Awareness-raising or knowledge transfer about conflict, conflict resolution and mediation. Rather, this. Need an update can refer to section B of this manual, where the basic principles and techniques of mediation. However, just as awareness-raising and knowledge transfer don't make a mediator, skill-building alone. MANUAL OF ANALYTICAL QUALITY CONTROL FOR PESTICIDES. Air Analysis. Water Analysis. Soil, House Dust, and Bottom Sediment. Polychlorinated Biphenyls (PCBs), and Other. Transfer of manually prepared samples onto a gas or liquid chromatography.

Adobe Flash Player is required to view this feature. If you are using an operating system that does not support Flash, we are working to bring you alternative formats. Original Article Reduced Mortality after Allogeneic Hematopoietic-Cell Transplantation Ted A.

Gooley, Ph.D., Jason W. Chien, M.D., Steven A. Pergam, M.D., M.P.H., Sangeeta Hingorani, M.D., M.P.H., Mohamed L. Sorror, M.D., Michael Boeckh, M.D., Paul J. Martin, M.D., Brenda M.

Sandmaier, M.D., Kieren A. Marr, M.D., Frederick R. Appelbaum, M.D., Rainer Storb, M.D., and George B. McDonald, M.D.

N Engl J Med 2010; 363:2091-2101 DOI: 10.1056/NEJMoa1004383. Methods We analyzed overall mortality, mortality not preceded by relapse, recurrent malignant conditions, and the frequency and severity of major complications of transplantation, including graft-versus-host disease (GVHD) and hepatic, renal, pulmonary, and infectious complications, among 1418 patients who received their first allogeneic transplants at our center in Seattle in the period from 1993 through 1997 and among 1148 patients who received their first allogeneic transplants in the period from 2003 through 2007. Components of the Pretransplant Assessment of Mortality (PAM) score were used in regression models to adjust for the severity of illness at the time of transplantation. Results In the 2003–2007 period, as compared with the 1993–1997 period, we observed significant decreases in mortality not preceded by relapse, both at day 200 (by 60%) and overall (by 52%), the rate of relapse or progression of a malignant condition (by 21%), and overall mortality (by 41%), after adjustment for components of the PAM score.

The results were similar when the analyses were limited to patients who received myeloablative conditioning therapy. We also found significant decreases in the risk of severe GVHD; disease caused by viral, bacterial, and fungal infections; and damage to the liver, kidneys, and lungs.

Infections, graft-versus-host disease (GVHD), and liver, kidney, and pulmonary complications have been associated with high mortality after allogeneic hematopoietic-cell transplantation since the introduction of this procedure 40 years ago. Changes in practice have decreased organ toxicity, and improved prevention and treatment strategies have decreased the severity of acute GVHD.

The control of infectious complications has improved since the development of molecular methods for the detection of viral and fungal infections, the use of preemptive treatments, the introduction of new antifungal agents, and the prevention of nosocomial infection. To examine the hypothesis that changes in the care of patients undergoing transplantation have improved outcomes, we compared the rates of death not preceded by relapse, recurrent malignant conditions, and overall deaths in two large cohorts of our patients from 1993 through 1997 and from 2003 through 2007. To explore correlates of improved outcomes, we analyzed the frequency and severity of acute GVHD and hepatic, renal, pulmonary, and infectious complications in these two time periods. Transplantation Techniques All patients received a conditioning regimen followed by infusion of donor cells. Although these regimens varied, the myeloablative conditioning regimens generally contained high-dose cyclophosphamide with busulfan or 12.0 to 13.2 Gy of total-body irradiation. Reduced-intensity regimens contained 2 to 3 Gy of total-body irradiation with or without fludarabine.

The patients received immunosuppressive drugs, usually a calcineurin inhibitor plus methotrexate or mycophenolate mofetil, to prevent GVHD. Prophylaxis against infections included low-dose acyclovir, trimethoprim−sulfamethoxazole or dapsone, an antifungal agent (fluconazole in both periods and drugs that are active against mold in patients with a pretransplantation mold infection), preemptive therapy with ganciclovir in patients with cytomegalovirus (CMV) infection (on the basis of antigen or DNA testing), and antibiotics in patients with neutropenia. In the second time period, all patients received ursodiol as prophylaxis against cholestasis. Outcome Measures Outcome measures included overall mortality, mortality not preceded by relapse, recurrent malignant conditions, and the frequency and severity of major complications. Death not preceded by relapse was defined as death after transplantation that was not preceded by a recurrent or progressive malignant condition. Data on overall mortality, mortality not preceded by relapse, and recurrent malignant conditions reflect events as of the date of the last contact before the database was locked on January 12, 2010.

Liver, Kidney, and Lung Complications through Day 100 Liver and kidney injuries were assessed according to the total serum bilirubin and creatinine concentrations. The severity of liver injury and liver GVHD was assessed according to the peak bilirubin concentration. Acute kidney injury was classified as a serum creatinine concentration that was two times as high as the baseline value or three times as high as the baseline value. Lung injury was defined by the need for diagnostic bronchoscopy and the development of respiratory failure.

The evaluation of pulmonary abnormalities included computed tomography to evaluate radiographic abnormalities and pulmonary consultation to determine whether fiberoptic bronchoscopy was indicated. Respiratory failure was defined by the need for more than 24 hours of mechanical ventilation for a nonelective reason. Viral, Bacterial, and Fungal Infections through Day 100 CMV infection was defined as the presence of viral pp65 antigen or DNA in plasma; CMV disease was defined as a dysfunction of an organ infected by CMV. Patients with one or more positive blood cultures for gram-negative organisms were considered to have gram-negative bacteremia. Invasive fungal infections were classified according to international consensus criteria. Only fungal infections that were proved or probable were included in this analysis.

Statistical Analysis The probability of overall survival was estimated with the use of the Kaplan−Meier method. Probabilities of death not preceded by relapse and of recurrent malignant conditions were estimated with the use of cumulative incidence curves, with recurrent malignant conditions viewed as a competing risk of death not preceded by relapse, and with death not preceded by relapse viewed as a competing risk of recurrent malignant conditions. Jaundice, GVHD, and doubling and tripling of baseline serum creatinine values were compared between the cohorts by means of logistic regression. Time to engraftment was defined as the first of 3 consecutive days during which the absolute neutrophil count was more than 500 cells per cubic millimeter. Mean times to engraftment, as well as the mean peak total serum bilirubin and creatinine values, were compared with the use of linear regression. The average daily total serum bilirubin and creatinine values were modeled with the use of generalized estimating equations.

