Global Health Training Collaborations: Lessons Learned and Best Practices.

Interest in global health training experiences among trainees from higher income countries has grown. The Working Group on Ethics Guidelines for Global Health Training (WEIGHT) clarified best practices in 2010 based on expert consensus. These guidelines address both balancing priorities in international partnerships and local sustainability concerns related to short-term experiences. However, the guidelines can be difficult to implement in actual practice. Because our organization predated the availability of these consensus guidelines, we reviewed our current set of practices for hosting service-learning programs at our rural Ugandan clinic for adherence to the WEIGHT guidelines. The discrete activities and standardized processes developed over 10 years of hosting experiences were grouped into broader hosting categories, with consensus among the hosting and sending volunteer coordinators of our non-governmental organization partnership. These practices were then mapped to the WEIGHT guidelines. We found our implementation strategies map these guidelines into a clear checklist of actions that can be used by coordinators involved in global health training programs. We include some of the historical reasons that led to our current processes, which may help other partnerships identify similar practice gaps. We anticipate that this action-oriented checklist with historical context will help accomplish the difficult implementation of best practices in global health training collaborations.

TLR3 agonist improves survival to secondary pneumonia in a double injury model.

BACKGROUND: Toll-like receptors (TLR) can initiate various immune responses and are therefore activated under diverse infectious states. Previous studies have focused on TLR3 primarily as an antiviral pathway. However, recent research has demonstrated its efficacy in bacterial infection. Having developed a murine double injury model of cecal ligation and puncture (CLP) followed by Pseudomonas aeruginosa (Pa), we hypothesized that targeted administration of Poly I:C, a TLR3 agonist, would protect mice against secondary pneumonia. MATERIAL AND METHODS: B6 mice underwent CLP followed 4 d afterward by an intranasal dose of Pa. Animals were given Poly I:C or vehicle (phosphate-buffered saline) intranasally 24 h post CLP and every day thereafter for a total of 6 d. For acute studies, mice were sacrificed at two time points, 4 d post CLP and 1 d post pneumonia (Pa). RESULTS: Poly I:C treatment led to a significant improvement in survival (69% versus 33%). Cytokine analysis from bronchioalveolar lavage displayed significant differences both immediately before and after pneumonia. Bronchioalveolar lavage cultures taken at 24 h post double injury showed significantly higher colony counts in the lungs of control animals compared with those of Poly I:C animals. Measurements of TLR3 expression showed significant increases within both the immune and lung epithelial cells of Poly I:C-treated mice. Finally, the lungs of treated animals had significant increases in lymphocytes and innate cells. CONCLUSIONS: The prophylactic treatment applied in this clinically relevant model further illustrates the overarching hypothesis of immune dysfunction and the possibility of corrective immune modulation within the setting of sepsis.

Immunosuppression in patients who die of sepsis and multiple organ failure.

