Interplay of H2A deubiquitinase 2A-DUB/Mysm1 and the p19(ARF)/p53 axis in hematopoiesis, early T-cell development and tissue differentiation.

Monoubiquitination of core histone 2A (H2A-K119u) has a critical role in gene regulation in hematopoietic differentiation and other developmental processes. To explore the interplay of histone H2A deubiquitinase Myb-like SWIRM and MPN domain containing1 (2A-DUB/Mysm1) with the p53 axis in the sequential differentiation of mature lymphocytes from progenitors, we systematically analyzed hematopoiesis and early T-cell development using Mysm1(-/-) and p53(-/-)Mysm1(-/-) mice. Mysm1(-/-) thymi were severely hypoplastic with <10% of wild-type cell numbers as a result of a reduction of early thymocyte progenitors in context with defective hematopoietic stem cells, a partial block at the double-negative (DN)1-DN2 transition and increased apoptosis of double-positive thymocytes. Increased rates of apoptosis were also detected in other tissues affected by Mysm1 deficiency, including the developing brain and the skin. By quantitative PCR and chromatin immunoprecipitation analyses, we identified p19(ARF), an important regulator of p53 tumor suppressor protein levels, as a potential Mysm1 target gene. In newly generated p53(-/-)Mysm1(-/-) double-deficient mice, anomalies of Mysm1(-/-) mice including reduction of lymphoid-primed multipotent progenitors, reduced thymocyte numbers and viability, and interestingly defective B-cell development, growth retardation, neurological defects, skin atrophy, and tail malformation were almost completely restored as well, substantiating the involvement of the p53 pathway in the alterations caused by Mysm1 deficiency. In conclusion, this investigation uncovers a novel link between H2A deubiquitinase 2A-DUB/Mysm1 and suppression of p53-mediated apoptotic programs during early lymphoid development and other developmental processes.

8-Isoprostane is a dose-related biomarker for photo-oxidative ultraviolet (UV) B damage in vivo: a pilot study with personal UV dosimetry.

BACKGROUND: Ultraviolet (UV) B irradiation causes visible erythema, which has been linked with DNA damage. However, besides such direct photochemical conformation changes, UVB also induces many indirect photochemical effects in the skin. Lipid peroxidation (LPO) is in this context one of the major pathways by which photo-oxidative stress disturbs cell signalling and promotes photocarcinogenesis and photoageing. So far we lack techniques for visualizing photo-oxidative stress in the skin. Furthermore, LPO has never been linked with individually acquired UVB doses measured by personal dosimetry. OBJECTIVES: Measuring the skin reaction and photo-oxidative damage by LPO in vivo after UVB exposure in a pilot study surveyed by personal dosimetry in order to allow for a correlation analysis of acquired dose, skin reaction and amount of LPO. METHODS: UVB exposure was measured with the opto-electronic X2000-1 (Gigahertz Optik, Puchheim, Germany) and the biological DLR Biofilm (German Aerospace Center DLR, Cologne, Germany) portable dosimeter. The skin reaction following UVB exposure was quantified with a Minolta chromameter (Minolta, Tokyo, Japan) and LPO in vivo was measured by 8-isoprostane generation by means of densitometric analysis of immunohistochemical samples obtained 30 min post-UVB irradiation. RESULTS: Regression analysis revealed significant linear relations between UVB exposures recorded by the dosimeters and colorimetry parameters of the skin reaction. Furthermore, an even better linear relation with higher significance was found between the generation of 8-isoprostane in the skin and the dosimeter readouts. CONCLUSIONS: LPO measured by the generation of 8-isoprostane provides a suitable intrinsic biomarker for photo-oxidative UVB damage in vivo. This study provides a new approach to visualizing photo-oxidative stress in the skin in vivo. Furthermore, future dosimeter readouts can now be set into relation to the expected increase of LPO that can be calculated within the limits of our study.

Relation between homocysteine and non-fatal stroke in peripheral arterial disease.

There are currently no data on whether high total serum homocysteine (tHcy) is predictive for cerebrovascular events in patients with symptomatic peripheral arterial disease (PAD). Therefore, the purpose of this study was to determine whether high tHcy levels were related to the evidence of non-fatal stroke in PAD. Evidence of non-fatal atherothrombotic stroke events was verified in 450 consecutive male patients, admitted for inpatient treatment of symptomatic PAD. The extent of carotid stenosis was evaluated by colour duplex Doppler measurement and fasting tHcy was determined by high-performance liquid chromatography. Within the population of 450 PAD patients a documented history of ischaemic stroke was evident in 50 subjects. The median tHcy values were significantly higher in PAD patients with stroke (18.6 micromol/l) than in PAD patients without stroke (15.1 micromol/l, P < 0.001). Logistic regression analysis revealed that tHcy was an independent and significant predictor (P=0.001) with an odds ratio (OR) of 1.37 for an increment of 5 micromol/l. In this multivariate model, diabetes mellitus (OR=2.34, P=0.011) and carotid stenosis > or =50% (OR=2.59, P=0.005) were also independently related to clinical cerebrovascular disease in PAD. In conclusion, the present study demonstrates an association of tHcy and evidence of non-fatal atherothrombotic stroke in patients with symptomatic PAD. This could be important, as a reduction of elevated tHcy concentrations by vitamin supplement might decrease the high frequency of cerebrovascular complications in PAD patients.

