A 1.1-kb duplication in the p67-phox gene causes chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency that is caused by a functional defect of the NADPH oxidase of phagocytes, and that leads to severe recurrent infections. CGD results from the absence or the dysfunction of various components of NADPH oxidase, and autosomal recessive CGD with the lack of p67-phox (A67 CGD) is the rarest form of the disease. Identifying familiar mutations in subjects with A67 CGD provides the most reliable method of detecting carriers and is the basis for prenatal diagnosis. In the present study, we report the detailed characterization of the first duplication in the p67-phox gene identified in a 30-year-old patient affected by systemic aspergillosis attributable to p67-phox deficiency. We show that this new mutation involving exons 9 and 10 is the result of a tandem duplication of approximately 1.1 kb, which resulted from the juxtaposition of intron 8 to intron 10. We have sequenced both the junction fragment of this duplication and the corresponding wild-type regions and have found that the breakpoint regions in intron 8 and in intron 10 show limited homology. Our result suggests that this interchange arose as an illegitimate recombination event. As in other non-homologous rearrangements previously reported, the duplication breakpoints are located within the sequence motif 5'-CCAG-3' and its complement 5'-CTGG-3'.     

Fungal infections in paediatric patients with chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency resulting from a disfunction of microbial capacity of phagocytes. Patients with this disease show great susceptibility to fungal and bacterial infections. Between 1988 and 1998, five paediatric patients with CGD who acquired mycotic infections were studied at the Paediatric Hospital Prof. Dr. J. P. Garrahan and their clinical and microbiological characteristics were described. The fungal infection appeared at the mean-age of 8.3 years (range: 1.1-17 years). All the patients had fever and lung involvement, three of them had suppurative abscesses of soft tissues. The mycological diagnosis was determined by microscopy, culture of clinical samples and serologic tests. There were three cases of disseminated aspergillosis, two cases of mixed infection: one due to Candida albicans and Nocardia asteroides and the other due to Scedosporium apiospermum and Cladosporium spp. Four out of the 5 patients died because of an infections process beyond control. Our conclusion is that new therapeutic measures must be considered along with the study of emerging pathogens in this group of patients.     

Autosomal recessive chronic granulomatous disease caused by novel mutations in NCF-2, the gene encoding the p67-phox component of phagocyte NADPH oxidase

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency disease that leads to severe recurrent infections. CGD is caused by defects in the phagocyte NADPH oxidase, a multiprotein enzyme that reduces oxygen to superoxide, a precursor of microbicidal oxidants. Less than 6% of CGD patients have an autosomal recessive form of the disease caused by mutations in NCF-2. This gene encodes p67-phox, a cytosolic oxidase subunit that associates with membrane-bound flavocytochrome b558 and regulates electron transfer. We studied six patients from five families with p67-phox deficiency and identified seven different mutant alleles. Patients from three of the kindreds were homozygous for their respective mutation, although the parents of only one family were known to be related. Five of the mutations have not previously been identified: (1) a missense mutation (383C-->T) in exon 5, (2) a nonsense mutation (196C-->T) in exon 3, (3) a missense mutation (230G-->A) in exon 3, (4) a nonsense mutation (298C-->T) in exon 4, and (5) a dinucleotide deletion (835-836 AC) from exon 9. Phagocytes from each of the patients analyzed failed to generate a measurable respiratory burst and had no detectable p67-phox protein. Our results further demonstrate that there is great heterogeneity among the mutations in p67-phox-deficient CGD patients, with no evidence for mutational hot-spots or a founder effect. Our data also support the hypothesis that the stability of p67-phox is particularly sensitive to missense mutations that cause amino acid substitutions within its N-terminal domain. In contrast, mutations predicting single amino acid changes elsewhere in the protein generally represent benign polymorphisms.     

