Planejamento, síntese e avaliação farmacológica de compostos híbridos potencialmente ativos para o tratamento da anemia falciforme

Sickle cell disease (SCD) is a hereditary hemolytic anemia caused by the inheritance of one S hemoglobin gene from each ancestor. Patients with SCD present increased circulating levels of cytokines, including TNF-alpha (TNF-α). Hydroxyurea (HU) is the available therapeutically strategy for treatment; it acts as a source of nitric oxide and benefits patients by increasing the levels of fetal hemoglobin (HbF). Thus, within one research line that aims at finding new drugs, a series of compounds with TNF-α inhibition and nitric oxide donation properties have been synthesized in order to explore possible synergism of actions beneficial in the treatment of the disease. Six compounds were synthesized: five derivatives of organic nitrates and one of sulfonamide. The compounds, (1,3-dioxo-1,3-dihydro-2Hisoindol-2-yl) methyl nitrate (compound I); (1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) ethyl nitrate (compound II); 3-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) benzyl nitrate (compound III);4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl)-N-hydroxybenzenesulfonamide (compound IV); 4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) benzyl nitrate (compound V) and 2-[4-(1,3-dioxo-1,3-dihydro-2H-isoindol-2-yl) phenyl]ethyl nitrate (compound VI), were synthesized using linear synthetic methodology, with excellent overall yields. All compounds showed anti-inflammatory and analgesic effects with a reduction in 43%-65% of ear edema in mice and a reduction of 25%-42% of writhing induced by acetic acid. All compounds showed comparable reductions in the leukocyte infiltration capacity and ability to generate nitric oxide. The aryl compounds (III, IV and V) presented less mutagenic activity compared to compounds I, II and VI according to the salmonella mutagenicity assay (Ames test). Compounds IV and VI showed activity in K562 culture cells, with increases in gamma globin gene expression to levels higher than with hydroxyurea suggesting a potential to increase fetal hemoglobin. This data set characterizes new potentially useful drug candidates for the treatment of symptoms of sickle cell anemia.

Effects of acute normovolemic anemia on hemodynamic parameters and acid-base balance in dogs

The aim of this study was to evaluate the hemodynamic and acid-base status of dogs subjected to acute normovolemic anemia. The dogs (n = 10) were evaluated 15 minutes and 24 hours after induction of anemia (hematocrit below 18) with blood withdrawal and simultaneously replacement of same volume of Ringer's lactate solution and hydroxyethyl starch-based solution in a 2:1 ratio. The cardiac output was measured by Doppler echocardiography and blood pressure by oscillometric device, and posteriorly hemodynamic parameters were calculated. The anemic groups had increase in cardiac index (P <.05) (3.82 ± 1.05 to 5.86 ± 1.49 and 5.81 ± 1.63 L/min m) and decreases (P <.05) in the indices of total peripheral resistance (6797.81 ± 3060.22 to 3220.14 ± 1275.02 and 3887.74 ± 1394.89 dinaseg/cm 5× m2) and oxygen delivery (7942.84 ± 3344.00 to 4021.68 ± 1627.00 and 4430.82 ± 1402.61 mL/min× m 2), respectively. There were no significant changes in pH, but PaO2 and SaO2 values were increased, and PaCO2 reduced in anemic dogs (P <.05). Therefore, acute normovolemic anemia can create significant hemodynamic changes and despite some hemogasometric changes, there were no changes in the acid-base status in dogs. Copyright © 2011 Tatiana Champion et al.

First defection of the equine herpesvirus 1 neuropathogenic variant in Brazil

This report describes the first detection of an equine herpesvirus 1 (EHV-1) neuropathogenic variant (G 2254/D 752) in Brazil from a case of fatal equine herpesvirus myeloencephalopathy (EHM) in a mare. The results of nucleotide sequencing of the EHV-1 ORF30 gene showed that two other Brazilian EHV-1 isolates from EHM cases are representatives of the non-neuropathogenic variant (A 2254/N 752), suggesting that other unidentified factors are probably also involved in the neuropathogenicity of EHV-1 in horses. These findings will contribute to the epidemiological knowledge of EHV-1 infection in Brazil.

