Single-exon approach to non-invasive fetal RHD screening in early pregnancy: An update after 10 years' experience.

BACKGROUND AND OBJECTIVES: Anti-D prophylaxis, administered to RhD-negative women, has significantly reduced the incidence of RhD immunization. Non-invasive fetal RHD screening has been used in Stockholm for more than 10 years to identify women who will benefit from prophylaxis. The method is based on a single-exon approach and is used in early pregnancy. The aim of this study was to update the performance of the method. MATERIALS AND METHODS: The single exon assay from Devyser AB is a multiplex kit detecting both exon 4 of the RHD gene and the housekeeping gene GAPDH. Cell-free DNA was extracted from 1 ml of plasma from EDTA blood taken during early pregnancy, weeks 10-12. The genetic RHD results were compared with serological typing of newborns for a determination of sensitivity and specificity. RESULTS: In total, 4337 pregnancies were included in the study; 44 samples (1%) were inconclusive either due to maternal RHD gene variants (n = 34) or technical reasons (n = 10). Of the remaining 4293 pregnancies, a total number of nine discrepant results were found. False positive results (n = 7) were mainly (n = 4) due to RHD gene variants in the child. False-negative results were found in two cases, of which one was caused by a technical error. None of the false-negative cases was due to RHD gene variants. Overall, the sensitivity of the method was 99.93% and specificity 99.56%. CONCLUSION: The single-exon assay used in this study is correlated with high sensitivity and specificity.

The importance of graft cell composition in outcome after allogeneic stem cell transplantation in patients with malignant disease.

BACKGROUND: Graft-versus-host disease (GVHD) and relapse remain majobstacles ftreatment success in allogeneic hematopoietic stem cell transplantation (HSCT). In the present study, we evaluated the immune cell profile of the graft to outcome after HSCT. STUDY DESIGN AND METHOD: Flow cytometry data of graft cell subsets [CD34+ , CD3+ , CD19+ , CD4+ , CD8+ , CD3-CD56+ CD16+ , CD4+ CD127low CD25high ] from G-CSF primed peripheral blood stem cell (PBSC) donors was collected retrospectively from 299 patients with hematological malignancies undergoing HSCT between 2006 and 2013. The association to overall survival, transplant-related mortality (TRM), GVHD and probability of relapse was analyzed. Patients outcome from HLA-identical sibling (Sib) (n = 97) and unrelated donors (URD) (n = 202) were analyzed separately as all URD patients received anti-thymocyte globulin (ATG). RESULTS: Five-year overall survival was similar in the two cohorts (68% (Sib) vs 65% (URD)). The relapse incidence was significantly lower in the Sib cohort (24% vs 35%, P = 0.04). Multivariate analysis in the URD group revealed an association between a higher CD8+ dose and less relapse (HR, 0.94; 95%CI, 0.90-0.98; P = 0.006) as well as an association between higher CD34+ dose and both higher TRM (HR, 1.09; 95%CI, 1.02-1.20; P = 0.02) and relapse (HR, 1.09; 95%CI, 1.01-1.17; P = 0.025). The Sib analysis showed an association between a higher graft CD19+ dose and more severe acute GVHD (HR, 1,09; 95%CI, 1.03-1.15; P = 0.003) and TRM (HR, 1.09; 95%CI, 1.01-1.17; P = 0.036). In addition, a higher CD4+ graft content was associated to an increased risk for chronic GVHD (HR, 1.02; 95%CI 1.00-1.04; P = 0.06). CONCLUSION: These data indicate an importance of PBSC dongraft composition in patients with a hematological malignancy.

Eleven percent intact PGM3 in a severely immunodeficient patient with a novel splice-site mutation, a case report.

