Camila Megale de Almeida-Leite

Virulence in Murine Model Shows the Existence of Two Distinct Populations of Brazilian Vaccinia virus Strains

Brazilian Vaccinia virus had been isolated from sentinel mice, rodents and recently from humans, cows and calves during outbreaks on dairy farms in several rural areas in Brazil, leading to high economic and social impact. Some phylogenetic studies have demonstrated the existence of two different populations of Brazilian Vaccinia virus strains circulating in nature, but little is known about their biological characteristics. Therefore, our goal was to study the virulence pattern of seven Brazilian Vaccinia virus strains. Infected BALB/c mice were monitored for morbidity, mortality and viral replication in organs as trachea, lungs, heart, kidneys, liver, brain and spleen. Based on the virulence potential, the Brazilian Vaccinia virus strains were grouped into two groups. One group contained GP1V, VBH, SAV and BAV which caused disease and death in infected mice and the second one included ARAV, GP2V and PSTV which did not cause any clinical signals or death in infected BALB/c mice. The subdivision of Brazilian Vaccinia virus strains into two groups is in agreement with previous genetic studies. Those data reinforce the existence of different populations circulating in Brazil regarding the genetic and virulence characteristics.

Nitric oxide synthase expression correlates with death in an experimental mouse model of dengue with CNS involvement

BackgroundThe clinical presentation of dengue is classified by the World Health Organization into dengue without warning signs, dengue with warning signs and severe dengue. Reports of neurological disease caused by Dengue virus (DENV) are becoming frequent, with symptoms that include reduced consciousness, severe headache, neck stiffness, focal neurological signs, tense fontanelle and convulsions. However, the immune mechanisms involved in neurovirulence remain poorly understood. Here we present a mouse model in which one genotype of DENV is inoculated by the intracranial route and infects C57/BL6 mice and replicates in the brain, causing death of mice.MethodsMice were infected with different serotypes/genotypes of DENV by the intracranial route to evaluate viral replication, host cytokine and nitric oxide synthase 2 (Nos2) expression in the brain via real-time PCR. Histological analysis of the brain tissues was also performed. An analysis of which cells were responsible for the expression of cytokines and Nos2 was performed using flow cytometry. Survival curves of infected animals were also generatedResultsDENV 3 genotype I infected mice and replicated in the brain, causing death in our murine model. The increased levels of NOS2 could be the cause of the death of infected mice, as viral replication correlates with increased Nos2 and cytokine expression in the brain of C57BL/6 mice. In Nos2−/− mice that were infected with DENV, no clinical signs of infection were observed and cytokines were expressed at low levels, with the exception of interferon gamma (Ifng). Additionally, the Ifng−/− mice infected with DENV exhibited a severe and lethal disease, similar to the disease observed in C57BL/6 mice, while the DENV- infected Nos2−/− mice did not display increased mortality. Analyses of the brains from infected C57BL/6 mice revealed neuronal degeneration and necrosis during histopathologic examination. IFNg and NOS2 were produced in the brains of infected mice by CD4+ T cells and macrophages, respectively.ConclusionThe neurovirulence of DENV 3 genotype I is associated with a deleterious role of NOS2 in the brain, confirming this murine model as an appropriate tool to study DENV neurovirulence.

Enteric Neuronal Damage, Intramuscular Denervation and Smooth Muscle Phenotype Changes as Mechanisms of Chagasic Megacolon: Evidence from a Long-Term Murine Model of Tripanosoma cruzi Infection

We developed a novel murine model of long-term infection with Trypanosoma cruzi with the aim to elucidate the pathogenesis of megacolon and the associated adaptive and neuromuscular intestinal disorders. Our intent was to produce a chronic stage of the disease since the early treatment should avoid 100% mortality of untreated animals at acute phase. Treatment allowed animals to be kept infected and alive in order to develop the chronic phase of infection with low parasitism as in human disease. A group of Swiss mice was infected with the Y strain of T. cruzi. At the 11th day after infection, a sub-group was euthanized (acute-phase group) and another sub-group was treated with benznidazole and euthanized 15 months after infection (chronic-phase group). Whole colon samples were harvested and used for studying the histopathology of the intestinal smooth muscle and the plasticity of the enteric nerves. In the acute phase, all animals presented inflammatory lesions associated with intense and diffuse parasitism of the muscular and submucosa layers, which were enlarged when compared with the controls. The occurrence of intense degenerative inflammatory changes and increased reticular fibers suggests inflammatory-induced necrosis of muscle cells. In the chronic phase, parasitism was insignificant; however, the architecture of Aüerbach plexuses was focally affected in the inflamed areas, and a significant decrease in the number of neurons and in the density of intramuscular nerve bundles was detected. Other changes observed included increased thickness of the colon wall, diffuse muscle cell hypertrophy, and increased collagen deposition, indicating early fibrosis in the damaged areas. Mast cell count significantly increased in the muscular layers. We propose a model for studying the long-term (15 months) pathogenesis of Chagasic megacolon in mice that mimics the human disease, which persists for several years and has not been fully elucidated. We hypothesize that the long-term inflammatory process mediates neuronal damage and intramuscular and intramural denervation, leading to phenotypic changes in smooth muscle cells associated with fibrosis. These long-term structural changes may represent the basic mechanism for the formation of the Chagasic megacolon.

