Mould-sensitized adults have lower Th2 cytokines and a higher prevalence of asthma than those sensitized to other aeroallergens.

BACKGROUND: Evidence suggests that specific allergen sensitizations are associated with different allergic diseases which may reflect different underlying immune profiles. We aimed to examine the cytokine profiles of individuals sensitized to eight common aeroallergens. METHODS: We used data from the Tasmanian Longitudinal Health Study a population-based cohort study of 45-year-olds. Serum cytokines (IL-4, IL-5, IL-6, IL-8, IL-10, TNF-α) were measured in 1157 subjects using the LINCOplex assays. Participants underwent skin prick testing for house dust mite, cat, grasses and moulds. Multivariable linear regression was used to compare serum cytokine levels between sensitized and nonatopic subjects. RESULTS: The prevalence of allergic sensitization to any aeroallergen was 51% (95% CI 47-54). Being sensitized to any aeroallergen was strongly associated with current asthma (OR = 3.7, 95% CI 2.6-5.3), and being sensitized to any moulds was associated with a very high risk of current asthma (OR = 6.40, 95% CI 4.06-10.1). The geometric mean (GM) levels of Th2 cytokines (IL-4, IL-5 and IL-6) for adults sensitized to Cladosporium were significantly lower than the levels for nonatopic individuals (IL-4 ratio of GMs = 0.25, 95% CI 0.10-0.62, P = 0.003; IL-5 GM = 0.55, 95% CI 0.30-0.99, P = 0.05; and IL-6 GM = 0.50, 95% CI 0.24-1.07, P = 0.07). Individuals sensitized to other aeroallergens all showed elevated Th2 cytokine levels. CONCLUSION: Our study is the first large population-based study to demonstrate reduced Th2 cytokines levels in people sensitized to mould. Underlying biological mechanisms driving allergic inflammatory responses in adults sensitized to moulds may differ from those sensitized to other aeroallergens. These findings suggest that it may be necessary to tailor treatments in individuals sensitized to moulds compared with other aeroallergens in order to optimize outcomes.

Attenuated and wild-type HIV-1 infections and long terminal repeat-mediated gene expression from plasmids delivered by gene gun to human skin ex vivo and macaques in vivo.

Gene expression from HIV-based gene therapy vectors or live-attenuated HIV-1 vaccines requires RNA transcription supported by the HIV-1 promoter, the long terminal repeat (LTR). Delivery of live-attenuated HIV-1 vaccines as plasmid DNA would overcome problems associated with production of attenuated HIV-1 strains. We investigated the expression of reporter plasmids and proviral HIV-1 constructs driven by either the HIV-1 LTR or LTRs with deletions in the U3 enhancer regions. LTR-driven plasmids were inoculated by gene gun into both human epidermis ex vivo and macaques in vivo. The HIV-1 LTR drove reporter gene expression in human and macaque skin, although with 15- to 20-fold less efficiency compared to the immediate-early cytomegalovirus promoter. A deleted LTR derived from a naturally attenuated HIV-1 strain infecting a member of the well-characterized Sydney Blood Bank Cohort of long-term nonprogressors was 5-fold less efficient in expression of the reporter gene compared to wild-type LTR. Delivery of proviral wild-type HIV-1 DNA constructs to human skin resulted in recovery of HIV-1 from cells emigrating from the epidermis, providing an ex vivo model of the infectivity of proviral HIV-1 DNA. However, delivery of proviral HIV-1 DNA containing deletions in either the LTR, Nef, or the secondary viral transcription activator,Vpr, significantly reduced HIV-1 replication in this model. The early coexpression of Tat from a second plasmid did not restore replication. Thus, although attenuated lentiviral vaccines might be deliverable as proviral DNA constructs in primate subjects, significant improvements are needed to enhance the efficiency of this method.

Uptake of HIV and latex particles by fresh and cultured dendritic cells and monocytes.

Blood dendritic cells (DC) efficiently carry HIV-1 and transmit infection to CD4+ T cells in the absence of productive infection of the APC. Fluorescent latex beads were used to define the endocytic pathways that may contribute to this non-infectious pathway of virus carriage. Beads between 14 nm and 2300 nm in diameter were taken up by uncultured blood DC, but uptake of beads larger than 280 nm was much reduced in the DC compared to monocytes. After culture, there was a reduction in bead carriage in DC compared to monocytes. In the DC, beads were found as small aggregates in class II containing compartments or as single beads just below the cell surface. Beads accumulated in monocytes as aggregates in class II negative compartments. Bead recycling occurred in DC, but not in the fresh or cultured monocytes. Electron microscopy of HIV-1-pulsed DC cultured with CD4+ T cells showed accumulation of apoptotic debris and virions within endosomes in the DC. The peripheral location and recycling of endocytosed material in DC provides a pathway for virion transfer from DC to T cells that does not occur in monocytes.

Efficacy and kinetics of glycerol inactivation of HIV-1 in split skin grafts.

Allogeneic split skin grafts are used widely in the treatment of burns. The relative simplicity of glycerol preservation of skin suggests it will be used increasingly in areas of high HIV-1 seroprevalence. The ability of glycerol preservation to inactivate HIV-1 present in skin graft infected in vitro was determined using a macrophage tropic strain HIV-1 as a cell-free virus suspension, within infected PBMCs, or within in vitro HIV-1 infected fresh cadaveric split skin. Different temperatures and concentrations of glycerol were used and infectivity determined by coculture with mitogen activated peripheral blood mononuclear cells (PBMCs) and measurement of reverse transcriptase activity after 7-10 days. Cell-free HIV-1 was inactivated within 30 min at 4 degrees C in glycerol concentrations of 70% or higher. During similar exposure cell- or skin-associated HIV-1 titer was reduced but not eliminated with 70% and 85% glycerol at 4 degrees C. HIV-1 was recovered consistently from skin stored in 85% glycerol at 4 degrees C for up to 72 hr but virus isolation was infrequent after storage for more than 5 days. At 20 degrees C or 37 degrees C, 70% or 85% glycerol could inactivate cell- or skin-associated HIV-1 within 8 hr. The initial glycerolization procedures and the storage at 4 degrees C eliminated effectively HIV-1 from skin.

