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Cardiomyocytes are a stable cell population with only limited potential for renewal after injury. Tissue regeneration may be due to infiltration of stem cells, which differentiate into cardiomyocytes. We have analysed the influx of stem cells in the heart of patients who received either a gender-mismatched BMT (male donor to female recipient) or a gender-mismatched cardiac transplant (HTX; female donor to male recipient). The proportion of infiltrating cells was determined by Y-chromosome in situ hybridization combined with immunohistochemical cell characterization. In BM transplanted patients and in cardiac allotransplant recipients, cardiomyocytes of apparent BM origin were detected. The proportions were similar in both groups and amounted up to 1% of all cardiomyocytes. The number of stem cell-derived cardiomyocytes did not alter significantly in time, but were relatively high in cases where large numbers of BM-derived Y-chromosome-positive infiltrating inflammatory cells were present. The number of Y-chromosome-positive endothelial cells was small and present only in small blood vessels. The number of BM-derived cardiomyocytes in both BMT and HTX is not significantly different between the two types of transplantation and is at most 1%.

The role of chimerism analysis as a prognostic indicator of relapse after hematopoietic stem cell transplantation (SCT) is controversial. We monitored chimerism status by short tandem repeat-based polymerase chain reaction (PCR) in T- and non-T-cell subsets and retrospectively evaluated clinical outcome in 96 patients with acute myeloid leukemia after myeloablative (MA) or reduced-intensity conditioning SCT. Fifty-six percent of 80 patients in the MA group demonstrated complete donor chimerism (CC) at all time points, whereas 6% had decreasing mixed chimerism (MC), 8% stable MC, 25% increasing MC and 3% increasing and decreasing MC. In 16 RIC patients, these percentages were 12, 50, 6, 6 and 19, respectively, together with 6% nonengraftment. Forty-three out of 96 patients experienced relapse. The last chimerism evaluation before relapse revealed increasing MC in only eight patients. In samples taken between 1 and 6 months post SCT, CC/decreasing MC was significantly related with a lower risk of relapse (31 versus 83%, P<0.000) and mortality (38 versus 83%, P<0.000) than with MC/increasing MC. However, the development of relapse was very rapid. Only very frequent monitoring of chimerism status by highly sensitive methods might identify impending relapse and allow early immunological intervention.

Objective: Suicide gene therapy for leukemia aims to benefit from T cells in the BM graft, by reducing the probability of leukemia relapse (GVL), while severe complications of graft-vs-host disease (GVHD) may be avoided. In an allogeneic rat BMT model we defined the conditions to induce a lethal GVHD with HSV-Tk gene-transduced T cells. We studied the feasibility to rescue the animals by conditional elimination of the T cells with ganciclovir (GCV) treatment., Methods: Allogeneic T cells transduced with a retroviral vector encoding the HSV-Tk suicide gene were added in varying numbers to a BM graft. Expression of HSV-Tk strongly increases the cytolytic effect of GCV, thereby allowing elimination of overreactive T cells at will. Various experimental conditions were tested in the rat model., Results: A relation between the number of HSV-Tk(+) T cells added to the BM graft and GVHD development was found. GCV treatment resulted in selective HSV-Tk(+) T-cell elimination in blood and tissues but not in abrogation of GVHD due to persistence of HSV-Tk(-) T cells. T cells in unmanipulated rat BM normally have a low risk to induce GVH but when they are administered in combination with high numbers of HSV-Tk(+) T cells there is an apparent increase in their GVH-inducing potential. When HSV-Tk(+) T cells are added to T cell-depleted BM a consequently developing GVH can be controlled by GCV treatment with 60-70% of the animals surviving., Conclusions: We show that T cell-mediated suicide gene therapy within the context of allo-BMT can be applied with success. The apparent limitation in the number of transduced as well as nontransduced T cells that can be safely added to the BM graft should be taken into consideration when designing human suicide gene therapy protocols.

