Autologous incubated macrophage therapy in acute, complete spinal cord injury: results of the phase 2 randomized controlled multicenter trial.

STUDY DESIGN: Randomized controlled trial with single-blinded primary outcome assessment. OBJECTIVES: To determine the efficacy and safety of autologous incubated macrophage treatment for improving neurological outcome in patients with acute, complete spinal cord injury (SCI). SETTING: Six SCI treatment centers in the United States and Israel. METHODS: Participants with traumatic complete SCI between C5 motor and T11 neurological levels who could receive macrophage therapy within 14 days of injury were randomly assigned in a 2:1 ratio to the treatment (autologous incubated macrophages) or control (standard of care) groups. Treatment group participants underwent macrophage injection into the caudal boundary of the SCI. The primary outcome measure was American Spinal Injury Association (ASIA) Impairment Scale (AIS) A-B or better at ≥6 months. Safety was assessed by analysis of adverse events (AEs). RESULTS: Of 43 participants (26 treatment, 17 control) having sufficient data for efficacy analysis, AIS A to B or better conversion was experienced by 7 treatment and 10 control participants; AIS A to C conversion was experienced by 2 treatment and 2 control participants. The primary outcome analysis for subjects with at least 6 months follow-up showed a trend favoring the control group that did not achieve statistical significance (P=0.053). The mean number of AEs reported per participant was not significantly different between the groups (P=0.942). CONCLUSION: The analysis failed to show a significant difference in primary outcome between the two groups. The study results do not support treatment of acute complete SCI with autologous incubated macrophage therapy as specified in this protocol.

Chlamydia pneumoniae aggravates vein graft intimal hyperplasia in a rat model.

BACKGROUND: Along with angioplasty, autologus vein grafts are commonly used for artery bypass grafting in patients with advanced arterial stenosis and drug-resistant angina pectoris. Although initially a successful procedure, long-term functionality is limited due to proliferation and migration of smooth muscle cells. Like in atherosclerosis, common chronic infections caused by viruses and bacteria may contribute to this process of vein graft failure. Here we investigated the possible role of Chlamydia pneumoniae (Cpn) in the pathogenesis of venous graft failure in an experimental animal model. In 2 groups (n = 10 rats/group), an epigastric vein-to-common femoral artery interposition graft was placed. Immediately thereafter, rats were infected with Cpn (5*108 IFU) or injected with control solutions. Rats were sacrificed three weeks after surgery and the grafts were harvested for morphometrical and immunohistochemical analysis. RESULTS: Cpn administration immediately after vein grafting resulted in a significant increase in medial cross-sectional area, wall thickness and total wall area. There were no significant differences in T-cell or macrophage influx. Likewise, although positive immunostaining for both HSP60 and CRP could be detected, no differences were found between groups. Based on the observation that the number of cells/microm2 was also not altered, we conclude that Cpn infection stimulates smooth muscle cell proliferation by hereunto unknown molecular mechanisms, resulting in a significant increase in intimal hyperplasia. CONCLUSION: In conclusion, in a well defined animal model we present here for the first time evidence for a role of Chlamydia pneumoniae in the process of venous graft failure.

Generation of histocompatible tissues using nuclear transplantation.

Nuclear transplantation (therapeutic cloning) could theoretically provide a limitless source of cells for regenerative therapy. Although the cloned cells would carry the nuclear genome of the patient, the presence of mitochondria inherited from the recipient oocyte raises questions about the histocompatibility of the resulting cells. In this study, we created bioengineered tissues from cardiac, skeletal muscle, and renal cells cloned from adult bovine fibroblasts. Long-term viability was demonstrated after transplantation of the grafts into the nuclear donor animals. Reverse transcription-PCR (RT-PCR) and western blot analysis confirmed that the cloned tissues expressed tissue-specific mRNA and proteins while expressing a different mitochondrial DNA (mtDNA) haplotype. In addition to creating skeletal muscle and cardiac "patches", nuclear transplantation was used to generate functioning renal units that produced urinelike fluid and demonstrated unidirectional secretion and concentration of urea nitrogen and creatinine. Examination of the explanted renal devices revealed formation of organized glomeruli- and tubule-like structures. Delayed-type hypersensitivity (DTH) testing in vivo and Elispot analysis in vitro suggested that there was no rejection response to the cloned renal cells. The ability to generate histocompatible cells using cloning techniques addresses one of the major challenges in transplantation medicine.

