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Stem Cells 2002;20:86-93 www.StemCells.com
© 2002 AlphaMed Press

RAPID COMMUNICATION

Localized Bone Marrow Transplantation Leads to Skin Allograft Acceptance in Nonmyeloablated Recipients: Comparison of Intra-Bone Marrow and Isolated Limb Perfusion

Nadir Askenasy

Center for Light Microscope, Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA; Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, USA; Present address: Frankel Laboratory of Bone Marrow Transplantation, Center of Pediatric Hematology Oncology, Schneider Children's Medical Center of Israel, Petach Tikva, Israel

Key Words. Bone marrow transplantation • Intra-bone marrow • Isolated limb perfusion • Hematopoietic chimerism • Skin grafts • Tolerance

Nadir Askenasy, Ph.D., Frankel Laboratory of Bone Marrow Transplantation, Center of Pediatric Hematology Oncology, Schneider Children's Medical Center of Israel, 14 Kaplan Street, Petach Tikva, 49202, Israel. Telephone: 9-723-925-3669; Fax: 9-723-925-3042; e-mail: anadir{at}012.net.il


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
It has been shown that engraftment of allogeneic bone marrow cells (BMC) induces tolerance to antigen-matched organs, and infusion of a megadose of cells improves the success of engraftment of T-cell-depleted BMC. This study explores intra-bone marrow injection (IB) and isolated limb perfusion (IL) as means of localized bone marrow transplantation (BMT) and assesses their tolerogenic effect. Intravenous (i.v.), IB, and IL infusion of syngeneic and allogeneic whole BMC rescued 90%-100% of myeloablated recipients. Tracing of PKH-labeled cells revealed early systemic dissipation after IB injection, indicating that it was equivalent to i.v. transplantation. In contrast, IL perfusion led to initial localization of donor BMC. BALB/c recipients conditioned with 70 µg/g busulfan had 58% ± 5% and 44% ± 4% donor lymphocytes at 4 weeks after i.v. and IL infusion, respectively, of 107 whole BMC from B10 donors. This suggests that cells migrated out of the IL femur and seeded other bones. All recipients accepted donor-matched skin grafts and acutely rejected third party grafts. T-cell depletion lowered the engraftment efficiency of i.v.-BMT by 35% (p < 0.001 versus whole BMC), but not when infused IL (p < 0.001). It is concluded that IL-BMT is a procedure for initial localization of donor cells, which is as efficient as i.v.- and IB-BMT in rescue of myeloablated mice, induction of hemopoietic chimerism, and donor-specific immune nonresponsiveness to secondary skin grafts without myeloablative conditioning. The megadose effect achieved by inoculation of a small hemopoietic space improved engraftment of T-cell-depleted BMC. This approach may have clinical applications.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Donor-specific tolerance evolves along with establishment of stable hematopoietic chimerism [1]. Wide clinical application of bone marrow transplantation (BMT) as a means of tolerance induction is restricted by the high morbidity and mortality rates associated with toxicity of conditioning, graft-versus-host disease (GVHD), and failure of engraftment [2]. Numerous studies have been devoted to designing nonlethal conditioning strategies and engineering of donor inoculum, attempting to minimize toxicity and maximize the success of engraftment [3–5]. Recently, injection into the portal vein and intra-bone marrow (IB) have been considered, aiming to optimize the site of donor cell inoculation [6,7]. IB infusion of fluids and cells, initially developed for pediatric treatment [8], has been evaluated in the clinical and experimental settings of BMT [7,9]. Conceptually, the advantages of IB injection of bone marrow cells (BMC) directly to their destination and natural site of engraftment include: A) elimination of the homing process; B) avoidance of depletion by host immune system during systemic circulation [10], and C) improved competitiveness of the relatively large number of donor BMC for vacant niches of engraftment [11].

