Quantitation of sentinel node metastatic burden and Her-2/neu over-expression accurately predicts residual axillary nodal involvement and extranodal disease in breast cancer.

BACKGROUND DATA: Recent literature has suggested that completion axillary lymph node dissection (ALND) in breast carcinoma patients with positive SLN may not be necessary. However, a method for determining the risk of non-SLN or extranodal disease remains to be established. AIMS: To determine if pathological variables from primary tumors and sentinel lymph node (SLN) metastases could predict the probability of non-sentinel lymph node (NSLN) metastases and extranodal disease in patients with breast carcinoma and SLN metastases. METHODS: 84 women with T1-3 breast cancer and clinically-negative axillae underwent completion ALND. Maximum diameter and width of SLN metastases were measured to calculate metastatic area. When multiple SLNs contained metastases, areas were summed to calculate the Total Metastatic Area (TMA). Multiple linear regression models were used to identify predictive factors. RESULTS: Her-2/neu over-expression increased the odds of NSLN metastases (OR 4.3, p = 0.01) and extranodal disease (OR 7.9, p < 0.001). Independent SLN predictors were ≥1 positive SLN (OR, 7.35), maximum diameter and area of SLN metastases (OR 2.26, 1.85 respectively) and TMA (OR, 2.12). Maximum metastatic diameter/SLN diameter (OR 3.71, p = 0.04) and the area of metastases/SLN area (OR 3.4, p = 0.04) were predictive. For every 1 mm increase in diameter of SLN metastases, the odds of NSLN extranodal disease increased by 8.5% (p = 0.02). TMA >0.40 cm(2) was an independent predictor for NSLN metastases and extranodal disease. CONCLUSION: Her-2/neu over-expression and parameters assessing metastatic burden in the SLN, particularly TMA, predicted the presence of NSLN involvement and extranodal disease in patients with breast carcinoma and SLN metastases.

Factors influencing rat survival in a warm renal ischemia model: time to adapt the protocols.

INTRODUCTION: Survival in warm renal ischemia models is not only dependent on the treatment or surgical technique being evaluated, but also on factors inherent to the model itself. Use of rats of various strains in previous studies makes interstudy comparison difficult when trying to design an appropriate model control that would yield intermediate survival. In this study, impact of rat strain on survival after prolonged warm renal ischemia in the setting of delivery-controlled inhalational anesthesia was evaluated. MATERIALS AND METHODS: Under general delivery-controlled inhalation anesthesia with isoflurane, Dahl salt-sensitive, Wistar-Furth, Sprague-Dawley, and spontaneously hypertensive rats (n = 66 rats) were subjected to 150 minutes of unilateral renal warm ischemia time, subsequent reperfusion, and contralateral nephrectomy. Animals were followed up for 1 month, after which survivors were euthanized and morphologic changes in kidneys were scored. RESULTS: Thirty-day survival was: Dahl salt sensitive, 78%; Wistar-Furth, 67%; Sprague-Dawley, 55%; and spontaneously hypertensive rats, 0% (P < .0001). Histologic acute injury scores were higher for non-survivors versus 30-day survivors (P < .0001). CONCLUSION: Our data strongly suggest that rat strain is a major factor influencing survival and that strain and warm ischemia time selections must be considered together when designing a model control yielding intermediate survival. Further study is warranted in order to compare the effect of delivery-controlled inhalational versus historical anesthesia methods on animal survival.

Incidence, mechanism and prognostic value of activated AKT in pancreas cancer.

When activated, the serine/threonine kinase AKT mediates an antiapoptotic signal implicated in chemoresistance of various cancers. The mechanism(s) of AKT activation are unknown, though overexpression of HER-2/neu has been implicated in breast cancer. Therefore, we determined the incidence of activated AKT in human pancreatic cancer, whether HER-2/neu is involved in AKT activation, and if AKT activation is associated with biologic behaviour. HER-2/neu expression and AKT activation were examined in seven pancreatic cancer cell lines by Western blotting. The in vitro effect of HER-2/neu inhibition on AKT activation was similarly determined. Finally, 78 pancreatic cancer specimens were examined for AKT activation and HER-2/neu overexpression, and correlated with the clinical prognostic variable of histologic grade. HER-2/neu was overexpressed in two of seven cell lines; these two cell lines demonstrated the highest level of AKT activation. Inhibition of HER-2/neu reduced AKT activation in vitro. AKT was activated in 46 out of 78 (59%) of the pancreatic cancers; HER-2/neu overexpression correlated with AKT activation (P=0.015). Furthermore, AKT activation was correlated with higher histologic tumour grade (P=0.047). Thus, it is concluded that AKT is frequently activated in pancreatic cancer; this antiapoptotic signal may be mediated by HER-2/neu overexpression. AKT activation is associated with tumour grade, an important prognostic factor.