Osteochondral autograft transplantation in the porcine knee.

BACKGROUND: Knee articular cartilage defects are not an uncommon problem. Because articular cartilage is limited in its ability to heal, these defects are difficult to manage. HYPOTHESIS: Osteochondral autografts will provide less of a cavitary defect and more viable hyaline articular cartilage than will control knees. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral autografts were grossly and microscopically evaluated in the porcine knee and compared with a control at 6 weeks, 3 months, and 6 months. In 18 porcine specimens, a 1-stage surgical procedure was performed to harvest an osteochondral graft from a nonweightbearing articular cartilage surface, and the graft was transplanted into a defect created in the weight-bearing region of the medial femoral condyle. In the opposite control knee, a similar defect was created in the medial femoral condyle; an osteochondral transplant was not performed. Six pigs each were sacrificed at 6 weeks, 3 months, and 6 months. RESULTS: Gross inspection of the control knees showed a cavitary defect. The defect grossly decreased in size with fibrous ingrowth seen on microscopic analysis. An increasing amount of fibrous tissue and fibrocartilage was present at the 3 time periods. Gross inspection of the graft knee showed a healed osteochondral plug with no obvious displacement, cavitary defects, or surrounding necrotic tissue at each time interval. Microscopic analysis revealed the graft knee contained viable hyaline cartilage and healed viable subchondral bone. At all time intervals, 75% to 100% of the hyaline cartilage was viable in all specimens. In 6-month specimens, bridging cartilage at the autograft-host junction was incomplete in 50%, partial in 33%, and complete in 17%. CONCLUSION: Osteochondral autografts in the porcine knee resulted in viable hyaline cartilage for up to 6 months; there was inconsistent bridging hyaline cartilage at the periphery. Grafts appeared to heal into existing subchondral bone without displacement or evidence of necrosis. CLINICAL RELEVANCE: This type of osteochondral transplant can be used as a reliable reconstructive alternative for osteochondral defects.

Comparison of fresh osteochondral autografts and allografts: a canine model.

BACKGROUND: Osteochondral autografts and allografts have been widely used in the treatment of isolated grade IV articular cartilage lesions of the knee. However, the authors are not aware of any study that has prospectively compared fresh osteochondral autografts to fresh allografts with regard to imaging, biomechanical testing, and histology. HYPOTHESIS: The imaging, biomechanical properties, and histologic appearance of fresh osteochondral autograft and fresh allograft are similar with respect to bony incorporation into host bone, articular cartilage composition, and biomechanical properties. STUDY DESIGN: Controlled laboratory study. METHODS: Eighteen adult dogs underwent bilateral knee osteochondral graft implantation after creation of an Outerbridge grade IV cartilage defect. One knee received an autograft, and the contralateral knee received a fresh allograft. Nine dogs were sacrificed at 3 months, and 9 dogs were sacrificed at 6 months. Graft analysis included gross examination, radiographs, magnetic resonance imaging, biomechanical testing, and histology. RESULTS: Magnetic resonance imaging demonstrated excellent bony incorporation of both autografts and allografts. Biomechanical testing demonstrated no significant difference between autografts versus allografts versus control at 3 or 6 months (P = .36-.91). A post hoc calculation showed 80% power to detect a 30% difference between allograft and control. Histologic examination showed normal cartilage structure for both autografts and allografts. CONCLUSION: Fresh osteochondral autograft and fresh allograft tissues are not statistically different with respect to bony incorporation, articular cartilage composition, or biomechanical properties up to 6 months after implantation. CLINICAL RELEVANCE: The use of fresh allograft tissue to treat osteochondral defects eliminates morbidity associated with harvesting autograft tissue without compromising the results of the surgical procedure.

