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1.  Effectiveness of Insecticide Spraying and Culling of Dogs on the Incidence of Leishmania infantum Infection in Humans: A Cluster Randomized Trial in Teresina, Brazil 
To evaluate the effect of insecticide spraying for vector control and elimination of infected dogs on the incidence of human infection with L. infantum, a randomized community intervention trial was carried out in the city of Teresina, Brazil.
Methods/Principal Findings
Within each of ten localities in the city, four blocks were selected and randomized to 4 interventions: 1) spraying houses and animal pens with insecticide; 2) eliminating infected dogs; 3) combination of spraying and eliminating dogs, and 4) nothing. The main outcome is the incidence of infection assessed by the conversion of the Montenegro skin test (MST) after 18 months of follow-up in residents aged ≥1 year with no previous history of visceral leishmaniasis (VL). Reactions were measured at 48–72 h, induration of ≥5 mm considered positive. Interventions were executed after the baseline interview and repeated 6 and 12 months later. The effects of each type of intervention scheme on the incidence of infection were assessed by calculating relative risks and 95% confidence intervals using Poisson population-averaged regression models with robust variance. Among the 1105 participants, 408 (37%) were MST positive at baseline. Of the 697 negatives, only 423 (61%) were reexamined at the end of the follow-up; 151 (36%) of them converted to a positive MST. Only dog culling had some statistically significant effect on reducing the incidence of infection, with estimates of effectiveness varying between 27% and 52%, depending on the type of analysis performed.
In light of the continuous spread of VL in Brazil despite the large scale deployment of insecticide spraying and dog culling, the relatively low to moderate effectiveness of dog culling and the non-significant effect of insecticide spraying on the incidence of human infection, we conclude that there is an urgent need for revision of the Brazilian VL control program.
Author Summary
Zoonotic visceral leishmaniasis (VL) constitutes a serious public health problem in the Americas, particularly in Brazil. The disease is caused by the protozoan parasite Leishmania infantum, which is transmitted by the bite of female sand flies, and dogs are the main source of infection. To decrease the risk of transmission, the Brazilian VL control program recommends residual insecticide spraying and environmental management for vector control, and culling of seropositive dogs in areas with moderate to high levels of transmission. Because there is a lack of scientific evidence supporting such interventions, we designed a study to assess the effectiveness of dog culling and residual insecticide spraying in the reduction of incidence of human VL infection. The results show that only dog culling had some statistically significant effect on reducing the incidence of infection, with estimates of effectiveness varying between 27% and 52%. In light of the continuous spread of VL in Brazil despite the large scale deployment of insecticide spraying and dog culling, the relatively low to moderate effectiveness of dog culling and the non-significant effect of insecticide spraying on the incidence of human infection, we conclude that there is an urgent need for revision of the Brazilian VL control program.
PMCID: PMC4214628  PMID: 25357122
2.  A Novel 785-nm Laser Diode-Based System for Standardization of Cell Culture Irradiation 
Photomedicine and Laser Surgery  2013;31(10):466-473.
Objective: The purpose of this study was to develop a novel device that concatenates alignment of infrared lasers and parallel procedure of irradiation. The purpose of this is to seek standardization of in vitro cell irradiation, which allows analysis and credible comparisons between outcomes of different experiments. Background data: Experimental data obtained from infrared laser therapies have been strongly dependent upon the irradiation setup. Although further optical alignment is difficult to achieve, in contact irradiation it usually occurs. Moreover, these methods eventually use laser in a serial procedure, extending the time to irradiate experimental samples. Methods: A LASERTable (LT) device was designed to provide similar infrared laser irradiation in 12 wells of a 24 well test plate. It irradiated each well by expanding the laser beam until it covers the well bottom, as occurs with unexpanded irradiation. To evaluate the effectiveness of this device, the spatial distribution of radiation was measured, and the heating of plain culture medium was monitored during the LT operation. The irradiation of LT (up to 25 J/cm2 – 20 mW/cm2; 1.250 sec) was assessed on odontoblast-like cells adhered to the bottom of wells containing 1 mL of plain culture medium. Cell morphology and metabolism were also evaluated. Results: Irradiation with LT presented a Gaussian-like profile when the culture medium was not heated >1°C. It was also observed that the LT made it 10 times faster to perform the experiment than did serial laser irradiation. In addition, the data of this study revealed that the odontoblast-like cells exposed to low-level laser therapy (LLLT) using the LT presented higher metabolism and normal morphology. Conclusions: The experimental LASERTable assessed in this study provided parameters for standardization of infrared cell irradiation, minimizing the time spent to irradiate all samples. Therefore, this device is a helpful tool that can be effectively used to evaluate experimental LLLT protocols.
PMCID: PMC3796329  PMID: 24102164
3.  Effects of Soft Denture Liners on L929 Fibroblasts, HaCaT Keratinocytes, and RAW 264.7 Macrophages 
BioMed Research International  2014;2014:840613.
