The importance of using computed tomographic scanning in breast irradiation after conservative treatment

Fanconi anaemia (FA) is an autosomal recessive disease characterized by bone marrow failure, variable congenital malformations and predisposition to malignancies. Cells derived from FA patients show elevated levels of chromosomal breakage and an increased sensitivity to bifunctional alkylating agents such as mitomycin C (MMC) and diepoxybutane (DEB). Five complementation groups have been identified by somatic cell methods, and we have cloned the gene defective in group C (FAC)(7). To understand the in vivo role of this gene, we have disrupted murine Fac and generated mice homozygous for the targeted allele. The -/- mice did not exhibit developmental abnormalities nor haematologic defects up to 9 months of age. However, their spleen cells had dramatically increased numbers of chromosomal aberrations in response to MMC and DEB. Homozygous male and female mice also had compromised gametogenesis, leading to markedly impaired fertility, a characteristic of FA patients. Thus, inactivation of Fac replicates some of the features of the human disease.     

Multiple inhibitory cytokines induce deregulated progenitor growth and apoptosis in hematopoietic cells from Fac-/- mice

We used a murine model containing a disruption of the murine homologue (Fac) of Fanconi Anemia group C (FAC) to evaluate the role of Fac in the pathogenesis of bone marrow (BM) failure. Methylcellulose cultures of BM cells from Fac-/- and Fac+/+ mice were established to examine the growth of multipotent and lineage-restricted progenitors containing inhibitory cytokines, including interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), and macrophage inflammatory protein-1alpha (MIP-1alpha). Clonogenic growth of Fac-/- progenitors was reduced by 50% at 50- to 100-fold lower concentrations of all inhibitory cytokines evaluated. We hypothesized that the aberrant responsiveness to inhibitory cytokines in clonogenic cells may be a result of deregulated apoptosis. To test this hypothesis, we performed the TUNEL assay on purified populations of primary BM cells enriched for hematopoietic progenitors or differentiated myeloid cells. After stimulation with TNF-alpha, accentuated apoptosis was observed in both populations of Fac-/- cells. In addition, deregulated apoptosis was also noted in the most immature phenotypic population of hematopoietic cells after stimulation with MIP-1alpha. Together these data suggest a role of Fac in affecting the signaling of multiple cytokine pathways and support cytokine-mediated apoptosis as a major mechanism responsible for BM failure observed in FA patients.     

Functional correction of Fanconi anemia group A hematopoietic cells by retroviral gene transfer

Fanconi anemia (FA) is an autosomal recessive genetic disorder characterized by a variety of physical anomalies, bone marrow failure, and an increased risk for malignancy. FA cells exhibit chromosomal instability and are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC). FA is a clinically heterogeneous disorder and can be functionally divided into at least five different complementation groups (A-E). We previously described the use of a retroviral vector expressing the FAC cDNA in the complementation of mutant hematopoietic cells from FA-C patients. This vector is currently being tested in a clinical trial of ex vivo hematopoietic progenitor cell transduction. The FA-A group accounts for over 65% of all FA cases, and the FAA cDNA was recently identified by both expression and positional cloning techniques. We report here the transduction and phenotypic correction of lymphoblastoid cell lines from four unrelated FA-A patients, using two amphotropic FAA retroviral vectors. Expression of the FAA transgene was adequate to normalize cell growth, cell-cycle kinetics, and chromosomal breakage in the presence of MMC. We then analyzed the effect of retroviral vector transduction on hematopoietic progenitor cell growth. After FAA transduction of mutant progenitor cells, either colony number or colony size increased in the presence of MMC. In addition, FAA but not FAC retroviral transduction markedly improved colony growth of progenitor cells derived from an unclassified FA patient. FAA retroviral vectors should be useful for both complementation studies and clinical trials of gene transduction.     