BACKGROUND: A novel immunodeficiency, frequently accompanied by high serum-IgE, and caused by mutations in the PGM3 gene was described in 2014. To date there are no unique phenotype characteristics for PGM3 deficiency. PGM3 encodes a carbohydrate-modifying enzyme, phosphoglucomutase 3. Null-mutations are quite likely lethal, and to date only missense mutations or small deletions have been reported. Such mutations frequently cause a combination of reduced enzyme activity and protein instability, complicating determination of the enzyme level needed for survival. Here we present the first patient with a homozygous splice-modifying mutation in the PGM3 gene. An A > G substitution at position c.871 + 3 (transcript NM_001199917) is causing a deletion of exon 7 in the majority of PGM3 transcripts. In addition, this case further increases the clinical phenotypes of immunodeficiency caused by PGM3 mutations. CASE PRESENTATION: We describe the symptoms of a 3-year-old girl who was severely growth retarded, had vascular malformations, extensive eczema, multiple food-allergies, and was prone to infections. Unlike the majority of reported PGM3 deficient patients she lacked skeletal dysplasia and had normal neurocognitive development. In addition to the high serum-IgE, she displayed altered T cell numbers with reduced naïve CD4+ and CD8+ T-cells, increased number of activated effector memory CD8+ T cells and aberrant T-cell functions. The patient was homozygous for a new hypomorphic, splice-modifying mutation in the PGM3 gene, causing severely reduced mRNA levels. In the patient's cells, we observed 5% intact mRNA and approximately 11% of the protein levels seen in healthy controls. Treatment with allogeneic hematopoietic stem cell therapy was planned, but unfortunately the clinical condition deteriorated with multi-organ failure, which led to her death at 3 years of age. CONCLUSIONS: There is still no specific phenotype identified that distinguishes immunodeficiency caused by PGM3 mutations from other forms of immunodeficiency. The patient described here yields new information on the phenotypic variability among these patients. In addition, since all the synthesized protein is wild-type, it is possible for the first time to estimate the enzyme activity in vivo. The results suggest that1/10 of the normal PGM3 level is sufficient for survival but that it is insufficient for accurate carbohydrate processing.

Improved overall survival for pediatric patients undergoing allogeneic hematopoietic stem cell transplantation - A comparison of the last two decades.

Pediatric protocols for allogeneic hematopoietic SCT have been altered during the last two decades. To compare the outcomes in children (<18 yr old), who underwent SCT at our center during 1992-2002 (P1) and 2003-2013 (P2). We retrospectively analyzed 188 patients in P1 and 201 patients in P2. The most significant protocol changes during P2 compared with P1 were a decrease in MAC protocols, particularly those containing TBI, an increase in RIC protocols, and altered GvHD prophylaxis. In addition, P2 had more patients with nonmalignant diagnoses (p = 0.002), more mismatched (MM) donors (p = 0.01), and more umbilical CB grafts (p = 0.03). Mesenchymal or DSCs were used for severe acute GvHD during P2. Three-yr OS in P1 was 58%, and in P2, it was 78% (p < 0.001). Improved OS was seen in both malignant disorders (51% vs. 68%; p = 0.05) and nonmalignant disorders (77% vs. 87%; p = 0.04). Multivariate analysis showed that SCT during P2 was associated with reduced mortality (HR = 0.57; p = 0.005), reduced TRM (HR = 0.57; p = 0.03), unchanged relapse rate, similar rate of GF, less chronic GvHD (HR = 0.49; p = 0.01), and more acute GvHD (HR = 1.77, p = 0.007). During recent years, OS has improved at our center, possibly reflecting the introduction of less toxic conditioning regimens and a number of other methodological developments in SCT.

T Cell Receptor Excision Circle (TREC) Monitoring after Allogeneic Stem Cell Transplantation; a Predictive Marker for Complications and Clinical Outcome.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a well-established treatment modality for a variety of malignant diseases as well as for inborn errors of the metabolism or immune system. Regardless of disease origin, good clinical effects are dependent on proper immune reconstitution. T cells are responsible for both the beneficial graft-versus-leukemia (GVL) effect against malignant cells and protection against infections. The immune recovery of T cells relies initially on peripheral expansion of mature cells from the graft and later on the differentiation and maturation from donor-derived hematopoietic stem cells. The formation of new T cells occurs in the thymus and as a byproduct, T cell receptor excision circles (TRECs) are released upon rearrangement of the T cell receptor. Detection of TRECs by PCR is a reliable method for estimating the amount of newly formed T cells in the circulation and, indirectly, for estimating thymic function. Here, we discuss the role of TREC analysis in the prediction of clinical outcome after allogeneic HSCT. Due to the pivotal role of T cell reconstitution we propose that TREC analysis should be included as a key indicator in the post-HSCT follow-up.