Extending these considerations, it was hypothesized that localized BMT could improve the efficiency of engraftment in nonmyeloablated recipients. There is mounting evidence that a megadose of donor cells improves engraftment across antigenic barriers, promotes engraftment of purified hemopoietic stem cells (HSC), and reduces the requirement for conditioning [12–15]. A "megadose" effect may be generated either by infusion of a large number of cells or inoculation of a limited hematopoietic space. It was recently demonstrated that IB-BMT reversed autoimmunity in MRL/lpr mice [7]. In this study, IB- and isolated limb perfusion (IL)-BMT were assessed as strategies for induction of localized BMT, and their tolerogenic efficiency was tested by skin grafting. The results indicated that IB inoculation is equivalent to i.v. injection of cells, while engraftment is localized after IL-BMT. Both routes of administration induced tolerance to skin grafts in nonmyeloablated recipients.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Recipient Preparation
B10 (C57Bl/10Sn, H2b), B10.BR (C57Bl/SgSn, H2k), and BALB/c (H2d) mice purchased from Jackson Laboratories (Bar Harbor, ME; http://www.jax.org) were housed in a pathogen-free facility. Mice, aged 8-10 weeks, were anesthetized with Avertin (12-17 µg/g, intraperitoneal [i.p.]; Sigma Chemical Co.; St. Louis, MO; http://www.sigma-aldrich.com). Recipients were conditioned with an i.p. injection of busulfan 36 hours before transplantation. Busulfan was dissolved in dimethylsulfoxide at a concentration of 24 mg/ml and was diluted fivefold in water at 40°C before injection. Donor BMC suspended in 0.2 ml phosphate buffered saline (PBS) or 2 ml Krebs-Henseleit physiological solution (KH) were injected into: A) lateral tail vein (i.v.) of warmed recipients; B) bone marrow (IB), and C) femoral artery (IL).

Intra-Bone Marrow Transplantation
The knee was exposed by a sharp skin incision, flexed, and a 24G needle was inserted into distal femoral epiphysis above the patella (Fig. 1Go). Donor BMC were injected into the femur using a miniperistaltic pump (P720; Instech; Plymouth Meeting, PA; http://www.instechlabs.com) via a double outlet system. Cells were infused through one outlet at a rate of sim;20-50 µl/minutes, and the other outflow was connected to a threshold pressure transducer and a PowerLab monitoring system (ADInstruments; Grand Junction, CO; http://www.adinstruments.com). When intraluminal pressure raised above a threshold value (8 or 20 mmHg), the medium was diverted into a drainage reservoir.



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  		Figure 1. Procedure for intra-bone marrow (IB) and isolated limb perfusion (IL). IB: Cells suspended in PBS were infused with a peristaltic minipump into the bone marrow through a needle inserted into distal femoral epiphysis. Infusion rate was monitored with a pressure transducer, and the medium was diverted into a reservoir when it reached a threshold of 8 or 20 mmHg. IL: In the isolated limb preparation, femoral artery and vein were occluded and were cannulated with 24G catheters. The limb was perfused with KH physiological solution, KH with donor cells, and KH at a rate of 0.2 ml/minute for 5, 10, and 10 minutes, respectively. After perfusion, blood flow in the femoral artery was restored and the vein remained occluded.

 
Isolated Limb Perfusion
Blood flow to the limb was occluded with a proximal tourniquet, and the femoral artery and vein were clamped and cannulated with needle-guided 24G catheters (Fig. 1Go). Surgery was performed under magnification using a Leica GZ6 surgical stereoscope (Leica; Northvale, NJ; http://www.leica.com). The KH perfusion medium was aerated with a mixture of CO2/O2 5/95% to give a final pH of 7.3, and was warmed to 37°C in a double-walled water-jacketed container [16]. The femoral artery was perfused in three stages at a rate of 0.2 ml/minute using a miniperistaltic pump: the first 5 minutes with KH, then with donor cells in KH (0.5 x 106-108 cells/ml), and the last 10 minutes with KH. The low infusion volume aimed to minimize extravasation and accumulation of cells in the soft tissues of the limb. Outflow from the femoral vein was collected into a container and was occluded after perfusion, diverting blood drainage from the limb to the deep veins. The femoral artery was restored either by a side-to-side anastomosis, ligation to a short segment of the 24G catheter, or repair of the site of cannule insertion. The limb was gradually reperfused by intermittent release of the arterial clamp.

