Biomedical subjects

C W Hewitt

Publications and source records attributed to C W Hewitt.

98 records · Page 6Linked to original sources

PubMedDate not supplied

Diamond-like carbon coating and plasma or glow discharge treatment of mechanical heart valves.

All mechanical heart valves (MHV) are thrombogenic. Application of surface modification technology to reduce the incidence of thrombus formation on MHV is a novel undertaking. This requires collaboration within the bioengineering and cardiothoracic surgery fields. From reviewing results of recent and past investigations, and our own preliminary study with diamond-like carbon coating (DLC) and plasma or glow discharge treatment (GDT) of MHV, we identify and discuss several potentially beneficial effects that may reduce the extent of valve-related thrombogenesis by surface modification. DLC and GDT may affect the surfaces of MHV in many ways, including cleaning of organic and inorganic debris, generating reactive and functional groups on the surface layers without affecting their bulk properties, and making the surfaces more adherent to endothelial cells and albumin and less adherent to platelets. These different effects of surface modification, separately or in combination, may transform the surfaces of MHV to be more thromboresistant in the vascular system.

Animals↗

PubMedDate not supplied

Burn depth evaluation with fluorometry: is it really definitive?

Clinical evaluation of burn depth soon after injury is subjective, based on gross visual assessment. Previous investigators have quantified this process using fluorometry. Their studies show fluorescein levels in full-thickness burns to be far below control levels and partial-thickness burns to be about 60% of nonburned skin. In both rat and human models, 59 burn sites (eight rats) and 37 burn sites (seven patients) were assessed. Readings were taken for three hours on the rats and one hour on the patients during the first 48 hours, and the procedure was repeated for five days postburn. Maximum values during these periods were determined for burn and nonburn sites, and background levels were subtracted from these values. The rate of fluorescein uptake and the peak times for burn and nonburn sites were then compared. Actual depth of burn was determined by whether or not healing had occurred. The results showed no significant difference between partial-thickness and full-thickness burns using fluorometry, as standard deviations in both models for both depths of burn were large. Therefore, fluorometry did not provide a definitive evaluation of burn depth. These results differ from those reported by previous investigators.

Animals↗

PubMedDate not supplied

Reconstructive allotransplantation: considerations regarding integumentary/musculoskeletal grafts, cyclosporine, wound coverage in thermal injury, and the immune response.

With the advent of cyclosporine, a powerful and selective immunosuppressant, comes resurgence of a long-sought goal: to transplant modules of allointegumentary/musculoskeletal tissues or components thereof for the repair of peripheral tissue defects. Because these modules of integumentary and/or musculoskeletal tissue are actually composites of various tissues, they are also known as composite tissue allografts. The immediate goal of the studies reviewed herein is to lay the foundation in transplant immunobiology for the clinical exploitation of composite tissue allografts. The objective of these continuing studies is to induce permanent acceptance of composite tissue allografts. The value of such grafts lies in their potential for complete functional and cosmetic restoration in the surgical reconstruction of tissue after full-thickness burn injury. The initial results of basic experiments with cyclosporine are extremely encouraging in regard to the clinical potential for integumentary/musculoskeletal grafts in reconstructive allotransplantation.

Burns↗

PubMedDate not supplied

The effect of leukocyte infiltration on apoptosis in an in vitro thermal injury bioartificial living skin equivalent model.

An in vitro bioartificial skin construct (BSC) model was studied to see how inflammatory infiltration affects apoptosis in skin that has been thermally injured. The BSC was used as a target organ. Control BSCs without leukocytes (CON) were burned (BCON) by scalding with phosphate-buffered saline heated to 70 degrees C for 6 seconds, and they were then cooled with room temperature phosphate-buffered saline for 15 seconds. Human alloimmunocytes were added to CON to create rejection cultures (REJ) and to BCON to create burned rejection cultures (BREJ). Slides were stained with hematoxylin and eosin and anti-Lewis antibody. In situ labeling of apoptosis was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Those sections that were immunostained for Lewis Y were analyzed for intensity stain index (ISI = [sigma P0 x I0]/ total tissue area in pixels). TUNEL was quantified with the following equation: no. of total apoptotic cells / total tissue area. Necrosis and blister formation in the epidermal layer were evident in BCON and BREJ. Pyknosis and nuclear fragmentation-indicators of apoptosis-were also present. Phagocytosis of keratinocytes by leukocytes was seen in REJ and BREJ. Immunostaining showed greater expression of Lewis Y antigen, as determined by ISI, in REJ as opposed to CON (58.2+/-2.3 vs. 36.4+/-2.3, respectively, P<.001), but no significant difference was found between BCON and BREJ (55.0+/-5.7 vs. 60.5+/-3.4, respectively) and REJ and BREJ (58.3+/-2.3 vs. 60.5+/-3.4, respectively). TUNEL staining indicated the presence of apoptosis as follows: REJ versus CON (0.0015+/-0.0002 vs. 0.0003+/-0.0001, respectively, P<.001); BREJ versus BCON (0.0031+/-0.0006 vs. 0.0018+/-0.0004, respectively, P<.05); REJ versus BREJ (0.0015+/-0.0002 vs. 0.0031+/-0.0006, respectively, P = .007). The presence of leukocytes and thermal injury induces apoptosis in BSC. The combination of these two variables results in increased apoptosis as determined by TUNEL. These findings suggest that a common pathway for skin injury may include inappropriate regulation of apoptosis exacerbated by a mechanism that includes inflammatory cellular infiltration.

Animals↗

PubMedDate not supplied

A rapid and sensitive cellular enzyme-linked immunoabsorbent assay (CELISA) for the detection and quantitation of antibodies against cell surface determinants. II. Optimal reagent concentrations and predictive analysis.

