A famÃlia de receptores de potencial transitÃrio (TRP) incluindo o receptor de potencial transitÃrio anquirina, subtipo 1 (TRPA1) tem mostrado ser um alvo terapÃutico potencial para o tratamento da dor aguda e crÃnica. Alguns estudos tÃm demonstrado que a resposta nociceptiva somÃtica se deve à ativaÃÃo dos receptores TRPA1 e sÃo efetivamente modulados atravÃs da ferramenta experimental, HC-030031, um antagonista seletivo. Contudo, existem poucos estudos que avaliam o papel dos receptores TRPA1 na dor visceral. Portanto, investigamos o papel do TRPA1 em modelos animais de nocicepÃÃo visceral induzido por diferentes substÃncias e tambÃm exploramos os possÃveis mecanismos envolvidos. Camundongos Swiss, machos (N=6) receberam carboximetilcelulose 0,5% (veÃculo CMC 0,5%, 1 mL/Kg, v.o.), HC-030031 (75, 150 ou 300 mg/Kg, v.o.), ou L-NAME (10 e 40 mg/Kg, s.c.) ou somente L-Arginina (600 mg/Kg, i.p.), 1 h apÃs foi administrado uma Ãnica injeÃÃo de IFO (400 mg/Kg, i.p.). A nocicepÃÃo visceral foi avaliada atravÃs do teste de Von Frey eletrÃnico previamente (T0) e 12 h (T1) apÃs a injeÃÃo de IFO com estimulaÃÃo abdominal atravÃs de um analgesÃmetro digital. Os resultados foram obtidos em gramas (T0-T1) pela variaÃÃo da hiperalgesia. Em seguida as bexigas dos animais foram removidas para pesagem, anÃlise e foram atribuÃdos escores macro e microscopicamente. Investigou-se, tambÃm, o efeito antinociceptivo visceral do HC-030031 atravÃs do modelo de nocicepÃÃo visceral induzido por Ãleo de mostarda (OM). Os animais foram tratados com CMC 0,5%, HC-030031 (18,75; 37,5 ou 75 mg/kg, v.o.) ou Morfina (5 mg/Kg, s.c.) isoladamente ou receberam Naloxona (2 mg/Kg, i.p.) previamente a estas drogas. Em seguida, OM 0,75% (50 &#956;L/colon) foi instilado localmente no cÃlon. A nocicepÃÃo visceral foi verificada atravÃs do teste de Von Frey previamente (T0) e 10 min. (T1) apÃs a injeÃÃo do OM. Em outro protocolo experimental, os animais foram tratados com CMC 0,5% (10 mL/kg, v.o.) ou HC-030031 (18,75; 37,5 ou 75 mg/Kg, v.o.) previamente a uma injeÃÃo intraperitoneal com Ãcido acÃtico 0,6% (AA, 10 mL/Kg,), zymosan (Zym, 1 mg/cavidade) ou misoprostol (MPT, 1 Âg/cavidade, um anÃlogo estÃvel de prostaglandinas). Imediatamente apÃs a injeÃÃo desses algogÃnicos, contabilizaram-se as contorÃÃes abdominais por 30 min. Adicionalmente, para investigar o papel de cÃlulas peritoneais residentes sobre o efeito do HC-030031, a cavidade peritoneal dos camundongos foi lavada com uma soluÃÃo de 30 mL (PBS + heparina) e os estÃmulos AA, Zym e MPT foram injetados i.p. Um grupo Sham foi incluÃdo tambÃm neste protocolo. Ao final do experimento, as contorÃÃes abominais foram registradas por 30 mim. Utilizou-se para a anÃlise estatÃstica, ANOVA/Student e Newman/Keul, foi considerado significativo um p < 0,05 (CEPA: Protocolo: 92/10). A IFO induziu significativa (p<0,05) nocicepÃÃo visceral (6,25Â1,08) e resposta inflamatÃria [escores edema 2(1-3); hemorragia 3(1-3) e peso bexiga (42,78 3,10)] comparado com o grupo salina (1,97Â0,89),[ 0(0-0); 0(0-0) e 20,01 0,7749] respectivamente. AlÃm disso, HC-030031(75 mg/Kg) e L-NAME (10 e 40 mg/Kg) preveniram de maneira significativa (p<0,05) da resposta nociceptiva (2,30Â1,07; 1,58Â0,86 e 0,2500  0,73) respectivamente quando comparado com o grupo IFO. O prÃ-tratamento com L-Arginina (6,844Â1,235) reverteu o efeito antinociceptivo do L-NAME 10 mg/Kg, (6,84Â1,23), mas foi ineficaz sobre o efeito do L-NAME 40 mg/Kg (1,500Â0,7361) e HC-030031 75 mg/Kg (0,7200Â0,6953). Contudo, o prÃ-tratamento com HC-030031 nÃo apresentou efeito antiinflamatÃrio. Adicionalmente, verificou-se que o OM induziu significativo (p<0,05) comportamento nociceptivo (6,333Â0,9458) quando comparado ao grupo salina (1,250Â0,9204). AlÃm disso, o HC-030031 preveniu de maneira significativa da resposta nociceptiva provocada pelo OM (1,536 Â0,7653). Avaliou-se tambÃm o envolvimento do sistema opiÃide no efeito antinociceptivo do HC-030031. Verificou-se que a morfina apresentou uma importante atividade antinociceptiva (0,07143Â0,07143) contra a nocicepÃÃo induzida por OM a qual foi significativamente revertida pelo prÃ-tratamento com naloxona (3,125 1,302). Por outro lado, o efeito antinociceptivo do HC-030031 nÃo foi afetado pela naloxona (2,240Â1,263). Adicionalmente, AA, Zym e MPT induziram respostas de contorÃÃes abdominais significativas (43,71Â4,43; 11,00Â2,11 e 9,00Â2.30, respectivamente) as quais foram significativamente inibidas com HC-030031 (18,75, 37,5 ou 75 mg/kg, v.o.) em todas as doses utilizadas no teste com AA (29,07%; 53,35% e 41,59%), no teste com Zym (55,85%; 61,03% e 71,20%) e no teste com MPT (63,88%; 83,33% e 88,88%). Uma vez que a prostaglandina ativa o nociceptor diretamente, demonstrou-se que o HC-030031 possivelmente inibe a nocicepÃÃo visceral atravÃs da estabilizaÃÃo direta de nociceptores. O efeito antinociceptivo do HC-030031 parece ser independente da inibiÃÃo de cÃlulas residentes inflamatÃrias, do Ãxido nÃtrico ou do