The Fanconi anemia proteins FAA and FAC function in different cellular compartments to protect against cross-linking agent cytotoxicity

Fanconi anemia (FA) is an autosomal recessive disease characterized by chromosomal instability, bone marrow failure, and a high risk of developing malignancies. Although the disorder is genetically heterogeneous, all FA cells are defined by their sensitivity to the apoptosis-inducing effect of cross-linking agents, such as mitomycin C (MMC). The cloned FA disease genes, FAC and FAA, encode proteins with no homology to each other or to any known protein. We generated a highly specific antibody against FAA and found the protein in both the cytoplasm and nucleus of mammalian cells. By subcellular fractionation, FAA is also associated with intracellular membranes. To identify the subcellular compartment that is relevant for FAA activity, we appended nuclear export and nuclear localization signals to the carboxy terminus of FAA and enriched its localization in either the cytoplasm or the nucleus. Nuclear localization of FAA was both necessary and sufficient to correct MMC sensitivity in FA-A cells. In addition, we found no evidence for an interaction between FAA and FAC either in vivo or in vitro. Together with a previous finding that FAC is active in the cytoplasm but not in the nucleus, our results indicate that FAA and FAC function in separate subcellular compartments. Thus, FAA and FAC, if functionally linked, are more likely to be in a linear pathway rather than form a macromolecular complex to protect against cross-linker cytotoxicity.     

Impaired bone marrow microenvironment and immune function in T cell protein tyrosine phosphatase-deficient mice

The T cell protein tyrosine phosphatase (TC-PTP) is one of the most abundant mammalian tyrosine phosphatases in hematopoietic cells; however, its role in hematopoietic cell function remains unknown. In this report, we investigated the physiological function(s) of TC-PTP by generating TC-PTP-deficient mutant mice. The three genotypes (+/+, +/-, -/-) showed mendelian segregation at birth (1:2:1) demonstrating that the absence of TC-PTP was not lethal in utero, but all homozygous mutant mice died by 3-5 wk of age, displaying runting, splenomegaly, and lymphadenopathy. Homozygous mice exhibited specific defects in bone marrow (BM), B cell lymphopoiesis, and erythropoiesis, as well as impaired T and B cell functions. However, myeloid and macrophage development in the BM and T cell development in the thymus were not significantly affected. BM transplantation experiments showed that hematopoietic failure in TC-PTP -/- animals was not due to a stem cell defect, but rather to a stromal cell deficiency. This study demonstrates that TC-PTP plays a significant role in both hematopoiesis and immune function.     

Overexpression of the fanconi anemia group C gene (FAC) protects hematopoietic progenitors from death induced by Fas-mediated apoptosis

Fanconi anemia is a rare, inherited disorder characterized by bone marrow failure, congenital malformations, and cancer susceptibility. The group C Fanconi anemia gene, FAC, identified by expression cloning methods, encodes a protein of unknown function that may be involved in the response to apoptotic stimuli. Hematopoietic progenitor cells from Fac knock-out mice are hypersensitive to IFN-gamma, a molecule that can induce apoptosis through up-regulation of the Fas death receptor. In this study, we used FAC-overexpressing transgenic mice to examine the relationship between FAC and Fas-triggered cell death. Hematopoietic progenitors from FAC-transgenic mice were up to 10-fold less sensitive to the cytolytic effect of Fas-ligation. Our experiments implicate FAC in the regulation of apoptosis mediated by the Fas death receptor.     

Empirically supported comprehensive treatments for young children with autism

Aplastic anemia can be either acquired or congenital. The paradigm for the congenital form is Fanconi's anemia (FA). FA is an autosomal recessive, genetic syndrome characterized by progressive bone marrow failure, developmental abnormalities, and a predisposition to malignancy. The clinical manifestations of FA are heterogeneous, but one common outcome in the majority of patients is the development of life-threatening hematologic disease. FA is thought to affect the hematopoietic stem cell, and the hematologic consequences of FA can be effectively treated by complete replacement of patient stem cells by those from a histocompatible donor. Unfortunately, allogeneic stem cell transplantation is currently limited to patients with an unaffected matched sibling donor. Transplantation from alternative donors, while successful in selected cases, is associated with a high risk of graft failure and must be carefully considered in terms of risk and benefit for each individual. For FA patients lacking an appropriate donor, new therapies need to be devised. This review summarizes both the scientific rationale and the progress of gene therapy strategies aimed at correcting the hematopoietic defect of FA.     

