A phase III randomized crossover trial of plerixafor versus G-CSF for treatment of WHIM syndrome.

BACKGROUNDWarts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function CXCR4 mutations. Myelokathexis is a kind of neutropenia caused by neutrophil retention in bone marrow and in WHIM syndrome is associated with lymphopenia and monocytopenia. The CXCR4 antagonist plerixafor mobilizes leukocytes to the blood; however, its safety and efficacy in WHIM syndrome are undefined.METHODSIn this investigator-initiated, single-center, quadruple-masked phase III crossover trial, we compared the total infection severity score (TISS) as the primary endpoint in an intent-to-treat manner in 19 patients with WHIM who each received 12 months treatment with plerixafor and 12 months treatment with granulocyte CSF (G-CSF, the standard of care for severe congenital neutropenia). The treatment order was randomized for each patient.RESULTSPlerixafor was nonsuperior to G-CSF for TISS (P = 0.54). In exploratory endpoints, plerixafor was noninferior to G-CSF for maintaining neutrophil counts of more than 500 cells/µL (P = 0.023) and was superior to G-CSF for maintaining lymphocyte counts above 1,000 cells/µL (P < 0.0001). Complete regression of a subset of large wart areas occurred on plerixafor in 5 of 7 patients with major wart burdens at baseline. Transient rash occurred on plerixafor, and bone pain was more common on G-CSF. There were no significant differences in drug preference or quality of life or the incidence of drug failure or serious adverse events.CONCLUSIONPlerixafor was not superior to G-CSF in patients with WHIM for TISS, the primary endpoint. Together with wart regression and hematologic improvement, the infection severity results support continued study of plerixafor as a potential treatment for WHIM syndrome.TRIAL REGISTRATIONClinicaltrials.gov NCT02231879.FUNDINGThis study was funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases.

Urine eosinophil-derived neurotoxin: A potential marker of activity in select eosinophilic disorders.

BACKGROUND: Biomarkers of eosinophilic disease activity, especially in the context of novel therapies that reduce blood eosinophil counts, are an unmet need. Absolute eosinophil count (AEC) does not accurately reflect tissue eosinophilia or eosinophil activation. Therefore, the aims of this study were to compare the reliability of plasma and urine eosinophil major basic protein 1, eosinophil cationic protein, eosinophil-derived neurotoxin (EDN), and eosinophil peroxidase measurement and to evaluate the usefulness of eosinophil granule protein (EGP) measurement for the assessment of disease activity in patients with eosinophil-associated diseases treated with mepolizumab, benralizumab, or dexpramipexole. METHODS: Eosinophil granule protein concentrations were measured in serum, plasma, and urine from healthy volunteers and patients with hypereosinophilic syndrome (HES), eosinophilic granulomatosis with polyangiitis (EGPA), and eosinophilic asthma using a multiplex assay. RESULTS: Urine EGP concentrations remained stable, whereas serum and plasma EGP concentrations increased significantly with delayed processing. Plasma (p) EDN, but not urine (u) EDN, concentration correlated with AEC and negatively correlated with prednisone dose. Both pEDN and uEDN decreased significantly following treatment of HES patients with benralizumab and EGPA patients with mepolizumab. uEDN appeared to increase with clinical relapse in both patient groups. CONCLUSIONS: Measurement of EGP in urine is noninvasive and unaffected by cellular lysis. Although plasma and urine EDN concentrations showed a similar pattern following benralizumab and mepolizumab treatment, the lack of correlation between AEC or prednisone dose and uEDN concentrations suggests that measurement of uEDN may provide a potential biomarker of disease activity in patients with HES and EGPA.

An International, Retrospective Study of Off-Label Biologic Use in the Treatment of Hypereosinophilic Syndromes.

