Phenotype-genotype analysis of cryopyrin-associated periodic syndromes (CAPS): Description of a rare non-exon 3 and a novel CIAS1 missense mutation

We describe in this paper the phenotype-genotype analysis of a Brazilian cohort of patients with cryopyrin-associated periodic syndromes (CAPS). Patient 1 presented with an urticarial rash and recurrent fever exacerbated by cold weather, arthritis, and anterior uveitis, thus, receiving a clinical diagnosis of familial cold autoinflammatory syndrome. CIAS1 sequencing identified the T436I mutation, previously associated to a clinical phenotype of chronic infantile neurological cutaneous and articular/neonatal onset multisystem inflammatory disease. Patient 2 developed a papular exanthema with daily fever shortly after birth, frontal bossing, patellae enlargement, and cognitive and motor impairments. Sequencing identified the exceedingly rare G755R CIAS1 mutation in exon 4. Patient 3 developed skin rash and articular symptoms 6 h after birth, followed by aseptic meningitis. He was found to have the novel C148Y missense mutation in CIAS1. This report expands the spectrum of CIAS1 mutations associated to clinical disease, suggests that the same mutation can be associated with different clinical syndromes, and supports the evidence that CAPS patients should always be screened for mutations outside exon 3.

Detection of enteroviruses in cases of neurological disorders in the State of Pará, Brazil

Eighty-one cerebrospinal fluid (CSF) samples mainly from cases of aseptic meningitis and motor deficiency syndrome were sent to the Virology Section of Evandro Chagas Institute, Belém Pará, in the period of January 1995 to January 1996 in order to isolate viruses. All samples were inoculated onto HEp-2 cell culture and newborn mice, with negative results. The probability of isolating viruses by these methods is reduced because of the low concentration of viral particles in these specimens. In order to obtain more information about the etiology of these cases, a group of 23 samples were selected to be tested by a more sensitive technique than the virus isolation - the reverse transcription polymerase chain reaction (RT-PCR). Specific primers directed to conserved regions in the enterovirus genome were used, considering that this group of viruses is frequently associated with these neurological disorder. The age of the patients ranged from 1 to 55 years and nearly all of them lived in Belém, State of Pará, North of Brazil. Of 15 samples analyzed by RT PCR nine (60%) were positive; of these, 6 (66.6%) had motor deficiency and 3 (33.3%) developed aseptic meningitis. These results show that it is important to investigate enterovirus as cause of these syndromes.

Echovirus 4 associated to hand, foot and mouth disease

Hand, foot and mouth disease (HFMD) is a contagious enteroviral infection occurring primarily in children and characterized by vesicular palmoplantar eruptions and erosive stomatitis. Echovirus 4 (EV-4) has been commonly associated with aseptic meningitis. The association of HFMD with EV-4 has not been reported previously. Two samples of a 14-month child who presented mild fever, sores in the mouth, rash with blisters on the palm of hands and soles of feet were sent to Enteric Viruses Laboratory of Adolfo Lutz Institute. Clinical samples were inoculated in three different cell lines, and those which presented cytopathic effect (CPE), were submitted to Indirect Immunofluorescence Assay (IFA) and "one step" RT-PCR. Agarose gel electrophoresis from RT-PCR product, showed a product with 437 bp, which is characteristic of Enterovirus group. Echovirus 4 was identified by IFA. Although HFMD is a viral infection associated mainly with Enterovirus 71 (HEV-71) and Coxsackievirus A16 (CV-A16), our results demonstrate a diversity of serotype related to HFMD and stress the importance of epidemiological surveillance to this disease and its complications.

Meningite Assética Associada à Terapêutica com Imunoglobulina Endovenosa

Aseptic meningitis can be an adverse drug reaction to intravenous immunoglobulin. We describe a previously healthy 4-yearold boy, admitted for idiopathic thrombocytopaenic purpura. He received two infusions of intravenous immunoglobulin. Four hours after the last administration the patient developed a meningeal syndrome. Analysis of cerebrospinal fluid revealed 500 cells/μl (predominantly neutrophils) and normal biochemistry. Bacteriological and virological tests were negative. After 48h he was asymptomatic. Given the absence of other aetiological factors and the temporal relationship between the administration of immunoglobulin and the development of symptoms, we believe the patient had an aseptic meningitis related to intravenous immunoglobulin. This therapy may cause headache, fever and vomiting; however, lumbar puncture is not usually performed, so this complication may be underdiagnosed.