Demyelination/remyelination and expression of interleukin-1β, substance P, nerve growth factor, and glial-derived neurotrophic factor during trigeminal neuropathic pain in rats.

The etiology of trigeminal neuropathic pain is not clear, but there is evidence that demyelination, expression of cytokines, neuropeptides, and neurotrophic factors are crucial contributors. In order to elucidate mechanisms underlying trigeminal neuropathic pain, we evaluated the time course of morphological changes in myelinated and unmyelinated trigeminal nerve fibers, expression of cytokine IL-1β, neuropeptide substance P (SP), nerve growth factor (NGF), and glial derived neurotrophic factor (GDNF) in peripheral and ganglion tissues, using a rat model of trigeminal neuropathic pain. Chronic constriction injury (CCI) of the infraorbital nerve (IoN), or a sham surgery, was performed. Mechanical allodynia was evaluated from day 3 to day 15 post-surgery. Trigeminal nerves were divided into 2 sections - distal to CCI and ganglion - for morphological analyses, immunohistochemistry (IL-1β, SP), and protein quantification by ELISA (NGF, GDNF). At early postoperative time points, decreased mechanical responses were observed, which were associated with demyelination, glial cell proliferation, increased immunoexpression of IL-1 β and SP, and impaired GDNF production. In the late postoperative period, mechanical allodynia was present with partial recovery of myelination, glial cell proliferation, and increased immunoreactivity of IL-1β and SP. Our data show that demyelination/remyelination processes are related to the development of pain behavior. IL-1β may have effects both in ganglia and nerves, while SP may be an important mediator at the nerve endings. Additionally, low levels of GDNF may produce impaired signaling, which may be involved in generation of pain.

A-type inclusion bodies: a factor influencing cowpox virus lesion pathogenesis

The family Poxviridae comprises the most complex animal DNA viruses. During some poxvirus infections, A-type inclusion bodies (ATIs), codified by the ati gene, are produced. Although some studies have compared poxviruses that encode these inclusion bodies with those that do not, the biological function of ATIs is poorly understood. A recombinant ati-deleted cowpox virus was constructed and compared with the wild-type virus in in vitro experiments including electron microscopy and plaque and viral growth assays. No significant differences were observed in vitro. This reinforces the conclusion that the inclusion body is not essential for in vitro viral replication and morphogenesis. Additionally, different lesion progressions in vivo were observed by macroscopic and histological analysis, suggesting that the presence or absence of ATIs could result in different healing dynamics. This is the first time that the role of ATIs during viral replication has been studied based solely on one variable, the presence or absence of ATIs.

Primary culture of glial cells from mouse sympathetic cervical ganglion: a valuable tool for studying glial cell biology.

Central nervous system glial cells as astrocytes and microglia have been investigated in vitro and many intracellular pathways have been clarified upon various stimuli. Peripheral glial cells, however, are not as deeply investigated in vitro despite its importance role in inflammatory and neurodegenerative diseases. Based on our previous experience of culturing neuronal cells, our objective was to standardize and morphologically characterize a primary culture of mouse superior cervical ganglion glial cells in order to obtain a useful tool to study peripheral glial cell biology. Superior cervical ganglia from neonatal C57BL6 mice were enzymatically and mechanically dissociated and cells were plated on diluted Matrigel coated wells in a final concentration of 10,000cells/well. Five to 8 days post plating, glial cell cultures were fixed for morphological and immunocytochemical characterization. Glial cells showed a flat and irregular shape, two or three long cytoplasm processes, and round, oval or long shaped nuclei, with regular outline. Cell proliferation and mitosis were detected both qualitative and quantitatively. Glial cells were able to maintain their phenotype in our culture model including immunoreactivity against glial cell marker GFAP. This is the first description of immunocytochemical characterization of mouse sympathetic cervical ganglion glial cells in primary culture. This work discusses the uses and limitations of our model as a tool to study many aspects of peripheral glial cell biology.

