Iliac Crest Marrow Research Articles

Acetabular Bone Marrow Aspiration During Total Hip Arthroplasty.

Biologically augmented surgical treatments of orthopaedic conditions are increasingly popular. Bone marrow aspirate concentrate is a key orthobiologic tissue source, and the field is moving from the standard iliac crest marrow aspiration toward local aspirations of marrow depots that are accessible during the standard-of-care procedures in an attempt to reduce morbidity, surgery time, and cost. Here, we present the aspiration of the standard iliac marrow depot, but through a novel acetabular approach during total hip arthroplasty. This procedure markedly simplifies biologic augmentation with bone marrow aspirate concentrate in this large patient cohort.

Fluorine-18 fluorodeoxyglucose positron emission tomography imaging of T-lymphoblastic lymphoma patients.

The purpose of the present study was to evaluate the fluorine-18 fluorodeoxyglucose positron emission tomography (FDG-PET) findings in patients with T-lymphoblastic lymphoma (T-LBL). In total, 9 patients with histopathologically confirmed T-LBL were included in the study. Bone marrow (BM) involvement and leukemic transformation (LT) were evaluated through iliac crest marrow biopsy and peripheral blood blast count. FDG-PET scans were performed at the initial pre-treatment point. Two experienced nuclear medicine physicians evaluated the FDG-PET images by visual analysis and using the maximum standardized uptake values (SUVmax) of the malignant lesions. Overall, 8 out 9 patients presented with BM involvement; 7 showed LT, while 1 showed BM involvement without LT. All involved T-LBL lesions were FDG-avid with variable uptake. The mean SUVmax was 6.4±3.3. T-LBL patients with BM involvement showed diffuse or nodular marrow uptake. In addition, all the patients with LT showed diffuse marrow FDG activity. However, the patient with BM involvement but no LT showed nodular FDG uptake in the marrow. In conclusion, the present study indicates that it is possible to use FDG-PET for the evaluation of the disease extent of T-LBL. Furthermore, the imaging technique could provide a diagnostic clue for determining BM involvement or LT.

Process automation in manufacturing of mesenchymal stromal cells.

Process automation in manufacturing of mesenchymal stromal cells.

Supercharging allografts with mesenchymal stem cells in the operating room during hip revision

Bone marrow derived mesenchymal stem cells (BM-MSCs) have been proposed to improve allografts used during hip revision. However, no study has reported the number of MSCs that could be associated with the allograft and the best technique to load MSCs in allografts. The optimal loading technique should combine methods to increase the initial cell density and create an appropriate environment to accelerate the efficiency of the cell-allograft constructs into clinically applicable grafts. We designed a study to evaluate the number of MSCs in an autograft femoral head considered as the gold standard and to determine the best operating room procedure for loading in allograft with MSCs to approach the same number as in an autograft femoral head. Therefore this study explored a potential of charging whole femoral head allografts with autologous MSCs from iliac crest aspirate for hip revision procedures. First, the study evaluated the total number of mesenchymal stem cells (MSCs) in 1 cc of an average autograft femoral head; this number then serves as a target for loading allografts, in order to achieve the same density of MSCs. For the loading technique itself, several questions were asked and hence several options were investigated. For example, is it better to load the whole allograft or break it up into several fragments? Which way of injecting works best for the whole femoral head allograft (through cartilage or femoral neck)? How concentrated (in terms of MSCs) should the injected iliac crest marrow be? Bone marrow for injection in allografts was obtained from residual marrow from patients undergoing surgical procedures with concentrated bone marrow. With this bone marrow (with and without concentration) we tested different techniques (injection and soaking) to load stem cells in allografts of different sizes: bulk allografts, pieces or blocks (8 or 1 cm(3) blocks) and morselized fragments (from 125 to 8 mm(3)) or particules (1 mm(3)). We also evaluated the release of MSCs from fragments of autografts and allografts loaded with MSCs in cultured medium. The femoral head autografts contained a lower concentration of MSCs than the iliac crests of the same patient. However, in absence of concentration, with bone marrow aspirated from the iliac crest, we were not able to load in the femoral head allograft the same number of MSCs as the number present in an autograft. The loaded volume of bone marrow (and the corresponding number of MSCs) depended on the technique (injecting, soaking) as well as on the volume and shape of the allografts. The seeding efficiency of loading MSCs in allografts increased with the concentration of MSCs in the bone marrow. With concentrated bone marrow, supercharging the allograft with MSCs (as compared with an autograft) was possible in the operating room, and the number of MSCs supercharged in allografts was predictable. The loaded volume of bone marrow depended on the technique (injecting, soaking) as well as on the volume and shape of the allografts. With concentrated bone marrow, the allograft could be charged with a similar or higher number of MSCs than the number present in a femoral head autograft.