sistema opiÃide. Este estudo fornece perspectivas para o manuseio da dor visceral atravÃs da modulaÃÃo dos canais TRPA1. / The description of the TRP family of receptors including TRPA1 has provided potential therapeutic targets for treating acute and chronic pain. Some studies have shown a somatic nociceptive response due to the TRPA1 receptors activation which is effectively modulated with the experimental tool, HC-030031, a TRPA1 antagonist. However, there are a few studies evaluating the role of TRPA1 receptors in visceral pain. Then aimed to investigate the role of TRPA1 in the animal models of visceral nociception induced by different substances and to explore the possible mechanisms involved. Swiss male mice (n=6) were given only Carboxymethyl cellulose (vehicle CMC 0.5%, 1 mL/kg, p.o.), the compound HC-030031 (75, 150 or 300 mg/Kg, p.o.) or L-NAME (10 or 40 mg/Kg, s.c.) alone or with L-arginine (600mg/Kg, i.p.) 1h previously a alone injection of IFO (400 mg/kg, i.p.). Visceral nociception was assessed through the von Frey test previously (T0) and 12h (T1) later IFO injection by the abdominal stimulation with a pressure meter. The results were obtained in grams (T0-T1). The bladder of these animals were also removed to weighted (BWW), analyzed and after given scores macro and microscopically. We also investigated the antinociceptive effect of HC-030031 in the model of mustard oil-induced visceral nociception. The animals were treated with CMC 0.5% or HC-030031 (18.75, 37,5 or 75 mg/kg) or Morphine (5 mg/Kg, s.c.) alone or with Naloxone (2 mg/Kg, i.p.) 1h previously the injection of Mustard oil (MO) 0,75% (MO, 50 ul/colon). Visceral nociception was assessed through the von Frey test previously (T0) and 10 min (T1) after MO injection by the abdominal stimulation with a pressure meter. The results were obtained in grams (T0-T1). In another experimental setting, the animals were treated with CMC 0.5% (1 mL/kg, p.o) or HC-030031 (18.75; 37.5 or 75mg/Kg, p.o.) previously an intraperitoneal injection with acetic acid 0.6% (AA, 10 mL/kg), zymosan (Zym, 1 mg/cavity) or misoprostol (MPT, a stable prostaglandin analogous, 1&#956;g/cavity) and immediately had the writhing responses counted for 30 min. In order to investigate the role of resident peritoneal cells on the effect of HC-030031, we washed the peritoneal cavity of mice with heparin added PBS (30 mL) and then AA, Zym or MPT were injected i.p. A Sham group was included. Eventually, the writhing responses were recorded. Statistical analysis was performed with ANOVA/Student Newman Keul as appropriate. p<0.05 was accepted. (CEPA: Protocol 92/10). IFO induced significant (p<0.05) visceral nociception (6.25Â1.08) and inflammatory response [scores to edema 2(1-3); hemorrhage 3(1-3); and bladder wet weight (42.78  3.1)] in comparison with saline treated group (1.97Â0.89), [0(0-0); 0(0-0); 20.01 0.7749] respectively. Moreover, HC-030031(75) and L-NAME (10 or 40 mg/Kg) prevented in a significant manner (p<0.05) the nociceptive response (2.30Â1.07; 1.58Â0.860 and 2500Â0.7361) respectively when compared with IFO-treated group. Although the pretreatment with L-arginine (6.844Â1.235) was able to reverse the antinoceceptive effect of L-NAME 10 mg/Kg, (6.84Â1.23), it failed to do the same (p>0.05) with L-NAME 40 mg/Kg (1.500Â0.7361) and HC-0300031 75 mg/Kg (0.72Â0.69). The same reversible effect of L-Arginine was observed for the anti-inflammatory activity of L-NAME (p<0.05). However, HC-030031 presented no anti-inflammatory effect. The antinociceptive activity of HC-030031 was also assessed in the MO nociception model. We verified that MO induced a significant (p<0.05) nociceptive behavior (6.333Â0.9458) when compared to saline injected mice (1.250Â0.9204). Moreover, HC-030031 prevented in a significant manner the nociceptive response elicited by MO (1.536Â0.7653). Furthermore, the involvement of opioid system in the antinociceptive effect of HC-030031 as tested. We observed that morphine presented an important antinociceptive activity (0.07143Â0.07143) against MO-induced nociception which was significantly reverted by naloxone pre-treatment (3.125 1.302). On the other hand, the antinociceptive effect of HC-030031 remained in spite the injection of naloxone (2.240Â1.263). In addition to that, AA, Zym and MPT induced significant writhing responses (43.71Â4.43; 11.00Â2.11; 9.00Â2.30; respectively) which was significantly inhibited with HC-030031(18.75, 37.5 e 75 mg/kg, p.o.) treated mice in all the doses tested (29.07%, 53.35% and 41.59%, in the AA test, 55.85%, 61.03% and 71.20%, in the Zym test, 63.88%, 83.33% and 88.88%, in the MPT induced nociception, respectively to 18.75, 37.5 and 75 mg/kg doses. Eventually, the reduction of cell population in the peritoneal cavity prevented the development of writhing responses in both AA and Zym injected mice, with no effect was visualized on MPT treated mice. We the conclude that, since prostaglandin activates the nociceptor directly, it was shown that HC-030031 inhibits visceral nociception possibly through the stabilization of the neuronal ends. The antinociceptive effect of HC-030031 seems to be independent of the inhibition of inflammatory resident cells, opioid and nitric oxide pathways. This study provides perspective for the effective management of visceral pain through the modulation of TRPA1 channels.

