CD34 Staining as a Useful Tool in Disorders of Collagen Degeneration.

ABSTRACT: The human progenitor-cell antigen CD34 is expressed in dermal dendritic cells and is lost in several disorders affecting dermal collagen. The loss of CD34 immunohistochemical staining has been demonstrated to be helpful in the histologic diagnosis of morphea, lichen sclerosus, and the classic pattern of granuloma annulare. This study characterized CD34 expression in 2 sclerosing disorders affecting the subcutis: lipodermatosclerosis (LDS) and the sclerodermoid form of chronic graft-versus-host disease (ScGVHD). In addition, we applied CD34 staining to the interstitial pattern of granuloma annulare (IGA), which is a diagnostically challenging entity with subtle amounts of dermal collagen degeneration. Fifteen cases of LDS, 6 cases of ScGVHD, and 4 cases of IGA were identified and stained with CD34. All cases of LDS showed loss of CD34 within subcutaneous septa, and 9 cases (60%) also exhibited full-thickness dermal loss of interstitial staining. All 6 cases of ScGVHD showed varying degrees of CD34 loss within the dermis and/or subcutaneous septa. The normal subcutis showed diffuse septal staining with CD34, with a density equal to that seen in the dermis. CD34 staining was lost in areas of dermal inflammation in half of the IGA cases. We conclude that CD34 staining is a useful ancillary test in disease processes affecting the subcutaneous collagen such as LDS and ScGVHD. Its utility also extends to diagnostically challenging disorders of dermal collagen degeneration such as IGA.

rAAV-compatible MiniPromoters for restricted expression in the brain and eye.

BACKGROUND: Small promoters that recapitulate endogenous gene expression patterns are important for basic, preclinical, and now clinical research. Recently, there has been a promising revival of gene therapy for diseases with unmet therapeutic needs. To date, most gene therapies have used viral-based ubiquitous promoters-however, promoters that restrict expression to target cells will minimize off-target side effects, broaden the palette of deliverable therapeutics, and thereby improve safety and efficacy. Here, we take steps towards filling the need for such promoters by developing a high-throughput pipeline that goes from genome-based bioinformatic design to rapid testing in vivo. METHODS: For much of this work, therapeutically interesting Pleiades MiniPromoters (MiniPs; ~4 kb human DNA regulatory elements), previously tested in knock-in mice, were "cut down" to ~2.5 kb and tested in recombinant adeno-associated virus (rAAV), the virus of choice for gene therapy of the central nervous system. To evaluate our methods, we generated 29 experimental rAAV2/9 viruses carrying 19 different MiniPs, which were injected intravenously into neonatal mice to allow broad unbiased distribution, and characterized in neural tissues by X-gal immunohistochemistry for icre, or immunofluorescent detection of GFP. RESULTS: The data showed that 16 of the 19 (84 %) MiniPs recapitulated the expression pattern of their design source. This included expression of: Ple67 in brain raphe nuclei; Ple155 in Purkinje cells of the cerebellum, and retinal bipolar ON cells; Ple261 in endothelial cells of brain blood vessels; and Ple264 in retinal Müller glia. CONCLUSIONS: Overall, the methodology and MiniPs presented here represent important advances for basic and preclinical research, and may enable a paradigm shift in gene therapy.