Attaques cérébrovasculaires ischémiques multiples dues à une méningovasculite à Borrelia garinii. [Multiple ischemic strokes due to Borrelia garinii meningovasculitis].

The most frequent clinical manifestation of borreliosis in Switzerland is erythema migrans, with about 2500 patients each year. Neurological manifestations are rare, mostly hyperalgesic radiculitis (Bannwarth syndrome), aseptic meningitis or cranial nerve involvement. We report the first Swiss patient with meningovasculitis due to neuroborreliosis, with recurrent multiple ischemic strokes in multiple vascular territories. The treatment with ceftriaxone stopped the progression, but the patient is still suffering from severe invalidating cognitive disorders. We also comment on the pathophysiology and review the literature of other clinical cases.

Neutralizing Antibodies to Enterovirus 71 in Belém, Brazil

Non-polio enteroviruses (Coxsackievirus A, Coxsackievirus B, Echovirus and EV 68-72) which belong to the enterovirus (EV) genus, Picornaviridae family, may be responsible for acute flaccid paralysis, aseptic meningitis, myocarditis, hepatitis, pleurodinia, neonatal sepsis, hand, foot and mouth disease (HFMD) even though 50-80% of infections are asymptomatic. EV 71 has been responsible for outbreaks and epidemics of HFMD and acute neurologic disease justifying its study in our country. The aim of this study was to detect neutralizing antibodies (NtAb) to EV 71 in individuals up to 15 years of age living in Belém, State of Pará, northern Brazil. Serum samples from 238 patients attending the Virology Sector of Evandro Chagas Institute in Belém, Brazil, were analyzed using microneutralization tests that included RD cells and BrCr strain. Overall 40.8% (97/238) of tested samples had NtAb to EV 71. Regarding the distribution per age group, 85.2% (92/108) of patients aged 0-3 years had no NtAb to this virus and 69.2% of those 12 to15 years of age were seropositive. These results confirm that EV 71 infection occurs in the city of Belém; and that a high rate of individuals in this study were infected aged 3 years and over and, when aged 15 years nearly 70% had EV 71 NtAb.

Analytical validation of a lymphocytic choriomeningitis virus real-time RT-PCR assay.

Lymphocytic choriomeningitis virus (LCMV) is a rare cause of central nervous system disease in humans. Screening by real-time RT-PCR assay is of interest in the case of aseptic meningitis of unknown etiology. A specific LCMV real-time RT-PCR assay, based on the detection of genomic sequences of the viral nucleoprotein (NP), was developed to assess the presence of LCMV in cerebrospinal fluids (CSF) sent for viral screening to a Swiss university hospital laboratory. A 10-fold dilution series assay using a plasmid containing the cDNA of the viral NP of the LCMV isolate Armstrong (Arm) 53b demonstrated the high sensitivity of the assay with a lowest detection limit of ≤50 copies per reaction. High sensitivity was confirmed by dilution series assays in a pool of human CSF using four different LCMV isolates (Arm53b, WE54, Traub and E350) with observed detection limits of ≤10PFU/ml (Arm53b and WE54) and 1PFU/ml (Traub and E350). Analysis of 130 CSF showed no cases of acute infection. The absence of positive cases was confirmed by a published PCR assay detecting all Old World arenaviruses. This study validates a specific and sensitive real-time RT-PCR assay for the diagnosis of LCMV infections. Results showed that LCMV infections are extremely rare in hospitalized patients western in Switzerland.

