An enzyme-mediated assay to quantify inoculation volume delivered by suture needlestick injury: two gloves are better than one.

BACKGROUND: Acquiring a blood-borne disease is a risk of performing operations. Most data about seroconversion are based on hollow-bore needlesticks. Some studies have examined the inoculation volumes of pure blood delivered by suture needles. There is a lack of data about the effect of double-gloving on contaminant transmission in less viscous fluids that are not prone to coagulation. STUDY DESIGN: We used enzymatic colorimetry to quantify the volume of inoculation delivered by a suture needle that was coated with an aqueous contaminant. Substrate color change was measured using a microplate reader. Both cutting and tapered suture needles were tested against five different glove types and differing numbers of glove layers (from zero to three). RESULTS: One glove layer removed 97% of contaminant from tapered needles and 65% from cutting needles, compared with the no-glove control data. Additional glove layers did not significantly improve contaminant removal from tapered needles (p > 0.05). For the cutting needle, 2 glove layers removed 91% of contaminant, which was significantly better than a single glove (p = 0.002). Three glove layers did not afford statistically significant additional protection (p = 0.122). There were no statistically significant differences between glove types (p = 0.41). CONCLUSIONS: With an aqueous needle contaminant, a single glove layer removes contaminant from tapered needles as effectively as multiple glove layers. For cutting needles, double-glove layering offers superior protection. There is no advantage to triple-glove layering. A surgeon should double-glove for maximum safety. Additionally, a surgeon should take advantage of other risk-reduction strategies, such as sharps safety, risk management, and use of sharpless instrumentation when possible.

Production of a novel fibroblast-populated platelet matrix cocultured with keratinocytes.

We have developed a new method for the production of a dermal matrix equivalent. Human platelets were used to dilute human fibroblasts. The platelet mix was placed in a cell culture well. Addition of 200 microL of a thrombin solution caused gel formation. Gels were overlaid with standard Iscove's growth medium supplemented with 10% fetal bovine serum, insulin, and N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer. Medium was exchanged regularly. Keratinocytes were plated on top of selected gels and elevated to the air-liquid interface. The gels were harvested weekly, fixed, cut, and stained with hematoxylin and eosin stains and immunostains for collagens I, III, and IV and cytokeratins. Digital image analysis was used to quantitate collagen production. Growth factors, including transforming growth factor-beta (TGF-beta), platelet-derived growth factor, and vitamin C were added. Staining identified fibroblasts within the gels with a surrounding fibrous matrix. Immunostaining for cytokeratin identified keratinocytes on the gel surface. Immunostaining revealed the fibrous matrix to be composed of collagen I and III and some collagen IV. Digital image analysis demonstrated that greater TGF-beta concentration resulted in greater collagen production. These differences were statistically significant. With development of this construct, a viable dermal/epidermal replacement may be possible. TGF-beta enhances collagen production by fibroblasts in this matrix.

Review of vascularized bone marrow transplantation: current status and future clinical applications.

In this review, we examine the applicability of the vascularized bone marrow transplant (VBMT) as an alternative to conventional bone marrow transplantation (BMT). As a new surgical approach, the VBMT is unique by transplantation of the stromal environment that eliminates the need for an engraftment period, provides critical signaling and modulatory functions, and may potentiate tolerance induction. Thus far, VBMT studies have demonstrated an absence of graft-versus-host disease (GVHD) and robust engraftment into nonmanipulated as well as irradiated recipients with evidence of immunological tolerance. Further investigation is needed to determine the applicability of VBMT as an alternative to BMT.

Pulse doppler and M-mode to assess viability of cardiac allografts using heterotopic femoral heart transplantation in rats.

Noninvasive assessment of heterotopic heart transplants using Doppler echocardiography was first described in two patients by Allen at Stanford in 1981. Since then, numerous experiments studying heterotopic heart transplantation in humans and large animals have confirmed its utility by employing either an intra-abdominal or cervical model. In rats, however, prior research investigating intra-abdominal heterotopic hearts has showed echocardiography to be ineffective. We have recently developed a new technique for heterotopic femoral heart transplantation in rats, which employs the novel use of trans-femoral echocardiography. Therefore, our goal was to re-examine the efficacy of echocardiography for detection of graft rejection.

A new modified technique for heterotopic femoral heart transplantation in rats.