Monocyte-derived dendritic cells as a model for the study of HIV-1 infection: productive infection and phenotypic changes during culture in human serum.

Dendritic cells (DC) have been implicated in the initial selection for macrophage-tropic HIV-1 during transmission and in the generation of high-level virus replication during interactions with CD4 T cells. The role of DC as viral reservoirs and the extent of productive infection is unclear, but the ability to generate large numbers of DC from blood monocytes has produced a tractable model for study of DC-HIV-1 interactions. When cultured in granulocyte-macrophage colony stimulating factor and IL-4, sorted CD14+ monocytes rapidly lost phagocytic function for both 93 nm and 977 nm latex particles and developed the surface markers and function of DC. After 7 days, when returned to medium containing human serum without cytokines, some monocyte-derived dendritic cells (MDDC) became adherent, but retained the costimulatory markers CD80 and CD86 and continued to express CD83 and CD40. The MDDC stimulated allogeneic CD4 T cells, did not express new macrophage markers and remained non-phagocytic. With or without TNF-alpha, MDDC generated in cytokines were infected by macrophage and T cell-tropic virus and produced higher reverse transcriptase levels than did the autologous monocyte-derived macrophages (MDM). When added to T cells, the infected MDDC were able to infect T cells with a wider range of viral isolates than were MDM.

HIV-1 selection by epidermal dendritic cells during transmission across human skin.

Macrophage tropic HIV-1 is predominant during the initial viremia after person to person transmission of HIV-1 (Zhu, T., H. Mo, N. Wang, D.S. Nam, Y. Cao, R.A. Koup, and D.D. Ho. 1993. Science. 261:1179-1181.), and this selection may occur during virus entry and carriage to the lymphoid tissue. Human skin explants were used to model HIV-1 selection that may occur at the skin or mucosal surface. Macrophage tropic, but not T cell line tropic strains of HIV-1 applied to the abraded epidermis were recovered from the cells emigrating from the skin explants. Dermis and epidermis were separated by dispase digestion after virus exposure to determine the site of viral selection within the skin. Uptake and transmission to T cells of all HIV-1 isolates was found with the dermal emigrant cells, but only macrophage tropic virus was transferred by emigrants from the epidermis exposed to HIV-1, indicating selection only within the epidermis. CD3+, CD4+ T cells were found in both the dermal and epidermal emigrant cells. After cell sorting to exclude contaminating T cells, macrophage tropic HIV-1 was found in both the dermal emigrant dendritic cells and in dendritic cells sorted from the epidermal emigrants. These observations suggest that selective infection of the immature epidermal dendritic cells represents the cellular mechanism that limits the initial viremia to HIV-1 that can use the CCR5 coreceptor.

Scanning for T helper epitopes with human PBMC using pools of short synthetic peptides.

Major T helper epitopes of medically important antigens can be located by measuring the proliferative responses of human peripheral blood mononuclear cells (PBMC) to pools of short synthetic peptides. The length and endings of the peptides used were shown to be critical for success in identifying Th cell epitopes. Many epitopes would be missed if either long (31mers) or short (less than 12mers) peptides were used. Pools of 14 and 16mers were more efficient than 12mers spanning the same region, however, for a promiscuous Th cell epitope of tetanus toxin (tt 947-967), two of three donors tested did not respond to 18mers or shorter peptides spanning this region. Although peptides with either unblocked or blocked ends were stimulatory, peptides with blocked ends were generally more efficient. The peptide concentration and number of available APC were also found affect the efficiency of the proliferation assay as a measure of peptide recognition by Th cells. Two screenings of the entire set of tetanus toxin peptide pools using different samples of PBMC from the same donor identified common major stimulatory regions. Thus, PBMC and peptide pools can be used for the reproducible identification of Th cell epitopes. After immunization with tetanus toxoid (TT), peptide-responsive cells increased in frequency in parallel to the increase in TT responsive cells, indicating that the peptide-responsive cells were primed by TT.

Mapping the major human T helper epitopes of tetanus toxin. The emerging picture.

Progress on the mapping of Th epitopes of tetanus toxin (tt) has been slow due to reliance on studies of clones. In this paper, human Th cell epitopes of tt were mapped using proliferation tests on PBMC in response to synthetic peptides. PBMC from nine donors were tested over the entire set of tt homologous overlapping dodecapeptides. The 1304 peptides were initially tested as 66 pools, each containing an average of 20 peptides. PBMC from individual donors responded to as few as 1 and as many as 17 of the 66 peptide pools. The sequences responsible for proliferation were identified for the two most frequently recognized pools, and for another two pools within a major immunodominant region. Three new epitope sequences were mapped in detail and based on their recognition by most individuals are likely to be promiscuous. A cocktail of peptides including the newly identified Th cell epitopes was able to induce proliferation in PBMC from 24 of 31 tetanus toxoid (TT)-responsive donors. This cocktail is a chemically defined reagent that can be used to quantitate in vitro Ag-specific Th cells in PBMC from most subjects, and may thus be useful for serial measurements of specific immunity such as in subjects undergoing immunotherapy or immunosuppressive treatment.