Tandem HLA-Mismatched Cadaveric Unrelated Donor Lung Transplant Followed by CD3-and CD19-Depleted Bone Marrow Transplant From the Same Donor Permits Withdrawal of Systemic Immunosuppression with Graft Acceptance and Functional Immune Reconstitution

Abstract 1006Here we report on the clinical course and immunologic recovery of a 17 year old patient with combined immunodeficiency disease (CID) requiring bilateral orthotopic lung tranplantaion (BOLT) followed by bone marrow transplant from the same unrelated cadaveric donor. Recurrent stenotrophomonas pneumonias since age 5 along with atypical mycobacterium and E. coli infections resulted in pulmonary failure by age 15 years. Chronic hypoxia and recurrent infectious gastroenteritis led to severe growth failure necessitating total parental nutrition. She presented in 2009 with the clinical necessity for lung and hematopoietic transplants to correct the underlying CID. At this age her lymphocytes had declined with severe lymphopenia of B cells and T cells (30–70 CD3+ T cells/mm3), but normal NK cell numbers.A single patient protocol was approved by Duke IRB and FDA (IDE #14206) proposing the use of a lung and bone marrow transplant from the same donor to reduce the probability of pulmonary graft rejection or GVHD of the lungs. A 4 of 8-HLA-matched unrelated donor was identified who underwent iliac crest marrow harvest yielding 5.4 × 10e8 TNC/kg. Lungs were procured and transplanted following the marrow harvest. The marrow was cryopreserved following CD3 and CD19 depletion on a CliniMacs device yielding 2×10e6/kg CD34+ cells and 4 × 10e4/kg CD3+ T cells/kg. Following BOLT in Dec 2009 (with the addition of pulse steroids, Basiliximab) she became free of supplemental oxygen and has remained ever since. In April 2010 while receiving FK506 and low dose prednisone she underwent conditioning with Rituximab, Alemtuzumab, ATGAM, Hydroxyurea, a dose of Thiotepa, and a single fraction of TBI (lung shielding) before infusion of the T cell depleted thawed bone marrow. She was discharged on Day +20 with full donor chimerism in whole blood, CD3+ T cell, and CD15+ myeloid cell fractions. She has remained 100% donor without detectable host cells - last tested at 15 months post BMT. Her immunosuppression remained FK506 monotherapy. Serial repeat lung biopsies have shown absence of immune rejection. After she returned to her home state with a KPS of 90% severe stage 4 gut GVHD developed in September 2010 following a bout of Norovirus gastroenteritis and an associated drop of FK506. GVHD resolved after a single dose of Infliximab and steroid therapy, however her course was complicated by VRE sepsis and DIC. A FK506 wean was started ∼1 year post BMT and completed by June 2011. Her pretransplant lymphopenia has resolved and since age 9 months post BMT her CD4+ cells are >250-cells/mm3. CD8+ cells have been >500 cells/mm3 since ∼3 months post BMT with associated significant anti-CMV proliferative responses (stimulation index >10). B cell recovery has been also noted with normal se IgA levels. Thymic recovery has been slow and at last testing (15 months post) it rose to 4% of CD4+ T cells co-expressing CD45RA and CD62L. 12 months post transplant, highly purified peripheral blood (donor) T cells demonstrate hyporeactivity (proliferation, IL2, TNF secretion, etc, see Table 1) against host-derived EBV-transformed B cell line compared to EBV-LCL generated from haplotype mismatched maternal or fully mismatched unrelated donor. This ‘tolerant’ state cannot be broken by in vitro T reg depletion (Denileukin difititox), and neither HLA class I or II blockade leads to further attenuation of self reactivity, data not shown. There is no measurable IL-10 in MLC culture supernatant. Taken together, the data supports central clonal deletion mechanism for lack of anti-self reactivity. Interestingly, lower proliferative and cytokine (IFNg, TNFa) secretion is noted against maternal EBV/LCL following HLA-class II blockade, data not shown. She demonstrates normal CMV -specific proliferative and cytokine responses but absent EBV- or tetanus -specific responses in the absence of prior antigenic exposure, priming. In sum, we demonstrate in the first successful human case the feasibility and immune consequences of tandem cadaveric lung and T cell depleted marrow transplantation from the same HLA-mismatched unrelated donor to create solid organ and recipient-specific tolerance in the absence of systemic immunosuppression.Mean valuesStimulatorsPatientMother3rd PartyProliferation (CPM)1,72546,51462,617IL-2 (pg/ml)<17.613.9IL-6 (pg/ml)1.12.54.3IL-13 (pg/ml)0.29.639.4IL-17 (pg/ml)0.21.41.4IFNg (pg/ml)12.160.963.5TNFa (pg/ml)3.213.521.6 Disclosures:No relevant conflicts of interest to declare.

