Search

The binding and opsonic properties of C-reactive protein (CRP) for various species of bacteria were investigated. CRP bound more avidly to killed than to live Streptococcus pneumoniae, the binding varying among various serotypes; CRP hardly bound to a number of other bacterial species studied. CRP enhanced complement-dependent phagocytosis of live S. pneumoniae by granulocytes but did not enhance the phagocytosis of live Staphylococcus aureus or group B streptococci. We suppose that CRP may serve as an opsonin for killed bacteria and bacterial debris but is probably not an important opsonin for live bacteria other than S. pneumoniae.

In this study, we have investigated the impact of deficient MHC class II expression on the use of TCRBV6 and TCRBJ gene elements, and on the pattern of amino acid incorporation exhibited in the N1-D-N2 segments of the third complementarity-determining region (CDR3) of these TCRBV6 rearrangements. To this end, we have analyzed circulating T cells from three, nonrelated MHC class II-deficient (bare lymphocyte syndrome (BLS)) patients and three MHC class II-expressing family members. The patients and healthy controls exhibited similar, nonrandom usage profiles of TCRBV6 and TCRBJ gene elements in both the CD4+CD8- and the CD4-CD8+ subsets of peripheral blood T cells. No statistically significant differences between patients and controls were detected in the length of CDR3, or in the amount of non-germline modification at the sites of recombination. However, detailed analysis of the TCRBV6 rearrangements derived from the CD4+CD8- subsets from the BLS patients revealed patterns of amino acid incorporation into the N1-D-N2 region of CDR3 that resulted in altered charge and hydropathicity properties of the presumed Ag binding site. In this way, we have been able to demonstrate that human T cell repertoire development in the absence of MHC class II expression results in a circulating CD4+CD8- T cell population bearing TCRs with altered CDR3 profiles. Such altered profiles are likely to be a direct reflection of the lack of MHC class II-mediated selection processes in these BLS patients.

Lethally irradiated C3H/Law mice were injected (i.v.) with C57BL/Rij allogeneic bone marrow cells to induce a delayed type graft-vs-host disease (GVHD). Signs of GVHD first became apparent in the third week after transplantation. The disease resulted in a mortality rate of 70% at 80 days. Treatment with IFN-gamma twice weekly, for a period of 6 wk, starting at the time of bone marrow transplantation (BMT), completely prevented overt GVHD, as evidenced by a lack of diarrhea and no mortality during the follow-up period of 100 days after BMT. Also, the histologic GVHD lesions in the gastrointestinal tract were almost completely abrogated by the IFN-gamma treatment. All long term survivors were proven to be chimeras. During the induction phase of GVHD, the number of Con A-induced, IFN-gamma-producing cells in the spleen was significantly reduced in the IFN-gamma-treated mice as compared with control mice. These results suggest that the normally enhanced production of endogenous IFN-gamma in the spleen at the time of hematopoietic reconstitution after BMT is down-regulated by exogenously administered IFN-gamma. This cytokine-mediated strategy to prevent GVHD might be an alternative to the current strategy of in vitro depletion of T cells for allogeneic BMT.

Bone marrow graft recipients suffer profound immunodeficiency during at least 3 months after transplantation. B-cell reconstitution following allogeneic bone marrow transplantation (BMT) in children was studied longitudinally by quantification of Ig (sub)class levels in serum and by investigation of numbers and characteristics of homogeneous Ig components (H-Ig); ie, monoclonal gammopathies (MG). For the latter purpose, a sensitive immunoblotting technique capable of detecting H-Ig of a concentration as low as 0.5 microgram/mL was used. Sera of 40 children grafted for a variety of diseases were investigated and followed up for 5 years. It was found that Ig (sub)classes reached normal levels from 3 months after BMT onward. The sequential increase of the different Ig isotypes was in accordance with that seen in normal ontogeny. This was especially clear following BMT for severe congenital immunodeficiency. H-Ig appeared from as early as 6 weeks after BMT in increasing numbers, beginning within IgM, IgG3, and IgG1, and afterward within other isotypes. After an initial increase of serum Ig levels, “overshooting” occurred accompanied by high frequency of H-Ig. H-Ig were still present at 5 years after BMT, when Ig levels normalized. Our data indicate that B-cell reconstitution after allogeneic BMT recapitulates normal ontogeny but in a clonally dysregulated fashion; that is, with overexpression of some clones and underexpression of others.

