Genetic and immunological assessment of a bone marrow transplantation in a patient with a primary immune defect: leukocyte adhesion deficiency

Leukocyte adhesion deficiency (LAD) was suspected in a three weeks old girl from a family with an established history of LAD with a lack (less then 1%) of the beta 2 integrins CD 11a, b/CD 18 expression at the leukocytes surface, was engrafted with her mother HLA identical bone marrow at the age of 14 months. Repeated post transplantation (up to 22 months). Immunological assessments showed a good engraftment with 97% of the lymphocytes expressing CD11a/CD18. Cells proliferated normally in response to PHA and to Tetanus toxo?d after revaccination. The level of serum immunoglobulins was normal. Investigation of the CD18 intragenic polymorphic marker Avall before and after bone marrow transplantation (BMT) showed a transition from the Avall +/+ genotype to the mother's Avall +/- genotype. Similarly DNA fingerprints obtained with the patient genomic DNA, prepared from PBMC, prior and after transplantation, showed that the patient's DNA fingerprints pattern matched the mother's one. These findings are consistent with the good engraftment observed clinically. This study emphasizes the usefulness of the molecular techniques to evaluate the degree of chimerism in monitoring the outcome of bon marrow transplantation.     

Identification of a defect in DNA ligase IV in a radiosensitive leukaemia patient.

The major mechanism for the repair of DNA double-strand breaks (DSBs) in mammalian cells is non-homologous end-joining (NHEJ), a process that involves the DNA-dependent protein kinase [1] [2], XRCC4 and DNA ligase IV [3] [4] [5] [6]. Rodent cells and mice defective in these components are radiation-sensitive and defective in V(D)J-recombination, showing that NHEJ also functions to rejoin DSBs introduced during lymphocyte development [7] [8]. 180BR is a radiosensitive cell line defective in DSB repair, which was derived from a leukaemia patient who was highly sensitive to radiotherapy [9] [10] [11]. We have identified a mutation within a highly conserved motif encompassing the active site in DNA ligase IV from 180BR cells. The mutated protein is severely compromised in its ability to form a stable enzyme-adenylate complex, although residual activity can be detected at high ATP concentrations. Our results characterize the first patient with a defect in an NHEJ component and suggest that a significant defect in NHEJ that leads to pronounced radiosensitivity is compatible with normal human viability and does not cause any major immune dysfunction. The defect, however, may confer a predisposition to leukaemia.     

Clonal expansion after bone marrow transplantation in a patient with chronic myelogenous leukemia

A patient with chronic myelogenous leukemia (CML) having the standard [t(9;22), Ph] translocation is presented where the Philadelphia (Ph) chromosome disappeared following bone marrow transplantation (BMT). The Ph chromosome reappeared in host cells after one year of stable hematologic remission. Three additional cell lines, all possessing the Ph chromosome with other abnormalities were consistently present in her marrow cells. Two years after BMT, ninety percent of her dividing bone marrow cells had become leukemic. The patient's clinical status remains unchanged, despite complex cytogenetic findings. The high incidence of multiple aberrant leukemic clones present in this case remains intriguing. Possible mechanisms for this unique transformation after BMT are discussed.     

Successful bone marrow transplantation from an unrelated donor in a child with AML in second remission

In February 1986 we transplanted a 10-year-old girl with AML in second remission with the bone marrow of an unrelated donor. HLA-types were different for one A- and one B-antigen between patient and donor. Conditioning regimen consisted of 14 Gy total body irradiation with lung shielding, 8 X 3 g/m2 cytosin arabinoside and 90 mg/kg cyclophosphamide. GVHD-prophylaxis was performed with cyclosporin A, methotrexate and prednisolone. Only mild GVHD I of the skin could be observed after rapid engraftment. 100 days after transplantation the patient was in good clinical condition and GVHD-prophylaxis was discontinued without any reactivation of acute or chronic GVHD. Engraftment was documented by sex chromosome and blood group typing. 120 days after transplantation leukemic blasts were detected in the peripheral blood and the child died 130 days after BMT from relapse of the leukemia. Despite the negative outcome, this was the first successful bone marrow transplantation from a unrelated donor in Germany.     