CONTEXT: Severe sepsis is typically characterized by initial cytokine-mediated hyperinflammation. Whether this hyperinflammatory phase is followed by immunosuppression is controversial. Animal studies suggest that multiple immune defects occur in sepsis, but data from humans remain conflicting. OBJECTIVES: To determine the association of sepsis with changes in host innate and adaptive immunity and to examine potential mechanisms for putative immunosuppression. DESIGN, SETTING, AND PARTICIPANTS: Rapid postmortem spleen and lung tissue harvest was performed at the bedsides of 40 patients who died in intensive care units (ICUs) of academic medical centers with active severe sepsis to characterize their immune status at the time of death (2009-2011). Control spleens (n = 29) were obtained from patients who were declared brain-dead or had emergent splenectomy due to trauma; control lungs (n = 20) were obtained from transplant donors or from lung cancer resections. MAIN OUTCOME MEASURES: Cytokine secretion assays and immunophenotyping of cell surface receptor-ligand expression profiles were performed to identify potential mechanisms of immune dysfunction. Immunohistochemical staining was performed to evaluate the loss of immune effector cells. RESULTS: The mean ages of patients with sepsis and controls were 71.7 (SD, 15.9) and 52.7 (SD, 15.0) years, respectively. The median number of ICU days for patients with sepsis was 8 (range, 1-195 days), while control patients were in ICUs for 4 or fewer days. The median duration of sepsis was 4 days (range, 1-40 days). Compared with controls, anti-CD3/anti-CD28-stimulated splenocytes from sepsis patients had significant reductions in cytokine secretion at 5 hours: tumor necrosis factor, 5361 (95% CI, 3327-7485) pg/mL vs 418 (95% CI, 98-738) pg/mL; interferon γ, 1374 (95% CI, 550-2197) pg/mL vs 37.5 (95% CI, -5 to 80) pg/mL; interleukin 6, 3691 (95% CI, 2313-5070) vs 365 (95% CI, 87-642) pg/mL; and interleukin 10, 633 (95% CI, -269 to 1534) vs 58 (95% CI, -39 to 156) pg/mL; (P < .001 for all). There were similar reductions in 5-hour lipopolysaccharide-stimulated cytokine secretion. Cytokine secretion in sepsis patients was generally less than 10% that in controls, independent of age, duration of sepsis, corticosteroid use, and nutritional status. Although differences existed between spleen and lung, flow cytometric analysis showed increased expression of selected inhibitory receptors and ligands and expansion of suppressor cell populations in both organs. Unique differences in cellular inhibitory molecule expression existed in immune cells isolated from lungs of sepsis patients vs cancer patients and vs transplant donors. Immunohistochemical staining showed extensive depletion of splenic CD4, CD8, and HLA-DR cells and expression of ligands for inhibitory receptors on lung epithelial cells. CONCLUSIONS: Patients who die in the ICU following sepsis compared with patients who die of nonsepsis etiologies have biochemical, flow cytometric, and immunohistochemical findings consistent with immunosuppression. Targeted immune-enhancing therapy may be a valid approach in selected patients with sepsis.

Increased susceptibility to Candida infection following cecal ligation and puncture.

Secondary infection following septic insult represents a significant cause of morbidity and mortality in hospitalized patients. Sepsis induced immunosuppression is a major factor in the host's susceptibility to nosocomial infections and Candida albicans accounts for a growing number of these. Given the importance of improving our understanding of the immune response to sepsis and the increasing rates of C. albicans infections, we sought to develop a murine model of double injury consisting of primary peritonitis, i.e., cecal ligation and puncture (CLP), followed by a secondary challenge of C. albicans. As observed in previous work, after primary injury the immune profile of the host changes over time. Therefore, while keeping the mortality rates from the respective individual injuries low, we altered the timing of the secondary injury between two post-CLP time points, day two and day four. Mice subjected to C. albicans infection following CLP have significantly different survival rates dependent upon timing of secondary injury. Animals challenged with C. albicans at two days post CLP had 91% mortality whereas animals challenged at four days had 47% mortality. This improvement in survival at four days was associated with restoration of innate cell populations and as evidenced by stimulated splenocytes, increases in certain inflammatory cytokines. In addition, we show that susceptibility to C. albicans infection following CLP is dependent upon the depth of immunosuppression. Although at four days post-CLP there is a partial reconstitution of the immune system, these animals remain more susceptible to infection compared to their single injury (C. albicans alone) counterparts. Collectively, these studies demonstrate that immunosuppression following initial septic insult changes over time. This novel, two hit model of CLP followed by Candida provides additional insight into the immune compromised state created by primary peritonitis, and thereby opens up another avenue of investigation into the causes and possible cures of an emerging clinical problem.

Characterization and modulation of the immunosuppressive phase of sepsis.