The effects of six soft liners (Ufi Gel P (UG), Sofreliner S (SR), Durabase Soft (D), Trusoft (T), Coe Comfort (CC), and Softone (ST)) on L929, HaCat, and RAW 264.7 cells were investigated. Eluates (24 and 48 h) from the materials were applied on the cells and the viability, type of cell death, and morphology were evaluated. Cells were also seeded on the specimens' surfaces (direct contact) and incubated (24 or 48 h), and viability was analyzed. Controls were cells in culture medium without eluates or specimens. For cell viability, no significant differences were found among materials or between extraction periods, and the liners were noncytotoxic or slightly cytotoxic. Morphology of RAW 264.7 cells was altered by the 24 h eluates from CC and D and the 48 h eluates from SR, CC, and D. The 24 and 48 h eluates from all materials (except T) increased the percentages of L929 necrotic cells. For direct contact tests, the lowest cytotoxicity was observed for UG and SR. Although eluates did not reduce viability, morphology alterations and increase in necrosis were seen. Moreover, in the direct contact, effects on viability were more pronounced, particularly for D, T, CC and ST. Thus, the use of UG and SR might reduce the risk of adverse effects.
PMCID: PMC4177820  PMID: 25295276
4.  A case of conventional treatment failure in visceral leishmaniasis: leukocyte distribution and cytokine expression in splenic compartments 
BMC Infectious Diseases  2014;14(1):491.
In this paper we study the distribution of leukocyte populations and of cytokine-producing cells in the spleen of a patient with visceral leishmaniasis resistant to clinical treatment. It is the first attempt to compare the distribution of leukocyte populations and cytokine-producing cells in the splenic compartments of a patient with visceral leishmaniasis with those observed in patients without the disease.
Case presentation
A 25-year-old male, farmer, was hospitalized on several occasions with diagnosis of visceral leishmaniasis and received all recommended treatments for the disease with only transient improvement followed by relapse. He was eventually subjected to splenectomy in order to control the effects of hypersplenism and to potentially overcome infection. After surgery and combined chemotherapy, the disease evolved to cure. In comparison with the spleens of the other two patients without visceral leishmaniasis, an increase was observed in the CD4/CD8 ratio and in the number of IL-10- and FoxP3-producing cells, while the number of IL-17-producing cells was lower in the spleen of the patient with visceral leishmaniasis.
This report confirms previous data on changes in the CD4/CD8 ratio in the spleens of patients with visceral leishmaniasis. Additionally the data presented herein suggests that splenic FoxP3- and IL-17-producing cells are involved in the chronicity of visceral leishmaniasis.
PMCID: PMC4175220  PMID: 25200768
Visceral leishmaniasis; Spleen; Cytokines; Leukocyte populations; Leishmania infantum; Leishmania chagasi
5.  Apolipoprotein E knockout mice have accentuated malnutrition with mucosal disruption and blunted insulin-like growth factor I responses to refeeding☆ 
Apolipoprotein E (apoE) is synthesized mainly in the liver and in the brain and is critical for cholesterol metabolism and recovery from brain injury. However, although apoE mRNA increases at birth, during suckling, and after fasting in rat liver, little is known about its role in early postnatal development. Using an established postnatal malnutrition model and apoE knock-out (ko) mice, we examined the role of apoE in intestinal adaptation responses to early postnatal malnutrition. Wild-type and apoE-ko mice were separated from their lactating dams for defined periods each day (4 hours on day 1, 8 hours on day 2, and 12 hours thereafter). We found significant growth deficits, as measured by weight gain or tail length, in the apoE-ko mice submitted to a malnutrition challenge, as compared with malnourished wild type, especially during the second week of postnatal development (P < .05). In addition, apoE-ko animals failed to show growth catch-up after refeeding, compared with wild-type malnourished controls. Furthermore, we found shorter crypts and reduced villus height and area in the apoE-ko malnourished mice, compared with controls, after refeeding. Insulinlike growth factor 1 expression was also blunted in the ileum in apoE-ko mice after refeeding, compared with wild-type controls, which exhibited full insulinlike growth factor 1 expression along the intestinal crypts, villi, and in the muscular layer. Taken together, these findings suggest the importance of apoE in coping with a malnutrition challenge and during the intestinal adaptation after refeeding.
PMCID: PMC4157828  PMID: 25210213
ApoE; Malnutrition; IGF-1; Small intestine; Growing mice
6.  Lipoprotein Lipase and PPAR Alpha Gene Polymorphisms, Increased Very-Low-Density Lipoprotein Levels, and Decreased High-Density Lipoprotein Levels as Risk Markers for the Development of Visceral Leishmaniasis by Leishmania infantum 
Mediators of Inflammation  2014;2014:230129.