Bone marrow transplantation in a patient with drug-induced aplastic anemia

A 23-year-old woman gravely ill with Pseudomonas septicemia secondary to presumed drug-induced bone marrow aplasia received marrow transplantation from two male HL-A identical sibling donors. She had a successful engraftment with excellent but temporary clinical improvement. Subsequently she succumbed to graft-versus-host disease manifested by Pseudomonas and Candida albicans septicemia, cytomegalovirus pneumonitis, three phases of dermatitis, nausea, vomiting, dysphagia, diarrhea, fever, edema and bone pain, with gradual but complete graft suppression by the 74th day after the transplantation. A second marrow transplant on the 70th day was unsuccessful.     

Successful allogeneic neonatal bone marrow transplantation devoid of myeloablation requires costimulatory blockade

A significant number of nonmalignant, progressive childhood disorders respond to bone marrow transplantation (BMT). Toxic myeloablative pretreatment regimens, graft failure, and graft-vs-host disease complicate the utility of BMT for neonatal treatment. We recently demonstrated high-dose BMT in neonatal animals enables chimeric engraftment without toxic myeloablation. Reagents that block T cell costimulation (anti-CD40L mAb and/or CTLA-4Ig) establish tolerant allogeneic engraftment in adult recipients. Donor lymphocyte infusion (DLI) re-establishes failing grafts and treats malignant relapse via a graft-vs-leukemia response. In this study, we tested the hypothesis that combining these approaches would allow tolerant allogeneic engraftment devoid of myeloablation in neonatal normal and mutant mice with lysosomal storage disease. Tolerant chimeric allogeneic engraftment was achieved before DLI only in the presence of both anti-CD40L mAb and CTLA-4Ig. DLI amplified allografts to full donor engraftment long-term. DLI-treated mice either maintained long-term tolerance or developed late-onset chronic graft-vs-host disease. This combinatorial approach provides a nontoxic method to establish tolerant allogeneic engraftment for treatment of progressive childhood diseases.     

Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

DNA ligase IV is an essential protein that functions in DNA non-homologous end-joining, the major mechanism that rejoins DNA double-strand breaks in mammalian cells. LIG4 syndrome represents a human disorder caused by mutations in DNA ligase IV that lead to impaired but not ablated activity. Thus far, five conserved motifs in DNA ligases have been identified. We previously reported G469E as a mutational change in a LIG4 syndrome patient. G469 does not lie in any of the previously reported motifs. A sequence comparison between DNA ligases led us to identify residues 468-476 of DNA ligase IV as a further conserved motif, designated motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly, however, such mutational changes do impact upon double-stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggest that residues 470-476 function as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.     

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

The nonhomologous end-joining (NHEJ) pathway is essential for radioresistance and lymphocyte-specific V(D)J (variable [diversity] joining) recombination. Defects in NHEJ also impair hematopoietic stem cell (HSC) activity with age but do not affect the initial establishment of HSC reserves. In this paper, we report that, in contrast to deoxyribonucleic acid (DNA)-dependent protein kinase catalytic subunit (DNA-PKcs)-null mice, knockin mice with the DNA-PKcs(3A/3A) allele, which codes for three alanine substitutions at the mouse Thr2605 phosphorylation cluster, die prematurely because of congenital bone marrow failure. Impaired proliferation of DNA-PKcs(3A/3A) HSCs is caused by excessive DNA damage and p53-dependent apoptosis. In addition, increased apoptosis in the intestinal crypt and epidermal hyperpigmentation indicate the presence of elevated genotoxic stress and p53 activation. Analysis of embryonic fibroblasts further reveals that DNA-PKcs(3A/3A) cells are hypersensitive to DNA cross-linking agents and are defective in both homologous recombination and the Fanconi anemia DNA damage response pathways. We conclude that phosphorylation of DNA-PKcs is essential for the normal activation of multiple DNA repair pathways, which in turn is critical for the maintenance of diverse populations of tissue stem cells in mice.     