Cox regression models were used to compare the hazards of failure for all other end points. Deaths after day 200 that were not preceded by relapse were not considered to be treatment failures for the end point of death by day 200 not preceded by relapse and were censored at day 200. Data for patients who survived without failure for the other end points were censored at the date of last contact.

Infectious and pulmonary complications occurring within the first 100 days were considered to be treatment failures; infectious and pulmonary events that occurred beyond this time were censored at day 100 and treated as nonfailures. Components of the Pretransplant Assessment of Mortality (PAM) score (on a scale of 8 to 50 when conditioning is included and 7 to 41 when conditioning is excluded, with higher scores indicating a higher risk of death) (Table 2 in the ) were used in regression models to adjust for the severity of illness at the time of transplantation, with the exception of the conditioning-regimen component.

All PAM components were treated as categorical variables, with a category for missing data included for each component. Additional adjustment for coexisting conditions, as captured by the Hematopoietic Cell Transplantation Comorbidity Index (HCT-CI) (on a scale of 0 to 29, with higher scores indicating a higher risk of death not preceded by relapse) (Table 3 in the ), was made in a subgroup of 1000 patients (409 patients from 1993 through 1997 and 591 patients from 2003 through 2007) who had previously been assessed with the use of this index by one of the authors. Two-sided P values were estimated by means of the Wald test; no adjustments were made for multiple comparisons. Characteristics of the Patients Table 1 Characteristics of Transplant Recipients According to Time Period., and Table 4 in the, show demographic, disease, and transplant characteristics, including components of the PAM score included in regression models. The adjusted average time to engraftment was 1.83 days less in the 2003−2007 period than in the 1993–1997 period among all patients with engraftment (P. Outcome Measures From the 1993−1997 period to the 2003−2007 period, significant decreases were seen in the hazard of death not preceded by relapse, both at day 200 (by 60%) and overall (by 52%), and in the rate of relapse or progression of a malignant condition (by 21%) and overall mortality (by 41%) ( Table 2 Comparison of Outcomes, Organ Dysfunction, Infection, and Acute GVHD after Transplantation between the Two Time Periods.

The probabilities of death not preceded by relapse at day 200 and of overall survival are shown in Figure 1 Probability of Death by Day 200 Not Preceded by Relapse and of Overall Survival during Two Time Periods. Panel A shows the probability of death not preceded by relapse, and Panel B shows the probability of overall survival. Data on patients who were alive after 7 years were censored at 7 years for graphic purposes only.. Among patients who received myeloablative regimens, significant reductions were seen in the hazard of death not preceded by relapse, at day 200 (by 56%) and overall (by 52%), as well as in the rate of recurrent malignant conditions (by 18%) and overall mortality (by 39%) ( ). Improvements in outcomes were consistent among various subgroups. The hazard ratios for death by day 200 that was not preceded by relapse were 0.62 among patients with acute lymphocytic leukemia, 0.38 among patients with acute myeloid leukemia, 0.60 among patients with chronic myeloid leukemia, and 0.42 among patients with the myelodysplastic syndrome; the hazard ratios for death from any cause were 0.67, 0.63, 0.67, and 0.65, respectively. Average PAM scores after exclusion of the conditioning-regimen component in patients receiving myeloablative regimens were 16.3 during the 1993−1997 period and 17.3 during the 2003−2007 period; the average PAM score after exclusion of the conditioning-regimen component was 22.1 in patients receiving reduced-intensity regimens.

Among patients with low PAM scores (. Liver Disease From the 1993−1997 period to the 2003−2007 period, the odds of jaundice were significantly reduced, by more than 70% ( ). The magnitude of the reduction was similar for patients who received only myeloablative regimens ( ). The average peak serum bilirubin level in the earlier period was 7.6 mg per deciliter (129.9 μmol per liter), as compared with 3.3 mg per deciliter (56.4 μmol per liter) in the later period (adjusted mean difference, 4.4 mg per deciliter [75.2 μmol per liter]; P.

Renal Injury The odds of acute kidney injury were significantly reduced between the first and second time periods. The magnitude of the reduction was similar among patients who received only myeloablative regimens ( ). Shows fitted average daily serum creatinine values to day 100.

The adjusted average difference in the daily serum creatinine level was 0.13 mg per deciliter (11.5 μmol per liter) (P. Infections Although the rate of CMV reactivation remained stable between the two time periods, the hazard of early CMV disease was reduced during the later period by 48% when all CMV-seropositive patients were considered, and by 47% when only CMV-seropositive patients receiving myeloablative regimens were considered ( ). The hazard of the development of bacteremia with a gram-negative organism decreased by 39%, the hazard of invasive mold infection decreased by 51%, and the hazard of invasive candida infection decreased by 88% between the two periods. The magnitude of the decreases in the hazards of these infections was similar among patients who received myeloablative conditioning regimens ( ). Acute GVHD The percentages of patients with mild, moderate, and severe acute GVHD decreased from the earlier period to the later period, with a 67% decrease in the odds of the development of grade 3 or 4 GVHD ( ). We found significant reductions in the frequency of stage 3 or 4 gut and especially stage 3 or 4 hepatic GVHD in the 2003−2007 period ( and ). The reduction in the odds of the development of grade 3 or 4 GVHD was consistently seen across donor types: odds ratios were 0.35 among patients who had a matched-sibling donor, 0.11 among patients who had a donor who was a relative but not a sibling or a mismatched-sibling donor, and 0.33 among patients who had an unrelated donor.

Discussion We found a substantial reduction in the hazard of death related to allogeneic transplantation and improved long-term survival from the 1993−1997 period to the 2003−2007 period. We also saw decreases in the hazard or probability of almost every transplantation complication that we examined. In these analyses, we adjusted our models for individual components of the previously validated PAM score and, when available, for HCT-CI scores.