Injectable Mesenchymal Stem Cell Therapy for Large Cartilage Defects—A Porcine Model

Current techniques in biological resurfacing of cartilage defects require an open arthrotomy or arthroscopy and involve the direct transplantation of isolated cells and/or scaffolds or whole tissue grafts with chondrogenic potential onto the cartilage defect. Our study investigates the possibility of direct intra-articular injection of mesenchymal stem cells suspended in hyaluronic acid (HA) as an alternative to the much more invasive methods currently available. A partial-thickness (without penetration of the subchondral bone) cartilage defect was created in the medial femoral condyle of an adult minipig. Mesenchymal stem cells from the iliac crest marrow of the same pig harvested in a separate procedure and suspended in 2 milliliters of hylan G-F 20 (Synvisc) were injected intra-articularly after the creation of the defect. This was followed by two more injections of hylan G-F 20 (HA) at weekly intervals. Either saline or HA was injected into the knees of the controls. The pigs were sacrificed at 6 and 12 weeks for morphological and histological analysis. The cell-treated groups showed improved cartilage healing both histologically and morphologically at 6 and 12 weeks compared with both controls. The use of intra-articular injections of mesenchymal stem cells suspended in HA is a viable option for treating large cartilage defects. This would be further explored in clinical trials.

Identification of Cardiomyogenic Lineage Markers in Untreated Human Bone Marrow–Derived Mesenchymal Stem Cells

Background Recent reports refute the classic paradigm by which human heart is unable to repair itself following disease or injury. Cardiac and noncardiac stem cells with cardiac regeneration potential have been documented. We studied whether untreated mesenchymal stem cells express markers of cardiomyogenic lineage in vitro. Methods Mesenchymal stem cells were obtained from human iliac crest marrow aspirates. Cells were isolated and characterized using flow cytometry by surface expression of CD105, CD166, CD29, CD44, CD14, and CD34. To evaluate their cardiomyogenic potential, presence of cardiac proteins (cardiac troponin I, sarcomeric α-actinin, β myosin heavy chain (β-MyHC), connexin-43, and SERCA-2), and transcription factors (GATA-4) were assessed. Results Mesenchymal stem cells expressed CD105 (4.25 ± 0.35), CD166 (27.83 ± 1.89), and CD29 (9.4 ± 0.57) and were negative for CD34, CD14, and CD45. In absence of additional stimuli in the culture media, these cells expressed connexin-43, α-actinin, and GATA-4, and were negative for SERCA-2, cardiac troponin I, and β-MyHC. Conclusions Human adult mesenchymal stem cells spontaneously exhibit markers of cardiac phenotype in vitro. In the appropiate myocardial environment, these cells may transdifferentiate into mature cardiomyocytes.

