Asymptomatic Visceral Leishmania infantum Infection in US Soldiers Deployed to Iraq.

BACKGROUND: Visceral leishmaniasis (VL), due to Leishmania infantum, is a persistent intracellular parasitic infection transmitted by the bite of infected sand flies. Symptomatic VL has been reported in U.S. soldiers with Iraq deployment. Untreated symptomatic VL can be fatal; asymptomatic VL (AVL) may establish a lifelong risk of reactivation. We report prevalence and AVL risk factors in Operation Iraqi Freedom (OIF) deployers during 2002-11. METHODS: Healthy soldiers exposed to VL endemic areas in Iraq and 50 controls who never traveled to endemic regions were recruited through military healthcare facilities (2015-17). Responses to a risk factor survey and blood samples were obtained. Leishmania research diagnostics utilized included enzyme-linked immunosorbent assay (ELISA), rk39 test strips, quantitative polymerase chain reaction (PCR), and interferon gamma release (IGRA) assays. Statistical analyses included Fisher exact test, Pearson χ2 test, Mann-Whitney U test, and logistic regression. RESULTS: 200 deployed subjects were enrolled, mostly males (84.0%), of white ethnicity (79.0%), and median age 41 (range 24-61) years. 64% were seropositive for Phlebotomus alexandri saliva antibodies. Prevalence of AVL (any positive test result) was 39/200 (19.5%, 95% confidence interval 14.4%-25.8%). Two (1.0%) PCR, 10 (5%) ELISA, and 28 (14%) IGRA samples were positive. Travel to Ninewa governorate increased risk for AVL (P = .01). CONCLUSION: AVL was identified in 19.5% of OIF deployers; travel to northwest Iraq correlated with infection. Further studies are needed to inform risk for reactivation VL in US veterans and to target additional blood safety and surveillance measures.

A new model of progressive visceral leishmaniasis in hamsters by natural transmission via bites of vector sand flies.

BACKGROUND: Visceral leishmaniasis (VL) is transmitted by sand flies. Protection of needle-challenged vaccinated mice was abrogated in vector-initiated cutaneous leishmaniasis, highlighting the importance of developing natural transmission models for VL. METHODS: We used Lutzomyia longipalpis to transmit Leishmania infantum or Leishmania donovani to hamsters. Vector-initiated infections were monitored and compared with intracardiac infections. Body weights were recorded weekly. Organ parasite loads and parasite pick-up by flies were assessed in sick hamsters. RESULTS: Vector-transmitted L. infantum and L. donovani caused ≥5-fold increase in spleen weight compared with uninfected organs and had geometric mean parasite loads (GMPL) comparable to intracardiac inoculation of 10(7)-10(8) parasites, although vector-initiated disease progression was slower and weight loss was greater. Only vector-initiated L. infantum infections caused cutaneous lesions at transmission and distal sites. Importantly, 45.6%, 50.0%, and 33.3% of sand flies feeding on ear, mouth, and testicular lesions, respectively, were parasite-positive. Successful transmission was associated with a high mean percent of metacyclics (66%-82%) rather than total GMPL (2.0 × 10(4)-8.0 × 10(4)) per midgut. CONCLUSIONS: This model provides an improved platform to study initial immune events at the bite site, parasite tropism, and pathogenesis and to test drugs and vaccines against naturally acquired VL.

Transition from gavage to nipple feeding for preterm infants with bronchopulmonary dysplasia.