BACKGROUND: Treatment of hypereosinophilic syndrome (HES) often requires the use of immunomodulators with substantial side effect profiles. The emergence of biologics offers an alternative treatment modality. OBJECTIVE: To examine real-world practice data to describe the safety and consequences of various biologics suspected to directly or indirectly affect eosinophilic inflammation for the treatment of HES. METHODS: Retrospective data from 13 centers were collected via an online Research Electronic Data Capture repository. Inclusion criteria included (1) peripheral eosinophil count of 1,500/mm3 or greater without a secondary cause; (2) clinical manifestations attributable to the eosinophilia; and (3) having received mepolizumab (anti-IL-5), benralizumab (afucosylated anti-IL-5 receptor α), omalizumab (anti-IgE), alemtuzumab (anti-CD52), dupilumab (anti-IL-4 receptor α), or reslizumab (anti-IL-5) outside a placebo-controlled clinical trial. RESULTS: Of the 151 courses of biologics prescribed for 121 patients with HES, 59% resulted in improved HES symptoms and 77% enabled tapering of other HES medications. Overall, 105 patients were receiving daily systemic glucocorticoids at the time of a biologic initiation and were able to reduce the glucocorticoid dose by a median reduction of 10 mg of daily prednisone equivalents. Biologics were generally safe and well-tolerated other than infusion reactions with alemtuzumab. Thirteen of 24 patients had clinical improvement after switching biologics and nine patients responded to increasing the dose of mepolizumab after a lack of response to a lower dose. CONCLUSIONS: Biologics may offer a safer treatment alternative to existing therapies for HES, although the optimal dosing and choice for each subtype of HES remain to be determined. Limitations of this study include its retrospective nature and intersite differences in data collection and availability of each biologic.

Bliss' and Loewe's additive and synergistic effects in Plasmodium falciparum growth inhibition by AMA1-RON2L, RH5, RIPR and CyRPA antibody combinations.

Plasmodium invasion of red blood cells involves malaria proteins, such as reticulocyte-binding protein homolog 5 (RH5), RH5 interacting protein (RIPR), cysteine-rich protective antigen (CyRPA), apical membrane antigen 1 (AMA1) and rhoptry neck protein 2 (RON2), all of which are blood-stage malaria vaccine candidates. So far, vaccines containing AMA1 alone have been unsuccessful in clinical trials. However, immunization with AMA1 bound with RON2L (AMA1-RON2L) induces better protection against P. falciparum malaria in Aotus monkeys. We therefore sought to determine whether combinations of RH5, RIPR, CyRPA and AMA1-RON2L antibodies improve their biological activities and sought to develop a robust method for determination of synergy or additivity in antibody combinations. Rabbit antibodies against AMA1-RON2L, RH5, RIPR or CyRPA were tested either alone or in combinations in P. falciparum growth inhibition assay to determine Bliss' and Loewe's additivities. The AMA1-RON2L/RH5 combination consistently demonstrated an additive effect while the CyRPA/RIPR combination showed a modest synergistic effect with Hewlett's [Formula: see text] Additionally, we provide a publicly-available, online tool to aid researchers in analyzing and planning their own synergy experiments. This study supports future blood-stage vaccine development by providing a solid methodology to evaluate additive and/or synergistic (or antagonistic) effect of vaccine-induced antibodies.

Single-Organ and Multisystem Hypereosinophilic Syndrome Patients with Gastrointestinal Manifestations Share Common Characteristics.

