RNA-Based Therapy for Cryptosporidium parvum Infection: Proof-of-Concept Studies.

Cryptosporidium is a leading cause of moderate-to-severe diarrhea in children, which is one of the major causes of death in children under 5 years old. Nitazoxanide is the only FDA-approved treatment for cryptosporidiosis. However, it has limited efficacy in immunosuppressed patients and malnourished children. Therefore, it is urgent to develop novel therapies against this parasite. RNA interference-mediated therapies are emerging as novel approaches for the treatment of infectious diseases. We have developed a novel method to silence essential genes in Cryptosporidium using single-stranded RNA (ssRNA)/Argonaute (Ago) complexes. In this work we conducted proof-of-concept studies to test the anticryptosporidial activity of these complexes by silencing Cryptosporidium parvum nucleoside diphosphate kinase (NDK) using in vitro and in vivo models. We demonstrated that a 3-day treatment with anti-sense NDK ssRNA/Ago decreased parasite burden by ~98% on infected cells. In vivo studies showed that ssRNA/Ago complexes encapsulated in lipid nanoparticles can be delivered onto intestinal epithelial cells of mice treated orally. In addition a cryptosporidiosis-mouse model showed that treatment with NDK ssRNA/Ago complexes reduced oocyst shedding in 4/5 SCID/beige mice during the acute phase of the infection. Our findings highlight the potential use of antisense RNA-based therapy as an alternative approach to cryptosporidiosis treatment.

Does heat acclimation improve exercise capacity at altitude? A cross-tolerance model.

New approaches to inducing altitude acclimation in a relatively short timeframe are needed, as it is not practical for many soldiers and athletes to gain access to specialized training facilities. Acclimation to one environmental stressor could enhance adaptation to various other stressors in animals and humans. This phenomenon has been described as cross-tolerance and involves the activation of common protective pathways. The purpose of this review is to discuss possible mechanisms involved in the cross-tolerance between heat and hypoxia. Future data could potentially support the use of a cross-tolerance model as a means for military personnel to prepare for deployment to high-altitude environments, as well as for athletes competing at high altitude.

Nonclassical velocity statistics in a turbulent atomic Bose-Einstein condensate.

In a recent experiment Paoletti [Phys. Rev. Lett. 101, 154501 (2008)]10.1103/PhysRevLett.101.154501 monitored the motion of tracer particles in turbulent superfluid helium and inferred that the velocity components do not obey the Gaussian statistics observed in ordinary turbulence. Motivated by their experiment, we create a small 3D turbulent state in an atomic Bose-Einstein condensate, compute directly the velocity field, and find similar nonclassical power-law tails. We obtain similar results in 2D trapped and 3D homogeneous condensates, and in classical 2D vortex points systems. This suggests that non-Gaussian turbulent velocity statistics describe a fundamental property of quantum turbulence. We also track the decay of the vortex tangle in the presence of the thermal cloud.

Supramaximal testing to confirm attainment of VO2max in sedentary men and women.

Supramaximal testing is widely used to verify VO2max attainment, yet its efficacy in sedentary subjects is unknown. The aim of the study was to test this hypothesis in men and women completing maximal cycle ergometry. Fifteen sedentary subjects (age=22.4+/-3.9 year) completed incremental exercise, and returned at least 24 h later to complete constant load exercise at 105% peak work rate (Wmax). Another group of nine sedentary men and women (age=21.8+/-5 year) completed supramaximal exercise at 115% Wmax 1-1.5 h after incremental exercise. During exercise, gas exchange data and heart rate (HR) were continuously obtained. VO2max was similar (p>0.05) between incremental and supramaximal exercise in subjects in the first (32.32+/-4.81 mL/kg/min vs. 31.80+/-5.35 mL/kg/min) and second subset (40.63+/-3.61 mL/kg/min vs. 41.66+/-5.55 mL/kg/min). Maximal HR was lower (p<0.05) with supramaximal exercise, yet respiratory exchange ratio was higher (p<0.05). Test-retest reliability (r=0.81-0.89, p<0.05) for VO2max was high during repeated bouts of supramaximal testing. Findings support use of this protocol to confirm VO2max attainment in healthy, sedentary men and women completing incremental cycle ergometry.

