Abstract: Mannose-binding lectin (MBL) is an activator of the lectin pathway of the C' system and hence an important component of the innate immune system. Although reports are conflicting, MBL deficiency has been reported to influence the infection susceptibility in hematology/oncology patients or recipients of allogeneic hematopoietic SCT (HSCT). MBL levels and the occurrence of infections were retrospectively analyzed in 98 pediatric HSCT patients. MBL deficiency in recipients was not corrected by HSCT using a donor with normal MBL production. In addition, low serum MBL concentrations were not associated with increased susceptibility to any type of infections post-HSCT in this cohort of pediatric HSCT recipients.
Abstract: A nine-yr-old boy with EPP suffered from severe skin burns and liver failure caused by progressive cholestasis and fibrosis. OLT was performed without major complications. Four months following liver transplantation he underwent parental haploidentical HSCT. The myeloablative conditioning regimen was relatively well tolerated and hematological engraftment was rapid (on day 10). Protoporphyrin concentrations returned to normal following HSCT. However, immune recovery was significantly delayed. Varicella zoster virus reactivation resulted in impaired vision, prolonged hospitalization and eventually in multiorgan failure and death. Sequential liver and haploidentical HSCT proved feasible though a high risk procedure in this EPP patient. The management of post-IST after these combined transplantations remains a challenge and needs to be further established.
Abstract: The transplant policy for unrelated donor (UD) BMT at Leiden Paediatrics' SCT-Centre consisted of the use of (1) fully HLA-matched donors or, if not available, HLA-class I matched and/or cytotoxic T-lymphocyte precursor (CTLp)-negative donors and (2) protective isolation of the recipient and antimicrobial suppression of his/her gut microflora to prevent infections and acute GVHD. Engraftment, GVHD, relapse in the case of malignancy and survival were studied retrospectively in 126 evaluable children, transplanted between 1988 and 2005. In addition to the effect of HLA-matching, that of other transplant-relevant variables on the outcome was also studied. Actuarial OS was 65% and the EFS was 59%, 13% graft failures occurred and 7.5% > or =grade II acute GVHD. HLA-class II mismatches combined with HLA-class I matches resulted in a superior OS of 92%, as did a negative vs positive CTLp test, that is, 65 vs 33%. Analysis of other variables showed a poorer OS in patients > or =10 yrs vs <10 yrs, that is, 54 vs 73%, and in male recipients of a female donor graft, that is, 53 vs 69% for other combinations. UD-BMT can be optimized by permitting HLA-class I-matched and/or CTLp-negative donors, and probably by choosing male donors for male recipients.
Abstract: Hematopoietic stem cell transplantation remains the only treatment for most patients with severe combined immunodeficiencies (SCIDs) or other primary immunodeficiencies (non-SCID PIDs).
Abstract: We describe a 15-year-old patient with X-linked agammaglobulinemia who developed malabsorption and bacteremia due to infection of Helicobacter pylori and Campylobacter jejuni. The Campylobacter bacteremia was only recognized after subculturing of blood culture bottles that failed to signal in the automated system. After 2Â weeks of treatment with meropenem and erythromycin for 4Â weeks, the patient developed a relapse of bacteremia 10Â months later with a high level erythromycin resistant C. jejuni. Sequencing revealed an A2058C mutation in the 23 S rRNA gene associated with this resistance. Treatment with doxycycline for 4Â weeks finally resulted in complete eradication. This case report illustrates the importance for physicians to use adapted culture methods and adequate prolonged therapy in patients with an immunodeficiency. A summary of published case reports and series of patients with hypogammaglobulinemia or agammaglobulinemia with Campylobacter or Helicobacter bacteremia is given.
Abstract: Cartilage-hair hypoplasia (CHH) is a rare autosomal recessive disease caused by mutations in the RMRP gene. Beside dwarfism, CHH has a wide spectrum of clinical manifestations including variable grades of combined immunodeficiency, autoimmune complications, and malignancies. Previous reports in single CHH patients with significant immunodeficiencies have demonstrated that allogeneic hematopoietic stem cell transplantation (HSCT) is an effective treatment for the severe immunodeficiency, while growth failure remains unaffected. Because long-term experience in larger cohorts of CHH patients after HSCT is currently unreported, we performed a European collaborative survey reporting on 16 patients with CHH and immunodeficiency who underwent HSCT. Immune dysregulation, lymphoid malignancy, and autoimmunity were important features in this cohort. Thirteen patients were transplanted in early childhood ( approximately 2.5 years). The other 3 patients were transplanted at adolescent age. Of 16 patients, 10 (62.5%) were long-term survivors, with a median follow-up of 7 years. T-lymphocyte numbers and function have normalized, and autoimmunity has resolved in all survivors. HSCT should be considered in CHH patients with severe immunodeficiency/autoimmunity, before the development of severe infections, major organ damage, or malignancy might jeopardize the outcome of HSCT and the quality of life in these patients.
Abstract: Allogeneic pediatric stem cell transplantation (SCT) is a very intensive treatment with a high mortality and morbidity. The objectives of this study were to assess the (1) self- and proxy-reported health-related quality of life (HRQoL) compared to a norm group, (2) levels of parenting stress compared to a norm group, (3) differences in HRQoL and parenting stress pre- and post-SCT, and (4) effect of child age and parenting stress on self- and proxy-reported HRQoL pre- and post-SCT.
Abstract: Busulfan, combined with therapeutic drug monitoring-guided dosing, is associated with higher event-free survival (EFS) rates due to fewer graft failures/relapses and lower toxicity. The optimal target area under the curve (AUC) and dosing schedule of intravenous busulfan in children undergoing hematopoietic stem cell transplantation (HSCT) remain unclear, however. We conducted a retrospective analysis of the association between busulfan exposure and clinical outcome in 102 children age 0.2 to 21 years who received busulfan 1 or 4 times daily before undergoing HSCT (46 malignant and 56 nonmalignant indications). EFS and overall survival after a median of 2 years of follow-up were 68% and 72%, respectively. EFS was optimal when the exposure of busulfan (AUC) was 78 mg x h/L (95% confidence interval=74 to 82 mg x h/L). Acute graft-versus-host disease (aGVHD) grade II-IV occurred more frequently with greater busulfan exposure. The addition of melphalan was an independent risk factor; melphalan use combined with high busulfan exposure (AUC >74 mg x h/L) was associated with high incidences of aGVHD (58%), veno-occlusive disease (66%), and mucositis grade III-IV (26%). Dosing frequency (1 or 4 times daily) was not related to any outcome. In conclusion, dose targeting of busulfan to a narrow therapeutic range was found to increase EFS in children. Adding melphalan to optimal busulfan exposure is associated with a high incidence of toxicity.