BACKGROUND: Eosinophilic gastrointestinal diseases (EGIDs) are defined by marked eosinophilia in the gastrointestinal (GI) tract resulting in a wide variety of GI symptoms. When accompanied by blood hypereosinophilia (HE; absolute eosinophil count ≥1500/mm3), EGID can occur as an isolated GI disorder (hypereosinophilic syndrome [HES]/EGID overlap) or as part of a multisystem hypereosinophilic syndrome (Multisystem HES). OBJECTIVE: To describe the GI disease of patients categorized as those with HES/EGID overlap versus those with Multisystem HES. METHODS: Consecutively enrolled patients on a natural history protocol to study eosinophilia with biopsy-proven EGID involving the esophagus, stomach, small-bowel, and/or colon were evaluated for clinical, histopathologic, and endoscopic features by retrospective chart review. RESULTS: Among the 56 patients with EGID and HE, 34 were categorized as HES/EGID overlap and 22 as Multisystem HES. Demographics, GI symptoms, and associated comorbidities were similar between the 2 groups. Multisegment GI eosinophilia was present in 20 of 30 (67%) patients who underwent tissue sampling of all 4 GI segments. Tissue eosinophilia in all 4 GI segments was found in 5 of 30 (17%) patients. Dietary therapy was more common in patients with HES/EGID overlap (65% vs 23%, P = .0028). Patients with Multisystem HES were more likely to receive glucocorticoids (100% vs 79%, P = .0349) and nonglucocorticoid systemic therapies (77% vs 38%, P = .0061). One-third (8 of 22) of patients with Multisystem HES presented with isolated GI symptoms before developing extraintestinal manifestations at a median of 1 year (range, 0.25-15 years). CONCLUSION: There are striking clinical similarities between patients with Multisystem HES and those with HES/EGID overlap, despite differing treatment approaches. Moreover, Multisystem HES can present with isolated GI involvement. Larger prospective studies are needed to confirm these findings.

Benralizumab for PDGFRA-Negative Hypereosinophilic Syndrome.

BACKGROUND: Hypereosinophilic syndrome is a group of diseases defined by marked eosinophilia in blood or tissue and eosinophil-related clinical manifestations. Benralizumab is a monoclonal antibody against interleukin-5 receptor α, which is expressed on human eosinophils. METHODS: In this randomized, double-blind, placebo-controlled, phase 2 trial, we administered a series of three monthly subcutaneous injections of either benralizumab (at a dose of 30 mg) or placebo in 20 symptomatic patients who had PDGFRA-negative hypereosinophilic syndrome and an absolute eosinophil count of at least 1000 cells per cubic millimeter; all the patients were receiving stable therapy (drugs or dietary changes) for this disease. This regimen was followed by an open-label phase, during which the patient's background therapy could be tapered as tolerated, and an extension phase. The primary end point of the randomized phase was a reduction of at least 50% in the absolute eosinophil count at week 12. RESULTS: During the randomized phase, the primary end point occurred in more patients in the benralizumab group than in the placebo group (9 of 10 patients [90%] vs. 3 of 10 patients [30%], P = 0.02). During the open-label phase, clinical and hematologic responses were observed in 17 of 19 patients (89%) and were sustained for 48 weeks in 14 of 19 patients (74%); in the latter group, in 9 of 14 patients (64%), background therapies could be tapered. Bone marrow and tissue eosinophilia were also suppressed with benralizumab therapy. The most common drug-related adverse events, headache and an elevated lactate dehydrogenase level, occurred in 32% of the patients after the first dose of benralizumab and resolved within 48 hours in all patients. Other adverse events occurred with similar frequency in the two groups. Of the many potential predictors of response that were examined, only clinical disease subtype appeared to be associated with the initial response or relapse. CONCLUSIONS: In this small phase 2 trial, patients with PDGFRA-negative hypereosinophilic syndrome who received benralizumab for 12 weeks had lower absolute eosinophil counts than those who received placebo. During the open-label phase, clinical and hematologic responses were sustained for 48 weeks in 74% of the patients. Adverse events did not limit treatment. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov numbers, NCT00001406 and NCT02130882.).

Dexpramipexole as an oral steroid-sparing agent in hypereosinophilic syndromes.