Sinus floor elevation applied tissue‐engineered bone

In the present study, we compared bone regeneration ability in sinus floor elevation between a tissue engineering method using mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP), and a promising new method using particulate cancellous bone and marrow (PCBM) and PRP. Bilateral sinus floor elevation procedures were performed in 18 adult Japanese white rabbits. MSCs/PRP or PCBM/PRP complexes were grafted to each maxillary sinus in the same rabbits. The MSCs were isolated from rabbit iliac crest marrow, and PRP was obtained from peripheral blood. PCBM were collected from the rabbit iliac crest and mixed with PRP. The animals were sacrificed at 2, 4, and 8 weeks after transplantation, and the bone formation ability of each implant was evaluated histologically and histometrically. According to the histological observations, both sites (MSCs/PRP and PCBM/PRP) showed well newly formed bone and neovascularization at 2 and 4 weeks. However, at 8 weeks, the lamellar bone was observed to be occupied by fatty marrow in large areas in both sites. There was no significant difference in bone volume or augmented height between MSCs/PRP and PCBM/PRP groups each week, but there were significant differences in bone volume and augmented height between 2 and 8 weeks in PCBM/PRP or MSCs/PRP groups and in bone volume between 4 and 8 weeks in the PCBM/PRP group (P<0.05). These results suggest that the MSCs/PRP complex may well be used for bone regeneration in sinus floor elevation, compared with the PCBM/PRP complex.

CELLULAR PHENOTYPES AFFECTED BY INDUCTION THERAPY WITH CAMPATH-1H VS THYMOGLOBULIN VS ZENAPAX IN KIDNEY ALLOGRAFT RECIPIENTS

P387 Introduction: Several independent studies have shown the safety and efficacy of antibody induction in renal transplantation using several different maintenance immunosuppressive protocols. This describes an 18 month follow-up in a randomized study, comparing the cytoablative effect of Campath-1H (C) 0.3 mg/kg-2 dosages, Thymoglobulin (T) 1mg/kg-5 dosages and Zenapax (Z) 1mg/kg- 5 dosages on peripheral blood and iliac crest bone marrow of kidney allograft recipients. Methods: In C half maintenance dosages of tacrolimus (tacro) and mycophenolate (MMF) but no corticosteroid were given. In T and Z higher maintenance doses of tacro, MMF as well as steroids were given. Depletion of discrete mononuclear cell phenotypes in peripheral blood and iliac crest bone marrow samples taken sequentially post-transplant at 10, 60, and 200 days were compared. The absolute number of mononuclear cells in peripheral blood or iliac crest bone marrow aspirates per mm3 and the CD3, 19, 56/16 (NK cells), 25, 14 (Monocytes) and 34 (Stem cells) phenotypes in flow cytometry were calculated. Results: In peripheral blood, when compared to normal values, in the C group, there was depletion of virtually all lymphocytes, T cells by 100%, B cells by 98%, NK cells by 95%, monocytes by 83% and Stem cells by 40%, with an earlier recovery (in descending order) of stem cells, B cells, monocytes, and NK cells, and later (at >6 months) with T cells. In contrast, T depleted T cells (99%) and NK cells (98%), but B cells, and monocytes were only moderately affected (20% and 45% respectively). Z depleted all CD25 positive cells with early modest depletion of T cells, B cells and NK cells by 50%, 72% and 62% respectively, and with no effect seen on monocytes. In iliac crest marrow, at 10 days C also strongly depleted all lymphocyte, CD3+, CD19+, CD56/16+, and CD14+ counts by 99%, 98%, 96%, and 68% respectively. However, virtually normal numbers of CD34+ cells were present. At 200 days post-transplant, an increased number of B cells (double the normal values) were seen. The early depletion of affected subsets by T in this compartment were 50% that of C with an earlier return to normal ranges in all phenotypes, while with Z there was only a minimal effect. Conclusion: C is the more potent lymphoablative induction agent (followed by T) allowing in most cases for early and total avoidance of corticosteroids and lower doses of maintenance immunosuppressive agents.

Clinical Tolerance: the end of the beginning.

Clinical Tolerance: the end of the beginning.