Antigen-induced regulation of T-cell motility, interaction with antigen-presenting cells and activation through endogenous thrombospondin-1 and its receptors.

Antigen recognition reduces T-cell motility, and induces prolonged contact with antigen-presenting cells and activation through mechanisms that remain unclear. Here we show that the T-cell receptor (TCR) and CD28 regulate T-cell motility, contact with antigen-presenting cells and activation through endogenous thrombospondin-1 (TSP-1) and its receptors low-density lipoprotein receptor-related protein 1 (LRP1), calreticulin and CD47. Antigen stimulation induced a prominent up-regulation of TSP-1 expression, and transiently increased and subsequently decreased LRP1 expression whereas calreticulin was unaffected. This antigen-induced TSP-1/LRP1 response down-regulated a motogenic mechanism directed by LRP1-mediated processing of TSP-1 in cis within the same plasma membrane while promoting contact with antigen-presenting cells and activation through cis interaction of the C-terminal domain of TSP-1 with CD47 in response to N-terminal TSP-1 triggering by calreticulin. The antigen-induced TSP-1/LRP1 response maintained a reduced but significant motility level in activated cells. Blocking CD28 co-stimulation abrogated LRP1 and TSP-1 expression and motility. TCR/CD3 ligation alone enhanced TSP-1 expression whereas CD28 ligation alone enhanced LRP1 expression. Silencing of TSP-1 inhibited T-cell conjugation to antigen-presenting cells and T helper type 1 (Th1) and Th2 cytokine responses. The Th1 response enhanced motility and increased TSP-1 expression through interleukin-2, whereas the Th2 response weakened motility and reduced LRP1 expression through interleukin-4. Ligation of the TCR and CD28 therefore elicits a TSP-1/LRP1 response that stimulates prolonged contact with antigen-presenting cells and, although down-regulating motility, maintains a significant motility level to allow serial contacts and activation. Th1 and Th2 cytokine responses differentially regulate T-cell expression of TSP-1 and LRP1 and motility.

Xeno-immunosuppressive properties of human decidual stromal cells in mouse models of alloreactivity in vitro and in vivo.

BACKGROUND AIMS: Human decidual stromal cells (hDSCs) may cure acute graft-versus-host disease (GVHD) in humans. We evaluated immunosuppression by xenogenic hDSCs in mice, both in vitro and in vivo. METHODS: hDSCs inhibited mouse lymphocyte proliferation in allo- and xeno-stimulation assays in mixed lymphocyte culture (MLC) and after mitogenic stimulation. The immunosuppressive effect of hDSCs was dose-dependent and strain-independent. Trans-well experiments showed that hDSCs needed cell-to-cell contact to be immunosuppressive. In a GVHD model, Balb/c mice were transplanted with bone marrow and splenocytes from C57BL/6 a donor. Varying doses of hDSCs (10(5)-10(6) per mouse) were infused at different time points. Recipient mice showed lower GVHD scores than untreated mice, but they did not have consistently improved survival. Histopathological investigation of liver, gastrointestinal tract and skin of animals with GVHD did not show any significant improvement from hDSC infusion. RESULTS: hDSCs were transduced with immunosuppressive genes including those encoding interleukin-10, prostaglandin-E2 receptor, indoleamine dioxygenase, interferon-γ and programmed death ligand-1. Transduced and untransduced hDSCs showed similar effects in vitro and in vivo. At a dose of 10(6)hDSCs per mouse, the majority of recipients died of embolism. CONCLUSIONS: hDSCs inhibit allo-reactivity, xeno-reactivity and mitogen-induced stimulation in mouse lymphocytes. Although the GVHD score was reduced by hDSC infusion, survival and GVHD histopathology were not improved. One reason for failure was fatal embolism.

Hypogammaglobulinemia in children after allogeneic hematopoietic stem cell transplantation: a cytokine mediated immunoglobulin isotype class switch arrest?