O efeito devastador da hipotensão na mortalidade relacionada ao trauma de crânio é bem estabelecido. Este estudo avalia a resposta hemodinâmica sistêmica e cerebral à reposição volêmica com Solução de Ringer Lactato (RL) ou Salina Hipertônica a 3% (SSH 3%), o comportamento da liberação de prostanóides encefálicos, aspectos anátomo-patológicos encefálicos e aspectos neurológicos, durante a fase aguda do choque hemorrágico (CH) associado ao traumatismo craniencefálico (TCE). MÉTODOS: Quinze cães foram distribuidos em três grupos (n=5, cada), após randomização, de acordo com o protocolo de reposição volêmica, realizada após TCE (fluido-percussão cerebral, 4 atm) e balão epidural para induzir hipertensão intracraniana (HIC) a 20-25 mmHg e CH, induzido por remoção sanguínea até pressão arterial média (PAM) de 40 mmHg em 5 minutos: grupo CH+TCE+HSS 3% (8ml/kg), CH+TCE+RL (16ml/kg) e grupo CH+TCE (controle, sem tratamento). Simulamos o tratamento durante a fase pré-hospitalar e hospitalar precoce. Os grupos tratados receberam infusão sanguínea para atingir hematócrito de 30%. As medidas incluiram volume sanguíneo removido, volume de cristalóide infundido para restabelecer PAM, PAM, índice cardíaco (IC), pressão intracraniana (PIC), pressão de perfusão cerebral (PPC), lactato sistêmico e cerebral, taxas de extração de oxigênio, concentração plasmática de tromboxane e prostaciclina. Avaliamos o padrão pupilar dos animais e realizamos análise anátomo-patológica dos encéfalos. RESULTADOS: A reposição volêmica com RL ou SSH 3% promoveu maior benefício hemodinâmico que o grupo controle sem tratamento. A pressão de perfusão cerebral foi maior que no grupo controle e similar entre os grupos tratados; entretanto, a infusão de SSH 3% foi associada com valores mais baixos de PIC durante a fase \"hospitalar precoce\" e com maior osmolaridade e concentração de sódio plasmático. Não houve diferença nos valores da concentração venosa cerebral de prostaciclina e tromboxane. Os encéfalos do grupo tratado com SSH 3% não demonstraram edema e os hipocampos dos cães do grupo controle se mostraram isquêmicos em relação ao grupo tratado com SSH 3%. A reversão pupilar após alteração pupilar estabelecida ocorreu mais precocemente no grupo tratado com SSH 3% que RL. CONCLUSÕES: No evento de um trauma de crânio grave e choque hemorrágico, o uso de SSH 3% ou o dobro do volume de RL promoveram benefícios hemodinâmicos sistêmicos e cerebrais. Entretanto, valores mais baixos de PIC foram observados após SSH 3%. / The devastating effects of hypotension on head-trauma-related mortality are well known. This study evaluates the systemic and cerebral hemodynamic responses to volume replacement with 3% hypertonic saline (HSS) or lactated Ringer\'s solution (LR), during the acute phase of hemorrhagic shock (HS) associated with traumatic brain injury (TBI). METHODS: Fifteen mongrel dogs were assigned to one of three groups (n=5, each) according to the volume replacement protocol, infused after TBI (brain fluid percussion, 4atm) and epidural balloon to an intracranial pressure (ICP) higher than 20 mm Hg and HS, induced by blood removal to a mean arterial pressure (MAP) of 40 mm Hg in 5 minutes: Group HS+TBI+HSS (8 mL/kg of 3% HSS), HS+TBI+LR (16 mL/kg LR) and Group HS+TBI (controls, no fluids). We simulated treatment during prehospital and early hospital admission. Groups HS+TBI+HSS and HS+TBI+LR received shed blood infusion to a target hematocrit of 30%. Measurements included shed blood volume, fluid volume infused to restore MAP, MAP, cardiac output, cerebral perfusion pressure, cerebral and systemic lactate, and oxygen extraction ratios, tromboxane and prostaciclin. We evaluated the dog\'s pupil pattern and the anatomopathological study of the brain. RESULTS: Fluid replacement with HSS or LR promoted major hemodynamic benefits over control animals without fluids. Cerebral perfusion pressure was higher than controls and similar between treated groups; however, HSS infusion was associated with lower ICP during the \"early hospital phase\" and a higher serum sodium and osmolarity. There were no differences between groups in cerebral venous concentration of tromboxane and prostaciclin. There was no edema in brains of HSS group, the hypocampi of control\'s group showed ischemia comparing to HSS group. The pupils reverted early in HSS than LR group. CONCLUSION: In the event of severe head trauma and hemorrhagic shock, the use of HSS and larger volumes of LR promote similar systemic and cerebral hemodynamic benefits. However, a lower ICP was observed after HSS 3% than after LR.

