Complete Reconstitution Research Articles

Haploidentical transplants from positively selected bone marrow haematopoietic stem cells: report of 20 cases

Genetic diseases affecting haematopoietic and immune systems can be cured by bone marrow transplantation (BMT). Haploidentical BMT is the treatment of choice in many cases since is uneven to have an healthy matched family donor in family trees affecte by a genetic disease. Moreover the search of matched unrelated donor can take long (3–6 months)and succeed in 70% of the cases. Since 1983 haploidentical traplants have been performed by T cell depletion. In our experience we have performed 40 haploidentical transplants by Campath 1M bone marrow T cell depletion in children affected by genetic diseases from 1991 to 1996. The overall engraftment rate was 60%, but the event free survival did not exceeded 40%. Camtpath 1M recognized CD52 positive cells, that meant T cells, B cells, monocytes, plasmacells and approximately 50% of CD34+ cells. Reasons of relative unsuccess were maybe related to a profound depltion of accesory cells within the bone marrow suspension. Since 1996 we utilized Clinimacs clinical device to perform positive selection of haetopoietic stem cells. Since we are treating genetic diseases the children affected have a mean age of 2,5 years, therefore the weight at transplant do not exceed 10 kg. We have performed 20 haploidentical transplants positively selecting haematopoietic stem cells from bone marrow. The children were affected by severe combined immunodeficinecies(SCID) and other primary immunodeficiencies. The mean yield of stem cells was 75% of the initial stem cell count, while the purity of the stem cells was 91. 8%. Engraftment rate was 90% with a mean time for complete immune reconstitution that did not exceed 3 months.

Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix.

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.

Production and loss of high‐density batholithic root, southern Sierra Nevada, California

Eclogites are commonly believed to be highly susceptible to delamination and sinking into the mantle from lower crustal metamorphic environments. We discuss the production of a specific class of eclogitic rocks that formed in conjunction with the production of the Sierra Nevada batholith. These high‐density eclogitic rocks, however, formed by crystal‐liquid equilibria and thus contrast sharply in their petrogenesis and environment of formation from eclogite facies metamorphic rocks. Experimental studies show that when hydrous mafic to intermediate composition assemblages are melted in excess of 1 GPa, the derivative liquids are typical of Cordilleran‐type batholith granitoids, and garnet + clinopyroxene, which is an eclogitic mineralogy, dominate the residue assemblage. Upper mantle‐lower crustal xenolith suites that were entrained in mid‐Miocene volcanic centers erupted through the central Sierra Nevada batholith are dominated by such garnet clinopyroxenites, which are shown further by geochemical data to be petrogenetically related to the overlying batholith as its residue assemblage. Petrogenetic data on garnet pyroxenite and associated peridotite and granulite xenoliths, in conjunction with a southward deepening oblique crustal section and seismic data, form the basis for the synthesis of a primary lithospheric column for the Sierra Nevada batholith. Critical aspects of this column are the dominance of felsic batholithic rocks to between 35 and 40 km depths, a thick (∼35 km) underlying garnet clinopyroxenite residue sequence, and interlayered spinel and underlying garnet peridotite extending to ∼125 km depths. The peridotites appear to be the remnants of the mantle wedge from beneath the Sierran arc. The principal source for the batholith was a polygenetic hydrous mafic to intermediate composition lower crust dominated by mantle wedge‐derived mafic intrusions. Genesis of the composite batholith over an ∼50 m.y. time interval entailed the complete reconstitution of the Sierran lithosphere. Sierra Nevada batholith magmatism ended by ∼80 Ma in conjunction with the onset of the Laramide orogeny, and subsequently, its underlying mantle lithosphere cooled conductively. In the southernmost Sierra‐northern Mojave Desert region the subbatholith mantle lithosphere was mechanically delaminated by a shallow segment of the Laramide slab and was replaced by underthrust subduction accretion assemblages. Despite these Laramide events, the mantle lithosphere of the greater Sierra Nevada for the most part remained intact throughout much of Cenozoic time. A pronounced change in xenolith suites sampled by Pliocene‐Quaternary lavas to garnet absent, spinel and plagioclase peridotites, whose thermobarometry define an asthenosphere adiabat, as well as seismic data, indicate that much of the remaining sub‐Sierran lithosphere was removed in Late Miocene to Pliocene time. Such removal is suggested to have arisen from a convective instability related to high‐magnitude extension in the adjacent Basin and Range province and to have worked in conjunction with the recent phase of Sierran uplift and a change in regional volcanism to more primitive compositions. In both the Mio‐Pliocene and Late Cretaceous lithosphere removal events the base of the felsic batholith was the preferred locus of separation.

