Membrane fatty acid heterogeneity of leukocyte classes is altered during in vitro cultivation but can be restored with ad-hoc lipid supplementation.

BACKGROUND: The cell membrane is a primary and fundamental player in most cellular processes, and fatty acids form a major structural component of cell membranes. The aim of this study was to compare the membrane fatty acid profiles of different human blood leukocytes and selected cell lines, to identify the effects of in vitro culture on fatty acid profiles, and to test medium supplements for their effect on fatty acid profiles. METHODS: Different classes of leukocytes were isolated from human blood and their membrane fatty acid profiles were analysed and compared. After culturing in vitro immortalised and primary leukocytes, membrane fatty acids were analysed and compared. Finally, different lipid formulations were developed and used for supplementing leukocytes in vitro in an effort to maintain the in vivo fatty acid profile. Descriptive and analytical tests were performed to compare the obtained fatty acid profiles. RESULTS: Membrane fatty acid profiles of primary human CD4(+) T-lymphocytes, CD8(+) T-lymphocytes, B-lymphocytes and monocytes differed. Moreover, there were differences among Jurkat, Raji and THP-1 cell lines and the corresponding primary leukocyte classes, as well as between freshly prepared and in vitro cultured primary lymphocytes. A lipid supplement was able to maintain cultured Jurkat cells with a membrane fatty acid profile almost identical to that of the primary CD4(+) T-lymphocytes. Finally, variations in the lipid supplement composition enabled the development of Jurkat cells with different membrane fatty acid profiles characterising different physiological or pathological human conditions. CONCLUSIONS: Each leukocyte class has its own specific membrane fatty acid profile in vivo. Cultured primary leukocytes and immortalized leukocytic cells display different membrane fatty acid profiles when compared to their respective in vivo counterparts. The membrane fatty acid composition of cultured cells can be restored to reflect that of the corresponding in vivo condition through use of optimised lipid supplementation. Typical physiological or pathological leukocyte membrane fatty acid profiles can be obtained by tuning in vitro fatty acid supplementation.

Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.

BACKGROUND: CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe. METHODS: We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. RESULTS: One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CONCLUSIONS: CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).

Unique and cross-reactive T cell epitope peptides of the major Bahia grass pollen allergen, Pas n 1.

BACKGROUND: Bahia grass pollen (BaGP) is a major cause of allergic rhinitis. Subcutaneous allergen-specific immunotherapy is effective for grass pollen allergy, but is unsuitable for patients with moderate to severe asthma due to the risk of anaphylaxis. T cell-reactive but IgE nonreactive peptides provide a safer treatment option. This study aimed to identify and characterize dominant CD4(+) T cell epitope peptides of the major BaGP allergen, Pas n 1. METHODS: Pas n 1-specific T cell lines generated from the peripheral blood of BaGP-allergic subjects were tested for proliferative and cytokine response to overlapping 20-mer Pas n 1 peptides. Cross-reactivity to homologous peptides from Lol p 1 and Cyn d 1 of Ryegrass and Bermuda grass pollen, respectively, was assessed using Pas n 1 peptide-specific T cell clones. MHC class II restriction of Pas n 1 peptide T cell recognition was determined by HLA blocking assays and peptide IgE reactivity tested by dot blotting. RESULTS: Three Pas n 1 peptides showed dominant T cell reactivity; 15 of 18 (83%) patients responded to one or more of these peptides. T cell clones specific for dominant Pas n 1 peptides showed evidence of species-specific T cell reactivity as well as cross-reactivity with other group 1 grass pollen allergens. The dominant Pas n 1 T cell epitope peptides showed HLA binding diversity and were non-IgE reactive. CONCLUSIONS: The immunodominant T cell-reactive Pas n 1 peptides are candidates for safe immunotherapy for individuals, including those with asthma, who are allergic to Bahia and possibly other grass pollens.

Immune response to a potyvirus with exposed amino groups available for chemical conjugation.

BACKGROUND: The amino terminus of the tobacco etch virus (TEV) capsid protein is located on the external surface of infectious TEV particles, as proposed by previous studies and an in silico model. The epsilon amino groups on the exposed lysine residues are available for chemical conjugation to any given protein, and can thus act as antigen carriers. The availability of amino groups on the surfaces of TEV particles was determined and the immune response to TEV evaluated. RESULTS: Using a biotin-tagged molecule that reacts specifically with amino groups, we found that the TEV capsid protein has amino groups on its surface available for coupling to other molecules via crosslinkers. Intraperitoneal TEV was administered to female BALB/c mice, and both their humoral and cellular responses measured. Different IgG isotypes, particularly IgG2a, directed against TEV were induced. In a cell proliferation assay, only spleen cells from vaccinated mice that were stimulated in vitro with TEV showed significant proliferation of CD3+/CD4+ and CD3+/CD8+ subpopulations and secreted significant amounts of interferon γ. CONCLUSIONS: TEV has surface amino groups that are available for chemical coupling. TEV induces both humoral and cellular responses when administered alone intraperitoneally to mice. Therefore, TEV should be evaluated as a vaccine adjuvant when chemically coupled to antigens of choice.