Anemia at one year is an independent risk factor of graft survival

Background Post-transplant anemia is multifactorial and highly prevalent. Some studies have associated anemia with mortality and graft failure. The purpose of this study was to assess whether the presence of anemia at 1 year is an independent risk factor of mortality and graft survival. Methods All patients transplanted at a single center who survived at least 1 year after transplantation and showed no graft loss (n = 214) were included. Demographic and clinical data were collected at baseline and at 1 year. Patients were divided into two groups (anemic and nonanemic) based on the presence of anemia (hemoglobin<130 g/l in men and 120 g/l in women). Results Baseline characteristics such as age, gender, type of donor, CKD etiology, rejection, andmismatches were similar in both groups. Creatinine clearance was similar in both anemic and nonanemic groups (69.32 ± 29.8 × 75.69 ± 30.5 ml/mim; P = 0.17). A Kaplan- Meier plot showed significantly poorer death-censored graft survival in the anemic group, P = 0.003. Multivariate analysis revealed that anemic patients had a hazard ratio for the graft loss of 3.85 (95% CI: 1.49-9.96; P = 0.005). Conclusions In this study, anemia at 1 year was independently associated with death-censored graft survival and anemic patients were 3.8-fold more likely to lose the graft. © 2010 Springer Science+Business Media, B.V.

The Effects of Refrigeration Temperature and Storage Time on Apoptotic Markers in Equine Semen

Evaluation of the damage caused by the sperm preservation process is crucial to improving fertilization rates. The objective of this study was to evaluate the effects of refrigeration temperature (5°C and 15°C) and storage time (0, 12, 24, 48, and 72 hours) on apoptotic markers in equine semen. Membrane phosphatidylserine translocation index, caspase activation index, and DNA fragmentation index were analyzed using epifluorescence microscopy. Analysis of variance was used for statistical analysis, and Tukey test was used to compare means. The significance level was set at P < .05. The results demonstrated that for transport duration shorter than 24 hours, semen quality was maintained when stored at either 5°C or 15°C. A storage temperature of 5°C should be used when it is necessary to transport semen for longer than 24 hours. There was a significant decrease in semen quality after 48 hours of refrigeration. © 2013 Elsevier Inc.

Phylogenetic and structural studies of a novel equine papillomavirus identified from aural plaques

Papillomaviruses (PVs) infect a wide range of animal species and show great genetic diversity. To date, excluding equine sarcoids, only three species of PVs were identified associated with lesions in horses: Equus caballus papillomavirus 1 (EcPV1-cutaneous), EcPV2 (genital) and EcPV3 (aural plaques). In this study, we identified a novel equine PV from aural plaques, which we designated EcPV4. Cutaneous samples from horses with lesions that were microscopically diagnosed as aural plaques were subjected to DNA extraction, amplification and sequencing. Rolling circle amplification and inverse PCR with specific primers confirmed the presence of an approximately 8. kb circular genome. The full-length EcPV4 L1 major capsid protein sequence has 1488 nucleotides (495 amino acids). EcPV4 had a sequence identity of only 53.3%, 60.2% and 51.7% when compared with the published sequences for EcPV1, EcPV2 and EcPV3, respectively. A Bayesian phylogenetic analysis indicated that EcPV4 clusters with EcPV2, but not with EcPV1 and EcPV3. Using the current PV classification system that is based on the nucleotide sequence of L1, we could not define the genus of the newly identified virus. Therefore, a structural analysis of the L1 protein was carried out to aid in this classification because EcPV4 cause lesion similar to the lesion caused by EcPV3. A comparison of the superficial loops demonstrated a distinct amino acid conservation pattern between EcPV4/EcPV2 and EcPV4/EcPV3. These results demonstrate the presence of a new equine PV species and that structural studies could be useful in the classification of PVs. © 2012 Elsevier B.V.

Evaluation of Sperm Kinetics and Plasma Membrane Integrity of Frozen Equine Semen in Different Storage Volumes and Freezing Conditions

