Identification and HLA-Tetramer-Validation of Human CD4(+) and CD8(+) T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4+ and CD8+ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8+ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4+ and CD8+ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4+ and CD8+ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4+ and 44 CD8+ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy

A prospective study of prognostic factors for duration of sick leave after endoscopic carpal tunnel release

<p>Abstract</p> <p>Background</p> <p>Endoscopic carpal tunnel release with a single portal technique has been shown to reduce sick leave compared to open carpal tunnel release, claiming to be a less invasive procedure and reducing scar tenderness leading to a more rapid return to work, and the purpose of this study was to identify prognostic factors for prolonged sick leave after endoscopic carpal tunnel release in a group of employed Danish patients.</p> <p>Methods</p> <p>The design was a prospective study including 75 employed patients with carpal tunnel syndrome operated with ECTR at two hospitals. The mean age was 46 years (SD 10.1), the male/female ratio was 0.42, and the mean preoperative duration of symptoms 10 months (range 6-12). Only 21 (28%) were unable to work preoperatively and mean sick leave was 4 weeks (range 1-4). At base-line and at the 3-month follow-up, a self-administered questionnaire was collected concerning physical, psychological, and social circumstances in relation to the hand problem. Data from a nerve conduction examination were collected at baseline and at the 3-month follow-up. Significant prognostic factors were identified through multiple logistic regression analysis.</p> <p>Results</p> <p>After the operation, the mean functional score was reduced from 2.3 to 1.4 (SD 0.8) and the mean symptom score from 2.9 to 1.5 (SD 0.7). The mean sick leave from work after the operation was 19.8 days (SD 14.3). Eighteen patients (24%) had more than 21 days of sick leave. Two patients (3%) were still unable to work after 3 months. Significant prognostic factors in the multivariate analysis for more than 21 days of postoperative sick leave were preoperative sick leave, blaming oneself for the hand problem and a preoperative distal motor latency.</p> <p>Conclusion</p> <p>Preoperative sick leave, blaming oneself for the hand problem, and a preoperative distal nerve conduction motor latency were prognostic factors for postoperative work absence of more than 21 days. Other factors may be important (clinical, demographic, economic, and workplace) in explaining the great variance in the results of sick leave after carpal tunnel release between studies from different countries.</p

Detection of large deletions in the LDL receptor gene with quantitative PCR methods

BACKGROUND: Familial Hypercholesterolemia (FH) is a common genetic disease and at the molecular level most often due to mutations in the LDL receptor gene. In genetically heterogeneous populations, major structural rearrangements account for about 5% of patients with LDL receptor gene mutations. METHODS: In this study we tested the ability of two different quantitative PCR methods, i.e. Real-Time PCR and Multiplex Ligation-Dependent Probe Amplification (MLPA), to detect deletions in the LDL receptor gene. We also reassessed the contribution of major structural rearrangements to the mutational spectrum of the LDL receptor gene in Denmark. RESULTS: With both methods it was possible to discriminate between one and two copies of the LDL receptor gene exon 5, but the MLPA method was cheaper, and it was far more accurate and precise than Real-Time PCR. In five of 318 patients with an FH phenotype, MLPA analysis revealed five different deletions in the LDL receptor gene. CONCLUSION: The MLPA method was accurate, precise and at the same time effective in screening a large number of FH patients for large deletions in the LDL receptor gene

Genomic characterization of five deletions in the LDL receptor gene in Danish Familial Hypercholesterolemic subjects

BACKGROUND: Familial Hypercholesterolemia is a common autosomal dominantly inherited disease that is most frequently caused by mutations in the gene encoding the receptor for low density lipoproteins (LDLR). Deletions and other major structural rearrangements of the LDLR gene account for approximately 5% of the mutations in many populations. METHODS: Five genomic deletions in the LDLR gene were characterized by amplification of mutated alleles and sequencing to identify genomic breakpoints. A diagnostic assay based on duplex PCR for the exon 7 – 8 deletion was developed to discriminate between heterozygotes and normals, and bioinformatic analyses were used to identify interspersed repeats flanking the deletions. RESULTS: In one case 15 bp had been inserted at the site of the deleted DNA, and, in all five cases, Alu elements flanked the sites where deletions had occurred. An assay developed to discriminate the wildtype and the deletion allele in a simple duplex PCR detected three FH patients as heterozygotes, and two individuals with normal lipid values were detected as normal homozygotes. CONCLUSION: The identification of the breakpoints should make it possible to develop specific tests for these mutations, and the data provide further evidence for the role of Alu repeats in intragenic deletions