Serosurvey Study of Toscana Virus in Domestic Animals, Granada, Spain

Toscana virus (TOSV) is transmitted by infected sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis in humans. It remains unclear if there are animal reservoirs able to maintain the virus through the cold months of the year, when the vector is not circulating. From May to October of 2006 and 2007, we conducted a serosurvey study on domestic animals from Granada province (southern Spain). TOSV was investigated in 1186 serum samples from horses, goats, pigs, cats, dogs, sheep, and cows by serology (indirect fluorescence assay), viral culture, and RT-polymerase chain reaction. Specific anti-TOSV antibodies were detected in 429 (36.2%) serum samples. The highest seropositivity rates were observed in cats (59.6%) and dogs (48.3%). These results suggest that an important percentage of the domestic animals have been infected by TOSV. Significantly different seroprevalence rates were detected in goats among distinct geographical areas. All viral cultures were negative. TOSV was detected by RT-polymerase chain reaction in only one serum sample from a goat. Thus, the studied animals do not seem to act as reservoirs for TOSV; otherwise, they could be amplifying hosts for the virus.

Receptors and endocytosis of coxsackievirus A9

Coxsackievirus A9 (CV-A9) belongs to human enteroviruses within family Picornaviridae, which are the main cause of aseptic meningitis. In addition, CV-A9 causes a wide range of other clinical manifestations of acute disease including respiratory infections, myocarditis, encephalitis and severe generalized infections in newborns. In this study, the functions of integrins αVβ6 and αVβ3 in the attachment and cellular entry of CV-A9 were analyzed. Further, virus and cell surface interactions and endocytosis of CV-A9 were studied in specific cell lines. Also, a method for production of GFP-expressing CV-A9 particles by long PCR-mediated mutagenesis and in vivo transcription was developed. The results indicated that RGD-motif (arginine-glycine-asparagine) that resides in the viral capsid is important for CV-A9 infection particularly in cell lines expressing integrin αVβ6 and that this integrin serves as a high affinity attachment receptor for the virus. CV-A9 is also capable of infecting certain cell lines independently of αV-integrins by binding to the cell surface HSPA5 protein. Regardless of the attachment stage, the internalization of the virus occurs via the same entry pathway and is dependent on β2M, dynamin, and Arf6 but independent of clathrin and caveolin-1. Furthermore, the virus internalization occurs within Arf6-containing vesicles suggesting that Arf6 is central mediator of CV-A9 endocytosis. While in this study the results of CV-A9 endocytosis were based on microscopical visualization within individual fixed cells, a rapid method for generation of a virus for real-time imaging was also described.

Análise do efeito de polimorfismos não-sinônimos em genes de reparo de DNA da via BER na resposta inflamatória da meningite

In vitro and in animal models, APE1, OGG1, and PARP-1 have been proposed as being involved with inflammatory response. In this work, we have investigated if the SNPs APE1 Asn148Glu, OGG1 Ser326Cys, and PARP-1 Val762Ala are associated to meningitis and also developed a system to enable the functional analysis of polymorphic proteins. Patients with bacterial meningitis (BM), aseptic meningitis (AM) and controls (non-infected) genotypes were investigated by PIRA-PCR or PCR-RFLP. DNA damages were detected in genomic DNA by Fpg treatment. IgG and IgA were measured from plasma and the cytokines and chemokines were measured from cerebrospinal fluid samples using Bio-Plex assays. The levels of NF-κB and c-Jun were measured in CSF by dot blot assays. A significant (P<0.05) increase in the frequency of APE1 148Glu allele in BM and AM patients was observed. A significant increase in the genotypes Asn/Asn in control group and Asn/Glu in BM group was also found. For the SNP OGG1 Ser326Cys, the genotype Cys/Cys was more frequent (P<0.05) in BM group. The frequency of PARP-1 Val/Val genotype was higher in control group (P<0.05). The occurrence of combined SNPs increased significantly in BM patients, indicating that these SNPs may be associated to the disease. Increasing in sensitive sites to Fpg was observed in carriers of APE1 148Glu allele or OGG1 326Cys allele, suggesting that SNPs affect DNA repair activity. Alterations in IgG production were observed in the presence of SNPs APE1Asn148Glu, OGG1Ser326Cys or PARP-1Val762Ala. Reductions in the levels ofIL-6, IL-1Ra, MCP-1/CCL2and IL-8/CXCL8 were observed in the presence of APE1148Glu allele in BM patients, however no differences were observed in the levels of NF-κB and c-Jun considering genotypes and analyzed groups. Using APE1 as model, a system to enable the analysis of cellular effects and functional characterization of polymorphic proteins was developed using strategies of cloning APE1 cDNA in pIRES2-EGFP vector, cellular transfection of the construction obtained, siRNA for endogenous APE1 and cellular cultures genotyping. In conclusion, we obtained evidences of an effect of SNPs in DNA repair genes on the regulation of immune response. This is a pioneering work in the field that shows association of BER variant enzymes with an infectious disease in human patients, suggesting that the SNPs analyzed may affect immune response and damage by oxidative stress level during brain infection. Considering these data, new approaches of functional characterization must be developed to better analysis and interactions of polymorphic proteins in response to this context

