Regeneration of T cell subpopulations after bone marrow transplantation: cytomegalovirus infection and lymphoid subset imbalance

Immune reconstitution was studied serially by using T lymphocyte cell surface differentiation antigens in 37 individuals receiving bone marrow transplants. Antigen expression was assessed by immunofluorescence analysis using monoclonal antibodies to T lymphocytes including Leu-3, which defines a T lymphocyte subpopulation in healthy individuals with T helper or inducer activity (L3+), and Leu-2, which defines a T lymphocyte subpopulation in healthy individuals with T helper or inducer activity (L3+), and Leu-2, which defines a T lymphocyte subpopulation with suppressor or cytotoxic activity (L2+). These studies demonstrated that the L2+ subpopulation regenerated more rapidly after transplant than did the L3+ subpopulation. Imbalances between these two T lymphocyte subpopulations, indicated by a decreased L3/L2 ratio, persisted for periods up to 12 mo post-transplant. Expression of a cell surface antigen associated with immature lymphocytes (OKT-10), and of HLA-DR (Ia-like) antigens was markedly increased during the post-transplant period. HLA-DR antigen expression did not appear related to immune activation in that increased reactivity was not detected with a monoclonal antibody (anti-TAC) specific for activated T cells. These observations in bone marrow transplant recipients and other disorders characterized by lymphoid restoration or immaturity indicate that inversion of the normal L3/L2 ratio and increased expression of OKT-10 and HLA-DR antigens may be features of a regenerating immune system. Furthermore, serial observation of individual patients indicated that infection with cytomegalovirus was associated with a progressive decrease in the L3/L2 ratio.     

Flt3 ligand expands dendritic cell numbers in normal and malignant murine prostate

We have developed a murine model that facilitates the structural and functional analysis in vivo of dendritic cell (DC)-mediated phagocytosis of prostate epithelial cells. Recombinant human Flt3 ligand (rhFL) expands the number of dendritic cells in lymphoid and non-lymphoid tissues of mice. We show that rhFL also induced the ingress of dendritic cells into murine prostate, which involutes via epithelial apoptosis after surgical castration. Intact or castrated C57BL/6 and syngeneic transgenic adenocarcinoma of mouse prostate (TRAMP) mice were treated with rhFL or PBS control. Prostate and spleen were then studied by flow cytometry and immunohistochemistry.The number of prostatic CD11c+ and CD11b+ dendritic cells increased significantly in rhFL-treated mice compared with PBS-treated control mice and this effect was greatly augmented by castration of the mice. The immunophenotype of rhFL-mobilized prostatic cells was consistent with that of Langerhans cells (MHC class II+, CD11c+,CD11b+, DEC-205+, CD8 alpha-).MHC class II+ and CD11c+ dendritic cells that were present in the prostate glands of rhFL-treated and castrated C57BL/6 mice were intimately associated with TUNEL+ inclusions, which suggests that Langerhans-type dendritic cells in prostate participated in the clearance of apoptotic cells. Expression of MHC class II, CD54, CD80 and CD86 by prostatic dendritic cells was not up-regulated after castration and freshly isolated rhFL-induced prostate cells were unable to prime allogeneicT cells unless they were activated by culture either on plastic or with recombinant soluble CD40 ligand. Our data suggest that rhFL-mobilized prostatic dendritic cells resemble the functionally immature dendritic cells, which reside in peripheral tissues and contribute to the maintenance of peripheral tolerance.     

Estradiol acts directly on bone marrow myeloid progenitors to differentially regulate GM-CSF or Flt3 ligand-mediated dendritic cell differentiation

Estrogen receptor (ER) ligands modulate hemopoiesis and immunity in the normal state, during autoimmunity, and after infection or trauma. Dendritic cells (DC) are critical for initiation of innate and adaptive immune responses. We demonstrate, using cytokine-driven culture models of DC differentiation, that 17-beta-estradiol exerts opposing effects on differentiation mediated by GM-CSF and Flt3 ligand, the two cytokines that regulate DC differentiation in vivo. We also show that estradiol acts on the same highly purified Flt3+ myeloid progenitors (MP) to differentially regulate the DC differentiation in each model. In GM-CSF-supplemented cultures initiated from MP, physiological amounts of estradiol promoted differentiation of Langerhans-like DC. Conversely, in Flt3 ligand-supplemented cultures initiated from the same MP, estradiol inhibited cell survival in a dose-dependent manner, thereby decreasing the yield of plasmacytoid and conventional myeloid and lymphoid DC. Experiments with bone marrow cells from ER-deficient mice and the ER antagonist ICI182,780 showed that estradiol acted primarily via ERalpha to regulate DC differentiation. Thus, depending on the cytokine environment, pathways of ER signaling and cytokine receptor signaling can differentially interact in the same Flt3+ MP to regulate DC development. Because the Flt3 ligand-mediated differentiation pathway is important during homeostasis, and GM-CSF-mediated pathways are increased by inflammation, our data suggest that endogenous or pharmacological ER ligands may differentially affect DC development during homeostasis and disease, with consequent effects on DC-mediated immunity.     