Hypereosinophilic syndromes (HESs) are a heterogeneous group of disorders characterized by peripheral eosinophilia and eosinophil-related end organ damage. Whereas most patients respond to glucocorticoid (GC) therapy, high doses are often necessary, and side effects are common. Dexpramipexole (KNS-760704), an orally bioavailable synthetic aminobenzothiazole, showed an excellent safety profile and was coincidentally noted to significantly decrease absolute eosinophil counts (AECs) in a phase 3 trial for amyotrophic lateral sclerosis. This proof-of-concept study was designed to evaluate dexpramipexole (150 mg orally twice daily) as a GC-sparing agent in HESs. Dual primary end points were (1) the proportion of subjects with ≥50% decrease in the minimum effective GC dose (MED) to maintain AEC <1000/µL and control clinical symptoms, and (2) the MED after 12 weeks of dexpramipexole (MEDD) as a percentage of the MED at week 0. Out of 10 subjects, 40% (95% confidence interval [CI], 12%, 74%) achieved a ≥50% reduction in MED, and the MEDD/MED ratio was significantly <100% (median, 66%; 95% CI, 6%, 98%; P = .03). All adverse events were self-limited, and none led to drug discontinuation. Affected tissue biopsy samples in 2 subjects showed normalization of pathology and depletion of eosinophils on dexpramipexole. Bone marrow biopsy samples after 12 weeks of dexpramipexole showed selective absence of mature eosinophils in responders. Dexpramipexole appears promising as a GC-sparing agent without apparent toxicity in a subset of subjects with GC-responsive HESs. Although the exact mechanism of action is unknown, preliminary data suggest that dexpramipexole may affect eosinophil maturation in the bone marrow. This study was registered at www.clinicaltrials.gov as #NCT02101138.

Long-Term Clinical Outcomes of High-Dose Mepolizumab Treatment for Hypereosinophilic Syndrome.

BACKGROUND: Conventional therapies for hypereosinophilic syndromes (HES) have variable efficacy and carry significant long-term toxicities. Anti-IL-5 (mepolizumab) therapy has a glucocorticoid (GC)-sparing effect in GC-sensitive HES, but the efficacy of mepolizumab in treatment-refractory HES patients with severe disease has not been examined to date. OBJECTIVE: To identify predictors of response to mepolizumab in subjects with severe treatment-refractory HES and compare long-term outcomes in these subjects with HES subjects treated with conventional therapies. METHODS: Retrospective analysis of clinical and laboratory data from 35 HES subjects treated with mepolizumab and 55 HES subjects on conventional therapy, all followed at a single center, was performed. RESULTS: Peak eosinophilia, GC sensitivity, pulmonary involvement, HES clinical subtype, and pretreatment serum IL-5 were correlated with mepolizumab response. Despite evidence of more severe disease at baseline, mepolizumab-treated subjects had comparable long-term clinical outcomes to HES subjects treated with conventional therapies and reported improvements in therapy-related comorbidities. Subjects managed with mepolizumab monotherapy had fewer disease flares than HES subjects on conventional therapies or mepolizumab-treated HES subjects requiring additional HES therapies. CONCLUSIONS: This study confirms that mepolizumab is an effective and well-tolerated therapy for HES, but suggests that response is more likely in GC-responsive subjects with idiopathic or overlap forms of HES. A primary benefit of treatment is the reduction of comorbidity due to discontinuation or the reduction of conventional HES therapies. Although subjects who completely discontinued GC had the most benefit, high-dose mepolizumab was a safe and effective salvage therapy for severe, treatment-refractory HES.

Hypereosinophilic Syndrome Subtype Predicts Responsiveness to Glucocorticoids.

BACKGROUND: Glucocorticoids (GCs) are considered first-line treatment for platelet-derived growth factor α (PDGFRA)-negative hypereosinophilic syndromes (HESs). Despite this, little is known about clinical predictors of GC responsiveness in HES. OBJECTIVE: Knowledge of clinical and laboratory predictors of GC response before initiation of GC could lead to more rational selection of subjects with HES for whom earlier institution of second-line and alternative therapies would be appropriate. METHODS: Response to GC, as defined by the reduction of the absolute eosinophil count to below 1000/mm3 and control of symptoms, was assessed by a retrospective chart review of subjects with PDGFRA-negative HES evaluated on an institutional review board-approved protocol. Demographic, clinical, and laboratory parameters obtained before institution of GC, as well as final diagnosis, were evaluated to determine predictors of GC response. Proportional odds models were used for univariate and multivariate assessment of predictors with permutation adjusted P values to correct for multiple comparisons. RESULTS: A total of 164 subjects with PDGFRA-negative HES were categorized according to GC response. Of them, 39% of the subjects responded to low dose (≤10 mg) prednisone, 9% did not respond to GC, and the remainder (52%) had variable responses to GC. The HES subtype diagnosis was the best predictor of response to GC with myeloid forms and lymphocytic variants of HES being the least responsive to GC. CONCLUSIONS: In a large cohort of well-characterized subjects with HES, the odds of response to GC was predicted by HES subtype. Using this model, clinicians may more readily proceed to second-line agents in subjects with confirmed lymphocytic or myeloid forms of HES.