Systematic gene silencing identified Cryptosporidium nucleoside diphosphate kinase and other molecules as targets for suppression of parasite proliferation in human intestinal cells.

Cryptosporidiosis is a major cause of diarrheal disease. The only drug approved for cryptosporidiosis has limited efficacy in high-risk populations. Therefore novel drugs are urgently needed. We have identified several enzymes as potential targets for drug development and we have optimized a rapid method to silence genes in Cryptosporidium. In this study, we knocked down expression of the four selected genes: Actin (Act), Apicomplexan DNA-binding protein (Ap2), Rhomboid protein 1 (Rom 1), and nucleoside diphosphate kinase (NDK). After gene silencing, we evaluated the role of each target on parasite development using in vitro models of excystation, invasion, proliferation, and egress. We showed that silencing of Act, Ap2, NDK, and Rom1 reduced invasion, proliferation, and egress of Cryptosporidium. However, silencing of NDK markedly inhibited Cryptosporidium proliferation (~70%). We used an infection model to evaluate the anticryptosporidial activity of ellagic acid (EA), an NDK inhibitor. We showed that EA (EC50 = 15-30 µM) reduced parasite burden without showing human cell toxicity. Here, we demonstrated the usefulness of a rapid silencing method to identify novel targets for drug development. Because EA is a dietary supplement already approved for human use, this compound should be studied as a potential treatment for cryptosporidiosis.

Reduced respiratory and skeletal muscle strength in survivors of sibling or unrelated donor hematopoietic stem cell transplantation.

We performed a retrospective analysis of muscle strength testing obtained following sibling or unrelated donor hematopoietic stem cell transplant (HSCT) between 1 January 1999 and 31 December 2003 in a cohort of 44 subjects at Tufts-New England Medical Center. Maximal inspiratory pressure (PI(max)) was

Molecular diagnosis of protozoan parasites by Recombinase Polymerase Amplification.

Infections caused by protozoan parasites affect millions of people around the world. Traditionally, diagnosis was made by microscopy, which is insensitive and in some cases not specific. Molecular methods are highly sensitive and specific, but equipment costs and personnel training limit its availability only to specialized centers, usually far from populations with the highest risk of infection. Inexpensive methods that can be applied at the point of care (POC), especially in places with limited health infrastructure, would be a major advantage. Isothermal amplification of nucleic acids does not require thermocyclers and is relatively inexpensive and easy to implement. Among isothermal methods, recombinase polymerase amplification (RPA) is sensitive and potentially applicable at POC. We and others have developed RPA diagnostic tests to detect protozoan parasites of medical importance. Overall, our results have shown high specificity with limits of detection similar to PCR. Currently, the optimization of RPA for use at the POC is under development, and in the near future the tests should become available to detect protozoan infections in the field. In this review we discuss the current status, challenges, and future of RPA in the field of molecular diagnosis of protozoan parasites.

Modulation of transforming growth factor-beta 1 antiproliferative effects on endothelial cells by cysteine, cystine, and N-acetylcysteine.

Early passaged bovine pulmonary artery endothelial cells exposed to 0.1-2.0 ng/ml transforming growth factor-beta 1 (TGF-beta 1) showed concentration-dependent growth inhibition, as assessed by [3H]thymidine labeling and cell counts, over a 96-h interval. Most of the inhibition of [3H]thymidine labeling measured at 96 h persisted when the medium was replaced with TGF-beta 1-free medium after 24 h, but the inhibition of labeling was prevented by the presence of anti-TGF-beta 1 antibody in the replacement medium. Additions of 2 mM cysteine, 1 mM cystine, or 2 mM N-acetylcysteine at the time of the initial addition of TGF-beta 1 blocked the inhibitory effect of TGF-beta 1 on [3H]-thymidine labeling when this was assessed after 72-96 h, but not at earlier times. Prevention of the inhibitory effect on cellular proliferation produced by cysteine, cystine and N-acetylcysteine was associated with elevation of cellular glutathione that was present at 48-96 h. There was no evidence for direct inactivation of TGF-beta 1 by the thiol-amino acids. Conditioned medium from TGF-beta 1-treated endothelial cells inhibited proliferation of mink lung carcinoma (CCL64) cells, supporting a previously reported concept of autocrine production of TGF-beta 1 by the endothelial cells. The inhibitory action of the conditioned medium was partially prevented when 1 mM cysteine was added during conditioning. Thus, TGF-beta 1 treatment of endothelial cells appears to set off autocrine production by these cells of TGF-beta 1 that perpetuates the inhibition of cellular proliferation. Replenishment of cellular glutathione with thiol-amino acids counteracts the growth-inhibitory effect of TGF-beta 1 through a currently undefined mechanism.