The kinetics of early T and B cell immune recovery after bone marrow transplantation in RAG-2-deficient SCID patients

The kinetics of T and B cell immune recovery after bone marrow transplantation (BMT) is affected by many pre- and post-transplant factors. Because of the profoundly depleted baseline T and B cell immunity in recombination activating gene 2 (RAG-2)-deficient severe combined immunodeficiency (SCID) patients, some of these factors are eliminated, and the immune recovery after BMT can then be clearly assessed. This process was followed in ten SCID patients in parallel to their associated transplant-related complications. Early peripheral presence of T and B cells was observed in 8 and 4 patients, respectively. The latter correlated with pre-transplant conditioning therapy. Cells from these patients carried mainly signal joint DNA episomes, indicative of newly derived B and T cells. They were present before the normalization of the T cell receptor (TCR) and the B cell receptor (BCR) repertoire. Early presentation of the ordered TCR gene rearrangements after BMT occurred simultaneously, but this pattern was heterogeneous over time, suggesting different and individual thymic recovery processes. Our findings early after transplant could suggest the long-term patients' clinical outcome. Early peripheral presence of newly produced B and T lymphocytes from their production and maturation sites after BMT suggests donor stem cell origin rather than peripheral expansion, and is indicative of successful outcome. Peripheral detection of TCR excision circles and kappa-deleting recombination excision circles in RAG-2-deficient SCID post-BMT are early markers of T and B cell reconstitution, and can be used to monitor outcome and tailor specific therapy for patients undergoing BMT.     

Flt3 ligand synergizes with granulocyte-colony-stimulating factor in bone marrow mobilization to improve functional outcome after spinal cord injury in the rat

Background aimsThe effect of granulocyte–colony-stimulating factor (G-CSF) and/or the cytokine fms-like thyrosin kinase 3 (Flt3) ligand on functional outcome and tissue regeneration was studied in a rat model of spinal cord injury (SCI).MethodsRats with a balloon-induced compression lesion were injected with G-CSF and/or Flt3 ligand to mobilize bone marrow cells. Behavioral tests (Basso-Beattie-Bresnahan and plantar test), blood counts, morphometric evaluation of the white and gray matter, and histology were performed 5 weeks after SCI.ResultsThe mobilization of bone marrow cells by G-CSF, Flt3 ligand and their combination improved the motor and sensory performance of rats with SCI, reduced glial scarring, increased axonal sprouting and spared white and gray matter in the lesion. The best results were obtained with a combination of G-CSF and Flt3. G-CSF alone or in combination with Flt3 ligand significantly increased the number of white blood cells, but not red blood cells or hemoglobin content, during and after the time–course of bone marrow stimulation. The combination of factors led to infiltration of the lesion by CD11b⁺ cells.ConclusionsThe observed improvement in behavioral and morphologic parameters and tissue regeneration in animals with SCI treated with a combination of both factors could be associated with a prolonged time–course of mobilization of bone marrow cells. The intravenous administration of G-CSF and/or Flt3 ligand represents a safe and effective treatment modality for SCI.     

B cell development and regulation after T cell-depleted marrow transplantation

Antibody secreting B lymphocytes from immunized donors can be adoptively transferred after T cell-depleted marrow transplantation to produce protective levels of antibody in the recipient. We have investigated whether these transferred lymphocytes remain subject to continued clonal selection and subsequently became memory B cells even in the initial absence of T cells. Twenty-eight donor/recipient pairs were randomized pretransplant to be immunized or not with tetanus toxoid (TT). The recipients were then vaccinated with TT at 3, 6, and 12 mo posttransplant, and the anti-TT antibody response (IgG and IgM) was measured. Only when both donor and recipient were immunized pretransplant could the recipient respond to antigen challenge within the first year posttransplant. Examination of the spectrotype pattern of the recipient anti-TT antibody shows that selection of B cell clones continues, so that T cell depletion does not prevent the appearance of oligoclonal antibody responses. However, because the spectrotype pattern of the recipient did not match the donors, B cell regulatory mechanisms in donor and recipient are nonidentical. These data contrast with observations made in recipients of non-T cell-depleted marrow and serve to illustrate the role of T lymphocytes in the induction and regulation of secondary antibody responses in man. The results also suggest that optimal humoral responses to any antigen after T cell depletion can only occur when both donor and recipient are immunized pretransplant, a prediction borne out by studies on the influence of donor cytomegalovirus status on the severity of cytomegalovirus infection in the recipient.     