Confidence intervals for directly standardized rates using mid-p gamma intervals.

Directly standardized rates continue to be an integral tool for presenting rates for diseases that are highly dependent on age, such as cancer. Statistically, these rates are modeled as a weighted sum of Poisson random variables. This is a difficult statistical problem, because there are k observed Poisson variables and k unknown means. The gamma confidence interval has been shown through simulations to have at least nominal coverage in all simulated scenarios, but it can be overly conservative. Previous modifications to that method have closer to nominal coverage on average, but they do not achieve the nominal coverage bound in all situations. Further, those modifications are not central intervals, and the upper coverage error rate can be substantially more than half the nominal error. Here we apply a mid-p modification to the gamma confidence interval. Typical mid-p methods forsake guaranteed coverage to get coverage that is sometimes higher and sometimes lower than the nominal coverage rate, depending on the values of the parameters. The mid-p gamma interval does not have guaranteed coverage in all situations; however, in the (not rare) situations where the gamma method is overly conservative, the mid-p gamma interval often has at least nominal coverage. The mid-p gamma interval is especially appropriate when one wants a central interval, since simulations show that in many situations both the upper and lower coverage error rates are on average less than or equal to half the nominal error rate.

Type 2 diabetes mellitus coincident with pulmonary tuberculosis is associated with heightened systemic type 1, type 17, and other proinflammatory cytokines.

RATIONALE: Type 2 diabetes mellitus is a major risk factor for the development of active tuberculosis, although the biological basis underlying this susceptibility remains poorly characterized. OBJECTIVES AND METHODS: To identify the influence of coincident diabetes mellitus on cytokine levels in pulmonary tuberculosis, we examined circulating levels of a panel of cytokines and chemokines in the plasma of individuals with tuberculosis with diabetes and compared them with those of individuals without diabetes. MEASUREMENTS AND MAIN RESULTS: Tuberculosis with diabetes is characterized by elevated circulating levels of type 1 (IFN-γ, tumor necrosis factor-α, and IL-2), type 2 (IL-5), and type 17 (IL-17A) cytokines but decreased circulating levels of IL-22. This was associated with increased systemic levels of other proinflammatory cytokines (IL-1ß, IL-6, and IL-18) and an antiinflammatory cytokine (IL-10) but not type 1 IFNs. Moreover, tuberculosis antigen-stimulated whole blood also showed increased levels of proinflammatory cytokines. Finally, type 1 and type 17 cytokines in plasma exhibit a significant positive correlation with hemoglobin A1C levels, indicating that impaired control of diabetes is associated with this proinflammatory milieu. Multivariate analysis revealed that the association of proinflammatory cytokines with diabetes mellitus was not influenced by age, sex, or other metabolic parameters. CONCLUSIONS: Our data reveal that tuberculosis with diabetes is characterized by heightened cytokine responsiveness, indicating that chronic inflammation underlying type 2 diabetes potentially contributes to increased immune pathology and poor control in tuberculosis infection.

Pointwise confidence intervals for a survival distribution with small samples or heavy censoring.