Donor Bone Marrow Infusion in Deceased and Living Donor Renal Transplantation

The infusion and persistence in a transplant recipient of donor-derived bone marrow cells (DBMC) of multi-lineage can lead to a state of permanent chimerism. In solid vascular organ transplantation, the donor bone marrow lineage cells can even be derived from the transplant organ, and these cells can be detected in very small numbers in the recipient. This has been called microchimerism. Much controversy has developed with respect to the function of chimeric cells in organ transplantation. One idea is that the occurrence of these donor cells found in microchimerism in the recipient are coincidental and have no long-term beneficial effect on engraftment. A second and opposing view, is that these donor cells have immunoregulatory function that affect both the acute and chronic phases of the recipient anti-donor responses. It follows that detecting quantitative changes in chimerism might serve as an indication of the donor-specific alloimmune or regulatory response that could occur in concert with or independent of other adaptive immune responses. The latter, including autoimmune native disease, need to be controlled in the transplant organ. The safety and immune tolerance potential of DBMC infusion with deceased and living donor renal transplants was evaluated in a non-randomized trial at this center and compared with non-infused controls given identical immunosuppression. Overall DBMC infusions were well tolerated by the recipients. There were no complications from the infusion(s), no episodes of graft-vs-host disease (GVHD) and no increase infections or other complications. In the deceased DBMC-kidney trial, actuarial graft survival at 5 years was superior especially when graft survival was censored for recipient death. Acute rejections were significant reduced in patients given two DBMC infusions, and chronic rejection was dramatically reduced in all DBMC treated patients. The most interesting finding was that the degree of microchimerism slowly increased over the years the DBMC group that had exhibited no rejection episodes. In the DBMC-living related trial, the incidence of acute rejection did not differ between groups. However, DBMC chimerism in recipient iliac crest marrow had increased more rapidly than might be predicted from results previously seen in the cadaver group, despite four times fewer DBMC infused, with the generation of T- regulartory cells in-vitro assays.

間葉系幹細胞とＰＲＰ複合体を用いた上顎洞底拳上術における骨造成法の実験的研究

Recently, minimally invasive surgery based on a tissue engineering has become possible. We used an animal model to evaluate a method for bone regeneration by maxillary sinus floor augmentation that uses mesenchymal stem cells (MSCs) and platelet-rich plasma (PRP) instead of an autologous bone graft. The MSCs were isolated from rabbit iliac crest marrow aspirates, and PRP was obtained from peripheral blood. MSCs and PRP complexes were used for grafting. In the control group, autogenous particulate cancellous bone and marrow (PCBM) was collected and mixed with PRP. Histological examination showed that new bone formation was good in both groups at 2 and 4 weeks. At 8 weeks, lamellar bone had formed, but fatty marrow was also observed in both groups.The present results suggest that MSCs/PRP complexes may be used in place of autogenous bone to promote bone regeneration after maxillary sinus floor augmentation.

Effect of living-related donor bone marrow infusion on chimerism and in vitro immunoregulatory activity in kidney transplant recipients.

In a previously reported series of donor-specific bone marrow cell (DBMC) infusions in cadaver kidney transplant recipients, there appeared to be an improvement in long-term graft survival (6 years) and fewer chronic rejections, which correlated with increasing DBMC chimerism (approximately 1.4% in the iliac crest bone marrow compartment now at 6 years). Prompted by this, we embarked on a study of DBMC infusion in living-related donor (LRD) kidney transplant recipients. Between November 1996 and May 2000, 47 LRD kidney transplant recipients received donor iliac crest marrow (1.8 x 10(8)+/-1.9 x 10(8) cells/kg body weight+/-SD) in a single infusion 4 days postoperatively. Either OKT3 (n=26) or daclizumab (n=21) were used for induction therapy, with maintenance tacrolimus, mycophenolate mofetil, and methylprednisolone immunosuppression. These recipients were prospectively compared with 39 noninfused LRD kidney transplants (control group), which received equivalent immunosuppression in the same time period. Clinical follow-up ranged from 19.0 months to 61.6 months (mean 33.2 months). Polymerase chain reaction-flow chimerism analysis and in vitro assays of immunoregulatory activity of chimeric cells were performed. The incidence of acute rejection over this period of time was 10.6% and 10.3%, respectively (i.e., did not differ between groups). Immunosuppressive dosages were somewhat (but not statistically) lower over time in the DBMC group. Four-year actuarial patient and graft survival for the DBMC-infused group was 98% and 98%, and 98% and 95% for the control group, respectively ( =NS). DBMC infusion was well tolerated, with no increase in infectious episodes. DBMC chimerism in recipient iliac crest marrow has increased more rapidly than might be predicted from results previously seen in the cadaver group, despite four times fewer DBMC infused. DBMCs and (donor) peripheral blood mononuclear cells purified by immunobeads from recipient blood or bone marrow (recipient-derived donor cells) inhibited mixed leukocyte responses of the recipient to the donor more strongly than freshly obtained peripheral blood cells drawn from the donors or even compared with bone marrow cells aspirated from the donors in a previously reported group of experiments. Additionally, similarly purified recipient-derived recipient cells from the same chimeric recipient more strongly inhibited the same mixed leukocyte response reactions autologously than a large group of nonchimeric (autologous) bone marrow modulating cells in similar reactions. These observations confirm that an immunoregulatory process appears to have been generated by DBMC infusion, encouraging a further decrease in immunosuppressive dosing using such assays in the future.