Abstract: The Wiskott-Aldrich syndrome (WAS) protein (WASp) is a regulator of actin cytoskeleton in hematopoietic cells. Mutations of the WASp gene cause WAS. Although WASp is involved in various immune cell functions, its role in invariant natural killer T (iNKT) cells has never been investigated. Defects of iNKT cells could indeed contribute to several WAS features, such as recurrent infections and high tumor incidence. We found a profound reduction of circulating iNKT cells in WAS patients, directly correlating with the severity of clinical phenotype. To better characterize iNKT cell defect in the absence of WASp, we analyzed was(-/-) mice. iNKT cell numbers were significantly reduced in the thymus and periphery of was(-/-) mice as compared with wild-type controls. Moreover analysis of was(-/-) iNKT cell maturation revealed a complete arrest at the CD44(+) NK1.1(-) intermediate stage. Notably, generation of BM chimeras demonstrated a was(-/-) iNKT cell-autonomous developmental defect. was(-/-) iNKT cells were also functionally impaired, as suggested by the reduced secretion of interleukin 4 and interferon gamma upon in vivo activation. Altogether, these results demonstrate the relevance of WASp in integrating signals critical for development and functional differentiation of iNKT cells and suggest that defects in these cells may play a role in WAS pathology.
Abstract: Human bocavirus (HBoV) is frequently detected in young children with respiratory symptoms. However, the prevalence and pathogenicity of HBoV in immunocompromised patients are largely unknown. This report describes a case of life-threatening hypovolemic shock due to diarrhea associated with disseminated HBoV infection in an immunocompromised child.
Abstract: Two adolescents, on immunosuppressive therapy for graft-versus-host disease, developed hemophagocytic lymphohistiocytosis (HLH) after varicella zoster virus (VZV) reactivation. In the absence of dermatome restricted characteristic skin lesions, VZV reactivation was not immediately recognized and treatment with acyclovir was delayed. The first patient developed optical neuritis and died 2 months after the VZV episode due to massive intracranial hemorrhage. The second patient presented with severe abdominal pain and pancreatitis, followed by atypical skin eruptions, which prompted a faster diagnosis. Both patients recovered from their HLH, the first patient being successfully treated with immunosuppressive agents and the second with VZV treatment only. These two cases demonstrate the difficulties in recognizing VZV reactivation, and in order to start adequate and timely treatment, the need to consider VZV as a possible cause of HLH in severely immunocompromised patients.
Abstract: Exposure to environmental factors during fetal life and infancy is thought to play an important role in the early development of innate and adaptive immunity. The immunological relationship between mother and infant and the effect that environmental exposures have during pregnancy and early childhood have not been studied extensively. Here the production of cytokines was measured in 146 pairs of mothers and their 2- month-old infants. The effect of place of residence, socio-economic variables, parasitic infections as well as maternal and child characteristics on measured cytokine production was determined. Mothers producing high levels of IL-10, IFN-gamma and IL-5 were more likely to have infants who also produced high levels of these cytokines either spontaneously (OR 2.6(95%CI 1.2-5.4), OR 2.9(CI 1.3-6.6), OR 11.2(CI 4.6-27.2), respectively) or in response to PHA (IL-10: OR 3.0(CI 1.4-6.6), IFN-gamma: OR 2.0(CI 1.0-4.2), respectively) even after adjustment for potential confounding variables. This was not the case for TNF-alpha. In response to LPS, place of residence was a strong determinant of infant IL-10 (OR 0.2(CI 0.1-0.9)) and TNF-alpha (OR 0.3(CI 0.1-0.9)) production. Maternal protozoan infections was independently associated with reduced infant IL10 in response to PHA and to LPS as well as reduced TNF-alpha and IFN-gamma in response to PHA. These results indicate strong relationship between maternal and infant's cellular immune responses even after taking into account many environmental influences that could affect infant's response directly or indirectly through uterine microenvironment. However, place of residence and intestinal infections may still directly affect the immune responses of the infant. Taken together, the study provides evidence for imprinted cytokine responses of an infant which may have implications for their reaction to incoming antigens, warranting further investigation into the role that genetics or epigenetics play in shaping the cytokine response by an infant to self or external antigens.
Abstract: Wiskott-Aldrich syndrome (WAS) is a rare X-linked immunodeficiency with microthrombocytopenia, eczema, recurrent infections, autoimmune disorders, and malignancies that are life-threatening in the majority of patients. In this long-term, retrospective, multicenter study, we analyzed events that occurred in 96 WAS patients who received transplants between 1979 and 2001 who survived at least 2 years following hematopoietic stem-cell transplantation (HSCT). Events included chronic graft-versus-host disease (cGVHD), autoimmunity, infections, and sequelae of before or after HSCT complications. Three patients (3%) died 2.1 to 21 years following HSCT. Overall 7-year event-free survival rate was 75%. It was lower in recipients of mismatched related donors, also in relation with an older age at HSCT and disease severity. The most striking finding was the observation of cGVHD-independent autoimmunity in 20% of patients strongly associated with a mixed/split chimerism status (P < .001), suggesting that residual-host lymphocytes can mediate autoimmune disease despite the coexistence of donor lymphocytes. Infectious complications (6%) related to splenectomy were also significant and may warrant a more restrictive approach to performing splenectomy in WAS patients. Overall, this study provides the basis for a prospective, standardized, and more in-depth detailed analysis of chimerism and events in long-term follow-up of WAS patients who receive transplants to design better-adapted therapeutic strategies.
Abstract: High busulfan exposure is associated with increased toxicity, for example veno-occlusive disease, whereas low exposure results in less efficacy such as lower engraftment rates. Despite adjusting dose to body weight, interindividual variability in pharmacokinetics and thus drug exposure remained rather large. In this report, the contribution of genetic polymorphisms in the glutathione-S-transferases (GST) isozymes GSTA1, GSTM1, GSTP1, and GSTT1 to the pharmacokinetics of busulfan is studied retrospectively. Seventy-seven children, undergoing myeloablative conditioning for allogeneic hematopoietic stem cell transplantation, were treated with busulfan (Busulvex) during 4 days, receiving busulfan either in one single dose or dived in four doses every 6 hours. Genetic variants of GSTA1, GSTM1, GSTP1, and GSTT1 were determined by pyrosequencing. Pharmacokinetic parameters were estimated by using nonlinear mixed-effect modeling (NONMEM). Subsequently, a combined population pharmacokinetic-pharmacogenetic model was developed describing the pharmacokinetics of busulfan taking into account the GST polymorphisms. In the presented pediatric population, body weight appeared to be the most important covariate and explained a major part of the observed variability in the pharmacokinetics of busulfan. None of the studied polymorphisms in the genes encoding GSTA1 GSTM1, GSTP1, and GSTT1 nor combinations of genotypes were significant covariates. It was concluded that in children, variability in pharmacokinetics of busulfan could not be related to polymorphisms in GST.