Add-back of allodepleted donor T cells to improve immune reconstitution after haplo-identical stem cell transplantation

Poor immune reconstitution after haplo-identical stem cell transplantation results in high mortality from viral infections and relapse. One approach to overcome this problem is to deplete alloreactive cells selectively by deleting T cells activated by recipient stimulators, using an immunotoxin directed against the activation marker CD25. However, the degree of depletion of alloreactive cells is variable following stimulation with recipient PBMC, and this can result in GvHD. We have shown that using recipient EBV-transformed LCL as stimulators to activate donor alloreactive T cells results in more consistent depletion of in vitro alloreactivity while preserving T-cell responses to viral and potential myeloid tumor Ag. Based on these data, we have embarked on a phase I clinical dose escalation study of add-back of allo-LCL-depleted donor T cells in the haplo-identical setting, to determine if the allodepletion we achieve to allow infusion of sufficient T cells to restore useful antiviral/anti-leukemic responses without causing GvHD. Fifteen patients have so far been treated. The incidence of significant acute or chronic GvHD has been low (2/15), as has mortality from infection (1/15). Preliminary data show accelerated immune reconstitution in dose level 2 patients. Infused allodepleted donor T cells appear able to expand significantly in the face of viral reactivations, and doses as low as 3 x 10(5)/kg may be sufficient to confer useful antiviral immunity in this setting. At a median follow-up of 19.5 months, nine of 15 patients are alive and disease-free. Five patients have relapsed, all of whom have died.     

Follicular dendritic cells are related to bone marrow stromal cell progenitors and to myofibroblasts

Follicular dendritic cells (FDC) are involved in the presentation of native Ags to B cells during the secondary immune response. Some authors consider FDC to be hemopoietic cells, whereas others believe them to be mesenchymal cells. The low proportion of FDC in the lymphoid follicle, together with technical difficulties in their isolation, make these cells difficult to study. We show that Fibroblast Medium can be used successfully to isolate and maintain FDC lines. In this culture medium, we obtained 18 FDC lines from human tonsils, which proliferated for as long as 18 wk and showed a stable Ag phenotype as detected by flow cytometry and RT-PCR. FDC lines were CD45-negative and expressed Ags associated to FDC (CD21, CD23, CD35, CD40, CD73, BAFF, ICAM-1, and VCAM-1) and Ags specific for FDC (DRC-1, CNA.42, and HJ2). These cell lines were also able to bind B cells and secrete CXCL13, functional activities characteristic of FDC. Nevertheless, the additional expression of STRO-1, together with CD10, CD13, CD29, CD34, CD63, CD73, CD90, ICAM-1, VCAM-1, HLA-DR, alkaline phosphatase, and alpha-smooth muscle actin (alpha-SM actin) indicated that FDC are closely related to bone marrow stromal cell progenitors. The expression of alpha-SM actin also relates FDC with myofibroblasts. Like myofibroblasts, FDC lines expressed stress fibers containing alpha-SM actin and were able to contract collagen gels under the effect of TGFbeta1 and platelet-derived growth factor. These findings suggest that FDC are a specialized form of myofibroblast and derive from bone marrow stromal cell progenitors.     

Early CD4+ T cell reconstitution as predictor of outcomes after allogeneic hematopoietic cell transplantation

