DNA probe array for the simultaneous identification of herpesviruses, enteroviruses, and flaviviruses

Viral infections of the central nervous system (CNS) are caused by a variety of viruses, namely, herpesviruses, enteroviruses, and flaviviruses. The similar clinical signs provoked by these viruses make the diagnosis difficult. We report on the simultaneous detection of these major CNS pathogens using amplification by PCR and detection of amplified products using DNA microarray technology. Consensus primers were used for the amplification of all members of each genus. Sequences specific for the identification of each virus species were selected from the sequence alignments of each target gene and were synthesized on a high-density microarray. The amplified products were pooled, labeled, and cleaved, followed by hybridization on a single array. This method was successfully used to identify herpesviruses, namely, herpes simplex virus type 1 (HSV-1), HSV-2, and cytomegalovirus; all serotypes of human enteroviruses; and five flaviviruses (West Nile virus, dengue viruses, and Langat virus). This approach, which used highly conserved consensus primers for amplification and specific sequences for identification, would be extremely useful for the detection of variants and would probably help solve some unexplained cases of encephalitis. The analytical sensitivity of the method was shown to be 500 genome equivalents ml(-1) for HSV-1, 0.3 50% tissue culture infectious doses (TCID50s) ml(-1) for the enterovirus coxsackievirus A9, and 200 TCID50s ml(-1) for West Nile virus. The clinical sensitivity of this method must now be evaluated.     

KCNJ11 activating mutations in Italian patients with permanent neonatal diabetes

Permanent neonatal diabetes mellitus (PNDM) is a rare condition characterized by severe hyperglycemia constantly requiring insulin treatment from its onset. Complete deficiency of glucokinase (GCK) can cause PNDM; however, the genetic etiology is unknown in most PNDM patients. Recently, heterozygous activating mutations of KCNJ11, encoding Kir6.2, the pore forming subunit of the ATP-dependent potassium (K(ATP)) channel of the pancreatic beta-cell, were found in patients with PNDM. Closure of the K(ATP) channel exerts a pivotal role in insulin secretion by modifying the resting membrane potential that leads to insulin exocytosis. We screened the KCNJ11 gene in 12 Italian patients with PNDM (onset within 3 months from birth) and in six patients with non-autoimmune, insulin-requiring diabetes diagnosed during the first year of life. Five different heterozygous mutations were identified: c.149G>C (p.R50P), c.175G>A (p.V59M), c.509A>G (p.K170R), c.510G>C (p.K170N), and c.601C>T (p.R201C) in eight patients with diabetes diagnosed between day 3 and 182. Mutations at Arg50 and Lys170 residues are novel. Four patients also presented with motor and/or developmental delay as previously reported. We conclude that KCNJ11 mutations are a common cause of PNDM either in isolation or associated with developmental delay. Permanent diabetes of non autoimmune origin can present up to 6 months from birth in individuals with KCNJ11 and EIF2AK3 mutations. Therefore, we suggest that the acronym PNDM be replaced with the more comprehensive permanent diabetes mellitus of infancy (PDMI), linking it to the gene product (e.g., GCK-PDMI, KCNJ11-PDMI) to avoid confusion between patients with early-onset, autoimmune type 1 diabetes.     

Patients with erythrophobia (fear of blushing) show abnormal autonomic regulation in mental stress conditions

OBJECTIVE: The objective of this study was to analyze the autonomic functions of patients with erythrophobia. METHODS: Forty patients with a diagnosis of erythrophobia (female/male ratio 18/22) without any other organic lesions and 20 healthy volunteers (female/male ratio 10/10) were assessed. Clinical evaluation was performed using a modified version of semistructured interviews. Autonomic testing was performed by means of spectral analysis of heart rate and continuous blood pressure by sparse discrete Fourier transformation at rest and under mental stress. RESULTS: There were no significant difference between the two samples in age, sex distribution, BMI, resting systolic, or diastolic blood pressure, nor was there a difference in autonomic baseline functioning between the 40 patients with erythrophobia and the control subjects. On the other hand, patients with erythrophobia consistently showed higher pulse rates (88 +/- 20 vs. 78 +/- 9 bpm, p <.05), higher total heart rate power values (8.40 +/- 0.63 vs. 8.07 +/- 1.02 p <.05), higher midfrequency spectral values (7.38 +/- 0.66 vs. 7.02 +/- 1.18, p <.01), higher high-frequency spectral values (6.89 +/- 0.86 vs. 6.48 +/- 1.44, p <.05), and lower baroreceptor sensitivity (8.62 +/- 8.16 vs. 11.65 +/- 4.42, p <.005) than the healthy subjects. ANOVA showed a significant group interaction (p <.0001) between the samples. CONCLUSIONS: This study provides evidence for abnormal autonomic functioning in patients with erythrophobia when under mental stress.     