Associação entre polimorfismos em enzimas de reparo de DNA e ocorrência de meningite bacteriana.

Despite advances in vaccine development and therapy, bacterial meningitis (BM) remains a major cause of death and long-term neurological disabilities. As part of the host inflammatory response to the invading pathogen, factors such as reactive oxygen species are generated, which may damage DNA and trigger the overactivation of DNA repair mechanisms. It is conceivable that the individual susceptibility and outcome of BM may be in part determined by non synonymous polymorphisms that may alter the function of crucial BER DNA repair enzymes as PARP-1, OGG-1 and APE-1. These enzymes, in addition to their important DNA repair function, also perform role of inflammatory regulators. In this work was investigated the non synonymous SNPs APE-1 Asn148Glu, OGG-1 Ser326Cys,PARP-1 Val762Ala, PARP-1 Pro882Leu and PARP-1 Cys908Tyr in patients with bacterial meningitis (BM), chronic meningitis (CM), aseptic meningitis (AM) and not infected (controls). As results we found increased frequency of variant alleles of PARP-1 Val762Ala (P = 0.005) and APE-1 Asn148Glu (P=0.018) in BM patients, APE-1 Asn148Glu in AM patients (P = 0.012) and decrease in the frequency of the variant allele OGG-1 Ser326Cys in patients with CM (P = 0.013), regarding the allelic frequencies in the controls. A major incidence of individuals heterozygous and/ or polymorphic homozygous in BM for PARP-1 Val762Ala (P= 0.0399, OD 4.2, 95% IC 1.213 -14.545) and PARP-1 Val762Ala/ APE-1 Asn148Glu (P = 0.0238, OD 11.111, 95% IC 1.274 - 96.914) was observed related to what was expected in a not infected population. It was also observed a major incidence of combined SNPs in the BM patients compared with the control group (P=0.0281), giving evidences that SNPs can cause some susceptibility to the disease. This combined effect of SNPs seems to regulate the principal cytokines and other factors related to BM inflammatory response and point the importance of DNA repair not only to repair activity when DNA is damaged, but to others essential functions to human organism balance.

Ativação local da rota da quinurenina por moduladores inflamatórios durante a meningite bacteriana.

