A model of corrective gene transfer in X-linked ichthyosis

Single gene recessive genetic skin disorders offer attractive prototypes for the development of therapeutic cutaneous gene delivery. We have utilized X-linked ichthyosis (XLI), characterized by loss of function of the steroid sulfatase arylsulfatase C (STS), to develop a model of corrective gene delivery to human skin in vivo. A new retroviral expression vector was produced and utilized to effect STS gene transfer to primary keratinocytes from XLI patients. Transduction was associated with restoration of full-length STS protein expression as well as steroid sulfatase enzymatic activity in proportion to the number of proviral integrations in XLI cells. Transduced and uncorrected XLI keratinocytes, along with normal controls, were then grafted onto immunodeficient mice to regenerate full thickness human epidermis. Unmodified XLI keratinocytes regenerated a hyperkeratotic epidermis lacking STS expression with defective skin barrier function, effectively recapitulating the human disease in vivo. Transduced XLI keratinocytes from the same patients, however, regenerated epidermis histologically indistinguishable from that formed by keratinocytes from patients with normal skin. Transduced XLI epidermis demonstrated STS expression in vivo by immunostaining as well as a normalization of histologic appearance at 5 weeks post-grafting. In addition, transduced XLI epidermis demonstrated a return of barrier function parameters to normal. These findings demonstrate corrective gene delivery in human XLI patient skin tissue at both molecular and functional levels and provide a model of human cutaneous gene therapy.      

Genesis of a new spleen focus-forming virus: possible role of MCF viruses

A Friend mink cell focus-inducing (Fr-MCF) virus isolated from a Friend tumor cell line was able to induce acute erythroleukemia associated with polycythemia when injected as a Friend murine ecotropic leukemia virus (F-MuLV) pseudotype into adult Swiss and ICFW mice. One virus isolate recovered from leukemic cells and designated as FV-F3 presented the following properties: (i) persistence of the same leukemogenic power when propagated in vivo and in vitro; (ii) in vivo spleen focus-forming (SFFV) capacity; (iii) presence of erythropoietin (EPO)-independent CFU-E in leukemic animals; (iv) expression of a 32 RNA specifically recognized by a SFFV probe, in FV-F3 infected cells; and (v) expression in FV-F3-infected cell of polypeptides in the range of gp52 SFFV. Peptide analysis of these products revealed close similarities with the parental MCF virus. These data suggest that a SFFV genome arose by genetic recombinational events involving MCF virus.     

Failure of TGF β1 and IL-12 to regulate human FasL and mTNF alloreactive cytotoxic T-cell pathways

Abstract: The effect of TGFβ1 and IL-12 on calcium-independent cytotoxic pathways was investigated. We have previously demonstrated that the regulatory effect of TGFβ1 and IL-12 on human alloreative CTL activity was associated with regulation of perform and granzyme B gene expression. To determine the effect of both cytokines on the alternative cytotoxic pathway involving FasL and mTNF, we first investigated the expression of both molecules on human primary alloactivated T cells. Our results show that human allostimulated T lymphocytes express FasL. Cell lysis experiments demonstrate that the FasL cytotoxic pathway is involved in the killing of specific target cells mediated by human alloreactive CTL. In addition, allogeneic stimulation induced significant mTNF expression on both CD4+ and CD8+ responder T cells. Using TNF-sensitive target cells, we also demonstrate that the mTNF-mediated cytotoxic pathway is involved in the cytotoxic activity of human primary allostimulated T lymphocytes. Neither TGFβ1 nor IL-12 had an effect on FasL or mTNF expression. Furthermore, addition of TGFβ1 or IL-12 at the initiation of the MLR had no significant effect on Fas- and mTNF-mediated cytotoxicity.
Taken together, our results provide a novel insight into the differences between regulation by cytokines of perforin-dependent and -independent cytotoxic mechanisms. Unlike their role in the perforin/granzyme B pathway, TGFβ1 and IL-12 do not appear to mediate any regulatory effect on FasL and mTNF cytotoxic pathways used by human alloreactive primary CTL.     

Genotypic differences in larval olfactory discrimination in twoDrosophila melanogaster strains

Wild-type (+B) and compound chromosome mutant(bB) Drosophila melanogaster larvae were tested in a U-maze. FreshDrosophila food or food and larvae were placed in each of the two goals (+B only in goal 1,bB in goal 2) and served as stimulus. Separate trials were conducted using +B andbB larvae to test for preference in the maze. Significantly more test larvae went to the arm of the maze containing their own strain as stimulus when (1) both goals contained larvae, (2) one goal contained homogenetic larvae and the other fresh food only, and (3) the goals contained biotic residues of stimulus larvae. The strength of the stimulus necessary to elicit the response differed for the two strains, the +B strain apparently being more sensitive. As the density of the stimulus larvae was increased, the choices of the test larvae became statistically nonsignificant and the number of larvae remaining in the starting arm of the maze increased. The data suggested that the strains of larvae utilized here have the capacity for olfactory discrimination.Part of this investigation was supported by National Institutes of Health Award 1 F 32 NS05155-01 CMS from the Institute of Neurological and Communicative Disorders and Stroke to A. P.     

