Neuroinflammation at single cell level: What is new?

Multiple sclerosis is a chronic and demyelinating disease of the central nervous system (CNS), most prevalent in women, and with an important social and economic cost worldwide. It is triggered by self-reacting lymphocytes that infiltrate the CNS and initiate neuroinflammation. Further, axonal loss and neuronal death takes place, leading to neurodegeneration and brain atrophy. The murine model for studying MS, experimental autoimmune encephalomyelitis (EAE), consists in immunizing mice with myelin-derived epitopes. APCs activate encephalitogenic T CD4 and CD8 lymphocytes that migrate mainly to the spinal cord resulting in neuroinflammation. Most of the knowledge on the pathophysiology and treatment of MS was obtained from EAE experiments, as Th17 cells, anti-alpha4 blocking Abs and the role of microbiota. Conversely, recent technology breakthroughs, such as CyTOF and single-cell RNA-seq, promise to revolutionize our understanding on the mechanisms involved both in MS and EAE. In fact, the importance of specific cellular populations and key molecules in MS/EAE is a constant matter of debate. It is well accepted that both Th1 and Th17 T CD4 lymphocytes play a relevant role in disease initiation after re-activation in situ. What is still under constant investigation, however, is the plasticity of the lymphocyte population, and the individual contribution of both resident and inflammatory cells for the progression or recovery of the disease. Thus, in this review, new findings obtained after single-cell analysis of blood and central nervous system infiltrating cells from MS/EAE and how they have contributed to a better knowledge on the cellular and molecular mechanisms of neuroinflammation are discussed.

Non-muscle myosin II as a predictive factor in head and neck squamous cell carcinoma.

BACKGROUND: The present study attempted to provide information regarding non-muscle myosin II (MII) isoforms immunoreactivity in patients with head and neck squamous cell carcinoma (HNSCC) and analysis of the patients' clinical status after 5 years of monitoring. MATERIAL AND METHODS: A semiquantitative analysis of the immunoreactivity of the MII isoforms was performed in 54 surgical specimens and its correlation with clinical and pathological variables and prognosis was verified. Data were analyzed using chi-square, Mann-Whitney and Kruskal-Wallis tests. To evaluate the survival over the total monitoring time and any connection with the proteins studied, the Kaplan-Meier analysis was used. P values ≤0.05 were considered statistically significant. RESULTS: In the advanced stages of pathological tumor-node-metastasis, the expression of MIIB in adjacent non-neoplastic epithelial tissues tended to increase (p = 0.057). In tumoral zones there was an association of high expression among the three isoforms (MIIA/MIIB p=0,001, MIIB/MIIC p=0,006 and MIIA/MIIC p=0,012). Negative clinical evolution in patients was directly correlated to increased MIIC expression in the tumoral zone of invasion in HNSCC (p = 0.017). Based on clinical evolution after the monitoring period, patients with tumors expressing MIIC had poorer prognoses (p = 0.048). CONCLUSIONS: The present study suggests that MIIB expression in non-neoplastic adjacent epithelial tissues may indicate a potential for regional metastasis and that MIIC expression in the tumoral zone of invasion is predictive of negative evolution of the disease.

Genetic diversity of Campylobacter jejuni and Campylobacter coli isolated from poultry meat products sold on the retail market in Southern Brazil.

Campylobacter is regarded as the most common bacterial cause of gastroenteritis throughout the world and most cases of human campylobacteriosis can be traced back to the consumption of poultry meat. In Brazil, few studies evaluated the genetic relatedness among Campylobacter isolates. The aim of this research was to evaluate the genetic diversity of Campylobacter spp. isolated from poultry meat products sold on the retail market in Southern Brazil. The presumptive identification of Campylobacter was performed using traditional microbiological analysis, followed by molecular confirmation by PCR. The genetic diversity of isolates was analyzed by pulsed-field gel electrophoresis (PFGE). Campylobacter spp. was isolated from 91.7% (33/36) of the samples, totaling 48 isolates. Campylobacter jejuni was the most prevalent species isolated (90.8%). PFGE data revealed 26 pulsotypes and 18 PFGE patterns composed of only 1 isolate. Campylobacter isolates exhibited high genetic diversity; however, some clones were recurrent in the poultry meat products sold on the retail market. As the south region of Brazil is an important producer and exporter of chicken meat, our results highlight the need to control this pathogen in the food chain in this area of the world to reduce the risks of exposing consumers to campylobacteriosis.