On average, patients who underwent transplantation during the 2003−2007 period were older, were more seriously ill, and had more advanced disease than those who underwent transplantation in the earlier period. In a subgroup of patients for whom HCT-CI scores were available, further adjustment for the HCT-CI score changed the hazard ratios for death by less than 2%. Both scores provide important prognostic information in this population. Several changes in our transplantation practice appear to have contributed to improved outcomes. We now treat patients who have coexisting medical conditions with less toxic conditioning regimens.

This shift in the intensity of the conditioning regimen resulted from data showing that higher-dose regimens resulted in more organ damage, without the commensurate benefit of a reduced risk of a recurrent malignant condition, and from data showing that the graft-versus-tumor activity of donor cells can have a dominant role in eliminating malignant cells. Lower-dose myeloablative regimens, which were used more often in the more recent period, are those in which the dose of total-body irradiation is limited or fludarabine is substituted for cyclophosphamide, or cyclophosphamide dosing is individualized; these regimens are based on data showing that aberrant metabolism of cyclophosphamide and high exposures to total-body irradiation were factors leading to fatal hepatic sinusoidal obstruction syndrome and multiorgan failure. Despite more frequent use of peripheral-blood hematopoietic cells instead of marrow during the 2003−2007 period, the odds of the development of grade 3 or 4 GVHD decreased by 67% from the earlier period, partly because of ursodiol's effect on GVHD-related cholestasis and the near disappearance of stage 4 hepatic GVHD. The role of more accurate HLA matching of unrelated donors in improving outcomes cannot be readily ascertained from our data, which show that the reduction in the rate of severe GVHD was similar in patients who received transplants from a matched sibling and in patients who received transplants from an unrelated donor. GVHD prophylaxis did not change substantially between the two time periods, but our approach to the treatment of GVHD did change. By 2003, two syndromes of gastrointestinal GVHD were apparent, one affecting mostly the upper gut (with anorexia, nausea, vomiting, and satiety), and the other mostly the midgut (with diarrhea, abdominal pain, and bleeding). The upper-gut syndrome occurs more frequently, seldom progresses to grade 4 GVHD, responds to prednisone therapy, and has a better prognosis.

Our past practice of treating all cases of acute GVHD with prednisone at a dose of 2 mg per kilogram of body weight per day was abandoned in favor of therapy based on clinical manifestations and the risk of death. This change in treatment philosophy was also prompted by data showing that the risks of CMV, fungal, and bacterial infections were significantly related to the prednisone dose. During the 2003−2007 period, most patients with the upper-gut GVHD syndrome were initially treated with prednisone at a dose of 1 mg per kilogram per day plus a topically active glucocorticoid, reducing average prednisone exposure by 48%. The increased use of peripheral-blood donor cells resulted in significantly faster neutrophil engraftment and earlier recovery of immunity against fungal and bacterial infections. The decreased hazards of gram-negative bacteremia and fungemia might be related to reduced gut toxicity from conditioning regimens and to reduced risks of multiorgan failure and midgut GVHD. Antibacterial prophylaxis in patients with neutropenia shifted from cephalosporins to quinolones between the two time periods.

Antifungal prophylaxis with fluconazole was used during the 1993−1997 period; with the advent of fungal antigen testing and new antifungal drugs, patients with positive blood tests or pulmonary nodules were more likely to receive mold-active azoles (e.g., itraconazole and voriconazole) or an echinocandin. Preemptive antiviral therapy is now based on a more sensitive diagnostic test for CMV viremia. The decrease in the degree of jaundice can be traced to less intense conditioning regimens, less frequent bacteremia and GVHD, and the use of ursodiol to prevent cholestasis. During the 2003−2007 period, patients who were at risk for the fatal sinusoidal obstruction syndrome underwent conditioning with fludarabine−busulfan or reduced-intensity regimens or 12 Gy of total-body irradiation plus individualized doses of cyclophosphamide, based on therapeutic drug monitoring, instead of high-dose cyclophosphamide. The adoption of ursodiol prophylaxis was based on data showing that ursodiol improved the results of liver tests in patients with GVHD, reduced the frequency of jaundice, and increased survival after transplantation.

The decreased frequencies of renal dysfunction and respiratory failure in the 2003−2007 period are intertwined with the increased use of lower-dose myeloablative regimens; reduced risks of the hepatic sinusoidal obstruction syndrome, gram-negative bacteremia, and invasive mold infections; and avoidance of amphotericin products. The decreased frequency of severe GVHD may also have had a protective effect on renal and pulmonary function, since both the kidneys and the lungs are affected by the inflammatory milieu of acute GVHD.

In conclusion, our data show clear improvement in outcomes of transplantation between the period from 1993 through 1997 and the period from 2003 through 2007. The data also indicate areas of transplantation biology and patient care in which research is needed to achieve further progress — specifically, GVHD and graft-versus-tumor effects, immunologic tolerance, and the management of infection and recurrent malignant conditions. Supported by grants (CA 18029, CA 15704, CA78902, HL36444, HL088201, HL088021, HL096831, and DK063038) from the National Institutes of Health. Gooley reports serving as a statistical consultant to DOR BioPharma; Dr. Pergam, receiving research funding from Chimerix and ViroPharma (through a sponsored American Society for Blood and Marrow Transplantation New Investigator Award), and consulting fees from ViroPharma; Dr. Boeckh, receiving grant support, consulting fees, or both from Roche/Genentech, ViroPharma, Astellas, Pfizer, Merck, Vical, Chimerix, AiCuris, Boehringer Ingelheim, and Theraclone Sciences; Dr. Martin, receiving grant support from Roche and Soligenix and payments for serving on data and safety monitoring boards for Pfizer; Dr.