We propose a beta product confidence procedure (BPCP) that is a non-parametric confidence procedure for the survival curve at a fixed time for right-censored data assuming independent censoring. In such situations, the Kaplan-Meier estimator is typically used with an asymptotic confidence interval (CI) that can have coverage problems when the number of observed failures is not large, and/or when testing the latter parts of the curve where there are few remaining subjects at risk. The BPCP guarantees central coverage (i.e. ensures that both one-sided error rates are no more than half of the total nominal rate) when there is no censoring (in which case it reduces to the Clopper-Pearson interval) or when there is progressive type II censoring (i.e. when censoring only occurs immediately after failures on fixed proportions of the remaining individuals). For general independent censoring, simulations show that the BPCP maintains central coverage in many situations where competing methods can have very substantial error rate inflation for the lower limit. The BPCP gives asymptotically correct coverage and is asymptotically equivalent to the CI on the Kaplan-Meier estimator using Greenwood's variance. The BPCP may be inverted to create confidence procedures for a quantile of the underlying survival distribution. Because the BPCP is easy to implement, offers protection in settings when other methods fail, and essentially matches other methods when they succeed, it should be the method of choice.

Weighted logrank tests for interval censored data when assessment times depend on treatment.

We consider weighted logrank tests for interval censored data when assessment times may depend on treatment, and for each individual, we only use the two assessment times that bracket the event of interest. It is known that treating finite right endpoints as observed events can substantially inflate the type I error rate under assessment-treatment dependence (ATD), but the validity of several other implementations of weighted logrank tests (score tests, permutation tests, multiple imputation tests) has not been studied in this situation. With a bounded number of unique assessment times, the score test under the grouped continuous model retains the type I error rate asymptotically under ATD; however, although the approximate permutation test based on the permutation central limit theorem is not asymptotically valid under every ATD scenario, we show through simulation that in many ATD scenarios, it retains the type I error rate better than the score test. We show a case where the approximate permutation test retains the type I error rate when the exact permutation test does not. We study and modify the multiple imputation logrank tests of Huang, Lee, and Yu (2008, Statistics in Medicine, 27: 3217-3226), showing that the distribution of the rank-like scores asymptotically does not depend on the assessment times. We show through simulations that our modifications of the multiple imputation logrank tests retain the type I error rate in all cases studied, even with ATD and a small number of individuals in each treatment group. Simulations were performed using the interval R package.

A valid formulation of the analysis of noninferiority trials under random effects meta-analysis.

A noninferiority (NI) trial is sometimes employed to show efficacy of a new treatment when it is unethical to randomize current patients to placebo because of the established efficacy of a standard treatment. Under this framework, if the NI trial determines that the treatment advantage of the standard to the new drug (i.e. S-N) is less than the historic advantage of the standard to placebo (S-P), then the efficacy of the new treatment (N-P) is established indirectly. We explicitly combine information from the NI trial with estimates from a random effects model, allowing study-to-study variability in k historic trials. Existing methods under random effects, such as the synthesis method, fail to account for the variability of the true standard versus placebo effect in the NI trial. Our method effectively uses a prediction interval for the missing standard versus placebo effect rather than a confidence interval of the mean. The consequences are to increase the variance of the synthesis method by incorporating a prediction variance term and to approximate the null distribution of the new statistic with a t with k-1 degrees of freedom instead of the standard normal. Thus, it is harder to conclude NI of the new to (predicted) placebo, compared with traditional methods, especially when k is small or when between study variability is large. When the between study variances are nonzero, we demonstrate substantial Type I error rate inflation with conventional approaches; simulations suggest that the new procedure has only modest inflation, and it is very conservative when between study variances are zero. An example is used to illustrate practical issues.

IL-5 receptor α levels in patients with marked eosinophilia or mastocytosis.