BACKGROUND: The transition from gavage to nipple feeding is difficult for preterm infants with bronchopulmonary dysplasia because of tachypnea and hypoxemia from chronic respiratory distress. OBJECTIVE: The aim of this study was to test the hypothesis that preterm infants with bronchopulmonary dysplasia who transitioned from gavage to nipple feeding with the semidemand method would achieve nipple feeding sooner and be discharged from hospital sooner than control infants who received standard care. METHODS: Forty-two infants were randomized to the control condition and 44 to the experimental protocol. Mean gestational ages and birth weights were 25 ± 1.5 weeks and 784 g for controls and 25 ± 1.4 weeks and 787 g for experimental infants. Control infants received standard care that included gradual increases in the number of nipple to gavage feedings per day. Experimental infants received the semidemand method that used infant behavioral and cardiorespiratory signs to regulate frequency, length, and volume of nipple feedings. General linear model procedures were used to compare study groups. RESULTS: Experimental infants achieved nipple feeding at M = 5.9 ± 0.7 days compared with control infants, M = 12.3 ± 0.8 (p < .0001). Length of hospitalization was not significantly different between groups. DISCUSSION: The semidemand method significantly shortened the time for infants to attain nipple feeding in a manner taking their respiratory distress into consideration.

Mobile learning module improves knowledge of medical shock for forward surgical team members.

OBJECTIVE: Acute trauma care is characterized by dynamic situations that require adequate preparation to ensure success for military health professionals. The use of mobile learning in this environment can provide a solution that standardizes education and replaces traditional didactic lectures. METHODS: A comparative evaluation with a pre-post test design regarding medical shock was delivered via either a didactic lecture or a mobile learning video module to U.S. Army Forward Surgical Team (FST) members. Participants completed a pretest, were randomly assigned to treatment group by FST, and then completed the post-test and scenario assessment. RESULTS: One-hundred and thirteen FST members participated with 53 in the mobile learning group and 60 in the lecture group (control). The percent mean score for the mobile learning group increased from 43.6 to 70 from pretest to post-test, with a scenario mean score of M = 56.2. The percent mean score for the control group increased from 41.5 to 72.5, with a scenario mean score of M = 59.7. The two-way analysis of variance mean score difference was 26.4 for the mobile learning group and 31.0 for the control, F = 2.18, (p = 0.14). CONCLUSIONS: Mobile learning modules, coupled with a structured assessment, have the potential to improve educational experiences in civilian and military settings.

Heart rate variability is an independent predictor of morbidity and mortality in hemodynamically stable trauma patients.

BACKGROUND: Reduced heart rate variability (HRV) reflects autonomic dysfunction and can triage patients better than routine trauma criteria or vital signs. However, there is questionable specificity and no consensus measurement technique. The purpose of this study was to analyze whether factors that alter autonomic function affect the specificity of HRV for assessing traumatic injury. METHODS: We evaluated 216 hemodynamically stable adults (3:1 M:F; 97:3 blunt:penetrating; age 49 years ± 1 year, mean ± standard error) undergoing computed axial tomography (CT) scan to rule out traumatic brain injury (TBI). All were prospectively instrumented with a Mars Holter system (GE Healthcare, Milwaukee, WI). HRV was determined offline using time domain (standard deviation of normal-normal intervals, root-mean-square successive difference) and frequency domain (very low frequency [VLF], LF, wideband frequency, high frequency [HF], low to HF index ratio) calculations from 15-minute electrocardiogram and correlated with routine vital signs, mortality, TBI, morbidity, length of stay (LOS), and comorbidities. Significance (p ≤ 0.05) was determined using nonparametric analysis, Student's t test, analysis of variance, or multiple logistic regression. RESULTS: VLF alone predicted survival, severity of TBI, intensive care unit LOS, and hospital LOS (all p < 0.05). Beta-blockers or diabetes had no effect, whereas age, sedation, mechanical ventilation, spinal cord injury, and intoxication influenced one or more of the variables with age being the most powerful confounder (all p < 0.05). Except for the Glasgow Coma Scale, no other routine trauma or hemodynamic criteria correlated with any of these outcomes. CONCLUSIONS: Decreased VLF is an independent predictor of mortality and morbidity in hemodynamically stable trauma patients. Other time and other frequency domain variables correlated with some, but not all, outcomes. All were heavily influenced by factors that alter autonomic function, especially patient age.

Osteopontin and interleukin-8 expression is independently associated with prostate cancer recurrence.