In visceral leishmaniasis (VL) endemic areas, a minority of infected individuals progress to disease since most of them develop protective immunity. Therefore, we investigated the risk markers of VL within nonimmune sector. Analyzing infected symptomatic and, asymptomatic, and noninfected individuals, VL patients presented with reduced high-density lipoprotein cholesterol (HDL-C), elevated triacylglycerol (TAG), and elevated very-low-density lipoprotein cholesterol (VLDL-C) levels. A polymorphism analysis of the lipoprotein lipase (LPL) gene using HindIII restriction digestion (N = 156 samples) (H+ = the presence and H− = the absence of mutation) revealed an increased adjusted odds ratio (OR) of VL versus noninfected individuals when the H+/H+ was compared with the H−/H− genotype (OR = 21.3; 95% CI = 2.32–3335.3; P = 0.003). The H+/H+ genotype and the H+ allele were associated with elevated VLDL-C and TAG levels (P < 0.05) and reduced HDL-C levels (P < 0.05). An analysis of the L162V polymorphism in the peroxisome proliferator-activated receptor alpha (PPARα) gene (n = 248) revealed an increased adjusted OR when the Leu/Val was compared with the Leu/Leu genotype (OR = 8.77; 95% CI = 1.41–78.70; P = 0.014). High TAG (P = 0.021) and VLDL-C (P = 0.023) levels were associated with susceptibility to VL, whereas low HDL (P = 0.006) levels with resistance to infection. The mutated LPL and the PPARα Leu/Val genotypes may be considered risk markers for the development of VL.
PMCID: PMC4163308  PMID: 25242866
7.  In Vitro and In Vivo Miltefosine Susceptibility of a Leishmania amazonensis Isolate from a Patient with Diffuse Cutaneous Leishmaniasis 
Miltefosine was the first oral compound approved for visceral leishmaniasis chemotherapy, and its efficacy against Leishmania donovani has been well documented. Leishmania amazonensis is the second most prevalent species causing cutaneous leishmaniasis and the main etiological agent of diffuse cutaneous leishmaniasis in Brazil. Driven by the necessity of finding alternative therapeutic strategies for a chronic diffuse cutaneous leishmaniasis patient, we evaluated the susceptibility to miltefosine of the Leishmania amazonensis line isolated from this patient, who had not been previously treated with miltefosine. In vitro tests against promastigotes and intracellular amastigotes showed that this parasite isolate was less susceptible to miltefosine than L. amazonensis type strains. Due to this difference in susceptibility, we evaluated whether genes previously associated with miltefosine resistance were involved. No mutations were found in the miltefosine transporter gene or in the Ros3 or pyridoxal kinase genes. These analyses were conducted in parallel with the characterization of L. amazonensis mutant lines selected for miltefosine resistance using a conventional protocol to select resistance in vitro, i.e., exposure of promastigotes to increasing drug concentrations. In these mutant lines, a single nucleotide mutation G852E was found in the miltefosine transporter gene. In vivo studies were also performed to evaluate the correlation between in vitro susceptibility and in vivo efficacy. Miltefosine was effective in the treatment of BALB/c mice infected with the L. amazonensis type strain and with the diffuse cutaneous leishmaniasis isolate. On the other hand, animals infected with the resistant line bearing the mutated miltefosine transporter gene were completely refractory to miltefosine chemotherapy. These data highlight the difficulties in establishing correlations between in vitro susceptibility determinations and response to chemotherapy in vivo. This study contributed to establish that the miltefosine transporter is essential for drug activity in L. amazonensis and a potential molecular marker of miltefosine unresponsiveness in leishmaniasis patients.
Author Summary
Leishmania amazonensis is the etiological agent of diffuse cutaneous leishmaniasis. The disease is extremely difficult to treat and frequently relapses once the treatment is interrupted. Although not yet approved in Brazil, miltefosine is an attractive alternative for leishmaniasis treatment due to its oral administration and low incidence of side effects. Here, we evaluated the efficacy of miltefosine against a L. amazonensis line that was isolated from a chronic diffuse cutaneous leishmaniasis patient to ascertain whether miltefosine could be considered as a therapeutic option in this case. Parasites isolated from this patient were less susceptible to miltefosine than a reference strain in vitro. The mechanisms underlying this decreased susceptibility were studied in this natural parasite isolate in parallel with mutant strains selected in vitro for miltefosine resistance. A mutation in the gene encoding the miltefosine transporter was identified in the mutants selected in vitro but not in the line isolated from the patient. Notwithstanding the decreased susceptibility in vitro, when used to treat infected mice, miltefosine was equally effective against the isolate from the patient and the type strain, but completely ineffective against the resistant line.
PMCID: PMC4102453  PMID: 25033218
8.  Biological effects of soft denture reline materials on L929 cells in vitro 
Journal of Tissue Engineering  2014;5:2041731414540911.
Soft denture reline materials have been developed to help patients when their oral mucosa is damaged or affected due to ill-fitting dentures or post-implant surgery. Although reports have indicated that these materials leach monomers and other components that do affect their biocompatibility, there is little information on what cell molecules may be implicated in these material/tissue interactions. The biocompatibility of six soft liners (Ufi Gel P, Sofreliner S, Durabase Soft, Trusoft, Softone and Coe Comfort) was evaluated using a mouse fibroblast cell line, L929. Within 2 h of material disc preparation, each of the materials was exposed by direct contact to L929 cells for periods of 24 and 48 h. The effect of this interaction was assessed by alamarBlue assay (for cell survival). The expression of integrin α5β1 and transforming growth factor β1 was also assessed using plate assays such as enzyme-linked immunosorbent assay. Trusoft, Softone and Coe Comfort showed significantly reduced cell survival compared with the other soft lining materials at each incubation period. Furthermore, there were significant differences with these same materials in the expression of both integrin α5β1 and transforming growth factor β1. Soft liner materials may affect cell viability and cellular proteins that have important roles in wound healing and the preservation of cell viability and function in the presence of environmental challenges and stresses.