Nitrergic Myenteric Neurons are Spared in Experimental Chagasic Megacolon

Chagas disease is a chronic disorder caused by the Trypanosoma cruzi protozoan. The infection causes alterations to the enteric nervous system such as megaesophagus and megacolon. There is evidence of denervation of myenteric ganglia. The intense parasitism of acute phase affects neuronal integrity but contrasts with the absence of parasites and the discreet inflammatory process of chronic phase, indicating a progressive injury mechanism that needs to be better understood in the megacolons. The potential selectivity of enteric neurons classes affected by the progression of the disease is not yet clear. Nitrergic neurons which co-localize other neurotransmitters represent the most common inhibitory neuron of the ENS. Recently a chronic stage of the Chagas disease was reproduced experimentally in a suitable murine model of megacolon. Considering the limitation of studying human intestine and the controversy on the pattern of nNOS involvement in chagasic megacolon, we decided to assess the nitrergic neurons in the myenteric plexus of mice. We used antibodies against structural protein gene product 9.5 (PGP 9.5) and functional neuronal nitric oxide synthase (n-NOS) at the acute and chronic phase of the disease to quantify myenteric ganglionar neurons in the colon of infected and non-infected mice. We found a reduction in the ganglionar number of neurons detected by anti-protein gene product 9.5 antibodies in colon from mice at the chronic stage. However, the number of nitrergic neurons per ganglia remained unchanged along the acute to phase chronic of the disease. Our findings indicate a long-term preservation of nitregic neurons detrimental to other classes of enteric in our model of experimental Chagas disease. We propose that differential loss of enteric neurons is at least one of the structural substrate for the development of the longterm morphfunctional changes that lead to the megacolon.

Sympathetic glial cells and macrophages develop different responses to Trypanosoma cruzi infection or lipopolysaccharide stimulation

Nitric oxide (NO) participates in neuronal lesions in the digestive form of Chagas disease and the proximity of parasitised glial cells and neurons in damaged myenteric ganglia is a frequent finding. Glial cells have crucial roles in many neuropathological situations and are potential sources of NO. Here, we investigate peripheral glial cell response to Trypanosoma cruzi infection to clarify the role of these cells in the neuronal lesion pathogenesis of Chagas disease. We used primary glial cell cultures from superior cervical ganglion to investigate cell activation and NO production after T. cruzi infection or lipopolysaccharide (LPS) exposure in comparison to peritoneal macrophages. T. cruzi infection was greater in glial cells, despite similar levels of NO production in both cell types. Glial cells responded similarly to T. cruzi and LPS, but were less responsive to LPS than macrophages were. Our observations contribute to the understanding of Chagas disease pathogenesis, as based on the high susceptibility of autonomic glial cells to T. cruzi infection with subsequent NO production. Moreover, our findings will facilitate future research into the immune responses and activation mechanisms of peripheral glial cells, which are important for understanding the paradoxical responses of this cell type in neuronal lesions and neuroprotection.

Effectiveness of Sequential Viscosupplementation in Temporomandibular Joint Internal Derangements and Symptomatology: A Case Series

Viscosupplementation is a minimally invasive technique that replaces synovial fluid by intra-articular injection of hyaluronic acid (HA). Although effective in some joints, there is not conclusive evidence regarding temporomandibular disorders. This case series described the efficacy of a viscosupplementation protocol in intra-articular temporomandibular disorders. Ten patients with a diagnosis of disc displacement and/or osteoarthritis by Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD) were submitted to four monthly injections of low or medium molecular weight HA. Pain, mandibular function, image analysis by tomography and magnetic resonance, and quality of life were assessed at baseline and follow-ups (1 and 6 months). Pain, jaw range-of-motion, mandibular function, and quality of life improved at follow-up evaluations. Osteoarthritis changes decreased, and 20% of patients improved mandibular head excursion after treatment. Resolution of effusion and improvement in disc morphology were observed for most patients. This viscosupplementation protocol reduced pain and symptoms associated with internal derangement of temporomandibular joint, improved quality of life, and showed benefits from both low and medium molecular weight HA in alternate cycles.

Sequential infiltration of sodium hyaluronate in the temporomandibular joint with different molecular weights. Case report

BACKGROUND AND OBJECTIVES: Temporomandibular disorders are diseases causing pain and dysfunction in joints and muscles controlling mandibular movements. Their etiology is multifactorial and multidisciplinary approaches are needed to reach a differential diagnosis and an adequate management plan. This case report proposes a management protocol, with monthly sodium hyaluronate infiltrations, with different molecular weights, to control such changes and promote improvement of temporomandibular joint biomechanics and pain. CASE REPORT: This study describes a case of a 48-year old patient with a 10-year history of temporomandibular pain with function loss since 2001. Patient has classified her pain as 9 according to analog visual scale. In addition, history and detailed physical evaluation have shown different signs and symptoms, such as localized pain (right side), and right temporomandibular joint arthralgia with noise. Diagnosis was disc displacement with reduction and possible synovitis/ capsulitis to the right. Right temporomandibular joint osteoarthritis was also diagnosed by cone beam CT-scan. Initially, a flat upper splint with total coverage and contact with all antagonist teeth was used. In the attempt to decrease temporomandibular arthralgia, non-steroid anti-inflammatory drugs and muscle relaxants were used. Since right temporomandibular joint pain was not effectively managed, we decided to use intra-joint sodium hyaluronate injections with different molecular weights, per month, in a total of four applications. At treatment completion, clinical evaluation has shown normal function, no pain with visual analog scale = zero, in addition to adequate interincisal distance. CONCLUSION: This report has suggested that viscosupplementation cycles with sodium hyaluronate of different molecular weights may provide excellent results in the long run, to control joint temporomandibular disorder symptoms. Therapeutic benefits were maintained for four years with no need for annual maintenance cycles.