We performed polymerase chain reaction-variable number of tandem repeats analysis of flow-sorted peripheral blood T-, B-, natural killer- , and myeloid cell populations (van Leeuwen et al, Br J Haematol 79:218, 1991) in 32 children following allogeneic bone marrow transplantation (BMT) for leukemia to evaluate the relationship between mixed lymphoid chimerism and leukemia relapse. Five patients showed a stable mixed chimerism pattern characterized by the presence of both recipient as well as donor type cells in all cell populations up to 1 year posttransplantation. Five others showed transient mixed chimerism in the T-lymphoid cell lineage. In one patient, host T cells persisted until leukemia relapse. The remaining 21 patients showed a complete chimerism throughout the period of investigation. Twenty-five of these patients were classified according to the presence (n = 10) or absence (n = 15) of recipient type T cells. Statistical analysis did not show significant differences in the distribution of a number of clinical variables between the two groups, nor in the actuarial survival (P = .11) and leukemia-free interval (P = .97). Therefore, these results suggest that persistence of recipient type T lymphoid cells after allogeneic BMT for hematologic malignancies is not correlated with leukemia relapse. In addition, we observed that persistence of host cells within the original leukemia cell lineage and at the correct maturational stage was predictive for leukemia relapse in one case.

The advances of molecular biological techniques of the last decade have made possible identification of a rapidly increasing number of congenital (or primary) immune deficiencies at the genetic level. As soon as an immune deficiency is becoming a likely conclusion from a series of diagnostic laboratory tests, these recent developments in molecular techniques allow us in principle to make a conclusive diagnosis on the basis of a well-defined genetic defect. The hereditary immune deficiencies can be divided in 4 groups according to the functional immune aberration: antigen recognition, communication between immune cells, adhesion and directional motility, and killing as an effector mechanism.

Severe congenital immunodeficiency diseases occur in approximately 1:10,000 newborns, in Western Europe and North America. They are characterised by recurrent infections, mostly caused by opportunistic micro-organisms, and autoimmune phenomena. In many patients the immune deficiency occurs as part of a syndrome. An immunological dysfunction may be caused by total absence, strong reduction or dysfunction of one or more cellular elements or of cell-associated or cellular secretion products. It is convenient to divide the immune system into a cellular compartment (with specific T and B cells, and non-specific natural killer cells and myelomonocytes), a compartment for cellular interactions (adhesion, costimulation and communication through cytokines) and an aspecific opsonization compartment. In cases of deficient humoral immunity treatment options are antimicrobial drugs and substitution therapy with immunoglobulins, in cases of phagocytic dysfunction the application of intracellularly active antibiotics and in cases of disturbances of cellular immunity allogeneic transplantation of haematopoietic stem cells and in the future perhaps the repair of the genetic defect by somatic gene therapy.

MHC class II deficiency or bare lymphocyte syndrome is a severe combined immunodeficiency caused by defects in MHC-specific transcription factors. In the present study, we show that fibroblasts derived from a recently identified bare lymphocyte syndrome patient, SSI, were mutated for RFX5, one of the DNA-binding components of the RFX complex. Despite the lack of functional RFX5 and resulting MHC class II-deficient phenotype, transfection of exogenous class II transactivator (CIITA) in these fibroblasts can overcome this defect, resulting in the expression of HLA-DR, but not of DP, DQ, and invariant chain. The lack of invariant chain expression correlated with lack of CIITA-mediated transactivation of the invariant chain promoter in transient transfection assays in SSI fibroblast cells. Consequently, these CIITA transfectants lacked Ag-presenting functions.

To study the effects of major histocompatibility complex (MHC) class II expression on T-cell development, we have investigated T-cell immune reconstitution in two MHC class II-deficiency patients after allogeneic bone marrow transplantation (allo-BMT). Our study showed that the induction of MHC class II antigen expression on BM graft-derived T cells in these allo-BMT recipients was hampered upon T-cell activation. This reduction was most striking in the CD8(+) T-cell subset. Furthermore, the peripheral T-cell receptor (TCR) repertoire in these graft-derived MHC class II-expressing CD4(+) and in the CD8(+) T-cell fractions was found to be restricted on the basis of TCR complementarity determining region 3 (CDR3) size profiles. Interestingly, the T-cell immune response to tetanus toxoid (TT) was found to be comparable to that of the donor. However, when comparing recipient-derived TT-specific T cells with donor-derived T cells, differences were observed in TCR gene segment usage and in the hydropathicity index of the CDR3 regions. Together, these results reveal the impact of an environment lacking endogenous MHC class II on the development of the T-cell immune repertoire after allo-BMT.