Background: An association between early CD4+ T cell immune reconstitution (CD4+ IR) and survival after T-replete allogeneic hematopoietic cell transplantation (HCT) has been previously reported. Here we report validation of this relationship in a separate cohort that included recipients of ex vivo T-cell-depleted (TCD) HCT. We studied the relationship between CD4+ IR and clinical outcomes.Methods: A retrospective analysis of children/young adults receiving their first allogeneic HCT for any indication between January 2008 and December 2017 was performed. We related early CD4+ IR (defined as achieving >50 CD4+ T cells/µL on two consecutive measures within 100 days of HCT) to overall survival (OS), relapse, non-relapse mortality (NRM), event-free survival (EFS) and acute graft-versus-host disease (aGVHD). Fine and Gray competing risk models and Cox proportional hazard models were used.Results: In this analysis, 315 patients with a median age of 10.4 years (interquartile range 5.0–16.5 years) were included. The cumulative incidence of CD4+ IR at 100 days was 66.7% in the entire cohort, 54.7% in TCD (N = 208, hazard ratio [HR] 0.47, P < 0.001), 90.0% in uCB (N = 40) and 89.6% in T-replete (N = 47) HCT recipients. In multi-variate analyses, not achieving early CD4+ IR was a predictor of inferior OS (HR 2.35, 95% confidence interval [CI] 1.46–3.79, P < 0.001) and EFS (HR 1.80, 95% CI 1.20–2.69, P = 0.004) and increased NRM (HR 6.58, 95% CI 2.82–15.38, P < 0.001). No impact of CD4+ IR on relapse or aGVHD was found. Within the TCD group, similar associations were observed.Conclusion: In this HCT cohort, including recipients of TCD HCT, we confirmed that early CD4+ IR was an excellent predictor of outcomes. Finding strategies to predict or improve CD4+ IR may influence outcomes.     

Tumor vaccination after allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned tumor-bearing murine host

Allogeneic bone marrow cell reconstitution of the nonmyeloablatively conditioned host is supposed to provide an optimized platform for tumor vaccination. We recently showed that an allogeneic T cell-depleted graft was well accepted if the tumor-bearing host was NK depleted. Based on this finding, a vaccination protocol in tumor-bearing, nonmyeloablatively conditioned, allogeneically reconstituted mice was elaborated. Allogeneically reconstituted mice, bearing a renal cell carcinoma, received tumor-primed donor lymph node cells (LNC), which had or had not matured in the allogeneic host. Primed LNC were supported by tumor lysate-pulsed dendritic cells, which were donor or host derived. Optimal responses against the tumor were observed with host-tolerant, tumor-primed LNC in combination with host-derived dendritic cells. High frequencies of tumor-specific proliferating and CTLs were recorded; the survival time of tumor-bearing mice was significantly prolonged, and in >50% of mice the tumor was completely rejected. Notably, severe graft-vs-host disease was observed in reconstituted mice that received tumor-primed LNC, which had not matured in the allogeneic host. However, graft-vs-host was not aggravated after vaccination with tumor-primed, host-tolerant LNC. Thus, the LNC were tolerant toward the host, but not toward the tumor. The finding convincingly demonstrates the feasibility and efficacy of tumor vaccination after allogeneic reconstitution of the nonmyeloablatively conditioned host.     

Apurinic/apyrimidinic endonuclease 2 is necessary for normal B cell development and recovery of lymphoid progenitors after chemotherapeutic challenge

B cell development involves rapid cellular proliferation, gene rearrangements, selection, and differentiation, and it provides a powerful model to study DNA repair processes in vivo. Analysis of the contribution of the base excision repair pathway in lymphocyte development has been lacking primarily owing to the essential nature of this repair pathway. However, mice deficient for the base excision repair enzyme, apurinic/apyrimidinic endonuclease 2 (APE2) protein develop relatively normally, but they display defects in lymphopoiesis. In this study, we present an extensive analysis of bone marrow hematopoiesis in mice nullizygous for APE2 and find an inhibition of the pro-B to pre-B cell transition. We find that APE2 is not required for V(D)J recombination and that the turnover rate of APE2-deficient progenitor B cells is nearly normal. However, the production rate of pro- and pre-B cells is reduced due to a p53-dependent DNA damage response. FACS-purified progenitors from APE2-deficient mice differentiate normally in response to IL-7 in in vitro stromal cell cocultures, but pro-B cells show defective expansion. Interestingly, APE2-deficient mice show a delay in recovery of B lymphocyte progenitors following bone marrow depletion by 5-fluorouracil, with the pro-B and pre-B cell pools still markedly decreased 2 wk after a single treatment. Our data demonstrate that APE2 has an important role in providing protection from DNA damage during lymphoid development, which is independent from its ubiquitous and essential homolog APE1.     