Aplastic anemia rescued by exhaustion of cytokine-secreting CD8+ T cells in persistent infection with lymphocytic choriomeningitis virus

Aplastic anemia may be associated with persistent viral infections that result from failure of the immune system to control virus. To evaluate the effects on hematopoiesis exerted by sustained viral replication in the presence of activated T cells, blood values and bone marrow (BM) function were analyzed in chronic infection with lymphocytic choriomeningitis virus (LCMV) in perforin-deficient (P0/0) mice. These mice exhibit a vigorous T cell response, but are unable to eliminate the virus. Within 14 d after infection, a progressive pancytopenia developed that eventually was lethal due to agranulocytosis and thrombocytopenia correlating with an increasing loss of morphologically differentiated, pluripotent, and committed progenitors in the BM. This hematopoietic disease caused by a noncytopathic chronic virus infection was prevented by depletion of CD8+, but not of CD4+, T cells and accelerated by increasing the frequency of LCMV-specific CD8+ T cells in T cell receptor (TCR) transgenic (tg) mice. LCMV and CD8+ T cells were found only transiently in the BM of infected wild-type mice. In contrast, increased numbers of CD8+ T cells and LCMV persisted at high levels in antigen-presenting cells of infected P0/0 and P0/0 x TCR tg mice. No cognate interaction between the TCR and hematopoietic progenitors presenting either LCMV-derived or self-antigens on the major histocompatibility complex was found, but damage to hematopoiesis was due to excessive secretion and action of tumor necrosis factor (TNF)/lymphotoxin (LT)-alpha and interferon (IFN)-gamma produced by CD8+ T cells. This was studied in double-knockout mice that were genetically deficient in perforin and TNF receptor type 1. Compared with P0/0 mice, these mice had identical T cell compartments and T cell responses to LCMV, yet they survived LCMV infection and became life-long virus carriers. The numbers of hematopoietic precursors in the BM were increased compared with P0/0 mice after LCMV infection, although transient blood disease was still noticed. This residual disease activity was found to depend on IFN-gamma-producing LCMV-specific T cells and the time point of hematopoietic recovery paralleled disappearance of these virus-specific, IFN-gamma-producing CD8+ T cells. Thus, in the absence of IFN-gamma and/or TNF/LT-alpha, exhaustion of virus-specific T cells was not hampered.     

A study of the changes during heating of paracetamol

Fanconi anaemia (FA) is an accepted indication for treatment with allogeneic HLA-identical BMT. Most patients, however, lack a suitable HLA-identical donor. In our centre, six FA patients were transplanted with a matched unrelated donor. Due to hypersensitivity to DNA cross-linking agents, a low-dose cyclophosphamide (CY) and thoraco-abdominal irradiation (TAI) regimen is recommended for conditioning in FA. We added Ara-C upfront and anti-T cell antibodies to enhance engraftment and to prevent GVHD, in combination with T cell depletion in four out of six of the first transplants. One patient did not engraft. In three patients rejection was observed. In three of these four patients a second BMT, using full bone marrow grafts, resulted in successful engraftment. The other patient died before a second BMT could be performed. The incidence and severity of acute GVHD was low: only one patient with grade III acute GVHD was seen. Two out of four surviving patients suffered from chronic GVHD. Four patients survived (median survival time 43 months after BMT), three with good and one with acceptable quality of life. Two patients died, one patient due to adenoviral reactivation with multi-organ failure, and one due to sepsis complicated by ARDS. In conclusion, MUD BMT is feasible in FA patients with bone marrow failure in whom no HLA-identical sibling donor is available. In our study group, the major problem was graft rejection, despite the administration of a combination of graft enhancing anti-T cell antibodies. Multicentre studies are needed to determine a more intensive, but still tolerable, conditioning regimen.     