Defective phagocytosis and clearance of Pseudomonas aeruginosa in the lung following bone marrow transplantation.

Bone marrow transplantation (BMT) is an important therapeutic option for a variety of malignant and nonmalignant disorders. Unfortunately, BMT recipients are at increased risk of infection, and in particular, pulmonary complications occur frequently. Although the risk of infection is greatest during the neutropenic period immediately following transplant, patients are still vulnerable to pulmonary infections even after neutrophil engraftment. We evaluated the risk of infection in this postengraftment period by using a well-established mouse BMT model. Seven days after syngeneic BMT, B6D2F(1) mice are no longer neutropenic, and by 3 wk, they demonstrate complete reconstitution of the peripheral blood. However, these mice remain more susceptible throughout 8 wk to infection after intratracheal administration of Pseudomonas aeruginosa; increased mortality in the P. aeruginosa-infected BMT mice correlates with increased bacterial burden in the lungs as well as increased systemic dissemination. This heightened susceptibility to infection was not secondary to a defect in inflammatory cell recruitment to the lung. The inability to clear P. aeruginosa in the lung correlated with reduced phagocytosis of the bacteria by alveolar macrophages (AMs), but not neutrophils, decreased production of TNF-alpha by AMs, and decreased levels of TNF-alpha and IFN-gamma in the bronchoalveolar lavage fluid following infection. Expression of the beta(2) integrins CD11a and CD11c was reduced on AMs from BMT mice compared with wild-type mice. Thus, despite restoration of peripheral blood count, phagocytic defects in the AMs of BMT mice persist and may contribute to the increased risk of infection seen in the postengraftment period.

Complete reconstitution of human lymphocytes from cord blood CD34+ cells using the NOD/SCID/γcnull mice model

AbstractEstablishment of an assay capable of generating all classes of human lymphocytes from hematopoietic stem cells (HSCs) will provide new insight into the mechanism of human lymphopoiesis. We report ontogenic, functional, and histologic examination results of reconstituted human lymphocytes in NOD/SCID/ γcnull mice after the transplantation of human cord blood (CB) CD34+ cells. After transplantation, human B, natural killer (NK), and T cells were invariably identified in these mice, even though no human tissues were cotransplanted. Immature B cells resided mainly in bone marrow (BM), whereas mature B cells with surface immunoglobulins were preferentially found in spleen. NK cells were identified in BM and spleen. T cells were observed in various lymphoid organs, but serial examinations after transplantation confirmed human T lymphopoiesis occurring in the thymus. These human lymphocytes were also functionally competent. Human immunoglobulin M (IgM), IgA, and IgG were detected in the sera of these mice. T cells showed a diverse repertoire of T-cell–receptor Vβ (TCR Vβ) chains, proliferated in response to phytohemagglutinin, and were cytotoxic against cell lines. NK activity was demonstrated using the K562 cell line. Immunohistochemical analysis revealed that human lymphocytes formed organized structures in spleen and thymus that were analogous to those seen in humans. In the thymus, CD4 and CD8 double-positive T cells were predominant and coexpressed CD1a and Ki-67, thereby supporting the notion that T lymphopoiesis was taking place. NOD/SCID/ γcnull mice provide a unique model to investigate human lymphopoiesis without the cotransplantation of human tissues.

Long-term CD4+ and CD8+ memory T cells developed in severe combined immunodeficiency mice during homoeostasis exhibit differences in sensitivity to antigen.

T cells transferred in small numbers to lymphopenic hosts proliferate spontaneously, and naïve T cells turn into memory cells without complete cellular reconstitution of the lymphoid compartment. In this study, neonatal severe combined immunodeficiency mice were treated with peripheral CD4+ or CD8+ T cells purified from the spleen of syngeneic C.B-17 mice. At 2 weeks and more pronounced at 10 weeks post treatment, a majority of the residing donor T cells showed memory phenotype, with high expression of CD44 and an early onset of proliferation and cytokine production upon stimulation. These memory type of donor cells were sustained in numbers for at least 1.5 years post treatment in a homoeostatic fashion, recognized by normal CD4/CD8 ratio and no bias towards type 1 or type 2 immune response. Furthermore, amongst the memory type of cells, there was a striking difference in their response, where the CD8+ donor cells had higher threshold for stimulation than the CD4+ donor cells.