As estratégias terapêuticas empregadas na ressuscitação do choque hemorrágico (CH) são capazes de evitar a morte, mas seus efeitos colaterais podem causar muitas alterações orgânicas, inclusive nos rins. Sabemos que a reposição volêmica feita com solução cristaloide restaura a hemodinâmica, diminui a mortalidade, mas pode levar a edema de órgãos e tecidos, entre outras alterações. Sendo assim, o objetivo do atual estudo foi comparar os efeitos da ressuscitação com Ringer lactato (RL) ou terlipressina (TLP) na função renal em modelo experimental de CH. Com este intuito, foi induzido o CH em suínos de 20-30 kg, por meio de sangramento pressão-controlada, até a obtenção e manutenção da pressão arterial média (PAM) em 40mmHg por 30 minutos. Os animais foram divididos em quatro grupos, sendo eles, o grupo Sham (feito apenas o procedimento anestésico), grupo Choque (indução de choque hemorrágico por 30 minutos) e os de instituição de estratégias de ressuscitação: grupo RL (indução de choque hemorrágico, e administração de RL de três vezes o volume de sangue retirado) e o grupo TLP (indução do choque e administração em bolus de 2 mg de TLP). Os parâmetros hemodinâmicos, de função renal e tubular foram avaliados nos momentos: basal; imediatamente após o CH; 30, 60, 90 e 120 minutos após tratamento. Ao final do estudo, os animais foram eutanasiados aos 60 ou aos 120 minutos. Foram coletadas amostras de tecido renal para avaliação de histologia e de western blott. Observamos uma melhora na microcirculação dos animais tratados com RL; entretanto, houve uma normalização da expressão da proteína NKCC2 e aquaporina 2 no grupo TLP. Observamos que o escore de lesão renal foi igual nos dois grupos de intervenção, mas a expressão de Bax estava mais diminuída no grupo TLP. Concluímos que a TLP mostrou ser uma estratégia tão eficaz quanto o RL no resgate do choque hemorrágico, com um provável potencial de maior proteção renal / Therapeutic strategies employed hemorrhagic shock (HS) resuscitation are able to prevent death, but its side effects can cause many organic dysfunction, including injury to the kidneys. It has been know that volume replacement with crystalloid solution can restore the hemodynamic status and decreases mortality; however, it can lead to organs and tissues edema, among other changes. Thus, the goal of this study was to compare the effects of resuscitation with lactated Ringer\'s (RL) or terlipressin (TLP) on renal function in an experimental model of HS. For this purpose, HS was induced in 20-30 kg swine, with pressure-controlled bleeding, to obtain and maintain the mean arterial pressure (MAP) of 40 mmHg for 30 minutes. The animals were divided into four groups, namely, the Sham group (made only the anesthetic procedure), Shock group (induction of hemorrhagic shock for 30 minutes) and the institution of resuscitation strategies: RL group (induction of hemorrhagic shock, and RL administration of three times the volume of removed blood) and TLP group (induction of shock and administration in bolus of 2 mg TLP). Hemodynamic parameters, renal and tubular function were evaluated at baseline; immediately after the HS; 30, 60, 90 and 120 minutes after treatment. At the end of the study, animals were euthanized after 60 or 120 minutes. Kidney tissue samples were collected for evaluation of histology and western blotting. We observed an improvement in microcirculation of animals treated with RL; however, there was a normalization of NKCC2 and aquaporin 2 protein expression in the TLP group. We observe that the kidney injury score was similar in both intervention groups, but Bax protein expression was more reduced than in the TLP group. We conclude that the TLP proved to be an effective strategy as RL in the rescue of hemorrhagic shock, with a likely potential for protect renal function

Geographically, Pakistan is in the western part of south Asia at about 24-37 °N latitudes and62-75 °E longitudes. Livestock and agriculture are two major sectors in Pakistan and play animportant role in the country economy.The tick infestation in livestock is not only devastating for animals and their products but alsobecome the cause of transmission of pathogens into humans. Crimean Congo fever (CCHF) isa viral tick-borne fatal disease. The dissemination of ticks and amplification of Crimean Congofever (CCHF) pathogen throughout the tick-animals-tick cycle, increases risk of transmissionto humans many times. In Pakistan, cases are reported in all areas, particularly those areaswhich lie on the border to CCHF endemic countries. There is a high prevalence of CCHF inboth Baluchistan and Khyber Pakhtunkhwa regions. Baluchistan is bordering with Afghanistanand Iran and Khyber Pakhtunkhwa with Afghanistan. Linear regression analysis revealed apositive significant association of high level of CCHF cases in livestock, with camels, goatsand sheep. The literacy rate is negatively significantly corelated with the numbers of cases.Statistical analysis of border effect revealed a high positive significant correlation of CCHFprevalence in areas near to borders. Both Baluchistan and Khyber Pakhtunkhwa (KPK) haslow literacy rate than other regions of Pakistan. Islamabad (capital city) has a higher literacyrate than all other regions but there is still a high CCHF prevalence. This is not only becauseof high population density but people from other regions, particularly from Baluchistan andKPK come here for animals selling or to seeking medical facilities in the large city hospitals.The study gives a proof that illiteracy and borders are the major respondent factors in theCCHF incidences and prevalence in an area. There is a need to raise awareness about ticksand tick-borne disease in the public and establishment of monitoring system across the bordersto prevent the spread of CCHF virus.