Comparison of detection of micrometostases in rib marrow aspirates compared to iliac crest aspirates

Iliac crest marrow remains the standard sampling site for detection of bone marrow micrometastases in breast cancer patients.

Six-year clinical effect of donor bone marrow infusions in renal transplant patients.

To date, several single- and multicenter clinical trials have attempted to induce specific immunological unresponsiveness using donor bone marrow cell infusions to augment solid organ transplantation, but the outcomes have not been definitive. Between September 1994 and May 1998, 63 cadaver (CAD) renal transplant recipients of either one or two postoperative donor bone marrow cell (DBMC) infusions were prospectively compared with 219 non-infused controls given equivalent immunosuppression. There was at least a 1 HLA DR antigen match present between donors and recipients. The immunosuppressive regimen included a 10-day course of OKT3 induction, and tacrolimus, mycophenolate mofetil, and methylprednisolone maintenance. A total 7.01x10(8)+/-1.9x10(8) (SD) DBMC/kg was infused into the CAD recipients on either days 4 and 11 (n=42) or one half of that dose on day 4 (n=21) postoperatively. Clinical follow-up has ranged from 2.9 to 6.3 years (mean, 4.7 years). Studies were also performed of humoral immunity and quantitative cellular chimerism. There is clear-cut equivalence in immunosuppressive dosaging and in the other major demographic variables in both groups. However, only 2/63 DBMC recipients had biopsy-proven chronic rejection, whereas 41/219 showed chronic rejection in the controls (P = <0.01). In both groups, mortality was not associated with rejection. The actuarial graft survival at 6.3 years in the CAD DBMC group was 84.3% compared with 72.2% in the control group (not statistically significant). However, if death with a functioning graft was excluded, graft survival was 94.1% in the DBMC group and 79.8% in the controls (P=0.039). Forty patients in the control group continue to have deteriorating renal function (increasing serum creatinine concentrations to 2 mg/dl and higher), compared with 2 patients in the DBMC group (P=0.04). In the DBMC group, chimerism in iliac crest marrow aspirates has increased 3-fold in yearly sequential measurements between 1 and 4 years postoperatively averaging 1.3+/-0.36% (SE) most recently. This has not occurred in the controls. There now appears to be more solid long-term evidence, in kidney transplant recipients prospectively receiving DBMC infusions, of an improvement in long-term graft survival, and of the degree of chimerism positively correlating with the absence of graft loss.

The human bone marrow as an immunoregulatory organ.

It was 45 years ago that in a virtual revolution in thinking in immunology there developed the acceptance and the subsequent expansion of two new dogmas: (1) that to eliminate toxins and pathogens as the major mode of defense, individual immune cells were, in their ontogeny of differentiation, internally programmed to react singly and then clonally against the virtually limitless individual stimuli of the outside world (1–3); (2) that before this programming was manifested the immune system would fail to recognize any antigenic stimulus as foreign, thereby not differentiating non-self from self-recognition. This allowed for non-self-antigens, if introduced in this early stage, to be immunologically tolerated on subsequent testing. In the chronology of the evolution of these two dogmas, there were the earlier descriptions of specific immunological paralysis and unresponsiveness to certain defined polysaccharides and haptens demonstrated in adult mice and guinea pigs respectively by Felton (4) and Chase (5). It remained for Medawar and his colleagues (6) in allotransplantation experiments to clearly define “acquired specific immunological tolerance” as an ontogenic concept involving the lack of maturation or differentiation were tolerance to be evoked. As early as 1953, Billingham, Brent, and Medawar demonstrated that allogeneic donor bone marrow-derived cells could confer such a specifically acquired tolerant state to the immune system of the murine recipient before self versus non-self recognition occurred in immune ontogeny, the test being subsequent acceptance of skin allografts from the same donors. The developmental processes of positive versus negative selection only a decade later began to be demonstrated to predominantly involve the thymus gland in maturational events of cells of the recipient immune system in studies first performed in neonatal rodents (7). By experimental manipulation of the immune system predominantly involving immunoablation by whole body x-irradiation, these findings of specific immunological tolerance as a result of the infusion of bone marrow-derived cells could be extended to adult animals as the system regenerated from stem cells in the bone marrow of either that of the donor (8), the recipient (9), or both (10). Nonetheless, except in the setting of clinical bone marrow transplantation using major (lethal) immunoablation followed by the salvage and replacement of the recipient immunohematopoietic system with donor cell lineages and only in the context of little or no donor-recipient MHC disparity, with few exceptions (11–15), to date, specific immunological tolerance in organ transplantation in humans has not been possible.