Common variable immunodeficiency - an update

Common variable immunodeficiency (CVID) describes a heterogeneous subset of hypogammaglobulinemias of unknown etiology. Typically, patients present with recurrent bacterial infections of the respiratory and gastrointestinal tract. A significant proportion of CVID patients develops additional autoimmune, inflammatory or lymphoproliferative complications. CVID is the most frequent symptomatic primary immunodeficiency encountered in adults. Informative monogenetic defects have been found in single patients and families but in most cases the pathogenesis is still elusive. Numerous immunological studies have demonstrated phenotypic and functional abnormalities of T cells, B cells and antigen-presenting cells. A hallmark is the impaired memory B-cell formation that has been taken advantage of for classifying CVID patients. Clinical multi-center studies have demonstrated a correlation between immunological markers and clinical presentation. Long-term outcome is significantly influenced by delay of diagnosis and treatment and the presence of chronic inflammatory complications. While immunoglobulin replacement therapy plus antibiotics can control infections in most cases, patients with non-infectious inflammatory complications such as granulomatous inflammation, interstitial lung disease, inflammatory bowel disease, lymphoproliferation and developing malignancies still represent a therapeutic challenge. In this review we provide a systematic overview of the immunological, clinical, diagnostic and therapeutic aspects of CVID and highlight recent developments in these fields.     

Muscle-fiber transdifferentiation in an experimental model of respiratory chain myopathy

Common variable immunodeficiency (CVID) describes a heterogeneous subset of hypogammaglobulinemias of unknown etiology. Typically, patients present with recurrent bacterial infections of the respiratory and gastrointestinal tract. A significant proportion of CVID patients develops additional autoimmune, inflammatory or lymphoproliferative complications. CVID is the most frequent symptomatic primary immunodeficiency encountered in adults. Informative monogenetic defects have been found in single patients and families but in most cases the pathogenesis is still elusive. Numerous immunological studies have demonstrated phenotypic and functional abnormalities of T cells, B cells and antigen-presenting cells. A hallmark is the impaired memory B-cell formation that has been taken advantage of for classifying CVID patients. Clinical multi-center studies have demonstrated a correlation between immunological markers and clinical presentation. Long-term outcome is significantly influenced by delay of diagnosis and treatment and the presence of chronic inflammatory complications. While immunoglobulin replacement therapy plus antibiotics can control infections in most cases, patients with non-infectious inflammatory complications such as granulomatous inflammation, interstitial lung disease, inflammatory bowel disease, lymphoproliferation and developing malignancies still represent a therapeutic challenge. In this review we provide a systematic overview of the immunological, clinical, diagnostic and therapeutic aspects of CVID and highlight recent developments in these fields.     

Clinical Characteristics and Immunogenetics of BCGosis/BCGitis in Chinese Children: A 6 Year Follow-Up Study

In this study, the clinical and immunogenetical features in a cohort of Chinese patients with BCGosis/BCGitis were investigated. For the patients with abnormal immunological functions, Sanger sequencing was used to identify the involved genes. There were 74 confirmed cases of BCGosis/BCGitis during 2007–2012. Classified by infected tissues and organs, no cases only had local infection, 39 patients had a regional infection, 21 patients had a distant infection and 14 patients had a disseminated infection. Thirty-two patients (43.2%) had definitive primary immunodeficiency diseases (PID) and chronic granulomatous disease (CGD) is the most common PID (n = 23, accounted for 71.9% of all PID patients). For CGD patients, based on the anti-tuberculosis treatment, administration of rhIFN-γ resulted in better control of BCGosis/BCGitis. The results indicate that PIDs are associated with susceptibility to BCG disease. For children with BCGosis/BCGitis, immune function evaluation is necessary, and IFN-γ treatment for BCGosis/BCGitis patients with CGD is effective.     

Primary hypogammaglobulinaemia and arthritis.

Arthritis may be the first clinical manifestation of primary hypogammaglobulinaemia. In 16 years of 281 patients who had immunodeficiency, 30 had arthritis at presentation. It was more common in Bruton''s disease (15 (22%) of 69 patients) than in other forms of immunodeficiency (15 (7%) of 212 patients). Non-septic arthritis was more prevalent than septic arthritis, particularly monoarticular arthritis in Bruton''s disease and pauciarticular disease in common variable immunodeficiency. Boys in whom a diagnosis of Bruton''s disease was delayed were likely to develop recurrent infections complicated by arthritis. The measurement of serum immunoglobulin concentrations readily differentiates immunodeficiency from conditions such as Still''s disease and dictates subsequent management.     

Increased nitric oxide production by neutrophils from patients with chronic granulomatous disease on trimethoprim-sulfamethoxazole.