An in vivo evaluation of a chondroitin sulfate solution to prevent postoperative intraperitoneal adhesion formation.

PURPOSE: The goal of this study was to determine the efficacy of a single intraperitoneal administration of a chondroitin sulfate solution in preventing postoperative adhesion formation. METHODS. Twenty-five Sprague-Dawley rats had a 1-cm(2) area of cecal serosa abraded. Controls (CON, n = 5) received no treatment, the chondroitin sulfate group (CS, n = 10) received chondroitin sulfate (0.013 g/kg) in 0.9% NaCl intraperitoneally (ip), and vehicle controls (VC, n = 10) received an equal volume of 0.9% NaCl solution ip before the abdomen was closed. All animals were sacrificed on postoperative day 10. The extent of adhesion was quantified according to Mazuji's adhesion grade (0 to 4: 0 = no adhesion and 4 = very dense adhesion) and quantitated after H&E, trichome, and immunohistochemical staining for fibrin and collagen type I and type III using digital image analysis. RESULTS: The mean Mazuji's adhesion grade in the CON was 4.0 +/- 0.0, in the VC 2.60 +/- 0.37, and in the CS 1.3 +/- 0.42 (P < 0.01 for CS vs CON and P < 0.05 for CS vs VC comparisons). The mean gray-scale intensity (0-255: 0 = dense amount and 255 = none) of adhesion density in the CON was 105. 5 +/- 5.5, in the VC 125 +/- 15.0, and in the CS 178.3 +/- 21.0 (P < 0.01 for CS vs CON and P < 0.05 for CS vs VC comparisons). The mean adjusted intensity stain indices (AISI) for fibrin and collagen type I in the CON were 59 +/- 17 and 53 +/- 19, in the VC 27 +/- 3 and 25 +/- 7, and in the CS 16 +/- 5 and 6 +/- 3, respectively (P < 0.05 between CS and CON comparisons). The AISI of collagen type III was not significant among all the groups (P > 0.1). CONCLUSIONS: The extent of early postoperative intra-abdominal adhesion formation as determined by gross assessment and from quantitation of fibrin and collagen type I deposition was significantly reduced by a single intraperitoneal administration of a chondroitin sulfate solution.

Use of three-dimensional microscopy with multiple oblique illumination in evaluating cytopathology preparations.

OBJECTIVE: To analyze whether the use of multiple oblique illumination (MOI), in contrast to axial illumination (AI), improves imagery in bright field microscopy. Specimens containing thick cell clusters, such as cervical Pap smears, are often misread because of out-of-focus cell clusters. We hypothesized that visualization of these specimens with MOI would enhance this information as compared with AI. STUDY DESIGN: Micrometer images and Pap smears were analyzed in focus, and 8 and 40 microns out of focus by MOI and AI. All fields were captured to a remote computer, histograms were constructed, and mean light intensity was calculated. Mathematical formulation was used to define the histogram distribution of the micrometer images. Statistical analysis was performed using one-way ANOVA and Newman-Keuls test. RESULTS: K(focus) was improved (P < .01) and at 20 and 40 microns out of focus, mean light intensities were greater (P < .003, P < .05, respectively) with MOI as compared to AI. CONCLUSION: MOI improves out-of-focus information by increasing light penetration through the specimen and enhancing contrast and resolution.

Composite tissue allografts in rats: IV. Graft-versus-host disease in recipients of vascularized bone marrow transplants.

