the phenotype of dendritic cells derived from discrete Peyer's patch macrophages of non-infected and Toxoplasma gondii infected mice.

A comparison of the expression of surface membrane antigens between dendritic cells (DC) derived from Peyer's patch macrophages (DPP-DC) of non-infected and Toxoplasma gondii (T. gondii) infected mice was performed. C57BL/6J mice aged 6-8 weeks of both sexes were infected orally with a 0.5 ml suspension containing 2 x 10(4) bradyzoites of the Beverley strain of T. gondii, sacrificed on day 8 and DC generated using discrete Peyer's patch macrophages (DPP-Mø) as progenitor cells. When a comparison of the expression of surface membrane antigens between the antigen presenting cells (APC) obtained from discrete Peyer's patches of non-infected and T. gondii infected mice was carried out, no significant differences were observed in the macrophage progenitor and DC populations expression of F4/80, DEC-205, CD11c, CD80 (B7-1) and CD34. However, a significant decrease in MHC class II antigen levels and a down regulation of the co-stimulatory molecule CD86 (B7-2) were noted. B7-1 appeared to be the dominant co-stimulatory ligand, whereas B7-2, which was down regulated during T. gondii infection, had a weak expression. Taken together, these results may help clarify the role of DC in the complex network regulating surface membrane antigens, as well as, their capacity for antigen uptake, processing and presentation during toxoplasmosis.

Phenotype and function of murine peritoneal cavity macrophage derived-dendritic cells.

The accessory activity was reported in murine peritoneal cavity macrophage derived dendritic cells (PEC-DC) in a mixed lymphocyte reaction (MLR). Here we continue the characterization of the generated PEC-DC using the criteria of morphology, phenotype and other accessory function. We have demonstrated that murine peritoneal cavity macrophages can be induced to differentiate in vitro into cells exhibiting typical dendritic cell (DC) morphology, phenotype and function. The proliferative capacity of the progenitors was amplified in the first step of the culture (day 0-7) using a combination of early cytokines: interleukin 4 and granulocyte-macrophage colony-stimulating factor. The second step of the culture started at day 7 with the removal of early growth factors to allow differentiation and final maturation of DC during 2 days of culture with interferon gamma plus either Toxoplasma lysate antigen (TLA) or lipopolysaccharide (LPS), a bacterial agent as a DC maturing agent. The resulting DC population exhibited typical DC morphology and expressed higher levels of MHC class II and the co-stimulatory molecules CD80 and CD86 compared to the untreated peritoneal cells. The generated DC cells efficiently presented soluble protein antigen to CD3(+) spleen T cells.

Phenotype and function of murine discrete Peyer's patch macrophage derived - dendritic cells.

The phenotype and function of peritoneal cavity macrophage-derived dendritic cells (PEC-DC) was previously reported. In this study we have gone further in using our established culture system to generated discrete Peyer's patch dendritic cells (DPP-DC) from murine discrete Peyer's patch macrophages (DPP-Mø), following stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4) for 7 days. DPP-Mø from murine small intestines were obtained by mechanical disruption of discrete Peyer's patches (DPP), followed by metrizamide density gradient centrifugation to remove Peyer's patch resident DC and debri, after which an overnight adherent step in tissue culture medium was carried out for macrophage enrichment. Characterization of the generated DPP-DC was carried out using well-established criteria of morphology, expression of membrane antigens and capacity for antigen presentation. Dendritic cells expressed DEC-205, F4/80 and CD34 at high levels, but exhibited very low CD11c levels. They were shown to present soluble protein antigen to CD3(+) spleen T cells. A comparison of the surface antigen expression in the progenitor DPP-Mø population and the generated DPP-DC showed a significant decrease in MHC class II levels and a marked down regulation of the co-stimulatory molecule CD86 (B7-2). High expression of the haemopoietic progenitor marker CD34 indicates that the generated DC, possess a haemopoietic rather than myeloid origin. Taken together, these results may provide a better understanding of the complex network regulating mucosal immune responses.

Immunology. Antigen-presenting cells in the gut.

It has been known for the past 85 years that mucosal responses can be stimulated by local presentation of antigen and that the gut immune system is capable of mounting both primary and secondary responses to potentially harmful antigens while avoiding the expression of damaging responses to harmless dietary proteins. How these types of responses are induced and regulated remains unclear. In the gut attention has for some time been focused on Peyer's patches (PP) due to evidence that they play a vital role in the induction of humoral and cellular responses. Moreover, it has been established that MHC class II molecules are found in the gut mucosa on a variety of cell types outside PP, namely the lamina propria (LP). Fed antigens have also been detected in the LP and studies have shown that LP cells can stimulate allogeneic mixed lymphocyte responses and are capable of presenting soluble protein antigen to naïve T cells. This article reviews the present understanding of the possible roles of PP and LP in intestinal immunity in terms of induction, regulation, surveillance of immune responses and the antigen presenting cell types involved.