The first report of a positive effect of allogeneic bone marrow transplantation (BMT) on the clinical course in a patient with a lysosomal storage disease was described in 1981. Since then, over 200 patients have been treated in this way but data are scarce and fragmentary. Allogeneic BMT involves replacement of the patient's haemopoietic system by that of a donor. The new cells that repopulate the body can correct the metabolic disturbance. Most experience with allogeneic BMT was gained in patients with mucopolysaccharidosis type I, metachromatic leukodystrophy and adrenoleukodystrophy. Allogeneic BMT reduces the amount of storage material in internal organs: skeletal abnormalities and neurological symptoms are at best stabilized. Transplantation-related mortality and morbidity are high. The applicability of allogeneic BMT is limited.

We found an unusual fc gamma receptor IIIa (CD16) phenotype on the natural killer (NK) cells of a 3-year-old boy, who suffered from recurrent viral respiratory tract infections since birth. He also had severe clinical problems after Bacille Calmette-Geérin (BCG) vaccination and following Epstein-Barr virus and Varicella Zoster virus infections. His peripheral blood lymphocytes contained a normal percentage and absolute number of CD3-CD7+ cells, which were positively stained with the CD16 monoclonal antibodies (MoAbs) 3G8 and CLBFcRgran1, but did marginally stain with the CD16 MoAb Lau11c/B73.1. Fc gamma RillIb expression on granulocytes appeared to be normal. NK cell function, analyzed in vitro by direct cytotoxicity on K562 target cells and ADCC-activity on P815 target cells, was normal compared with an age-matched healthy control. Sequence analysis of the Fc gamma RIIIA gene, encoding CD16 on NK cells and macrophages, showed a T to A nucleotide substitution at position 230 on both alleles, predicting a leucine (L) to histidine (H) amino acid change position 48 in the first extracellular lg-like domain of Fc gamma RIIIa, which contains the Leu11c/B73.1 epitope. The combined use of CD16 and CD56 MoAbs labeled with the same fluorescent dye, as often applied in routine immunophenotyping procedures, will leave these homozygotes undiagnosed. The pattern of infections in this patient is in agreement with the postulated function of NK cells in the immunological defense against viruses and other intracellular microorganisms. Further analysis of the NK cell function in vitro and follow-up of the clinical course of Fc gamma RIIIA-48H/H homozygotes is required to ascertain whether this genotype is causally related to an NK cell immunodeficiency.

B cell lymphoproliferative disorders (BLPD) are relatively frequent after genotypically non-HLA-identical BMT. We performed univariate analysis to study which BMT-related variables were associated with an increased risk of developing BLPD. Sixty-five recipients of other than genotypically HLA-identical BM grafts were included in the study. Seventy-seven recipients of genotypically HLA-identical BM grafts served as a comparison group. BLPD occurred in nine of 65 children after non-HLA-identical BMT (14%) and in none of the 77 children after HLA-identical BMT (0%). In all cases, BLPD was proven to be EBV-related. Our data suggest that the combined use of Campath 1G and anti-LFA1 was associated with an increased risk of developing BLPD, particularly children who had received a T cell-depleted BM graft, using albumen density gradient sedimentation followed by E-rosetting, and who were conditioned with Ara-C, CY and TBL. In addition, T cell numbers below 50/microliters at 1 month and below 100/microliters at 2 months after BMT, respectively, were associated with an increased risk of developing BLPD. Longitudinal determination of T cell numbers after non-HLA-identical BMT is a simple method for identifying patients at risk of developing BLPD. In addition to monitoring levels of circulating EBV-infected lymphocytes, monitoring T cell numbers may allow early intervention to prevent progression of BLPD.

After allogeneic BMT, transient homogeneous Ig components (H-Ig) can be detected in the sera of most graft recipients. So far, data on the antigen-specificity and therefore the function of these H-Ig are not available. Such information may be important for our understanding of the underlying mechanisms that are responsible for these excessive clonal B cell expansions, and it may help to delineate the functional antibody repertoire after BMT. In the present study, sera of 98 paediatric BM graft recipients were investigated for the potential presence of H-Ig of IgG isotype (H-IgG) with specificity towards a panel of antigens, including vaccine and herpes virus antigens, auto-antigens and allo-antigens. The vast majority of H-IgG in sera of BM graft recipients were unreactive when tested for this panel of antigens. However, in four cases, antigen-specificity of H-IgG to tetanus toxoid could be demonstrated after vaccination with that antigen. An explanation for the negative findings may be either that a restricted antibody production had been elicited by other non-tested antigens, eg substances of colonizing and translocating bacteria or of food antigens, or that the H-IgG components may have anti-idiotype or anti-'self' specificity.