Abstract: To establish the prevalence of elevated liver enzymes in children transplanted in a Dutch haematopoietic stem cell transplantation (HSCT) centre, we retrospectively assessed AST and ALT values at 2 years after HSCT. Age, sex, diagnosis, type of transplant, conditioning regimen and early post-transplant complications involving the liver (veno-occlusive disease, acute GVHD, viral reactivation) were analysed as risk factors. AST and ALT values were available at 2 years after HSCT in 216 of 290 patients (75%) alive at that time and were above normal in 53 (25%) and at least twice normal in 17 (8%) patients. Older age at HSCT and a diagnosis of benign haematological disease are risk factors for abnormal liver enzymes late after HSCT. In half of the patients with benign haematological disease, iron overload is the most likely aetiological factor. Chronic hepatitis B or C is uncommon in our centre. In conclusion, the prevalence of abnormal liver enzymes late after HSCT in our centre is lower than reported in previous studies. Abnormal liver enzymes occur more often in children who are older at HSCT and transplanted for benign haematological disease. Long-term follow-up is crucial to establish if elevated liver enzymes precede clinical liver disease.
Abstract: Because of intra- and interindividual variability, bioavailability, and pharmacokinetics of busulfan (Bu) in children, oral busulfan without therapeutic drug monitoring (TDM) is assumed to be associated with higher graft failure rates as well as higher toxicity (eg, veno-occlusive disease [VOD]). This study compares the outcome of hematopoietic stem cell transplantation (HSCT) of 2 groups: 1) 30 patients who received myeloablation with once-daily intravenous (i.v.) dose-targeted busulfan (BUdtIV) based on TDM and 2) 30 patients who received the current practice of untargeted oral busulfan (BUPO). Patients received a 3-hour infusion of Bu at a first dose of 120 mg/m(2) (age >or=1 year) or 80 mg/m(2) (<1 year), or BUPO 1 mg/kg 4 times daily. Both regimens were continued for 4 days. The target area under the curve (AUC) was defined as 17,500 microg *h/l. BUdtIV resulted in higher event-free survival (EFS) and survival rates compared to BUPO (EFS: 30% versus 83%, P < .001, survival: 53% versus 83%, P = .016). BUdtIV was associated with more cases of VOD. TDM was feasible in routine clinical practice. The results show that i.v. Bu using TDM is preferable over oral Bu in children undergoing allogeneic stem cell transplantation, especially in those at high risk for graft failure/relapse.
Abstract: Autosomal recessive osteopetrosis is usually associated with normal or elevated numbers of nonfunctional osteoclasts. Here we report mutations in the gene encoding RANKL (receptor activator of nuclear factor-KB ligand) in six individuals with autosomal recessive osteopetrosis whose bone biopsy specimens lacked osteoclasts. These individuals did not show any obvious defects in immunological parameters and could not be cured by hematopoietic stem cell transplantation; however, exogenous RANKL induced formation of functional osteoclasts from their monocytes, suggesting that they could, theoretically, benefit from exogenous RANKL administration.
Abstract: Immunodeficiency-centromeric instability-facial dysmorphism syndrome, characterized by variable immunodeficiency, centromeric instability, and facial anomalies caused by epigenetic dysregulation resulting in hypomethylation, is caused in many patients by mutations in DNMT3B, a DNA methyltransferase gene; associated infections are a major cause of serious sequelae and death. Hematopoietic stem cell transplantation may improve the clinical course in immunodeficiency-centromeric instability-facial dysmorphism syndrome. We report 3 unrelated patients with persistent infections and intestinal complications who successfully underwent hematopoietic stem cell transplantation after nonmyeloablative or myeloablative conditioning regimens using HLA-matched donors. In all cases, donor chimerism led to resolution of intestinal complications and infections, growth improvement, and correction of the immunodeficiency.
Abstract: Epstein-Barr virus (EBV) reactivation is a frequent event after allogeneic stem cell transplantation and may progress to life-threatening lymphoproliferative disease (EBV-LPD) in the absence of adequate EBV-specific T cell immunity. Quantification of EBV DNA load in asymptomatic individuals who are at risk is a useful (although not entirely predictive) indicator of progression to EBV-LPD and guide for preemptive treatment with CD20 antibodies.
Abstract: The immune system of patients with severe combined immunodeficiency (SCID) reconstitutes to a large extent during the first years after hematopoietic stem cell transplantation (HSCT). It was suggested, however, that accelerated loss of thymus output may cause impaired immune function at the long term. To address this issue, we studied patients with SCID who underwent allogeneic HSCT 5 to 32 years earlier and identified early determinants of long-term T-cell reconstitution. A variety of immune parameters were analyzed both early (1-4 years) and late (5-32 years) after HSCT. Late after HSCT, a clear distinction could be made between a group of 8 patients with impaired T-cell reconstitution and 11 patients with good immune reconstitution. Importantly, in patients with decreased long-term T-cell reconstitution, T-cell recovery was already poor early after HSCT, demonstrating that long-term immune failure was not caused by accelerated loss of thymus output or long-term graft failure, but resulted from poor early grafting. The number of T-cell receptor excision circles (TRECs) early after HSCT was most predictive for long-term T-cell reconstitution. Frequent monitoring of T-cell immunity and TREC numbers early after HSCT may thus serve to timely identify patients who will fail to reconstitute properly and who may need additional treatment.
Abstract: We studied the pharmacokinetics and clinical outcome of a new once-daily intravenous area under the curve-targeted dosing scheme for busulfan based on body surface area. Eighteen children undergoing busulfan-based conditioning for allogeneic stem cell transplantation were enrolled. The age of the children ranged from 0.5 to 16 years. For all children, the starting dose was 80 mg/m. Unlimited dose adjustment was allowed to reach the target area under the curve (3800 micromol/l . min). This target area under the curve was determined on the basis of a previous study in our hospital. Pharmacokinetic studies were performed after the first dose. The median area under the curve on day 1 was 2616 (range 1781-5040) micromol/l . min at a dose of 80 mg/m. This resulted in a median dose increment to 114 (range 62-168) mg/m to reach the target area under the curve. In only one patient, the dose was decreased. Donor engraftment was established in 14 out of 18 patients (78%). Two of the four patients were successfully retransplanted. Relapse occurred in two patients (one died, one received additional treatment). Fourteen patients survived with a median follow-up of 1.6 years (1.0-2.2 years). The disease-free survival was 66% (12 of 18 patients). Despite the high systemic peak levels, there was no new unexpected or unusual toxicity. Moderate veno-occlusive disease was seen in one patient only. We conclude that intravenous busulfan in children administered once daily is safe, convenient and feasible, and can be dosed surface-based, independent of age. There was very limited (liver) toxicity, but the rejection rate was relative high, which can be probably overcome by a higher exposure to busulfan. Future investigations should be aimed at further optimizing the target area under the curve of intravenous busulfan for specific patient groups.
Abstract: For many children with life-threatening hematological diseases, hematopoietic stem cell transplantation (HSCT) is the only curative option. In children lacking a matched related or unrelated donor and with the certainty that, left untreated, death will ensue alternative donors must be sought. Haplo-identical peripheral blood stem cell transplantation (PBSCT) from a healthy parent is a feasible alternative. To reduce the risk of fatal graft-versus-host disease (GvHD) as a complication of transplant across major histocompatibility antigens, intense T-cell depletion is required. Large numbers of purified, cytokine mobilized peripheral stem cells (the so-called mega-dose concept) are required to compensate for the significantly increased risk of either graft failure or early rejection. In our unit, despite this approach, graft dysfunction has, in a significant group of children, proved problematic and, despite salvage attempts at re-transplantation, usually fatal. In children with hematological malignant disease, our overall relapse-free survival is 41%. However, successful transplant outcome has been associated with considerable delays in immune reconstitution that can be implicated in subsequent viral reactivation. We are investigating new strategies to improve the outcome of haplo-identical PBSCT, which may allow us to offer this form of treatment to more children requiring urgent HSCT.