Factors associated with rerupture of intracranial aneurysms after endovascular treatment: A retrospective review of 11years experience at a single institution and review of the literature.

Aneurysm rebleeding following initial endovascular management is uncommon, and the factors associated with its occurrence are poorly defined. We retrospectively analyzed a consecutive series of patients presenting with aneurysmal subarachnoid hemorrhage who underwent endovascular management to determine factors associated with rebleeding. Rebleeding occurred in 7/183 (3.8%) patients, 6 of which had an adjacent hematoma on initial neuroimaging. Aneurysms were located on the ACoA (n=5), PCoA (n=1), and MCA (n=1). Sizes ranged from 3.5 to 13.0mm (mean 8.0), with neck sizes ranging from 1.8 to 4.6mm (mean 3.2). Time-to-rerupture ranged from hours to years, with 3/7 cases rebleeding within 30days and 4/7 cases rebleeding later than 30days. Initial incomplete angiographic occlusion occurred in 2/3 cases of early rebleeding. The presence of adjacent intracerebral hematoma (ɸ=0.354, p<0.005), increasing Fisher Grade (t(9.4)=7.72, p<0.005), and aneurysmal outpouching (ɸ=0.265, p<0.005) were found to be the only factors associated with rerupture status. Recurrent hemorrhage following endovascular management of ruptured intracranial aneurysms is an uncommon but important source of morbidity, particularly in the early post-embolization period. The presence of high-risk features, such as an adjacent intracerebral hematoma or aneurysm outpouching, warrant early and frequent angiographic follow up to document stability and mitigate rupture risk.

Revised diagnostic criteria for neurocysticercosis.

BACKGROUND: A unified set of criteria for neurocysticercosis (NCC) has helped to standardize its diagnosis in different settings. METHODS: Cysticercosis experts were convened to update current diagnostic criteria for NCC according to two principles: neuroimaging studies are essential for diagnosis, and all other information provides indirect evidence favoring the diagnosis. Recent diagnostic advances were incorporated to this revised set. RESULTS: This revised set is structured in absolute, neuroimaging and clinical/exposure criteria. Absolute criteria include: histological confirmation of parasites, evidence of subretinal cysts, and demonstration of the scolex within a cyst. Neuroimaging criteria are categorized as major (cystic lesions without scolex, enhancing lesions, multilobulated cysts, and calcifications), confirmative (resolution of cysts after cysticidal drug therapy, spontaneous resolution of single enhancing lesions, and migrating ventricular cysts on sequential neuroimaging studies) and minor (hydrocephalus and leptomeningeal enhancement). Clinical/exposure criteria include: detection of anticysticercal antibodies or cysticercal antigens by well-standardized tests, systemic cysticercosis, evidence of a household Taenia carrier, suggestive clinical manifestations, and residency in endemic areas. Besides patients having absolute criteria, definitive diagnosis can be made in those having two major neuroimaging criteria (or one major plus one confirmative criteria) plus exposure. For patients presenting with one major and one minor neuroimaging criteria plus exposure, definitive diagnosis of NCC requires the exclusion of confounding pathologies. Probable diagnosis is reserved for individuals presenting with one neuroimaging criteria plus strong evidence of exposure. CONCLUSIONS: This revised set of diagnostic criteria provides simpler definitions and may facilitate its more uniform and widespread applicability in different scenarios.

Plasma antioxidants in subjects before hematopoietic stem cell transplantation.