Osteo-odonto-keratoprosthesis: a human model of autotransplant.

We evaluated the microscopical changes that occurred when bone and dental tissue were exposed to such a foreign environment as the ocular surface and anterior chamber in 17 osteo-odonto-keratoprostheses removed from the recipient's eye after 1 to 20 years. Histochemical methods were performed to demonstrate elastic and precursor fibers, while immunohistochemical procedures were used to study the distribution of collagen types I to VI. Islands of heterotopic, newly formed bone were observed in the dentin and the periodontal space, leading to focal dentoalveolar ankylosis. Remodelling and disappearance of the periodontal ligament was never diffuse.

Autologous chondrocyte implantation: natural history of postimplantation periosteal hypertrophy and effects of repair-site debridement on outcome.

PURPOSE: Our purposes were to report the clinical outcome of autologous chondrocyte implantation (ACI) patients with graft hypertrophy compared with that of unoperated ACI patients and to longitudinally assess the effects of graft hypertrophy debridement. METHODS: We divided 170 knee ACI patients with a minimum of 2 years' follow-up into groups according to the need for reoperation after ACI and the findings at surgery. Group A (n = 73) comprised patients who did not undergo reoperation, group B (n = 61) comprised patients who underwent reoperation and had findings unrelated to the repair, and group C (n = 36) comprised patients who underwent reoperation and had isolated graft hypertrophy. The International Knee Documentation Committee, modified Cincinnati knee rating, and Short Form 36 physical component scores for the 3 groups were compared. Of the repairs debrided because of graft hypertrophy, 41 were longitudinally assessed with arthroscopy or magnetic resonance imaging. RESULTS: The mean follow-up was 42.2 months. Patch-related problems were seen in 73.7% of cases undergoing reoperation less than 2 years after implantation, whereas cartilage-related problems were the dominant finding more than 2 years after implantation (70.2%). Group A patients fared significantly better than group B or C patients with regard to all 3 parameters measured, with no difference between groups B and C. Longitudinal assessment of 41 hypertrophied repairs revealed 18 with signs of pathology after graft debridement. CONCLUSIONS: This study shows that reoperation is frequent after ACI and is associated with a less satisfying outcome. Furthermore, debridement of a hypertrophied ACI graft appears to be detrimental as shown by longitudinal assessment of repairs. LEVEL OF EVIDENCE: Level IV, therapeutic case series.

Chimerism and minimal residual disease monitoring after reduced intensity conditioning (RIC) allogeneic transplantation.

Since graft-versus-leukemia (GVL) is the main weapon for disease eradication after reduced intensity conditioning (RIC) allogeneic SCT, the availability of sensitive and specific techniques to monitor changes in tumor load after transplant are especially helpful. These minimal residual disease techniques would allow an early intervention in the event of low tumor burden, for which immunotherapy is highly effective. Some authors have found an association between persistence of MRD, mixed chimerism and risk of relapse. Nevertheless, data from the literature remain contradictory and further correlations should be established, especially in RIC transplants. In this study we have analyzed the impact of MRD and chimerism monitoring on the outcome of 34 patients undergoing RIC allogeneic SCT who were considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions. At day +100 25 (75%) patients reached complete remission (CR), there were five (15%) partial responses and three patients progressed. Incidence of grade 2-4 aGVHD and extensive cGVHD were 35% and 58%, respectively. Sixteen percent of patients developing aGVHD relapsed as compared to 47% in those without aGVHD (P = 0.03) and also 10% of patients developing cGVHD relapsed as compared to 50% relapses in those without cGHVD (P = 0.03). Four patients (12%) died due to early (n = 1) and late (n = 3) transplant-related mortality. After a median follow-up of 15 months, 24 out of the 34 patients remain alive. Projected overall survival and disease-free survival at 3 years are 68% and 63%, respectively. Early chimerism analysis showed 67% of patients with complete chimerism (CC) in bone marrow (BM), 86% in peripheral blood (PB), 89% in granulocytes and 68% in T lymphocytes. On day +100, these figures were 68%, 79%, 90% and 73%, respectively, and on day +180 there were 83% patients with CC in BM, 100% in PB, 100% in granulocytes and 100% in T lymphocytes. We observed a trend to a higher incidence of relapse in patients with mixed chimerism (MC) as compared to patients with CC. MRD monitoring by flow cytometry and/or RT-PCR analysis was performed in 23 patients. MRD assessment on days +21 to +56 after transplant allowed identification of patients at risk of relapse. In this sense, seven out of 12 patients (58.3%) who had positive MRD on days +21 to +56 relapsed as compared to none out of 11 patients who had negative MRD (P = 0.002). Of the seven patients with criteria to monitor MRD who relapsed after transplant, all but one remained MRD positive until relapse. By contrast, 10 patients remained MRD negative and all of them are in continuous CR. In nine additional patients, persistence of MRD or mixed chimerism was observed after transplant and withdrawal of cyclosporin with or without DLI was performed. Only two out of these nine patients relapsed. MRD clearance was preceded by CC and GVHD. In conclusion, in our study we found that RIC allogeneic transplantation can be used in patients considered poor candidates for conventional transplantation due to advanced age or other concurrent medical conditions with both low toxicity and low transplant-related mortality. Simultaneous studies of both chimerism and MRD are a useful tool in order to predict risk of relapse in patients undergoing RIC transplants and so can be helpful for individualizing treatment strategies after transplant.