Abstract: Many patients do not reach haematopoietic stem cell transplantation. Shortage of unrelated donors (UDs) is still seen as the main cause. However, with a worldwide UD pool containing more than 8 million donors, it is possible that other impediments are becoming more important. We analysed 549 UD searches for Dutch patients, performed between 1987 and 2000, in order to find the reasons for failure or success to reach transplantation. Between 1996 and 2000, 59% of the patients of Northwest European origin received a graft from an UD with a median time span of 4.4 months from the start of the search. In all, 11% of the patients lacked a compatible donor, while 30% became medically unfit for transplantation. This is in contrast to the patients of non-Northwest European origin for whom UD shortage is still the most important impediment; only 32% were transplanted while 50% lacked a compatible donor. We conclude that the shortage of donors is no longer the biggest constraint in unrelated stem cell transplantation for patients of Northwest European origin. It may be more effective to optimize the chance on transplantation by making the search process more efficient.
Abstract: Juvenile myelomonocytic leukemia (JMML) is a childhood leukemia for which allogeneic BMT is the only curative therapy. At our pediatric stem cell transplantation unit, we performed 26 BMTs in 23 children (age 0.5-12.7 years). Conditioning was CY/TBI based (1980-1996, n=14) or BU/CY/melphalan based (1996-2001, n=9). Donors were HLA-identical siblings (n=11), unrelated volunteers (n=9) or mismatched family members (n=3). A total of 10 patients survive in CR (median follow-up 6.8 years, range 3.1-22.2 years). Relapse or persistent disease was observed in eight and two patients, respectively. Nine of these patients died, one achieved a second remission following acute nonlymphatic leukemia chemotherapy (duration to date 5.3 years). Transplant-related mortality occurred in four patients. Overall survival at 5 and 10 years was 43.5%. Using T-cell-depleted, one-antigen mismatched unrelated donors was the only significant adverse factor associated with relapse in multivariate analysis (P=0.039, hazard ratio 4.9). Together with a trend towards less relapse in patients with graft-versus-host-disease and in patients transplanted with matched unrelated donors, this suggests a graft-versus-leukemia effect of allogeneic BMT in JMML.
Abstract: We studied the pharmacokinetics of intravenous busulfan (Bu) in children in order to further optimize intravenous Bu dosing in relation to toxicity and survival. A total of 31 children undergoing Bu-based conditioning for allogeneic SCT were enrolled in a study. The starting dose was 1.0 mg/kg (age < 4 years) and 0.8 mg/kg (age > or =4 years), four doses per day during 4 days. Dose adjustment was allowed up to a maximum dose of 1.0 mg/kg per dose if the target area under the serum concentration-time curve (AUC) was not reached. Pharmacokinetic studies were performed after the first dose. Donor engraftment was established in 28 out of 31 patients. The average AUC after the first dose was the same in children < 4 years as in children > or =4 years. Mean clearance was higher in children < 4 years than in children > or =4 years. In 35% of all patients, total AUC was within the target AUC. The other children's AUCs were below the target range. No relationships were found between systemic exposure to Bu and toxicity or clinical outcome. We concluded that, in accordance with previous data, within the observed AUCs no clear relationship was observed between Bu AUC and outcome with respect to toxicity, engraftment and relapse.
Abstract: Paroxysmal nocturnal haemoglobinuria (PNH) is characterized by intravascular haemolysis, nocturnal haemoglobinuria, thrombotic events, serious infections and bone marrow failure. This acquired disease, caused by a deficiency of glycosylphosphatidylinositol (GPI) anchored proteins on the haematopoietic cells, is rare in children. We describe 11 Dutch paediatric PNH patients (median age: 12 years, range 9-17 years) diagnosed since 1983, seven cases associated with aplastic anaemia (AA), four with myelodysplastic syndrome (MDS). Presenting symptoms were haemorrhagic diathesis (n = 10), palor/tiredness (n = 8), dark urine (n = 1), fever (n = 1) and serious weight loss (n = 1). Treatment consisted of prednisolone (n = 7), anti-thymocyte globulin (n = 3) and/or androgens (n = 5). Eventually, five patients received a bone marrow transplantation (BMT) (three matched unrelated donors/two matched family donors), of whom four are still alive. PNH, diagnosed by immunophenotypic GPI-linked anchor protein analysis, should be considered in all children with AA or MDS. BMT should be considered as a therapeutic option in every paediatric PNH patient with BM failure.
Abstract: We report on a case of osteoclast-poor osteopetrosis who received a hematopoietic stem cell graft and, despite hematological engraftment, showed little signs of response in the skeletal defect. Clinical and laboratory studies supported the concept that the bone microenvironment remained abnormal, thus reducing the clinical response to transplantation.
Abstract: This report assessed the results of allogeneic stem cell transplantation (allo-SCT) in 26 patients with Shwachman-Diamond disease (SDS) and severe bone marrow abnormalities. The conditioning regimen was based on busulphan (54%), total body irradiation (23%), fludarabine (15%) or other chemotherapy combinations (8%). Standard prevention of graft versus host disease (GVHD) with cyclosporin +/- methotrexate was adopted in 54% of the patients whilst in vivo or in vitro T-cell depletion was used in 17 and four patients respectively. Neutrophil and platelet engraftment were achieved in 21 (81%) and 17 (65%) of 26 patients after a median time of 18 days and 29 days respectively. The incidence of grade III and IV acute GVHD was 24% and of chronic GVHD 29%. Nine patients died after a median time of 70 d, post-SCT. After a median follow-up of 1.1 years, the transplant-related mortality was 35.5% (95% CI 17-54) whilst the overall survival was 64.5% (95% CI 45.7-83.2). Allo-SCT was found to be successful in more than half of SDS patients with severe bone marrow dysfunction. Further improvements would be anticipated by a better definition of the optimum time in the course of disease to transplant and by the adoption of less toxic conditioning regimens.
Abstract: X-linked lymphoproliferative disease (XLP) is a rare immunodeficiency caused by mutations in the signaling lymphocyte activating molecule-associated protein/SH2D1A gene and characterized by a dysregulated immune response to Epstein-Barr virus and other pathogens. The clinical presentation is heterogeneous and includes fulminant infectious mononucleosis, lymphoma, hypogammaglobulinemia and aplastic anemia. XLP is associated with a high morbidity and overall outcome is poor. At present, allogeneic stem cell transplantation (alloSCT) is the only curative treatment. XLP patients may be recognized in various stages of disease and even when symptoms are not yet evident. We here present two related XLP patients in different stages of disease that were both treated successfully with alloSCT using a matched unrelated donor. In addition, we have reviewed all reported cases of alloSCTs in XLP patients. Based on these results and in order to improve the final outcome, we conclude that alloSCT should be recommended in both symptomatic and asymptomatic XLP patients.
