Dvora Beach

Enhancing the survival of aspirated human fat injected into nude mice

Injection of aspirated fat is now the most commonly used technique for the filling of depressed areas. Partial absorption of the injected fat is the main limitation of this procedure. Cariel T.M. is an enriched serum-free cell culture medium, its ability to enhance the survival of human aspirated fat grafts was investigated in the nude mouse model. A volume of 0.75-cc Cariel preprocessed fat was injected under the scalp skin of 16 nude mice in the experimental group, and the same volume of saline preprocessed fat was injected to 15 control group of mice. Significant maintenance of the weight, 46 percent in the experimental group compared with 29 percent in the control group (p < 0.008), and the volume, 44 percent in the experimental group compared with 31 percent in the control group (p < 0.026), was observed, after 15 weeks, in this newly used model. It seems that addition of the nutrients enriched with anabolic hormones enabled the survival and take of more adipose cells in the graft.

SLP-76 mediates and maintains activation of the Tec family kinase ITK via the T cell antigen receptor-induced association between SLP-76 and ITK.

ITK (IL-2-inducible T cell kinase), a Tec family protein tyrosine kinase (PTK), is one of three PTKs required for T cell antigen receptor (TCR)-induced activation of phospholipase C-γ1 (PLC-γ1). Like Src and Abl family PTKs, ITK adopts an inactive, “closed” conformation, and its conversion to the active conformation is not well understood, nor have its direct substrates been identified. In a side-by-side comparison of ITK and ZAP-70 (ζ chain-associated protein kinase of 70 kDa), ITK efficiently phosphorylated Y783 and Y775 of PLC-γ1, two phosphorylation sites that are critical for its activation, whereas ZAP-70 did not. SLP-76 (SH2-domain-containing leukocyte protein of 76 kDa), an adaptor required for TCR-induced activation of PLC-γ1, was required for the phosphorylation of both PLC-γ1 sites in intact cells. Furthermore, this event depended on the N-terminal tyrosines of SLP-76. Likewise, SLP-76, particularly its N-terminal tyrosines, was required for TCR-induced tyrosine phosphorylation and activation of ITK but was not required for the phosphorylation or activation of ZAP-70. Both ZAP-70 and ITK phosphorylated SLP-76 in vitro; thus, both PTKs are potential regulators of SLP-76, but only ITK is regulated by SLP-76. Upon TCR stimulation, a small fraction of ITK bound to SLP-76. This fraction, however, encompassed most of the catalytically active ITK. Catalytic activity was lost upon mild elution of ITK from the SLP-76-nucleated complex but was restored upon reconstitution of the complex. We propose that SLP-76 is required for ITK activation; furthermore, an ongoing physical interaction between SLP-76 and ITK is required to maintain ITK in an active conformation.

Dual Role of SLP-76 in Mediating T Cell Receptor-induced Activation of Phospholipase C-γ1

Phospholipase C-γ1 (PLC-γ1) activation depends on a heterotrimeric complex of adaptor proteins composed of LAT, Gads, and SLP-76. Upon T cell receptor stimulation, a portion of PLC-γ1 is recruited to a detergent-resistant membrane fraction known as the glycosphingolipid-enriched membrane microdomains (GEMs), or lipid rafts, to which LAT is constitutively localized. In addition to LAT, PLC-γ1 GEM recruitment depended on SLP-76, and, in particular, required the Gads-binding domain of SLP-76. The N-terminal tyrosine phosphorylation sites and P-I region of SLP-76 were not required for PLC-γ1 GEM recruitment, but were required for PLC-γ1 phosphorylation at Tyr783. Thus, GEM recruitment can be insufficient for full activation of PLC-γ1 in the absence of a second SLP-76-mediated event. Indeed, a GEM-targeted derivative of PLC-γ1 depended on SLP-76 for T cell receptor-induced phosphorylation at Tyr783 and subsequent NFAT activation. On a biochemical level, SLP-76 inducibly associated with both Vav and catalytically active ITK, which efficiently phosphorylated a PLC-γ1 fragment at Tyr783 in vitro. Both associations were disrupted upon mutation of the N-terminal tyrosine phosphorylation sites of SLP-76. The P-I region deletion disrupted Vav association and reduced SLP-76-associated kinase activity. A smaller deletion within the P-I region, which does not impair PLC-γ1 activation, did not impair the association with Vav, but reduced SLP-76-associated kinase activity. These results provide new insight into the multiple roles of SLP-76 and the functional importance of its interactions with other signaling proteins.

Improved vitality of experimental random dorsal skin flaps in rats treated with enriched cell culture medium

A defined, serum-free cell culture medium supplemented with nonsteroidal anabolic hormones, insulin, thyroxin, and growth hormone was found to accelerate wound healing by stimulating vascularized granulation tissue formation, epithelialization, and angiogenesis. The aim of this work was to study the effect of cell culture medium on the survival rate of cephalically based random dorsal skin flaps in an animal model. A total of 77 Sprague-Dawley rats were randomized into five treatment groups: pharmacologic delay with cell culture medium, flap enhancement with cell culture medium, surgical delay, biological delay with saline, and control. Statistically significant differences in distal flap necrosis were found among all groups (p<0.003). The rats treated with cell culture medium before flap elevation showed a significant increase in flap viability: a survival rate of 83 percent, compared with the control group, which demonstrated a survival rate of only 58 percent (p<0.0001). The surgical delay and the groups treated with cell culture medium yielded similar results with no significant difference between them. This study indicates that preoperative injection of cell culture medium may play a role in decreasing skin flap necrosis.