Bone marrow failure by cytomegalovirus is associated with an in vivo deficiency in the expression of essential stromal hemopoietin genes

Bone marrow (BM) failure associated with cytomegalovirus (CMV) infection is a feared complication after clinical BM transplantation. Experiments in long-term BM cultures have indicated that BM stromal cells (BMSC) are targets of productive CMV infection, but an in situ infection of BM stroma remained to be documented, and the pathomechanism is open to question. Here we describe a murine in vivo model of lethal CMV aplastic anemia (CMV-AA). The reconstitution of hematopoietic progenitor cells expressing stem cell factor (SCF) receptor was found to be defective in CMV-AA. While murine CMV replication in permissive parenchymal tissues is cytolytic, the hematopoietic cord was found to be a site of very limited virus production with foci of reticular BMSC expressing the intranuclear viral IE1 protein, but with only a few BMSC positive for viral genome in the in situ hybridization. XX-XY BM chimeras were established in order to quantitate Y-chromosome-tagged BMSC by a PCR specific for the male-sex-determining gene Tdy. This approach revealed that murine CMV infection is not associated with a significant loss of BMSC. Despite the physical integrity of the stromal network, the functional integrity of the stroma was impaired. While housekeeping genes were expressed normally in BMSC of infected mice, the expression of genes encoding the essential hemopoietins SCF, granulocyte colony-stimulating factor, and interleukin-6 was markedly reduced. In conclusion, the mechanism of BM failure is not a stromal lesion but an insufficient stromal function. These findings explain CMV-AA as a manifestation of multiple hemopoietin deficiency.     

Identification of human and mouse hematopoietic stem cell populations expressing high levels of mRNA encoding retrovirus receptors

One obstacle to retrovirus-mediated gene therapy for human hematopoietic disorders is the low efficiency of gene transfer into pluripotent hematopoietic stem cells (HSC). We have previously shown a direct correlation between retrovirus receptor mRNA levels in mouse HSC and the efficiency with which they are transduced. In the present study, we assayed retrovirus receptor mRNA levels in a variety of mouse and human HSC populations to identify HSC which may be more competent for retrovirus transduction. The highest levels of amphotropic retrovirus receptor (amphoR) mRNA were found in cryopreserved human cord blood HSC. The level of amphoR mRNA in Lin- CD34(+) CD38(-) cells isolated from frozen cord blood was 12-fold higher than the level in fresh cord blood Lin- CD34(+) CD38(-) cells. In mice, the level of amphoR mRNA in HSC from the bone marrow (BM) of mice treated with stem cell factor and granulocyte-colony stimulating factor was 2.8- to 7.8-fold higher than in HSC from the BM of untreated mice. These findings suggest that HSC from frozen cord blood and cytokine-mobilized BM may be superior targets for amphotropic retrovirus transduction compared with HSC from untreated adult BM.     

Evidence for at least eight Fanconi anemia genes

Fanconi anemia (FA) is an autosomal recessive chromosomal breakage disorder with diverse clinical symptoms including progressive bone marrow failure and increased cancer risk. FA cells are hypersensitive to crosslinking agents, which has been exploited to assess genetic heterogeneity through complementation analysis. Five complementation groups (FA-A through FA-E) have so far been distinguished among the first 20 FA patients analyzed. Complementation groups in FA are likely to represent distinct disease genes, two of which (FAC and FAA) have been cloned. Following the identification of the first FA-E patient, additional patients were identified whose cell lines complemented groups A-D. To assess their possible assignment to the E group, we introduced selection markers into the original FA-E cell line and analyzed fusion hybrids with three cell lines classified as non-ABCD. All hybrids were complemented for cross-linker sensitivity, indicating nonidentity with group E. We then marked the three non-ABCDE cell lines and examined all possible hybrid combinations for complementation, which indicated that each individual cell line represented a separate complementation group. These results thus define three new groups, FA-F, FA-G, and FA-H, providing evidence for a minimum of eight distinct FA genes.     