Isolation of BMC
BMC were harvested from femurs and tibia crushed in Hank' balanced salt solution (HBSS; GIBCO Laboratories; Grand Island, NY; http://www.invitrogen.com). BMC were suspended using an 18G needle, filtered with a 30 µm sterile nylon mesh, collected by centrifugation (400 g, 10 minutes, 4°C) and resuspended in HBSS containing 2% fetal calf serum (FCS). Erythrocytes were lysed by incubation with ammonium chloride for 4 minutes at room temperature. Nucleated cells (whole BMC) were counted after being washed twice with excess medium.

T-cell depletion was performed by incubation for 30 minutes at 4°C with rat-anti-mouse CD4 and CD8 monoclonal antibodies (mAb; Pharmingen; San Diego, CA; http://www.pharmingen.com). Excess mAb were removed by washing twice with PBS containing 2% FCS. Then, cells were gently mixed for 20 minutes at 4°C with goat-anti-rat immunoglobulin G conjugated to M-450 magnetic beads at a ratio of four beads per cell (Dynal Inc.; Lake Success, NY; http://www.dynal.no). T cells rosetted with beads were precipitated by exposure to a magnetic field, and supernatant containing nonlymphocytes was collected. Depletion of T cells was assessed by flow cytometry (Coulter Elite; Miami, FL; http://www.coulter.com) using fluorescein isothiocyanate (FITC)-labeled anti-{alpha}ßT-cell receptor mAb.

PKH Staining
2 x 107 cells were suspended in 1 ml of Diluent C, and freshly prepared PKH67 membrane linker was added to a final concentration of 2 µM (provided by Dr. K. Muirhead; SciGro Co.; Malvern, PA; http://www.maconsultants.com/scigro). Samples were incubated at room temperature for 5 minutes with gentle mixing. Staining was terminated by addition of 4 volumes HBSS containing 10% FCS, cells were collected by centrifugation (400 g, 10 minutes, 4°C) and washed twice with HBSS. The average recovery of the procedure was 90%, with a viability of 95% as determined with the trypan blue exclusion test.

Kinetics of BMC Systemic Scattering
Blood samples were collected into heparinized serum vials in 200 µl HBSS, washed twice, and collected by centrifugation (400 g, 10 minutes, 4°C). Femurs were harvested from mice euthanized by CO2 asphyxiation under anesthesia, and BMC were collected from bones crushed in HBSS. After lysis of erythrocytes, the fraction of PKH+ cells was determined by flow cytometry using the blast cell gate. When the number of PKH+ cells was low, samples were screened with a fluorescence microscope (Axiophot; C. Zeiss; Thornwood, NY; http://www.zeiss.com).

Characterization of Donor Chimerism
Blood samples were collected into heparinized serum vials in 200 µl HBSS and were incubated with fluorochrome-labeled mAb: anti-H2b-PE mAb for B10 donors, anti-H2k-FITC and H2d-FITC mAb for B10.BR and BALB/c recipients, respectively. Blood cells were layered over 1.5 ml lymphocyte separation media (1.087 g/ml; CedarLane; Hornby, Ontario, Canada; http://www.cedarlanelabs.com). After centrifugation (1,000 g, 20 minutes, 4°C), low-density cells were collected, washed twice with HBSS, and fixed with 0.5% paraformaldehyde. The percentage of phycoerythrin (PE)+-donor (H2b) peripheral blood lymphocytes (PBL) was determined by flow cytometry on the lymphocyte gate.

Skin Grafting
Full-thickness tail skin from BMC-matched and third party donors was grafted in the inter-scapular region of anesthetized chimeras [17]. Grafts were inspected on a daily basis for signs of rejection. Disappearance of the epidermis was considered as complete rejection.

Statistical Analysis
Data are presented as means ± standard deviation for each experimental protocol. Within the groups, reproducibility was evaluated by linear regression of duplicate measurements. Differences between the experimental protocols were estimated with a post hoc Scheffe t-test at a 5% level of significance.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
In preliminary experiments, the procedure of double cannulation of the femoral artery and vein was established. The difficult stage of the surgical procedure was restoration of arterial blood supply. To avoid ischemic injury to the bone marrow, limbs were perfused with oxygenated KH physiological solution; the average minimal duration was 1 hour. Low perfusion rates used in this study aimed to prevent accumulation of cells in soft tissues. The perfusion rates could be increased several fold without apparent injury to vascular endothelium, as assessed by perfusion with KH solution containing methyl blue. Limbs were flushed with medium to wash cells from the vasculature. Initial studies were performed in a donor-recipient pair of B10 (H2b) and B10.BR (H2k), respectively. Recipients were BALB/c (H2d), which are white, for easier monitoring of the skin allografts from B10 mice, which are black.