Avian tibial dyschondroplasia. III. Electron probe analysis.

Tibial dyschondroplastic (TD) lesions and their associated growth plates, obtained from chickens, were prepared by freeze-drying and embedding in an anhydrous epoxy resin. Quantitative electron probe analysis was performed on dry, unstained sections. Levels of Na, Mg, P, S, Cl, K, and Ca were determined in cytoplasm (endoplasmic reticulum), mitochondria, and extracellular matrix of the proliferative, prehypertrophic, and early hypertrophic zones of the growth plate and in the proximal, mid, and distal regions of the lesion. A zone of calcification in the growth plate was absent. The concentration of elements in all regions of the TD growth plate was the same as found in an earlier study for normal growth plate. The cytoplasm of proximal lesion chondrocytes was similar to that of early hypertrophic chondrocytes. However, in the remainder of the lesion there was a progressive increase in cellular Na, S, Cl, and Ca and a progressive loss of P. In matrix, there was less S and K than expected in all regions of growth plate and lesion, except in the proliferating zone. Also, in matrix of the distal lesion there was less Na and Cl. The levels of Na, S, Cl, and K in matrix may have been lowered by their adsorption into the condensed masses of dead cells. Mitochondria acquire only half as much Ca and P as normal and release it earlier than usual (ie, early prehypertrophic cells, rather than chondrocytes of the lower hypertrophic zone). There were no granules in mitochondria of the cells at all levels of the lesion, even though anhydrous methods were used. The first sign of the disease appears in the matrix of the growth plate, where it seems that S and K are in abnormally low amounts. Although there are sufficient levels of Ca and P present, the matrix does not calcify. The cartilage remains avascular, and the cells appear to be dying. The event that triggers the chondrocytes of the growth plate to form an abnormal uncalcified matrix is not known.     

Optimal donation of kidney transplants after controlled circulatory death

Controlled donation after circulatory death (cDCD) is used for “extended criteria” donors with poorer kidney transplant outcomes. The French cDCD program started in 2015 and is characterized by normothermic regional perfusion, hypothermic machine perfusion, and short cold ischemia time. We compared the outcomes of kidney transplantation from cDCD and brain-dead (DBD) donors, matching cDCD and DBD kidney transplants by propensity scoring for donor and recipient characteristics. The matching process retained 442 of 499 cDCD and 809 of 6185 DBD transplantations. The DGF rate was 20% in cDCD recipients compared with 28% in DBD recipients (adjusted relative risk [aRR], 1.43; 95% confidence interval [CI] 1.12–1.82). When DBD transplants were ranked by cold ischemia time and machine perfusion use and compared with cDCD transplants, the aRR of DGF was higher for DBD transplants without machine perfusion, regardless of the cold ischemia time (aRR with cold ischemia time <18 h, 1.57; 95% CI 1.20–2.03, vs aRR with cold ischemia time ≥18 h, 1.79; 95% CI 1.31–2.44). The 1-year graft survival rate was similar in both groups. Early outcome was better for kidney transplants from cDCD than from matched DBD transplants with this French protocol.     

Paradigm shift in gastrointestinal surgery − combating sarcopenia with prehabilitation: Multimodal review of clinical and scientific data

A growing body of evidence has demonstrated the prognostic significance of sarcopenia in surgical patients as an independent predictor of postoperative complications and outcomes. These included an increased risk of total complications, major complications, re-admissions, infections, severe infections, 30 d mortality, longer hospital stay and increased hospitalization expenditures. A program to enhance recovery after surgery was meant to address these complications; however, compliance to the program since its introduction has been less than ideal. Over the last decade, the concept of prehabilitation, or “pre-surgery rehabilitation”, has been discussed. The presurgical period represents a window of opportunity to boost and optimize the health of an individual, providing a compensatory “buffer” for the imminent reduction in physiological reserve post-surgery. Initial results have been promising. We review the literature to critically review the utility of prehabilitation, not just in the clinical realm, but also in the scientific realm, with a resource management point-of-view.     

The skeleton as a unique environment for breast cancer cells

Bone is a favored location for several cancer metastases especially breast, prostate and myeloma. This review evaluates various properties of the skeleton that contribute to its successful colonization by breast cancer cells. The first consideration is the unique aspects of the vasculature of metaphyseal bone, which may account for the initial lodging of breast cancer cells in specific regions of the skeleton. Metasphyseal bone, found at the ends of long bone, in ribs and in vertebrae, is comprised of trabecular bone interspersed with marrow and a rich vasculature. The chemotactic factors that arise from bone marrow and bone cells are discussed in terms of cancer cell migration out of the vasculature and entry of cancer cells into the marrow cavity. Once the breast cancer cells have migrated into the metaphysis, they interact both directly and indirectly with bone cells and other cells in the marrow. As tumor growth progresses, functional bone cells are lost, most likely through apoptosis. This revised version was published online in July 2006 with corrections to the Cover Date.