Chronic granulomatous disease (CGD) is an inherited disease characterized by severe and recurrent bacterial and fungal infections. Phagocytic cells of CGD patients are unable to produce superoxide anion, and their efficiency in bacterial killing is significantly impaired. In these patients, the prophylactic and therapeutic validity of a long-term use of trimethoprim-sulfamethoxazole (TMP-SMX) has been well established. However a role of nitric oxide (NO) produced by phagocytic cells from CGD patients is unknown, and the mechanism of TMP-SMX in CGD is unclear. We have directly measured NO production in whole human blood by using 4,5-diaminofluorescein as a novel fluorescent indicator for intracellular NO. Intracellular NO production of gated neutrophils increased time dependently when stimulated by lipopolysaccharide (LPS) and calcium ionophore. Although all polymorphonuclear leukocyte (PMN) specimens from patients with CGD failed to generate hydrogen peroxide, NO production by CGD PMNs was significantly increased compared with that of control PMNs (p<0.05). TMP-SMX with LPS significantly increased compared with LPS-stimulated samples at clinical (n=5, p<0.05) and 10-fold clinical concentrations (n=5, p<0.01). TMP-SMX with LPS in CGD PMNs significantly increased the production of NO in comparison with the LPS stimulation at 10-fold clinical concentrations (n=5, p<0.05). In conclusion, our data indicate the possibility that NO production by neutrophils from patients with CGD treated with TMP-SMX has a role of bactericidal activity instead of O(2)(-) in host defense mechanism.     

Human polymorphonuclear leukocytes inhibit Aspergillus fumigatus conidial growth by lactoferrin-mediated iron depletion

Aspergillus fumigatus, a common mold, rarely infects humans, except during prolonged neutropenia or in cases of chronic granulomatous disease (CGD), a primary immunodeficiency caused by mutations in the NADPH oxidase that normally produces fungicidal reactive oxygen species. Filamentous hyphae of Aspergillus are killed by normal, but not CGD polymorphonuclear leukocytes (PMN); however, the few studies on PMN-mediated host defenses against infectious conidia (spores) of this organism have yielded conflicting results, some showing that PMN do not inhibit conidial growth, with others showing that they do, most likely using reactive oxygen species. Given that CGD patients are exposed daily to hundreds of viable A. fumigatus conidia, yet considerable numbers of them survive years without infection, we reasoned that PMN use ROS-independent mechanisms to combat Aspergillus. We show that human PMN from both normal controls and CGD patients are equipotent at arresting the growth of Aspergillus conidia in vitro, indicating the presence of a reactive oxygen species-independent factor(s). Cell-free supernatants of degranulated normal and CGD neutrophils both suppressed fungal growth and were found to be rich in lactoferrin, an abundant PMN secondary granule protein. Purified iron-poor lactoferrin at concentrations occurring in PMN supernatants (and reported in human mucosal secretions in vivo) decreased fungal growth, whereas saturation of lactoferrin or PMN supernatants with iron, or testing in the presence of excess iron in the form of ferritin, completely abolished activity against conidia. These results demonstrate that PMN lactoferrin sequestration of iron is important for host defense against Aspergillus.     

Study of naïve and memory cells in a cohort of Egyptian chronic granulomatous disease patients

Abstract

Context: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder caused by inherited defects in the NADPH oxidase complex which may be involved in important pathways that connect innate and adaptive immunity. Objectives: Characterize the naive and memory compartment of B and T lymphocytes in patients with CGD. Methods: Twenty CGD patients and twenty healthy controls matched for age and sex were enrolled in this study. Flow cytometric assessment of the naïve and memory compartments of peripheral blood lymphocytes was done using cell surface markers CD45RO, CD45RA, CD27, CD3 and CD19. Results: There were 15 (79%) autosomal recessive CGD patients (8 females (53%) and 7 males (47%), 100% positive parental consanguinity) and four (21%) X-linked CGD patients. On comparing the 3 groups; AR CGD, X-linked CGD and controls, there was a positive statistical significant difference for the percentage and absolute count of CD19?+?CD27+ memory B cell (p?=?0.028 and p?=?0.047 respectively), CD45RA cells (with p values of p?=?0.000 and 0.033, respectively), the naïve compartment CD3?+?CD45RA+ cells percentage and absolute counts (p?=?0.005, 0.01respectively), CD3?+?CD27?+?cells percentage and absolute counts (p?=?0.001, 0.012 respectively), CD3?+?CD45RA?+?CD27+ cells percentage and absolute counts (p?=?0.015, 0.005, respectively). The significance was mainly attributed to the decrease in the X-linked group than control group. Conclusion: There was an altered naïve and memory B profile in CGD patients, this may increase susceptibility of the patients to opportunistic infections and autoimmune disorders. T-cell alterations have to be interpreted cautiously especially in the presence of infections.     