Bei IL-10 handelt es sich um ein antiinflammatorisches Zytokin, dessen immunmodulatorische Effekte bereits in zahlreichen Studien aufgezeigt werden konnten. Ziel dieser Studie war, die Unterschiede in der systemischen Inflammation und Organdysfunktion an Mäusen zu untersuchen, die nach Induktion eines hämorrhagischen Schocks, entweder inhalativ oder systemisch, mit IL-10 behandelt wurden. Männliche C57/BL6 Mäuse (6 Tiere pro Gruppe) wurden für 1.5 Stunden blutdruckkontrolliert in einen hämorrhagischen Schock versetzt. Nach anschließender Volumensubstitution wurde ihnen inhalativ oder intraarteriell rekombiniertes Maus - IL-10 verabreicht. Nach einer Gesamtversuchsdauer von 6 - bzw. 24 Stunden erfolgte die Tötung der Tiere. Die Ergebnisse der Studie zeigen, dass die lokale und systemische Verabreichung von IL-10 das Zytokinprofil der systemischen Inflammationsantwort unterschiedlich beeinflusst. Die Lunge kann durch inhalative Gabe von IL-10 geschützt werden, ohne die systemische Inflammationsantwort zu beeinflussen.

The four serotypes of dengue virus (DENV 1-4) have a significant and growing impact on global health. Dengue disease encompasses a wide range of clinical symptoms, usually presenting as an uncomplicated febrile illness lasting 5-7 days; however, a small percentage of infections are associated with plasma leakage and bleeding tendency (called dengue hemorrhagic fever, DHF), which can result in shock. Epidemiological studies indicate that severe dengue disease most often occurs during secondary heterotypic DENV infection. Additionally, plasma leakage (the hallmark of DHF) coincides with defervescence and viral clearance, suggesting that severe disease arises from the immune response to infection rather than a direct effect of the virus. A number of studies have found increased levels of markers of immune cell activation in patients with DHF compared to patients with the less severe form of disease (DF). These markers include IFNγ, TNFα, soluble CD8, soluble IL-2 receptor, soluble TNF receptor, and CD69, which support a role for T cells in mediating immunopathology. Because of the high homology of DENV 1-4, some degree of serotype-cross-reactivity is seen for most T cell epitopes. A high percentage of DENV-specific T cells recognize multiple DENV serotypes, as demonstrated by peptide-MHC (pMHC) tetramer binding and in vitro functional assays performed on PBMC from subjects vaccinated with an experimental DENV vaccine or naturally-infected subjects with secondary (>1) DENV infection. This thesis sought to address several gaps in the literature, specifically whether T cell responses differ in primary versus secondary (natural) infection. We studied the frequency, phenotype, and function of DENV-specific T cells. We demonstrated substantial serotype-cross-reactivity of antigen-specific T cells generated in response to naturally-acquired primary as well as secondary DENV infection. The frequency of A11-NS3133 epitope-specific T cells during acute infection did not correlate with disease severity. However, the peak frequency occurred earlier in primary infection while the frequency of CD45RA+ T cells declined quicker in secondary infection, suggesting the expansion of DENV-specific memory T cells. DENV-immune T cells exhibited different functional capabilities that were dependent on the particular serotype of infection. Specifically, DENV-1 or -3 stimulation of A11-NS3133 epitope-specific T cell lines resulted in robust function that included IFNγ production, whereas DENV-2 stimulation resulted in limited function that often included MIP-1β but not IFNγ production. These data support a role for T cells in DENV infection and offer new insights into their potential contribution to dengue pathology.