PURPOSE: Lack of reliable biomarkers limits accurate prediction of prostate-specific antigen biochemical recurrence (disease progression) in prostate cancer. The two inflammatory chemokines, osteopontin and interleukin-8 (IL-8), are associated with tumor angiogenesis and metastasis. We investigated whether osteopontin and IL-8 expression in prostate cancer correlates with disease progression. EXPERIMENTAL DESIGN: Archival prostatectomy specimens (n = 103) were obtained from patients with minimum 72-month follow-up. Osteopontin and IL-8 expression was evaluated by immunohistochemistry and graded for intensity and the area. Association of osteopontin and IL-8 staining with biochemical recurrence was evaluated by univariate and multivariate models. RESULTS: In tumor cells, osteopontin and IL-8 staining was higher in the recurred group (203.2 +/- 78.4; 181.1 +/- 89.3) than in the nonrecurred group (122.7 +/- 76.6; 96.4 +/- 85.6; P < 0.001). Higher osteopontin and IL-8 staining was also observed in benign areas adjacent to tumor in the recurred group, than in nonrecurred group. In univariate analysis, except age, all preoperative and postoperative variables and osteopontin and IL-8 staining scores were significantly associated with biochemical recurrence (P < 0.05). In multivariate analysis, margin status and osteopontin staining independently associated with biochemical recurrence within 72 months. Osteopontin, either alone or with IL-8 and seminal vesicle invasion, was a significant variable in predicting biochemical recurrence within 24 months. Osteopontin and IL-8 staining predicted recurrence with high sensitivity (75.5%; 73.6%) and specificity (76%; 70.6%). CONCLUSION: In prostatectomy specimens, osteopontin expression is independently associated with biochemical recurrence. Both osteopontin and IL-8 may be predictors of early disease progression.

Detecting an association between socioeconomic status and late stage breast cancer using spatial analysis and area-based measures.

OBJECTIVES: To assess the relationship between socioeconomic status (SES) and late stage breast cancer using the cluster detection software SaTScan and U.S. census-derived area-based socioeconomic measures. MATERIALS AND METHODS: Florida's 18,683 women diagnosed with late stage breast cancer (regional or distant stage) between 1998 and 2002 as identified by Florida's population-based, statewide, incidence registry were analyzed by SaTScan to identify areas of higher-than-expected incidence. The relationship between SES and late stage breast cancer was assessed at the neighborhood (block group) level by combining the SaTScan results with area-based SES data. RESULTS: SaTScan identified 767 of Florida's 9,112 block groups that had higher-than-expected incidence of late stage breast cancer. After controlling for patient level insurance status, county level mammography prevalence, and urban/rural residence in the logistic regression model, women living in neighborhoods of severe and near poverty were respectively 3.0 and 1.6 times more likely to live in areas of higher-than-expected incidence of late stage breast cancer when compared with women living in nonpoverty. Additionally, areas in the lowest quartile of mammography usage were almost seven times more likely to have higher-than-expected incidence than areas in the higher quartiles. CONCLUSIONS: In addition to confirming the importance of mammography, results from the present study suggest that "where" you live plays an important role in defining the risk of presenting with late stage breast cancer. Additional research is urgently needed to understand this risk and to leverage the strengths and resources present in all communities to lower the late stage breast cancer burden.

Heart rate variability index in trauma patients.