Combined sentinel lymph-node mapping and bone-marrow micrometastatic analysis for improved staging in breast cancer

In a prospective study of 29 patients with clinically node-negative breast cancer, sampling of rib marrow was superior to sampling of iliac-crest marrow for detection of micrometastases. Combination of marrow micrometastatic analysis with sentinel lymph-node mapping may improve accuracy of staging in breast cancer.

CLINICAL AND IN VITRO SUPPORT FOR IMMUNOREGULATION BY DONOR SPECIFIC BONE MARROW INFUSIONS IN RENAL TRANSPLANTATION

11 Are there clinical effects of augmenting kidney transplantation with putatively immunoregulatory donor specific bone marrow cell (DBMC) infusions? Recipients of DBMC and kidney transplants from 63 cadaveric (CAD) and 30 living related (LR) donors have been studied beginning September, 1994, and November, 1996 respectively, through December 31, 1998. Recipients of 220 CAD kidneys, without bone marrow, treated with similar immunosuppression (OKT3 induction, Tacrolimus, Mycophenolate and Methylprednisolone) were considered controls. The actuarial 4 year clinical results, to date, demonstrate patient and graft survival in the DBMC CAD group of 93% and 92% respectively, versus that of the controls of 95% and 91%. Although there were still no essential differences in these parameters between the 2 groups, the effects of DBMC on chronic rejection (CR) with additional follow-up is now even more prominent than previously reported, i.e. 3 of 63 (5%) versus 35 of 220 (16%) (p=<0.01), in which, as of 12/31/97, one graft has now been lost in surviving patients in the DBMC group, in contrast to the controls in which 11 were lost. If only the 42 patients receiving 2 DBMC infusions were considered (as opposed to 21 who received one infusion due to insufficient numbers of DBMC) there were no graft losses. (p=<0.01). Quantitative donor cell chimerism has now increased three-fold in the iliac crest marrow of the DBMC infused CAD recipients using PCR-Flow analysis, i.e. 0.30%±0.07 at 1 year versus 1.30%±0.27 at 3 years in serial yearly studies on 19 patients. (p=<0.0001) The 2 year actuarial patient and graft survival in the LR DBMC group is 100%, (and no CR). Moreover, PCR-Flow assays of donor cell chimerism in recipient iliac crest bone marrow and peripheral blood detected even higher numbers of donor cells (>2 fold) than in the CAD group 1 year post-operatively (p=<0.001). This group, by contrast, received 25% of the total number of CAD marrow cells infused. A powerful proof of immunoregulatory reactions operative in LRD DBMC recipients was seen when after 1 year non-chimeric donor iliac crest marrow aspirates were simultaneously compared with chimeric recipient samples, both samples equivalently treated by isolation of donor Class I expressing cells using specific alloreactive monoclonal antibodies and immunomagnetic beads. (n=5) Donor specific MLC and CML reactions were inhibited using responding recipient PBL and serial dilutions of the isolated chimeric versus non-chimeric donor bone marrow cells as third party regulatory cells. There were ten-fold fewer of the chimeric donor cells (than non-chimeric donor cells) needed to regulate (inhibit by >50%) such reactions in vitro. We conclude that 4 years into this project, there is now substantial support for the immunoregulatory role of DBMC infusions in kidney transplant recipients in facilitating graft survival.

