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Abstract: Although cryopreservation of peripheral blood mononuclear cells (PBMC) is a commonly used technique, the degree to which it affects subsequent functional studies has not been well defined. Here we demonstrate that long-term cryopreservation has detrimental effects on T cell IFN-γ responses in human immunodeficiency virus (HIV) infected individuals. Long-term cryopreservation caused marked decreases in CD4+ T cell responses to whole proteins (HIV p55 and cytomegalovirus (CMV) lysate) and HIV peptides, and more limited decreases in CD8+ T cell responses to whole proteins. These losses were more apparent in cells stored for greater than one year compared to less than six months. CD8+ T cell responses to peptides and peptide pools were well preserved. Loss of both CD4+ and CD8+ T cell responses to CMV peptide pools were minimal in HIV-negative individuals. Addition of exogenous antigen presenting cells (APC) did not restore CD4+ T cell responses to peptide stimulation and partially restored T cell IFN-γ responses to p55 protein. Overnight resting of thawed cells did not restore T cell IFN-γ responses to peptide or whole protein stimulation. A selective loss of phenotypically defined effector cells did not explain the decrement of responses, although cryopreservation did increase CD4+ T cell apoptosis, possibly contributing to the loss of responses. These data suggest that the impact of cryopreservation should be carefully considered in future vaccine and pathogenesis studies. In HIV-infected individuals short-term cryopreservation may be acceptable for measuring CD4+ and CD8+ T cell responses. Long-term cryopreservation, however, may lead to the loss of CD4+ T cell responses and mild skewing of T cell phenotypic marker expression. [Copyright &y& Elsevier]

We optimized a whole blood intracellular cytokine assay to quantitate the frequency of specific CD4+ and CD8+ T cells in small volumes of whole blood from infants from developing countries. The assay is performed in two steps. First, whole blood is stimulated in the presence of specific antigens for 6–18 h, ending with cryopreservation of fixed white cells. These stimulation steps were specifically adapted to be practical and reliable in a rural, developing country field setting. Later, in a more resourceful setting, interferon-gamma producing CD4+ or CD8+ T cells are detected by flow cytometry. The assay proved sensitive and specific for detecting mycobacteria-specific immunity 10 weeks after Bacillus Calmette-Guerin (BCG) vaccination of newborns from a rural field site. [Copyright &y& Elsevier]

The mechanisms responsible for oral tolerance have been analyzed using various antigens. The induction of oral nonresponsiveness to low doses of Staphylococcal Enterotoxin B (SEB), in which SEB-reactive clonal anergy and deletion are involved, has also been confirmed. However, few reports have examined the cytokine milieu of the mucosal and peripheral lymphoid tissue during tolerance induction after the oral administration of SEB. In this study, to elucidate the mechanism responsible for the oral tolerance to low dose of SEB, the cytokine responses of the gut-associated lymphoid tissues (GALT) and the spleen were examined using the reverse transcription-polymerase chain reaction (RT-PCR) method. After the oral administration of a single low dose of SEB, the expressions of Th2 and TGF-β, and to a lesser extent Th1 mRNA were observed in the mucosa. In contrast, following repeated oral low doses of SEB, in a manner different from previous results using conventional antigens, no cytokine enhancements were demonstrated in the GALT or the spleen. In conclusion, the involvement of unknown inhibitory or regulatory cytokines, together with SEB-reactive clonal anergy and deletion, is suggested in the induction of oral tolerance to low dose SEB. [Copyright &y& Elsevier]

The integrity of the skin is an important aspect of QOL. Whether caused by genetic deficiencies or environmental insults, disruption of the surface barrier allows irritants and allergens to penetrate the skin, which initiates inflammatory responses by immune cells that often lead to life-long allergies. In this study, eczema was induced on depilated mouse skin with topical lipopolysaccharide or a mixture of Staphylococcal enterotoxin B and an extract of house dust mites, which resulted in thickening of the epidermis, epidermal disruption, and abundant neutrophils in the dermis. Within 14 days of topical treatment with 1 μM svL4, a tetravalent peptide, neutrophils were absent, and the epidermis had returned to a normal morphology. The sequence of svL4 contains glutamine residues that serve as a cross-linking substrate for transglutaminase 2, which gains access to the skin surface where the epidermis becomes disrupted. In contrast, topical application of 1 μM svH1C, a peptide mimetic of sialic acid that lacks glutamine residues, or 1 μM dexamethasone was ineffective in restoring normal epidermal morphology. The data suggest that svL4 would be a powerful treatment for resolving severe eczema., (© 2022 The Authors.)