BACKGROUND: Heart rate variability (HRV) changes often reflect autonomic dysfunction with high sensitivity, but the specificity is also low. There are several different methods for measuring HRV, but interpretation is often complex, and the units are not interchangeable. For these reasons, HRV monitoring is not routinely used in many clinical situations. We hypothesized that the specificity of HRV as a screening tool for trauma patients could be improved by controlling some of the confounding influences using multiple logistic regression. METHODS: A prospective observational trial with waiver of consent was performed in 243 healthy student volunteers and 257 trauma patients, in the resuscitation bay and intensive care units of a Level I trauma center, who received computed axial tomography (CT) scans of the head as part of the initial work up. Electrocardiogram results were recorded for 5 minutes. HRV was defined by SD of normal R-R intervals (SDNN5) and by root mean square of successive differences of R-R intervals (RMSSD5). A head CT scan was considered positive (+) if there were abnormalities in the parenchyma (diffuse axonal injury or contusion), vasculature (intraparenchymal, subdural, or epidural hemorrhage), and/or structural or bony components (fractures of the face or cranium). RESULTS: In volunteers, SDNN5 was 73 +/- 15 (M +/- SD) milliseconds, compared with 42 +/- 22, 31 +/- 19, 28 +/- 17, and 12 +/- 8 milliseconds in, CT(-) patients with no sedation (n = 82), CT(-) with sedation (n = 60), CT(+) with no sedation (n = 55), and CT(+) with sedation (n = 60), respectively. The differences between trauma, sedation, and CT categories were significant (all p < 0.001). RMSSD5 differences were similar and also highly significant (all p < 0.001). For both SDNN5 and RMSSD5, in each category, there was wide overlap in the range of values, and strong inverse correlations with heart rate (all p < 0.001). Using multiple logistic regression in a subset with no missing data (n = 194), an index was derived from ln(SDNN5) adjusted for six confounding factors. With a negative predictive value held constant at 0.90, compared with ln(SDNN5) alone, the stepwise addition of heart rate, sedation, age, gender, and blood pressure progressively improved the specificity of the HRV index from 0.56 to 0.77, positive predictive value from 0.55 to 0.68, and efficiency from 0.68 to 0.80. This index was then normalized (0-100 scale) for ease of interpretation. CONCLUSIONS: (1) Several factors alter HRV in patients; (2) when HRV was indexed for some of these factors, its specificity and efficiency were improved for predicting a discrete pathologic state in trauma patients, i.e. (+) or (-) cranial CT scans; (3) the algorithm can incorporate other factors to further refine the diagnostic and/or prognostic ability of HRV as a noninvasive clinical tool; (4) this concept should be applicable to any other HRV measurement technique or outcome.

An assessment of the virtual conversations method for prostate cancer patient education.

INTRODUCTION: Virtual dialogue is a specific educational methodology that employs speech recognition, digital video, and computer technologies in a symbiotic relationship to allow users to have the illusion of a face-to-face conversation with a person in cyberspace. A voice-activated, interactive, virtual dialogue with a prostate cancer specialist was developed and tested in a clinical setting at the Walter Reed Army Medical Center (WRAMC). OBJECTIVES: The purpose of this study was two-fold: (a) to create a prototype virtual dialogue program on the subject of prostate cancer and (b) to evaluate the effectiveness of this method to educate men about their disease and treatment. METHOD: Participants were recruited using a convenience sample of patients attending the Center for Prostate Disease Research multidisciplinary clinic at WRAMC. An automated pretest and post-test instrument was developed by the investigators to assess patients' knowledge before and after the virtual dialogue session. RESULTS: A total of 33 patients volunteered for the study. Results from this convenience sample showed an increase in patients' knowledge and positive acceptance of this innovative method of patient education. CONCLUSIONS: The data resulting from this study provide persuasive evidence that a virtual dialogue with a knowledgeable health professional can be a useful and highly effective method for educating men about prostate cancer. This method may also offer an effective way for health professionals to systematically provide their patients with comprehensive and reliable information.

Upregulation of surface proteins in Leishmania donovani isolated from patients of post kala-azar dermal leishmaniasis.