PMCID: PMC4221910  PMID: 25383166
Soft denture reline material; biocompatibility; fibroblast; integrin α5β1; transforming growth factor β1
9.  Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene 
Memórias do Instituto Oswaldo Cruz  2014;109(3):379-383.
An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.
PMCID: PMC4131795  PMID: 24821056
blood meal analysis; cytochrome B; PCR-RFLP
10.  Long-term survival in multiple myeloma 
Clinical Case Reports  2014;2(5):173-179.
Key Clinical Message
The survival of multiple myeloma patients has improved very significantly over the last decade. Still median overall survival is inferior to 5 years. A small proportion of patients survive longer than 10 years. In this paper we discuss four cases illustrating the nonhomogeneous clinical presentation and evolution of this subset of patients. Surprisingly, these long survivors do not always have deep responses and some require frequent treatments, which include autologous stem cell transplantation and novel drugs. The authors discuss several aspects of these clinical histories, including treatment options, raising hypothesis on their relation with long survivorship which may be important to have in consideration when studying this subject.
PMCID: PMC4302619  PMID: 25614805
Clinical characteristics in multiple myeloma; cytogenetics; multiple myeloma long survivors; toxicities
11.  Inactivation of Matrix-bound MMPs by Cross-linking Agents in Acid Etched Dentin 
Operative dentistry  2013;39(2):152-158.
Published TEM analysis of in vivo resin-dentin bonds shows that in 44 months almost 70% of collagen fibrils from the hybrid layer disappear. Matrix metalloproteinases (MMPs) play an important role in that process and are thought to be the main factor responsible for the solubitization of dentin collagen. Therefore, this study aimed to evaluate the inactivation of matrix-bound MMPs by carbodiimide (EDC) or proanthocyanidin (PA) both cross-linking agents, or the MMP-inhibitor, chlorhexidine (CHX), on acid-etched dentin using a simplified MMP assay method.
Dentin beams (1×1×6mm) were obtained from mid-coronal dentin of sound third molars and randomly divided into 6 groups (G) according to the dentin treatment: G1: Deionized water (control), G2: 0.1M EDC, G3: 0.5M EDC, G4: 0.5M EDC+35% HEMA, G5: 5% Proanthocyanidin (PA) and G6: 2% CHX. The beams were etched for 15s with 37% phosphoric acid, rinsed and then immersed for 60s in one of the treatment solutions. The total MMP activity of dentin was analyzed for 1 h by colorimetric assay (Sensolyte). Data were submitted to Wilcoxon non-parametric test and Mann-Whitney tests (p>0.05).
All experimental cross-linking solutions significantly reduced MMP activity compared to control, except 0.1M EDC (53.6% ±16.1). No difference was observed between cross-linking agents and 2% CHX 0.5M EDC + 35% HEMA (92.3% ±8.0) was similar to 0.5M EDC (89.1% ±6.4), 5% PA (100.8% ±10.9) and 2% CHX (83.4% ±10.9).
Dentin treatment with cross-linking agents is effective to significantly reduce MMP activity. Mixing 0.5M EDC and 35% HEMA did not influence EDC inhibitor potential.
PMCID: PMC3989999  PMID: 23786610
MMPs; collagen; dentin; cross-linker
12.  Serum cytokines associated with severity and complications of kala-azar 
Pathogens and Global Health  2013;107(2):78-87.
Recent clinical data suggest that severe kala-azar (or visceral leishmaniasis) is an exaggerated innate immune response mediated by inflammatory cytokines, leading to a systemic inflammatory syndrome similar to what is observed in malaria, sepsis and other diseases. We tested this hypothesis by measuring serum cytokines in individuals with kala-azar.
We compared patients with severe kala-azar (i.e. hemorrhagic manifestations, n = 38) with patients without evidence of hemorrhage (n = 96). We conducted a detailed clinical and laboratory evaluation, measuring serum IL-1beta, IL-6, IL-8, IL-10, IL-12, interferon-gamma, and TNF-alpha, and markers of disseminated intravascular coagulation (DIC).
Infants had higher levels of inflammatory cytokines, while HIV-infected patients had lower concentrations of IL-10 and interferon-gamma. Higher levels of IL-6, interferon-gamma, and IL-8 were found among deceased patients. IL-8 and interferon-gamma were independently associated with bleeding. Several cytokines were associated with different signs of severe clinical and laboratory manifestations, including DIC. IL-6 was highly positively and independently associated with IL-1beta, IL-8, IL-10, and negatively associated with TNF-alpha. IL-1beta and TNF-alpha were also highly independently associated with disease severity.