In this study, we analysed the chimaeric status of peripheral blood leucocytes (PBLs) in recipients of allogeneic bone marrow transplantation (BMT) with the use of short tandem repeat (STR) microsatellite markers for monitoring the efficacy of BMT and donor leucocyte infusions (DLIs). A set of four STR markers was used with a highly discrimative capacity between individuals. STRs were detected by polymerase chain reaction (PCR) and were analysed by gene scanning (STR-GS). Between June 1990 and December 1998, 52 patients treated with BMT for chronic myeloid leukaemia (CML) were analysed. Seventeen patients relapsed after BMT and two patients never achieved remission after BMT. Fourteen of the 17 patients achieved a complete donor chimaerism after BMT, as detected by the presence of only donor STR-GS fragments, and in three cases a weak recipient STR-GS signal remained persistently detectable after BMT. A reappearance or increase of recipient STR-GS signals was indicative of relapse, which was mostly detected by STR-GS several months before relapse was diagnosed clinically. Nineteen patients were treated with DLI for reappearance of CML after BMT which resulted in complete remission in 17 patients, concordant with the disappearance of recipient STR-GS signals. More importantly, DLI treatment could be guided based upon the STR-GS data, which prevented unnecessary extra DLI courses that could cause toxicity. This study indicates that STR-GS is an effective and reliable method for monitoring BMT recipients.

We evaluated the efficacy and toxicity of different doses of donor T cells given with donor leukocyte infusions (DLI) as treatment for relapse of various hematologic malignancies after allogeneic bone marrow transplantation (BMT). We also studied whether DLI treatment was more effective if circulating T cells were exclusively of donor origin (complete donor T cell chimeras) as compared with T cells originating from both donor and recipient (mixed T cell chimeras). Twenty-eight patients were studied of whom 24 had a complete donor T cell chimerism. The malignancies were as follows: chronic myeloid leukemia (CML) in chronic phase (CP) (n = 9); more advanced CML (n = 5); multiple myeloma (MM) (n = 5); acute leukemia (AL) (n = 9). T cell doses varied from 0.1 x 10(7) to 33 x 10(7) T cells/kg. Eight patients received two to four DLI courses because they failed to respond to one course. Thirteen of 14 patients with CML, including four patients with more advanced CML, achieved complete remission (CR). All five patients with MM responded, including three CRs. Six patients (three with CML, three with MM) responded only after two to four DLI courses. Patients with CML-CP were likely to respond to as few as 1 x 10(7) T cells/kg whereas patients with MM generally responded when they received > or = 10 x 10(7) T cells/kg. However, despite the infusion of high T cell doses (up to 32 x 10(7) T cells/kg), practically all patients with AL failed to respond. The likelihood of response was strongly related to the occurrence of graft-versus-host disease (GVHD) in patients with CML and MM (P = 0.0002), although GVHD was not helpful for patients with AL. Higher T cell doses (> or = 10 x 10(7)/kg) induced serious GVHD (n = 17) and marrow aplasia (n = 5), and GVHD was directly or indirectly the cause of death for six patients. Finally, there were no obvious differences in responses between complete donor T cell chimeras and mixed T cell chimeras.

T cell depletion of the bone marrow graft, the most effective method to prevent severe graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), has resulted in approximately three times more relapses of the disease post-transplant than after non-T cell-depleted BMT. It has been hypothesized that this is caused by the development of mixed T cell chimerism, often observed after T cell depleted BMT, whereas non-T cell-depleted BMT generally results in complete donor T cell chimerism. In order to find an approach of T cell depletion which may avoid the high relapse rate but prevent severe GVHD, we gave marrow recipients a partial T cell-depleted marrow graft containing 1 x 10(5) donor T cells/kg recipient's weight. To investigate whether our approach results in complete donor T cell chimerism, we analyzed post-transplant the origin of purified T cells in 56 patients with hematologic malignancies, including 15 patients at the time they relapsed. The T cells were studied for being of host or donor origin by amplification of four loci of variable number of tandem repeats (VNTR) by PCR. From 6 months post-BMT, all 45 patients who could be analyzed in remission (five had died and six had relapsed within 6 months) had T cells that were exclusively of donor origin. Furthermore, the T cells of 15 patients who had relapsed post-BMT were also exclusively of donor origin. Severe GVHD was never observed. Thus, this approach seems to combine the favorable aspects of both T cell-depleted and non-T cell-depleted BMT.