Abstract: Glomerular function of all long-term survivors who underwent hemopoietic stem cell transplantation (HSCT) from 1991 to 1998 (study I, n=121) was studied retrospectively. In addition, we prospectively analyzed glomerular and tubular function of all long-term surviving children who received an HSCT between 1998 and 2000 (study II, n=41). We found a lower prevalence of children with chronic renal failure (CRF) post-HSCT in our more recent cohort (study II: 10%) as compared to the older cohort (study I: 24%) 5.0 (0.7 s.d.) and 7.6 (2.4 s.d.) year's post-HSCT, respectively. Furthermore, it seems that renal function may stabilize after 1-year post-HSCT. None of the patients required dialysis or antihypertensive medication at long-term follow-up. The sole predictor of CRF in our study was high serum creatinine pre-HSCT (P=0.007), while acute renal failure within 3 months after HSCT (P=0.08) only showed a trend towards predicting CRF. We could not confirm a relation of conditioning with irradiation with CRF post-HSCT, as was shown in several other pediatric and adult studies. Proximal and distal tubular dysfunction only occurred in a minority of long-time survivors of HSCT (3-12 and 9-13%, respectively) and had no clinical consequences.
Abstract: The aetiology of several autoimmune diseases has not yet been elucidated. Microchimerism, the persistence of small numbers ofallogeneic cells in an individual, has been mentioned recently in connection with the occurrence of autoimmune diseases such as systemic sclerosis and juvenile inflammatory idiopathic myopathy. These allogeneic cells can originate from mutual foeto-maternal exchange of blood cells during pregnancy or from a donor after blood transfusion or (organ) transplantation. In some cases, a syndrome then develops that resembles the chronic graft-versus-host reaction after stem-cell transplantation, in which allogeneic cells react with autologous cells. Studies on microchimerism in patients with systemic sclerosis and juvenile inflammatory idiopathic myopathy, compared to controls, sometimes reveal a clearly increased prevalence of microchimerism in patients. However, microchimerism can also be found in healthy individuals. Direct proof of a causal relation between microchimerism and autoimmune diseases does not exist. Additional genetic or environmental factors may be partly responsible for a disturbed balance between tolerance and aggression.
Abstract: Sequential herpes simplex virus type 1 (HSV-1) isolates were obtained from a paediatric haematopoietic stem cell transplant (HSCT) patient who received prolonged therapy with acyclovir (ACV) followed by foscarnet (PFA) and topical cidofovir (HPMPC) for severe persistent mucocutaneous HSV-1 infection. The isolates were retrospectively studied for drug resistance. The first resistant isolate associated with clinical failure of antiviral therapy emerged 44 days post-ACV treatment initiation. Susceptibility testing revealed an ACV-resistant HSV strain that demonstrated cross resistance to PFA in the absence of any previous PFA treatment. The observed cross resistance was conferred by a single amino acid substitution, Ser724Asn, in the HSV DNA polymerase (DNA pol) gene. During the subsequent course of ACV therapy, the ACV/PFA-cross-resistant isolates were replaced by ACV-resistant, PFA-sensitive isolates. These isolates carried no DNA pol mutations, but had an Arg163His substitution in the thymidine kinase gene. Upon subsequent switching of antiviral therapy from ACV to PFA, the original ACV/PFA-cross-resistant DNA pol mutant re-appeared. Our study shows the emergence of different drug-resistant HSV variants during ongoing, unchanged ACV therapy. Furthermore, a rapid re-selection of the original resistant variant was observed after switch. For optimal antiviral management of HSV infections in HSCT recipients, therapeutic decisions should be guided by drug susceptibility results whenever therapeutic failure is observed and/or when changes in antiviral treatment are considered.
Abstract: A 5-year-old boy with acute lymphoblastic leukaemia (ALL) received a haematopoietic stem cell transplant (HSCT) from his father and was monitored for the presence of respiratory viruses.
Abstract: CD40 ligand (CD40L) deficiency causes recurrent sinopulmonary infection, Pneumocystis carinii pneumonia, and Cryptosporidium parvum infection. Approximately 40% to 50% of patients survive to the third decade: long-term survival is unclear. Hematopoietic stem cell transplantation (HSCT) is curative. We present a retrospective analysis of 38 European patients undergoing HSCT for CD40L deficiency in 8 European countries between 1993 and 2002. Donor stem cell source included 14 HLA-identical siblings, 22 unrelated donors, and 2 phenotypically matched parental stem cells (12 T-cell depleted). Of the patients, 34 engrafted and 26 (68%) survived; 3 had autologous reconstitution, 22 (58%) were cured, and 1 engrafted but has poor T-cell immune reconstitution. There were 18 evaluated patients who responded to vaccination. Of the patients, 12 (32%) died from infection-related complications, with severe cryptosporidiosis in 6. Grades 2 to 4 graft-versus-host disease (GvHD) associated with infection occurred in 6 of 12 fatal cases. HSCT cured 58% of patients, 72% of those without hepatic disease. Early T-cell function following whole marrow HSCT may limit cryptosporidial disease, but survival was similar after T-cell-depleted HSCT. Preexisting lung damage was the most important adverse risk factor. Further studies will determine optimal timing and type of HSCT.
Abstract: Adenovirus (AdV) infections are an increasingly frequent and potentially fatal complication in allogeneic stem cell transplant recipients. To determine the antiviral potential of ribavirin in an unbiased way, 4 patients without immune recovery were prospectively analyzed by quantitative measurement of plasma AdV DNA load. Administration of ribavirin at the first signs of AdV dissemination was not accompanied by a decrease in the plasma AdV DNA load in any of these patients, and an increase in the AdV load was even documented in 3. These observations question the potential of ribavirin to improve the outcome for patients with disseminating AdV infection and support a critical evaluation of antiviral treatments for AdV infection that involves the kinetics of virus DNA load as an objective parameter of viral replication.
Abstract: Community-acquired respiratory viruses are an important cause of respiratory disease in pediatric patients undergoing hemopoietic stem cell transplantation. However, there are no studies examining the impact of more rapid and sensitive diagnosis by real time polymerase chain reaction (PCR) in this population. We performed a prospective study to assess the impact of real time PCR diagnosis as well as protective isolation for community-acquired respiratory virus infections in pediatric patients undergoing hemopoietic stem cell transplantation.
Abstract: Aplastic anemia was diagnosed at birth for a first child from healthy nonconsanguineous parents. The girl had hypoglycemia, which normalized within 2 months. Cow milk allergy was suspected initially, because of skin lesions and diarrhea, followed by severe growth retardation. Clinical and radiologic symptoms gradually became typical for Shwachman-Diamond syndrome. Two common mutations in the SBDS gene (183-184TA-->CT [K62X] and IVS2(258)+2T--> C [C84fs]) were found.