Marr, receiving grant support from Astellas and Merck and consulting fees from Astellas, Basilea, Evolva, Merck, and Pfizer; and Dr. McDonald, being a consultant to and holding an equity position with Soligenix and receiving consulting fees from Gentium. Provided by the authors are available with the full text of this article at NEJM.org. No other potential conflict of interest relevant to this article was reported. We thank the many physicians, nurses, physician assistants, pharmacists, and support staff who cared for our patients during the two periods of the study; the patients who allowed us to care for them and who participated in our ongoing clinical research; and David Myerson, Gary Schoch, Chris Davis, Margaret Au, Miwa Sakai Vernon, and Emily Pao for help with data abstraction. Source Information From the Clinical Research Division (T.A.G., J.W.C., S.A.P., S.H., M.L.S., M.B., P.J.M., B.M.S., K.A.M., F.R.A., R.S., G.B.M.) and the Vaccine and Infectious Disease Institute (S.A.P., M.B.), Fred Hutchinson Cancer Research Center; and the Departments of Medicine (J.W.C., S.A.P., M.L.S., M.B., P.J.M., B.M.S., K.A.M., F.R.A., R.S., G.B.M.), Pediatrics (S.H.), and Biostatistics (T.A.G.), University of Washington — both in Seattle. Address reprint requests to Dr.

McDonald at the Gastroenterology/Hepatology Section (D2-190), Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109. References • 1 Appelbaum FR, Forman SJ, Negrin RS, Blume KG, eds.

Thomas' hematopoietic cell transplantation. Oxford, England: Wiley-Blackwell Publishing, 2009. • 2 McDonald GB, Slattery JT, Bouvier ME, et al. Cyclophosphamide metabolism, liver toxicity, and mortality following hematopoietic stem cell transplantation.

Blood 2003;101:2043-2048 • 3 Hingorani SR, Guthrie K, Batchelder A, et al. Acute renal failure after myeloablative hematopoietic cell transplant: incidence and risk factors. Kidney Int 2005;67:272-277 • 4 Clark JG, Madtes DK, Martin TR, Hackman RC, Farrand AL, Crawford SW. Idiopathic pneumonia after bone marrow transplantation: cytokine activation and lipopolysaccharide amplification in the bronchoalveolar compartment. Crit Care Med 19-1806 • 5 Patriarca F, Skert C, Sperotto A, et al.

Incidence, outcome, and risk factors of late-onset noninfectious pulmonary complications after unrelated donor stem cell transplantation. Bone Marrow Transplant 2004;33:751-758 • 6 Martin PJ, McDonald GB, Sanders JE, et al. Increasingly frequent diagnosis of acute gastrointestinal graft-versus-host disease after allogeneic hematopoietic cell transplantation. Biol Blood Marrow Transplant 2004;10:320-327 • 7 Mielcarek M, Storer BE, Boeckh M, et al.

Initial therapy of acute graft-versus-host disease with low-dose prednisone does not compromise patient outcomes. Blood 2009;113:2888-2894 • 8 Hockenbery DM, Cruickshank S, Rodell TC, et al. A randomized, placebo-controlled trial of oral beclomethasone dipropionate as a prednisone-sparing therapy for gastrointestinal graft-versus-host disease.

Blood 2007;109:4557-4563 • 9 Lee SJ, Klein J, Haagenson M, et al. High-resolution donor-recipient HLA matching contributes to the success of unrelated donor marrow transplantation. Blood 2007;110:4576-4583 • 10 Upton A, Kirby KA, Carpenter P, Boeckh M, Marr KA.

Invasive aspergillosis following hematopoietic cell transplantation: outcomes and prognostic factors associated with mortality. Clin Infect Dis 2007;44:531-540 • 11 Yokoe D, Casper C, Dubberke E, et al. Infection prevention and control in health-care facilities in which hematopoietic cell transplant recipients are treated. Bone Marrow Transplant 2009;44:495-507 • 12 Boeckh M, Bowden RA, Gooley T, Myerson D, Corey L. Successful modification of a pp65 antigenemia-based early treatment strategy for prevention of cytomegalovirus disease in allogeneic marrow transplant recipients. Blood 19-1782 • 13 Nakamae H, Kirby KA.

The Plasmodium mitochondrial electron transport chain has received considerable attention as a potential target for new antimalarial drugs. Atovaquone, a potent inhibitor of Plasmodium cytochrome bc 1, in combination with proguanil is recommended for chemoprophylaxis and treatment of malaria. The type II NADH:ubiquinone oxidoreductase (NDH2) is considered an attractive drug target, as its inhibition is thought to lead to the arrest of the mitochondrial electron transport chain and, as a consequence, pyrimidine biosynthesis, an essential pathway for the parasite. Using the rodent malaria parasite Plasmodium berghei as an in vivo infection model, we studied the role of NDH2 during Plasmodium life cycle progression. NDH2 can be deleted by targeted gene disruption and, thus, is dispensable for the pathogenic asexual blood stages, disproving the candidacy for an anti-malarial drug target. After transmission to the insect vector, NDH2-deficient ookinetes display an intact mitochondrial membrane potential. However, ndh2(−) parasites fail to develop into mature oocysts in the mosquito midgut.

We propose that Plasmodium blood stage parasites rely on glycolysis as the main ATP generating process, whereas in the invertebrate vector, a glucose-deprived environment, the malaria parasite is dependent on an intact mitochondrial respiratory chain. Introduction Apicomplexan parasites of the genus Plasmodium are the causative agents of malaria. Worldwide, malaria accounts for >200 million infections and nearly 1 million deaths every year (). The emergence and ongoing spread of resistance to antimalarial drugs calls for the development of new antimalarial compounds (). Cases of resistance to almost all quinolone and antifolate drugs have been reported as well as decreased susceptibility to artemisinin drugs (). The Plasmodium mitochondrial electron transport chain (mtETC) is a validated target for the development of antimalarial drugs (–). The effects of atovaquone on cytochrome bc I () and candidate inhibitors on dihydroorotate dehydrogenase (DHOD) (–), succinate dehydrogenase (, ), and the NADH:ubiquinone oxidoreductase (NDH2) (–) have been studied.