Toward protein biomarkers for allergy: CD4+ T cell proteomics in allergic and nonallergic subjects sampled in and out of pollen season.

Allergy is an immunological disorder of the upper airways, lung, skin, and the gut with a growing prevalence over the last decades in Western countries. Atopy, the genetic predisposition for allergy, is strongly dependent on familial inheritance and environmental factors. These observations call for predictive markers of progression from atopy to allergy, a prerequisite to any active intervention in neonates and children (prophylactic interventions/primary prevention) or in adults (immunomodulatory interventions/secondary prevention). In an attempt to identify early biomarkers of the "atopic march" using minimally invasive sampling, CD4+ T cells from 20 adult volunteers (10 healthy and 10 with respiratory allergies) were isolated and quantitatively analyzed and their proteomes were compared in and out of pollen season (± antigen exposure). The proteome study based on high-resolution 2D gel electrophoresis revealed three candidate protein markers that distinguish the CD4+ T cell proteomes of normal from allergic individuals when sampled out of pollen season, namely Talin 1, Nipsnap homologue 3A, and Glutamate-cysteine ligase regulatory protein. Three proteins were found differentially expressed between the CD4+ T cell proteomes of normal and allergic subjects when sampled during pollen season: carbonyl reductase, glutathione S-transferase ω 1, and 2,4-dienoyl-CoA reductase. The results were partly validated by Western blotting.

Caffeic acid phenethyl ester inhibits nuclear factor-kappaB and protein kinase B signalling pathways and induces caspase-3 expression in primary human CD4+ T cells.

Caffeic acid phenethyl ester (CAPE), an active component in propolis, is known to have anti-tumour, anti-inflammatory and anti-oxidant properties. In this study, the effects of CAPE on the functions of primary human CD4+ T cells were evaluated in vitro. CAPE significantly suppressed interferon (IFN)-gamma and interleukin (IL)-5 production and proliferation of CD4+ T cells stimulated by soluble anti-CD3 and anti-CD28 monoclonal antibodies in both healthy subjects and asthmatic patients. CAPE inhibited nuclear factor (NF)-kappaB activation and protein kinase B (Akt) phosphorylation, but not p38 mitogen-activated protein kinase (MAPK) phosphorylation in T cells. CAPE also induced active caspase-3 expression in CD4+ T cells; CCR4+CD4+ T cells were more sensitive to CAPE induction than CXCR3+CD4+ T cells. Together, these results indicate that CAPE inhibits cytokine production and proliferation of T cells, which might be related to the NF-kappaB and Akt signalling pathways, and that CCR4+CD4+ T cells are more sensitive to CAPE inhibition. This study provides a new insight into the mechanisms of CAPE for immune regulation and a rationale for the use of propolis for the treatment of allergic disorders.

How can (-)-epigallocatechin gallate from green tea prevent HIV-1 infection? Mechanistic insights from computational modeling and the implication for rational design of anti-HIV-1 entry inhibitors.

Possible inhibitors preventing human immunodeficiency virus type 1 (HIV-1) entry into the cells are recognized as hopeful next-generation anti-HIV-1 drugs. It is highly desirable to develop a potent inhibitor blocking binding of glycoprotein CD4 of the cell with glycoprotein gp120 of HIV-1, because the gp120-CD4 binding is the initial step of HIV-1 entry into the cells. It has been recently reported that (-)-epigallocatechin gallate (EGCG) from green tea is an inhibitor blocking gp120-CD4 binding. But the inhibitory mechanism remains unknown. For understanding the inhibitory mechanism, extensive molecular docking, molecular dynamics simulations, and binding free-energy calculations have been performed in this study to predict the most favorable structures of CD4-EGCG, gp120-CD4, and gp120-CD4-EGCG binding complexes in water. The results reveal that EGCG binds with CD4 in such a way that the calculated binding affinity of gp120 with the CD4-EGCG complex is negligible. So, the favorable binding of EGCG with CD4 can effectively block gp120-CD4 binding. The calculated CD4-EGCG binding affinity (DeltaG(bind) = -5.5 kcal/mol, K(d) = 94 microM) is in excellent agreement with available experimental data suggesting IC(50) approximately 100 microM for EGCG-blocking CD4-gp120 binding. These results and insights provide a rational basis for future design of novel, more potent inhibitors to block gp120-CD4 binding.

Changes in the leukocyte distribution and surface expression of adhesion molecules induced by hypothalamic stimulation in the cat.

Emotions and the neuroendocrine system are known to affect leukocyte distribution. However, there have so far been few reports on the relationship between hypothalamically induced emotional behavior and the endocrine-immune response. We previously reported changes in the leukocyte distribution and adhesion molecules induced by anteromedial hypothalamus stimulation (AH stimulation), which elicits restlessness behaviors in the cat. In this study, we examined ventromedial hypothalamus stimulation (VMH stimulation), which elicits threat behaviors. In addition, the endocrine responses after VMH stimulation were evaluated. VMH stimulation as well as AH stimulation induced elevations of plasma cortisol and epinephrine levels and granulocytosis and lymphopenia. In contrast, VMH stimulation induced only an elevation of plasma norepinephrine and elicited an opposite pattern of CD62L expression on the leukocyte subpopulations. The different endocrine-immunological reactions between VMH stimulation and AH stimulation were thus associated with different types of behavioral responses.