Allogeneic BMT using a non-genotypically HLA-identical donor may be curative for children suffering from lethal haematological diseases, who lack a genotypically HLA-identical donor. Unfortunately, graft failures are often seen, especially after T cell depletion of the graft. We studied whether untimely decreased counts of leucocytes and reticulocytes in peripheral blood might predict graft failure at an early stage. Fifty-five recipients of a non-genotypically HLA-identical BM graft were included in the study; data from these children were compared with those of 77 recipients of a genotypically HLA-identical BM graft. Time-related reference values of peripheral blood leucocyte and reticulocyte counts were established in graft recipients with proven donor-origin recovery after BMT. Graft failure after nonHLA-identical BMT was observed in 16 out of 55 children (29%) and after HLA-identical BMT in one out of 77 (1.3%). With respect to early graft failure, the predictive value of granulocyte numbers falling below the lower limit of the reference values and a rapid decline of reticulocyte numbers after their appearance in peripheral blood was 100% (95% confidence intervals of 83-100% and of 80-100%, respectively). Early immunosuppressive intervention was applied in six patients and was successful in three of them.

Pediatric recipients (n = 25) of an allogeneic bone marrow (BM) graft were selected on the basis of informative IgG allotype (Gm) differences between the BM donor and the recipient. To investigate the kinetics of the appearance of IgG of donor origin and the disappearance of IgG of recipient origin, G1m and G2m allotype levels were quantified in sera obtained at regular intervals between 3 months and 5 years after BM transplantation (BMT). For this quantification, a dot immunobinding assay (DIBA) has been developed. In 19 of 22 informative recipients, the Gm allotype distribution had reached the range of values expected on the basis of the Gm phenotype of the donor within 6 months after BMT. Remarkably, IgG of recipient origin persisted in 15 of 18 informative recipients until last follow up, ie, for several years after BMT. In addition to the origin of total IgG production, the origin of homogeneous IgG components (H-IgG) appearing after BMT was investigated. H-IgG of donor origin could be detected as early as 3 weeks after BMT, but also H-IgG of recipient origin were present in 8 of 13 informative recipients for a period of up to 1 year after BMT. We conclude that host-type IgG-producing cells were not eradicated by the (myeloablative) conditioning regimen and persisted in a high number of graft recipients. It is our hypothesis that lack of graft-versus-host disease (GVHD) in the majority of these recipients results in the persistence of IgG-producing cells of host origin. These observations may be relevant for the evaluation of patients who received allogeneic BMT for the treatment of multiple myeloma.

Inventory of patients with immunodeficiency in the Netherlands.Retrospective over the period 1970-1983.Dutch Working Group on Immunodeficiency, University Children's Hospital, 'Het Wilhelmina Kinderziekenhuis', Utrecht.A questionnaire with specific information on immunodeficient patients was completed by clinical members of the Working Group i.e. paediatricians and internists. Diagnosis of the immunodeficiency was according to WHO recommendations.The data of 336 patients could be included and analysed. Defects in humoral immunity were the most common (62.9%), followed by combined defects (18.75%), T cell defects (7.4%), phagocytic disorders (6.8%) and complement component deficiencies (2.7%). Sixty-two patients (15.5%) died, notably those with combined T and B cell defects, selective T cell defects and those with immunodeficiency based on DNA repair defects. Autoimmunity was relatively frequent in immunodeficiency (7.7%) as was cancer (2.7%).Results were similar to those of other European countries.

To determine the variability and the natural course of children suffering from autosomal recessive osteopetrosis to allow optimal counseling and decision making with respect to therapeutic intervention.Retrospective and longitudinal evaluation of clinical symptoms and natural course with emphasis on survival and sensoneurologic and hematologic findings.Two large referral-based pediatric bone marrow transplantation units in Europe.Thirty-three patients with autosomal recessive osteopetrosis admitted to units in Paris and Leiden between 1972 and 1988 were analyzed until last follow-up or the time at which bone marrow transplantation was performed. The great number of patients and unprecedented amount of data make this report one of the single largest clinical studies on autosomal recessive osteopetrosis.Ocular involvement occurring at a median age of 2 months was the symptom at initial examination in half of the patients. A striking variability between patients was found. Retinal degeneration was present in three patients and associated generalized neurodegeneration was present in two. The probability of survival until the age of 6 years was about 30% for the group as a whole. The cumulative risk of developing visual or hematologic impairment in the first year of life was about 75% and leveled off afterward. Patients with early hematologic impairment, ie, before 3 months of age, especially when combined with early visual impairment, had a very poor prognosis regarding life expectancy.Autosomal recessive osteopetrosis seems to be a variable disorder with a poor prognosis, especially in children with early visual and hematologic impairment. Allogenic bone marrow transplantation remains the only curative approach.