Three Approaches to BMT: Myeloablated Recipients
None of the myeloablated B10.BR mice (busulfan 145 µg/g) injected with medium (without BMC) survived for 30 days. Injection of 5 x 107 syngeneic whole BMC (B10.BR-> B10.BR) i.v., IB, and IL rescued all myeloablated recipients. Survival of myeloablated B10.BR recipients after i.v. injection of allogeneic BMC (B10->B10.BR) was 100% and 90% after IL and IB injection (n = 10). At 4 weeks, the chimeras had 96% ± 4%, 91% ± 5%, and 78% ± 4% donor lymphocytes in the i.v., IB, and IL protocols, respectively. The data demonstrated the capacity of IL- and IB-BMT to reconstitute hematopoiesis in myeloablated recipients.

The Fate of BMC: IB Injection Versus IL Perfusion
The early systemic distribution of BMC labeled with PKH membrane linkers was assessed in peripheral blood and femurs of the recipients. Preliminary studies showed that PKH dyes did not affect, qualitatively or quantitatively, cellular homing and seeding in recipient BM. Figure 2AGo compares the dissipation of PKH+ cells in peripheral blood after i.v., IB, and IL transplantation of syngeneic BMC in myeloablated B10.BR recipients. The fraction of PKH+ blast cells was not significantly different between the i.v. and IB protocols (n = 5). In both cases, PKH+ cells almost disappeared from the blood within 2 hours after transplantation. Even distribution of transplanted cells could result either from their systemic scattering during IB injection, or early mobilization of cells from the injected femur and secondary systemic dispersion. Lowering the injection pressure to 8 mmHg (n = 3) did not change the profile of PKH+ cells in peripheral blood. In contrast, very few PKH+ cells were detected in peripheral blood of mice injected IL (p < 0.05 versus i.v.- and IB-BMT), demonstrating limited systemic dissipation.



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  		Figure 2. Comparison of intravenous (i.v.), intra-bone marrow (IB), and isolated limb perfusion (IL). Myeloablated B10.BR recipients were injected with 5x107 syngeneic whole BMC labeled with PKH67 (n = 5). PKH+ BMC were detected by flow cytometry on the blast gate. A) Blood levels of circulating PKH+ BMC; B) Contents of PKH+ BMC of injected (IB or IL) and contralateral femurs. Contralateral femurs after i.v.-BMT represent the mean of the two femurs. Data represent means ± standard deviation. *p <0.05.

 
Femurs were harvested 2 hours after BMT, and their contents of PKH+ cells were quantified (Fig. 2BGo). First, there were no significant differences between the number of PKH+ cells in the injected femur (IB) and the contralateral naïve femur (Fig. 3Go). Second, the fraction of PKH+ BMC in the IB protocol was similar to the contents of both femurs after i.v.-BMT. Third, the profile did not change when the IB injection pressure was lowered to 8 mmHg. These data indicated that, in our experimental conditions, IB and i.v. infusions were equivalent in terms of systemic BMC dissipation.



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  		Figure 3. Dissipation of PKH-labeled BMC as determined by flow cytometry on the blast gate 2 hours after transplantation of PKH-labeled BMC. After intra-bone marrow (IB) injection, PKH+ cells were detected in the contralateral femur at the same incidence observed after injection into a peripheral vein. Isolated limb perfusion (IL) resulted in localized seeding of PKH+ cells, with a very low incidence in the contralateral femur.

 
In contrast, the femur of the perfused limb (IL) contained sim;65% PKH+ BMC (p < 0.001 versus i.v. and IB), whereas flow cytometry did not detect PKH+ cells in the contralateral femur (Fig. 3Go). Detailed examination of the contralateral femur by fluorescence microscopy revealed 12 ± 3 (range 8-37) PKH+ cells. Considering the low levels of PKH+ BMC in the blood after IL-BMT, it was evident that systemic scattering was markedly reduced in the IL protocol. In a separate group of mice, femurs were harvested 48 hours after IL-BMT (n = 4), and sim;70 PKH+ BMC were identified by fluorescence microscopy in the noninjected femur.