A cellular enzyme-linked immunosorbent assay (CELISA) was developed for the detection and quantification of antibodies elicited against allogeneic cell surface determinants. The technique uses a solid-phase cell matrix created by fixing cells with a mild formalin solution onto the bottom of a 96-well microtiter plate. A primary layer of alloantisera is first reacted against rat target cells. The secondary antibody, peroxidase conjugated antirat IgG, is then added to each well and serves as the second sandwich layer. Optimal reagent concentrations were determined by serial dilution analysis of various cell concentrations and secondary antibody dilutions. It was found that 200,000 cells per well was the optimal target cell concentration. However, 100,000 cells per well was also sufficient to run the assay with acceptable performance characteristics. Even lower cell concentrations of 10,000 and 20,000 cells/well, although not optimal, also produced acceptable results. Secondary antibody concentration with respect to the optimal cell concentration was determined to be 1:500. At 200,000 cells per well and a 1:500 secondary antibody dilution, the assay presented excellent coefficients of determination and high positive to negative ratios. The reaction was found to be very sensitive in yielding high antibody titers with low background levels and could be defined mathematically as a linear-log function. Titers of multiple unknown alloantibody samples were easily and accurately predicted in an automated manner by regression analysis form known standards. This immunoassay will be useful in studies of cell surface determinant expression and quantitation of antibodies reactive to such markers.

Animals↗

PubMedDate not supplied

Use of regression analysis and flow cytometry for determining levels of mixed semiallogeneic immune chimerism.

It has been shown that tolerance or specific immunologic nonresponsiveness in various lymphohemopoietic transplant models can be associated with the development of mixed lymphoid chimerism. As a specific example, composite tissue (limb) allografts were studied as a model for vascularized bone marrow transplantation (VBMT) and it was demonstrated that development of stable cellular immune chimerism is associated with long-term allograft survival. Recently, studies were initiated using a new parental to hybrid VBMT model, but the detection of donor cells is complicated, due to the fact that they share one parental allotypic determinant. Therefore, regression analysis with a flow cytometric immunofluorescent staining assay was evaluated for the assessment of cellular lymphoid chimerism in donor parental to hybrid (P-->F1) lymphohemopoietic transplant models. Standard curves consisting of known mixed populations of parental donor (Lewis, LEW) and hybrid host F1 (Lew x BN, LBN) lymphocytes were established. Standard curves were analyzed by linear regression statistics and excellent coefficients of determination (r > .881) were obtained for all standard curves. A highly statistically significant (p < .016) linear relationship between level of donor cell chimerism (independent variable) and percent stained (dependent variable) was determined. The technique was then evaluated using the parental to hybrid VBMT model. Levels of donor LEW lymphoid chimerism in all VBMT LBN recipients were successfully assessed by regression analysis and inverse prediction using distinct recipient allodeterminant markers. In conclusion, this technique was proven to be reliable and accurate for the detection of of chimerism in parental to F1 lymphohemopoietic allograft models.

Animals↗

PubMedDate not supplied

Site-specific immunosuppression: mechanisms of cellular immunosuppression that are operative at local and systemic levels.

The cellular mechanisms by which topical cyclosporine A (tCsA) induces site-specific immunosuppression were investigated. Experiments were designed to elucidate how cyclosporine A (CsA) suppresses activated immunocytes in animals that are undergoing local alloactivation and concomitant tCsA immune suppression. Lewis rats received dual Lewis x Brown Norway rat skin allografts; the rats were treated with systemic CsA (sCsA) at 8 mg/kg/day for 10 days after grafting and then tCsA and vehicle thereafter. CsA added to mixed lymphocyte reactions 24 hours after culture initiation modeled the local effects of CsA on alloactivated immunocytes, and tCsA in conjunction with limited sCsA prolonged local skin allograft survival. CsA inhibited both antigen-specific and nonspecific activated alloresponses of immunocytes from animals that had received allografts and that underwent limited sCsA treatment only in a dose-dependent manner. When tCsA had been applied, immunocyte responses to a nonspecific antigen were extremely CsA-resistant as compared with those induced by antigen-specific suppression. However, this nonspecific alloresponse was fully suppressible with the use of elevated CsA doses (66 microg/mL); thus alloresponding immunocytes were significantly more sensitive to CsA if they were challenged with the donor antigen and preexposed to limited sCsA followed by tCsA in vivo.

Administration, Topical↗

PubMedDate not supplied

Cyclosporine-induced long-term allograft survival and its potential in posttrauma tissue replacement.

This report is a partial review of our work to date concerning the use of cyclosporine and integumentary/musculoskeletal allografts for posttrauma tissue replacement. Our ultimate goals for such allografts are their lifesaving capabilities in addition to their utilization in functional and aesthetic surgical reconstructions. The potential for such a treatment regimen to produce anatomical replacement of lost parts is a primary motivating reason to pursue such studies. Permanent host-accepted integumentary/musculoskeletal allografts would appear to offer much greater promise in comparison with recent synthetic and cultured tissue replacements. Summarized topics covered in this review include short- and long-term use of cyclosporine in a 30% body surface area rat burn model; bacterial studies in this model; primary wound excision and use of cyclosporine in a massive 80% body surface area rat burn model; pathological skin alterations in cyclosporine-treated rats; the synergistic immunosuppressive effects of prior blood transfusions and cyclosporine; long-term residual cyclosporine levels assayed in various long-term surviving allograft-recipients; and the use of cyclosporine and cadaver skin allografts to treat massive full-thickness burns in patients.

Animals↗