CD4 T cell-mediated alloresistance to fully MHC-mismatched allogeneic bone marrow engraftment is dependent on CD40-CD40 ligand interactions, and lasting T cell tolerance is induced by bone marrow transplantation with initial blockade of this pathway

Costimulatory blockade can be used to promote allogeneic marrow engraftment and tolerance induction, but on its own is not 100% reliable. We sought to determine whether one or the other of the CD4 or CD8 T cell subsets of the recipient was primarily responsible for resistance to allogeneic marrow engraftment in mice receiving costimulatory blockade, and to use this information to develop a more reliable, minimal conditioning regimen for induction of mixed chimerism and transplantation tolerance. We demonstrate that a single anti-CD40 ligand mAb treatment is sufficient to completely overcome CD4 cell-mediated resistance to allogeneic marrow engraftment and rapidly induce CD4 cell tolerance, but does not reliably overcome CD8 CTL-mediated alloresistance. The data suggest that costimulation, which activates alloreactive CTL, is insufficient to activate alloreactive CD4 cells when the CD40 pathway is blocked. The addition of host CD8 T cell depletion to anti-CD40 ligand treatment reliably allows the induction of mixed chimerism and donor-specific skin graft tolerance in 3 Gy-irradiated mice receiving fully MHC-mismatched bone marrow grafts. Thus, despite the existence of multiple costimulatory pathways and pathways of APC activation, our studies demonstrate an absolute dependence on CD40-mediated events for CD4 cell-mediated rejection of allogeneic marrow. Exposure to donor bone marrow allows rapid tolerization of alloreactive CD4 cells when the CD40 pathway is blocked, leading to permanent marrow engraftment and intrathymic tolerization of T cells that develop subsequently.     

Immune tolerance to combined organ and bone marrow transplants after fractionated lymphoid irradiation involves regulatory NK T cells and clonal deletion

Immune tolerance to organ transplants has been reported in laboratory animals and in humans after nonmyeloablative conditioning of the host and infusion of donor bone marrow cells. We examined the mechanisms of immune tolerance to mouse cardiac allografts in MHC-mismatched hosts that developed mixed chimerism after posttransplant conditioning with a 2-wk course of multiple doses of lymphoid tissue irradiation, depletive anti-T cell Abs, and an infusion of donor bone marrow cells. When CD1(-/-) or J(alpha)281(-/-) hosts with markedly reduced NK T cells were used instead of wild-type hosts, then the conditioning regimen failed to induce tolerance to the heart allografts despite the development of mixed chimerism. Tolerance could be restored to the CD1(-/-) hosts by infusing enriched T cells from the bone marrow of wild-type mice containing CD1-reactive T cells but not from CD1(-/-) host-type mice. Tolerance could not be induced in either IL-4(-/-) or IL-10(-/-) hosts given the regimen despite the development of chimerism and clonal deletion of host T cells to donor MHC-Ags in the IL-10(-/-) hosts. We conclude that immune tolerance to bone marrow transplants involves clonal deletion, and tolerance to heart allografts in this model also involves regulatory CD1-reactive NK T cells.     

Allogeneic bone marrow stromal cell transplantation after cerebral hemorrhage achieves cell transdifferentiation and modulates endogenous neurogenesis

Background aimsWhen a severe neurologic lesion occurs as a consequence of intracerebral hemorrhage (ICH), there is no effective treatment available for improving the outcome. However, cell therapy has opened new perspectives on reducing neurologic sequels subsequent to this diseaseMethodsIn this study, ICH was induced by stereotactic injection of 0.5 U collagenase type IV in the striatum of adult Wistar rats, and 2 h later a group of animals (n = 48) was subjected to intracerebral injection of 2 × 10⁶ allogeneic bone marrow stromal cells (BMSC), while a control group (n = 48) received saline only. Eight animals from each group were killed at 48 h, 72 h, 7 days, 14 days, 21 days and 28 days. At these time-points, endogenous neurogenesis and survival of transplanted BMSC were studied.ResultsOur findings show that after allogeneic BMSC transplantation, donor cells can survive in the brain tissue expressing neuronal and astroglial markers. Furthermore, BMSC transplantation enhances endogenous neurogenesis and inhibits apoptosis of newborn neural cells.ConclusionsAlthough these results should be extrapolated to human disease with caution, it is obvious that cell therapy using allogeneic BMSC transplantation offers great promise for developing novel and efficacious strategies in patients suffering ICH.     

