Hannah Ben-Bassat

Early nonsurgical removal of chemically injured tissue enhances wound healing in partial thickness burns

Chemical burns are slow healing injuries and their depth is difficult to assess. Tissue destruction continues as long as active material is present in the wound site. The routine therapy for treatment of full thickness chemical burns is early excision; it shortens hospitalization and reduces morbidity. However, presently there is no specific treatment for chemical burns of partial thickness. This study examined several treatment modalities for partial thickness chemical burns: surgical excision; laser ablation and chemical debridement with Debridase or trypsin-linked to gauze. Chemical burns were inflicted with nitrogen mustard (NM -- a nitrogen analog to sulfur mustard -- mustard gas) in an experimental guinea pig model. Debridase was most effective and reduced significantly lesion area of burns after humid exposure to 2 mg NM. The healing action of Debridase was also evident in the significantly higher histopathological score of biopsies from local tissue obtained on day 5. Laser ablation was most effective and accelerated healing of burn lesions after dry exposure to 5 mg NM. The histopathology score of the laser treated burns was higher on day 4 compared to untreated controls. It is concluded that for partial thickness chemical burns early nonsurgical removal of the damaged tissues accelerates wound healing.

Structurally different bisphosphonates exert opposing effects on alkaline phosphatase and mineralization in marrow osteoprogenitors.

Bisphosphonates (BPs) are inhibitors of bone resorption and soft tissue calcification. The biological effects of the BPs in calcium‐related disorders are attributed mainly to their incorporation in bone, enabling direct interaction with osteoclasts and/or osteoblasts through a variety of biochemical pathways. Structural differences account for the considerable differences in the pharmacological activity of BPs. We compared the effects of two structurally different compounds, alendronate and 2‐(3′‐dimethylaminopyrazinio)ethylidene‐1,1‐bisphosphonic acid betaine (VS‐6), in an osteoprogenitor differentiation system. The BPs were examined in a bone marrow stromal‐cell culture system, which normally results in osteoprogenitor differentiation. The drugs were present in the cultures from days 2 to 11 of osteogenic stimulation, a period estimated as being comparable to the end of proliferation and the matrix‐maturation stages. We found that the two different BPs have opposing effects on specific alkaline phosphatase (ALP) activity, on stromal‐cell proliferation, and on cell‐mediated mineralization. These BPs differentially interact with cell‐associated phosphohydrolysis, particularly at a concentration of 10−2 of ALP Km, in which alendronate inhibits whereas VS‐6 did not inhibit phosphatase activity. VS‐6 treatment resulted in similar and significantly increased mineralization at 10 and 1 μM drug concentrations, respectively. In contrast, mineralization was similar to control, and significantly decreased at 10 and 1 μM drug concentrations, respectively, under alendronate treatment. J. Cell. Biochem. 68:186–194, 1998.

Ataxia telangiectasia: chromosomal stability in continuous lymphoblastoid cell lines

Chromosomal breakage in peripheral lymphocytes, cultured fibroblasts and long-term lymphoblastoid cell lines was investigated in five hitherto undescribed patients with ataxia telangiectasia (AT). Increased chromosomal instability was observed in lymphocytes and fibroblasts, and clones possessing a Dq+ marker were observed. Breakage rates were significantly higher in the fibroblasts than in the lymphocytes of AT patients or in similar tissues from patients with Bloom syndrome or Fanconi anemia. However, chromosome breakage in lymphoblastoid lines established from these five AT patients and six others did not differ from controls. These observations suggests that selection pressures, in vivo or in vitro, or both, act differently on the expression of chromosomal instability in these various cell types.

Characterization of hereditary inclusion body myopathy myoblasts: possible primary impairment of apoptotic events.