UNLABELLED: Chemo-irradiation induced oxidative damage to vascular endothelium may contribute to pulmonary complications of hematopoietic stem cell transplantation (HSCT). We measured antioxidants, markers of oxidative stress and plasma antioxidant capacity in plasma or serum from 24 subjects at day 7 before HSCT and 20 control subjects. The plasma concentration of extracellular glutathione peroxidase (GPX-3) was significantly reduced in the HSCT subjects compared with controls (HSCT: 98+/-42 microg/ml, control: 169+/-56 microg/ml, P<0.0001). The concentration of gamma-tocopherol was significantly higher in the HSCT subjects compared with controls (HSCT: 207+/-103 microg/dl; CONTROL: 98+/-52 microg/dl; P=0.0002). The plasma concentrations of protein carbonyl, nitrotyrosine, malondialdehyde, alpha-tocopherol, vitamin A, homocysteine, cysteine and cysteinylglycine did not differ between HSCT and control subjects. Plasma from HSCT subjects was as effective as control plasma in quenching menadione-induced intracellular reactive oxygen species production in human microvascular endothelial cells. In summary, subjects before HSCT have significantly reduced plasma concentrations of GPX-3, elevated plasma gamma-tocopherol yet retains the ability to quench an acute oxidative stress. These changes may play a role in chronic oxidative stress in the HSCT population.

Effects of delta9-tetrahydrocannabinol in Lewis lung adenocarcinoma cells in tissue culture.

We found a dose-related decrease in DNA synthesis in transformed cell cultures treated with delta9-tetrahydrocannabinol (delta9-THC). The decrease, observed over a 4-hour period, was not accompanied by a change in the radioactive precursor pool as compared to that of control culture. The distribution of labeled products clearly differed from that observed after treatment with cytosine arabinoside. delta9-THC inhibited DNA synthesis at some point beyond the uptake of 3H-thymidine.

Site-selective growth of a hormone-responsive human breast carcinoma in athymic mice.

MCF-7 is a human breast cancer line which requires estradiol supplementation for growth in s.c. tissues of athymic mice. In order to evaluate the influence of host site on hormone-dependent tumorigenicity and growth, MCF-7 cells were inoculated into tissues varying in ability to concentrate exogenous estradiol. Tumorigenicity was defined in terms of latency, threshold inoculum size, and tumor growth and progression. We observed that sites such as lung rarely supported MCF-7 tumors. However, moderately estrophilic sites such as mammary fat and adjacent subcutaneum and dermis supported the growth of small MCF-7 tumors from large tumor cell inocula, but only in estrogenized mice. In contrast, the highly estrophilic sites, brain and periuterine tissues, produced rapidly growing tumors from small tumor cell inocula. Only in periuterine tissues did tumors develop in the absence of exogenous estradiol. These studies demonstrate that tumorigenicity and growth rates of estrogen-dependent MCF-7 tumors vary as a function of tissue implantation site.

Aspergillus fumigatus epidural abscess in a renal transplant recipient.

An epidural abscess caused by Aspergillus fumigatus occurred in a recipient of a cadaveric, renal allograft. The patient had persistent back pain and a peripheral neuropathy that involved the lower extremities. Signs of spinal cord compression evolved. No definite portal of entry was found. Diagnosis was made by histologic examination and culture of a biopsy specimen. Therapy, consisting of aggressive surgical debridement, intravenous amphotericin B, and oral flucytosine was unsuccessful in eradicating the organism. At postmortem examination, Aspergillus was identified at the abscess site. To our knowledge, aspergillosis presenting as an epidural abscess in the immunosuppressed, renal transplant recipient has not previously been reported and should be considered in the differential diagnosis of back pain and peripheral neuropathy in such a patient.

Advice on malaria and yellow fever prevention provided at travel agencies in Cuzco, Peru.