Development of immunity in human severe primary T cell deficiency following haploidentical bone marrow stem cell transplantation

Recent advances in the prevention of graft-vs-host disease (GVHD) have allowed the use of haploidentical bone marrow cells for correction of lethal genetic defects of the immune system. Sequential analyses of blood lymphocyte phenotypes and functions were done before and after transplantation of haploidentical marrow stem cells into 17 infants with severe primary T cell deficiencies. The marrow was depleted of post-thymic T cells and most other mature marrow cells by soy lectin agglutination and sheep erythrocyte rosetting. The studies were performed to define the time course and extent of appearance of immune function, and to identify factors leading to resistance to engraftment. No pretransplant immunosuppression was used. T cell function was detected between 34 and 287 days after transplantation, but a sharp rise usually occurred between 84 and 115 days, and normal function was reached between 113 and 210 days. Fifteen of the patients are alive from 6 to 41 mo post-transplantation, 12 have improved or have normal T lymphocyte function, and nine have proven T cell chimerism. Increased immunoglobulins of several isotypes have been noted in 11 patients and specific antibodies in seven patients, although B cell chimerism has been detected in only one patient. B cell function required 2 to 2.5 yr for normalization. No GVHD occurred in 14 patients, and the other three had only transient mild skin rashes. Two patients died of viral infections. Failure to engraft was correlated with some pre-transplant lymphocyte responses to mitogens and allogeneic cells (three cases), but not with the presence of pre-transplant natural killer cell function (five cases) nor with the presence of purine salvage pathway enzyme deficiencies (four cases). The latter, however, was associated with poor lymphoid function in two patients. These studies indicate that the thymic microenvironment of most infants with severe combined immunodeficiency disease is capable of differentiating donor stem cells to mature and functioning T lymphocytes which can cooperate with apparently normal host B cells for antibody production.     

Bone marrow cell dose and kinetics of recovery following allogeneic marrow transplantation in man

In 50 patients transplanted for acute or chronic leukemia we studied the correlation between the number of infused bone marrow cells/kg recipient body weight and the time needed for engraftment. Engraftment was arbitrarily defined as the first day of 1 X 10(9) leukocytes/l and of 20 X 10(9) reticulocytes/l after the posttransplantation nadir. There is a negative non-linear correlation between the duration of leukopenia following marrow transplantation and the amount of transfused nucleated cells (p = 0.01). Since the incidence of infectious or hemorrhagic complications depends directly on the duration of aplasia it is justifiable to give a maximal cell dose.     

Formation of bone marrow organs following transplantation of cell suspensions to sponges

Heterotopic transplantation of bone marrow monocellular suspensions in gelatin and collagenous sponges is highly effective in promoting osteal tissue formation and organization of hemopoietic microenvironment. This depends on the presence in the transplant of a framework for the growth of stromal mechanocytes. Formation of bone marrow organs proceeds at a greater efficacy on using trypsinized bone marrow cells versus those isolated by mechanical techniques.     

Estimating the size of the dividing stem cell pool after allogeneic bone marrow transplantation

Currently the problem of estimation of the number of pluripotent stem cells reconstituting marrow grafts following bone marrow transplantation, or studies looking at questions of clonal dominance in hematopoietic cell populations, rely on indirect measurement and a simple application of the formula for the sampling variation of a binomial proportion. This approach, from a statistical viewpoint, can be seen to be flawed. It is very easily remedied though and only requires appropriate use of variance stabilizing transformations. These lead to a very simple estimator for the number of hematopoietic stem cells involved in repopulating the marrow and require little in the way of additional calculation. We give the distribution theory for this estimator as well as simple approximations for practical application. As an illustration we rework data recently gathered to address the question as to whether or not reconstitution of marrow grafts in the clinical setting is oligoclonal.     

Urothelial cell changes due to busulfan and cyclophosphamide treatment in bone marrow transplantation

Since the administration of cyclophosphamide and busulfan can cause hemorrhagic cystitis and changes in urothelial cells, an investigation was carried out to see whether patients undergoing bone marrow transplantation (BMT) who were treated with these drugs showed such urothelial changes and whether exfoliative urinary cytology can contribute to the early diagnosis and monitoring of such changes. Morphologic and morphometric analyses were performed on cytocentrifuged, Papanicolaou-stained preparations of 700 samples from 107 patients. Various degrees of urothelial cell changes were found in 30.8% of the cases. These changes consisted mainly of a considerable increase in the size of the nucleus and of a cytoplasm that was often bizarrely shaped. Even the structures of the nucleus and the cytoplasm changed. The results of this study showed that exfoliative urinary cytology permits an early diagnosis and monitoring of urothelial cell changes related to the administration of busulfan and cyclophosphamide in connection with BMT.