However, the in vivo functions and physiological relevance of the mtETC components are still not understood. In Plasmodium parasites NDH2 is one of five mitochondrial dehydrogenases that feed electrons into the mtETC (). Typically, eukaryotes possess a multicomponent rotenone-sensitive NADH:ubiquinone oxidoreductase, also termed complex I, which is located in the inner mitochondrial membrane. It catalyzes the transfer of electrons from NADH to ubiquinone (coenzyme Q (Q)) leading to the reduced form, ubiquinol (QH 2), and is involved in establishing the electropotential across the inner mitochondrial membrane (Δψ m) by pumping hydrogen ions out of the mitochondrial matrix (, ). Plasmodium spp., however, has a rotenone-insensitive, single subunit NADH:quinone oxidoreductase, also termed alternative complex I or NDH2 ().

This enzyme is found in some bacteria and archaea as well as in yeast and plants (). It is characterized by the lack of a transmembrane domain, by conserved triglyceride nucleotide binding motifs (G XG XXG), and by the apparent lack of any detectable proton pump activity. Although in general NDH2 sequences are highly divergent, this protein is relatively well conserved among Plasmodium species ().

Structural information remains elusive, but a first prediction of a Plasmodium falciparum NDH2 structure has been proposed based on sequence and similarity data (,, ). Sensitivity of P. Falciparum NDH2 to various inhibitors, such as dibenziodolium chloride (CID 16219231), diphenyliodonium chloride (CID 73870), and the quinolone derivative HDQ (2-dodecylquinolin-4-ol,1-oxide; CID 600305), has been reported (). However, the drugs proved to be neither effective nor specific for NDH2 (,, ). Hypothetical model of the mitochondrial NADH:oxidoreductase NDH2 in the mtETC of the malarial parasite.

Shown is a schematic of the localization and biochemical function of PbNDH2 ( dark blue ellipse). Return Of The Durruti Column Rar. The precise localization to either the internal (mitochondrial.

The electron transfer from NADH to Q has been shown to follow a ping-pong mechanism, possibly to maintain a pool of oxidized NADH, which in turn can be used for metabolic processes, such as glycolysis (). At the same time, NDH2 feeds electrons into the mtETC, thereby contributing indirectly to the Δψ m.

However, the relevance of the plasmodial NDH2 as well as of the mtETC and the mitochondrial membrane potential in vivo, in particular throughout the Plasmodium life cycle, is not yet elucidated. In other eukaryotes, one of the main functions of mitochondria is the generation of ATP through the respiratory chain. In Plasmodium blood stages, however, the energy metabolism relies largely on glycolysis (, ), and the gain of ATP through the mtETC is considered only marginal (). It has been suggested that the main function of the mtETC might be to provide the electron acceptor CoQ (ubiquinone) for DHOD, a key enzyme for the essential de novo biosynthesis of pyrimidine (). The Δψ m, in turn, is essential for transport processes, for example in the heme biogenesis pathway (). However, Δψ m does not rely exclusively on the mtETC ().

Atovaquone, a Q analog, binds to cytochrome bc I, thereby inhibiting the reoxidization of QH 2 to coenzyme Q and interrupting the mtETC. Atovaquone alone, however, does not lead to the collapse of the mitochondrial membrane potential. Furthermore, it has recently been shown that short time treatment with atovaquone and proguanil exerts a stage-specific and cytostatic effect on P. Falciparum blood stages in vitro () with ring- and schizont stages being able to survive the interruption of the mtETC as well as the collapse of the Δψ m for as long as 48–96 h, whereas trophozoites proved to be more sensitive. In this study we addressed the in vivo role of NDH2 in the rodent malaria model parasite Plasmodium berghei. We could identify an essential function for parasite growth and, as a consequence, sporozoite formation inside the mosquito vector.

Our data suggest that the Plasmodium alternative type I complex is dispensable for life cycle progression in the vertebrate host. Gene Expression Profiling For quantitative real-time-PCR analyses, poly(A)+ RNA was isolated using oligo-dT columns (Invitrogen) from mixed blood stages, ookinetes, oocysts, salivary gland-associated sporozoites, and 24-h liver stages of P. Berghei wild-type (WT) parasites. After DNase treatment, the mRNA of each sample was reverse-transcribed to cDNA using oligo-dT primers (Ambion).

Specificity of the amplification products was confirmed by melting curve analysis; a no-template control was added in every run. For primers, see. In our experiments, each RT-PCR run was carried out in triplets of one mRNA pool, and the whole series was reproduced in a second independent experiment. Gene Targeting Vectors and P. Berghei Transfection For mCherry tagging of PbNDH2, we cloned a C-terminal fragment of 1194 bp into a standard transfection vector, which contained the mCherry coding sequence using primers NDH2-mCherry_for and NDH2_mCherry_rev. After linearization of the plasmid, transfection, and a single crossover event, we obtained parasites with the tagged full-length NDH2.

Genotyping was performed using primers 5′integration_for and 5′KO_flank_rev and WT_for and WT_rev for transgenic and WT parasites, respectively. For gene deletion of PbNDH2, a replacement vector was generated by cloning two fragments, 5′ KO flank (649 bp) and 3′ KO flank (651 bp), into the standard transfection vector () using P. Berghei genomic DNA as a template and the following primer combinations (see ): 5′_KO_flank_for and 5′_KO_flank_rev, and 3′_KO_flank_for and 3′_KO_flank_rev. For replacement-specific amplification of the ndh2(−) locus, the following primer pairs were utilized: in the 5′ region, 5′_integration_for and 5′_integration_rev; the 3′ region, 3′_integration_for and 3′_integration_rev. To validate the purity of the clonal ndh2(−) population, an NDH2-specific amplification using WT_for and WT_rev was performed.

Two independent ndh2(−) parasite populations, termed ko1 and ko2, from two consecutive transfections and subsequent in vivo clonings were obtained. After verification of the phenotypical identity, one representative clone of each ko1 and ko2 was used for a detailed analysis.

The Southern blot was done using the PCR DIG probe synthesis kit and the DIG Luminescent Detection kit (Roche Applied Science) according to the manufacturer's instructions. Plasmodium Life Cycle and Phenotypic Analysis of Mutant Parasites Anopheles stephensi mosquitoes were kept at 21 °C, 80% humidity, and daily feeding on 10% sucrose. Asynchronous blood stages of P. Berghei ANKA-GFP (WT) () and ndh2(−) parasites were maintained in NMRI mice and checked for gametocyte formation and exflagellation of microgametes before mosquito feeding. Exflagellation events were found to be similar, with ∼3900/μl (WT), ∼7200/μl ( ndh2(−) ko1), and ∼2700/μl ( ndh2(−) ko2) observed within a period from ∼10 to 16 min after taking blood from the infected mouse. For the subsequent mosquito infection, age-matched A.