Five to fifteen percent of visceral leishmaniasis (VL) patients in India develop post kala-azar dermal leishmaniasis (PKDL), usually 1-2 years after apparent clinical cure. There is evidence pointing to a role played by the host immune responses in the disease pathogenesis, however, the contribution of changes in parasite gene expression has not been explored. Highly sensitive gene expression microarray technology was employed to identify genes that are differentially expressed in Leishmania parasites isolated from PKDL patients in comparison with those from VL. Hybridization on Leishmania donovani genomic microarray comprised of unique clones allowed us to identify 46/2268 (2%) clones that showed statistically significant (P<0.05) changes in expression (1.5-3.5-fold) in parasites of PKDL origin compared to those of VL origin. Sequence analysis of six genomic clones, consistently showing approximately 2-fold higher expression in PKDL parasites, revealed significant homology with gp63, gp46, putative amastin, a putative reductase and a possible calpain-like protein. The gene products showing upregulated expression in PKDL isolates may be candidates playing a role in the altered clinical manifestation in PKDL. Such differentially expressed genes hold the key to understanding the parasite genetic factors that contribute to the persistence after clinical cure of VL.

Evaluation of the prognostic potential of hyaluronic acid and hyaluronidase (HYAL1) for prostate cancer.

Despite the development of nomograms designed to evaluate the prognosis of a patient with prostate cancer (CaP), the information has been limited to prostate-specific antigen (PSA), clinical stage, Gleason score, and tumor volume estimates. To improve our ability to predict prognosis, information regarding the molecular properties of CaP is needed. Hyaluronic acid (HA) is a glycosaminoglycan that promotes tumor metastasis. Hyaluronidase (HAase) is an enzyme that degrades HA into angiogenic fragments. We recently showed that in CaP tissues, whereas HA is localized mostly in the tumor-associated stroma, HYAL1 type HAase is exclusively localized in CaP cells (Lokeshwar et al. J. Biol. Chem., 276: 11922-11932, 2001). We evaluated the prognostic potential of HA and HYAL1 in CaP by immunohistochemistry. Archival CaP specimens were obtained from patients who underwent radical retropubic prostatectomy for clinically localized CaP. Group 1 (n = 25) included patients who showed biochemical recurrence (PSA >0.4 ng/ml; mean recurrence: 21.3 months). Group 2 included patients with no clinical or biochemical recurrence (n = 45; mean follow-up: 80.9 months). For HA and HYAL1 staining, a biotinylated HA-binding protein and an anti-HYAL1 antibody were used. The staining was evaluated on the basis of intensity (0 to 3+) and as dense or sparse (for HA staining only) and then grouped as low grade and high grade. In CaP specimens, HYAL1 was exclusively expressed in tumor cells. Although the stroma was stained positive for HA, 40% of tumor cells also expressed HA. HA, HYAL1, and combined HA-HYAL1 staining predicted progression with 96%, 84%, and 88% sensitivity, 55.5%, 80%, and 84.4% specificity, and 70%, 81.4%, and 85.7% accuracy, respectively. In the univariate analysis, preoperative PSA, Gleason sum, stage, margin, seminal vesicle, extra-prostatic extension (EPE), HA, HYAL1, and HA-HYAL1 were significant in predicting progression (P < 0.05). However, in the multiple logistic regression analysis, only EPE [odds ratio (OR) = 33.483; P = 0.002), HYAL1 (OR = 12.42; P = 0.009), HA-HYAL1 (OR = 18.048; P = 0.0033), and margin (OR = 26.948; P = 0.006)] were significant. Thus, in this 5-year follow-up study, HYAL1, together with EPE and margin, was found to be an independent prognostic indicator.

The application of gene expression microarray technology to kinetoplastid research.

Protozoan parasites in the order Kinetoplastida cause severe disease primarily in tropical and subtropical areas. Vaccines to control these diseases have shown some promise, but none are in active clinical use. Drug treatments are available for all of the acute infections, but the emergence of resistance and an unresponsive chronic phase are current problems. Rapid advances in genomic technology open the possibility of discovering new genes that can contribute to vaccine initiatives or serve as targets for development of new drugs. The DNA microarray is a genomic technology, which is being applied to new gene discovery in kinetoplastid parasites. Both cDNA and genomic microarrays for Leishmania major have identified a number of new genes that are expressed in a stage-specific fashion and preliminary results from a L. donovani genomic microarray also demonstrated new gene discovery. A microarray of Trypanosoma brucei genomic fragments identified new genes whose expression differs between the insect borne stage and the human infectious stage of the parasite. The next few years, building on this foundational work, should witness the most exciting stage as microarrays are applied to questions such as the basis of drug resistance, post kala azar dermal leishmaniasis, the regulation of differentiation to infectious stages, linking coordinately regulated pathways of genes and development of genetically defined parasites that may have potential as live attenuated vaccines.