Hematopoietic reconstitution of irradiated, stem cell-injected mice: early dynamics of restoration of the cell lineages of the spleen and bone marrow.

Hematopoietic stem cells, numbering approximately 1/100,000 cells in mammalian bone marrow, are capable of complete hematopoietic and immune reconstitution upon injection into a myeloablated host. The present study aimed to analyze the earliest events in reconstitution of lethally irradiated, host murine bone marrow and spleen, after injecting purified Thy 1(lo)Lin(-)Sca-1(+) stem cells. Thy-1(lo)Lin(-)Sca-1(+) cells were isolated by fluorescence-activated cell sorting (FACS) from the bone marrow of 4-week-old C57BL(Thy1.1, Ly5.1) mice and injected into preirradiated, syngeneic hosts. These stem cells were also injected into congenic hosts, i.e., C57BL(Thy1.2, Ly5.2), and confirmed the donor origin of hematopoietic cells in the reconstituted host mice. Hematologically stained smears of the spleen and bone marrow of stem cell-injected recipients were prepared at 11, 14, 17, 21, 24, and 28 days after stem cell injection, and nucleated erythroid cells, mature granulocytes, and their myeloid precursors, monocytes, and large and small lymphocytes were recorded as a proportion of all nucleated cells in each organ at each time interval. The results indicated that in the earliest post stem cell injection intervals, both organs were predominantly erythroid and myeloid. Only at the later intervals did both organs show high proportions of large lymphoid cells and their progeny, small lymphocytes. Thus, early (<1 month) dynamics of hematopoietic reconstitution after transplantation of purified hematopoietic stem cells, is cell lineage specific.

Kinetics of Liver Repopulation after Bone Marrow Transplantation

Recent work has convincingly demonstrated that adult bone marrow contains cells capable of differentiating into liver epithelial cells in vivo. However, the frequency and time course with which fully functional hepatocytes emerge after bone marrow transplantation remained controversial. Here, we used the fumarylacetoacetate hydrolase knockout mouse to determine the kinetics of hepatocyte replacement after complete hematopoietic reconstitution. Single donor-derived hepatocytes were first detected 7 weeks after lethal irradiation and bone marrow transplantation. Liver disease was not required for this transdifferentiation. In the presence of selective pressure the single cells evolved into hepatocyte nodules by 11 weeks after transplantation and resulted in >30% overall liver repopulation by 22 weeks. The frequency with which hepatocytes were produced was between 10(-4) and 10(-6), resulting in only 50 to 500 repopulation events per liver. Hepatic engraftment was not observed without previous hematopoietic reconstitution even in the presence of liver injury. In addition, significant liver repopulation was completely dependent on hepatocyte growth selection. We conclude that hepatocyte replacement by bone marrow cells is a slow and rare event. Significant improvements in the efficiency of this process will be needed before clinical success can be expected.

Retroviral transduction of IL2RG into CD34+ cells from X-linked severe combined immunodeficiency patients permits human T- and B-cell development in sheep chimeras

AbstractX-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain of cytokine receptors, γc. Because bone marrow transplantation (BMT) for XSCID does not provide complete immune reconstitution for many patients and because of the natural selective advantage conferred on lymphoid progenitors by the expression of normal γc, XSCID is a good candidate disease for therapeutic retroviral gene transfer to hematopoietic stem cells. We studied XSCID patients who have persistent defects in B-cell and/or combined B- and T-cell function despite having received T cell–depleted haploidentical BMT. We compared transduction of autologous B-cell lines and granulocyte colony-stimulating factor–mobilized peripheral CD34+ cells from these patients using an MFGS retrovirus vector containing the γc gene IL2RG pseudotyped with amphotropic, gibbon ape leukemia virus, or RD114 envelopes. Transduced B-cell lines and peripheral CD34+ cells demonstrated provirus integration and new cell-surface γc expression. The chimeric sheep model was exploited to test development of XSCID CD34+ cells into mature myeloid and lymphoid lineages. Transduced and untransduced XSCID CD34+ cells injected into developing sheep fetuses gave rise to myeloid cells. However, only transduced γc+ progenitors from XSCID patients developed into T and B cells. These results suggest that gene transfer to autologous peripheral CD34+ cells using MFGS-gc retrovirus may benefit XSCID patients with persistent T- and B-cell deficits despite prior BMT.

