The goal of the present study was to design a vaccine that would provide universal protection against infection of humans with diverse influenza A viruses. Accordingly, protein sequences from influenza A virus strains currently in circulation (H1N1, H3N2), agents of past pandemics (H1N1, H2N2, H3N2) and zoonotic infections of man (H1N1, H5N1, H7N2, H7N3, H7N7, H9N2) were evaluated for the presence of amino acid sequences, motifs, that are predicted to mediate peptide epitope binding with high affinity to the most frequent HLA-DR allelic products. Peptides conserved among diverse influenza strains were then synthesized, evaluated for binding to purified HLA-DR molecules and for their capacity to induce influenza-specific immune recall responses using human donor peripheral blood mononuclear cells (PBMC). Accordingly, 20 epitopes were selected for further investigation based on their conservancy among diverse influenza strains, predicted population coverage in diverse ethnic groups and capacity to recall influenza-specific responses. A DNA plasmid encoding the epitopes was constructed using amino acid spacers between epitopes to promote optimum processing and presentation. Immunogenicity of the DNA vaccine was measured using HLA-DR4 transgenic mice and the TriGrid™ in vivo electroporation device. Vaccination resulted in peptide-specific immune responses, augmented HA-specific antibody responses and protection of HLA-DR4 transgenic mice from lethal PR8 influenza virus challenge. These studies demonstrate the utility of this vaccine format and the contribution of CD4 + T cell responses to protection against influenza infection.

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Our objective was to determine whether treatment-naive HIV-infected adolescents manifest abnormalities in thymus function and peripheral T cell repertoire, and to assess relationships of these immunologic characteristics with each other, with plasma HIV virus load, and T cell surface markers. TCR Vbeta repertoire was determined by CDR3 length spectratyping in purified CD4(+) and CD8(+) T cells of high-risk, HIV-negative adolescents and of treatment-naive, HIV-infected adolescents. Thymus function was investigated by the simultaneous examination of T cell receptor excision circles (TRECs) in the CD4(+) and CD8(+) T cell subsets. HIV-infected adolescents exhibited significantly greater perturbations in their TCR Vbeta repertoire in comparison with HIV-negative subjects. Perturbations in the CD8(+) T cell compartment were more profound in comparison with CD4(+) T cells. The CD4(+) TCR Vbeta perturbations were negatively correlated with the total and phenotypically naive CD4(+) T cells, and with CD4(+) TRECs. CD8(+) TRECs, although not correlated with CD8(+) TCR Vbeta perturbations, showed negative correlation with memory and activated CD8(+) T cells. Interestingly, TRECs in CD4(+) and CD8(+) T cells were not significantly different between HIV-infected and uninfected adolescents. The TCR Vbeta repertoire in adolescents is profoundly perturbed even in early stages of HIV infection, when total CD4(+) cell counts in most subjects are within normal limits. The correlative analyses demonstrating negative association of CD4(+) cell TRECs with CD4(+) TCR Vbeta perturbations and of CD8(+) TRECs with CD8(+) cell activation markers provide evidence of the intense activation of the central and peripheral immune compartments in this study population.

Children with chronic HIV infection manifest CD8 T cell clonal dominance, which appears to be dependent upon the adequacy of the CD4 cells. With optimization of therapy, a gain in clonal dominance is the predominant response, except in situations of failure to contain viral replication.

The epidermal keratinocytes express two major pairs of keratin polypeptides. One pair (K5/K14) expressed specifically in basal generative compartment and the other (K1/K10) expressed specifically in the differentiating suprabasal compartment. The switch in the expression of the keratins from proliferating to differentiating compartment indicates the changes that occur in the keratin filament organization which in turn influences the functional properties of the epidermis. Proper regulation of keratin gene expression and the filament organization are absolutely necessary for normal functioning of the skin. Keratin gene mutations can influence the filament integrity thereby causing several heritable blistering disorders of the skin such as epidermolysis bullosa, bullous icthyosiform erythroderma, etc. Changes in the keratin gene expression may lead to incomplete differentiation of the epidermal keratinocyte, causing hyperproliferative diseases of the skin such as psoriasis, carcinomas, etc. This review briefly describes the changes in keratin structure or gene expression that are known to result in various disorders of the skin.

Rationale: evaluation of the T-cell receptor (TCR) V␤-chain repertoire by PCR-based CDR3 length analysis allows fine resolution of the usage of the TCR V␤ repertoire and is a sensitive tool to monitor changes in the T-cell compartment. A multiplex PCR method employing 24 labeled upstream V␤ primers instead of the conventionally labeled downstream C␤ primer is described. Method: RNA was isolated from purified CD4 and CD8 T-cell subsets from umbilical cord blood and clinical samples using TRI reagent followed by reverse transcription using a C␤ primer and an Omniscript RT kit. The 24 V␤ primers were multiplexed based on compatibility and product sizes into seven reactions. cDNA was amplified using 24 V␤ primers (labeled with tetrachloro-6-cardoxyfluorescein, 6-carboxyfluorescein, and hexachloro-6-carboxyfluorescein), an unlabeled C␤ primer, and Taqgold polymerase. The fluorescent PCR products were resolved on an automated DNA sequencer and analyzed using the Genotyper 2.1 software. Results: V␤ spectratypes of excellent resolution were obtained with RNA amounts of 250 ng using the labeled V␤ primers. The resolution was superior to that obtained with the labeled C␤ primer assay. Also the numbers of PCRs were reduced to 7 from the 12 required in the C␤ labeling method, and the sample processing time was reduced by half. Conclusion: The method described for T-cell receptor V␤-chain repertoire analysis eliminates tedious dilutions and results in superior resolution with small amounts of RNA. The fast throughput makes this method suitable for automation and offers the feasibility to perform TCR V␤ repertoire analyses in clinical trials.

The intactness of monocyte function in chronic myeloid leukemia (CML) patients as assessed by their ability to secret tumor necrosis factor was evaluated. Monocytes from CML patients continued to respond to lipopolysaccharide (LPS) stimulation even during the refractory period, suggesting different pathways for stimulation by LPS and malignant processes.

Multiplexing immunoassays have become a very powerful and economic way to measure multiple analytes from a single sample. This is especially important when sample volumes are limited, such as for rodent samples. Life Technologies has recently launched a set of luminex bead-based immunoassays that can conveniently quantify between 10 and 30 cytokines from human or mouse tissue culture, serum and plasma samples. These assays use magnetic beads which offer a convenient way to automate these assays using commercially available plate washers, such as BioTek ELx405 or Tecan HydroSpeed. The mouse analytes in these kits are: GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 p40/p70, TNF-α (10-plex), supplemented with FGF basic, IL-1α, IL-13, IL-17, IP-10, KC, MCP-1, MIG, MIP-1α and VEGF (mouse 20-plex). Human analytes are GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10 and TNF-α (10-plex), supplemented with IL-1RA, IL-2R, IL-7, IL-12 p40/p70, IL-13, IL-15, IL-17, IFN-α, MIP-1α, MIP-1β, IP-10, MIG, eotaxin, RANTES, MCP-1, VEGF, G-CSF, FGF basic and HGF (human 30-plex). All the kits have been extensively validated to include sample recoveries routinely between 80 and 120%, intra-assay and inter-assay precision of <10%, and linearity of dilution with tissue culture medium and serum with R2 values of 0.98 and greater. Benchmarking of the kits has been performed against our ELISA kits to ensure equal results between the two immunoassay platforms.