In the present study, different freezing systems (Styrofoam box and Mini Digitcool ZH 400) and storage volumes (0.5- and 0.25-mL straws) were compared with regard to sperm kinetics and plasma membrane integrity of frozen and thawed semen. For that, three ejaculates from four animals were frozen in Styrofoam box and Mini Digitcool ZH 400 machine. The 0.5-mL straws were thawed at 46°C for 20 seconds, and the 0.25-mL straws were thawed at 46°C for 12 seconds. Statistical analysis was performed using program R of descriptive analysis box plot, followed by analysis of variance using PROC MIXED of SAS 9.1 package. Variances of 5% were considered as different. There was no interaction between the straw sizes and volumes; however, statistical differences were observed between the semen storage volumes. The 0.5-mL straws had higher total motility (%), progressive motility (%), average path velocity (μm/s), straight-line velocity (μm/s), curvilinear velocity (μm/s), and rapid sperm percentage (%) than the 0.25-mL straws. However, plasma membrane integrity analysis did not differ between the two straws. Thus, it is possible to conclude that equine sperm cryopreserved in 0.5-mL straws has better sperm kinetics than when stored in 0.25-mL straws. Additionally, it is possible to conclude that automated systems that enable faster freezing rates result in a seminal quality that is similar to the one obtained by the conventional system using Styrofoam boxes. © 2013 Elsevier Inc.

Effect of Storage Time and Temperature of Equine Epididymis on the Viability, Motion Parameters, and Freezability of Epididymal Sperm

The recovery of sperm from the epididymal cauda may be the last chance to obtain genetic material when sudden death or serious injuries occur in valuable stallions. However, the lack of technical knowledge regarding the storage and transportation of the epididymis often prevents the preservation of the sperm. Therefore, the aim of this study was to compare sperm parameters of sperm obtained immediately after orchiectomy with sperm recovered from epididymal cauda at different times after storage at 5°C and at room temperature (RT). For that, 48 stallions of different breeds were used. In group 1 (control group), eight stallions were used, and the harvest of the epididymal sperm was performed immediately after orchiectomy. In group 2, 40 stallions were used, which were divided into five groups according to the storage time of the epididymis after orchiectomy (6, 12, 18, 24, or 30 hours), making a total of eight stallions per group. One epididymis of each stallion was stored at 5°C, and the contralateral epididymis was stored at RT, both for the same period. The sperm parameters of total motility, progressive motility, progressive linear velocity, curvilinear velocity, percentage of rapid sperm, and plasma membrane integrity were evaluated in all the groups after sperm recovery, resuspension in a sperm freezing diluent, and thawing. In conclusion, the storage of the testis-epididymis complex at 5°C provided better preservation of epididymal sperm than the storage at RT, and regardless of the temperature, the progressive motility is the sperm parameter that is most sensitive to storage time. © 2013 Elsevier Inc.

Mesenchymal stem cell therapy for equine tendinitis

Tendinitis is an important disease that leads to lameness and decreased performance in equine athletes and results in high costs associated with therapy due to a long recovery period and a high rate of recurrence. Although, several treatments for equine tendinitis have been described, few are effective in significantly improving the quality of the extracellular matrix and reducing the rate of recurrence. The use of cell therapy with mesenchymal stem cells (MSCs) derived from various sources has received much attention because of its therapeutic potential for equine tendinitis. In this paper, we review patents on stem cell therapy and the specific use of MSCs for the treatment of equine tendinitis. © 2013 Bentham Science Publishers.

Immunophenotypic, immunocytochemistry, ultrastructural, and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n=64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. © 2013 Wiley Periodicals, Inc.

Effects of Pentoxifylline on Equine Epididymal Sperm

This study evaluated whether pentoxifylline (PTX) present in the flushing extender influenced the function of equine epididymal spermatozoa after recovery and after thawing. For this experiment, 58 testicles from 29 Brazilian Jumping Horses were used. Cauda epididymides of each stallion were separated and flushed with a skim milk extender, with or without 7.18 mM PTX and then subjected to the freezing process. Samples flushed with the extender containing PTX showed a significant increase in total motility, progressive motility, straight line velocity, curvilinear velocity, and percentage of rapid sperm immediately after the recovery of epididymal sperm and after 15 minutes of incubation at 37°C (P < .05). However, the presence of PTX in the flushing extender did not affect the post-thaw motility parameters or plasma membrane integrity (P > .05). The results of this study showed that the PTX present in the flushing extender improved motility parameters of recently recovered epididymal sperm and had no deleterious effects on plasma membrane integrity and freezability of equine epididymal sperm. © 2013 Elsevier Inc. All rights reserved.