Immunohistochemical phenotypic alterations of rabbit autologous vein grafts implanted under arterial circulation with or without poor distal runoff-implications of vein graft remodeling.

Although intimal hyperplasia is a major cause limiting the long-term patency of the vein grafts, its precise mechanisms, including the effect of poor runoff, has not yet been well characterized. We thus designed the present study to try to determine the effect of poor runoff arterial flow to the phenotypic alterations of the graft wall by immnohistochemistry using anti-intermediate filaments (alpha-SM actin, desmin, and vimentin) and anti-myosin heavy chain (SM1, SM2, and SMemb) specific antibodies. Vein grafts implanted under the poor runoff hind limb of rabbits showed enhanced intimal hyperplasia, however, no apparent difference in the cytoskeleton expression, including intermediate filaments and MHC, between two groups until 4 weeks. Interestingly, six of eight vein grafts at 2 weeks after implantation in both groups showed the accumulations of perivascular fibroblast-like phenotype (negative for SM1, alpha-SM actin, and desmin) in some parts of the outer neointima, whereas the inner neointima at 2 weeks and the whole neointima at 4 weeks were mainly occupied by a smooth muscle phenotype (positive for these three). Although the cellular origin of these cells is still unknown, these results suggest that the migration of non-muscle mesenchymal cells is involved in the neointima and thus may provide a clue for better understanding vein graft remodeling.

Alterations in bronchoalveolar lavage and plasma endothelin-1 levels early after lung transplantation.

A temporary increase in pulmonary vascular resistance is observed during the first 24 hr following lung allotransplantation. We hypothesized that such early vascular changes are secondary to endothelial injury by ischemia-reperfusion, and that this may be mediated by an increased pulmonary endothelin-1 production/release. To test this hypothesis, radioimmunoassay was used to analyze endothelin-1 levels in bronchoalveolar lavage and plasma taken before surgery and at 1 hr, 4 hr, 24 hr, and 1 week after transplantation. The study was carried out on 2 groups of mongrel dogs. One group was subjected to left single-lung allotransplantation and the other to autotransplantation. Endothelin-1 levels in the bronchoalveolar lavage samples from the lung allografts were significantly increased at 1 (0.70 +/- 0.18 pg/ml) and 4 (1.84 +/- 0.65 pg/ml) hr after transplantation compared with the preoperative value (0.14 +/- 0.05 pg/ml), and declined at 24 (0.85 +/- 0.84 pg/ml) hr after transplantation. Similarly, plasma endothelin-1 levels in the allografts were significantly increased at 1 (2.0 +/- 0.80 pg/ml) and 4 (2.0 +/- 0.71 pg/ml) hr after transplant when compared with preoperative levels (0.54 +/- 0.09 pg/ml). Plasma endothelin-1 levels, however, remained significantly high after 24 hr (1.4 +/- 0.4 pg/ml; P < 0.007) and decreased after 1 week after transplant (0.89 +/- 0.32 pg/ml). On the other hand, endothelin-1 levels in bronchoalveolar lavage from the autograft group remained relatively unchanged; however, plasma levels showed a significant increase at 4 hr (6.6 +/- 1.8 pg/ml) after transplantation compared with preoperative levels (2.8 +/- 0.38 pg/ml). Elevation of endothelin-1 levels early after lung transplantation may play an important role in early high pulmonary vascular resistance and temporary graft dysfunction.