IL-BMT in Nonmyeloablated Recipients
An attractive application of localized BMT is induction of hemopoietic chimerism to achieve tolerance to donor-matched organs. To evaluate the characteristics of IL-BMT, three variables were assessed: nonablative recipient preconditioning, size and composition of donor inoculum, and timing of BMC and skin grafting. Survival of BALB/c recipients conditioned with doses of 35-145 µg/g busulfan 36 hours before i.v. infusion of medium (without cells) is presented in Figure 4AGo. Injection of 107 whole BMC from B10 donors rescued mice conditioned with all doses of busulfan (n = 5). Overall, higher doses of busulfan increased the levels of donor PBL at 4 weeks post-transplantation (Fig. 4BGo).



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  		Figure 4. Comparison of intravenous (i.v.) and isolated limb perfusion (IL). A) Survival of BALB/c mice conditioned with busulfan, injected i.v. with medium without cells. B) Percent donor PBL in BALB/c recipients conditioned with various doses of busulfan, 4 weeks after injection of 107 whole BMC from B10 donors. C) Percent donor PBL in recipients conditioned with 70 µg/g busulfan 4 weeks after injection of 5x106-5x107 whole BMC. D) Percent donor PBL in recipients conditioned with 70 µg/g busulfan after injection of 107 whole or TCD-BMC.

 
Size and Composition of Donor Inoculum
Injection of increasing numbers of BMC (B10->BALB/c) into recipients conditioned with 70 µg/g busulfan increased the levels of donor chimerism (Fig. 4CGo). A sharper increase in donor PBL occurred in the range of 106-107 whole BMC injected i.v., and 1-5 x 106 BMC injected IL (n = 5). The lower levels of chimerism, and the smaller numbers of BMC that increased the yield of engraftment, may reflect a hemopoietic space limitation during IL infusion.

The less efficient engraftment of T-cell-depleted BMC (TCD-BMC) may be improved by infusion of large numbers of cells [12,14]. To assess potential advantages of the megadose effect, BALB/c recipients conditioned with 70 µg/g busulfan were injected with 107 whole and TCD-BMC (n = 10). While the fraction of donor PBL decreased by 35% when TCD-BMC were injected i.v. (p < 0.001 versus whole BMC), there was a nonsignificant decrease of 10% in mice perfused IL (Fig. 4DGo). Overall, IL infusion of TCD-BMC resulted in superior levels of donor PBL compared with i.v. injection (p < 0.05). The 30-day survival rates were 80% and 90% after i.v. and IL transplantation of TCD-BMT, respectively.

Acceptance of Secondary Skin Grafts
The tolerogenic effect of IL-BMT was assessed by secondary transplantation of skin grafts 3 weeks after BMT (B10->BALB/c). All recipients conditioned with 70 µg/g busulfan injected i.v., IB, and IL with 107 whole BMC accepted donor-matched skin grafts for periods exceeding 16 weeks (n = 5). In a similar manner, skin grafts were accepted by all viable chimeras transplanted with TCD-BMC either i.v. or IL (n = 5). In contrast, third party skin allografts (B10.BR) were acutely rejected in these experimental groups (n = 3-4).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Aiming to generate a protocol of localized BMT, IB and IL transplantation were assessed for their efficiency at inducing hemopoietic chimerism. IB injection rescued myeloablated recipients, as efficiently as i.v.-BMT, corroborating recent reports [7,9]. However, assessment of the kinetics of systemic BMC dispersion revealed that the IB route was not efficient in creation of localized BMT. Injected blasts were found in peripheral blood and contralateral femurs within minutes, indicating that IB-BMT is, in fact, a systemic route of injection. The conceivable reason was massive mobilization of BMC from recipient femur at the time of infusion, most likely caused by high intraluminal pressures. Evidently, spillage of small amounts of BMC into the soft tissues could not account for the large numbers of circulating donor blasts found in peripheral blood. Precautions taken to minimize local spill of BMC into the blood stream, by lowering the injection pressure, did not prevent systemic dispersion. Apparently, a pressure of 8 mmHg was not below the critical threshold that entraps cells in the bone. Possibly, work in larger animals will facilitate such attempts.