In its severe form, kala-azar, a neglected tropical disease, initiates a systemic inflammatory response that leads to DIC and other manifestations. Children may have higher risk of death due to the more intense cytokine release. The data supports the notion that IL-6 is the central cytokine that is associated with lethal disease, but interferon-gamma, IL1beta, IL-8, and TNF-alpha are also involved with disease severity. Inhibition of IL-6 is a potential target of adjuvant therapy for severe or pediatric forms of this disease.
PMCID: PMC4001482  PMID: 23683334
Cytokines; Kala-azar; Leishmania infantum; Disseminated intravascular coagulation; Systemic inflammatory response
13.  Transdentinal cytotoxicity of experimental adhesive systems of different hydrophilicity applied to ethanol-saturated dentin 
The aim of this study was to evaluate the transdentinal cytotoxicity of experimental adhesive systems (EASs) with different hydrophilicity and dentin saturation solutions (ethanol and water) on odontoblast-like cells. One hundred 0.4-mm-thick dentin discs were mounted in in vitro pulp chambers and assigned to 10 groups. Odontoblast-like cells MDPC-23 were seeded onto the pulpal side of the discs, incubated for 48h. The EASs with increasing hydrophilicity (R2, R3, R4 and R5) were applied to the occlusal side of the discs after acid etching and saturation of demineralized dentin with water or ethanol. R0 (water and ethanol- no adhesive) served as controls. After 24h, cell metabolism was evaluated by SDH enzyme production (MTT assay; n=8 discs) and cell morphology was examined by SEM (n=2 discs). The type of cell death was identified by flow cytometry and the degree of monomer conversion (%DC) was determined by infrared spectroscopy (FTIR) after two photoactivation times (10 s or 20 s). Data were analyzed statistically by the Kruskal-Wallis and Mann-Whitney tests (α=0.05). Dentin saturation with ethanol resulted in higher necrotic cell death ratios for R3, R4 and R5 compared with water saturation, although R3 and R4 induced higher SDH production. Photoactivation for 20 s significantly improved the %DC of all EASs compared with 10 s. A significant positive correlation was observed between the degree of hydrophilicity and %DC, for both photoactivation times. In conclusion, except for R2, dentin saturation with ethanol increased the cytotoxicity of EASs, as expressed by the induction of necrotic cell death.
PMCID: PMC3896916  PMID: 23906501
adhesive systems; cytotoxicity; ethanol; dentin; odontoblast-like MDPC-23 cells
14.  Protective Effect of Alpha-Tocopherol Isomer from Vitamin E against the H2O2 Induced Toxicity on Dental Pulp Cells 
BioMed Research International  2014;2014:895049.
The aim of this study was to evaluate the protective effects of different concentrations of vitamin E alpha-tocopherol (α-T) isomer against the toxicity of hydrogen peroxide (H2O2) on dental pulp cells. The cells (MDPC-23) were seeded in 96-well plates for 72 hours, followed by treatment with 1, 3, 5, or 10 mM α-T for 60 minutes. They were then exposed or not to H2O2 for 30 minutes. In positive and negative control groups, the cells were exposed to culture medium with or without H2O2 (0.018%), respectively. Cell viability was evaluated by MTT assay (Kruskal-Wallis and Mann-Whitney tests; α = 5%). Significant reduction of cell viability (58.5%) was observed in positive control compared with the negative control. Cells pretreated with α-T at 1, 3, 5, and 10 mM concentrations and exposed to H2O2 had their viability decreased by 43%, 32%, 25%, and 27.5%, respectively. These values were significantly lower than those observed in the positive control, thereby showing a protective effect of α-T against the H2O2 toxicity. Overall, the vitamin E α-T isomer protected the immortalized MDPC-23 pulp cells against the toxic effects of H2O2. The most effective cell protection was provided by 5 and 10 mM concentrations of α-T.
PMCID: PMC3918697  PMID: 24587995
15.  A Framework for Integration of Heterogeneous Medical Imaging Networks 
Medical imaging is increasing its importance in matters of medical diagnosis and in treatment support. Much is due to computers that have revolutionized medical imaging not only in acquisition process but also in the way it is visualized, stored, exchanged and managed. Picture Archiving and Communication Systems (PACS) is an example of how medical imaging takes advantage of computers. To solve problems of interoperability of PACS and medical imaging equipment, the Digital Imaging and Communications in Medicine (DICOM) standard was defined and widely implemented in current solutions. More recently, the need to exchange medical data between distinct institutions resulted in Integrating the Healthcare Enterprise (IHE) initiative that contains a content profile especially conceived for medical imaging exchange: Cross Enterprise Document Sharing for imaging (XDS-i). Moreover, due to application requirements, many solutions developed private networks to support their services. For instance, some applications support enhanced query and retrieve over DICOM objects metadata.
This paper proposes anintegration framework to medical imaging networks that provides protocols interoperability and data federation services. It is an extensible plugin system that supports standard approaches (DICOM and XDS-I), but is also capable of supporting private protocols. The framework is being used in the Dicoogle Open Source PACS.
PMCID: PMC4181172  PMID: 25279021
Cloud computing; data integration; DICOM; medical imaging; PACS and XDS-I.