Dengue viruses (DV) are re-emerging mosquito-borne pathogens for which four distinct lineages, grouped based on serology and referred to as serotypes 1-4 (DIV-D4V), have been described. Epidemiological data imply that re-infection with a "heterologous" serotype, i.e, one other than that to which the individual was originally exposed, enhances the risk for development of severe disease, dengue hemorrhagic fever (DHF). The hallmark of DHF is a transient capillary leakage syndrome of rapid onset, temporally associated with the resolution of fever and viremia. In its most grave form, the vascular permeability phenomenon in DHF may progress to dengue shock syndrome (DSS), which is often fatal in the absence of appropriate medical care. Despite the fulminant nature of vascular leakage during DHF/DSS, this phenomenon does not appear to be due to direct cytopathic effects of DV. Rather, inappropriate reactivation and/or regulation of dengue-specific memory are the prevailing theorized (immunopathological) etiologies. Traditional vaccine development techniques have proven insufficient for DV, since any vaccine must offer complete protection against all four serotypes to avoid enhanced pathology on natural viral challenge. Understanding the underlying mechanisms that contribute to dengue disease, particularly the development of dengue-specific memory, is therefore of critical importance. Dengue immunopathology and the specific aspects of immunological memory that determine disease severity are heatedly debated. Previous research in our lab has suggested that T cell responses contribute to the severity of dengue illness. Clinical data indicate enhanced immune activation in more grave cases of DV infection, and serotype cross-reactive T cells from multiple individuals are present after both primary and secondary dengue infections. However, little is known about the conditions under which T cells are primed and dengue-specific memory is generated. Dendritic cells (DCs) are bone marrow-derived cells that play a central role in directing activity within the immune system. DCs shape quantitative and qualitative aspects of adaptive immunity, and therefore the intrinsic characteristics of host memory to a pathogen. DCs are essential in generating primary immune responses, due to their particular effectiveness in stimulating naïve T cells. DCs also play important roles in the reactivation of memory to an infectious agent, and as reservoirs for the dissemination of invading microorganisms. Exposure to pathogens or their products initiates a series of phenotypic and functional changes in DCs, termed maturation. DC maturation involves a coordinated response of immunomodulatory surface molecule elaboration and cytokine production, culminating in antigen presentation to, and co-stimulation of, T cells specific for the invading agent. The DC response is ostensibly tailored to facilitate effective elimination by regulating effective downstream interactions of the DC with T cells. A number of viruses have evolved to infect DCs and alter their functional behavior, facilitating their own survival within the host, and the herd. DV readily infects DCs both in primary cell cultures and in vivo. However, reports on the effects of DV infection on DC maturation vary both with regard to some of the cytokines produced, and the phenotypes of infected versus bystander cells. Although DCs appear to be activated following DV exposure, responses on the single-cell level appear to depend on the infection state of the cell, hypothetically driven by intracellular virus-mediated effects. Therefore, downstream responses to these divergent populations - i.e., actively infected cells versus uninfected bystander cells - are likely to be the consequence of at least two modes of DC behavior. Because DCs play a pivotal role in adaptive immune development, and because the resulting memory response appears to be critical in affecting disease pathology after heterologous DV re-infection, I sought to explore the phenomena of DC maturation in response to dengue exposure, and to begin to answer the question of how active infection alters the functional capabilities of DCs. Notably, primary dengue infection is generally well-controlled with minimal pathology. Therefore, this thesis addresses the hypothesis that DV infection of DCs results in cellular activation and stimulation of antiviral immunity, despite virus-mediated alteration of DC maturation. In order to address this hypothesis, I examined both DV infection-dependent and independent effects on DC functional responses including surface molecule regulation secretory activity, and CD4 T cell allostimulatory priming. DCs derived from human peripheral blood monocytes were readily infected with multiple strains of DV. DV infection of DCs derived from separate donors was dose-dependent, with substantial variability in DC susceptibility to infection. Exposure to live DV activated surface molecule expression in DCs, similar to the effects of defined maturation stimuli including a combination of TNF-α and IFN-α, or LPS. In addition, UV-inactivated DV induced expression of cell surface molecules, albeit to a lesser extent than did live virus demonstrating inherent stimulatory properties of DV particles. Using intracellular staining for DV envelope (E) protein, I detected increased surface molecule expression on both infected DCs and uninfected bystander DCs from the same culture, as