Repair of orbital floor fractures: Application of biomater als in plastic surgery

The perfect reconstructive material for orbital floor fractures remains elusive. However, recent advances in the development of bioresorbable materials has broadened the surgeon's choice of options for the clinical use and application of such materials. Previously, fractures of this nature were reconstituted with a variety of metallic plating devices or silicone. Today, reconstructive surgeons have a number of additional different options to support repaired orbital contents. The identification and application of noninflammatory and nonallergic, sufficiently rigid biomaterials (that persist over a finite period of time permitting complete bony reconstitution) remains an exciting future topic for clinicians and researchers.

Partial reconstitution of human DNA mismatch repair in vitro: characterization of the role of human replication protein A.

DNA mismatch repair (MMR) is a critical genome-stabilization system. However, the molecular mechanism of MMR in human cells remains obscure because many of the components have not yet been identified. Using a functional in vitro reconstitution system, this study identified three HeLa cell fractions essential for in vitro MMR. These fractions divide human MMR into two distinct stages: mismatch-provoked excision and repair synthesis. In vitro dissection of the MMR reaction and crucial intermediates elucidated biochemical functions of individual fractions in human MMR and identified hitherto unknown functions of human replication protein A (hRPA) in MMR. Thus, one fraction carries out nick-directed and mismatch-dependent excision; the second carries out DNA repair synthesis and DNA ligation; and the third provides hRPA, which plays multiple roles in human MMR by protecting the template DNA strand from degradation, enhancing repair excision, and facilitating repair synthesis. It is anticipated that further analysis of these fractions will identify additional MMR components and enable the complete reconstitution of the human MMR pathway with purified proteins.

Single channel recordings of α-hemolysin reconstituted in S-layer-supported lipid bilayers

Previous studies demonstrated that lipid membranes attached to a proteinaceous crystalline surface-layer (S-layer) revealed a prolonged lifetime and showed a reduced tendency to rupture in the presence of membrane active molecules. In addition, comparative studies on folded and S-layer-supported lipid membranes (SsLM) revealed an uniform capacitance of 0.64±0.04 μF/cm 2 for both composite membranes. In the present study, the feasibility to reconstitute the channel-forming protein α-hemolysin (αHL) into SsLM at single channel resolution was investigated. Single αHL channels could be recorded and the intrinsic properties like unitary conductance, current–voltage characteristics, and closure was found to be similar at both membranes. Thus, the tightly attached S-layer allowed complete reconstitution of αHL channels in SsLM.

Selective Degradation of Ubiquitinated Sic1 by Purified 26S Proteasome Yields Active S Phase Cyclin-Cdk

Selective degradation of single subunits of multimeric complexes by the ubiquitin pathway underlies multiple regulatory switches, including those involving cyclins and Cdk inhibitors. The machinery that segregates ubiquitinated proteins from unmodified partners prior to degradation remains undefined. We report that ubiquitinated Sic1 (Ub-Sic1) embedded within inactive S phase cyclin-Cdk (S-Cdk) complexes was rapidly degraded by purified 26S proteasomes, yielding active S-Cdk. Mutant proteasomes that failed to degrade Ub-Sic1 activated S-Cdk only partially in an ATP-dependent manner. Whereas Ub-Sic1 was degraded within ∼2 min, spontaneous dissociation of Ub-Sic1 from S-Cdk was ∼200-fold slower. We propose that the 26S proteasome has the intrinsic capability to extract, unfold, and degrade ubiquitinated proteins while releasing bound partners untouched. Activation of S-Cdk reported herein represents a complete reconstitution of the regulatory switch underlying the G1/S transition in budding yeast.

In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase Enzymes catalyze the conversion of 2-methylcitrate to 2-methylisocitrate.