This laboratory has used a composite tissue allograft model as a vehicle for studies on a new type of bone marrow transplant, the vascularized bone marrow transplant. The model consists of a rat hind limb transplant that incorporates integumentary musculoskeletal, and lymphopoietic tissues. These transplants, in comparison with conventional marrow transplants, have the advantage of providing a syngeneic microenvironment and immediate engraftment of both mature and progenitor hemopoietic cells at the time of transplantation. The characteristics of graft-versus-host disease were studied in this model. Lewis X Brown Norway F1 (LBN RT-1(1+n)) rats received hind limbs from Lewis (LEW RT-1(1)) donors (n = 19). Animals were observed daily for signs of graft-versus-host disease. Necropsies were performed. A minority of animals developed lethal disease (7 of 19 recipients) and demonstrated cachexia with concomitant histopathologic changes of the disease. Acute and chronic groups emerged with distinct clinical courses, which are similar to other models of this disease. Recipients of vascularized bone marrow transplants (limb transplants) showed clinical and histopathologic changes of the disease. The transplants may be used as a model of graft-versus-host disease in humans. Most interestingly, the transplant has a lower incidence of disease compared with other methods of bone marrow transplantation and represents an alternative to conventional bone marrow transplantation, which deserves further exploration. It may be possible to develop a new technique for bone marrow transplantation based on this surgical approach. It is proposed that the transfer of vascularized blocks of bone/marrow into prospective recipients as opposed to cellular bone marrow transplants may be preferable.

A novel in vitro model for xenorejection and immune mechanisms using bioengineered living skin equivalents.

We hypothesized that an in vitro bioengineered skin (LSE) could be used for the study of xenogeneic inflammatory or immunosuppressive mechanisms. Murine fibroblasts (10(4)/mL) were mixed with type 1 rat-tail collagen to form a matrix (approximately 5 days) on which human keratinocytes (NHEK, 10(5)/75 microL) were seeded. The xeno-LSE was used as a rejection target organ in vitro. Rejection was achieved by the addition of human lymphocytes (10(6)/75 microL). At various intervals of growth, control LSEs without lymphocytes (CON) and rejecting LSEs with immunocytes (REJ) were analyzed. Slides were stained with hematoxylin & eosin and examined under light microscopy. REJ xenocomposite LSEs showed classic histologic signs of rejection. There was separation of the D-E junction with the presence of dyskeratotic and necrotic cells with a concomitant inflammatory infiltrate. The CON LSEs showed essentially normal dermis (collagen fibroblast matrix) and keratinocytes. A significant finding was the separation of the D-E junction in the absence of an inflammatory infiltrate. Previous studies have shown that human keratinocytes can be grown with human fibroblasts that are histologically similar to natural human skin. This discohesive nature of the murine fibroblasts and human keratinocytes may represent an important intercellular interaction associated with xeno-cellular interactions and is worthy of further study. Furthermore, the ability to grow xeno-composite tissues has profound implications in the face of organ donor shortages. They may represent an eventual in vitro replacement of skin and even solid organs.

Sclerosing epithelioid fibrosarcoma: short T2 on MR imaging.

A case of sclerosing epithelioid fibrosarcoma and its appearance on MRI is presented. The tumor showed a zonal architecture on MRI with a large central core of very low signal intensity and a peripheral rim of intermediate to high signal intensity on T1- and T2-weighted spin echo pulse sequences. The core showed decreased cellularity with dense collagen deposition on histologic examination, and the peripheral zone increased cellularity with increased nuclear atypia. The presence of a prominent region of very low signal intensity on T1- and T2- weighted images can be seen with neural tumors, giant cell tumor of the tendon sheath, aggressive fibromatosis, and, in rare instances, with soft tissue sarcomas rich in collagen.

Donor treatment with the lazeroid U74389G reduces ischemia-reperfusion injury in a rat lung transplant model.

