Molecular epidemiology of tick-borne encephalitis virus in Ixodes ricinus ticks in Lithuania

In Lithuania, 171-645 serologically confirmed cases of tick-borne encephalitis occurred annually [Mickiene et al. (2001): Eur J Clin Microbiol Infect Dis 20:886-888] in 1993-1999, and the tick-borne encephalitis virus (TBEV) seroprevalence in the general population was found previously to be 3.0% [Juceviciene et al. (2002): J Clin Virol 25:23-27]. To assess the risk for TBEV virus infection in Lithuania and to characterize the agent a panel of 3,234 ticks combined into 436 pools [Juceviciene et al., 2005] were tested for presence of TBEV RNA by a nested RT-PCR targeting at the NS5 gene. Six pools were confirmed positive and the prevalence of the infected ticks was 0.2% (if one tick per pool [Juceviciene et al., 2005] was considered positive) and the proportion of positive tick pools was 1.4%. The prevalence of the infected ticks in the Panevezys, Siauliai, and Radviliskis regions (in central Lithuania) was 0.1%, 0.4%, and 1.7% corresponding with a higher TBE disease burden in these regions. The 252-nucleotide NS5-region amplicons, and a longer sequence (737 nucleotides) obtained from one sample from the PrM-E gene region, were sequenced. Phylogenetic analysis of the latter showed that all western type TBEV PrM-E sequences, including the Lithuanian strains, were monophyletic, showed no clustering and had very little variation. The NS5 sequences, although identical within one locality, did not show any mutations common to strains from the two Lithuanian regions, nor could any geographical clustering be found among western type TBEV strains from other areas.     

Suboptimal recognition of a T cell epitope of the major dog allergen Can f 1 by human T cells

We have previously proposed that mammalian lipocalin allergens are recognized suboptimally by the human immune system due to their homology with endogenous lipocalins. Here, we have characterized in detail the human T cell recognition of one of the previously identified T cell epitopes of the major dog allergen Can f 1, contained in peptide p105–120. A panel of peptide analogues (altered peptide ligands, APLs) of p105–120 was tested on two specific T cell clones restricted by different human leukocyte antigen (HLA) alleles. Interestingly, we identified for both of the clones several heteroclitic APLs that were capable of stimulating them at 10–30-fold lower concentrations than the natural peptide. Moreover, one of the heteroclitic APLs identified with the T cell clones, L115F, was observed to induce a stronger polyclonal T cell response than the natural allergen peptide from the peripheral blood mononuclear cells (PBMCs) of six Can f 1-allergic subjects studied. The heteroclitic APLs bound with the same affinity as p105–120 to common HLA-DR- and HLA-DP-alleles, suggesting that their improved stimulatory capacity is attributable to a more efficient T cell receptor (TCR) recognition rather than increased HLA binding. Collectively, our data suggest that p105–120 is recognized suboptimally by human T cells. This may contribute to the allergenicity of Can f 1.     

Early diagnosis of dengue in travelers: Comparison of a novel real-time RT-PCR,NS1 antigen detection and serology

BackgroundThe increased traveling to dengue endemic regions and the numerous epidemics have led to a rise in imported dengue. The laboratory diagnosis of acute dengue requires several types of tests and often paired samples are needed for obtaining reliable results. Although several diagnostic methods are available, proper comparative data on their performance are lacking.ObjectivesTo compare the performance of novel methods including a novel pan-DENV real-time RT-PCR and a commercially available NS1 capture-EIA in regard to IgM detection for optimizing the early diagnosis of DENV in travelers.Study designA panel of 99 selected early phase serum samples of dengue patients was studied by real-time RT-PCR, NS1 antigen ELISA, IgM-EIA, IgG-IFA and cell culture virus isolation.ResultsThe novel real-time RT-PCR was shown specific and sensitive for detection of DENV-1-4 RNA and suitable for diagnostic use. The diagnostic rate using combination of RNA and IgM detection was 99% and using NS1 and IgM detection 95.9%. The results of RNA and NS1 antigen detection disagreed in 15.5% of samples that had only RNA or NS1 antigen detected.ConclusionsThe diagnostic rates of early samples are higher when either RNA or NS1 antigen detection is combined with IgM detection. Besides the differences in the RNA and NS1 detection assays, the observed discrepancy of results could suggest individual variation or differences in timing of these markers in patient serum.     