IMMUNOREGULATION OF BONE MARROW INFUSED LRD RENAL TRANSPLANT RECIPIENTS BY CHIMERIC CELLS OF DONOR PHENOTYPE.

120 A number of transplant centers, including our own, have adopted donor specific bone marrow cell infusions of organ transplant recipients to enhance donor chimerism with the aim of inducing allograft acceptance. However, to date there has been no direct evidence linking chimerism with specific tolerance induction in the transplant recipients. In this study in renal transplant recipients from living related donors (LRD) who had received perioperative donor bone marrow cell infusions, chimeric cells of donor phenotype were isolated from the peripheral blood and the iliac crest bone marrow one year post-operatively, using monoclonal antibodies to donor mismatched HLA class 1 antigens and magnetic microbeads. They were then characterized phenotypically and functionally. In contrast freshly isolated and similarly treated cells from blood and iliac crest marrow of the donors (non-chimeric donor cells) were similarly treated and tested at the same 1 year interval. The yield of such chimeric cells from the recipient marrow by this method of purification ranged as high as 0.5% at this time. Phenotypic characterizations of similarly isolated chimeric cells from the iliac crest marrow of cadaveric kidney recipients 1-3 years postoperatively (the purified chimeric cells from LRD recipients were sufficient only for the functional studies) showed that 51.8±6.3% of the chimeric cells were TcRαβ+ CD2+ CD3-dimly positive. 28.3±7.5% cells were also CD4− CD8−. In addition, 20.4±11.8% were CD4+ CD3+ and 15.0±4.9% were CD8+ CD3+ (n = 4). The regulatory activity of chimeric and non-chimeric donor cells was assessed by testing them as third party cells in MLC and CML responses of the recipients to the allogeneic LRD PBL as stimulating cells. Of the seven cases analyzed so far, two recipients did not respond to the donor in MLC, although this reaction was present preoperatively, so that the role of chimeric cells could not be ascertained. In the five patients with positive anti-donor MLC responses, the purified chimeric cells showed significantly stronger inhibition than non-chimeric donor marrow cells similarly treated (p < 0.01). Modulation experiments on CML responses could be performed only in three cases because of the low yield of the chimeric cells. The chimeric cells were also significantly more potent inhibitors (p < 0.05) of the generation cytotoxic T cells than the non-chimeric donor marrow cells. The inhibitions occurred with chimeric (regulatory) cell concentrations as low as 8% of the responder cells. These results clearly showed that donor bone marrow derived chimeric cells present in transplant recipients have evolved into even more potent immunoregulatory cells by 1 year postoperatively, supporting the notion that chimerism plays a major role in the induction and/or maintenance of transplant tolerance.

Micrometastases in esophagogastric cancer: High detection rate in resected rib segments

Background & Aims: Micrometastases within bone marrow indicate a poor prognosis. We prospectively examined micrometastases in patients undergoing resection of esophagogastric cancers for (1) prevalence in rib marrow; (2) comparative detection rates in rib and iliac crest marrow; (3) responsiveness to neoadjuvant therapy; and (4) viability and tumorigenicity. Methods: In 50 consecutive patients, marrow was obtained before manipulation of the primary tumor. Micrometastatic cells were detected by staining contaminant cytokeratin-18–positive cells. Viability and tumorigenicity were determined by culture and xenograft. Results: Micrometastases were detected in rib marrow from 88% of patients (44 of 50). When bilateral iliac crest marrow was also obtained, micrometastases were found in 15% (4 of 27) compared with 89% (24 of 27) for ribs ( P < 0.001). Detection rates were independent of histological type or nodal status and were similar in patients with and without neoadjuvant therapy. Metastatic cells were cultured from rib marrow of 5 of 7 patients and were tumorigenic in nude mice. Conclusions: Most patients undergoing resection of esophagogastric malignancies have micrometastases in rib marrow. Detection rates based on iliac crest marrow are underestimates. Hematogenous spread of metastatic cells is independent of histological type or nodal status. The metastatic cells are viable, tumorigenic, and resistant to neoadjuvant therapy. GASTROENTEROLOGY 1999;116:543-548