compared to mock-infected DCs. These data indicate that activation was not prevented in cells undergoing active viral replication. However, the degree of surface molecule induction depended on the infection state of the cell. Infected DCs had enhanced PD-L2 and MHC II expression relative to uninfected bystander cells, while PD-L1, CD80, CD86, and MHC I expression were suppressed with active infection. Therefore, intracellular DV replication altered the process of cell surface molecule regulation within these cells. DV infection of DCs also resulted in the secretion of a broad array of cytokines and chernokines. These included the antiviral cytokine IFN-α, inflammatory cytokines TNF-α, IL-6, and IL-1α, and inflammatory chemokines IP10, MCP-1, MIP-1α, and RANTES. DV infection did not induce DC production of the IL-12 p70 heterodimer, and secretion of the immunosuppressive cytokine IL-10 was low in most experiments. Similar to the results seen with surface molecule induction, UV inactivation of DV reduced, but did not eliminate, cytokine and chemokine responses. At the single-cell level, TNF-α and IP10 production profiles of infected DCs and uninfected bystander DCs were distinct. DV infection in DCs reduced production of IP10, but stimulated TNF-α as compared to uninfected bystander cells in the same culture. Blocking experiments demonstrated that IFN-α/β produced by DCs in response to infection actively inhibited viral protein expression and drove IP10, but not TNF-α, production. DV infection of DCs did not consistently suppress DC stimulation of allogeneic CD4 T cell proliferation. In cases where infection enhanced DC stimulatory function, T cell proliferation was less pronounced than that induced by DCs activated with exogenous TNF-α plus IFN-α. Increasing multiplicity of infection (MOI) of DCs with DV resulted in increasing DC infection rates, but a statistically significant trend at the highest MOIs for decreased T cell alloproliferation, suggesting that direct infection of DCs reduces their CD4 T cell priming function. MOI-dependent reduction in DC stimulatory function depended on replication-competent virus. Increased MOIs during DV infection of DCs did not cause an elevation in detectable IL-10 in supernatants derived from T-DC co-cultures. In addition, increased DV MOI of DCs was not associated with increased levels of either IL-13 or IFN-γ in supernatants from T-DC co-culture, suggesting that actively infected DC do not skew CD4 T cells towards a specific Th phenotype. These data demonstrate that DV infection induces functional maturation of DCs that is modified by the presence of virus through both IFN-dependent and independent mechanisms. However, the allostimulatory phenotype of DCs was not universally enhanced, nor was it skewed towards antiviral (Th1)-type responses. These data suggest a model whereby dengue infection during primary illness results in controlled immune stimulation through activation of bystander DCs, and the generation of mixed Th-type responses. Direct DV infection of DCs appears to attenuate activation of, and potentially clearance by, antiviral mechanisms. During secondary infection, reduced IP10 production and enhanced TNF-α secretion by infected cells coupled with MHC I downregulation and enhanced PD-L2 expression, would subvert both Th1 CD4 T cell recruitment and result in CD8 T cell suppression and death. Furthermore, DV-specific effects on DCs would allow for continued viral replication in the absence of effective clearance. These DV-mediated effects would modify T cell memory responses to infected DC, and potentially facilitate the expansion of pathologic T cell subsets. Contributing to this pathological cascade, antibody-dependent enhancement of infection in monocytic cells and macrophages would shift antigen presentation and cytokine production paradigms, increasing the risk of DHF.

Background: Rare genetic mutations cause familial early-onset stroke disorders, known as “Mendelian strokes”. The broader relevance of rare mutations in unrelated young stroke patients is uncertain. We hypothesize that rare mutations in known and novel genes are important risk factors for stroke. Methods: Exome sequencing was used to characterize rare disruptive protein-altering mutations in 185 young cases and 185 matched controls from INTERSTROKE, a large and globally representative stroke study. The major objectives were: 1) to precisely define the role of known Mendelian stroke genes and 2) to discover novel gene and pathway associations. Results: A focused assessment of known Mendelian stroke genes revealed a significant contribution from NOTCH3, the causal gene for Cerebral Autosomal Dominant Arteriopathies with Subcortical Infarcts and Leucoencephalopathies (CADASIL). CADASIL mutations were identified in six cases and no controls (P=0.03). The clinical presentation of CADASIL mutation carriers deviated from known symptomatology, consisting of small-vessel ischemic strokes (SVIS) accompanied by secondary features including migraine and depression. A novel role for non-CADASIL NOTCH3 mutations in ICH was also elucidated (OR=2.86; 95% CI, 1.13 to 7.93, P=0.02). Such mutations were present in 22% of ICH cases and 8% of matching controls. An agnostic evaluation of all genes did not reveal any genome-wide significant associations. However, NOTCH3 was among the top ICH genes out of 13,706 tested, and many others were also biologically relevant, notably, AARS2 and NBEAL2. A protective association was identified for the renin angiotensin system (P=8.1x10-4), whereas type II diabetes mellitus was associated with increased risk (P=1.9x10-2). Conclusion: Rare mutations influence risk of early-onset stroke. CADASIL mutations play an important role in unrelated stroke patients. Beyond CADASIL, a novel role was uncovered for other NOTCH3 mutations as common and significant risk factors for ICH. Novel biologically relevant genes and pathways may also affect stroke susceptibility. / Thesis / Master of Science in Medical Sciences (MSMS)