Sepsis continues to cause significant morbidity and mortality in critically ill patients. Studies of patients and animal models have revealed that changes in the immune response during sepsis play a decisive role in the outcome. Using a clinically relevant two-hit model of sepsis, i.e., cecal ligation and puncture (CLP) followed by the induction of Pseudomonas aeruginosa pneumonia, we characterized the host immune response. Second, AS101 [ammonium trichloro(dioxoethylene-o,o')tellurate], a compound that blocks interleukin 10 (IL-10), a key mediator of immunosuppression in sepsis, was tested for its ability to reverse immunoparalysis and improve survival. Mice subjected to pneumonia following CLP had different survival rates depending upon the timing of the secondary injury. Animals challenged with P. aeruginosa at 4 days post-CLP had approximately 40% survival, whereas animals challenged at 7 days had 85% survival. This improvement in survival was associated with decreased lymphocyte apoptosis, restoration of innate cell populations, increased proinflammatory cytokines, and restoration of gamma interferon (IFN-gamma) production by stimulated splenocytes. These animals also showed significantly less P. aeruginosa growth from blood and bronchoalveolar lavage fluid. Importantly, AS101 improved survival after secondary injury 4 days following CLP. This increased survival was associated with many of the same findings observed in the 7-day group, i.e., restoration of IFN-gamma production, increased proinflammatory cytokines, and decreased bacterial growth. Collectively, these studies demonstrate that immunosuppression following initial septic insult increases susceptibility to secondary infection. However, by 7 days post-CLP, the host's immune system has recovered sufficiently to mount an effective immune response. Modulation of the immunosuppressive phase of sepsis may aid in the development of new therapeutic strategies.

Early neural and vascular dysfunctions in diabetic rats are largely sequelae of increased sorbitol oxidation.

These experiments were undertaken to assess the importance of cytoplasmic (c) sorbitol oxidation versus mitochondrial (m) pyruvate oxidation in mediating neural and vascular dysfunction attributable to hyperglycemia in diabetic rats. Increased oxidation of sorbitol is coupled to enzymatic reduction of free oxidized NAD(+)c to reduced NADHc, manifested by an increased ratio of NADH to NAD(+)c. Likewise, increased oxidation of pyruvate is coupled to reduction of NAD(+)m to NADHm, which increases the NADH/NAD(+)m ratio. Specific inhibitors of sorbitol production or sorbitol oxidation normalized: increased diabetic nerve NADH/NAD(+)c, impaired nerve-conduction velocity, and vascular dysfunction in sciatic nerve, retina, and aorta; however, they had little or no impact on increased NADH/NAD(+)m. These observations provide, for the first time, strong in vivo evidence for the primacy of sorbitol oxidation versus. pyruvate oxidation in mediating the metabolic imbalances, impaired nerve conduction, and vascular dysfunction evoked by diabetes. These findings are consistent with (a) the fact that oxidation of sorbitol produces "prooxidant" NADHc uncoupled from subsequent production of "antioxidant" pyruvate required for reoxidation of NADHc to NAD(+)c by lactate dehydrogenase, and (b) the hypothesis that neural and vascular dysfunction in early diabetes are caused primarily by increased NADHc, which fuels superoxide production by NADH-driven oxidases.

Anti-apoptotic peptides protect against radiation-induced cell death.

The risk of terrorist attacks utilizing either nuclear or radiological weapons has raised concerns about the current lack of effective radioprotectants. Here it is demonstrated that the BH4 peptide domain of the anti-apoptotic protein Bcl-xL can be delivered to cells by covalent attachment to the TAT peptide transduction domain (TAT-BH4) and provide protection in vitro and in vivo from radiation-induced apoptotic cell death. Isolated human lymphocytes treated with TAT-BH4 were protected against apoptosis following exposure to 15Gy radiation. In mice exposed to 5Gy radiation, TAT-BH4 treatment protected splenocytes and thymocytes from radiation-induced apoptotic cell death. Most importantly, in vivo radiation protection was observed in mice whether TAT-BH4 treatment was given prior to or after irradiation. Thus, by targeting steps within the apoptosis signaling pathway it is possible to develop post-exposure treatments to protect radio-sensitive tissues.

An in vivo selection system for homing endonuclease activity.

Homing endonucleases are enzymes that catalyze the highly sequence-specific cleavage of DNA. We have developed an in vivo selection in Escherichia coli that links cell survival with homing endonuclease-mediated DNA cleavage activity and sequence specificity. Using this selection, wild-type and mutant variants of three homing endonucleases were characterized without requiring protein purification and in vitro analysis. This selection system may facilitate the study of sequence-specific DNA cleaving enzymes, and selections based on this work may enable the evolution of homing endonucleases with novel activities or specificities.