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.

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.

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.

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.

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.

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.

Stem cell quiescence acts as a tumour suppressor in squamous tumours.

In some organs, adult stem cells are uniquely poised to serve as cancer cells of origin. It is unclear, however, whether tumorigenesis is influenced by the activation state of the adult stem cell. Hair follicle stem cells (HFSCs) act as cancer cells of origin for cutaneous squamous cell carcinoma and undergo defined cycles of quiescence and activation. The data presented here show that HFSCs are unable to initiate tumours during the quiescent phase of the hair cycle, indicating that the mechanisms that keep HFSCs dormant are dominant over the gain of oncogenes (such as Ras) or the loss of tumour suppressors (such as p53). Furthermore, Pten activity is necessary for quiescence-based tumour suppression, as its deletion alleviates tumour suppression without affecting proliferation. These data demonstrate that stem cell quiescence is a form of tumour suppression in HFSCs, and that Pten plays a role in maintaining quiescence in the presence of tumorigenic stimuli.

Amplification-free detection of Cryptosporidium parvum nucleic acids with the use of DNA/RNA-directed gold nanoparticle assemblies.

This study describes the development and evaluation of an amplification-free molecular assay for detection of Cryptosporidium parvum oocysts. The assay employed a pair of oligonucleotide-functionalized gold nanoparticle (AuNP) probes that were complementary to adjacent sequences on C. parvum 18s rRNA. Hybridization of the probes to the target RNA resulted in the assembly of AuNPs into target-linked networks, which were detected both visibly and spectroscopically, by a redshift in the wavelength of light scattered by the gold nanoparticles. The limit of detection was between 4 × 10(5) and 4 × 10(6) copies of RNA per microliter reaction mix, when a short synthetic target or full-length in vitro transcribed target was employed. With total nucleic acids purified from C. parvum oocysts spiked into 100-mg stool, as few as 670 oocysts/µl reaction mix were detected. The ability to detect the nucleic acids of C. parvum oocysts in stool, without the need for complex amplification, offers unique advantages for such AuNP aggregation assays to be extended toward use in resource-limited settings where protozoan detection is needed most.

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.

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

Treatment of neurocysticercosis: current status and future research needs.

Here we put forward a roadmap that summarizes important questions that need to be answered to determine more effective and safer treatments. A key concept in management of neurocysticercosis is the understanding that infection and disease due to neurocysticercosis are variable and thus different clinical approaches and treatments are required. Despite recent advances, treatments remain either suboptimal or based on poorly controlled or anecdotal experience. A better understanding of basic pathophysiologic mechanisms including parasite survival and evolution, nature of the inflammatory response, and the genesis of seizures, epilepsy, and mechanisms of anthelmintic action should lead to improved therapies.

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.

Purification and characterization of a metacestode cysteine proteinase from Taenia solium involved in the breakdown of human IgG.

Infection of the central nervous system by Taenia solium cysticerci is the cause of human neurocysticercosis, a major neurological infection in the Third World and an emerging infectious disease in the United States. We previously isolated a cysteine proteinase from cysticerci of Taenia crassiceps and demonstrated that it degrades human IgG in vitro. We have now isolated a 48 kDa thiol-dependent proteinase from T. solium. The T. solium enzyme also degrades human IgG, but does not significantly degrade albumin. IgG degradation was inhibited by cysteine proteinase inhibitors, but not significantly by inhibitors of aspartic, serine, or metalloproteinases. The peptide substrate specificity and pH optimum resemble cathepsin L. The Km for the peptide substrate Z-Phe-Arg-AFC was calculated to be 7.0 x 10(-6) M, the Kcat was 1.98 x 10(-5) s(-1), and the Kcat/Km 2.84 x 10(9) M(-1) s(-1), a value which is within the diffusion control limit for highly catalytic enzymes. We propose that immunoglobulin degradation by the T. solium cysteine proteinase may play a key role in the host-parasite interface and could be employed as a target for chemotherapy.

Determinants of sustained virological suppression in indigent, HIV-infected patients: is single protease inhibitor-based antiretroviral therapy truly highly active?