Molecular quality control machinery contributes to the leukocyte NADPH oxidase deficiency in chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease caused by defects in leukocyte NADPH oxidase. Various inherited defects in one of the membrane-bound components of NADPH oxidase, gp91-phox, cause X-linked (X91) CGD. Analysis of three patients with X91 CGD revealed that different mechanisms of molecular quality control lead to the common phenotype of absence of mature membrane-bound NADPH oxidase complex in leukocytes. In the first patient, aberrant intron splicing created a premature stop codon. However, the mutant mRNA was degraded prematurely, which prevented the production of truncated protein. In the second patient, a frameshift mutation with the potential to generate a gp91-phox polypeptide, with an aberrant and elongated C-terminus, led to barely detectable levels of gp91-phox, even though the reported functional domains of the protein appeared unaffected. In the third patient, a point mutation created a single amino acid change in the predicted FAD-binding site of gp91-phox. Although gp91-phox was detectable with Western blotting, no cytochrome b(558) was expressed on the cell surface. These analyses showed that molecular quality control machinery plays an important role in the pathogenesis of CGD, not only in the X910 but also in the X91- form of this X-linked disease.     

Phenotypic diagnosis of primary immunodeficiencies in Antioquia,Colombia, 1994-2002

Recurrent infections are a frequent cause of medical visits. They can be due to a heterogeneous group of dysfunctions that increase the susceptibility to pathogenic and opportunistic microorganisms, such as immunological deficiencies. To define an opportune rational treatment and to guide the molecular diagnosis of primary immunodeficiency diseases, we establish a program for the phenotypic diagnosis of these illnesses in Antioquia, Colombia, including clinical and laboratory evaluations of patients who present recurrent infections with abnormal evolution. Between August 1, 1994 and July 31, 2002, phenotypic diagnosis of primary immunodeficiency was made in 98 patients. Similar to data reported in the literature, antibody deficiencies were the most frequent (40.8%), followed by combined deficiencies (21.4%). This phenotypic characterization has allowed for appropriate treatments for each patient and, in some cases, functional and molecular studies that can lead to a definite molecular diagnosis.     

Gene therapy for inherited diseases using heamatopoietic stem cells--gene therapy for patients with chronic granulomatous disease

The possibility of gene therapy for inherited diseases with a single gene mutation in Figure 1 had been verified by the successful treatment with bone marrow transplantation. As the gene therapy method and theory has been progressing rapidly, it is expected that gene therapy will overcome the complications of bone marrow transplantation. Of these inherited diseases, chronic granulomatous disease (CGD) is the one of the most expected disease for gene therapy. CGD is an inherited immune deficiency caused by mutations in any of the following four phox genes encoding subunits of the superoxide generating phagocyte NADPH oxidase. It consists of membranous cytochrom b558 composed of gp91 phox and p22 phox, and four cytosolic components, p47 phox, p67 phox, rac p21 and p40 phox, which translocate to the membrane upon activation. In our group study, more than 220 CGD patients has been enrolled. The incidence of CGD patients was estimated as 1 out of 250,000 births. The expected life span of the CGD patients is 25 to 30 years old by the Kaplan Meier analysis. Comparing with the ratio of CGD subtype in US and Europe, that with p47phox deficiency is lower (less than 10%/o vs. 23%) and that of gp91 phox deficiency is higher (more than 75% vs. 60%). Prophylactic administration of ST antibiotics and IFN-gamma and bone marrow transplantation have been successfully employed in our therapeutic strategy. However, it is necessary to develop the gene therapy technology for CGD patients as more promising treatment. In the current study we constructed two retrovirus vectors; MFGS-gp91/293 SPA which contains only the therapeutic gp91 phox gene, a bicistronic retrovirus pHa-MDR-IRES-gp91/PA317 which carries a multi drug resistant gene (MDR1) and the gp91phox gene connected with an internal ribosome entry site (IRES). We demonstrate high efficiency transduction of gp 91 phox to CGD EB virus established cell line with high levels of functional correction of the oxidase by MFGS-gp91 and by pHa-MDR-IRES-gp91, respectively. We also demonstrate sufficient transduction of gp91 phox to CD34+ hematopoietic stem cell from the patients with gp91 phox deficiency by MFGS-gp91/293 SPA. Our current studies suggest that the combination of the 293-SPA packaging system and the bicistronic retrovirus system inserted MDR1 gene make our CGD gene therapy more feasible for clinical application.     