Stephensi mosquitoes were allowed to blood-feed on the anesthetized mice for 15 min. For mosquito infection with cultured ookinetes, age-matched mosquitoes were membrane fed with 3,500,000 ookinetes of each WT, ndh2(−) ko1, and ndh2(−) ko2. Dissection of mosquitoes was conducted at days 10, 14, 17, and 21 to determine infectivity and sporozoite numbers in midguts and salivary glands, respectively. For transmission electron microscopy, infected midguts were fixed in 2.5% glutaraldehyde. To obtain exo-erythrocytic forms, the hepatocytes of the cell line HuH7 were infected with salivary gland sporozoites and cultured for 24–48 h.

Immunofluorescence and Mitochondrial Staining Parasites were fixed with 4% paraformaldehyde and permeabilized with 1% Triton X-100. Immunofluorescence was done with previously described monoclonal antibodies against P. Berghei circumsporozoite protein () and Hsp70 (). Oocysts were stained with either antibodies against circumsporozoite protein or GFP (Abcam), and the NDH2-mCherry signal was enhanced using anti-mCherry antibodies (Chromotek). Mitochondria were stained with MitoTracker Green FM (Invitrogen) according to the manufacturer's instructions.

MitoTracker Green FM is not retained well after fixation, but in our hands at a concentration of 500 n m, subsequent fixation of parasites with 4% paraformaldehyde and permeabilization with methanol led to satisfying results. To detect the mitochondrial membrane potential, JC-1 (Sigma) was used on WT and ndh2(−) ookinetes according to the manufacturer's instructions. Ookinetes were incubated in JC-1 staining solution for 20 min at 20 °C and observed under the fluorescent microscope within 20 min. NDH2 Is Expressed in Multiple Plasmodium Life Cycle Stages and Localizes to Mitochondria We initiated our analysis by profiling the expression of NDH2 transcripts in different extra- and intracellular stages of the P.

Berghei life cycle ( A). Using cDNA as template we could readily identify NDH2 mRNA in mixed blood stages, purified ookinetes, midgut-associated oocysts, and liver stage trophozoites (24 h after infection). Although transcript levels varied slightly in these stages, the most prominent exception of NDH2 expression was purified salivary gland-associated sporozoites, which had a significantly reduced (>300-fold) level as compared with all other stages tested. The type II NDH2 is a mitochondrial protein in the malaria parasite.

A, expression profiling of NDH2 by quantitative real time PCR is shown. Data were normalized to GFP, which is constitutively expressed under the EF1α promoter.

Note the abundant. We next generated a parasite line, termed NDH2mCherry, which was obtained by insertional tagging of the endogenous NDH2 open reading frame by in-frame fusion of mCherry at the C terminus ( B). For this purpose we designed a targeting vector that contained a 5′ deleted copy of NDH2 fused to the mCherry protein (). Upon single crossover by restriction endonuclease-mediated homologous recombination and positive selection with the antifolate pyrimethamine, this vector is predicted to result in a functional, mCherry-tagged 5′ and a non-functional, promoter-less and N-terminal-deleted 3′ copy, separated by the positive selection marker ( B). Genotyping of the pyrimethamine-resistant parasite line after in vivo cloning by limited dilution revealed the desired parasite clone, which no longer contained wild-type parasites ( C). We used the NDH2mCherry parasites to monitor expression in the mammalian and insect hosts. Live cell imaging based on mCherry signals revealed only faint signals.

We, therefore, fixed and stained parasites with an anti-mCherry antibody ( D). This approach revealed a number of important findings with respect to the expression and localization of NDH2. Unexpectedly, no robust mCherry signal was detected in asexual blood stages, as exemplified in intracellular trophozoites.

In contrast, gametocytes, the sexual forms of the blood stage development, consistently displayed an intense punctuate or branched 3 staining, indicative of abundant NDH2 protein that localizes to a cellular organelle, most likely the Plasmodium mitochondrion. NDH2 expression in gametocytes also explains the expression obtained by mRNA profiling in mixed blood stages ( A). Expression continued in ookinetes, in good agreement with the transcript analysis.

In ookinetes, a prominent concentration of the signal in a branched structure adjacent to the parasite nucleus was detectable. NDH2 protein was prominent in oocysts and late liver stages but absent in sporozoites, further corroborating the transcript profiles ( A). Together, we conclude that NDH2 is most prominently expressed in gametocytes, ookinetes, oocysts, and liver stages and virtually absent in asexual blood stages and sporozoites. The immunofluorescence analysis of the NDH2-mCherry parasites was already suggestive of the expected localization of NDH2 to the parasite mitochondria. To unequivocally show mitochondrial localization, we used MitoTracker, an organelle-specific fluorescent dye ( E). We analyzed gametocytes and ookinetes, where NDH2 expression is robust. The mCherry signal showed perfect overlap with the MitoTracker, corroborating proper localization of NDH2 to the mitochondria.

Ablation of NDH2 Does Not Affect Growth of Asexual Blood Stage Parasites We next wanted to test in vivo essentiality of NDH2 by targeted gene deletion. For this purpose, we employed experimental genetics and targeted the gene by double cross-over recombination ( A). As predicted from the expression analysis ( D) but in stark contrast with the proposed candidacy as an anti-malaria drug target (,,, ), we were able to recover recombinant parasites that contained the deletion of NDH2, as confirmed by PCR-based genotyping ( B) and quantitative RT-PCR ( C). This finding already indicated that loss of NDH2 function is compatible with parasite survival inside host erythrocytes in vivo. Berghei NDH2 is dispensable for asexual blood stages. A, the wild-type NDH2 locus is targeted with a linearized replacement plasmid containing the 5′- and 3′-UTRs of PbNDH2, GFP, and the positive selection marker Tgdhfr/ts. After double.