Sequential phosphorylation of SLP-76 at tyrosine 173 is required for activation of T and mast cells

Cooperatively assembled signalling complexes, nucleated by adaptor proteins, integrate information from surface receptors to determine cellular outcomes. In T and mast cells, antigen receptor signalling is nucleated by three adaptors: SLP‐76, Gads and LAT. Three well‐characterized SLP‐76 tyrosine phosphorylation sites recruit key components, including a Tec‐family tyrosine kinase, Itk. We identified a fourth, evolutionarily conserved SLP‐76 phosphorylation site, Y173, which was phosphorylated upon T‐cell receptor stimulation in primary murine and Jurkat T cells. Y173 was required for antigen receptor‐induced phosphorylation of phospholipase C‐γ1 (PLC‐γ1) in both T and mast cells, and for consequent downstream events, including activation of the IL‐2 promoter in T cells, and degranulation and IL‐6 production in mast cells. In intact cells, Y173 phosphorylation depended on three, ZAP‐70‐targeted tyrosines at the N‐terminus of SLP‐76 that recruit and activate Itk, a kinase that selectively phosphorylated Y173 in vitro. These data suggest a sequential mechanism whereby ZAP‐70‐dependent priming of SLP‐76 at three N‐terminal sites triggers reciprocal regulatory interactions between Itk and SLP‐76, which are ultimately required to couple active Itk to its substrate, PLC‐γ1.

Novel hyperactive mitogen to endothelial cells: human decidual NKG5.

Langer N, Beach D, Lindenbaum ES. Novel hyperactive mitogen to endothelial cells: human decidual NKG5. AJRI 1999; 42:263–272

Effect of decidua angiogenic factors on experimental dermis allografts

Meshed human dermis allografts containing human uterine angiogenic factor were implanted over full thickness surgical skin wounds in rats. Enhanced angiogenesis of the wound bed, three times more than that observed in control grafts, was found in experimental grafts at 3 days postimplantation. This was accompanied by a greater amount of granulation tissue formation, enhanced granulation tissue penetration into the dermal grafts and accelerated incorporation of the grafts. Angiogenesis of the wound bed regressed to control levels at 7 days postimplantation. The growth and penetration of the granulation tissue and the accelerated incorporation of the dermis grafts, however, continued ahead in the experimental grafts while necrosis appeared in the control grafts. The relevance of these results for acceleration of wound healing and improvement of the wound bed for subsequent application of cultured epidermal grafts is discussed. This method may be extended to the treatment of ulcers and burns.

Stimulated healing of recalcitrant wounds by topical application of enriched cell culture medium: a clinical report.

This study was designed to test the efficacy of enriched cell culture medium as a wound dressing. The rationale was to create within the wound space an optimal microenvironment, conducive to cellular proliferation, vascular granulation tissue formation, and epithelialization. This study was performed on various wounds that failed to respond to previous conventional treatments. A total of 288 wounds were within the inclusion criteria, with only contaminated and neoplastic wounds excluded. Most of the patients (80 percent) were ambulatory, and the wounds were examined by the attending physician once every 7 to 14 days at an outpatient clinic. The remaining 20 percent of patients were admitted to the study while hospitalized. Cell culture medium MCDB, supplemented with insulin, thyroxin, and growth hormone, was gelled. The gel was self‐applied once a day to freshly washed wounds, covered with a gauze pad, and anchored with netting. Healing started 7 to 14 days after the initiation of treatment with enriched cell culture medium. However, the criterion for success of the treatment was determined on complete wound closure, which was achieved in 189 of 288 wounds (65.6 percent). Wound closure was correlated with the initial wound volume, stage, and origin. The average time required for closure of wounds caused by systemic pathologies (n = 181) and those based on regional status (n = 107) were 12.0 and 4.4 weeks, respectively, compared with 290 and 10.3 weeks of the previous conventional treatment. In 19 extensive wounds, when vascularized granulation tissue was established, a successful surgical closure was attained. Most wounds of patients who did not continue the enriched cell culture medium treatment (34.4 percent) manifested reduced wound volume, ranging from 11 to 98 percent of initial volume. Discontinuation of treatment was associated with difficulties in reaching the clinic for the weekly examination, rather than for reasons directly related to the treatment itself, and occurred significantly earlier during the treatment period. Thus, enriched cell culture medium was effective in stimulating wound healing in recalcitrant wounds. The healing was rapid with minimum scarring and pain. No side effects or allergic reactions were reported or observed. (Plast. Reconstr. Surg. 108: 104, 2001.)

Preliminary clinical trials of serum free cell culture medium as a treatment for chronic atrophic ulcers

Among the common treatments for chronic atrophic wounds, moist dressings are used to create optimal environment and prevent desiccation of the wound bed. Modified serum free cell culture medium supplemented with nonsteroidal anabolic hormones thyroxine, growth hormone, insulin and transferrin was gelled in 1% Agarose. The gel was applied to the surface of chronic atrophic wounds (1 ml/cm2) once a day. All the wounds included in this study were clear of infection following treatment with topical antiseptics and antibiotics. The study was performed on 92 patients suffering from chronic leg ulcers. Etiologies of the ulcers varied from venous insufficiency, arterial insufficiency, insulin-dependent and non-dependent diabetic ulcers or combinations of the above. Complete wound closure was obtained in 50 patients. In the remaining 42 patients, the treatment was discontinued because of unrelated medical or psychiatric problems — 13 patients, noncompliance —16 patients, relocation — 12 patients, and infection — 1 patient.