Are burn patients really at risk for thrombotic events?

There continues to be debate about the routine use of deep vein thrombosis (DVT) prophylaxis in burn patients. The concern is that routine prophylaxis may lead to adverse events. The debate hinges on the incidence of DVT and its relation to the risk-benefit ratio. This study seeks to estimate the true rate of DVT in burn patients, and to evaluate possible risk factors to its development. The Nationwide Inpatient Sample was queried for all patients with age ≥18 years with ICD-9 codes for burn injuries. Demographic data, comorbidities, burn data, length of stay, total charges, procedures, presence of central venous catheter, and mortality were recorded. Patients were classified based on the presence of DVT. Student's t-test, χ test, and logistic regression were performed. 36,638 burn patients were identified. DVT rate was 0.8%. Patients with DVT were older, had longer hospitalizations, more procedures, and higher charges. On logistic regression, black race, TBSA ≥20%, history of previous venous thrombotic events, blood transfusion, and mechanical ventilation were the significant factors associated with DVT. Patients with DVT were almost twice as likely to die during the admission (P = .011). This is the largest series to date examining the risk factors for DVT in burn patients. DVT developed in approximately 0.8% of burn patients. Black race, TBSA ≥20%, blood transfusions, and mechanical ventilation were associated with approximately 2-fold odds of developing DVT. Identification of these additional risk factors may allow targeted patient prophylaxis. Additionally, patients with DVT incurred higher total charges and longer hospitalization.

Factors affecting late survival after surgical remodeling of left ventricular aneurysms.

OBJECTIVES: Surgical remodeling of the left ventricle has involved various techniques of volume reduction. This study evaluates factors that influence long-term survival results with 3 operative methods. METHODS: From 1979 to 2000, 157 patients (134 men, mean age 61 years) underwent operations for class III or IV congestive heart failure, angina, ventricular tachyarrhythmia, and sudden death after anteroseptal myocardial infarction. The preoperative ejection fraction was 28% +/- 0.9% (mean +/- standard error), and the pulmonary artery occlusive pressure was 15 +/- 0.07 mm Hg. Cardiogenic shock was present in 26 patients (16%), and an intra-aortic balloon pump was used in 48 patients (30%). The type of procedure depended on the extent of endocardial disease and was aimed at maintaining the ellipsoid shape of the left ventricle cavity. In group I patients (n = 65), radical aneurysm resection and linear closure were performed. In group II patients (n = 70), septal dyskinesis was reinforced with a patch (septoplasty). In group III patients (n = 22), ventriculotomy closure was performed with an intracavitary oval patch. RESULTS: Hospital mortality was 16% (25/157) and was similar among the groups. Actuarial survival up to 18 years was better with a preoperative ejection fraction of 26% or greater (P =.004) and a pulmonary artery occlusive pressure of 17 mm Hg or less (P =.05). Survival was worse in patients who had intra-aortic balloon pump support (P =.03). Five-year survival for all patients in group III was higher than for patients in group II (67% vs 47%, P =.04). CONCLUSIONS: Factors that improved long-term survival after left ventricular surgical remodeling were intraventricular patch repair, preoperative ejection fraction of 26% or greater, and pulmonary artery occlusive pressure of 17 mm Hg or less without the need for balloon pump assist.

Multiplex screening for blood-borne viral, bacterial, and protozoan parasites using an OpenArray platform.