DNA ligase IV as a new molecular target for temozolomide

Temozolomide (TMZ) is a methylating agent used in chemotherapy against glioblastoma. This work was designed to clarify details in repair pathways acting to remove DNA double-strand breaks (DSBs) induced by TMZ. Cultured mouse embryonic fibroblasts were used which were deficient in DSB repair genes such as homologous recombination repair-related genes X-ray repair cross-complementing group 2 (XRCC2)and radiation sensitive mutant54 (Rad54), non-homologous end joining repair-related gene DNAligase IV (Lig4). Cell sensitivity to drug treatments was assessed using colony forming assays. The most effective molecular target which was correlated with TMZ cell sensitivity was Lig4. In addition, it was found that small interference RNAs (siRNA) for Lig4 efficiently enhanced cell lethality induced by TMZ in human glioblastoma A172 cells. These findings suggest that down regulation of Lig4 might provide a useful tool for cell sensitization during TMZ chemotherapy.     

Frontiers in bone marrow transplantation

The early murine experiments and human studies that indicated the potential of marrow transplantation are reviewed. The results of marrow grafting for a variety of human diseases are summarized. Current directions of research are indicated.     

Interleukin-2 induces chemotactic deficiency in patients with onco hematologic malignancies and autologous bone marrow transplantation.

Unusual gram positive bacteremia has been reported in non granulopenic patients receiving recombinant human interleukin-2 (IL-2) suggesting a beneficial effect of anti gram positive prophylaxis in such patients. We report here studies on granulocyte functions examined during the course of high dose IL-2 therapy (16 to 24 million IU/m2/days for 11 to 18 days) administered during a period of 35 days in 14 patients including 4 solid tumors, 5 chronic myeloid leukemias, 4 recipients of autologous bone marrow transplant (ABMT) and 1 recipient of syngeneic bone marrow transplant. Neutrophils functions were studied before IL-2 administration (d 0), after the first cycle (d 8) and after the third cycle (d 36). Nylon fiber adherence, superoxide production, random migration, phagocytosis, nitroblue tetrazolium reduction, lysozyme and elastase release were not impaired significantly throughout therapy. However N-Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) stimulated chemotaxis of granulocytes, normal before therapy, was significantly impaired as early at d 8 and severely inhibited at d 36 (p less than 0.001). Three septicemia, one corynebacteria parvum septicemia and two gram-negative septicemia despite normal neutrophil counts and oxacillin or Penicillin G plus Pefloxacin prophylaxis, occurred among the 14 patients studied. Although neutrophil functions were not more depressed in transplanted patients than in the other non transplanted patients, special attention should be paid to such patients in whom delayed immune reconstitution could increase the risk of sepsis.     

Preparing the patient for bone marrow transplantation: nursing care issues

The phases of bone marrow transplantation can be identified as the pre-transplant period, the immediate post-transplant period, and the late post-transplant period. The pre-transplant period is characterized by identification of the appropriate type of transplant to be done and, if necessary, finding an appropriate donor; entry of the patient into the transplant unit; administration of the preparative chemotherapy/irradiation regime; management of early toxicities; and pre-transplant supportive care. Nurses play an integral role during the entire transplant process. During the pre-transplant phase, nursing expertise is exemplified in the administration of chemotherapy, management of side effects, teaching of transplant procedures to patient and family, and supportive care. This paper reviews the patient care issues during the pre-transplant phase of bone marrow transplantation and identifies nursing management strategies.     

DNA ligase IV deficiency in mice leads to defective neurogenesis and embryonic lethality via the p53 pathway

DNA ligase IV (LIG4) is a nonhomologous end-joining (NHEJ) protein used for V(D)J recombination and DNA repair. In mice, Lig4 deficiency causes embryonic lethality, massive neuronal apoptosis, arrested lymphogenesis, and various cellular defects. Herein, we assess potential roles in this phenotype for INK4a/ARF and p53, two proteins implicated in apoptosis and senescence. INK4a/ARF deficiency rescued proliferation/senescence defects of Lig4-deficient fibroblasts but not other phenotypic aspects. In contrast, p53 deficiency rescued embryonic lethality, neuronal apoptosis, and fibroblast proliferation/senescence defects but not lymphocyte development or radiosensitivity. Young Lig4/p53 double null mice routinely died from pro-B lymphomas. Thus, in the context of Lig4 deficiency, embryonic lethality and neuronal apoptosis likely result from a p53-dependent response to unrepaired DNA damage, and neuronal apoptosis and lymphocyte developmental defects can be mechanistically dissociated.     