The material from 59 and 134 cerebrospinal fluid (CSF) samples, taken in the week prior to grafting and during the first 100 days after grafting respectively, were examined from 37 children undergoing allogeneic BMT. All CSF samples were obtained from lumbar punctures performed for regular administration of methotrexate (MTX). Prior to bone marrow transplantation (BMT) the increased protein levels and increased cell numbers noted in two children were probably caused by arachnoiditis from MTX administration 5 days earlier. After BMT, cell numbers increased in 54% of children and in 27% of children increased protein levels were found in the CSF. No correlation with clinical findings including neurological findings, infections and prior intrathecal MTX administration was found. The CSF changes observed are probably induced by the conditioning regimen (i.e. cyclophosphamide and total body irradiation). We conclude that the normal ranges for CSF components seen in healthy individuals do not apply in BMT recipients during the first 100 days after grafting.

All Dutch academic and non-academic pediatric training centres were requested to give a survey of all scientific publications produced from 1986 to 1990. The initiative came from the board of the Dutch Pediatric Association. During the observation period (1986-1990) the number of international publications with an external reference system was twice when compared with 1981-1985. The quality of the publications was similar. In at least four of the participating academic pediatric hospitals most publications came from the subspecialty area of metabolic diseases, oncology/hematology, immunology/infectious diseases, and cardiology, both during the first and the second observation period. From 1986 to 1990 the total number of theses in which a pediatrician was the main promoter or promoter in charge came to 84.

Growth characteristics of stromal cells, assessed as adherent cells in long-term bone marrow cultures, and of hematopoietic progenitor cells, prior to and shortly after allogeneic bone marrow transplantation (BMT), were investigated, more specifically with regard to their possible correlations. The main constituent cells of the bone marrow stroma, that is, endothelial cells, reticular cells/fibroblasts, and monocytes/macrophages, showed an as yet inexplicable increased growth in samples taken from recipients prior to BMT, as compared with the growth in samples from their healthy donors and those taken after BMT. In the third week after BMT the in vitro outgrowth of hematopoietic precursors was severely depressed, but cell numbers in the adherent layer were normal. No relationship between in vitro growth of hematopoietic precursor cells and stromal cells was observed at that time. Probably the precursor cells growing in vitro are committed progenitor cells, relatively independent of stromal influences. In the eight week after grafting, endothelial cell outgrowth in vitro was highly correlated with granulocyte-macrophage colony-forming unit (CFU-GM) colony formation and to a lesser extent with mixed-lineage colony-forming unit (CFU-Mix) colony formation. This may indicate the reappearance of cytokine-mediated influences or the reappearance of a direct interaction, for example, by cell-cell contact between stromal cells and hematopoietic progenitor cells at that time.

One of the major complications of allogeneic bone marrow transplantation (BMT) is graft-versus-host disease (GvHD), which is caused by donor type lymphocytes which react against the recipient's tissues. An important factor which influences GvHD is the recipient's gastrointestinal microflora. This was originally observed in gnotobiotic mice. Infusion of 10(7) H-2 incompatible bone marrow cells into lethally irradiated (9.0 Gy X-rays) conventional mice results in a late onset type GvHD which causes the death of the majority of the recipients during the first two months after BMT. This mortality can be completely prevented if the recipients are germfree mice, or when they are conventional animals which have been subjected to complete or selective gastrointestinal decontamination (GID). In a mouse model, the mechanism responsible for the influence of the microflora on GvHD after allogeneic BMT was investigated. These studies indicate that GvHD can be induced by activated T-lymphocytes from donor origin reacting against bacterial antigens which might be cross-reactive with the recipient's epithelial tissue antigens. Activation of these T-lymphocytes is confined to antigens of certain bacterial species of the recipient which are not present in the indigenous microflora of the donor mice. These bacteria most likely belong to the anaerobic flora of the recipient. The latter hypothesis is strongly supported by the observation in human patients that, in contrast to complete GID, selective decontamination of the gastrointestinal tract did not have any beneficial effect on moderately severe to severe GvHD after transplantation with MHC-matched sibling donor bone marrow grafts.