Salmonella enterica serovar Typhimurium LT2 catabolizes propionate through the 2-methylcitric acid cycle, but the identity of the enzymes catalyzing the conversion of 2-methylcitrate into 2-methylisocitrate is unclear. This work shows that the prpD gene of the prpBCDE operon of this bacterium encodes a protein with 2-methylcitrate dehydratase enzyme activity. Homogeneous PrpD enzyme did not contain an iron-sulfur center, displayed no requirements for metal cations or reducing agents for activity, and did not catalyze the hydration of 2-methyl-cis-aconitate to 2-methylisocitrate. It was concluded that the gene encoding the 2-methyl-cis-aconitate hydratase enzyme is encoded outside the prpBCDE operon. Computer analysis of bacterial genome databases identified the presence of orthologues of the acnA gene (encodes aconitase A) in a number of putative prp operons. Homogeneous AcnA protein of S. enterica had strong aconitase activity and catalyzed the hydration of the 2-methyl-cis-aconitate to yield 2-methylisocitrate. The purification of this enzyme allows the complete reconstitution of the 2-methylcitric acid cycle in vitro using homogeneous preparations of the PrpE, PrpC, PrpD, AcnA, and PrpB enzymes. However, inactivation of the acnA gene did not block growth of S. enterica on propionate as carbon and energy source. The existence of a redundant aconitase activity (encoded by acnB) was postulated to be responsible for the lack of a phenotype in acnA mutant strains. Consistent with this hypothesis, homogeneous AcnB protein of S. enterica also had strong aconitase activity and catalyzed the conversion of 2-methyl-cis-aconitate into 2-methylisocitrate. To address the involvement of AcnB in propionate catabolism, an acnA and acnB double mutant was constructed, and this mutant strain cannot grow on propionate even when supplemented with glutamate. The phenotype of this double mutant indicates that the aconitase enzymes are required for the 2-methylcitric acid cycle during propionate catabolism.

Lymph nodes during antiretroviral therapy.

Highly active antiretroviral therapy rapidly reduces virus replication in the lymphoid tissue. Production of viral RNA, however, may still be detected in the lymphoid tissue despite negative plasma viremia. Continuing virus production and latent infection in resting cells seem to be important factors for viral rebound following treatment interruption. In parallel with viral suppression, immune activation is decreased and CD4+ T cell counts in the lymphoid tissue increase. It is still not known if there is a potential for complete viral suppression and immune reconstitution. Analyses of the lymphoid tissue during therapy may be helpful in addressing these issues.

Comparison of T-Cell Subsets' Reconstitution After 12 Months of Highly Active Antiretroviral Therapy Initiated During Early Versus Advanced States of HIV Disease

This research comprised a pilot open prospective clinical study comparing T-cell subset reconstitution in antiretroviral-naive patients, after 12 months of HAART when treatment was initiated in early stages (ES; n = 18) of infection versus advanced stages (AS; n = 20). Control group: 10 healthy HIV-negative individuals. Immunophenotypic markers were evaluated before and after 6- and 12-months' therapy. Functional assays were performed in one subset. Results: Viral load (VL) was < 200 copies/ml in all patients. Median percentages of CD4+ pretherapy were 33% and 6%, respectively, in the ES and AS groups, increment after 12 months of therapy was +15% and +13% respectively. Only the ES group achieved normal values. Declines of CD8+ percentage were significantly higher in the ES (-18%) than in the AS group (-2%). CD4+ memory and naive cells in the ES group were similar to those of controls before treatment and did not change after therapy. In contrast, CD4+ memory and naive cells in the AS group did not reach normal levels despite treatment. In the ES group, there was a significant increment in CD8+ naive cells (+8%) and a decrement in CD8+CD38+ cells (-17%), both populations reached values close to normal, whereas these subsets remained far from normal in the AS group. Improvement of lymphoproliferative response after therapy was observed in both groups. One patient in the ES group showed positive LPR against p24 after treatment. After 12 months' highly active antiretroviral therapy, only those who began such therapy in ES disease reached values within the range of healthy controls. To achieve a more complete immunologic reconstitution, early initiation of potent antiretroviral therapy should be recommended.