Catalytically inactive DNA ligase IV promotes DNA repair in living cells

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo et al. propose that NHEJ fidelity can be maintained by restricting end-processing to a ligation competent short-range NHEJ complex that ‘maximizes the fidelity of DNA repair’. These in vitro studies show that although this short-range NHEJ complex requires DNA ligase IV (Lig4), its catalytic activity is dispensable. Here using cellular models, we show that inactive Lig4 robustly promotes DNA repair in living cells. Compared to repair products from wild-type cells, those isolated from cells with inactive Lig4 show a somewhat increased fraction that utilize micro-homology (MH) at the joining site consistent with alternative end-joining (a-EJ). But unlike a-EJ in the absence of NHEJ, a large percentage of joints isolated from cells with inactive Lig4 occur with no MH – thus, clearly distinct from a-EJ. Finally, biochemical assays demonstrate that the inactive Lig4 complex promotes the activity of DNA ligase III (Lig3).     

No narcosis for bone marrow harvest in autologous bone marrow transplantation

A prospective study with mild general analgesia and sedation together with local anesthesia during bone marrow harvest was performed. Thirty-one patients underwent 33 bone marrow collections. Pretreatment consisted of 100 mg meperidine i.m. and 20 mg diazepam i.m. 1 h before start of procedure. Eight patients got additional meperidine and diazepam during the procedure, all patients got lidocaine 1% locally. A mean volume of 1.321 was obtained with 42.5 punctures. Twenty-two patients had no complications, 4 vomited, 4 had easily correctable hypotension of short duration, one got oxygen for cyanosis of short duration. Acceptance was good in 23 patients, in 6 reasonably well, in two bad. Only one patient experienced pain problems, due to suction. Anxiety was no major problem due to good information before the procedure and mild sedation. This form of anesthesia for bone marrow collection is a safe procedure, it is generally well accepted by the patient and it can be performed on an out-patient basis.     

Human DNA ligase IV and the ligase IV/XRCC4 complex: analysis of nick ligation fidelity

In addition to linking nicked/fragmented DNA molecules back into a contiguous duplex, DNA ligases also have the capacity to influence the accuracy of DNA repair pathways via their tolerance/intolerance of nicks containing mismatched base pairs. Although human DNA ligase I (Okazaki fragment processing) and the human DNA ligase III/XRCC1 complex (general DNA repair) have been shown to be relatively intolerant of nicks containing mismatched base pairs, the human DNA ligase IV/XRCC4 complex has not been studied in this regard. Ligase IV/XRCC4 is the sole DNA ligase involved in the repair of double strand breaks (DSBs) via the non-homologous end joining (NHEJ) pathway. During the repair of DSBs generated by chemical/physical damage as well as the repair of the programmed DSB intermediates of V(D)J recombination, there are scenarios where, at least conceptually, a capacity for ligating nicks containing mismatched base pairs would appear to be advantageous. Herein we examine whether ligase IV/XRCC4 can contribute a mismatched nick ligation activity to NHEJ. Toward this end, we (i) describe an E. coli-based coexpression system that provides relatively high yields of the ligase IV/XRCC4 complex, (ii) describe a unique rate-limiting step, which has bearing on how the complex is assayed, (iii) specifically analyze how XRCC4 influences ligase IV catalysis and substrate specificity, and (iv) probe the mismatch tolerance/intolerance of DNA ligase IV/XRCC4 via quantitative in vitro kinetic analyses. Analogous to most other DNA ligases, ligase IV/XRCC4 is shown to be fairly intolerant of nicks containing mismatched base pairs. These results are discussed in light of the biological roles of NHEJ.     

Supportive haemotherapy in bone marrow transplantation

The intensive supportive haemotherapy which has to be used in bone marrow transplantation is discussed, taking mainly into account platelet transfusions. Ways to avoid alloimmunizations against platelet antigens, especially HLA-ABC antigens, are shown (use of HLA-AB homozygous donors or of cross-reacting groups).     

Legal issues in bone marrow transplantation

The article discusses some of the more common legal issues involved in bone marrow transplantation. These include malpractice claims, testing prospective donors for AIDS, sale of bone marrow, informed consent for both donor and recipient, and questions that arise when the donor is a child.