BACKGROUND: Hypogammaglobulinemia (hypo-IgG) is common early post-HSCT in children, occasionally necessitating long-term immunoglobulin (Ig) G replacement therapy. IgG replacement may not reduce mortality, although infectious complications are decreased PROCEDURE: Clinical data and samples from 86 children were analyzed retrospectively with the aim to identify risk factors for developing long-term hypo-IgG (i.e., requiring ≥ 3 months IgG replacement) post-HSCT and studying the underlying biology. Laboratory studies covered serum cytokines, IGHG2 genotyping and routine laboratory investigations. Results were analyzed statistically. RESULTS: Forty-eight percent of the children developed long-term hypo-IgG. These children were younger (<5 years) and had higher acute GvHD incidence, but had better overall survival (88% vs. 69%, P = 0.036). Significantly lower Ig levels post-HSCT but equal immune cell recovery were seen in patients with long-term hypo-IgG compared with those of transient or no hypo-IgG. Pre-HSCT IL-6 and -7 and post-HSCT BAFF and APRIL levels were significantly higher in the long-term hypo-IgG group. CONCLUSIONS: Findings suggests an unfavorable cytokine milieu for graft-derived immune recovery, possibly inducing Ig isotype class switch arrest. Younger age, acute GvHD, and higher pre-HSCT IL-6 levels were identified as significant risk factors for long-term hypo-IgG. Long-term hypo-IgG post-HSCT does not need to be unfavorable and could be an effect of deteriorated cytokine signaling.

Fetal membrane cells for treatment of steroid-refractory acute graft-versus-host disease.

The placenta protects the fetus from the mother's immune system. We have previously found that fetal membrane cells (FMCs) isolated from term placenta prevent alloreactivity in vitro. FMCs share many features with bone marrow-derived mesenchymal stromal cells (MSCs), which we previously introduced to treat severe acute graft-versus-host disease (GVHD). Here, we tested FMCs for treatment of steroid-refractory acute GVHD. After two passages in culture, approximately 10(9) FMCs were obtained from one single placenta, although not all cells from passage 0 and passage 1 were used for expansion. The FMCs were positive for CD29, CD44, CD73, CD90, CD105, and CD49d but were negative for hematopoietic, endothelial, and epithelial markers. Microsatellite polymorphism analysis showed that FMCs were of maternal origin. All FMCs used showed normal karyotype. Nine patients who had undergone hematopoietic stem cell transplantation (HSCT) and who had developed steroid-refractory grade III-IV acute GVHD were given 0.9-2.8 × 10(6) FMCs per kg at 15 infusions. Median age was 57 years. There was no toxicity from infusion of FMCs in eight patients. One patient had seizures after infusion. Two of eight evaluable patients had a complete response and four had a partial response, giving an overall response rate of 75%. Two patients showed no response at all. Three patients are alive from 6 to 21 months after HSCT. One patient is well and two have chronic GVHD. Thus, FMCs may be successfully used for immune modulation and tissue repair.

Rapid salvage treatment with virus-specific T cells for therapy-resistant disease.

BACKGROUND: Viral infections are major complications after allogeneic hematopoietic stem cell transplantation (HSCT). During posttransplant immunosuppression the regular T-cell control is compromised. Even if treatment strategies against infections caused by herpes viruses such as cytomegalovirus, Epstein-Barr virus, and adenovirus have improved, the mortality rate is still considerable. If primary antiviral therapy fails or cannot be tolerated, adoptive therapy with virus-specific cytotoxic T cells (CTL) can be utilized. METHODS: In this study, we used virus-specific CTLs to treat 8 patients suffering from severe viral infections after allogeneic HSCT. Using positive selection with HLA multimers and magnetic beads, we isolated CTLs from both frozen donor material as well as third-party donors within hours. RESULTS: At 90 days after CTL infusions 7 out of 8 patients were still living. CTLs infused from third-party donors were detected in 5 of 6 patients up to 76 days after infusion. No graft-versus-host disease associated with CTL infusions was observed. CONCLUSIONS: Our separation approach offers a rapid alternative for adoptive CTL therapy if primary antiviral treatment strategies fail. Because no prolonged expansion steps are needed, this method may be used for early treatment of patients suffering from life-threatening infectious complications.