Marjolin's ulcer in two horses with hereditary equine regional dermal asthenia

Two Quarter Horse mares with hereditary equine regional dermal asthenia (HERDA) were diagnosed with metastatic squamous cell carcinoma (SCC) associated with chronic nonhealing wounds. The lesions were similar to the development of SCC from chronic nonhealing ulcers, known as Marjolin's ulcers in humans. The horses showed recurrent skin wounds in the saddle and paralumbar regions and were confirmed by molecular techniques as having HERDA. Both horses were maintained as research animals for prolonged periods and received regular veterinary care and wound treatment. Both horses were ultimately euthanized because of their chronic progressive wounds, coupled with declining health. At necropsy, the nonhealing wounds were found to be complicated by infiltrative SCC; both horses had metastasis to lungs. Chronically inflamed, recurrent skin wounds that heal slowly and incompletely as a consequence of HERDA are proposed as a major pathogenetic factor in tumorigenesis. Consistent findings with respect to proliferation index (Ki-67) and mutations of p53 tumor suppressor gene were confirmed by immunohistochemistry in one horse. SCC consistent with Marjolin's ulcer has been previously suggested in association with chronic ulcers or burn scars in horses, but this is the first report of an association with chronic poor healing wounds in HERDA horses. © 2013 Elsevier Inc.

Equine tendonitis therapy using mesenchymal stem cells and platelet concentrates: A randomized controlled trial

Introduction. Tendon injury is a major cause of lameness and decreased performance in athletic equines. Various therapies for tendonitis have been described; however, none of these therapies results in complete tissue regeneration, and the injury recurrence rate is high even after long recovery periods involving rest and physiotherapy. Methods. A lesion was induced with collagenase gel in the superficial digital flexor tendon in the center portion of the metacarpal region of eight equines of mixed breed. After two weeks, the lesions of the animals in the treated and control groups were treated through the intralesional administration of mesenchymal stem cells derived from adipose tissue (adMSCs) suspended in platelet concentrate (PC) and with phosphate buffered saline (PBS), respectively. Serial ultrasound analyses were performed every two weeks. After 16 weeks of therapy, a biopsy was performed for histopathological, immunohistochemical and gene expression (type I collagen (COL1A1), type III collagen (COL3A1), tenascin-C (TNC), tenomodulin (TNMD), and scleraxis (SCX)) analyses. Results: Differences in the ultrasound and histopathological analyses were observed between the groups. Improved results were reported in the group treated with adMSCs suspended in PC. There was no difference in the gene expression levels observed after the different treatments. The main results observed from the histopathological evaluation of the treated group were as follows: a prevention of the progression of the lesion, a greater organization of collagen fibers, and a decreased inflammatory infiltrate. A lack of progression of the lesion area and its percentage was observed in the ultrasound image, and increased blood flow was measured by Power Doppler. Conclusions: The use of adMSCs combined with PC for the therapy of experimentally induced tendonitis prevented the progression of the tendon lesion, as observed in the ultrasound examination, and resulted in a greater organization and decreased inflammation, as observed in the histopathological evaluation. These data demonstrate the therapeutic potential of this therapy for the treatment of equine tendonitis. © 2013 Carvalho et al.; licensee BioMed Central Ltd.

Humoral and cell-mediated immune responses to different doses of attenuated vaccine against avian infectious bronchitis virus

The antibody and cellular immune responses against infectious bronchitis virus (IBV) were evaluated at mucosal sites of chickens after immunization with various doses of an attenuated vaccine at 1 day of age. The correlation of these immune responses with protection of tracheal tissues was evaluated after experimental infection of these birds. Significantly reduced tracheal pathologic effects, measured according to ciliostasis and histology lesions, and reduced viral load were observed only in the full-dose vaccinated group at 5 days post-infection (dpi), while incomplete protection was observed for the subdose vaccinated groups. Moreover, birds of vaccinated groups, especially with full dose, developed higher levels of lachrymal IBV-specific IgG and IgA and increased the expression of cell-mediated immunity (CMI) genes, such as gamma interferon (IFNγ), CD8+ T cell marker, and granzyme homolog A more rapidly. In addition, these humoral and cellular immune responses evaluated at mucosal sites correlated significantly with tracheal protection against homologous IBV challenge in a vaccine dose-dependent manner. The results indicate that IgG, IgA and CD8+ T cell responses developed at mucosal sites after IBV vaccination of day-old chicks, could be taken as good correlates of protection against this virus. © 2013, Mary Ann Liebert, Inc.