BACKGROUND: IL-5 plays a central role in the development and maintenance of eosinophilia (EO) and eosinophil activation in a wide variety of eosinophilic disorders. Although IL-5, IL-3, and GM-CSF can modulate the expression of IL-5 receptor α (IL-5Rα) on eosinophils in vitro, little is known about soluble and surface IL-5Rα levels in vivo. OBJECTIVE: To assess soluble and surface IL-5Rα levels in patients with EO and/or mastocytosis. METHODS: Surface IL-5Rα expression was assessed by flow cytometry in blood and/or bone marrow from subjects with EO (n = 39) and systemic mastocytosis (n = 8) and from normal volunteers (n = 28). Soluble IL-5Rα (sIL-5Rα) level was measured in a cohort of 177 untreated subjects and correlated with EO, eosinophil activation, and serum tryptase and cytokine levels. RESULTS: IL-5Rα expression on eosinophils inversely correlated with EO (r = -0.48; P < .0001), whereas serum levels of sIL-5Rα increased with the eosinophil count (r = 0.56; P < .0001) and serum IL-5 (r = 0.40; P < .0001) and IL-13 (r = 0.29; P = .004) levels. Of interest, sIL-5Rα level was significantly elevated in patients with systemic mastocytosis without EO. Although sIL-5Rα levels correlated with serum tryptase levels in these patients, eosinophil activation, assessed by CD69 expression on eosinophils and serum eosinophil-derived neurotoxin levels, was increased compared with that in normal subjects. CONCLUSIONS: These data are consistent with an in vivo IL-5Rα regulatory pathway in human eosinophils similar to that described in vitro and involving a balance between soluble and surface receptor levels. This may have implications with respect to the use of novel therapeutic agents targeting IL-5 and its receptor in patients with EO and/or mastocytosis.

Disruption of a Plasmodium falciparum multidrug resistance-associated protein (PfMRP) alters its fitness and transport of antimalarial drugs and glutathione.

ATP-binding cassette transporters play an important role in drug resistance and nutrient transport. In the human malaria parasite Plasmodium falciparum, a homolog of the human p-glycoprotein (PfPgh-1) was shown to be involved in resistance to several drugs. More recently, many transporters were associated with higher IC(50) levels in responses to chloroquine (CQ) and quinine (QN) in field isolates. Subsequent studies, however, could not confirm the associations, although inaccuracy in drug tests in the later studies could contribute to the lack of associations. Here we disrupted a gene encoding a putative multidrug resistance-associated protein (PfMRP) that was previously shown to be associated with P. falciparum responses to CQ and QN. Parasites with disrupted PfMRP (W2/MRPDelta) could not grow to a parasitemia higher than 5% under normal culture conditions, possibly because of lower efficiency in removing toxic metabolites. The W2/MRPDelta parasite also accumulated more radioactive glutathione, CQ, and QN and became more sensitive to multiple antimalarial drugs, including CQ, QN, artemisinin, piperaquine, and primaquine. PfMRP was localized on the parasite surface membrane, within membrane-bound vesicles, and along the straight side of the D-shaped stage II gametocytes. The results suggest that PfMRP plays a role in the efflux of glutathione, CQ, and QN and contributes to parasite responses to multiple antimalarial drugs, possibly by pumping drugs outside the parasite.

In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies.

Infection with the obligate intracellular protozoan Leishmania is thought to be initiated by direct parasitization of macrophages, but the early events following transmission to the skin by vector sand flies have been difficult to examine directly. Using dynamic intravital microscopy and flow cytometry, we observed a rapid and sustained neutrophilic infiltrate at localized sand fly bite sites. Invading neutrophils efficiently captured Leishmania major (L.m.) parasites early after sand fly transmission or needle inoculation, but phagocytosed L.m. remained viable and infected neutrophils efficiently initiated infection. Furthermore, neutrophil depletion reduced, rather than enhanced, the ability of parasites to establish productive infections. Thus, L.m. appears to have evolved to both evade and exploit the innate host response to sand fly bite in order to establish and promote disease.

Changes in the plasmodial surface anion channel reduce leupeptin uptake and can confer drug resistance in Plasmodium falciparum-infected erythrocytes.