Abstract: Thirty-one herpes simplex virus type one (HSV-1) isolates from 12 haematopoietic stem cell transplant recipients with persistent HSV infections despite acyclovir (ACV) prophylaxis or treatment, were genotypically and phenotypically characterized. The relationship between drug susceptibility of the isolates and mutations in thymidine kinase (TK) and DNA polymerase (DNA pol) genes was examined. In all 12 patients, HSV infections were due to ACV-resistant, foscarnet-sensitive viruses. Out of 31 isolates examined, 23 were resistant and eight were sensitive to ACV; eight patients carried viruses with frameshift mutations in the TK gene (due to addition or deletion of single nucleotides in homopolymeric repeats). These mutations were found at codon 61 (G deletion, one patient), 146 (G insertion, five patients) and 153 or 185 (C deletion, one patient each). In four patients, viruses were selected during ACV therapy that contained novel amino acid substitutions in the TK gene (H58R, G129D, A189V, R216H, R220C). Their possible role in ACV resistance was further confirmed phenotypically and by the absence of any resistance-associated mutations in the DNA pol gene. These substitutions were located in ATP- or nucleoside-binding sites or in conserved regions of the TK gene. In addition, a single mutation, Q570R, in the delta-region C of the DNA pol gene, was identified in an isolate from a single patient with resistance to ACV. Our study confirms and expands previous data on genotypic changes associated with ACV resistance of HSV-1 clinical isolates.
Abstract: X-linked agammaglobulinaemia (XLA) is a primary immunodeficiency caused by a mutation in the gene encoding Bruton's tyrosine kinase (BTK). The classical presentation of XLA consists of the almost complete absence of B-lymphocytes and immunoglobulins in the peripheral blood leading to severe, mainly bacterial, upper and lower respiratory-tract infections already in early childhood. Irrespective of the kind of BTK-gene mutation the phenotype of XLA can be very diverse. Two 9-year-old cousins with the same BTK-gene mutation illustrate this phenotypical diversity. One boy had a classical presentation and was on maintenance treatment with intravenous immunoglobulins and prophylactic antibiotics to control his infections. Without any prophylactic treatment, his cousin had no abnormal infectious course despite very low B-lymphocyte counts and immunoglobulin levels in the blood. The mechanisms underlying the phenotypical diversity of XLA have not been clarified. Gene polymorphisms affecting the innate immune system may play a role.
Abstract: Both innate and adaptive immune responses are dependent on activation of nuclear factor kappaB (NF-kappaB), induced upon binding of pathogen-associated molecular patterns to Toll-like receptors (TLRs). In murine models, defects in NF-kappaB pathway are often lethal and viable knockout mice have severe immune defects. Similarly, defects in the human NF-kappaB pathway described to date lead to severe clinical disease. Here, we describe a patient with a hyper immunoglobulin M-like immunodeficiency syndrome and ectodermal dysplasia. Monocytes did not produce interleukin 12p40 upon stimulation with various TLR stimuli and nuclear translocation of NF-kappaB was impaired. T cell receptor-mediated proliferation was also impaired. A heterozygous mutation was found at serine 32 in IkappaBalpha. Interestingly, his father has the same mutation but displays complex mosaicism. He does not display features of ectodermal dysplasia and did not suffer from serious infections with the exception of a relapsing Salmonella typhimurium infection. His monocyte function was impaired, whereas T cell function was relatively normal. Consistent with this, his T cells almost exclusively displayed the wild-type allele, whereas both alleles were present in his monocytes. We propose that the T and B cell compartment of the mosaic father arose as a result of selection of wild-type cells and that this underlies the widely different clinical phenotype.
Abstract: Transplantation of allogeneic haemopoietic stem cells can cure several primary immunodeficiencies. This European report focuses on the long-term results of such procedures done between 1968 and December, 1999, for primary immunodeficiencies.
Abstract: X-linked hyper-IgM syndrome (XHIM) is a rare congenital immunodeficiency disorder, characterised by a defect in both humoral and cellular immune responses. In XHIM, the membrane glycoprotein CD40 ligand (expressed on activated T-cells) is deficient, which compromises T-cell interactions with antigen-presenting cells. Patients with XHIM present with severe, recurrent infections, predominantly of the respiratory and gastrointestinal tract. The gene encoding the CD40 ligand is located on the long arm of the X chromosome.
Abstract: We reviewed the results of the Dutch paediatric bone marrow transplant (BMT) program for children receiving HLA-identical BMT for beta-thalassaemia major over an 18-year period. In all, 19 patients underwent a total of 21 transplants in our treatment centre between July 1984 and February 2002. Eight females (age 0.3-12 years; median 5 years) and 11 males (age 0.8-18 years; median 6 years) were included. Information, prospectively collected, included molecular defects, donor genotype, beta/alpha-globin expression rates, serum ferritin levels, hepato-splenomegaly, chelation history, virology screening, liver pathology together with post-transplant outcome inclusive of leucocyte chimerism. In total, 11 patients received standard busulphan/cyclophosphamide (Bu/Cy) conditioning, with or without ATG. Stable engraftment was seen in 5/11 with late rejection occurring in six patients. Of these, two children underwent a second successful SCT. For this group, overall event-free survival (EFS) and disease-free survival (DFS) were 90 (10/11) and 64% (7/11), respectively. The probability of rejection was 55%. Subsequent addition of melphalan to the conditioning regimen resulted in long-term stable engraftment in all patients with an EFS/DFS for this group of 90% (9/10). Treatment-related mortality, irrespective of conditioning, was low at 5% (1/19 patients). Veno-occlusive disease (VOD) occurred in 19% (4/21 transplants) and acute GvHD in 19% (4/21 transplants). Post-BMT beta/alpha synthetic ratio measurement monitored donor erythroid engraftment and predicted rejection with a return to transfusion dependency. Maintained full donor chimerism is indicative of stable engraftment both for leucocyte and erythroid lineages, whereas mixed donor chimerism is not. Our results emphasise the importance of the conditioning regimen and post-transplant chimerism surveillance predictive of rejection or long-term stable engraftment.
Abstract: A boy suffered from severe recurrent intestinal infections from the age of 8 months onwards; investigation into an immune disorder ultimately resulted in the diagnosis of 'hyper-IgM syndrome'. He was treated successfully with bone marrow transplantation, using an HLA-matched donor. Another boy had severe recurrent respiratory tract infections from the age of 3 months onwards. At the age of 6.5 years, 'hyper-IgM syndrome' was diagnosed. No suitable donor was available. In addition, he developed sclerosing cholangitis and end-stage liver disease, making a combined bone marrow and liver transplantation too risky. He died at 10.5 years of age. X-linked hyper-IgM syndrome is a rare congenital immunodeficiency disorder, characterised by a defect in both humoral and cellular immune responses. Deficiency in the membrane glycoprotein CD40 ligand (expressed on activated T-cells) compromises T-cell interactions with antigen-presenting cells. In a child with severe recurrent infections, and with dysgammaglobulinaemia with a normal or increased IgM level, the diagnosis of 'X-linked hyper-IgM syndrome' should be considered.