Chromosomal studies were performed on 114 blood samples and 117 bone marrow samples, taken systematically over a period of 4 months after bone marrow transplantation (BMT) in 42 children grafted for acute lymphoblastic leukaemia (ALL) (n = 20), acute myeloid leukaemia (AML) (n = 16), non-Hodgkin's lymphoma (NHL) (n = 2) and myelodysplastic syndrome (MDS) (n = 4). In some cases, follow-up investigations were performed. In the first 4 months following BMT, mixed chimerism was frequently observed in blood of AML (25%), ALL (30%), NHL (100%) cases and in bone marrow samples of ALL (35%). The presence and relative number of a patient's own metaphases found shortly after transplantation was not related to leukaemia relapse and probably represents residual non-malignant haematopoietic precursor cells of the host. In only one child grafted for MDS with 45,XY, -7 karyotype was the marker clone still detectable in bone marrow at day +437 post-BMT. This patient shows no recurrence of the MDS and has a sustained haematological recovery at the time of writing, i.e. 4.5 years post-BMT. In 11 other patients, various structural chromosomal abnormalities (not related to the original leukaemia) were found in both peripheral blood and bone marrow. In three different patients structural anomalies were also found in bone marrow and blood samples from donor-derived cells. This indicates that, besides irradiation, there are other as yet unidentified factors (e.g. drugs), which are capable of inducing chromosomal anomalies in the post-BMT period.

Discrimination between donor and recipient peripheral blood cells through detection of the Y chromosome can be useful to document chimerism and engraftment after sex-mismatched bone marrow transplantation. Currently applied methods are hampered by the selection of cells (e.g., karyotyping of cells in metaphase) or by the fact that the detection of Y chromosome by in situ hybridization with specific probes does not allow further characterization of the cells. Although quinacrine staining of Y chromosomes can be performed on cells previously marked for membrane antigens, this staining is not fully discriminative between male and female cells. To circumvent this, a technique has been developed, in which mononuclear cells in suspension were stained for membrane antigens by the consecutive use of monoclonal antibodies and tetramethylrhodamine isothiocyanate conjugates. After the cells were spun down on slides and fixed with methanol/acetic acid and formaldehyde, in situ hybridization with a biotinylated Y-chromosome-specific DNA probe was performed. The probe was detected with avidin-fluorescein isothiocyanate and the signal was amplified by consecutive incubation with biotinylated anti-avidin and avidin-fluorescein isothiocyanate. The membrane staining for various antigens remained undisturbed during the hybridization procedure and the Y probe discriminated almost completely between male and female cells. Therefore, this approach allowed us to determine the chimerism within different subpopulations of unseparated mononuclear cells after sex-mismatched bone marrow transplantation with a sensitivity of 98% and a specificity of 100%.

We report two different disorders of the beta-globin gene cluster segregating in a Belgian family: a novel deletion that results in (G) gamma + ((A) gamma delta beta)(0)-thalassemia (thal) and a heterocellular hereditary persistence of foetal hemoglobin of the Swiss type linked to a delta(0)-thal gene (delta (0)-HPFH). Heterozygosity for the heterocellular HPFH brings about a moderate (3.4% to 8.24%) increase of hemoglobin (Hb) F having a G gamma/A gamma ratio of 4:1, whereas carriers of the G gamma + ((A) gamma delta beta)(0)-thal deletion show in their peripheral blood a considerably higher (15%) percentage of Hb F. Both defects interact in the compound heterozygotes for G gamma + ((A) gamma delta beta)(0)-thal and delta(0)-HPFH producing a further increase (up to 24%) of fetal Hb consisting entirely of G gamma chains. Molecular characterization of the (G) gamma + ((A) gamma delta beta)(0)-thal by means of Southern analysis showed that the deletion spans about 50 kb, removing the 3' end of the A gamma-gene, the psi beta-, delta-, and beta-genes. A number of possible mechanisms leading to the overproduction of Hb F in HPFH and (G) gamma + ((A) gamma delta beta)(0)-thal will be discussed.

The relative distribution of T cell subsets, as defined by the monoclonal antibodies OKT, was analyzed in peripheral blood lymphocytes of 8 children after bone marrow transplantation for aplastic anemia. The percentage of peripheral blood lymphocytes binding OKT3 (directed against a common T cell surface antigen and defining most peripheral T cells) reached normal values shortly after transplantation. In 5 patients, lymphocytes binding OKT8 (directed against an antigen present on the suppressor/cytotoxic T cell subset) were found in high proportion, and lymphocytes binding OKT4 (detecting an antigen present on inducer/helper T cells) in low percentage. This resulted in an inverted ratio OKT4/OKT8 compared with that of lymphocytes from normal individuals. In all patients the lymphocytes bound abnormally high amounts of OKT10 (directed against an antigen present on all thymocytes but not on mature peripheral T cells). In the course of time, a trend towards normalization was observed for all parameters investigated; however, the kinetics of the recovery showed a marked heterogeneity. From the analysis of this phenomenon, it is likely that, among other conditions yet unknown, a minimum of 20% of OKT4-positive lymphocytes is required for a normal proliferative response to T cell mitogens in vitro. No other correlation was found between any lymphocyte phenotype, as defined by the OKT antibodies, and proliferative response in vitro. Furthermore, lymphocytes from the patient with chronic graft-vs-host disease (greater than 50% OKT8 positive) failed to suppress the proliferative response to mitogens and antigens of the lymphocytes of the histo-identical bone marrow donor.