Influence of activation and differentiation of cells on the effectiveness of photodynamic therapy.

Photodynamic therapy combines photosensitizers absorbing light in the red spectral region and irradiation with light of the corresponding wavelength. To analyse the influence of cell differentiation on susceptibility to photodynamic therapy, we compared the proliferation inhibition induced by photodynamic therapy on normal human keratinocytes, spontaneously transformed human keratinocyte cell line HaCat and on squamous cell carcinoma lines. Cells were irradiated with polychromatic methylene blue as well as the precursor of protoporphyrin, 5-aminolevulinic acid. When incubated with Photosan-3, normal human keratinocytes exhibited and ED50 about 10-fold lower than the other cell lines studied. When methylene blue and 5-aminolevulinic acid were used, photodynamic therapy had comparable effects on all cell types. Stimulation of normal human keratinocytes with either EGF-alpha or IFN-gamma resulted in an increase susceptibility to photodynamic therapy when 5-aminolevulinic acid was used. This effect was more pronounced in the case of EGF-alpha. Our experiments suggest that activation and differentiation are two important parameters determining susceptibility to photodynamic therapy.

Stable engraftment of human female genital mucous membrane xenografts on SCID mice.

We developed a model in which full-thickness human genital mucous membranes (fallopian tubes, endometrium) were heterotopically xenografted into the skin of severe combined immunodeficient (SCID) mice. The transplanted tissue retained its human phenotype for at least 4 weeks including the glandular epithelium, the lamina propria, and main parts of the grafted vessels. By using an occlusive chamber filled with covering phosphate-buffered saline we created a system that protected the moist human epithelial surface. This system will allow the study of the interaction of test substances, or of invasive, pathogenic microorganisms, with epithelial cells and other cellular components of the human genital mucosa under in vivo conditions.

Psoriasiform architecture of murine epidermis overlying human psoriatic dermis transplanted onto SCID mice.

Preliminary observations in a xenogeneic SCID mouse transplantation model indicated that murine epidermis overgrows human dermis from psoriatic skin but not that form normal skin. To investigate the effect of peripheral blood mononuclear cells on the differentiation of murine keratinocytes, we transplanted involved and uninvolved full-thickness skin from patients with psoriasis onto SCID mice and followed this with repeated subcutaneous injections of cells suspended in patient serum. After 6 weeks grafts were analysed morphologically and immunohistochemically. The epidermis in grafts from clinically uninvolved skin appeared normal. The persistence of a psoriasiform epidermis was noted in all grafts from affected sites despite a lack of lymphocytic infiltration. Staining for human and mouse MHC class I antigens revealed the murine origin of keratinocytes forming the psoriasiform epidermis, while the human dermis was retained. Our observations indicate that the defect underlying the pathogenesis of psoriasis is most likely located in the dermal rather than the epidermal compartment. This xenogeneic transplantation model may be useful for future studies of the pathogenesis and treatment of psoriasis.

In vivo targeting of integrin receptors in human skin xenografts by intravenously applied antibodies.

We examined whether systemically injected monoclonal antibodies (mAbs) directed to cell-surface glycoproteins of human keratinocytes reach their target antigens in xenograft transplants of normal human skin on SCID mice. The integrins alpha 6 beta 4, expressed in the basal cell layer of human epidermis, and glycoprotein T16 (gp 40/50), expressed in terminally differentiating keratinocytes of the stratum spinosum, were selected as targets. It was found that all injected mAbs selectively localized to their antigens and bound and saturated their targets even in the uppermost layers of the stratum malpighii. This could easily be monitored by direct immunofluorescence staining since SCID mice lack endogenous production of significant amounts of immunoglobulins. After a single injection, mAbs could still be detected at the target site after 14 days. Our results proved that heterologous immunoglobulins pass systemic capillary filters in this xenograft model and specifically bind to their target molecules. Thus, xenografted SCID mice provide a versatile model for studying cell-surface glycoprotein-mediated interactions by the use of functionally interfering antibodies under in vivo conditions in human skin.

Cell surface glycoproteins define distinct states of eccrine gland differentiation.

The capability to distinguish eccrine gland cells of the ductal compartment from the secretory portion on the molecular level, as well as from epidermal keratinocytes and other skin cells, is of importance for the study of eccrine differentiation and function. Furthermore, the assessment of differences between the cell systems is useful for the characterization of benign and malignant neoplasms derived from eccrine glands. In the present study, we analysed the expression of four selected epithelial cell surface glycoproteins (gp 80, gp 38, gp 115 and gp 200) in eccrine glands using specific monoclonal antibodies. We found that the glycoproteins are differentially expressed in the ductal compartments and in the secretory portion of the glands, as well as in normal epidermis and other skin cells. This permitted the assignment of specific phenotypes to cells of the ductal compartment and to those of the secretory portion.