Thymic function after allogeneic stem cell transplantation is dependent on graft source and predictive of long term survival.

T-cell deficiency after allogeneic stem cell transplantation (ASCT) is common and has major impact on clinical outcome. In this retrospective study 210 patients were analyzed with regards to levels of T-cell receptor excision circles (TRECs) during the first 24 months after transplantation. We could for the first time show a significant correlation between the use of bone marrow grafts and higher TREC levels >6 months post-ASCT (p<0.001). Treatment with anti-thymocyte globulin was correlated with lower TREC levels ≤6 months post-ASCT (p<0.001). Patients with TREC levels above median at 3 months had a superior overall survival, 80% vs. 56% (p=0.002), and lower transplantation-related mortality, 7% vs. 21% (p=0.01). We conclude that graft source and conditioning regimen may have a significant effect on T-cell reconstitution after ASCT and can thus affect outcome. These results strongly support the use of TREC measurement as part of the standard repertoire of immunological monitoring after ASCT.

Improved survival after allogeneic hematopoietic stem cell transplantation in recent years. A single-center study.

We analyzed the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) over the past 2 decades. Between 1992 and 2009, 953 patients were treated with HSCT, mainly for a hematologic malignancy. They were divided according to 4 different time periods of treatment: 1992 to 1995, 1996 to 2000, 2001 to 2005, and 2006 to 2009. Over the years, many factors have changed considerably regarding patient age, diagnosis, disease stage, type of donor, stem cell source, genomic HLA typing, cell dose, type of conditioning, treatment of infections, use of granulocyte-colony stimulating factor (G-CSF), use of mesenchymal stem cells, use of cytotoxic T cells, and home care. When we compared the last period (2006-2009) with earlier periods, we found slower neutrophil engraftment, a higher incidence of acute graft-versus-host disease (aGVHD) of grades II-IV, and less chronic GVHD (cGHVD). The incidence of relapse was unchanged over the 4 periods (22%-25%). Overall survival (OS) and transplant-related mortality (TRM) improved significantly in the more recent periods, with the best results during the last period (2006-2009) and a 100-day TRM of 5.5%. This improvement was also apparent in a multivariate analysis. When correcting for differences between the 4 groups, the hazard ratio for mortality in the last period was 0.59 (95% confidence interval [CI]: 0.44-0.79; P < .001) and for TRM it was 0.63 (CI: 0.43-0.92; P = .02). This study shows that the combined efforts to improve outcome after HSCT have been very effective. Even though we now treat older patients with more advanced disease and use more alternative HLA nonidentical donors, OS and TRM have improved. The problem of relapse still has to be remedied. Thus, several different developments together have resulted in significantly lower TRM and improved survival after HSCT over the last few years.

Sirolimus and tacrolimus as immune prophylaxis compared to cyclosporine with or without methotrexate in patients undergoing allogeneic haematopoietic stem cell transplantation for non-malignant disorders.

For prevention of graft-versus-host disease (GVHD), treatment of 24 haematopoietic stem cell transplantation (HSCT) patients with sirolimus and tacrolimus was compared to treatment of matched controls with cyclosporine-based regimens. The patients mainly had non-malignant disorders. Two-thirds of the donors were unrelated, and bone marrow was the most common source of stem cells. Rejection occurred in four patients in the sirolimus group and three in the control group. Donor chimerism for CD3, CD19 and CD33 was similar in the two groups. The cumulative incidence of grade II acute GVHD was 22% in the sirolimus patients and 17% in the controls (P=0.78). No patients developed acute GVHD of grades III-IV. The cumulative incidence of chronic GVHD was 25% and 37% in the two groups, respectively (P=0.40). Two patients in the sirolimus group developed Epstein-Barr virus lymphoma, and none in the controls. Side effects and toxicity were similar in the two groups. There was no transplant-related mortality at 5 yr in the sirolimus group, as opposed to 8% in the controls (P=0.47). Survival at 5 yr was 95% and 92%. Thus, sirolimus combined with tacrolimus is a promising immunosuppressive regimen in HSCT, also for non-malignancies, and its efficacy should be confirmed in prospective clinical trials.