Infection with human cytomegalovirus (HCMV) after allogeneic bone marrow transplantation (BMT) was studied in 12 HCMV seronegative recipients of marrow from seropositive donors by weekly monitoring of cultures, expression of HCMV antigenemia (pp65) in granulocytes, polymerase chain reaction (PCR) on HCMV-DNA in granulocytes and IgM and IgG anti-HCMV antibodies. Eight patients remained negative in all tests as did 33 HCMV seronegative recipients of marrow from seronegative donors. In four patients, a transient expression of HCMV antigen pp65 in granulocytes from peripheral blood, together with a positive PCR on HCMV-DNA from the same samples were found without positive cultures, seroconversion or expression of other HCMV antigens in granulocytes. The data indicate the presence of an abortive HCMV infection in these four patients.

The antigenemia assay, polymerase chain reaction (PCR) and rapid culture technique on buffy coat cells (DEAFF test) were used to monitor 37 cytomegalovirus (CMV) seropositive bone marrow transplant (BMT) recipients for active CMV infection during the first 3 months after BMT. The antigen assay and PCR demonstrated a comparable sensitivity for the detection of CMV in blood: discordant results were only obtained in the early or late phase of infection when the viral load was low. The antigen assay was more sensitive than the DEAFF test. Only 12 out of 40 antigen-positive samples yielded a positive result with DEAFF test, whereas viremia without antigenemia was never found. The discordance between these two tests increased further during antiviral therapy with ganciclovir. A correlation was observed between the duration of antigenemia during treatment and the recurrence of systemic CMV reactivation. Ten out of 11 patients with antigen-positive leukocytes present for more than 1 week after starting the treatment subsequently exhibited a relapse of active infection, whereas only three out of nine patients who resolved their antigenemia within 1 week did so. In conclusion, the antigen assay and PCR are useful techniques for detection of CMV infection in BMT patients. Test results obtained during therapy give reliable information regarding the viral load and the possibility of recurrence of antigenemia, and can be taken into account when prolonged administration of ganciclovir is considered.

Autopsy tissues of 19 patients with complications after bone marrow transplantation (BMT) were analysed for the presence of cytomegalovirus (CMV) using histochemical methods. CMV antigens were detected by antibodies to CMV Immediate Early Antigen (IEA) or CMV Late Antigen (LA). CMV-DNA was detected by DNA in situ hybridization (DISH). IEA was detected in one or more tissues in 79% of 14 patients from whom frozen tissue was available. CMV-DNA was detected on paraffin sections in 84% of all 19 patients. CMV components were present in all organs studied; the highest incidence was found in lung, gastrointestinal tract and kidney. In histology, only 37% of patients showed signs of CMV infection by the presence of cytomegalic cells with nuclear inclusions (or so called "owl eye cells"). In tissue culture, only 33% of 15 patients were CMV positive. Serologically, 68% of all patients had active CMV infection, as indicated by a rise in antibody titres. We conclude that the quick detection of CMV IEA and CMV-DNA has a high sensitivity and predictive value, which is comparable to or exceeds the serological detection of CMV.

We studied the effect of cytomegalovirus immunoglobulin alone, or combined with ganciclovir, on the outcome of biopsy proven cytomegalovirus pneumonia after bone marrow transplantation. Treatment with cytomegalovirus immunoglobulin alone had no effect on the cytomegalovirus nor on clinical outcome. The combined treatment of cytomegalovirus immunoglobulin with ganciclovir suppressed the cytomegalovirus but all patients died because of ongoing pulmonary deterioration. These results may suggest that this combined treatment has limited value on the outcome of an established cytomegalovirus pneumonia after marrow transplantation.

B lymphoid cells with monotypic immunoglobulin (Ig) expression, i.e., a single Ig heavy chain isotype combined with a single light chain type, were observed in five of nine patients after allogeneic bone marrow transplantation. Three patients exhibited plasmacellular hyperplasia of lymph nodes and spleen; in one case the monotypic cells were immunoblasts in a lymph node, and one patient suffered from an immunoblastic lymphoma along the gastrointestinal tract. We present the clinical history and the detailed (immuno) histologic analysis of lymphoid tissue from three patients. In DNA analysis of lymph node using DNA probes specific for the JH-gene segment of Ig heavy chains and for light chains, bands indicative of single Ig gene rearrangements were not observed. Thus, the monotypic B lymphoid elements represent polyclonal B cell expansion.