Thymidine kinase 2 mutations in autosomal recessive progressive external ophthalmoplegia with multiple mitochondrial DNA deletions

Autosomal-inherited progressive external ophthalmoplegia (PEO) is an adult-onset disease characterized by the accumulation of multiple mitochondrial DNA (mtDNA) deletions in post-mitotic tissues. Mutations in six different genes have been described to cause the autosomal dominant form of the disease, but only mutations in the DNA polymerase gamma gene are known to cause autosomal recessive PEO (arPEO), leaving the genetic background of arPEO mostly unknown. Here we used whole-exome sequencing and identified compound heterozygous mutations, leading to two amino acid alterations R225W and a novel T230A in thymidine kinase 2 (TK2) in arPEO patients. TK2 is an enzyme of the mitochondrial nucleotide salvage pathway and its loss-of-function mutations have previously been shown to underlie the early-infantile myopathic form of mtDNA depletion syndrome (MDS). Our TK2 activity measurements of patient fibroblasts and mutant recombinant proteins show that the combination of the identified arPEO variants, R225W and T230A, leads to a significant reduction in TK2 activity, consistent with the late-onset phenotype, whereas homozygosity for R225W, previously associated with MDS, leads to near-total loss of activity. Our finding identifies a new genetic cause of arPEO with multiple mtDNA deletions. Furthermore, MDS and multiple mtDNA deletion disorders are manifestations of the same pathogenic pathways affecting mtDNA replication and repair, indicating that MDS-associated genes should be studied when searching for genetic background of PEO disorders.     

Dysregulation of innate immunity in hepatitis C virus genotype 1 IL28B-unfavorable genotype patients: impaired viral kinetics and therapeutic response

Recent studies have shown that a single-nucleotide polymorphism upstream of the interleukin-28B (IL28B) gene plays a major role in predicting therapeutic response in hepatitis C virus (HCV)-infected patients treated with pegylated interferon (PEG-IFN)/ribavirin. We sought to investigate the mechanism of the IL28B polymorphism, specifically as it relates to early HCV viral kinetics, IFN pharmacokinetics, IFN pharmacodynamics, and gene expression profiles. Two prospective cohorts (human immunodeficiency virus [HIV]/HCV-coinfected and HCV-monoinfected) completing treatment with IFN/ribavirin were enrolled. Patients were genotyped at the polymorphic site rs12979860. In the HIV/HCV cohort, frequent serum sampling was completed for HCV RNA and IFN levels. DNA microarray of peripheral blood mononuclear cells and individual expression of IFN-stimulated genes (ISGs) were quantified on IFN therapy. The IL28B-favorable (CC) genotype was associated with improved therapeutic response compared with unfavorable (CT or TT) genotypes. Patients with a favorable genotype had greater first- and second-phase viral kinetics (P = 0.004 and P = 0.036, respectively), IFN maximum antiviral efficiency (P = 0.007) and infected cell death loss (P = 0.009) compared with unfavorable genotypes. Functional annotation analysis of DNA microarray data was consistent with depressed innate immune function, particularly of natural killer cells, from patients with unfavorable genotypes (P <0.004). Induction of innate immunity genes was also lower in unfavorable genotypes. ISG expression at baseline and induction with IFN was independent of IL28B genotype. CONCLUSION: Carriers of the IL28B-favorable genotype were more likely to have superior innate immune response to IFN therapy compared with unfavorable genotypes, suggesting that the unfavorable genotype has aberrant baseline induction of innate immune response pathways resulting in impaired virologic response. IL28B genotype is associated with more rapid viral kinetics and improved treatment response outcomes independent of ISG expression.     

Audio Vestibular Status in CML Patients on Imatinib Mesylate with Review of Literature

Chronic myeloid leukemia (CML) is a myeloproliferative neoplasm occasionally presenting with features of leukostasis as primary symptoms. Hearing loss is occasionally reported in CML patients. Further whether Imatinib mesylate has any effect on vestibular functions is not known. We conducted a preliminary study to assess hearing pattern in patients with CML on long term TKI therapy. This is a single center, cross sectional study from northern-India. Patients of CML who were on regular Tyrosine kinase inhibitors (TKI) therapy for at least 6 months underwent audiovestibular evaluation. A total of 44 CML patients on TKI therapy were assessed over a period of 6 months. The median age of the patients was 41 years, the mean duration of TKI therapy was 36 months. Four patients were found to have otological disorder clinically. On pure tone audiometry of 88 ears normal hearing pattern were found with at low and mid frequencies. There was a down sloping type of curve at higher frequencies in PTA in most of the patients. Cold caloric tests in 42 patients were found as equal response in both ears. We conclude from this preliminary study that there are no audio vestibular dysfunctions amongst patients of CML on TKI. It’s a negative study wherein we have ruled out any auditory deficits secondary to Imatinib therapy. Further studies are required to evaluate the audiometric profile in CML patients before Imatinib therapy and to be compared with the patients already on Imatinib in a large cohort.