Low-level laser therapy improves peri-implant bone formation: resonance frequency, electron microscopy, and stereology findings in a rabbit model.

Previous studies have reported positive effects of low-level laser therapy (LLLT) on bone healing. This study evaluated the effects of LLLT on peri-implant healing in vivo. Thirty-two rabbits had their mandibular left incisors removed, followed by immediate insertion of a dental implant into the fresh socket. Animals were assigned randomly to four groups: control (non-irradiated) or LLLT at three different doses per session: 5J/cm(2), 10J/cm(2), and 20J/cm(2). A GaAlAs laser (830nm, 50mW) was applied every 48h for 13 days, starting immediately after surgery. The implant stability quotient (ISQ) was measured using resonance frequency analysis upon implant insertion and immediately after death, 30 days after the last application. Tissues were prepared for scanning electron microscopy (SEM) and stereology. Variables measured were bone-implant contact (BIC) and bone neoformation within implant threads at three different sites. The results showed better ISQ for the 20J/cm(2) group (P=0.003). BIC values were significantly higher (P<0.05) in the 20J/cm(2) group, on both SEM and stereology. Bone area values were better in the 10J/cm(2) (P=0.036) and 20J/cm(2) (P=0.016) groups compared to the control group. Under these conditions, LLLT enhanced peri-implant bone repair, improving stability, BIC, and bone neoformation. The findings support and suggest parameters for the design of clinical trials using LLLT after implant placement.

Evaluation of anti-nociceptive and anti-inflammatory activity of low-level laser therapy on temporomandibular joint inflammation in rodents.

The aim of this study was to investigate the analgesic and anti-inflammatory activity of low-level laser therapy (LLLT) on the nociceptive behavioral as well as histomorphological aspects induced by injection of formalin and carrageenan into the rat temporomandibular joint. The 2.5% formalin injection (FRG group) induced behavioral responses characterized by rubbing the orofacial region and flinching the head quickly, which were quantified for 45 min. The pretreatment with systemic administration of diclofenac sodium-DFN group (10 mg/kg i.p.) as well as the irradiation with LLLT infrared (LST group, 780 nm, 70 mW, 30 s, 2.1 J, 52.5 J/cm(2), GaAlAs) significantly reduced the formalin-induced nociceptive responses. The 1% carrageenan injection (CRG group) induced inflammatory responses over the time-course of the study (24 h, and 3 and 7 days) characterized by the presence of intense inflammatory infiltrate rich in neutrophils, scanty areas of liquefactive necrosis and intense interstitial edema, extensive hemorrhagic areas, and enlargement of the joint space on the region. The DFN and LST groups showed an intensity of inflammatory response that was significantly lower than in CRG group over the time-course of the study, especially in the LST group, which showed exuberant granulation tissue with intense vascularization, and deposition of newly formed collagen fibers (3 and 7 days). It was concluded that the LLLT presented an anti-nociceptive and anti-inflammatory response on the inflammation induced in the temporomandibular joint of rodents.

Assessment of the systemic effects of low-level laser therapy (LLLT) on thyroid hormone function in a rabbit model.

The aim of this study was to assess the effects of low-level laser therapy (LLLT) applied to a dental extraction socket on thyroid gland function in a rabbit model, based on serum triiodothyronine and thyroxine levels. Sixteen male New Zealand rabbits were randomly distributed into two groups: a control group (non-irradiated animals) and an experimental group (irradiated animals: one irradiation point in the extraction socket of the lower incisor). Animals in the experimental group were irradiated with an aluminium gallium arsenide diode laser (AlGaAs; wavelength 830 nm, 40 mW, CW laser), for 13 days, every 48 h, at a dose of 6 J/cm(2) per session, resulting in a total dose of 42 J/cm(2). Serum triiodothyronine and thyroxine levels were measured in both groups before extraction and on the last day of observation (day 15). There were no statistically significant differences between the groups in pre- and post-irradiation triiodothyronine and thyroxine values. With the irradiation protocol used in this study, LLLT did not affect thyroid function in rabbits as assessed by circulating serum triiodothyronine and thyroxine levels.