Abstract: Panoramic radiographs (orthopantomogram [OPT]) are, beside clinical examination, helpful in detecting possible dental foci of infections before bone marrow transplantation (BMT). The value of an OPT in paediatric BMT-recipients was assessed by the results of dental evaluation, consisting of an OPT and a consultation by an oral surgeon, in 161 children between 1987 and 1999. Eleven out of 161 children had at least one oral focus of infection that required treatment before myeloablative therapy. In seven children the foci were detectable both clinically and radiographically and in two children the foci were visible only clinically. Only two children had a focus that was only detectable by means of radiographs. In both patients it concerned erupting third molars. In conclusion, we recommend an OPT only when the child has clinically obvious abnormalities. In addition, in older children an OPT will be indicated to evaluate the third molars.
Abstract: Intravenous formulations of busulfan have recently become available. Although busulfan is used frequently in children as part of a myeloablative regimen prior to bone marrow transplantation, pharmacokinetic data on intravenous busulfan in children are scarce. The aim was to investigate intravenous busulfan pharmacokinetics in children and to suggest a limited sampling strategy in order to determine busulfan systemic exposure with the minimum of inconvenience and risk for the patient.
Abstract: In two Turkish brothers familial haemophagocytic lymphohistiocytosis (FHLH) was diagnosed at 3 years and 2.5 months, respectively. FHLH is a rare autosomal recessive condition with a typical clinical presentation including prolonged fever, failure to thrive, irritability and hepatosplenomegaly. Laboratory evaluations show cytopenia (at least two out of the three cell lines), hypertriglyceridaemia and hypofibrinogenaemia. A pathognomonic sign is haemophagocytosis in bone marrow or tissue biopsy. Both patients were treated with stem-cell transplants using bone marrow and peripheral blood stem cells, respectively, from one unrelated donor. They showed a good haematological recovery, with minor complications, and at follow-up after one year were free of disease. Immune suppression can induce prolonged remission in FHLH, but cure is only achieved after a successful allogeneic stem-cell transplantation. Without transplantation, the prognosis is very poor.
Abstract: Since more and more children survive allogeneic bone marrow transplantation (BMT), knowledge of acute and late complications becomes increasingly important. Besides the major complications [(opportunistic) infections, veno-occlusive disease, graft versus host disease, and recurrence of primary disease], acute and chronic renal insufficiency are significant post-transplant complications that may contribute to transplant-related mortality. To elucidate risk factors for acute and chronic renal insufficiency post BMT, we performed a prospective study of all 66 children who received a BMT in a 2-year period at our institution; 21% had acute renal insufficiency post BMT. Risk factors for acute renal insufficiency were veno-occlusive disease, high cyclosporin serum levels, and foscarnet therapy. Of surviving patients, 11% developed chronic renal insufficiency 1 year post BMT. Acute renal insufficiency was the sole predictor of chronic renal insufficiency. In contrast to studies in adults, we did not find total body irradiation to be a risk factor for chronic renal insufficiency. Future long-term studies are needed to assess incidence and morbidity of chronic renal insufficiency in children following allogeneic BMT.
Abstract: Individuals with either a late (C5-9) complement component deficiency (LCCD) or properdin deficiency are at increased risk to develop meningococcal disease, often due to serogroups W135 and Y. Anti-meningococcal defence in both LCCD persons and properdin-deficient individuals without bactericidal antibodies depends mainly on phagocytosis. Three types of opsonin receptors are involved in phagocytosis by polymorphonuclear cells (PMN). These represent the polymorphic FcgammaRIIa (CD32) and FcgammaRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcgammaRIIa and FcgammaRIIIb allotypes was assessed in 15 LCCD and in 15 properdin-deficient patients with/without previous meningococcal disease, we found the combination of FcgammaRIIa-R/R131 with FcgammaRIIIb-NA2/NA2 allotypes to be associated with previous meningococcal disease (odds ratio 13.9, Fisher's test P = 0.036). No such relation was observed in the properdin-deficient patients. The importance of FcgammaRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcgammaRIIa-R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0.05) less than PMN from FcgammaRIIa-H/H131 donors. When properdin-deficient serum was tested, it was observed that reconstitution with properdin resulted in enhanced PMN phagocytosis of the W135 meningococci (P = 0.001). This enhanced phagocytosis was parallelled by an increase in C3 deposition onto the opsonized meningococci W135 (r = 0.6568, P = 0. 01). We conclude that the occurrence of meningococcal disease in LCCD patients is associated with certain FcgammaR allotypes. Properdin-deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.
Abstract: Since nonencapsulated Haemophilus influenzae persists in the lower respiratory tracts of patients with chronic bronchitis despite the presence of specific antibodies, complement, and polymorphonuclear leukocytes (PMNs), opsonophagocytosis of H. influenzae was analyzed. Nonencapsulated H. influenzae isolated from the sputa of chronic bronchitis patients was labeled with fluorescein isothiocyanate and incubated with human PMNs in the presence of complement and antibodies for 30 min at 37 degrees C. Candida albicans was added to each sample as an internal standard, and the reduction of the number of bacteria was determined by flow cytometry. Fluorescence quenching with ethidium bromide was used to discriminate between intracellular and extracellular bacteria. Opsonophagocytosis of viable H. influenzae d1 was 17% +/- 29% in the presence of complement and human pooled sera containing high titers of strain-specific antibodies. Opsonophagocytosis of six other H. influenzae strains was also poor. Under the same conditions, opsonophagocytosis of Staphylococcus aureus was 90% +/- 5%, and opsonophagocytosis of C. albicans was 55% +/- 23%. About half of the number of H. influenzae bacteria associated with PMNs was internalized. Opsonophagocytosis of heat-killed H. influenzae d1 (41% +/- 20%) was higher than that of viable bacteria of the same strain (P < 0.05). This result suggests that the accessibility of epitopes on H. influenzae for opsonizing antibodies is better on killed than on viable bacteria. We conclude that viable nonencapsulated H. influenzae is poorly opsonophagocytized in the presence of strain-specific antibodies and complement.
Abstract: Antibodies are essential in host defense against Neisseria meningitidis. Therefore, interactions among IgG and Fc receptors (Fc gamma R) on phagocytes may be crucial. Genetic polymorphic forms of Fc gamma RIIa (CD32) express different functional activities. In a retrospective study, Fc gamma R polymorphisms were determined in 25 children who survived fulminant meningococcal septic shock: 11 had Fc gamma RIIa-R/R131, the poor IgG2-binding allotype, which is a significantly more frequent rate than found in a healthy white population (44% vs. 23%; P = .028; odds ratio = 2.67; 95% confidence interval, 1.09-6.53). The relevance of this finding was further supported by the fact that neutrophils with the Fc gamma RIIa-R/R131 allotype phagocytized N. meningitidis opsonized with polyclonal IgG2 antibodies less effectively than did IIa-H/H131 neutrophils. Our findings suggest an important role for anti-N. meningitidis IgG2 and the Fc gamma RIIa polymorphism in host defense against systemic meningococcal infections.