BACKGROUND: Antioxidant treatment with lazeroids has proven beneficial for the amelioration of reperfusion injury in experimental lung transplantation. This study compares the effect of donor versus recipient treatment on immediate postoperative graft function. METHODS: A model of acute double-lung transplantation in rats was used to assess graft function. Transplanted controls after 2 (group I) and 16 hours of ischemia (group II) were compared to a recipient (group III; 16-hour ischemia) and a donor treatment group (group IV; 16-hour ischemia) using the lazeroid U74389G (6 mg/kg). Serial assessment of alveolar-arterial oxygen difference, dynamic lung compliance, airway and pulmonary vascular resistance was obtained during a 2-hour reperfusion period. Final analysis included survival, weight gain, and histologic examination. RESULTS: Graft function was significantly better after 2 hours of ischemia than in any of the three 16-hour ischemia groups (II, III, IV). After 16 hours of ischemia, donor treatment provided superior graft function with respect to dynamic lung compliance, airway resistance, and alveolar-arterial oxygen difference when compared with groups II and III. The pulmonary vascular resistance was significantly higher in group III when compared with groups II and IV. Graft weight increase reflecting edema was highest in groups III (104%) and II (98%). CONCLUSIONS: After prolonged ischemia only donor treatment with the lazeroid U74389G was able to significantly reduce ischemia-reperfusion-related graft dysfunction.

Exogenous surfactant treatment before and after sixteen hours of ischemia in experimental lung transplantation.

OBJECTIVE: A syngeneic, acute, double lung transplant model in the rat was used to determine the impact of exogenous surfactant treatment on graft function after prolonged cold storage. METHODS: The donor grafts were flush perfused, preserved for 16 hours, and then reperfused for 120 minutes. Untreated lungs served as controls (group I). In group II the recipient received a 200 mg/kg dose of surfactant (CuroSurf) before reperfusion. In groups III and IV, surfactant was administered before perfusion and harvesting (III, 20 mg/kg; IV, 200 mg/kg). Serial measurements of graft pulmonary vascular resistance, alveolar-arterial oxygen difference, and compliance were obtained. Final graft assessment included weight gain and histologic study. RESULTS: Repeated-measures analysis of variance showed significant improvement of graft performance in respect to compliance, alveolar-arterial oxygen difference, and pulmonary vascular resistance in donor surfactant treatment group IV (200 mg/kg) in comparison with recipient treatment (group II) and untreated controls (group I). Reducing the donor surfactant supplementation from 200 mg/kg to 20 mg/kg (group III) improved oxygenation and lung compliance as compared with untreated controls. Grafts in groups I and II had significantly more weight gain after 2 hours of reperfusion. Recipient treatment resulted in significantly more pulmonary hemorrhage in histologic sections. CONCLUSION: Donor treatment with exogenous surfactant is advantageous for preservation of graft function after extended ischemia. Positive effects may be seen with as little as 20 mg/kg of exogenous surfactant given before donor organ perfusion.

Donor pretreatment with intravenous prostacyclin versus inhaled nitric oxide in experimental lung transplantation.

The pulmonary vasodilatory effects of prostacyclin (PGI2) were compared with inhaled nitric oxide (NO) for donor treatment in an acute double lung transplantation model in the rat. The PGI2 group (n=10) received 35 microg/kg PGI2 both intravenously and into the flush solution. The NO group (n=10) was ventilated before and during perfusion with nitric oxide for an expiratory NO concentration of 20 ppm. Both groups were compared with untreated controls (n=10). Following cold ischemia of 16 hr the donor lungs were implanted in syngeneic recipients via specially designed stents to the left pulmonary artery and vein. Separate graft ventilation permitted determination of compliance and resistance. During 120 min of reperfusion serial measurements of graft pulmonary vascular resistance (PVR) and alveolar arterial oxygen difference (AAD02) were obtained. Final graft assessment included weight gain and histological analysis. Data are listed as mean+/-SE. The type of donor pretreatment had a definite and negative impact on survival (NO: 106+/-6, controls: 116+/-4, PGI2: 120+/-0 min; P<0.02) and overall graft function. During reperfusion the compliance was significantly reduced in NO (23+/-4) in comparison with controls (34+/-3) and PGI2 (50+/-4 ml/cmH2O; P<0.01). The PVR was 785+/-238 in NO, 240+/-60 in controls and 181+/-71 mmHg/ml/min in PGI2 (P<0.02). The AaD02 was compromised in NO (486+/-44) compared with controls (396+/-53) and PGI2 (108+/-34 mmHg; P<0.02). The weight increase at the end of reperfusion amounted to 101+/-17% in NO, 98+/-13% in controls, and 69+/-7% in PGI2 (P<0.05). Histological analysis showed significantly more interstitial edema in the NO group. In conclusion, PGI2 administration significantly improves global lung function while the inhalation of nitric oxide before and during donor perfusion has a detrimental effect on the quality of graft preservation.