The use of nucleic acid tests for detection of pathogens has improved the safety of blood products. However, ongoing pathogen emergence demonstrates a need for development of devices testing for multiple pathogens simultaneously. One approach combines two proven technologies: Taqman chemistry for target identification and quantification and the OpenArray nanofluidic real-time PCR platform for spatial multiplexing of assays. A panel of Taqman assays was developed to detect nine blood-borne pathogens (BBPs): four viral, two bacterial, and three protozoan parasites. The custom BBP OpenArray plate with 18 assays was tested for specificity and analytical sensitivity for nucleic acid from each purified pathogen and with pathogen-spiked human blood and plasma samples. For most targets, the limits of detection (10 to 10,000 copies/mL) were comparable with existing real-time platforms. The testing of the BBP OpenArray with pathogen-spiked coded human plasma or blood samples and negative control specimens demonstrated no false-positive results among the samples tested and correctly identified pathogens with the lowest concentration detected ranging from 10 cells/mL (Trypanosoma cruzi) to 10,000 cells/mL (Escherichia coli). These results represent a proof of concept that indicated the BBP OpenArray platform in combination with Taqman chemistry may provide a multiplex real-time PCR pathogen detection method that points the way for a next-generation platform for infectious disease testing in blood.

Leishmania donovani argininosuccinate synthase is an active enzyme associated with parasite pathogenesis.

BACKGROUND: Gene expression analysis in Leishmania donovani (Ld) identified an orthologue of the urea cycle enzyme, argininosuccinate synthase (LdASS), that was more abundantly expressed in amastigotes than in promastigotes. In order to characterize in detail this newly identified protein in Leishmania, we determined its enzymatic activity, subcellular localization in the parasite and affect on virulence in vivo. METHODOLOGY/PRINCIPAL FINDINGS: Two parasite cell lines either over expressing wild type LdASS or a mutant form (G128S) associated with severe cases of citrullinemia in humans were developed. In addition we also produced bacterially expressed recombinant forms of the same proteins. Our results demonstrated that LdASS has argininosuccinate synthase enzymatic activity that is abolished using an ASS specific inhibitor (MDLA: methyl-D-L-Aspartic acid). However, the mutant form of the protein is inactive. We demonstrate that though LdASS has a glycosomal targeting signal that binds the targeting apparatus in vitro, only a small proportion of the total cellular ASS is localized in a vesicle, as indicated by protection from protease digestion of the crude organelle fraction. The majority of LdASS was found to be in the cytosolic fraction that may include large cytosolic complexes as indicated by the punctate distribution in IFA. Surprisingly, comparison to known glycosomal proteins by IFA revealed that LdASS was located in a structure different from the known glycosomal vesicles. Significantly, parasites expressing a mutant form of LdASS associated with a loss of in vitro activity had reduced virulence in vivo in BALB/c mice as demonstrated by a significant reduction in the parasite load in spleen and liver. CONCLUSION/SIGNIFICANCE: Our study suggests that LdASS is an active enzyme, with unique localization and essential for parasite survival and growth in the mammalian host. Based on these observations LdASS could be further explored as a potential drug target.

Mitochondrial associated ubiquitin fold modifier-1 mediated protein conjugation in Leishmania donovani.

In this report, we demonstrate the existence of the ubiquitin fold modifier-1 (Ufm1) and its conjugation pathway in trypanosomatid parasite Leishmania donovani. LdUfm1 is activated by E1-like enzyme LdUba5. LdUfc1 (E2) specifically interacted with LdUfm1 and LdUba5 to conjugate LdUfm1 to proteinaceous targets. Mass spectrometry analysis revealed that LdUfm1 is conjugated to Leishmania protein targets that are associated with mitochondria. Immunofluorescence experiments showed that Leishmania Ufm1, Uba5 and Ufc1 are associated with the mitochondria. The demonstration that all the components of this system as well as the substrates are associated with mitochondrion suggests it may have physiological roles not yet described in any other organism. Overexpression of a non-conjugatable form of LdUfm1 and an active site mutant of LdUba5 resulted in reduced survival of Leishmania in the macrophage. Since mitochondrial activities are developmentally regulated in the life cycle of trypanosomatids, Ufm1 mediated modifications of mitochondrial proteins may be important in such regulation. Thus, Ufm1 conjugation pathway in Leishmania could be explored as a potential drug target in the control of Leishmaniasis.