Host response toBothrops asper snake venom

As part of the characterization of the host reactivity to the venom ofBothrops asper, we investigated the inflammatory responses in the mouse footpad model. The subcutaneously injected venom induced a rapid increase of serum IL-6 concentration, which peaked between 3 and 6 h and returned to normal values at 12 h. In contrast, serum TNF- and IL-1 were not detectable at any time point studied. A myotoxic phospholipase A₂ isoform purified from this venom, myotoxin II, was also able to induce a systemic IL-6 release when injected into the footpad. Both venom and myotoxin induced local edema and a leukocyte infiltrate accumulating in the muscle and subdermal tissue within 6 h. The infiltrate consisted predominantly of neutrophils at 6 and 24 h, but at later times, mononuclear cells also appeared. The edema, leukocyte infiltration, and IL-6 responses did not depend on the hemorrhagic activity of venom, since all three effects were seen after injection of (1) preneutralized venom, devoid of hemorrhagic activity, and (2) purified myotoxin II. Circulating platelet numbers were significantly decreased 30 min after venom injection and returned to normal after 12 h. The venom also induced a rapid inversion in the ratio of neutrophils to lymphocytes in peripheral blood, which did not normalize until 12 h later. The present observations suggest that venom, besides its cytotoxic properties, induces early hematologic and immunologic alterations. These findings may be of relevance in future treatment modalities.     

A two-stage study on multiple sclerosis susceptibility and chromosome 2q33

We have performed a two-stage study to analyse the association of polymorphism on chromosome 2q33 with multiple sclerosis (MS). In all, 17 markers were analysed in stage-1 in 134 Finnish MS families and the observed associations were tested in stage-2 in 186 MS families. We did not find previously reported allelic or haplotype associations with CTLA4. We obtained a weak signal of two distinct predisposing genes, one proximal the other distal of CTLA4. The putative proximal gene was associated with the marker rs3977 in families lacking HLA-DR2 (P=0.02 and 0.02) and the other distal gene was associated with D2S1271 in families from a high-risk region in western Finland (P=0.02 and 0.01). Based on the >3 cM distance and the lack of linkage disequilibrium between these loci, we conclude that the two association signals are independent. Our results provide preliminary evidence for two distinct MS susceptibility genes on 2q33 outside of CTLA4.     

Absence of Correlation Between Delayed-Type Hypersensitivity and Protection in Experimental Systemic Candidiasis in Immunized Mice

We found that in mice which had been immunized intraperitoneally with 2 × 10⁸ heat-killed Candida albicans cells there was a striking temporal relationship between resistance to systemic challenge with 10⁶ living C. albicans cells and a number of measurable cellular parameters of the host response. These included the emergence of delayed-type hypersensitivity and the development of granulocytosis. Since it had been shown in previous work that granulocytosis was associated with an increase in resistance when nonspecific immunostimulation was used, we performed experiments to induce delayed-type hypersensitivity without any measurable modification of the granulocyte population. Adoptive transfer of delayed-type hypersensitivity with spleen cells from immune and resistant donor mice did not produce any increase in resistance in normal recipients. When separate groups of mice were immunized intraperitoneally or subcutaneously with varying doses of heat-killed C. albicans, we found that doses of less than 10⁸ cells did induce significant delayed-type hypersensitivity without any increase in granulocytosis. In such mice, as well as in animals pretreated with immunomodulators before immunization with heat-killed C. albicans, the presence of cell-mediated immunity, as measured by the delayed-type hypersensitivity test, was not associated with an increase in resistance against systemic candidiasis. On the contrary, the results suggested that cell-mediated immunity was associated with an increase in the susceptibility of these mice. The same effect on candidiasis susceptibility was observed when animals were immunized with heat-killed filamentous C. albicans.