The importance of static lung inflation during organ storage: the impact of varying ischemic intervals in a double lung rat transplantation model.

To determine the importance of static lung inflation during storage, graft performance was evaluated at different levels of intratracheal pressure and varying ischemic intervals. Lewis rat lungs were perfused with low-potassium Euro-Collins and stored for 4 or 8 hr either in atelectasis (4 hr: group I; 8 hr: group IV, respectively) or 13 (group II; V) or 26 cmH2O of airway pressure (groups III, VI). Following implantation continuous measurement of alveolar-arterial oxygen difference (AaDO2*) and pulmonary vascular resistance (PVR) were performed. Separate ventilation allowed assessment of mechanical lung function of the graft. At the end of reperfusion (120 min) weight gain, histology, and phospholipid and protein content in the pulmonary lavage were compared between the groups. Despite significant differences in survival at 4 hr of ischemia graft function did not differ in groups I to III. In contrast, static inflation had a significant impact after 8 hr of ischemia. Lungs stored in atelectasis (group IV) could not be reperfused and failed immediately. Survival in group V was 83+/-11 versus 107+/-7 min in group VI (P<0.05). Compliance at 80 min was 27+/-3 in group V and 52+/-6 ml/cmH2O in group VI (P<0.02). Corresponding values for PVR were 232+/-92 and 112+/-16 mmHg/ml/min, respectively (P<0.05). Less inflation and longer ischemia resulted in a reduction of the large to small phospholipid aggregate ratio and deterioration of surfactant function in the bubble surfactometer. In conclusion, while the amount of static lung inflation may not be critical following short ischemia, the performance of the graft improves significantly with full inflation (26 cmH2O) following extended ischemia (8 hr).

Graft-versus-host disease in limb transplantation: digital image analysis of bone marrow and TGF-beta expression in situ using a novel 3-D microscope.

A subpopulation of parental to hybrid VBMT recipients developed characteristic clinical and histopathologic manifestations of GVHD. These changes are similar to those seen in human GVHD secondary to bone marrow transplantation. Human GVHD also manifests itself in an acute and chronic manner. Only a minority (30% to 40%) of animals developed lethal GVHD in our model. Those animals developing GVHD had a significantly (P < .0001) higher expression of TGF-beta in situ compared to the tolerant subpopulation. The differential expression of TGF-beta may represent an important mechanism of immune dysregulation associated with GVHD in CTA recipients.

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.

Cellular mechanisms of alloimmune non-responsiveness in tolerant mixed lymphocyte chimeras induced by vascularized bone marrow transplants.

It has been demonstrated that the development of stable mixed lymphocyte chimerism is associated with alloimmune tolerance induction in vascularized bone marrow transplant (VBMT) recipients. The underlying mechanisms of immune non-responsiveness in tolerant VBMT chimeras remains unclear. Our VBMT model involves the transplantation of a parental donor limb (Lewis rats) onto a hybrid (Lewis x Brown Norway) F1 recipient. Tolerogenic mechanisms and cellular immune regulation to self and host allodeterminants were investigated during the early post-transplant phase of tolerance induction. Flow cytometric analysis of sIg+-depleted experimental peripheral blood lymphocytes from tolerant VBMT recipients demonstrated low level stable mixed immune chimerism. Chimeric cells tested for responsiveness against self-LEW determinants showed activated proliferation and immune dysregulation 30 days post-transplantation. However, direct immunocytolytic activity against LEW determinants was not found. Tolerant chimeras also demonstrated elevated cellular proliferation and cytolytic responses against host-specific BN allodeterminants at 30 days. Consistent with these in vitro findings, limited clinical signs compatible with GVH reactivity were evident in vivo at this time. Following this initial period, the tolerant VBMT animals returned to normal clinical condition and remained otherwise healthy throughout the study. Consistent with these results, VBMT chimeras then showed declining proliferative responses from the elevated values seen at 30 days against self-LEW determinants. Proliferative and immunocytolytic responses also decreased against host-specific BN allodeterminants from peak levels at 30 days. In conclusion, these results provide evidence that the initial phases of tolerance induction in VBMT chimeras consist of self- and alloimmune regulation that follow an early period of immune dysregulation. Sequential phases of immune dysregulation and re-regulation elucidated in VBMT stable mixed chimeras within the first 100-day period may represent important mechanisms of tolerance induction.