Evaluation of nitric oxide (NO) and nitric oxide synthases (NOS) in the amniotic fluid in an experimental gastroschisis rat model.

UNLABELLED: Intestinal damage due to gastroschisis (G), an anomaly found with increasing incidence by pediatric surgeons, is intimately associated with endogenous nitric oxide (NO) production and NO synthase (NOS) expression. AIM: Aim of the study was to evaluate NO production and NOS isoforms in the intestine and amniotic fluid (AF) using a rat model of gastroschisis. METHODS: A gastroschisis rat model was surgically created at 18.5 days of gestation (term=22 days). 3 groups of 12 fetuses each were studied: control (C), sham (S) and (G). Morphometric data of body weight (BW), intestinal weight (IW) and the IW/BW ratio were evaluated and compared. Indirect quantification of NO (nitrite and nitrate - NOx) was analyzed by chemiluminescence, and the expression of the 3 isoforms was analyzed by Western blotting. RESULTS: Group G showed an increase in IW and IW/BW compared with groups C and S. IW: G=0.27 ± 0.06, C=0.20 ± 0.02, S=0.20 ± 0.02 (p<0.01); IW/BW: G=4.11 ± 0.57, C=5.21 ± 1.04, S=5.18 ± 1.23 (p<0.05). NO in the G group was lower in the intestine and higher in AF, as opposed to C and S, where it had increased in the intestine and decreased in AF. Intestinal NOx: G=0.85 ± 0.28, C=1.86 ± 0.82, S=1.80 ± 0.69 (p<0.05); NOx in AF: G=161.87 ± 52.11, C=6.99 ± 5.45, S=48.73 ± 13.183 (p<0.001). CONCLUSION: The intestinal inflammation in gastroschisis promotes the release of nitric oxide to the environment (AF). Perhaps NO in the AF may be an inflammatory marker for G.

Topical S-nitrosoglutathione-releasing hydrogel improves healing of rat ischaemic wounds.

Topical application of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO) is known to exert beneficial effects on wound healing. The aim of this study was to evaluate, for the first time, the effect of topical application of GSNO on the healing of ischaemic wounds. Wistar rats were submitted to two parallels incisions on their backs; the skin was separated from the underlying tissue, the incisions were sutured and an excisional wound was made between the parallel incisions to create an ischaemic condition surrounding the wound. The animals were separated into a control group, which received a hydrogel vehicle without GSNO, and a GSNO-treated group, which received a GSNO-containing hydrogel. The animals were treated for 7 days consecutively with one daily application. The GSNO-treated group displayed higher rates of wound contraction and re-epithelization, lower amounts of inflammatory cells, an increase in collagen fibre density and organization and a decrease in the neovascularization compared to control. These results show that topical application of GSNO is effective in the treatment of ischaemic wounds, leading to a significant improvement in the wound healing. Therefore, topical GSNO-containing hydrogels have potential for the therapeutic treatment of ischaemic diabetic and venous ulcers.

S-nitrosoglutathione-containing hydrogel accelerates rat cutaneous wound repair.

BACKGROUND: Nitric oxide (NO) plays a key role in wound repair and S-nitrosothiols like S-nitrosoglutathione (GSNO) are well known NO donors. METHODS: Animals were separated in two groups and submitted to excisional wounds on the dorsal surface at the first day. GSNO (100 microm)-containing hydrogels were topically applied on the wound bed in the GSNO group, daily, during the first 4 days. Control group was topically treated with hydrogel without GSNO for the same period. Wound contraction and re-epithelialization were measured. Animals were sacrificed 21 days after wounding. Samples of lesion and normal tissue were formalin-fixed, paraffin embedded for histological analysis. RESULTS: Wound contraction, measured 14 and 21 days after wounding, was greater in the GSNO group than in the control group (P<0.05 for both). The re-epithelialized wound area, measured 14 days after wounding, was higher in the GSNO group than in the control group (P<0.05). A higher amount of inflammatory cells was observed in superficial and deep areas of the granulation tissue of the control group compared to the GSNO group. Twenty-one days after wounding, thin red-yellow collagen fibers arranged perpendicularly to the surface were found in the granulation tissue of the control group, whereas in the GSNO-treated group collagen fibers were thicker and arranged parallel to the surface. Increased number of mast cells was observed in the GSNO group compared with that in the control group. Vascularization and myofibroblast distribution were similar in both groups. CONCLUSION: Topical application of GSNO-containing hydrogel during the early phases of rat cutaneous wound repair accelerates wound closure and re-epithelialization and affects granulation tissue organization.