Transplantation of autologous iris pigment epithelium to the subretinal space in rabbits.

BACKGROUND: Transplantation of autologous iris pigment epithelium (IPE) into the subretinal space has been suggested as one approach for the treatment of age-related macular degeneration. Autologous rabbit IPE cells were transplanted to the subretinal space to define the technique of transplantation and examine the survival of the transplanted cells. METHODS: Autologous IPE cells were harvested by iridectomy and transplanted directly to the subretinal space of the fellow eye in 25 rabbits, using the parsplana approach. Animals were killed over a period of 5 months, and the retinas were examined morphologically by light and electron microscopy. RESULTS: Autologous IPE cells survived and formed a polarized monolayer above the retinal pigment epithelium in the subretinal space, with apical microvilli adjacent to photoreceptors. Fragments of phagocytosed photoreceptor rod outer segments were observed in phagosomes in the cytoplasm of IPE cells. Adjacent rod outer segments remained healthy throughout the experimental period. No signs of a cell-mediated immunologic response were observed. CONCLUSIONS: Our results show that in rabbits, autologous IPE cells transplanted to the subretinal space survive and do not adversely affect the photoreceptors. These results suggest that in humans, IPE cells might provide a substitute for retinal pigment epithelium cells as autologous transplants for the treatment of age-related macular degeneration.

Suppression of acute rejection prevents graft arteriosclerosis after allogeneic aorta transplantation in the rat.

Rat aortic allografts develop arteriosclerotic alterations in the vascular wall that are histologically similar to those observed in chronic rejecting vascular allografts. We used cyclosporine and rapamycin (RAPA) in two different rat strain aorta transplantation models to investigate the effect of immunosuppression on posttransplant graft arteriosclerosis. High dose CsA (25 mg/kg, 3 times/week) treatment significantly inhibited intimal proliferation in the "strong" WAG-BN model (P < 0.01) as well as in the "weak" BN-WAG combination (P < 0.001), compared with untreated allogeneic controls. In the latter combination, even fewer intimal lesions were present than in WAG autotransplants, suggesting that CsA may also inhibit the arteriosclerotic response to mechanical injury. RAPA (3 mg/kg, 3 times/week) was as effective as CsA in reducing intimal lesions (P < 0.01 and P < 0.001 in the BN-WAG and WAG-BN models, respectively). Low dose CsA (5 mg/kg, 3 times/week) was only partially effective in preventing intimal lesions. Histology of the nontreated allografts showed ongoing acute rejection in the adventitial layer. The degree of cellular infiltration correlated with the severity of arteriosclerotic lesions. High dose CsA and RAPA treatment prevented adventitial infiltration in both models, while low dose CsA was only moderately effective. In conclusion, in the present study, the degree of arteriosclerotic involvement after allogeneic aorta transplantation was related to the severity of cellular adventitial infiltration. The myointimal thickening was inhibited by effective immunosuppressive treatment. These observations may imply that chronic rejection develops after ineffective immunosuppression.

Allo- and autotransplantation of carotid artery--a new model of chronic graft vessel disease: evaluation by magnetic resonance imaging and histology.