To test whether other mitochondrial NADH-dependent dehydrogenases are up-regulated in ndh2(−) parasites and can potentially compensate for loss of NDH2 function, we performed quantitative PCR in WT and ndh2(−) mixed blood stages for glycerol-3-phosphate dehydrogenase ( G3PDH), DHOD, malate:quinone oxidoreductase ( MQO), and succinate dehydrogenase ( SDH) ( D). This analysis confirmed complete absence of NDH2 transcripts in the knock-out parasite line and showed no compensatory up-regulation of any of the dehydrogenases tested. To corroborate our findings and detect potential minor growth defects, we next infected C57bl/6 mice by intravenous inoculation of 1,000,000 asexual blood stages of either WT or ndh2(−) parasites ( E).

NDH2 loss-of-function parasites replicated with identical kinetics as compared with WT parasites, suggesting that ablation of NDH2 is tolerated by Plasmodium parasites during the intra-erythrocytic replication cycle. Moreover, all animals ultimately displayed symptoms of cerebral malaria (), suggesting that ndh2(−) parasites retained virulence and the capacity to induce experimental cerebral malaria in vivo. We conclude that targeted design of potential specific NDH2 inhibitors would not aid in development of new anti-malarial drugs.

Arrested Oocyst Maturation in ndh2(−) Parasites We next examined oocyst development that occurs at the outer side of the mosquito midgut (). When we isolated midguts from A. Stephensi mosquitoes that had fed on WT- or ndh2(−)-infected mice 10 days earlier, we noticed abundant oocysts that were reactive with anti-circumsporozoite protein antibodies ( A).

Infectivity, i.e. The proportion of oocyst-positive insects, was similar in WT- and ndh2(−) -infected mosquitoes irrespective of natural transmission or membrane feedings with cultured ookinetes and remained constant over time ( B). When we scored the total numbers of oocysts, again no differences were detected between mosquitoes infected with the two parasite populations ( C and ). As predicted, the number of oocysts in mosquitoes after membrane feeding was lower for both WT and ndh2(−) parasites compared with after natural feeding ().

We conclude that ndh2(−) parasites establish infections in the insect vector similar to WT parasites. Ndh2(−) parasites are arrested in oocyst maturation. A, shown is successful colonization of midguts after transmission to A.

Mosquitoes were allowed to feed on WT- and ndh2(−)-infected mice, and midguts were removed 17 days. Upon closer inspection we noticed that ndh2(−) oocysts were considerably smaller than oocysts from WT-infected mosquitoes ( D). Quantification of oocyst sizes revealed largely reduced ndh2(−) oocysts ( E). Prolonged development did not improve sporogony, resulting in the complete failure to produce sporozoites (). We conclude that NDH2 is vital for sporogony. In the absence of this mitochondrial protein, life cycle progression is completely blocked inside the insect vector.

Developmental Defects in Oocysts Lacking NDH2 To gain further insight into oocyst development of ndh2(−) parasites, we employed transmission electron microscopy (). We observed fully rounded oocysts with apparently intact oocyst walls, indicating that initial transition from ookinete to oocyst is not impaired by the absence of NDH2. We could also consistently identify mitochondria with the typical tubular cristae ( and ).

However, the overall level of organization in mutant oocysts was low compared with WT oocysts. We could not detect any sporozoite formation. Strikingly, we repeatedly detected remnants of ookinete-related structures in ndh2(−) oocysts (), such as the apical complex, microtubules, and micronemes. This finding is unprecedented and bears resemblance to an ookinete surrounded by oocyst cytoplasm. We detected these structures up until day 14 after infection, indicative of a defect in proper ookinete disintegration.

We conclude that NDH2 is vital for multiple developmental processes during oocyst maturation in the mosquito vector. Ndh2(−) Parasites Are Infectious to the Mammalian Host When Complemented during Sporogony We finally wanted to test whether ndh2(−) parasites display an additional defect after transmission to the mammalian host, i.e.

During preerythrocytic mammalian stages. Ookinetes and sporozoites are tetraploid and haploid, respectively. Previous work established that heterozygous oocysts, obtained by crossing mutant and WT parasites in vivo, can rescue loss-of-function mutants if defects are restricted to sporogony (, ). For the present study we crossed ndh2(−) and WT blood stage parasites ( input) and genotyped the mixed parasites in comparison with clonal parasites before and after mosquito transmission ().

After bite back of mosquitoes infected with the mixed population, the ndh2(−) genotype was recovered from blood stage infection in mice ( output), strongly suggesting that the essential in vivo function of Plasmodium NDH2 is restricted to the insect vector stages. DISCUSSION Our data show that NDH2 plays a vital role for sporogony inside the invertebrate definitive host but not during asexual blood stage development in the warm-blooded intermediate host.

Plasmodium parasites encounter considerable environmental changes upon transmission from the vertebrate to the insect vector, reflected by fundamental changes in morphology and gene expression of the parasite (, ). The sexual phase of Plasmodium ends with fertilization of the macrogamete in the midgut lumen and subsequent development into a motile zygote, the so-called ookinete.

The ookinete then traverses the midgut wall and settles between the midgut epithelium and the basal lamina, where it develops into the sessile oocyst. Parasites are now extracellular, in contrast to replicative stages inside the mammalian host. They are exposed to ambient temperatures, more oxygen, and of particular importance in view of their energy metabolism, to a glucose-deprived environment. NDH2 Is Most Abundant in Cristate Mitochondria Data based on microarray and proteomic studies have shown before that some members of the mitochondrial electron transport chain are up-regulated in oocysts compared with asexual blood stages, such as the FAD-dependent glycerol-3-phosphate dehydrogenase and cytochrome c (, ).

We found that NDH2 transcripts can be detected in most phases of the parasite lifecycle, and using an endogenous tagging approach, we show that NDH2 is most abundant in gametocytes, ookinetes, oocysts, and liver stages. The notion that the NDH2-mCherry fusion protein is functional is supported by localization to parasite mitochondria and functional complementation of the developmental arrest of NDH2 loss of function parasites. Remarkably, abundance of NDH2 correlates with the cristate stage of the mitochondrion. Berghei, cristae have been reported for pre-erythrocytic stages (), gametocytes, sporogonic stages (), and oocysts (, ), whereas trophozoites were termed acristate (). Similarly, few cristae were found in asexual stages of P.