Activation of the kynurenine (KYN) pathway (KP) by modulators of immune system has been observed during several neurological diseases. Here we assessed the association of chemo-/cytokine levels with the concentration of KP metabolites in cerebrospinal fluid (CSF) and plasma samples from patients with bacterial meningitis (BM). All samples were collected from 42 patients diagnosed with acute bacterial meningitis (ABM), aseptic meningitis, tuberculous meningitis and patients without infection neurological disorders. CSF and plasma concentration of metabolites from the KP was assessed by high pressure liquid chromatography (HPLC) and cytokines and chemokines by Bio-plex 200 suspension array system. Concentrations of the KP metabolites KYN and kynurenic acid (KYNA) were significantly higher in CSF of patients with ABM compared to other groups. Tryptophan (TRP), anthranilic acid (AA), 3-hydroxykynurenine (3HK) and 3-hydroxyanthranilic acid (3HAA) did not show statistical significance, although some of them presented a good accumulation during ABM. The expression of TNF-alpha, IL-6, IL-1beta, IFN-gamma, IL-10, IL-1 receptor antagonist (IL-1Ra), MIP-1alpha, MIP-1beta, MCP-1 and G-CSF was about 100-fold higher in CSF from ABM patients than other infected groups. In all CSF and plasma samples, the concentration of IL-2, IL-12(p70), IL-4, IL-8 and GM-CSF was not significant. ABM still showed significant concentrations of IL-6, IL-10, IL-1Ra and MCP-1 in plasma samples. Based on the comparison of KP metabolites concentrations between plasma and CSF samples we conclude that the activation of the tryptophan pathway upon BM occurs within the brain. This increase in KP metabolites is most due to activation of the KP by molecules as IFN-gamma and TNF-alpha in response to infection.

Complicações neurológicas determinadas pela anestesia subaracnóidea

JUSTIFICATIVA E OBJETIVOS: Complicações neurológicas da anestesia subaracnóidea, apesar de raras, podem determinar seqüelas importantes. O objetivo deste estudo é apresentar estas complicações com a finalidade de esclarecer os fatores desencadeantes, o que facilita o diagnóstico das lesões. CONTEÚDO: São apresentadas as seguintes complicações: lesão de nervo desencadeada pela agulha e cateter, cefaléia pós-punção, síndrome da artéria espinhal anterior, hematoma espinhal, meningite bacteriana, meningite asséptica, aracnoidite adesiva, síndrome da cauda eqüina e sintomas neurológicos transitórios. CONCLUSÕES: O conhecimento dos fatores desencadeantes de complicações neurológicas determinadas pela anestesia subaracnóidea pode prevenir as lesões, diagnosticar e tratar mais precocemente e, desse modo, mudar o prognóstico das mesmas.

Atypical manifestations in Brazilian patients with neuro-Behcet's disease

Type and frequency of systemic and neurologic manifestations of Beh double dagger et's disease (BD) vary with ethnicity. In Brazil, BD occurs as sporadic cases. We describe clinical and radiological features of 36 Brazilian patients of mixed ethnicity with neuro-Beh double dagger et's disease (NBD). Medical records of 178 BD patients were reviewed and 36 (20%) NBD patients were identified. Twenty-one NBD patients (58.3%) were female and 27 (75%) presented with parenchymal manifestations. Brainstem involvement was the most common neurologic syndrome (41.7%). Seizures (27.8%), isolated aseptic meningitis (16.7%), optic neuropathy (ON) (16.7%), cerebral venous thrombosis (CVT) (8.3%), peripheral neuropathy (2.8%), and spinal cord involvement (5.6%) were other neurologic manifestations observed among Brazilian NBD patients. Eighteen (50%) had at least one relapse, and isolated aseptic meningitis was the most common relapsing manifestation. No significant differences concerning the number of relapses between parenchymal and non-parenchymal groups were found. A multivariate model including disease duration, cell count in spinal fluid, cyclosporine use, immunosuppressive use at disease onset, age at NBD onset, and ON did not reveal any significant associations with NBD relapse. There was a low frequency of CVT and an unexpected higher number of isolated aseptic meningitis. Brazilian NBD patients present more parenchymal and atypical manifestations, and relapse more often than NBD patients from other populations.

Coxsackie B viruses and the kidney--a neglected topic

Coxsackie B viruses types 1-6 (CVB1-6) occur worldwide and cause a broad spectrum of diseases, including myocarditis and aseptic meningitis. Although renal damage due to CVB has been suspected since the 1950s, these agents are only rarely searched for in today's clinical nephrological practice. Nevertheless, CVB can infect mesangial cells. Furthermore, infections with these viruses lead to a histological picture resembling mesangioproliferative glomerulonephritis and IgA-nephropathy in mice. In the present article, we provide an overview of this largely neglected topic, and of the slowly and steadily increasing evidence suggesting a link between coxsackieviral infections and kidney diseases.