BACKGROUND: Graft vessel disease is a special form of accelerated arteriosclerosis. Because immunological and nonimmunological factors can contribute to graft vessel disease, we developed a model that enables the study of both factors simultaneously. METHODS: A carotid artery was allografted from DA to Lewis rats, with the excised native artery autografted on the contralateral side. Five groups of six to seven rats were treated for 8 weeks with vehicle (placebo) or cyclosporine (CsA) (0.3, 1, 3, and 10 mg x kg(-1) x day(-1)), which was administered using subcutaneous osmotic minipumps. The carotid lumen area was estimated in vivo at 2, 4, and 8 weeks by magnetic resonance imaging (MRI); CsA blood levels were determined twice. Carotid neointimal thickening and medial and luminal area were measured with histological techniques. RESULTS: MRI showed bulging of the allografts but not autografts. Bulging disappeared over time with narrowing of the allograft lumina estimated by both MRI and histology. Histologically, vehicle-treated animals developed a massive neointima, which was inhibited in a dose-dependent manner by CsA. Autografts remained normal except for minimal subintimal thickening of two of four arteries in the group given the highest dose of CsA. Cellular rejection was detected in the allografts of all but the highest-dose group. The CsA blood levels were similar to those used in man at the two lower doses and about 10-fold higher at the highest dose. CONCLUSIONS: Subintimal thickening did not correlate with in vivo lumen size, a phenomenon that we have previously described for balloon catheter-induced lesions. CsA blood concentrations similar to those used in patients suppressed neointima formation in part, and 10-fold higher concentrations almost completely suppressed neointima formation.

The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin.

BACKGROUND: Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. Epidermal regeneration obtained with autologous cultured keratinocytes (cultured autografts) can be life-saving for patients suffering from massive full-thickness burns. However, the widespread use of cultured autografts has been hampered by poor clinical results that have been consistently reported by different burn units, even when cells were applied on properly prepared wound beds. This might arise from the depletion of epidermal stem cells (holoclones) in culture. Depletion of holoclones can occur because of (i) incorrect culture conditions, (ii) environmental damage of the exposed basal layer of cultured grafts, or (iii) use of new substrates or culture technologies not pretested for holoclone preservation. The aim of this study was to show that, if new keratinocyte culture technologies and/or "delivery systems" are proposed, a careful evaluation of epidermal stem cell preservation is essential for the clinical performance of this life-saving technology. METHODS: Fibrin was chosen as a potential substrate for keratinocyte cultivation. Stem cells were monitored by clonal analysis using the culture system originally described by Rheinwald and Green as a reference. Massive full-thickness burns were treated with the composite allodermis/cultured autograft technique. RESULTS: We show that: (i) the relative percentage of holoclones, meroclones, and paraclones is maintained when keratinocytes are cultivated on fibrin, proving that fibrin does not induce clonal conversion and consequent loss of epidermal stem cells; (ii) the clonogenic ability, growth rate, and long-term proliferative potential are not affected by the new culture system; (iii) when fibrin-cultured autografts bearing stem cells are applied on massive full-thickness burns, the "take" of keratinocytes is high, reproducible, and permanent; and (iv) fibrin allows a significant reduction of the cost of cultured autografts and eliminates problems related to their handling and transportation. CONCLUSION: Our data demonstrate that: (i) cultured autografts bearing stem cells can indeed rapidly and permanently cover a large body surface; and (ii) fibrin is a suitable substrate for keratinocyte cultivation and transplantation. These data lend strength to the concept that the success of cell therapy at a clinical level requires cultivation and transplantation of stem cells. We therefore suggest that the proposal of a culture system aimed at the replacement of any severely damaged self-renewing tissue should be preceded by a careful evaluation of its stem cell population.

A long-term study of allogeneic rat hindlimb transplants immunosuppressed with RS-61443.