S-nitrosoglutathione-containing hydrogel increases dermal blood flow in streptozotocin-induced diabetic rats.

BACKGROUND: Endothelial dysfunction is characterized by decreased vasodilatory capacity of the arterioles mainly due to the reduced release of nitric oxide (NO). Application of NO donors may prevent or even reverse the consequences of endothelial dysfunction, such as diabetic leg ulcers. OBJECTIVES: To investigate the vasodilatory capacity and the possible side-effects of topical application of an NO donor-containing hydrogel in diabetic rats. METHODS: S-nitrosoglutathione (GSNO) was incorporated in Pluronic F127 hydrogel and applied on the foot sole skin of healthy and streptozotocin-induced diabetic rats. Blood flow was monitored using a laser-Doppler probe. Nitrotyrosine formation, a possible side-effect of GSNO action, was evaluated by Western blotting of skin protein extracts. Systemic circulatory side-effects were investigated by monitoring blood pressure and heart rate during the application. RESULTS: The hydrogel alone did not induce any changes in microvascular flow, while GSNO-containing hydrogel caused a twofold increase in perfusion. This effect was similar in diabetic and healthy animals. Topical GSNO application did not increase the nitrotyrosine content of skin proteins, nor did it have any effect on blood pressure or heart rate. CONCLUSIONS: Dermal application of GSNO may be an effective treatment for promoting the local vasodilation in both healthy and diabetic states, without inducing protein nitration or alterations in blood pressure or heart rate.

Characteristics of hammer stones and anvils used by wild bearded capuchin monkeys (Cebus libidinosus) to crack open palm nuts.

Capuchins living in Boa Vista (Piauì, Brazil) crack open hard palm nuts on hard, level surfaces (anvils) using stones (hammers) as percussive tools. This activity leaves diagnostic physical remains: distinctive shallow depressions (pits) on the surface of the anvil, cracked shells, and stone hammers on the anvil. To initiate comparison of percussive stone tool use and interpretation of the artifacts it produces across capuchins, chimpanzees, and hominins, we describe a sample of the anvils and hammer stones used by capuchin monkeys at our site. Anvils (boulders and logs) were located predominantly in the transition zone between the flat open woodland and ridges, in locations that offered some overhead coverage, and with a tree nearby, but not necessarily near palm trees. Anvils contained shallow, hemispherical pits with smooth interiors. Hammers represent a diverse assemblage of ancient rocks that are much harder than the prevailing sedimentary rock out of which they eroded. Hard stones large enough to serve as hammers were more abundant on the anvils than in the surrounding area, indicating that capuchins transport them to the anvils. Capuchins use hammers weighing on average more than 1 kg, a weight that is equivalent to 25-40% of the average body weight for adult males and females. Our findings indicate that capuchins select stones to use as hammers and transport stones and nuts to anvil sites. Wild capuchins provide a new reference point for interpreting early percussive stone tool use in hominins, and a point of comparison with chimpanzees cracking nuts.

[Medication errors in two Brazilian hospitals]. / Errores de medicación de dos hospitales de Brasil.

OBJECTIVE: To determine medication errors in a public and in a private hospital. METHOD: Cross-sectional. 638 dosis opportunities for errors (administered dosis + omitted dosis) were assessed in January, 2005. Medication error was defined as any given dose different from a legible prescription on patient chart. The error rate was calculated by the following equation: number of dosis/error opportunities. The errors were classified according to the categories: omission, unordered dose, extra-dose, wrong dose, wrong route, wrong form, wrong time. RESULTS: Out of 638 opportunities of error, 209 (32,9%) were wrong in some way. When wrong time errors were excluded, this rate decreased to 156 (25%). The most frequent types of errors were omission and unordered dosis, 67 (10,5%) and 65 (10,2%), respectively. There was no significant difference on the total error rate according to the type of hospital (public or private). CONCLUSIONS: The public hospital showed a double-fold unordered dose error rate as compared to the private hospital. Inversely, the private hospital showed a double-fold wrong time error rate than the public hospital.