16.  Carbohydrate use and reduction in number of balance beam falls: implications for mental and physical fatigue 
Artistic Gymnastics is a sport where athletes are frequently fatigued. One element that might influence this aspect is carbohydrate, an important energy substrate for the muscles and the CNS. Our goal was to investigate the influence of fatigue over artistic gymnastics athlete’s performance and the effects of a carbohydrate supplementation on their performance.
We evaluated 15 athletes divided in 2 groups (control and fatigue) from 12 to 14 years old in two different experimental days. On the first day (water day), they did 5 sets of exercises on the balance beam (experimental protocol) ingesting only water, CG (control group) warmed up before the experimental protocol and FG (fatigue group) did a fatigue circuit, warm up exercises and then the experimental protocol. On the second day (carbohydrate day), we used the same protocol but CG ingested a sugar free flavored juice and FG ingested a 20% concentration maltodextrin solution before the protocol on the balance beam.
We observed a greater number of falls from the balance beam from the FG on the first day (5.40 ± 1.14 FG vs 3.33 ± 1.37 CG; p = 0.024) and a decrease in the number of falls on the second day (2.29 ± 1.25 FG water day vs 5.40 ± 1.14 FG carbohydrate day; p = 0.0013). Carbohydrate solution was able to supply muscle demands and improve the athlete’s focus showed by the reduced number of falls.
PMCID: PMC3733607  PMID: 23875791
Maltodextrin supplementation; Artistic gymnastics; Mental fatigue
17.  Identification of L. infantum chagasi proteins in VL patients' urine: a promising antigen discovery approach of vaccine candidates 
Parasite immunology  2012;34(7):360-371.
Visceral leishmaniasis (VL) is a serious lethal parasitic disease caused by Leishmania donovani in Asia and by Leishmania infantum chagasi in Southern Europe and South America. VL is endemic in 47 countries with an annual incidence estimated to be 500,000 cases. This high incidence is due in part to the lack of an efficacious vaccine. Here, we introduce an innovative approach to directly identify parasite vaccine candidate antigens that are abundantly produced in vivo in humans with VL. We combined RP-HPLC and mass spectrometry and categorized three L. infantum chagasi proteins, presumably produced in spleen, liver, and bone marrow lesions and excreted in the patients’ urine. Specifically, these proteins were the following: Li-isd1 (XP_001467866.1), Li-txn1 (XP_001466642.1), and Li-ntf2 (XP_001463738.1). Initial vaccine validation studies were performed with the rLi-ntf2 protein produced in E. coli mixed with the adjuvant BpMPLA-SE. This formulation stimulated potent Th1 response in BALB/c mice. Compared to control animals, mice immunized with Li-ntf2 + BpMPLASE had a marked parasite burden reduction in spleens at 40 days post-challenge with virulent L. infantum chagasi. These results strongly support the proposed antigen discovery strategy of vaccine candidates to kala-azar and opens novel possibilities for vaccine development to other serious infectious diseases.
PMCID: PMC3400926  PMID: 22443237
Leishmania infantum chagasi; vaccine; ntf2; kala-azar; visceral leishmaniasis; urine
18.  Definition of a core set of quality indicators for the assessment of HIV/AIDS clinical care: a systematic review 
Several organizations and individual authors have been proposing quality indicators for the assessment of clinical care in HIV/AIDS patients. Nevertheless, the definition of a consensual core set of indicators remains controversial and its practical use is largely limited. This study aims not only to identify and characterize these indicators through a systematic literature review but also to propose a parsimonious model based on those most used.
MEDLINE, SCOPUS, Cochrane databases and ISI Web of Knowledge, as well as official websites of organizations dealing with HIV/AIDS care, were searched for articles and information proposing HIV/AIDS clinical care quality indicators. The ones that are on patient’s perspective and based on services set were excluded. Data extraction, using a predefined data sheet based on Cochrane recommendations, was done by one of the authors while a second author rechecked the extracted data for any inconsistency.
A total of 360 articles were identified in our search query but only 12 of them met the inclusion criteria. We also identified one relevant site. Overall, we identified 65 quality indicators for HIV/AIDS clinical care distributed as following: outcome (n=15) and process-related (n=50) indicators; generic (n=36) and HIV/AIDS disease-specific (n=29) indicators; baseline examinations (n=19), screening (n=9), immunization (n=4), prophylaxis (n=5), HIV monitoring (n=16), and therapy (=12) indicators.
There are several studies that set up HIV clinical care indicators, with only a part of them useful to assess the HIV clinical care. More importantly, HIV/AIDS clinical care indicators need to be valid, reliable and most of all feasible.
PMCID: PMC3735478  PMID: 23809537
Performance Measures; Quality Indicator; Infectious Disease
19.  Familial Transmission of Human T-cell Lymphotrophic Virus: Silent Dissemination of an Emerging but Neglected Infection 
HTLV-1 is a retrovirus that causes lymphoproliferative disorders and inflammatory and degenerative diseases of the central nervous system in humans. The prevalence of this infection is high in parts of Brazil and there is a general lack of public health care programs. As a consequence, official data on the transmission routes of this virus are scarce.