A novel haplo-identical adoptive CTL therapy as a treatment for EBV-associated lymphoma after stem cell transplantation.

Epstein-Barr virus (EBV)-related malignancies such as post-transplant lymphoproliferative disease (PTLD) are severe complications after allogeneic stem cell transplantation and solid-organ transplantation. In immunosuppressed transplant recipients, the activity of EBV-specific CTLs are often decreased or absent which leads to an increased risk of developing PTLD. If primary treatment modalities of PTLD fail, the most efficient way of treating the malignancy is adopting EBV-specific CTLs from the donor or, more recently, third-party donors. However, both are time consuming and expensive and often it is too late to administer cells to the patient. We have for the first time, using a rapid isolation protocol of EBV-specific T cells, treated and cured a patient suffering from PTLD with multiple-associated tissue lesions, using her haplo-identical mother as a donor. This treatment approach paves way for a new possibility to within-days treat patients with life-threatening EBV-associated malignancies.

Mesenchymal stem cells exert differential effects on alloantigen and virus-specific T-cell responses.

Mesenchymal stem cells (MSCs) suppress alloantigen-induced T-cell functions in vitro and infusion of third-party MSCs seems to be a promising therapy for graft-versus-host disease (GVHD). Little is known about the specificity of immunosuppression by MSCs, in particular the effect on immunity to pathogens. We have studied how MSCs affect T-cell responses specific to Epstein-Barr virus (EBV) and cytomegalovirus (CMV). We found that EBV- and CMV-induced proliferation and interferon-gamma (IFN-gamma) production from peripheral blood mononuclear cells (PBMCs) was less affected by third-party MSCs than the response to alloantigen and that MSCs had no effect on expansion of EBV and CMV pentamer-specific T cells. Established EBV-specific cytotoxic T cells (CTL) or CMV-CTL cultured with MSCs retained the ability to proliferate and produce IFN-gamma in response to their cognate antigen and to kill virally infected targets. Finally, PBMCs from 2 patients who received MSCs for acute GVHD showed persistence of CMV-specific T cells and retained IFN-gamma response to CMV after MSC infusion. In summary, MSCs have little effect on T-cell responses to EBV and CMV, which contrasts to their strong immunosuppressive effects on alloreactive T cells. These data have major implications for immunotherapy of GVHD with MSCs and suggest that the effector functions of virus-specific T cells may be retained after MSC infusion.

Maternal microchimerism in juvenile tonsils and adenoids.

During pregnancy small amounts of cells pass between the mother and the fetus, and this transfer may give rise to a chimeric state that persist for years in both individuals. Both fetal and maternal microchimerism (MMc) have been associated with different autoimmune disorders. Information about MMc in tissues of healthy individuals is sparse but is important when looking for maternal cells within affected tissues of certain diseases. The aim of this study was to investigate the occurrence of maternal cells in tonsils and adenoids of 20 healthy children between the ages of 2 and 15 years. All the children underwent surgery because of recurrent tonsillitis or respiratory obstruction. MMc was detected using an RT-PCR assay based on differences in gene polymorphisms between mother and child. We found maternal cells in the tonsils and/or adenoids in four of 20 children. This frequency is less than the frequency of maternal cells found in the peripheral blood of healthy adults but in agreement with the previously reported frequency of maternal chimerism in control tissues

Evidence for no relevance of anti-major histocompatibility complex class I-related chain a antibodies in liver transplantation.

The polymorphic major histocompatibility complex class I-related chain A (MICA) antigen is being increasingly recognized as a potential target molecule for immune cells during allograft rejection. Here we studied whether MICA is a target antigen for antibodies in liver transplant patients. Eighty-four patients were investigated for the presence of MICA antibodies before and after liver transplantation with MICA-transfected cells and flow cytometry. MICA typing was performed by polymerase chain reaction. Expression of MICA in liver cells was determined by reverse-transcription polymerase chain reaction, Western blotting, and flow cytometry. Liver biopsy specimens from liver transplant patients were examined for MICA expression. A total of 22 of 84 (26%) patients had MICA antibodies either pre-transplant (8/84, 9.5%) or post-transplant (14/84, 17%). No correlation between rejection frequencies (14/22, 63%) or other clinical parameters was observed in patients with MICA antibody versus those without MICA antibody (29/62, 47% P = not significant). We found weak messenger RNA expression for MICA in liver cells but no protein or cell surface expression. In addition, no MICA expression in liver biopsy sections from liver transplant patients was observed at any time point, including rejections. Thus, our preliminary results demonstrate no causal relationship between the presence of MICA antibodies and liver allograft rejections. Therefore, it is likely that MICA may not be an important target antigen during liver allograft rejections.