Hereditary inclusion body myopathy (HIBM) is a unique muscular disorder caused by mutations in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene. GNE encodes a bi-functional enzyme acting in the biosynthetic pathway of sialic acid. Since the underlying myopathological mechanism leading to the disease phenotype is poorly understood, we have established human myoblasts cultures, derived from HIBM satellite cells carrying the homozygous M712T mutation, and identified cellular and molecular characteristics of these cells. HIBM and control myoblasts showed similar heterogeneous patterns of proliferation and differentiation. Upon apoptosis induction, phosphatidylserine externalization was similar in HIBM and controls. In contrast, the active forms of caspase-3 and -9 were strongly enhanced in most HIBM cultures compared to controls, while pAkt, downregulated in controls, remained high in HIBM cells. These results could indicate impaired apoptotic signaling in HIBM cells. Since satellite cells enable partial regeneration of the post-mitotic muscle tissue, these altered processes could contribute to the muscle mass loss seen in patients. The identification of survival defects in HIBM affected muscle cells could disclose new functions for GNE in muscle cells.

How long can cryopreserved skin be stored to maintain adequate graft performance

Skin graft preservation for the purpose of delayed application is still a basic tool in burn treatment and plastic and reconstructive surgery. As the demand for skin allografts has increased the responsibility for processing, storage and evaluation of graft performance of preserved skin has become an important issue of banking organizations. The present experiments were undertaken to determine how long can cryopreserved cadaveric skin be stored to maintain adequate graft performance? We applied a mouse recipient model, developed by us: Human cadaveric skin cryopreserved and stored for 5,6 or 7 years was grafted on Balb/c mice, and primary take was evaluated by gross observation and predetermined histologic criteria after 7 days. The results demonstrate that graft performance of cryopreserved skin decreased with time, as reflected in the lower percent of samples with high score of separate histologic criteria after prolonged storage. Nevertheless, paired comparison analysis between cryopreserved and fresh skin indicated that this decrease was not significant for storage of 5 years; whereas it was highly significant for 6 years of storage. Linear regression analysis indicated that there was no correlation between the score of the histologic criteria and storage period for upto 65 months. These results are in line with the paired comparison analysis. We feel that our in vivo model and analysis may be used as an evaluation procedure for transplantation performance of banked skin.

Superoxide dismutase (SOD) for mustard gas burns.

Mustard gas (MS) has been used in chemical warfare since World War I. The blistering skin lesions are slow to heal. Secondary inflammation might occur, as well as damage to organs distant from the original wound. Presently there is no specific antidote for burns and poisoning by MS. This study examined treatment modalities with free oxygen radical scavengers, copper-zinc, and manganese superoxide dismutase (SOD), for MS skin burns in an experimental guinea pig model. Each of the SOD compounds reduced dramatically burn lesion area when administered intraperitoneally/intralesionally (i.p./i.l.) before wound infliction. The protective action of the SODs was also evident in the significantly higher histopathological score of biopsies obtained on day 7 from local tissue, caused with the lower dose of MS. When the SOD compounds were administered i.p. 1 hour after burn infliction, and repeated daily for 7 days, no protective effect could be detected under the present experimental conditions.

The Proteomic Profile of Hereditary Inclusion Body Myopathy

Hereditary inclusion body myopathy (HIBM) is an adult onset, slowly progressive distal and proximal myopathy. Although the causing gene, GNE, encodes for a key enzyme in the biosynthesis of sialic acid, its primary function in HIBM remains unknown. The goal of this study was to unravel new clues on the biological pathways leading to HIBM by proteomic comparison. Muscle cultures and biopsies were analyzed by two dimensional gel electrophoresis (2-DE) and the same biopsy extracts by isobaric tag for relative and absolute quantitation (iTRAQ). Proteins that were differentially expressed in all HIBM specimens versus all controls in each analysis were identified by mass spectrometry. The muscle cultures 2-DE analysis yielded 41 such proteins, while the biopsies 2-DE analysis showed 26 differentially expressed proteins. Out of the 400 proteins identified in biopsies by iTRAQ, 41 showed altered expression. In spite of the different nature of specimens (muscle primary cultures versus muscle biopsies) and of the different methods applied (2D gels versus iTRAQ) the differentially expressed proteins identified in each of the three analyses where related mainly to the same pathways, ubiquitination, stress response and mitochondrial processes, but the most robust cluster (30%) was assigned to cytoskeleton and sarcomere organization. Taken together, these findings indicate a possible novel function of GNE in the muscle filamentous apparatus that could be involved in the pathogenesis of HIBM.