BACKGROUND: Abbott developed the first experimental accessory heart transplant rat model in 1964. This intra-abdominal model required a labor-intensive aortic anastomosis. In 1971, Heron modified the operation by using sutureless cervical vessel anastomoses. Rao and Lisitza developed a femoral heart accessory transplant model in 1985. Our goal was to improve this femoral model for the study of cardiac transplantation between both syngeneic and allogeneic rats. METHODS: ACI and Lewis rats weighing 150 to 350 g were used as donors and recipients (n = 12). The left common carotid and left pulmonary arteries were anastomosed to the femoral artery and vein in an end-to-end fashion, respectively. Improved modifications included the use of hemostatic vessel clips, heparinization of both donor and recipient, a ventricular prolene stay-suture for secure graft placement, and transfemoral echocardiography (TFE). Total operative time averaged 61 +/- 12 minutes. RESULTS: Femoral accessory transplanted hearts (FATHs) allowed easier pulse palpation and access for TFE versus previously described cervical and intra-abdominal models. This modification allows precise detection of acute graft rejection (AGR) and is defined as absent ventricular contraction in the presence of anastomostic patency. CONCLUSIONS: Our new modified technique for heterotopic femoral heart transplantation in rats is a relatively easily learned and reproduced procedure that allows superior allograft access for palpation and improved echocardiographic assessment. Femoral heterotopic heart transplantation remains an effective model for allograft transplantation study.

From experimental rat hindlimb to clinical face composite tissue allotransplantation: historical background and current status.

The purpose of this article is to review the historical background and clinical status of composite tissue allotransplantation and to discuss the scientific evolution of clinical face transplantation. Composite tissue allotransplantation (CTA) rapidly progressed in the 1980s with the discovery of cyclosporine. Although the most success has been achieved with hand transplantation, others have made progress with allografts of trachea, peripheral nerve, flexor tendon apparatus, vascularized knee, larynx, abdominal wall, and most recently, partial face. The world's first partial face allotransplantation occurred in November 2005 in France. In April of 2006, there was a second performed in China. As of today, there are now multiple institutions with plans to attempt the world's first full facial/scalp transplant. Complete facial/scalp allotransplantation offers a viable alternative for unfortunate individuals suffering severe facial disfigurement and is a product of many decades of experimental research, beginning with rat hindlimb allografts.

Vascularized lymph node transplantation induces graft-versus-host disease in chimeric hosts.

BACKGROUND: The role of lymph nodes (LNs) in adaptive immune responses has been the subject of extensive research. In previous studies, the surgical removal of lymph nodes from rat hind limbs prevented the development of lethal graft-versus-host disease (GVHD) after allogeneic hind limb transplantation to chimeric recipient rats. The purpose of this study was to establish the role of the cellular fraction versus the microenvironment of LNs in the development of GVHD in this model. METHODS: A rat model for vascularized LN transplantation was developed and graft-versus-host responses were compared after: 1) naive ACI LN cells were infused into Wistar-Furth (WF) rats as chimeric recipients (e.g. [ACI-->WF]); 2) vascularized WF lymph nodes were transplanted to syngeneic WF recipients; 3) nonvascularized ACI lymph nodes were transplanted to [ACI-->WF] chimeric recipients; 4) vascularized ACI lymph nodes were transplanted to [ACI-->WF] chimeric recipients. RESULTS: Transplantation of vascularized ACI lymph nodes to [ACI-->WF] chimeric recipient rats resulted in severe and sometimes lethal GVHD. In contrast, neither the infusion of purified ACI LN cells nor the transplantation of nonvascularized LNs led to GVHD in chimeric recipients. CONCLUSIONS: When introducing allogeneic cells into chimeric recipients, concomitant transplantation of the vascularized LN microenvironment makes a manifest difference between induction and absence of GVHD. This illustrates the important role of the LN microenvironment in adaptive immune responses.

Nicotine induces mononuclear leukocyte adhesion and expression of adhesion molecules, VCAM and ICAM, in endothelial cells in vitro.