BACKGROUND: Travelers receive medical advice from a variety of sources, including travel agencies. The aim of this study is to describe the quality of pre-travel advice provided by travel agencies in Cuzco to travelers interested in visiting malaria and yellow fever endemic areas. METHODS: Trained medical students posed as tourists and visited travel agencies in Cuzco requesting travel advice for a trip to the southern Amazon of Peru, recording advice regarding risk and prevention of malaria and yellow fever. RESULTS: A total of 163 registered travel agencies were included in the study. The mean proposed tour duration was 6.8 days (±1.4 days) with a median time to departure of 3 days and a median tour cost of 805 US dollars (USD) [interquartile range (IQR) 580-1,095]. Overall, 45% employees failed to mention the risk for any illness. Eighteen percent of the employees acknowledged risk of malaria and 53% risk of yellow fever. However, 36% denied malaria risk and 2% denied risk of yellow fever in the region. The price of tours from travel agencies that did not mention any health risk was significantly lower [1,009.6 ± 500.5 vs 783.9 ± 402 USD, t (152) = 3, p < 0.01] compared with the price from agencies that did mention health risks. Almost all who acknowledged malaria (97%) and/or yellow fever (100%) were able to provide at least one recommendation for prevention. However, advice was not always accurate or spontaneously volunteered. Only 7% of the employees provided both correct scheduling and location information for administration of the yellow fever vaccine. CONCLUSIONS: The majority of registered travel agencies in Cuzco did not provide sufficient and accurate information regarding risk and prevention of malaria and yellow fever to travelers inquiring about trips to the southern Amazon of Peru.

ABT-378/ritonavir plus stavudine and lamivudine for the treatment of antiretroviral-naive adults with HIV-1 infection: 48-week results.

OBJECTIVE: To evaluate the safety and antiviral activity of different dose levels of the HIV protease inhibitor ABT-378 combined with low-dose ritonavir, plus stavudine and lamivudine in antiretroviral-naive individuals. DESIGN: Prospective, randomized, double-blind, multicenter. METHODS: Eligible patients with plasma HIV-1 RNA > 5000 copies/ml received ABT-378 200 or 400 mg with ritonavir 100 mg every 12 h; after 3 weeks stavudine 40 mg and lamivudine 150 mg every 12 h were added (group I, n = 32). A second group initiated treatment with ABT-378 400 mg and ritonavir 100 or 200 mg plus stavudine and lamivudine every 12 h (group II, n = 68). RESULTS: Mean baseline HIV-1 RNA was 4.9 log10 copies/ml in both groups and CD4 cell count was 398 x 10(6)/l and 310 x 10(6)/l in Groups I and II respectively. In the intent-to-treat (ITT; missing value = failure) analysis at 48 weeks, HIV-1 RNA was < 400 copies/ml for 91% (< 50 copies/ml, 75%) and 82% (< 50 copies/ml, 79%) of patients in groups I and II respectively. Mean steady-state ABT-378 trough concentrations exceeded the wild-type HIV-1 EC50 (effective concentration to inhibit 50%) by 50-100-fold. The most common adverse events were abnormal stools, diarrhea and nausea. No patient discontinued before 48 weeks because of treatment-related toxicity or virologic rebound. CONCLUSIONS: ABT-378 is a potent, well-tolerated protease inhibitor. The activity and durable suppression of HIV-1 observed in this study is probably attributable to the observed tolerability profile and the achievement of high ABT-378 plasma concentrations.

Comparative studies of two-times-daily versus three-times-daily indinavir in combination with zidovudine and lamivudine.

OBJECTIVES: To compare the efficacy and safety of two-times-daily versus three-times-daily indinavir in combination with zidovudine and lamivudine. DESIGN: Two multicenter, open-label, randomized 24-week studies. METHODS: Adults HIV-1 infection, HIV-1 RNA greater than 10000 copies/ml, and no prior lamivudine or protease inhibitor therapy were eligible. In a pilot study (Study A), patients received indinavir at 800 mg every 8 h, 1000 mg every 12 h, or 1200 mg every 12 h. In a subsequent study (Study B), patients received indinavir at 800 mg every 8 h or 1200 mg every 12 h. All subjects received zidovudine (300 mg) and lamivudine (150 mg) every 12 h. An intent-to-treat analysis was used. RESULTS: In Study A, which enrolled 88 patients, neither HIV-1 RNA nor CD4 cell responses differed significantly between treatment groups at 24 weeks when corrected for multiple comparisons. Study B enrolled 433 patients, but was prematurely discontinued when interim analysis suggested greater efficacy of three-times-daily indinavir. Of the first 87 patients reaching week 24, HIV-1 RNA was less than 400 copies/ml in 91% receiving three-times-daily versus 64% receiving two-times-daily indinavir (P < 0.01). CONCLUSION: Three-times-daily indinavir appears more efficacious than two-times-daily dosing when administered with zidovudine and lamivudine. Two-times-daily indinavir dosing should only be considered in situations characterized by favorable pharmacokinetic drug-drug interactions.