To demonstrate familial aggregation of HTLV infections in the metropolitan region of Belém, Pará, Brazil.
A cross-sectional study involving 85 HTLV carriers treated at an outpatient clinic and other family members. The subjects were tested by ELISA and molecular methods between February 2007 and December 2010.
The prevalence of HTLV was 43.5% (37/85) for families and 25.6% (58/227) for the family members tested (95% CI: 1.33 to 3.79, P = 0.0033). Sexual and vertical transmission was likely in 38.3% (23/60) and 20.4% (29/142) of pairs, respectively (95% CI: 1.25 to 4.69, P = 0.0130). Positivity was 51.3% (20/39) and 14.3% (3/21) in wives and husbands, respectively (95% CI: 0.04 to 0.63, P = 0.0057). By age group, seropositivity was 8.0% (7/88) in subjects <30 years of age and 36.7% (51/139) in those of over 30 years (95% CI: 0.06 to 0.34, P<0.0001). Positivity was 24.1% (7/29) in the children of patients infected with HTLV-2, as against only 5.8% (4/69) of those infected with HTLV-1 (95% CI: 0.05 to 0.72, P = 0.0143).
The results of this study indicate the existence of familial aggregations of HTLV characterized by a higher prevalence of infection among wives and subjects older than 30 years. Horizontal transmission between spouses was more frequent than vertical transmission. The higher rate of infection in children of HTLV-2 carriers suggests an increase in the prevalence of this virus type in the metropolitan region of Belém.
Author Summary
Human T-cell lymphotropic virus (HTLV) has a slow replication rate and infection is characterized by low morbidity and mortality, as well as silent transmission within the population. While rare, HTLV-associated diseases are usually debilitating and life-threatening. The virus is endemic in the state of Pará (Brazil), although there have been no studies of the distribution of the virus within the local population. The results of the present study confirm the existence of familial aggregations of HTLV infection in the metropolitan region of the state capital, Belém. Considerably higher rates of sexual transmission of HTLV from men to women were also demonstrated. Rates of infection were similar for the two virus types, although HTLV-2 appears to be increasing in the population. The frequency of positivity among family members increased in direct proportion to age and was associated with a relatively large proportion of asymptomatic carriers. In addition, widespread ignorance of the virus increases the risk of transmission. The available evidence indicates that significant human suffering is caused by this virus in patients suffering complications, and this is little justification for the lack of intervention on the part of public health authorities, which might impede the ongoing proliferation of this infection in the population.
PMCID: PMC3681619  PMID: 23785534
20.  Dicoogle, a Pacs Featuring Profiled Content Based Image Retrieval 
PLoS ONE  2013;8(5):e61888.
Content-based image retrieval (CBIR) has been heralded as a mechanism to cope with the increasingly larger volumes of information present in medical imaging repositories. However, generic, extensible CBIR frameworks that work natively with Picture Archive and Communication Systems (PACS) are scarce. In this article we propose a methodology for parametric CBIR based on similarity profiles. The architecture and implementation of a profiled CBIR system, based on query by example, atop Dicoogle, an open-source, full-fletched PACS is also presented and discussed. In this solution, CBIR profiles allow the specification of both a distance function to be applied and the feature set that must be present for that function to operate. The presented framework provides the basis for a CBIR expansion mechanism and the solution developed integrates with DICOM based PACS networks where it provides CBIR functionality in a seamless manner.
PMCID: PMC3646026  PMID: 23671578
21.  Identification and Diagnostic Utility of Leishmania infantum Proteins Found in Urine Samples from Patients with Visceral Leishmaniasis 
Despite the clear need to control visceral leishmaniasis (VL), the existing diagnostic tests have serious shortcomings. Here, we introduce an innovative approach to directly identify Leishmania infantum antigens produced in vivo in humans with VL. We combined reverse-phase high-performance liquid chromatography (RP-HPLC) with mass spectrometry and categorized three distinct L. infantum proteins presumably produced in bone marrow/spleen/liver and excreted in the urine of patients with VL. The genes coding for these proteins (L. infantum iron superoxide dismutase, NCBI accession number XP_001467866.1; L. infantum tryparedoxin, NCBI accession number XP_001466642.1; and L. infantum nuclear transport factor 2, NCBI accession number XP_001463738.1) were cloned, and the recombinant molecules were produced in Escherichia coli. Antibodies to these proteins were produced in rabbits and chickens and were used to develop a capture enzyme-linked immunosorbent assay (ELISA) designed to detect these L. infantum antigens in the urine of VL patients. Specificity of the antibodies was confirmed by a Western blot analysis using both recombinant proteins and whole parasite extract. Importantly, a urinary antigen detection assay assembled with pairs of antibodies specific for each of these antigens identified 17 of 19 patients with VL. These results indicate that an improved antigen detection assay based on L. infantum proteins present in the urine of patients with VL may represent an important new strategy for the development of a specific and accurate diagnostic test that has the potential to both distinguish active VL from asymptomatic infection and serve as an important tool to monitor therapy efficacy.
