A flowcytometric analysis to efficiently quantify multiple innate immune cells and T Cell subsets in human blood.

The balance of inflammation and immunosuppression driven by changed ratios in diverse myeloid and T cell subsets, as well as their state of activation and ability to migrate to lymphoid compartments or inflammatory sites, has emerged as a highly active area of study across clinical trials of vaccines and therapies against cancer, trauma, as well as autoimmune and infectious diseases. There is a need for effective protocols which maximally use the possibilities offered by modern flow cytometers to characterize such immune cell changes in peripheral blood using small volumes of human blood. Additionally, longitudinal clinical studies often use cryopreserved samples, which can impact flow cytometric results. To efficiently gauge both the innate and the adaptive immune response, two novel 15-color antibody panels to identify key myeloid and T cell subsets and their functional potential were established. This approach was used to compare cellular immune profiles in fresh whole blood and in matched cryopreserved peripheral blood mononuclear cells (PBMCs). Cocktail I was designed to identify and characterize myeloid cell populations including dendritic cells (DCs), monocytic monocyte-derived suppressor cells (MO-MDSC), and monocytes, determining further core aspects of their state of maturity, T cell stimulatory (or inhibitory) potential, and migration capability. Cocktail II was used for phenotyping diverse T cells subsets, and their key migration and functional regulatory capabilities. The two 15-color antibody panels for the evaluation of both immune-stimulating and immunosuppressive processes presented herein allowed for efficient evaluation of the balance of immune activation versus immunosuppression across key blood cells, with good resolution for all 15 markers stained for in each panel. Gating strategies for the myeloid and T cells are presented to further support specific subset identification. This protocol was shown to be reproducible across donors and useful to study both RBC-lysed whole blood and cryopreserved PBMCs. © 2017 International Society for Advancement of Cytometry.

Basic fibroblast growth factor and tumour necrosis factor alpha in vitiligo and other hypopigmented disorders: suggestive possible therapeutic targets.

BACKGROUND: In healthy skin, there is a molecular microenvironment that favours the survival of melanocytes and regulates their function. Keratinocytes synthesize and secrete several cytokines that have stimulatory and inhibitory effects on melanocytes. AIM OF THE WORK: This work was conducted to evaluate the expression of basic fibroblast growth factor (bFGF) and tumour necrosis factor alpha (TNF-α) mRNA levels in lesional skin of vitiligo, hypopigmented mycosis fungoides and hypopigmented tinea versicolor. PATIENTS AND METHODS: Forty eight patients (25 vitiligo, 14 hypopigmented mycosis fungoides, 9 hypopigmented tinea versicolor) and 10 healthy controls were included. A 4 mm punch skin biopsy was taken from lesional skin of patients, and the normal skin of controls for quantitative PCR examination of TNF-α and bFGF mRNA. RESULTS: The level of TNF-α mRNA in lesional skin of the three studied disorders was significantly higher than in the control group, while the level of bFGF mRNA was significantly lower in lesional skin of the three diseases than the control skin. A significant inverse correlation was demonstrated between the mRNA levels of the two studied cytokines in vitiligo and hypopigmented MF lesions. CONCLUSION: The study's findings demonstrate that the studied hypopigmented (vitiligo, hypopigmented MF, hypopigmented TV) disorders show similar changes in their cutaneous microenvironment with increased TNF-α and decreased bFGF mRNA expression. This cytokine microenvironment change may be implicated in the pigment loss and hence these cytokines may have future therapeutic implications.

Narrow band Ultraviolet B 311 nm in the treatment of vitiligo: two right-left comparison studies.

AIM: Evaluation of narrow band ultraviolet B (NB UVB 311 nm) in the treatment of vitiligo by two independent studies. The first study compared NB UVB with a well-established therapeutic modality, psoralen ultraviolet A (PUVA), and the second study was conducted to find out whether psoralen might add to its efficacy. METHODS: In the first study, 15 patients were exposed on the left half of their body to UVB 311 nm and then exposed on their right half to UVA after ingestion of psoralen. In the second study, 20 patients were exposed to UVB 311 nm on the left side of the body, followed by ingestion of psoralen and exposure to NB UVB 311 nm 90 min later to the right side of the body. In both studies, while exposing one side, the other was protected by an UV-proof gown. Thus two right-left comparative studies were carried out simultaneously, namely: UVB 311 nm vs. PUVA and UVB 311 nm vs. PUVB 311 nm. RESULTS: In the first study, comparison of PUVA and NB UVB 311 nm showed no difference either in the degree of response or in the incidence of complications. In the second study, comparison of PUVB and UVB showed equal clinical improvement on both sides. The cumulative dose needed to achieve the same response on the PUVB side was lower than that on the UVB side, but the difference was not statistically significant. The incidence of phototoxic reactions was significantly higher on the PUVB treated body half. CONCLUSION: NB UVB 311 nm has similar repigmentary effects as PUVA. The addition of psoralen does not increase its efficacy.