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Ο ιός του αιμορραγικού πυρετού Κριμαίας-Κογκό (Crimean-Congo Hemorrhagic Fever Virus, CCHFV), καθώς και οι χανταϊοί (hantaviruses) προκαλούν στον άνθρωπο αιμορραγικό πυρετό. Αυτοί παρουσιάζουν ευρεία γεωγραφική κατανομή και αποτελούν απειλή για τη δημόσια υγεία, λόγω του υψηλού ποσοστού θνητότητας που σημειώνουν και της απουσίας αποτελεσματικής θεραπευτικής αγωγής. Παρότι επιδημιολογικές μελέτες δείχνουν την παρουσία αντισωμάτων στον ελληνικό πληθυσμό, περιορισμένες είναι οι αναφορές κλινικών περιστατικών CCHF και HFRS στην Ελλάδα. Σκοπός της παρούσας μελέτης είναι να προσδιορίσει τον επιπολασμό της μόλυνσης με τον CCHFV και τους χανταϊούς στον Ν. Αχαΐας, που αν και παρουσιάζει ευνοϊκές συνθήκες για την κυκλοφορία των δύο ιών, δεν έχει μελετηθεί στο παρελθόν. Σχεδιάσθηκε διατμηματική μελέτη και συγκεντρώθηκαν προοπτικά 207 δείγματα ορού φαινομενικά υγιών ατόμων-κατοίκων της περιοχής, τα οποία εξετάστηκαν με τη μέθοδο ELISA και έμμεσου ανοσοφθορισμού για την ύπαρξη αντισωμάτων έναντι του CCHFV και των χανταϊών. Ο επιπολασμός για τη μόλυνση με CCHFV βρέθηκε 3,4% και 9,7% για τη μόλυνση με χανταϊούς, ενώ κανένα από τα οροθετικά άτομα δεν ανακαλούσε συμπτώματα παρόμοια με αυτά του CCHF ή του HFRS. Για τον CCHFV, βρέθηκε ότι η ηλικία, η αγροτοκτηνοτροφική ενασχόληση, η κατοχή/εκτροφή αιγοπροβάτων, το ιστορικό νύγματος κρότωνα, η μόνιμη διαμονή σε υψόμετρο ≥400μ., η μόνιμη διαμονή σε μη αρδευόμενες αρόσιμες εκτάσεις ή σε αγροτικές εκτάσεις με σημαντικό ποσοστό φυσικής βλάστησης, καθώς και η μόνιμη διαμονή σε αγροτική περιοχή είναι σημαντικοί παράγοντες κινδύνου. Από αυτούς, το νύγμα κρότωνα, η αγροτοκτηνοτροφική ενασχόληση και η μόνιμη διαμονή σε υψόμετρο ≥400μ. βρέθηκαν να προβλέπουν καλύτερα την οροθετικότητα ενός ατόμου. Επίσης, βρέθηκε ότι παράγοντες που σχετίζονται με τη μόλυνση με χανταϊούς είναι: η ηλικία, η θέαση τρωκτικών σε ακτίνα <200μ. γύρω από την οικία και η ιδιοκτησία υπόγειας αποθήκης. Από αυτούς, μόνο η ηλικία βρέθηκε να προβλέπει καλύτερα την οροθετικότητα ενός ατόμου. Επιπλέον, παρατηρήθηκε ότι σχεδόν το 75% των θετικών ατόμων για αντισώματα έναντι των χανταϊών παρουσίαζαν ήπια επηρεασμένη νεφρική λειτουργία. Εντοπίστηκαν, επίσης, ενδημικές εστίες των ιών στον νομό: ο Δ. Ερυμάνθου για τον CCHFV και ο Δ. Δυτικής Αχαΐας για του χανταϊούς. Λαμβάνοντας υπόψη τα παραπάνω αποτελέσματα, θα πρέπει οι κλινικοί γιατροί της περιοχής να συμπεριλαμβάνουν τον CCHF και τον HFRS στη διαφορική διάγνωση εμπύρετων νοσημάτων, ιδίως όταν αυτά συνοδεύονται από θρομβοπενία ή επηρεασμένη νεφρική λειτουργία. / Crimean-Congo hemorrhagic fever virus (CCHFV) and hantaviruses cause to humans fever with hemorrhagic manifestations. These viruses present wide geographic distribution and represent major threats for public health, because of the high fatality rate that they present and the lack of appropriate treatment. Although seroprevalence studies show the presence of antibodies against CCHFV and hantaviruses in the greek population, only some reports of human cases have been reported to date in Greece. The aim of the present study is to estimate seroprevalence for CCHFV and hantaviruses in humans in the prefecture of Achaia, where the local conditions potentially favor the circulation of these viruses and which has not been previously studied. A cross-sectional study was designed and 207 human sera were collected from apparently healthy individuals living in Achaia, which were tested for CCHFV and hantaviruses IgG antibodies by ELISA and by indirect immunofluorescence assay (IFA). Seroprevalence for CCHFV infection was estimated at 3.4%, whereas for hantaviruses at 9.7%; none recalled any illness resembling CCHF or HFRS. For CCHFV, it was found that age, agro-pastoral occupation, tending sheep and/or goats, tick bite, living in areas at an altitude of ≥400m., living at rural areas, living on non-irrigated arable land or on land principally occupied by agriculture, with significant areas of natural vegetation are significantly related to seropositivity. Among them, tick bite, agro-pastoral occupation and living in areas at an altitude of ≥400m. better predict seropositivity of an individual. For hantaviruses, it was found that age, rodent sighting around home and the ownership of an underground shed are significantly related to seropositivity. Among them, it seems that only age can predict seropositivity of an individual. Moreover, it was observed that almost 75% of the seropositive for hantaviruses individuals presented mild renal dysfunction. In this study, endemic foci were also detected: the municipality of Erimanthos for CCHFV and the municipality of Western Achaia for hantaviruses. Clinicians should include CCHF and HFRS in the differential diagnosis of an acute febrile case, especially when thrombocytopenia or impaired renal function is encountered.