Reticulocytes in fixed human blood samples were stained for RNA with the fluorescent dye pyronin Y and measured by flow cytometry. The resulting relative frequency distributions of the RNA fluorescence intensities concurred with the different stages in maturation from early reticulocytes to mature red cells. A computer program was written to calculate from these frequency distributions the relative number of reticulocytes, their relative RNA content, and the median of the reticulocyte population (RNA index). This method was applied to 30 healthy blood bank donors (control group), as well as to patients with various hematologic disorders showing abnormal erythropoietic activity. The measured percentage of reticulocytes, RNA content, and RNA index were found to correlate well with the various hematologic disorders. Changes in erythropoiesis could be clearly followed, as was demonstrated by analyzing blood samples from children with aplastic anemia or acute myeloid leukemia, who were treated with allogeneic bone marrow transplantation. Measurements on blood samples from healthy blood bank donors showed that with this method, small changes in the reticulocyte population, such as the appearance of polychromatic erythrocytes in the peripheral blood 5–8 hr after donation, can be detected. The statistical reliability and the information provided on the maturation stage of the entire reticulocyte population make flow cytometry of peripheral blood reticulocytes a more informative method for the study of hematologic abnormalities than conventional methods for reticulocyte counting and classification.

Acute graft-versus-host disease (GVHD), a major complication of allogeneic bone marrow transplantation (BMT), is probably mediated by T lymphocytes present in the marrow graft. In this study, the repopulation of the peripheral blood with T4+ and T8+ T cells was investigated during the period preceding the occurrence of acute GVHD. Twenty-four allogeneic and 11 autologous BMT recipients were monitored from day 4 post-BMT onward by the use of monoclonal antibodies, indirect immunofluorescence, and flow cytometry. The recipients of allogeneic transplants received methotrexate as GVHD prophylaxis. Similar recovery patterns for T4+ and T8+ T cells were found following autologous and allogeneic BMT. However, lymphoid repopulation occurred at a clearly faster rate after autologous BMT. T4+ T cells were the first to reappear in the peripheral blood, followed by T8+ T cells 4–7 days later. The T8+ T cell reconstitution occurred at an even faster rate in patients who were to develop grade II-IV GVHD, as compared with those with grade O-I GVHD, thus leading to an earlier decrease in the T4/T8 ratio. Of 10 patients with a T4/T8 ratio less than 2.5 at day 19, 9 developed grade II-IV GVHD and 1 showed no GVHD. Of 14 patients with a ratio greater than 2.5 at that time, only 2 developed grade II-IV and 12 grade O-I GVHD (p less than 0.001). In the 11 patients developing grade II-IV GVHD, the T4/T8 ratio decreased to values less than 2.5 before the first clinical symptoms of GVHD in 9; it coincided in one and occurred later in another patient. Thus, early monitoring of the T4/T8 ratio can distinguish patients at risk of developing grade II-IV GVHD.

SUMMARYArguments in favour of biotyping of Enterobacteriaceae excreted in the faeces of isolated patients, as a method of investigating the efficiency of the isolation procedures, are presented as well as a technical outline of the procedure. The study included three kidney transplantation patients, five acute myeloid leukaemia patients and four healthy persons as controls.The results show, apart from new colonizations during isolation, a difference in the mean number of contaminations and colonizations with different Enterobacteriaceae biotypes. It is concluded from these results, that the isolation procedures were not completely effective and that the AML patients studied had a decreased colonization resistance of their digestive tract. This was less evident in the kidney transplant group.

In der Periode von 1958 bis 1968 wurden in der ganzen Welt mehr als 200 Knochenmarktransplantationen bei Patienten mit verschiedenen Krankheiten vorgenommen, vor allem bei aplastischer Anamie und Leukamie. In den meisten Fallen wurde kein „take“ des Knochenmarks erreicht und die Resultate waren minimal (BORTIN 1970). Wahrend der letzten funf Jahre haben sich die Resultate jedoch deutlich verbessert, was vor allem der Zunahme unseres Wissens zu danken ist auf dem Gebiet der „matching“ von Donor und Empfanger mit Hilfe der HL-A Typisierung und gemengter Lymphozytenkultur („mixed lymphocyte culture“ oder MLC), und auf dem Gebiet der unterstutzenden Therapie.