The pathology of atherosclerotic cardiovascular disease (ASCVD) has been characterized as an inflammatory response to vessel injury. The initial steps of this response involve mononuclear leukocyte (MNL) attachment and infiltration into the vessel wall. Leukocyte adhesion is potentiated by expression of cellular adhesion molecules. Vascular cell adhesion molecule-1 (VCAM) and intracellular adhesion molecule-1 (ICAM) are markers of cellular activation and have the ability to attach leukocytes to the endothelium, which is an initial event in the inflammatory response in the vessel wall. Human umbilical vein endothelial cells (HUVEC) were plated in endothelial growth medium (EGM) on plastic coverslips and grown until cells were 75% confluent. Free base nicotine (FBN) was diluted in EGM to a concentration of 10(-8) M and added to experimental cells. At 3 hr, coverslips were removed and fixed. Immunohistochemical staining (IHCS) was performed using a monoclonal antibody to human ICAM and VCAM. Digital image analysis (DIA) was performed to quantify the expression of ICAM and VCAM. An intensity stain index (ISI) measuring area and intensity of stain/total cellular area was determined. Additional HUVEC grown in a similar manner were either exposed to 10(-8) M FBN in EGM or EGM control for 4 hr, then were exposed to MNL suspension for 10 min. Coverslips were removed, rinsed, and fixed. Hematoxylin and eosin staining was performed and cells examined under light microscopy. Leukocyte number per high power field (HPF) was counted and compared to controls. Data were analyzed using analysis of variants (ANOVA) and Student's t-test. Differences were considered significant if p < 0.05. ICAM and VCAM expression was absent in control cells. Nicotine exposure at 3 hr induced expression of VCAM (ISI = 30.85+/-0.77) and to a lesser extent ICAM (ISI = 16.6+/-1.39) (p < 0.001). MNL adhesion was markedly increased in cells exposed to nicotine (79.4+/-16.9/HPF) when compared to control cells (1.8+/-0.91/HPF) exposed to MNL (p < 0.01). These data show nicotine's ability to activate HUVEC as evidenced by induction of ICAM and VCAM expression in vitro. The biological effects of these adhesion molecules are demonstrated by a marked increase in MNL adhesion to HUVEC as demonstrated by leukocyte adhesion assay (LAA). MNL adhesion and subsequent migration into the intima, if occurring in vivo, may be a vital step in the pathogenesis of ASCVD associated with nicotine exposure.

Lymphadenectomy prior to rat hind limb allotransplantation prevents graft-versus-host disease in chimeric hosts.

In previous rat studies, the use of mixed allogeneic chimerism (MAC) to induce host tolerance to hind limb allografts has resulted in severe graft-versus-host disease (GVHD). The purpose of this study was to determine if immunocompetent cells in bone marrow (BM) and/or lymph nodes (LNs) of transplanted limbs were responsible for inducing GVHD in mixed chimeric hosts. [ACI-->Wistar Furth] chimeric rats received ACI hind limbs that were non-irradiated, irradiated (1050 cGy) or lymphadenectomized. Rejection, GVHD and donor chimerism was assessed. Chimeric hosts rejected none of their limbs. However, hosts of non-irradiated hind limbs succumbed to GVHD 22.4+/-0.8 days after transplantation. In contrast, chimeras that received irradiated or lymphadenectomized ACI hind limbs showed no clinical or histological signs of GVHD at 5 months. We conclude that mixed chimeric hosts are susceptible to GVHD due to the immunocompetent cell load provided by the LNs, not the BM, of hind limb allografts.

Absence of graft-versus-host disease in the isolated vascularized bone marrow transplant.

An isolated vascularized bone marrow transplant (iVBMT) model was developed to study the contribution of the bone marrow component in a composite tissue allograft. We hypothesized that the iVBMT would be functional and cause graft-versus-host disease (GVHD) in a fraction of the recipients. Lewis iVBMT grafts were transplanted to Lewis-Brown Norway recipients. Animals were sacrificed at various times from 1 to 14 weeks. Polymerase chain reaction for microchimerism was performed on the host's marrow. No animals exhibited signs of GVHD at death. Histologic examination of the grafts showed a normal mix of hematopoietic and fatty elements and appeared to be functional. Tissues usually affected-tongue, ear, liver, and gut-also showed no evidence of disease. Polymerase chain reaction demonstrated microchimerism in both groups. These findings suggest that the vascularized bone marrow within a composite tissue allograft is not the component that causes GVHD; rather, it may serve an immunomodulatory function for tolerance induction.

Composite tissue allotransplantation.

Composite tissue allotransplantation (CTA) recently took its first steps in the clinical arena in 1998 with the successful hand transplant performed in Lyons, France. That single operation represented a culmination of many years of laboratory research in multiple fields involving integumentary/musculoskeletal transplantation. Here we review the prerequisite developments in the field of immunology, microsurgery, and pharmacotherapy that helped bring CTA to clinical reality. This new field still has many unanswered questions which are addressed below. Additionally, new evolving research in CTA is also discussed.

An extraperitoneal isolated vascularized bone marrow transplant model in the rat.