Although technically possible, limb allotransplantation has not been applied clinically. The skin component is especially antigenic, requiring high immunosuppressant doses with an unacceptable toxicity profile. RS-61443, an experimental mycophenolic acid ester that inhibits lymphocyte proliferation without major systemic toxicity, was tested as an immunosuppressant to prevent rejection of rat hindlimb allotransplants. Utilizing Brown-Norway donors and F344 recipients to provide a major mismatch at the MHC, midfemur orthotopic limb transfer was performed with microsurgical repair of femoral vessels and sciatic nerve. Four primary groups were studied: autografts (n = 4); untreated allografts (n = 6); allografts receiving CsA 10 mg/kg for 20 days, then twice per week (n = 6); and allografts receiving RS-61443 30 mg/kg/day (n = 6). Skin and soft tissues were biopsied to assess rejection. Autografts had indefinite limb survival, while untreated allografts had complete acute rejection within 10-12 days. Five of the six CsA rats developed delayed mild-moderate acute rejection within 6 months. In contrast, 5 of the 6 RS-61443 rats had no rejection after at least 32 weeks, while the sixth rat developed only slight rejection on skin biopsy. All animals regained full sensation and partial functional return. RS-61443 is highly effective as a primary immunosuppressant for hindlimb allotransplantation. The disturbing moderate rejection observed in CsA animals, which was absent with RS-61443, may significantly hamper function of transplanted limbs.

The effect of small bowel transplantation on the morphology and physiology of intestinal muscle: a comparison of autografts versus allografts in dogs.

The effects of acute (AR) and chronic rejection (CR) on intestinal smooth muscle that are responsible for the dysmotility following small bowel transplantation (SBTX) are incompletely understood. Jejunal and ileal specimens from normal control dogs (n=7), and autotransplanted dogs were examined at 7 days (n=6) and 1 (n=7), 3 (n=6), 6 (n=6), and 12 months (n=6). Allotransplanted dogs that developed AR (n=8) and CR (n=5) were examined for gross and microscopic morphology (muscle thickness, the number and size of myocytes, and inflammatory infiltrate), and for contractile and intracellular electrical function in vitro. Auto-SBTX did not alter morphology at any period, but contractile function was impaired at 7 days (73.6%) compared with normal intestine. Acute rejection did not influence myocyte number or size, but was associated with a prominent infiltrate of neutrophils and lymphocytes, and severely impaired contractile function (20.6%) compared with auto-SBTX controls. Acute rejection also significantly inhibited the amplitude of slow waves and of inhibitory junction potentials. Chronic rejection caused thickening of muscularis propria by both hyperplasia (175.5%) and hypertrophy (202.6%) accompanied by moderate inflammatory cell infiltrate compared with auto-SBTX controls. We conclude that the marked inflammatory infiltrate into the muscularis propria indicates that the graft muscle is injured by both acute and chronic rejection; impaired function of intestinal smooth muscle following SBTX results from both rejection and the injury associated with transplantation, and chronic rejection following SBTX is associated with both hyperplasia and hypertrophy of the muscularis propria.

Characteristics of endothelin receptors in acutely rejecting transplanted lungs.

Experiments were designed to characterize endothelin receptors in bronchi and parenchyma of transplanted lungs during acute rejection. Third-order bronchi from autografted or allografted lungs were either cut into rings and suspended in organ chambers for the measurement of isometric force or frozen for isolation of membrane proteins. Lung parenchyma was prepared for histology or isolation of membrane protein. The grade of rejection was 2.74+/-0.17 (n= 19) in allotransplanted lungs; evidence of infection was present in 58% of the transplanted lungs. In organ chamber experiments, endothelin 1 (which stimulates endothelin A receptors) caused comparable contraction of bronchi from autotransplanted and allotransplanted rejecting lungs. Endothelin 3 (which stimulates endothelin A and B receptors) caused contractions of bronchi from autotransplanted lungs which were not different from those caused by endothelin 1. In contrast, contractions caused by endothelin 3 were reduced in bronchi from rejecting allotransplanted lungs. The magnitude of contractions caused by endothelin 3 was reduced further when infection was present with rejection. Competitive inhibition of 125I-endothelin 1 by endothelin 3 was significant for a two-site binding model in membranes prepared from all bronchi and lung parenchyma. The total number of binding sites (Bmax) was reduced significantly in bronchi and parenchyma from rejecting lungs with or without infection. The relative proportions of high-affinity and low-affinity binding sites did not change. Affinities of both high- and low-affinity receptors were not altered with rejection. These results indicate that at least two subtypes of endothelin receptors are present on canine bronchial smooth muscle and parenchyma. The number of endothelin receptors associated with bronchial contractions is reduced with rejection of lung allografts.