Dose evaluation in paediatric radiology and adult bone densitometry examinations.

Dose measurements are acknowledged to be a vital part of the quality assurance process in diagnostic radiology, and the use of thermoluminescence dosemeters (TLDs) is a recommended method of entrance dose evaluation. Measurement of doses in radiographic examinations is widely adopted in clinical practice for adults as well as for children. Phantoms can be used to simulate different parts of the body, depending on the materials used to build them. In this work two different sets of phantoms have been prepared with acrylic blocks. The first set was used to simulate children of different ages. The second set was used to simulate the adult spine. The dosimetric measurements have been carried out using TLD and an ionising chamber. Measurements were performed in three X-ray equipments in Aracaju, Brazil. The entrance, half thickness and exit surface doses were analysed.

Effects of the LactoSorb bioabsorbable plates on the craniofacial development of rabbits: direct morphometric analysis using linear measurements.

The use of plates for the treatment of fractures can affect craniofacial bone development. This study investigated the effect of bioabsorbable plates and titanium microscrews on the growth of the craniofacial skeleton of rabbits (Oryctolagus cuniculus), in the neonatal period. A LactoSorb plate and PROMM titanium microscrews were positioned across the coronal suture in animals in the study group. In the control group, only PROMM titanium microscrews were attached to the cranium. Anteroposterior linear measurements were obtained using 3 different gauging devices: digital precision caliper, EKG caliper and nylon string. Frontal-nasal (FN) distances were statistically different between the left and right side when the digital caliper (P

Antiatherogenic effects of S-nitroso-N-acetylcysteine in hypercholesterolemic LDL receptor knockout mice.

BACKGROUND: The pathophysiology of the NO/NO synthase system and dysfunctional changes in the endothelium in the early phases of the atherogenic process are incompletely understood. In this study, we investigated the effects of the nitrosothiol NO donor S-nitroso-N-acetylcysteine (SNAC) in the early prevention of plaque development in the hypercholesterolemic LDLr-/- mice as well as the changes in endothelium-dependent relaxation and NO synthase expression. METHODS AND RESULTS: LDLr-/- mice were fed a 1.25% cholesterol-enriched diet for 15 days. Plasma cholesterol/triglyceride levels increased and this increase was accompanied by the development of aortic root lesions. Aortic vasorelaxation to acetylcholine was increased, although endothelium-independent relaxation in response to sodium nitroprusside did not change, which suggest stimulated NO release enhanced. This dysfunction was associated with enhanced aortic superoxide production and with increased levels of constitutive NOS isoform expression, particularly neuronal NOS. SNAC (S-nitroso-N-acetylcysteine) administration (0.51 micromol/kg/day i.p. for 15 days) decreased the extent of the plaque by 55% in hypercholesterolemic mice, but had no effects on vasomotor changes. It did, however, lead to a decrease in constitutive NOS expression. The SNAC induced only minor changes in plasma lipid profile. CONCLUSION: The present study has shown that, in early stages of plaque development in LDLr-/- mice, specific changes in NO/NO synthase system develop, that are characterized by increased endothelium-dependent vasorelaxation and increased constitutive NOS expression. Since the development of plaque and the indicator of endothelial cell dysfunction were prevented by SNAC, such treatment may constitute a novel strategy for the halting of progression of early plaque.

Topically applied S-nitrosothiol-containing hydrogels as experimental and pharmacological nitric oxide donors in human skin.