Differences in the ConA-induced redistribution and agglutination patterns of EBV genome-free and EBV-carrying human lymphoma lines.

Summary EBV-carrying human lymphoid lines showed a reduced redistribution of concanavalin A (ConA) receptors after ligand contact, compared with EBV negative lines. ConA agglutinability showed the opposite pattern, high in EBV positive, low in EBV negative lines. The same differences were also found in a comparison between two EBV negative lines and their in vitro EBV-converted, viral genome-carrying sublines, suggesting that the viral genome is directly responsible.

Opposing effects on mitochondrial membrane potential by malonate and levamisole, whose effect on cell-mediated mineralization is antagonistic.

The act of chondrocyte preparation for primary, enchondral, mineralization is associated with a decline in mitochondrial respiration toward the end of the proliferative zone and the hypertrophic zone in the growth plate. Dexamethasone (Dex)‐stimulated cultures of rat marrow stroma constitute a differentiation model simulating, in its energy metabolism, chondrocyte mineralization. In this model, early inhibition of succinate dehydrogenase (SDH) enriches the culture with mineralizing cells, whereas levamisole inhibits mineralization. Dex also increases mitochondrial membrane potential in stromal cells, especially on days 7–8 of stimulation. In the present study, suicide inhibition of SDH, by nitropropionic acid (NPA), in Dex‐stimulated cells showed a dose‐dependent increase in day 21 mineralization; the maximal effect was induced on days 2–4 of stimulation. Mineralization under 2‐day‐long exposure to NPA showed a similar trend to the previously studied effect of continuous exposure to malonate applied between days 3–11. Unlike malonate, the effect of NPA required its presence in the cultures for only 2 days and resulted in higher mineralization than that seen under 8 days of malonate. NPA delineated a period, days 2/4 to 7/9, in which inhibition of succinate oxidation is necessary to augment mineralization. During this period, NPA also exhibited OPC selection capacity. Early application of levamisole, under conditions previously shown to decrease day 21 mineralization, maintained mitochondrial membrane potential at the beginning of Dex stimulation but decreased or had little effect on it during days 5–10. By contrast, malonate previously found to increase day 21 mineralization decreased the membrane potential at the beginning of Dex stimulation but increased it later on day 7, or during days 5–10. These results indicate that during osteoprogenitor differentiation, before the mineralization stage, a surge in mitochondrial inner membrane potential during late matrix maturation may be a marker that heralds the extracellular matrix mineralization.

Optimization of Energy-Consuming Pathways towards Rapid Growth in HPV-Transformed Cells

Cancer is a complex, multi-step process characterized by misregulated signal transduction and altered metabolism. Cancer cells divide faster than normal cells and their growth rates have been reported to correlate with increased metabolic flux during cell transformation. Here we report on progressive changes in essential elements of the biochemical network, in an in vitro model of transformation, consisting of primary human keratinocytes, human keratinocytes immortalized by human papillomavirus 16 (HPV16) and passaged repeatedly in vitro, and the extensively-passaged cells subsequently treated with the carcinogen benzo[a]pyrene. We monitored changes in cell growth, cell size and energy metabolism. The more transformed cells were smaller and divided faster, but the cellular energy flux was unchanged. During cell transformation the protein synthesis network contracted, as shown by the reduction in key cap-dependent translation factors. Moreover, there was a progressive shift towards internal ribosome entry site (IRES)-dependent translation. The switch from cap to IRES-dependent translation correlated with progressive activation of c-Src, an activator of AMP-activated protein kinase (AMPK), which controls energy-consuming processes, including protein translation. As cellular protein synthesis is a major energy-consuming process, we propose that the reduction in cell size and protein amount provide energy required for cell survival and proliferation. The cap to IRES-dependent switch seems to be part of a gradual optimization of energy-consuming mechanisms that redirects cellular processes to enhance cell growth, in the course of transformation.