Maternal microchimerism in human fetal tissues.

OBJECTIVE: The aim of the present study was to analyze the presence of maternal cells in human fetal tissues in the second trimester. STUDY DESIGN: Tissues from 11 second-trimester fetuses terminated because of social reasons or because of malformations and/or trisomy were investigated. By cell sorting and polymerase chain reaction amplification, we studied the presence of maternal CD3+, CD19+, CD34+, and CD45+ in different fetal tissues and in placenta. RESULTS: In the group of fetuses with normal karyotype and normal autopsy findings, 4 of 5 fetuses were positive for maternal microchimerism. In the group in which the fetuses were diagnosed with trisomy 21 and/or malformations, we found cells of maternal origin in 3 of 6 fetuses. CONCLUSION: The results from this study indicate that maternal microchimerism is a common phenomenon in the second-trimester fetuses. Maternal cells of lymphoid and myeloid lineages and hematopoietic progenitors are widely distributed in the second-trimester fetuses.

Unrelated versus related allogeneic stem cell transplantation after reduced intensity conditioning.

BACKGROUND: The use of reduced intensity conditioning (RIC) regimens in allogeneic hematopoietic stem cell transplantation (HSCT) has increased over the past five years. PATIENTS: In this study, involving 137 patients, we compared the outcome after RIC in patients receiving grafts from matched unrelated donors (MUD; n=74) and sibling donors (n=63). The MUD and sibling groups were comparable regarding diagnosis, including solid tumors and hematological malignancies, and conditioning regimens. RESULTS: Engraftment was successful in most patients (88%), with no significant difference between MUD and sibling transplants. Cytomegalovirus (CMV) infection was more common in the MUD group (65%) than in the sibling group (46%) (P=0.04). No difference in severe acute graft-versus-host disease (GVHD) was found between the groups. However, the incidence of chronic GVHD was higher after sibling transplants. This was probably due to higher donor age in this group, since this was the only significant risk factor for chronic GVHD in multivariate analysis. The incidence of transplant related mortality (TRM) was significantly higher after MUD transplantation (40%) than after sibling transplantation (16%) (P<0.01). Because relapse/disease progression was more common after sibling transplantation, there was no significant difference in overall survival between the two groups. CONCLUSION: Using unrelated donors after RIC is feasible, but it resulted in more CMV infection and increased transplant-related mortality. Survival was comparable to that of sibling transplants.

Mesenchymal stem cells for treatment of therapy-resistant graft-versus-host disease.

BACKGROUND: Mesenchymal stem cells (MSC) have immunomodulatory effects. The aim was to study the effect of MSC infusion on graft-versus-host disease (GVHD). METHODS: We gave MSC to eight patients with steroid-refractory grades III-IV GVHD and one who had extensive chronic GVHD. The MSC dose was median 1.0 (range 0.7 to 9)x10(6)/kg. No acute side-effects occurred after the MSC infusions. Six patients were treated once and three patients twice. Two patients received MSC from HLA-identical siblings, six from haplo-identical family donors and four from unrelated mismatched donors. RESULTS: Acute GVHD disappeared completely in six of eight patients. One of these developed cytomegalovirus gastroenteritis. Complete resolution was seen in gut (6), liver (1) and skin (1). Two died soon after MSC treatment with no obvious response. One of them had MSC donor DNA in the colon and a lymph node. Five patients are still alive between 2 months and 3 years after the transplantation. Their survival rate was significantly better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSC during the same period (P = 0.03). One patient treated for extensive chronic GVHD showed a transient response in the liver, but not in the skin and he died of Epstein-Barr virus lymphoma. CONCLUSION: MSC is a very promising treatment for severe steroid-resistant acute GVHD.