PMCID: PMC3370439  PMID: 22518013
22.  Dicoogle - an Open Source Peer-to-Peer PACS 
Journal of Digital Imaging  2010;24(5):848-856.
Picture Archiving and Communication Systems (PACS) have been widely deployed in healthcare institutions, and they now constitute a normal commodity for practitioners. However, its installation, maintenance, and utilization are still a burden due to their heavy structures, typically supported by centralized computational solutions. In this paper, we present Dicoogle, a PACS archive supported by a document-based indexing system and by peer-to-peer (P2P) protocols. Replacing the traditional database storage (RDBMS) by a documental organization permits gathering and indexing data from file-based repositories, which allows searching the archive through free text queries. As a direct result of this strategy, more information can be extracted from medical imaging repositories, which clearly increases flexibility when compared with current query and retrieval DICOM services. The inclusion of P2P features allows PACS internetworking without the need for a central management framework. Moreover, Dicoogle is easy to install, manage, and use, and it maintains full interoperability with standard DICOM services.
PMCID: PMC3180530  PMID: 20981467
PACS; Digital Imaging and Communications in Medicine (DICOM); Medical imaging; Peer-to-peer; Computer communication networks; Open source; PACS implementation; Information storage and retrieval
23.  Post-Weaning Protein Malnutrition Increases Blood Pressure and Induces Endothelial Dysfunctions in Rats 
PLoS ONE  2012;7(4):e34876.
Malnutrition during critical periods in early life may increase the subsequent risk of hypertension and metabolic diseases in adulthood, but the underlying mechanisms are still unclear. We aimed to evaluate the effects of post-weaning protein malnutrition on blood pressure and vascular reactivity in aortic rings (conductance artery) and isolated-perfused tail arteries (resistance artery) from control (fed with Labina®) and post-weaning protein malnutrition rats (offspring that received a diet with low protein content for three months). Systolic and diastolic blood pressure and heart rate increased in the post-weaning protein malnutrition rats. In the aortic rings, reactivity to phenylephrine (10−10–3.10−4 M) was similar in both groups. Endothelium removal or L-NAME (10−4 M) incubation increased the response to phenylephrine, but the L-NAME effect was greater in the aortic rings from the post-weaning protein malnutrition rats. The protein expression of the endothelial nitric oxide isoform increased in the aortic rings from the post-weaning protein malnutrition rats. Incubation with apocynin (0.3 mM) reduced the response to phenylephrine in both groups, but this effect was higher in the post-weaning protein malnutrition rats, suggesting an increase of superoxide anion release. In the tail artery of the post-weaning protein malnutrition rats, the vascular reactivity to phenylephrine (0.001–300 µg) and the relaxation to acetylcholine (10−10–10−3 M) were increased. Post-weaning protein malnutrition increases blood pressure and induces vascular dysfunction. Although the vascular reactivity in the aortic rings did not change, an increase in superoxide anion and nitric oxide was observed in the post-weaning protein malnutrition rats. However, in the resistance arteries, the increased vascular reactivity may be a potential mechanism underlying the increased blood pressure observed in this model.
PMCID: PMC3329540  PMID: 22529948
24.  Application of Serial Analysis of Gene Expression to the Study of the Gene Expression Profile of Leishmania infantum chagasi Promastigote 
This study describes the application of the LongSAGE methodology to study the gene expression profile in promastigotes of Leishmania infantum chagasi. A tag library was created using the LongSAGE method and consisted of 14,208 tags of 17 bases. Of these, 8,427 (59.3%) were distinct. BLAST research of the 1,645 most abundant tags showed that 12.8% of them identified the coding sequences of genes, while 82% (1,349/1,645) identified one or more genomic sequences that did not correspond with open reading frames. Only 5.2% (84/1,645) of the tags were not aligned to any position in the L. infantum genome. The UTR size of Leishmania and the lack of CATG sites in some transcripts were decisive for the generation of tags in these regions. Additional analysis will allow a better understanding of the expression profile and discovering the key genes in this life cycle.
PMCID: PMC3336188  PMID: 22570533
25.  Who Is a Typical Patient with Visceral Leishmaniasis? Characterizing the Demographic and Nutritional Profile of Patients in Brazil, East Africa, and South Asia 
Drug-dosing recommendations for visceral leishmaniasis (VL) treatment are based on the patients' weight or age. A current lack of demographic and anthropometric data on patients hinders (1) the ability of health providers to properly prepare for patient management, (2) an informed drug procurement for disease control, and (3) the design of clinical trials and development of new drug therapies in the different endemic areas. We present information about the age, gender, weight, and height of 29,570 consecutive VL patients presenting to 20 locations in six geographic endemic regions of Brazil, East Africa, Nepal, and India between 1997 and 2009. Our compilation shows substantial heterogeneity in the types of patients seeking care for VL at the clinics within the different locations. This suggests that drug development, procurement, and perhaps even treatment protocols, such as the use of the potentially teratogenic drug miltefosine, may require distinct strategies in these geographic settings.
PMCID: PMC3062446  PMID: 21460007

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