An isolated vascularized bone marrow transplant (iVBMT) model was previously developed in the rat to specifically study the role of bone marrow and its environment in a composite tissue allotransplant. An extraperitoneal model was successfully created to avoid laparotomy and cross-clamping of the great vessels. The extraperitoneal iVBMT model consisted of a left donor femur that was harvested with its nutrient vessels, anastomosed to the right femoral vessels in a syngeneic host, and then placed subcutaneously in the abdominal wall. At explant, the graft vessels were grossly patent, and histology of the graft bones showed a viable marrow compartment. Polymerase chain reaction demonstrated peripheral chimerism in the recipients. This model is technically simple with minimal morbidity in the recipient animals. By using the iVBMT, future studies across semiallogeneic and allogeneic barriers will help define the role of the bone marrow compartment in composite tissue allotransplants to potentially induce immune tolerance.

Real-time, high-definition, three-dimensional microscopy for evaluating problematic cervical Papanicolaou smears classified as atypical squamous cells of undetermined significance.

BACKGROUND: The perceived inadequacies of the cervical Papanicolaou (Pap) smear have been attributed to sampling, screening, or interpretive errors. Within this type of cytologic preparation, there are thick cell clusters in which the cells are obscured. It may not possible to evaluate these areas by conventional microscopy. The authors clinically tested the hypothesis that high-definition, three-dimensional (3-D) microscopy based on multiple oblique illumination (MOI), with its ability to penetrate into thick areas, would be useful in evaluating problematic cervical Pap smears, particularly those diagnosed as atypical squamous cells of undetermined significance (ASCUS). METHODS: ASCUS Pap smears and corresponding surgical biopsy specimens were evaluated prospectively using standard, axially illuminated microscopes and a new high-definition, 3-D microscope employing MOI. The Pap smears were reviewed in a blinded fashion with both types of microscopy. The rendered diagnoses were then compared with the subsequent tissue biopsies, which also were blinded, as the definitive end point. RESULTS: It was immediately apparent that the high-definition, 3-D MOI microscope had better resolution compared with the standard microscopes. Pap smears and biopsy diagnoses were correlated significantly for MOI (P < 0.001), and there were significant improvements (P = 0.0108) in accuracy when 3-D, high-definition microscopy was compared with conventional microscopy. The authors found no statistically significant correlation between ASCUS diagnoses that were rendered by using standard microscopes compared with the subsequent biopsy. CONCLUSIONS: Due to enhanced visualization through thick cell clusters, an increased depth of field, light penetration, and resolution, high-definition, 3-D microscopy based on MOI produced superior accuracy compared with conventional light microscopy in evaluating cervical Pap smears.

Engineering a composite neotrachea with surgical adhesives.

BACKGROUND/PURPOSE: Reconstructive surgery often is limited by the availability of normal tissue. Tissue engineering provides promise in the development of "artificial tissues." The purpose of this study was to test the efficacy and viability of the use of a biologic surgical adhesive TISSEEL in combining engineered bronchial epithelium with engineered cartilage. METHODS: Using isolated human cells, bronchial epithelium and mature cartilage were engineered. Using a contact adhesive technique, TISSEEL was used to biologically fuse the bronchial epithelium and the cartilage. The fused composite then was supported for 5 days in tissue culture. The mechanical properties of the adhesion were tested, and the construct was studied morphologically to assess viability of the cartilage and the bronchial epithelium. The bronchial epithelium showed a normal cell size (337.2 microm2) and epithelial thickness (46.47 microm). RESULTS: TISSEEL was effective in fusing the epithelium to the cartilage. The construct remained viable for 5 days in culture. There was no difference in the dimensions of the bronchial epithelium or the epithelial cells. Mechanical adhesion was achieved. CONCLUSIONS: Biologically compatible fibrin glue is an effective surgical adhesive that allows the tissue types to be fused while remaining viable and morphologically accurate. Surgical adhesives may show promise in the development of composite tissue development in the field of bioengineering.

Mechanisms of alloimmune tolerance associated with mixed chimerism induced by vascularized bone marrow transplants.

Rat limb allograft recipients represent surgically induced, immediately vascularized bone marrow transplant (VBMT) chimeras. The majority of these chimeras undergo tolerance while a minority develop graft versus host disease (GVHD). T-cell chimerism and associated mechanisms of cellular immune nonresponsiveness were investigated in tolerant VBMT chimeras. A strong correlation (p < 0.01) was observed between the clinical onset of GVHD and levels of donor T-cell chimerism approximating or greater than 50%. However, stable mixed chimerism was associated with tolerance. In conclusion, three major sequential mechanisms of immune nonresponsiveness were elucidated in tolerant VBMT chimeras over time and included development of nonspecific suppressor cells (which potentially represent natural suppressor cells), maturation of antigen-specific suppressor cell circuits, and eventually putative clonal inactivation.