Effect of vitamin supplementation on cytokine response and on muscle damage after strenuous exercise.

The present double-blinded, placebo-controlled study investigated whether antioxidant vitamin supplementation was able to modulate the cytokine and lymphocyte responses after strenuous eccentric exercise. Furthermore, muscle enzyme release was examined to see whether antioxidant treatment could reduce muscle damage. Twenty male recreational runners randomly received either antioxidants (500 mg of vitamin C and 400 mg of vitamin E) or placebo for 14 days before and 7 days after a 5% downhill 90-min treadmill run at 75% .VO(2 max). Although the supplemented group differed significantly with regard to plasma vitamin concentration before and after exercise when compared with the placebo group, the two groups showed identical exercise-induced changes in cytokine, muscle enzyme, and lymphocyte subpopulations. The plasma level of interleukin (IL)-6 and IL-1 receptor antagonist increased 20- and 3-fold after exercise. The plasma level of creatine kinase was increased sixfold the day after exercise. The concentrations of CD4+ memory T cells, CD8+ memory and naïve T cells, and natural killer cells increased at the end of exercise. The total lymphocyte concentration was below prevalues in the postexercise period. In conclusion, the present study does not support the idea that exercise-induced inflammatory responses are induced by free oxygen radicals.

Intragraft events preceding chronic renal allograft rejection in a modified tolerance protocol.

BACKGROUND: Inbred miniature swine treated for 12 days with high-dose cyclosporine A develop tolerance to histocompatibility complex (MHC) class I-mismatched renal allografts. When this protocol was modified by adding thymectomy before transplant, all animals developed acute rejection. Thereafter, by day 100, one half developed chronic rejection (progression group) and the other half recovered (recovery group). This provides an excellent experimental model to identify the mechanisms of chronic rejection as well as the early changes that may predict chronic rejection. METHODS: We assessed the cellular infiltration, immune activation, humoral immunity, and cell- and antibody-mediated graft injury in the progression and the recovery groups. In addition, we also examined circulating donor reactive cytotoxic T lymphocyte (CTL) and antidonor antibody in both groups. RESULTS: From days 8 to 18 after transplantation, the two groups were indistinguishable. Both showed acute rejection with endarteritis (type II); had IgG and IgM deposition in glomeruli and small vessels; had an infiltrate with similar numbers of T cells, proliferating (PCNA+) and activated (interleukin-2 receptor+) cells; and had a similar degree of parenchymal cell apoptosis [in situ DNA nick-end labeling (TUNEL)+]. However, by days 30 to 60, the two groups could be distinguished by several intragraft features. The recovery group became tolerant and had diminished T-cell infiltration, activation and proliferation, and no detectable antibody deposition. The number of TUNEL+-injured parenchymal cells decreased. In contrast, the progression group showed persistent cell infiltration with activation and proliferation. Significantly prominent TUNEL+ apoptotic parenchymal cells in tubules, glomeruli, peritubular capillaries and arteries were seen from day 30 to day 100. Circulating donor reactive CTL and antidonor class I IgG were detected in the progression group at higher levels than in the recovery group from days 30 to 60. CONCLUSION: In tolerance-induction protocols, unstable tolerance induction is associated with the persistent immunologic activation that mediates immunologic destruction of graft parenchymal cells and chronic rejection. Certain of the described immunopathologic findings (activation, proliferation, apoptosis, and antibody deposition) may be useful in distinguishing the type of rejection, that is, whether the allograft will progress to chronic rejection or recovery.

Effect of ascorbic acid supplementation on the immune response of chickens vaccinated and challenged with infectious bursal disease virus.

One-day-old chickens were divided into two groups and reared under similar conditions. One group was fed a diet supplemented with 1000ppm ascorbic acid and the other group was fed an identical diet, but not supplemented with ascorbic acid. Both groups were vaccinated against infectious bursal disease (IBD) at 7 days of age and challenged orally with 4x10(5) of 50% embryo-lethal-dose IBDV 14 days later. The number of anti-IBDV antibody secreting cells, production of interleukin-2 (IL-2) by splenocytes, number of CD4(+), CD8(+) and IgM(+) cells in spleen and IgM(+) cells in bursa of Fabricius were compared between the two groups at 7 days (prior to vaccination), 21 days (14 days post-vaccination and prior to challenge) and 31 days (10 days post-challenge) of age. The number of CD8(+) in spleen at 7 days of age and IgM(+) cells in bursa at 7, 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Production of IL-2 by splenocytes was higher as indicated by higher stimulation indices in ascorbic acid supplemented group. The number of anti-IBDV IgG antibody secreting cells in spleen at 21 and 31 days of age were significantly higher in ascorbic acid supplemented group (P<0.05). Dietary supplementation of ascorbic acid may ameliorate the immunosuppression caused by IBDV vaccination and improve humoral and cellular immune responses.