Overexpression of histone H2A modulates drug susceptibility in Leishmania parasites.

Resistance to antimonials has emerged as a major hurdle to the treatment and control of visceral leishmaniasis (VL), also know as kala-azar (KA), the disease caused by Leishmania donovani, in India where >60% of KA patients are unresponsive to sodium antimony gluconate (SAG) treatment. Determinants of resistance in laboratory strains are partly known, however the mechanism operating in field isolates is not well understood. In microarray-based expression profiling with RNA isolated from field isolates of drug-resistant and -sensitive L. donovani parasites, genes encoding histone 1 (H1), histone 2A (H2A), histone 4 (H4), mitogen-activated protein kinase 1 (MAPK1) and two hypothetical proteins showed significantly higher expression in antimony-resistant parasites, whilst genes encoding an amino acid transporter showed higher expression in sensitive parasites. The expression level of these genes was validated by semiquantitative polymerase chain reaction (PCR). Furthermore, the higher expression of H1, H2A and MAPK1 was confirmed at the protein level in resistant isolates. Overexpression of H2A in a drug-sensitive laboratory strain as well as a field isolate of L. donovani resulted in conversion of SAG-sensitive Leishmania parasites into a resistant phenotype. Moreover, H2A overexpression resulted in a significant decrease in susceptibility towards other antileishmanial drugs currently in use, i.e. amphotericin B and miltefosine, pointing to its role in drug resistance.

HYAL-1 hyaluronidase: a potential prognostic indicator for progression to muscle invasion and recurrence in bladder cancer.

BACKGROUND: For bladder cancer (BCa) patients undergoing bladder-sparing treatments, molecular markers may aid in accurately predicting progression to muscle invasion and recurrence. Hyaluronic acid (HA) is a glycosaminoglycan that promotes tumor metastasis. Hyaluronoglucosaminidase 1 (HYAL-1)-type hyaluronidase (HAase) promotes tumor growth, invasion, and angiogenesis. Urinary HA and HAase levels are diagnostic markers for BCa. OBJECTIVE: We evaluated whether HA and HYAL-1 can predict progression to muscle invasion and recurrence among patients with non-muscle-invasive BCa. DESIGN, SETTING, AND PARTICIPANTS: : Based on tissue availability, tissue microarrays were prepared from a cohort of 178 BCa specimens (144 non-muscle invasive, 34 muscle invasive). Follow-up information was available on 111 patients with non-muscle-invasive BCa (mean follow-up: 69.5 mo); 58 patients recurred and 25 progressed to muscle invasion (mean time to progress: 22.3 mo). MEASUREMENTS: HA and HYAL-1 expression was evaluated by immunohistochemistry and graded for intensity and area of staining. Association of HA and HYAL-1 staining with BCa recurrence and muscle invasion was evaluated by univariate and multivariate models. RESULTS AND LIMITATIONS: HA and HYAL-1 expression correlated with tumor grade, stage, and multifocality (p<0.05). In non-muscle-invasive BCa specimens, HYAL-1 staining was higher (234.3+/-52.2; 200.6+/-61.4) if patients experienced progression to muscle invasion or recurrence when compared with no progression or recurrence (164.1+/-48.2; 172.1+/-57; p<0.001). HA staining correlated with muscle invasion (p<0.001). In univariate analysis, age (p=0.014), multifocality (p=0.023), and HYAL-1 staining (p<0.001) correlated with muscle invasion, whereas only HYAL-1 correlated with recurrence (p=0.013). In multivariate analysis, HYAL-1 significantly associated with muscle invasion (p<0.001; 76.8% accuracy) and recurrence (p=0.01; 67.8% accuracy). CONCLUSIONS: HYAL-1 is a potential prognostic marker for predicting progression to muscle invasion and recurrence.