Shielding of augmented tendon--tendon repair.

Strength and function of autogenic and xenogenic reconstruction of digital extensor tendons was examined in an ovine model. In this study, tendon-graft junctions were formed by either suture augmented with a woven polyester tube (A), or augmented and shielded from surrounding tissues by chemically-treated bovine pericardium (S). By 12 wk, both A and S sheep had returned to full range of motion. Mechanical strength of both the autograft-host and xenograft-host repair sites was similar, with a pooled strength of 131 +/- 25 N (n = 15). Similarly, the mid-portion xenograft strengths were constant at approximately 366 +/- 97 N (n = 7). In contrast, mid-portion autograft strengths decreased from 380 +/- 110 N (N = 4) to 120 +/- 66 N (n = 4) if shielding was omitted. The loss in autograft strength was attributed to loss of function associated with adhesions. The use of the augmentation device coupled with an adhesion barrier gives higher initial reconstruction strength and improved function during the host repair period up to 12 wk.

Effects of collagen gel mixed with hydroxyapatite powder on interface between newly formed bone and grafted achilles tendon in rabbit femoral bone tunnel.

The Achilles tendon was implanted into a bone tunnel made in the femoral condyle of 20 rabbits. In the left femur, collagen gel mixed with hydroxyapatite powder (C-HAp) was injected between the graft and the bone tunnel. On the other hand, as a control, simple saline was injected in the right femur. Five rabbits were sacrificed at 4, 8, 12, and 16 weeks after surgery. Histological findings showed that in the C-HAp group, the grafted tendon came in direct contact with new bone, and Sharpey-like collagen fibers arising from the grafted tendon were observed to penetrate new bone by 4 weeks after surgery. In the control group, however, fibrous tissue was observed between new bone and the grafted tendon, but no penetrating fibers from the grafted tendon into the new bone were observed until 16 weeks. The area of new bone in the C-HAp group was significantly greater than that in the control group 4, 8, and 12 weeks after surgery (p < 0.0001, p < 0.0007, p < 0.0013, respectively).

Interphase FISH: a rapid method for detecting malignant plasma cells in multiple myeloma patients submitted to autologous transplantation.

Fluorescence in situ hybridization (FISH) on interphase nuclei has been shown to be an efficient method for detecting aneuploidy in multiple myeloma (MM). The aim of this study was to test the feasibility of FISH techniques for detecting malignant cells in the harvests of MM patients submitted to autologous transplantation. As trisomy 9 (T9) is a frequent event in MM, we used it as a genetic marker of malignant plasma cells. T9 was detected in 45 out of 55 MM bone marrow samples (81.8%) using a chromosome 9 centromeric (C9C) probe. Twenty-four of the 55 MM patients were subjected to high-dose therapy followed by autologous unselected progenitor cell transplantation. Trisomy 9 was detected in 20 patients and was used as a marker of malignant cells. Upon karyotypic analysis, three of the four remaining patients without T9 showed an unbalanced translocation leading to a complete trisomy of the long arm of chromosome 1 (T1q). We thus used a 1q juxtacentromeric probe, pUC1.77, as another genetic marker of malignant plasma cells in these three further patients. FISH with C9C or pUC1.77 probes was performed on the harvests of these 23 patients and detected clonal cells in 11 transplants. The disease-free survival from graft was significantly longer for the patients who had no malignant cells in their transplant (P=0.009). The median disease-free survival was 23 months in these patients, as compared to 12 months in the patients whose transplant was contaminated. As almost all MM are cytogenetically abnormal, FISH with adequate probes represents a simple, quantitative tool for rapid detection of malignant cells in the harvests. Our results also suggest that the presence of MM cells in the transplant may be predictive of poor outcome.