Decreased dihydropyridine receptor number in hypertensive rat vascular muscle cells.

To further investigate the altered function of Ca2+ channels in vascular muscle cells in hypertension, a novel fluorescently labeled dihydropyridine was used with ultrahigh-sensitivity photometry to study dihydropyridine binding sites on the surface membrane of living vascular muscle cells from stroke-prone spontaneously hypertensive rats and their normotensive controls. Fluorescent nitrobenzoxadiazol-6-dihydropyridine in concentrations of 1 to 100 nmol/L bound specifically to vascular muscle cells' Ca2+ channels, and was displaced by the unlabeled dihydropyridine analogue or nisoldipine (10 mumol/L). Stroke-prone spontaneously hypertensive rat vascular muscle cells showed significantly decreased binding of nitrobenzoxadiazol-6-dihydropyridine compared with normotensive National Institutes of Health rats. Decreased binding of dihydropyridine by vascular muscle cells from stroke-prone spontaneously hypertensive rats (cells that in other studies show increased Ca2+ channel function) indicates a change in channel regulation that is possibly due to a deficiency in the inactivation mechanism, consistent with our earlier electrophysiological studies reporting deficiencies in Ca(2+)-dependent inactivation in genetic hypertension. These data demonstrate decreased numbers of localized sites of dihydropyridine binding on the sarcolemma of living vascular muscle cells, and support the hypothesis that Ca2+ channel alterations may significantly contribute to the molecular etiology of genetic hypertension.

Cryptosporidiosis in Houston, Texas. A report of 95 cases.

Cryptosporidiosis is an important cause of diarrhea. We identified 95 patients with cryptosporidiosis over a 6-year period in our county hospital system, including 9 children and 86 adults infected with the human immunodeficiency virus (HIV). Risk factors included male-to-male sexual practices and Hispanic race. Diarrhea, weight loss, and gastrointestinal complaints were the most common symptoms at presentation. Among the HIV-infected adults, 20 (23%) developed biliary tract disease. Biliary involvement was associated with low CD4 counts. Treatment with paromomycin and antimotility agents was effective in reducing diarrheal symptoms in 54 of 70 (77%) patients with the acquired immunodeficiency syndrome (AIDS), although there was a high rate of relapse. Paromomycin did not prevent the development of biliary disease. Biliary disease responded to cholecystectomy or sphincterotomy with stent placement. Though often a cause of morbidity, cryptosporidiosis was only rarely the cause of death, even among patients with HIV. Cryptosporidiosis continues to be an important medical problem even in developed-countries. Current methods of prevention and treatment are suboptimal.

Neurocysticercosis in Houston, Texas. A report of 112 cases.

Neurocysticercosis (NCC) is a common disease among Hispanic immigrants to the American Southwest. The experience in this series suggests a higher incidence of disease than has been reported by public health personnel. Symptoms at presentation correlate with the anatomic site of disease. Parenchymal disease and inactive calcifications present primarily with seizures; ventricular disease with headaches and symptoms of acute hydrocephalus; and cisternal disease with basilar meningitis, hydrocephalus, or seizures from coexisting parenchymal disease. Current treatment regimens are suboptimal, although symptoms may resolve in the absence of therapy. Patients may not respond to a single course of praziquantel therapy. Procedures to divert cerebrospinal fluid are often needed and frequently require revision. We found an association between corticosteroid treatment and the need for repeat therapy with praziquantel, but controlled studies are needed to clarify the role of antiparasitic agents in all forms of neurocysticercosis.