Falciparum and more in gametocytes (, ). Apparently, Plasmodium mitochondria undergo distinct switches from cristate (sexual and mosquito stages) to acristate stages (asexual erythrocytic stages) and back (). Cristae are invaginations of the inner membrane, providing a greater surface for the complexes of the mtETC, thus allowing for a higher rate of ATP metabolism. For instance, it has been shown that the activity of succinate dehydrogenase is high in cristate but low in acristate mitochondria (). Therefore, abundant expression of NDH2mCherry might reflect the increase in surface of the mitochondrial inner membrane, which in turn would lead to a higher capacity to integrate NDH2.

We found that mitochondria in ndh2(−) oocysts, the point of life cycle arrest, are clearly cristate, similar to those of WT oocysts. NDH2 Is Vital Only for Oocyst Maturation It has been proposed that the mtETC might be particularly important in non-erythrocytic stages (, ). We now provide genetic evidence to support this hypothesis. Our findings that ablation of NDH2 is tolerated in disease-causing asexual blood stages but lethal during oocyst maturation and that transient trans-complementation during sporogony leads to successful life cycle progression suggests that NDH2 is needed, most likely as an electron donor, in oocysts. A plausible assumption is that NDH2 assures sufficient supply of ATP during sporogony in the insect vector.

However, NDH2 does not seem to be essential for the branching or segregation of mitochondria. Dynaguard Dvr Software. The latter has to take place not only during the sporozoite budding process in oocysts but also in liver and blood schizogony to assure that merozoites receive a single mitochondrion. None of these processes was affected by the deletion of NDH2, as we could show by complementing ndh2(−) with WT parasites; once ndh2(−) parasites had been rescued from arrest during oocyst maturation, they were able to complete the life cycle and establish blood stage infections. In a preliminary experiment we used HDQ to test its effect on P.

Berghei WT and ndh2(−) blood stages in vivo. HDQ was thought to target P. Falciparum NDH2 () but was more recently shown to likely inhibit DHOD (, ). We speculated that the absence of NDH2 in combination with HDQ treatment might show a stronger effect than HDQ treatment alone. However, at a concentration of 50 mg/kg bodyweight, HDQ had no effect on parasite growth in vivo () either in WT or ndh2(−) parasites. Interestingly, 32 mg of HDQ/kg bodyweight has been shown to affect Toxoplasma gondii growth in vivo ().

We interpret these findings as an indication for potential differences in energy requirements and ATP generation between Plasmodium parasites and T. NDH2 Is Not Vital for Maintenance of the Mitochondrial Membrane Potential We show in ookinetes that the mitochondrial membrane potential was not abolished in the absence of NDH2. Although this finding casts doubt on the proposed role of NDH2 as one of the main suppliers of electrons to the mtETC (), a possible explanation is that only a marginal electron flux is necessary to maintain the mitochondrial membrane potential because of the overall low level of ATP synthesis. Alternatively, reverse action of the F0F1 ATP synthase, i.e. ATP hydrolysis, might maintain the mitochondrial membrane potential for some time. Such a reverse role has for instance been demonstrated for Trypanosoma brucei and Tetrahymena thermophila (, ). Although reverse action of F0F1 ATPase has not yet been shown for Plasmodium, the components of the enzyme have been identified ().

We were unable to employ JC-1 in ndh2(−) oocysts, mainly due to difficulties to unequivocally detect them among the metabolically active insect cells without fixation and immunofluorescence analysis. 3 We hypothesize that the membrane potential would be substantially impaired during sporogony, where presumably more ATP needs to be generated through the mtETC, resulting in insufficient supply of electrons. However, we observed viable, i.e. GFP expressing, ndh2(−) oocysts for extended periods (day 21 after infection and beyond), which would suggest that the membrane potential is not completely abolished even in these stages. Ablation of NDH2 Leads to Impairment of ATP-dependent Processes, Such as Organelle Disposal The maturing oocyst needs ATP for a number of tasks, such as protein synthesis, mitosis, and processes involving actin polymerization. Within the first days of oocyst maturation, the parasite has to dispose of and possibly recycle ookinete-related organelles, such as micronemes and microtubules. The pathways of organelle clearance remain elusive.

It was suggested that exocytosis, ubiquitylation, and autophagy all might play a role in the disposal of sporozoite organelles in early liver stages (). Using electron microscopy, in ndh2(−) oocysts we have observed signature ookinete structures, such as the apical complex, micronemes, and microtubules, as late as 14 days after mosquito infection. We have also repeatedly observed spindle fibers on day 21, indicating viable oocysts but a delay in mitosis and/or the ATP-dependent process of chromosome segregation. We postulate that ablation of NDH2 results in delay and, ultimately, arrest of ATP-dependent processes needed for parasite remodeling during oocyst development. Our findings raise the possibility that in the mammalian host ATP synthesis through the mtETC is dispensable for parasite development, whereas in the insect host this evolutionary conserved function is essential. Elegant genetic experiments established an essential role for the mtETC, via DHOD, for pyrimidine biosynthesis (,, ), during asexual blood stage development.

However, this function was independent of ATP generation, which is mostly derived from glycolysis. NDH2 in turn, appears to be even dispensable as an electron donor for complex III to recycle ubiquinone for DHOD.

In conclusion, we provide experimental genetic evidence that NDH2 is needed in a glucose-deprived environment, such as the mosquito. Candidacy of NDH2 as a promising drug target seems highly unlikely. However, at this stage we cannot formally exclude distinct roles, such as additional non-overlapping functions, of NDH2 paralogs in different Plasmodium species.

Complementation of our ndh2(−) parasites with P. Falciparum NDH2 can only partially address this notion.

In the absence of in vivo models for P. Falciparum, essentiality of P. Falciparum NDH2 throughout the entire life cycle may be extrapolated from the findings described herein. In extension of our study, systematic analysis of the other mitochondrial dehydrogenases by experimental genetics in the rodent malaria model will inform design and preclinical research toward the identification of novel partner drugs for atovaquone.