BACKGROUND: Nitric oxide (NO) has a wide range of functions in the skin, and topical NO donors have several potential clinical applications. However, currently available donors are either unstable on the skin surface, release low concentrations of NO, or have a short duration of action. Endogenous S-nitrosothiols (RSNOs) store and transport NO within the body and can be used as exogenous sources of NO. OBJECTIVES: To study in vitro and in vivo the chemical and biological behaviour of two RSNO species, S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC), in an easily applied hydrogel, and to correlate dermal nitrite concentration with erythema following application of the RSNOs. To assess the suitability of GSNO and SNAC as biologically effective NO donors for clinical research and as potential therapeutic agents. METHODS/PATIENTS: GSNO (0.3 mol g(-1)) and SNAC (0.6 mol g(-1)) were incorporated in Synperonic F-127 hydrogels (Uniquema, Belgium). The in vitro kinetics of decomposition were measured by spectrophotometry at 37 degrees C. The RSNO-containing hydrogels were applied to the forearm skin of eight subjects. Blood flow was measured by laser Doppler for 3 h following application of NO donors and dermal nitrite simultaneously measured in microdialysate in four subjects. RESULTS: The mean peak blood flow achieved was 250. At blood flow values of < 250, dermal nitrite correlated closely with blood flow and could be defined by the equation: blood flow = (nitrite concentration x 0.66) + 120, (P = 0.013). At higher blood flows there was a paradoxical fall in dermal nitrite concentration. CONCLUSIONS: Topical RSNOs produce a consistent, sustained and biologically effective release of NO on human skin in vivo, which offers advantages over currently available topical NO donors. Dermal nitrite concentration--the oxidation product of NO--is directly correlated with blood flow at low and moderate levels of blood flow. At high levels of blood flow, there is a reduction in dermal nitrite, which is presumed to be due to increased blood scavenging.

Effect of vitamin E supplementation on antibody levels against malondialdehyde modified LDL in hyperlipidemic hamsters.

OBJECTIVE: The aim of this work was to investigate the effect of vitamin E (VE) supplementation on the formation of autoantibodies against oxidized low density lipoproteins (LDL) in a hyperlipidemic animal model. METHODS: Thirty-four male hamsters (Mesocricetus auratus), (4 weeks old) were divided into three groups: Group A (n=9) was fed with standard rodent chow; group B (n=13) was fed with a standard rodent chow plus 2% cholesterol and 10% butter and group C (n=12) was fed with the same diet plus 0.2% (w/w) VE. Blood samples were collected by intracardiac puncture and antibody levels were determined in each animal at 4 weeks of age and after 20 weeks of experimental diet. A modified ELISA technique was used to analyze the modulation of autoantibody titers against an epitope of oxidized LDL in serum samples. Antigens prepared for the ELISA tests were characterized using spectrofluorimetry. Serum VE levels were determined in the lipidic fractions by HPLC. RESULTS: The groups fed with cholesterol-fat enriched diet presented a three-fold increase in total serum cholesterol and two-fold increase in serum triglycerides compared to the control group. VE supplementation played no role in serum cholesterol and serum triglyceride concentrations but led to a decreased autoantibody (anti-LDL-malondialdehyde) formation (P<0.05). CONCLUSIONS: Our results show that VE supplementation leads to a lower production of autoantibodies against oxidized LDL, suggesting a protective effect of VE against in vivo oxidation of LDL particles, in a dose-dependent manner.

Study of the evolution of the placenta and fetal pancreas in the pathophysiology of growth retardation intrauterine due to restricted maternal diet.

CONTEXT: Intrauterine growth retard (IUGR) continues to be a significant perinatology problem at the end of this century. The nature of the etiologic agent, the time when the attack occurred during pregnancy and its duration affect the type of IUGR. OBJECTIVE: To study the evolution of fetal pancreas and placenta between the 18th and 21st day of pregnancy in rats submitted to maternal protein-calorie restriction. DESIGN: Randomized controlled trial on laboratory animal. SAMPLE: Forty-one normoglycemic pregnant Wistar rats. INTERVENTION: Rats were divided into six experimental groups according to their access to food and date of cesarean section (18th or 21st day): control with free access to food; diet restricted to 25% introduced on 1st day of pregnancy; and diet restricted to 25% after the 3rd day of pregnancy. MAIN MEASUREMENTS: Newborn weight, placenta weight, histopathological study (morphological histochemistry). RESULTS: Maternal protein-calorie malnutrition caused intrauterine growth retard (IUGR) after the 18th day of pregnancy. Dietary restriction did not interfere with the morphology of the fetal pancreas and the immunohistochemical study of the placenta showed that glycogen stores were decreased between the 18th and 21st day in the control group and in a diet restricted to 25% from the first day of pregnancy. Dietary restriction after the 3rd day of pregnancy led to low placental glycogen concentrations on the 18th day and disappearance on the 21st day. CONCLUSION: The pathophysiology of IUGR due to maternal protein-calorie restriction in rats is related to lower placental weight and low placental glycogen stores.