Cysteine protease inhibitors kill malaria parasites and are being pursued for development as antimalarial agents. Because they have multiple targets within bloodstream-stage parasites, workers have assumed that resistance to these inhibitors would not be acquired easily. In the present study, we used in vitro selection to generate a parasite resistant to growth inhibition by leupeptin, a broad-profile cysteine and serine protease inhibitor. Resistance was not associated with upregulation of cysteine protease activity, reduced leupeptin sensitivity of this activity, or expression level changes for putative cysteine or serine proteases in the parasite genome. Instead, it was associated with marked changes in the plasmodial surface anion channel (PSAC), an ion channel on infected erythrocytes that functions in nutrient and bulky organic solute uptake. Osmotic fragility measurements, electrophysiological recordings, and leupeptin uptake studies revealed selective reductions in organic solute permeability via PSAC, altered single-channel gating, and reduced inhibitor affinity. These changes yielded significantly reduced leupeptin uptake and could fully account for the acquired resistance. PSAC represents a novel route for the uptake of bulky hydrophilic compounds acting against intraerythrocytic parasite targets. Drug development based on such compounds should proceed cautiously in light of possible resistance development though the selection of PSAC mutants.

Relapse following discontinuation of imatinib mesylate therapy for FIP1L1/PDGFRA-positive chronic eosinophilic leukemia: implications for optimal dosing.

Although imatinib is clearly the treatment of choice for FIP1L1/PDGFRA-positive chronic eosinophilic leukemia (CEL), little is known about optimal dosing, duration of treatment, and the possibility of cure in this disorder. To address these questions, 5 patients with FIP1L1/PDGFRA-positive CEL with documented clinical, hematologic, and molecular remission on imatinib (400 mg daily) and without evidence of cardiac involvement were enrolled in a dose de-escalation trial. The imatinib dose was tapered slowly with close follow-up for evidence of clinical, hematologic, and molecular relapse. Two patients with endomyocardial fibrosis were maintained on imatinib 300 to 400 mg daily and served as controls. All 5 patients who underwent dose de-escalation, but neither of the control patients, experienced molecular relapse (P < .05). None developed recurrent symptoms, and eosinophil counts, serum B12, and tryptase levels remained suppressed. Reinitiation of therapy at the prior effective dose led to molecular remission in all 5 patients, although 2 patients subsequently required increased dosing to maintain remission. These data are consistent with suppression rather than elimination of the clonal population in FIP1L1/PDGFRA-positive CEL and suggest that molecular monitoring may be the most useful method in determining optimal dosing without the risk of disease exacerbation. This trial was registered at http://www.clinicaltrials.gov as no. NCT00044304.

KIT D816V-associated systemic mastocytosis with eosinophilia and FIP1L1/PDGFRA-associated chronic eosinophilic leukemia are distinct entities.

BACKGROUND: The broad and overlapping clinical manifestations of D816V KIT-associated systemic mastocytosis with eosinophilia and FIP1L1/PDGFRA-associated chronic eosinophilic leukemia (CEL), coupled with the increase in activated eosinophils and mast cells seen in both disorders, have led to confusion in the nomenclature. It is of paramount importance, however, to distinguish between these 2 groups of patients because of differences in clinical sequelae, prognoses, and selection of treatment. OBJECTIVE: We thus sought to identify clinical and laboratory features that could be used to distinguish these 2 diagnoses. METHODS: We compared 12 patients with D816V-positive systemic mastocytosis with eosinophilia with 17 patients with FIP1L1/PDGFRA-positive CEL. Distinguishing features were used to create a risk factor scoring system. RESULTS: This system correctly classified 16 of 17 FIP1L1/PDGFRA-positive patients with CEL and all 12 patients with systemic mastocytosis with eosinophilia. Thirty-four FIP1L1/PDGFRA-positive patients described in the literature were also classified using this system, and although a complete set of data was not available for any of the historical patients, 21 were correctly classified. CONCLUSION: These results reinforce the hypothesis that the FIP1L1/PDGFRA gene fusion and D816V-KIT mutation cause distinct clinical syndromes. CLINICAL IMPLICATIONS: This novel diagnostic approach should prove helpful in clinical practice in the evaluation of patients with increased mast cells and peripheral eosinophilia.