Invasive Fungal Infections in the Child with Chronic Granulomatous Disease

Invasive fungal infections (IFI) are a major threat for patients with chronic granulomatous disease (CGD) which is an inherited disorder of NADPH oxidase. The absence of a functional NADPH oxidase complex affects the display of an efficient antimicrobial effect as well as a controlled inflammatory response. Invasive aspergillosis caused by either Aspergillus fumigatus or A. nidulans is the most common IFI. Aspergillus nidulans infections seem to display a unique interaction with the CGD host and are seldom reported in other immunocompromised hosts. The occurrence of mucormycosis in the CGD host is mainly noted in the setting of treatment of inflammatory complications with immunosuppressive drugs. Candida infections are infrequently seen and show an age-dependent clinical presentation mainly affecting infants and young children. Furthermore, the child with CGD is susceptible to a wide range of fungal pathogens, indicating the need to determine the causative fungus, often by invasive diagnostic approaches, to guide optimal and rational treatment. Currently, it is becoming more and more clear that the exaggerated inflammatory response to fungal infection in the CGD host is leading in the pathogenesis, and antiinflammatory treatment might become as important as antifungal treatment in this specific host.     

Phagocyte NADPH oxidase,chronic granulomatous disease and mycobacterial infections

Infection of humans with Mycobacterium tuberculosis remains frequent and may still lead to death. After primary infection, the immune system is often able to control M. tuberculosis infection over a prolonged latency period, but a decrease in immune function (from HIV to immunosenescence) leads to active disease. Available vaccines against tuberculosis are restricted to BCG, a live vaccine with an attenuated strain of M. bovis. Immunodeficiency may not only be associated with an increased risk of tuberculosis, but also with local or disseminated BCG infection. Genetic deficiency in the reactive oxygen species (ROS)‐producing phagocyte NADPH oxidase NOX2 is called chronic granulomatous disease (CGD). CGD is among the most common primary immune deficiencies. Here we review our knowledge on the importance of NOX2‐derived ROS in mycobacterial infection. A literature review suggests that human CGD patient frequently have an increased susceptibility to BCG and to M. tuberculosis. In vitro studies and experiments with CGD mice are incomplete and yielded – at least in part – contradictory results. Thus, although observations in human CGD patients leave little doubt about the role of NOX2 in the control of mycobacteria, further studies will be necessary to unequivocally define and understand the role of ROS.     

Reactive oxygen species produced by the NADPH oxidase 2 complex in monocytes protect mice from bacterial infections

Chronic granulomatous disease (CGD) is an inherited disorder characterized by recurrent life-threatening bacterial and fungal infections. CGD results from defective production of reactive oxygen species by phagocytes caused by mutations in genes encoding the NADPH oxidase 2 (NOX2) complex subunits. Mice with a spontaneous mutation in Ncf1, which encodes the NCF1 (p47(phox)) subunit of NOX2, have defective phagocyte NOX2 activity. These mice occasionally develop local spontaneous infections by Staphylococcus xylosus or by the common CGD pathogen Staphylococcus aureus. Ncf1 mutant mice were more susceptible to systemic challenge with these bacteria than were wild-type mice. Transgenic Ncf1 mutant mice harboring the wild-type Ncf1 gene under the human CD68 promoter (MN(+) mice) gained the expression of NCF1 and functional NOX2 activity specifically in monocytes/macrophages, although minimal NOX2 activity was also detected in some CD11b(+)Ly6G(+) cells defined as neutrophils. MN(+) mice did not develop spontaneous infection and were more resistant to administered staphylococcal infections compared with MN(-) mice. Most strikingly, MN(+) mice survived after being administered Burkholderia cepacia, an opportunistic pathogen in CGD patients, whereas MN(-) mice died. Thus, monocyte/macrophage expression of functional NCF1 protected against spontaneous and administered bacterial infections.     