Abstract: The four subclasses of IgG have different biological activities associated with their Fc regions. Fc gamma receptors on leucocytes (Fc gamma R) mediate binding and phagocytosis of opsonized particles. Two structurally and functionally distinct allelic polymorphisms of the Fc gamma R have been defined: the H/R131 forms of Fc gamma RIIa (CD32), and the neutrophil antigen 1 (NA1)/NA2 forms of Fc gamma RIIIb (CD16). In this study the activities of allotypes of CD16 are analysed with antibacterial IgG subclass antibodies and with IgG1 and IgG3 anti-Rhesus D, and the activities of CD32 with IgG1 and IgG3 anti-Rhesus D. With respect to the allotypes of CD16, polymorphonuclear leucocytes (PMN) homozygous for Fc gamma RIIb-NA2 exhibited a lower (21-25%) IgG1-mediated phagocytosis of Staphylococcus aureus strain Wood (STAW), Haemophilus influenzae type b (Hib), and Neisseria meningitidis group B (NMen) than IIIb-NA1 PMN. The difference was apparent only when the micro-organisms were opsonized in the absence of complement, and was furthermore enhanced (34-52%) upon blockade of Fc gamma RIIa. In addition, monoclonal IgG3 anti-D-mediated rosette formation and phagocytosis was consistently found to be lower (16%) with Fc gamma RIIIb-NA2 than with IIIb-NA1 PMN. For the allotypes of CD32 we now show that IgG3 anti-D sensitized erythrocytes formed more (50%) rosettes and were phagocytosed at a higher rate with PMN carrying Fc gamma RIIa-H131 than with PMN carrying IIa-R131. Heterozygous Fc gamma RIIa-H/R131 PMN exhibited intermediate phagocytic activity in this respect. This study illustrates a critical role of Fc gamma R allotypes in functional interactions with biologically relevant IgG subclass antibodies.
Abstract: To assess the function of IgG subclass antibodies we studied the opsonization of Staphylococcus aureus (STAW) and Haemophilus influenzae type b (Hib) by natural IgG1 and IgG2 antibodies from five healthy individuals. Phagocytosis by polymorphonuclear leukocytes (PMN) was analyzed using FITC-labeled bacteria and flow cytometry. All PMN donors were typed for the High- and Low-Responder phenotype of the human Fc gamma receptor (hFc gamma) type IIa (CD32) and the NA1/NA2 allotype of the hFc gamma RIIIb (CD16). When PMN were used that were heterozygous for hFc gamma RIIa and hFc gamma RIIIb, phagocytosis of STAW opsonized with IgG1 antibodies was similar to that with IgG2, both in the presence and absence of a source of complement (agammaglobulinemic serum). Phagocytosis of Hib opsonized with IgG2 anti-Hib proved significantly lower (p < 0.05) than with IgG1 anti-Hib using preparations from 4 of the 5 individuals tested. IgG2 anti-Hib from one donor, however, proved more effective than IgG1 anti-Hib. The properties of PMN with hFc gamma RIIaLR,LR and hFc gamma RIIaHR,HR were compared, using hFc gamma RIIIb NA1/NA2-matched PMN. hFc gamma RIIaHR,HR PMN were virtually incapable of phagocytosing STAW and Hib opsonized with IgG2 anti-bodies without complement, in contrast to hFc gamma RIIaLR,LR PMN. Phagocytosis of IgG2-oposonized bacteria by hFc gamma RIIaLR,LR PMN was effectively inhibited by mAb against hFc gamma RIIa (IV.3). IgG1-mediated phagocytosis was blocked by mAb against hFc gamma RIIIb (CLB/FcRGran 1) to a greater extent than by anti-hFc gamma RIIa mAb. Inhibition studies with mAb against CR3 (B2.12) and hFc gamma RIIa showed that both receptors cooperated in phagocytosis. We conclude that PMN phagocytosis can be effectively mediated by antibacterial IgG2 anti-STAW and anti-Hib. Thus, IgG2 may have a critical role in the immune defense against these bacteria. However, the role of antibacterial IgG2 in opsonization and phagocytosis may be limited in individuals homozygous for hFc gamma RIIaHR.
Abstract: We describe a patient with leucocyte adhesion deficiency (LAD). Clinically, the patient had delayed umbilical cord detachment, omphalitis, impaired wound healing and persistent leucocytosis. The patient had the severe form of LAD, with a total absence of leucocyte cell adhesion molecules (LeuCAMs) and undetectable mRNA for the beta chain, the common subunit of the LeuCAMs. In vitro neutrophil chemotaxis, aggregation and oxygen consumption were severely impaired. In vitro incubation of neutrophils with recombinant human interferon-gamma (rIFN-gamma) showed an increase in oxygen consumption, but no effect on the expression of the LeuCAMs, or the beta chain mRNA. In vivo treatment with IFN-gamma was started. The Fc gamma RI receptor appeared on the neutrophils, the LeuCAMs remained undetectable, while the neutrophil functions remained disturbed. The patient died of surgical complications after 10 weeks of rIFN-gamma treatment. No new infections or side-effects due to rIFN-gamma were observed.
Abstract: To obtain information on effector functions of human immunoglobulin G2 (IgG2), we have measured the complement-activating properties of polyclonal IgG subclass antibodies against bacterial antigens. IgG1 and IgG2 were purified from serum samples from five healthy individuals, and complement activation was measured with different bacterial antigens. We used Staphylococcus aureus Wood 46 (STAW), which is a common antigen, Haemophilus influenzae type b (Hib), which is a common pathogenic microorganism in children, and formaldehyde-inactivated tetanus toxin (TT). Bacteria were incubated with antibodies and then incubated with sera from agammaglobulinemic patients as a complement source, and C3c deposition was measured by enzyme-linked immunosorbent assay. We found that anti-STAW IgG2 activated complement to a level similar to that of anti-STAW IgG1. Anti-Hib IgG1 complement activation was as much as seven times higher than that of anti-Hib IgG2 in four individuals. In one individual, anti-Hib IgG2 was more effective in complement activation than anti-Hib IgG1. Anti-TT antibodies showed patterns similar to those of anti-Hib. Our results indicate that IgG2 antibodies may contribute significantly to antibacterial defense. Also, individual differences in antibody effector functions should be taken into account when evaluating the immune status of patients and during early phase 1 studies of new vaccines.
Abstract: Leukocyte adhesion deficiency (LAD) is a rare autosomal recessive disorder which leads to recurrent severe infections due to impaired leukocyte functions. The disorder is caused by an absence or deficiency of leukocyte cell adhesion molecules (LeuCAMs) on the leukocyte membranes. The diagnosis is established with monoclonal antibodies against the LeuCAMs. We have carried out a prenatal diagnostic procedure by means of cordocentesis in a mother who was 20 weeks pregnant and had previously given birth to a child with LAD. This previous child had the severe form of LAD with undetectable mRNA for the beta chain, the common subunit of the LeuCAMs. We found that the fetal granulocytes expressed the LeuCAMs normally. At birth, the baby was physically normal and showed no signs of impaired leukocyte functions.