A rapid and sensitive cellular enzyme-linked immunoabsorbent assay (CELISA) for the detection and quantitation of antibodies against cell surface determinants. I. A comparison of cell fixation and storage techniques.

A solid phase cellular ELISA was designed and evaluated for the detection of antibodies specific for cell surface determinants. It was hypothesized that certain fixation and freezing procedures would result in stabilization of cell structures for prevention of antigen diffusion and extraction during washing procedures. This would assure assay accuracy and convenient sample management. It was hypothesized that fixation with certain reagents prior to analysis would not alter antigenicity of antibody targeted epitopes. In order to improve the preservation of the cells following cell binding to the solid phase matrix while still retaining antigenicity and morphology, a series of fixatives and storage procedures were screened to determine which were best suited for CELISA. Methanol, washing buffer (WB), Hanks' balanced salt solution (HBSS), and 0.5% formalin in HBSS were examined by comparing their relative cell binding capacity and the subsequent cell morphology. In consideration of all variables, fixation in 0.5% formalin provided the best maintenance of cell antigenicity, morphology, binding, and was associated with consistent results. Cells used immediately after fixation and fixed cells used after storage at -80 degrees C for up to 12 months were compared to determine if long term storage affected antigenicity. Since frozen cells and fresh cells demonstrated statistically identical positive to negative ratios and consistency of antibody binding, it was determined that long term frozen storage of formalin-fixed cells did not adversely affect antibody binding capacity to cell surface determinants.

Mechanisms of prior blood transfusion-cyclosporine-induced tolerance: a potential role for immune-cellular chimerism.

Skin allografts were not enhanced by prior conditioning of blood and CyA (5 or 10 mg/kg/d). However, when BM-CyA pretreatment was used, SA survival was significantly prolonged (CyA, 5 or 10 mg/kg/d). In examining differences between the BT-CyA and BM-CyA protocols, equivocal levels of donor microchimerism (1.5%) were found in the spleens of BT-CyA conditioned recipients at the time of transplantation (day 0). In contrast, highly significant levels of splenic donor chimerism (17.2%) developed at day 0 for the BM-CyA pretransplant recipients. Skin-allograft prolongation under the BM-CyA protocol implied that the effect may be linked to the existence of a donor-specific stem-cell population in the recipient animal.

Vascularized bone marrow transplantation (VBMT): induction of stable mixed T-cell chimerism and transplantation tolerance in unmodified recipients.

In this preliminary report, our model of VBMT across a semiallogeneic barrier consistently brings about antigen-specific host tolerance with absence of GVHD in the majority of recipients. No immunologic or radiologic intervention was utilized. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention.

Development of stable mixed T cell chimerism and transplantation tolerance without immune modulation in recipients of vascularized bone marrow allografts.

A consistent majority (62.5%) of immunologically unmodified rat recipients transplanted with vascularized hind-limb bone marrow allografts across a semiallogeneic transplant barrier developed tolerance with absence of graft-versus-host disease. A minority of recipients (37.5%) demonstrated lethal GVHD. Transplantation tolerance in the majority was associated with the induction of stable low-level mixed T cell chimerism, including donor CD5+, CD4+, and CD8+ lymphocytes. Chimeras were specifically immune nonresponsive to host alloantigenic determinants. These results emphasized a potentially important mechanism for low-level stable mixed lymphoid chimerism (SMLC) in tolerance induction, independent of immune suppressive effects due to irradiation or immunopharmacologic intervention. These vascularized bone marrow transplantation (VBMT) results may establish the experimental foundation for a novel approach to stem cell transfer and bone marrow transplantation.