In contrast, IL perfusion led to localized engraftment in the limb and progressive secondary systemic seeding of transplanted cells. IL transplantation shared some of the characteristics of i.v.- and IB-BMT. Infusion of syngeneic and allogeneic BMC rescued myeloablated recipients, and the levels of donor PBL chimerism varied with conditioning and the size of donor inoculum. At 4 weeks, the IL and i.v. protocols induced comparable levels of donor PBL chimerism, which were sufficient for acceptance of skin allografts by nonmyeloablated recipients. Creation of localized BMT was evident from the remarkable increase in femur cellularity in the perfused limb, and the extremely low incidence of PKH+ cells in peripheral blood and contralateral femurs. Considering that IL-BMT created localized engraftment and systemic distribution of few cells, it is likely that BMC were mobilized from the injected limb at a later time [18]. Secondary systemic redistribution was apparent in the levels of donor chimerism at 30 days (24%-76%) that exceeded by far the contribution of one femur and tibia (7%-9%) to the hemopoietic space in the mouse [19].

Infusion of a large number of cells into a small hematopoietic space creates a local "megadose effect" that may provide significant advantages for initial HSC engraftment [12–15]. It may be argued that if transplanted cells have to compete with host HSC for vacant niches for engraftment, most prominent in nonablated recipients, then there may be a limitation in number of BMC that can be effectively transplanted into a limb [11,13]. However, there was no apparent limitation in stromal niches for initial seeding when large numbers of BMC (2 x 108) were injected directly into the lumen of isolated femurs [20].

We found several advantages of IL, as a route of administration of donor cells. First, the results support the prediction that the efficiency of localized engraftment would increase at a smaller donor inoculum in the IL- versus i.v.-BMT [13–15]. Second, there was a significant improvement in efficiency of engraftment of TCD-BMC infused into the isolated limb compared with injection into a peripheral vein. Depletion of T cells decreased the incidence of GVHD, however, more conditioning was required for engraftment [21,22]. Infusion of a megadose of donor cells was shown to overcome the absence of T cells and antigenic barriers between donor cells and recipient BM stroma [7,12]. It will be interesting to further assess whether IB- and IL-BMT reduce the reactivity of T cells that leads to GVHD, as recently reported for IB marrow infusion [7]. Third, immunoreactivity against third party allografts was preserved, suggesting that acceptance of the skin grafts was mediated by hemopoietic chimerism [1–5], and was not a limited consequence of chemical conditioning. Finally, considering the relative safety of IL [23], IL-BMT may have significant clinical applications for induction of tolerance and treatment of nonmalignant disorders.

In this study, busulfan was chosen as a single conditioning agent for assessment of the localized BMT approach, because its cytoreductive effect is more pronounced than its immunosuppressive activity. The assumption was that localized BMT significantly reduces the need for systemic immunosuppression at the time of transplantation. Busulfan is a cytoreductive agent that selectively affects slow-cycling and quiescent BMC, predominantly primitive stem cells in G0 [24]. It is likely that the systemic preconditioning procedure used in this study promoted the rates of secondary systemic seeding of locally transplanted BMC [18,25].

In summary, this study reports a novel approach for localized BMT by IL. The data demonstrate that IL-BMT: A) resulted in initial localization of donor cells and secondary expansion to give high levels of donor chimerism; B) reconstituted hematopoiesis in myeloablated recipients; C) induced hemopoietic chimerism as efficiently as systemic BMT, and D) led to donor-specific tolerance to secondary skin grafts across major and minor antigenic barriers, while preserving immunoreactivity against third party alloantigens. This approach may have clinical applications for treatment of nonmalignant disorders and induction of tolerance without myeloablative conditioning.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We thank Mrs. Judy Montibeller and Mrs. Lisa McGow for the excellent technical assistance, Dr. Kathy Muirhead, SciGro Co., Philadelphia, for providing PKH membrane linkers, Dr. Sallie S. Boggs, University of Pittsburgh Medical Center for the stimulating discussions and critical assessment of the manuscript, and Dr. Daniel L. Farkas, Center for Light Microscope, Imaging and Biotechnology, Carnegie Mellon University, for the outstanding support of this work. This study was partially supported by AHA grant 9960386V.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received October 12, 2001; accepted for publication October 23, 2001.



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