BACKGROUND: Effective virological suppression with HAART is dependent on strict adherence to therapy. Compliance with therapy is influenced by clinical and psychosocial factors. METHOD: We performed a retrospective study investigating determinants of effective virological suppression, defined as <400 RNA at 11-13 months of HAART, in an urban indigent population. The study included 366 new patients presenting for care to the Thomas Street Clinic, Houston, Texas, between April and December 1998. Median age, CD4 count, and viral load (VL) of the study population were 37.5 years, 189 cells/mm(3), and 53,000, respectively. Thirty-nine percent had AIDS, 20% had cocaine-positive drug screens, and 64% were antiretroviral naïve. Two hundred and sixty-seven patients were started on HAART. Thirty-four percent showed virological suppression. RESULTS: In multivariate analysis, adherence to HAART, care by experienced primary provider, baseline VL <100,000 copies/mL, age >35 years, and no active substance use were associated with virological suppression. Rates of virological suppression with HAART are unacceptably low in this urban indigent population. CONCLUSION: Low rates of virological suppression are primarily due to lack of adherence rather than late utilization of care among ethnic minorities. Single protease-inhibitor-based antiretroviral therapy does not appear to be highly active in this patient population.

Response of HIV-infected patients with asymptomatic syphilis to intensive intramuscular therapy with ceftriaxone or procaine penicillin.

The objective of this prospective pilot study was to evaluate the response of HIV-infected patients with asymptomatic syphilis to one of two intensive antibiotic treatment regimens. Thirty-one HIV-infected patients with serum rapid plasma reagin titre > or =1:4 and no clinical findings of syphilis were randomized to receive daily intramuscular injections of ceftriaxone or procaine penicillin (plus oral probenecid) for 15 days; 24 returned for follow-up study. Seven of 10 (70%) procaine penicillin-treated patients and 10 of 14 (71%) ceftriaxone-treated patients had a > or =4-fold decline in RPR (P=0.94); two penicillin-treated and one ceftriaxone-treated patient relapsed. Two patients failed ceftriaxone therapy. Three penicillin-treated, and two ceftriaxone-treated patients were serofast. Serological responses were similar in those patients with and without asymptomatic neurosyphilis. There was no difference in the serologic response to daily treatment with ceftriaxone vs that with procaine penicillin plus probenecid; both treatments were associated with comparatively high rates of serological non-response and relapse.

Management of neurocysticercosis.

Neurocysticercosis is a common cause of neurological disease in developing countries and a major cause of epilepsy worldwide. A unique characteristic of human neurocysticercosis is that the living parasite is very well tolerated in human brain, so symptoms and clinical disease primarily result from death of the organism and accompanying inflammatory reaction in the human CNS. Among the diverse clinical manifestations of human neurocysticercosis, seizures are the most common, but other clinical problems occur, depending upon the localisation and viability of the parasite. Although both praziquantel and albendazole are effective agents, there is controversy about their role in several forms of the disease. Systematic reviews have pointed out the limited quality of available data on therapy. At a recent international conference convened to develop guidelines for treatment of this disease, areas of consensus and disagreement on the role of antiparasitic therapy were discussed. It was clear to all that cysticercosis cannot be regarded as a single disorder; treatment needs to be modified based on the location and number of cysticerci and the host response. There was a strong consensus that there is no role for antiparasitic drugs in patients with only calcified lesions. Studies suggest that patients with single enhancing lesions will do well regardless of antiparasitic therapy. Antiparasitic drugs are contraindicated in patients with cerebral oedema (cysticercal encephalitis). Most experts strongly recommend antiparasitic therapy in patients with multiple subarachnoid cysticerci or giant cysticerci. In patients with ventricular cysticerci, endoscopic removal is the preferred therapy. However, recent evidence suggests that placement of a ventricular shunt followed by antiparasitic therapy is an acceptable alternative. Standard treatment for localization-related epilepsy is effective for seizures caused by cysticercosis. In general, seizures are easily controlled in this illness. While many controversies regarding the treatment of patients with neurocysticercosis were not resolved at the international consensus conference, participants did conclude that controlled prospective studies are required to define optimal therapy for the infection and that treatment of infected individuals must be individualised.

Guidance on standards of health for clinical health care workers.

Judgements about the health of clinical health care workers in relation to fitness to practice are made by a variety of doctors. These guidelines have been written to assist with such judgements and to facilitate equitable decision making in matters of employment.