Regulation of innate immunity by NADPH oxidase

NADPH oxidase is a critical regulator of both antimicrobial host defense and inflammation. Activated in nature by microbes and microbial-derived products, the phagocyte NADPH oxidase is rapidly assembled, and generates reactive oxidant intermediates (ROIs) in response to infectious threat. Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent and severe bacterial and fungal infections, and pathology related to excessive inflammation. Studies in CGD patients and CGD mouse models indicate that NADPH oxidase plays a key role in modulating inflammation and injury that is distinct from its antimicrobial function. The mechanisms by which NADPH oxidase mediates killing of pathogens and regulation of inflammation have broad relevance to our understanding of normal physiological immune responses and pathological states, such as acute lung injury and bacterial or fungal infections.     

A novel bacterium associated with lymphadenitis in a patient with chronic granulomatous disease

Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system causing defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections. We identified a novel gram-negative rod in excised lymph nodes from a patient with CGD. Gram-negative rods grew on charcoal-yeast extract, but conventional tests could not identify it. The best 50 matches of the 16S rRNA (using BLAST) were all members of the family Acetobacteraceae, with the closest match being Gluconobacter sacchari. Patient serum showed specific band recognition in whole lysate immunoblot. We used mouse models of CGD to determine whether this organism was a genuine CGD pathogen. Intraperitoneal injection of gp91(phox -/-) (X-linked) and p47 (phox -/-) (autosomal recessive) mice with this bacterium led to larger burdens of organism recovered from knockout compared with wild-type mice. Knockout mouse lymph nodes had histopathology that was similar to that seen in our patient. We recovered organisms with 16S rRNA sequence identical to the patient's original isolate from the infected mice. We identified a novel gram-negative rod from a patient with CGD. To confirm its pathogenicity, we demonstrated specific immune reaction by high titer antibody, showed that it was able to cause similar disease when introduced into CGD, but not wild-type mice, and we recovered the same organism from pathologic lesions in these mice. Therefore, we have fulfilled Koch's postulates for a new pathogen. This is the first reported case of invasive human disease caused by any of the Acetobacteraceae. Polyphasic taxonomic analysis shows this organism to be a new genus and species for which we propose the name Granulobacter bethesdensis.     

Deconstructing common variable immunodeficiency by genetic analysis

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency. Patients have recurrent bacterial infections and an increased risk of developing autoimmune diseases, lung damage, and selected cancers. Since 2003, four genes have been shown to be mutated in CVID patients: ICOS, TNFRSF13B (encoding TACI), TNFRSF13C (encoding BAFF-R) and CD19. Heterozygous mutations in TNFRSF13B are also associated with CVID, whereas the other three genes are purely recessive. Recent genetic linkage studies have also identified possible loci for dominant CVID genes on chromosomes 4q, 5p and 16q. These findings markedly improved the genetic diagnosis of CVID and point towards new strategies for future genetic studies. In addition, some CVID genes might be relevant to more common diseases such as asthma and stroke.     

Bone marrow transplantation in Canada.

Bone marrow transplantation is an established form of therapy for aplastic anemia and severe combined immunodeficiency. It is also a therapeutic option for acute leukemia in remission. Unfortunately, compatible donors are not available for most patients who could benefit from it. Further refinement of the techniques involved may make it suitable for more patients. Graft rejection, recurrent leukemia, graft-versus-host disease and interstitial pneumonia continue to be the main unsolved complications of bone marrow transplantation, but recent advances have decreased their frequency and severity. Most of the complications of allogeneic bone marrow transplantation may be eliminated with the use of autologous stem cells. For further refinement bone marrow transplantation should continue to be performed in large centres that combine treatment with research.