Acute lymphoblastic leukemia (ALL) is the commonest malignancy in children and adolescents. It comprises about 83% of all leukemias in that age-group (Van Steensel-Moll, 1983). In the last decade the progress with the management of ALL has been slow, the most important innovations in the conventional cytoreductive regimens occurred in the intensification of the treatment protocols for the groups of patients discerned as having a poor prognosis (Chessels, 1982; Henze et al., 1982). Despite the overall rate of about 95% of complete remissions following “standard” remission-induction therapy, and the use of more intensive maintenance programs for bad-risk patients, a longlasting complete continuous remission (CCR) was only attained in 43% of the patients with poor prognostic factors in the study of the Children’s Cancer Study Group (CCSG), comprising 880 patients (Miller et al., 1983). The overall cure rate of children with ALL, i.e. a disease-free survival at 4 years and later after stopping chemotherapy is about 50% with the current treatment protocols.

One hundred and seventyeight children above the age of three months were treated for purulent meningitis (68 X N.meningitidis, 60 X H.influenzae, 19 X D.pneumoniae, 9 X other bacteria and 22 X with negative culture results) with a combination of either two or three antimicrobial drugs i.e. penicillin, sulphonamides and chloramphenicol, according to the causative microorganisms. The number of deaths was four (2%). Early neurological complications were seen in two (1%) and severe long-term sequelae in one (0.6%) of the patients. Antagonism between chloramphenicol and penicillin was not found in vivo, probably because the level of the latter drug was too low in the cerebrospinal fluid. These results contrast favourable with those after single-drug treatment i.e. with ampicillin, as reported in the literature. A possible explanation for this finding is the low level of beta-lactam antibiotics in the cerebrospinal fluid and the emergence of strains of H.influenzae and D.pneumoniae insensitive to such levels.

A case of leukemia relapse in a 15-year-old girl occurring 5 years after successful allogeneic bone marrow transplantation for acute non-lymphocytic leukemia is reported. Laboratory findings demonstrated that this relapse was in host cells and had the same phenotype as the original leukemia. Incomplete lymphoid chimerism after bone marrow transplantation might have resulted in an inappropriate graft-versus-leukemia effect.

The primary immune response in vivo of 20 patients with selective IgA deficiency was studied and compared to controls. The primary cellular immune response tested by dinitrochlorobenzene (DNCB) was decreased in many patients. The primary humoral immune response was elicited by immunization with the test immunogen Helix pomatia haemocyanin (HPH). Using a direct ELISA technique antibodies against HPH of the IgA, IgG and IgM class were measured. Two weeks after immunization no response of IgA anti-HPH was seen except in three patients who showed a low but detectable antibody level. In spite of normal or even elevated serum IgG and IgM levels there was a significantly lower response of the IgG and IgM anti-HPH antibodies at 2 weeks after immunization as compared to the controls followed by a further decline at 6 weeks. We conclude that selective IgA deficiency is often accompanied by more general disturbances in humoral and cellular immunity to newly encountered antigens.

In 15 children transplanted with allogeneic bone marrow for acute leukemia and in complete remission, regeneration of the early stages of the B cell system was studied. Bone marrow aspirates taken before and longitudinally after BMT were investigated for pre-B and B cells by immunofluorescence techniques; in some cases, TdT+ cells were also determined. Normal values were derived from bone marrow samples taken from 23 healthy individuals who served as bone marrow donors. In normal bone marrow, B cells outnumber pre-B cells and the latter are more numerous than TdT+ cells. Before BMT, the numbers of BM pre-B were outside the normal range in all cases; B cell numbers were abnormal in most of the 11 patients studied, probably due to the antileukemic remission induction/consolidation therapy. After BMT, two distinct patterns of regeneration of the B cell system were observed. In 9 patients, TdT+ cells were considerably increased early after BMT. This was followed by a rise in pre-B cells, with values well above the normal range, and resulting in ratios of TdT+:pre-B cells and of pre-B cells:B cells that were transiently greater than 1. In the other 6 patients, the regeneration of TdT+ cells varied and the reconstitution of the pre-B cells was more gradual than in the first group, with pre-B-to-B cell ratios less than 1 during the whole observation period. The only consistent difference between the patients of the two groups, possibly relevant to the regeneration of the B cell lineage, was the duration of corticosteroid therapy, which was much longer in the 6 patients with slow-pace reconstitution. The pace of regeneration of the B cell system in the bone marrow was correlated with the recovery of the humoral immunity, as indicated by a significant increase in specific antibody titers after the second vaccination with diphtheria-tetanus-poliomyelitis vaccine in 7 of 9 patients in the rapid-pace group, versus 2 of 6 patients in the slow-pace group.