Reconstitution of the Pore-Forming Toxin α-Hemolysin in Phospholipid/18-Octadecyl-1-thiahexa(ethylene oxide) and Phospholipid/n-Octadecanethiol Supported Bilayer Membranes

We are studying the functional reconstitution of membrane-bound proteins into supported bilayer membranes (SBMs). Here, we describe the physical properties of SBMs formed by a layer of egg-phosphatidyl choline deposited on a monolayer of either 18-octadecyl-1-thiahexa(ethylene oxide) [THEO-C18] or n-octadecanethiol on gold. We also show that the pore-forming protein α-hemolysin (αHL) self-assembles in these thin films. The insulating properties and the stability of the THEO-C18 self-assembled monolayers were characterized by ac impedance spectroscopy and voltammetry. An impedance model, including constant phase elements, was determined for THEO-C18 monolayers and the SBMs. Cyclic voltammetry measurements demonstrated virtually full blockage of ferricyanide oxidation and reduction by the THEO-C18 monolayers. The monolayer stability test showed that, at applied potentials between ±400 mV versus Ag/AgCl in 3 M KCl, the electrical properties of THEO-C18 SAMs did not change with time. The reconstitution of αHL in SBMs caused a decrease in impedance and an increased permeability to redox ions. The impedance model parameters suggest that αHL partially penetrates into the SBMs, increasing the dielectric constant of the alkane portion of the monolayers. The complete reconstitution of αHL that could provide the free access of the redox ions to the metal surface was not observed in these thin films.

Defining the requirements for peptide recognition in gene therapy-induced T cell tolerance.

Expression of a retrovirally transduced MHC class I Ag, H-2K(b) (K(b)), in bone marrow-derived cells leads to specific prolongation of K(b) disparate skin grafts. To examine the extent to which peptides derived from K(b) contribute to the induction of tolerance, retroviruses carrying mutant K(b) genes designed to enter separate pathways of Ag presentation were constructed. Thymectomized and CD8 T cell-depleted mice that had been irradiated and reconstituted with bone marrow cells expressing a secreted form of K(b) showed prolongation of K(b) disparate skin graft survival. Skin graft prolongation was not observed when similar experiments were performed using mice that were not CD8 T cell depleted. This suggests that hyporesponsiveness can be induced in CD4 T cells, but not CD8 T cells by Ags presented via the exogenous pathway of Ag processing. Modest prolongation of skin allografts was observed in mice reconstituted with bone marrow cells transduced with retroviruses carrying a gene encoding a mutant K(b) molecule expressed only in the cytoplasm. Prolongation was also observed in similar experiments in mice that were thymectomized and CD4 T cell depleted following complete reconstitution, but not in mice that were reconstituted and then thymectomized and CD8 T cell depleted. Thus, hyporesponsiveness can be induced in a subset of CD8 T cells by recognition of peptides derived from K(b) through both the direct and indirect pathways of Ag recognition, while CD4 T cell hyporesponsiveness to MHC class I disparate grafts occurs only through the indirect pathway of Ag recognition.

Sites in the A2 Subunit Involved in the Interfactor VIIIa Interaction

Sites in the A2 Subunit Involved in the Interfactor VIIIa Interaction

Negative immunomagnetic purging of peripheral blood stem cell harvests from breast carcinoma patients reduces tumor cell contamination while not affecting hematopoietic recovery.

Because tumor contamination of hematopoietic stem cell grafts may influence the outcome in breast carcinoma (BC) patients undergoing high dose chemotherapy (HDC), several ex vivo procedures for the purging of autologous harvests have been investigated. The authors studied the presence of epithelial tumor cells and the growth of hematopoietic progenitors in peripheral blood stem cell (PBSC) collections from patients with metastatic breast carcinoma before and after a purging procedure performed by a negative immunomagnetic BC cell separation. Eighteen patients entered the study. Tumor contamination was assessed by conventional immunocytochemistry (ICC) and by a liquid culture assay developed in the study laboratory. Committed and more primitive hematopoietic progenitors were quantitated before and after the negative selection. Ten patients received HDC with purged PBSC support. Before purging, 4 of 18 PBSC collections were found to be contaminated by liquid culture; among these samples, only 1 was positive by ICC. Three of the four positive collections, including the ICC positive sample, became negative after immunomagnetic selection whereas BC cells still were present after the procedure in one harvest. A high recovery of both primitive and mature hematopoietic progenitors was found after the purging procedure. Patients receiving purged PBSC after myeloablation had a prompt and complete hematopoietic reconstitution, and no graft failure was observed at a median follow-up of 1 year. The preliminary results of the current study suggest that negative selection of BC cells is able to purge PBSC effectively while having no apparent affect on hematopoietic progenitor recovery in vitro and in vivo.