Endothelial selectins and vascular cell adhesion molecule-1 promote hematopoietic progenitor homing to bone marrow

The adhesive mechanisms allowing hematopoietic progenitor cells (HPC) homing to the bone marrow (BM) after BM transplantation are poorly understood. We investigated the role of endothelial selectins and vascular cell adhesion molecule-1 (VCAM-1) in this process. Lethally irradiated recipient mice deficient in both P-and E-selectins (P/E-/-), reconstituted with minimal numbers (相似文献   

Positive diepoxybutane test in only one of two brothers found to be compound heterozygotes for Fanconi's anaemia complementation group C mutations

Fanconi's anaemia (FA) is an autosomal recessive disorder characterized by diverse congenital abnormalities, the development of progressive bone marrow failure, and an increased predisposition to malignancy, particularly acute leukaemia. The FA phenotype is so variable that diagnosis on the basis of clinical manifestations alone can be difficult. The modern diagnosis of FA no longer rests entirely on the constellation of clinical and haematological abnormalities first described by Fanconi, but depends on finding elevated chromosomal breakage after incubation of peripheral blood lymphocytes with the chemical clastogens diepoxybutane (DEB) or mitomycin-C (MMC). The cloning of the gene for FA complementation group C [FAC] provides an opportunity to test the validity of the "DEB test' which in recent times has become the main arbiter as to whether a patient is classified as FA or non-FA. We report on two brothers with similar clinical and haematological features who have both been identified as compound heterozygotes for the FAC mutations L554P and delta G322, but only one of the brothers has a positive DEB test. On the basis of the DEB test one would be classified as FA and the other as non-FA. The time has come to re-evaluate the diagnostic criteria of "Fanconi's anaemia'.     

Flt-3 ligand increases microchimerism but can prevent the therapeutic effect of donor bone marrow in transiently immunosuppressed cardiac allograft recipients

C3H (H2k) mice received 50 x 10(6) B10 (H2b) bone marrow (BM) cells either alone or with flt-3 ligand (FL) (10 microg/day), tacrolimus (2 mg/kg/day), or both agents for 7 days. Donor MHC class II+ (IAb+) cells were quantitated in spleens by immunohistochemical analysis, and donor class II DNA detected in BM by PCR. Donor cells were rare in the BM alone and BM + FL groups, whereas there was a substantial increase in chimerism in the BM + tacrolimus group. Addition of FL to BM + tacrolimus led to a further eightfold increase in donor cells and enhanced donor DNA compared with the BM + tacrolimus group. This increase in donor cells was almost 500-fold compared with BM alone. C3H recipients of B10 heart allografts given perioperative B10 BM and tacrolimus (days 0-13) exhibited a markedly extended median graft survival time (MST, 42 days) compared with those given tacrolimus alone (MST, 22 days). Addition of FL (10 microg/day; 7 days) to BM + tacrolimus prevented the beneficial effect of donor BM (MST, 18 days). BM alone or BM + FL resulted in uniform early heart graft failure (MST < 8 days). Functional studies revealed maximal antidonor MLR and CTL activities in the BM- and BM + FL-treated groups, with minimal activity in the tacrolimus-treated groups. Thus, dramatic growth factor-induced increases in chimerism achieved under cover of immunosuppression may result in augmented antidonor T cell reactivity and reduced graft survival after immunosuppressive drug withdrawal. With FL, this may reflect striking augmentation of immunostimulatory dendritic cells.     

Prolonged expression of c-fos suppresses cell cycle entry of dormant hematopoietic stem cells

The proto-oncogene c-fos was transiently upregulated in primitive hematopoietic stem (Lin-Sca-1(+)) cells stimulated with stem cell factor, interleukin-3 (IL-3), and IL-6. To investigate a role of the c-fos in hematopoietic stem cells, we used bone marrow (BM) cells from transgenic mice carrying the c-fos gene under the control of the interferon-alpha/beta-inducible Mx-promoter (Mx-c-fos), and fetal liver cells from c-fos-deficient mice. Prolonged expression of the c-fos in Lin-Sca-1(+) BM cells inhibited factor-dependent colony formation and hematopoiesis on a stromal cell layer by keeping them at G0/G1 phase of the cell cycle. These Lin-Sca-1(+) BM cells on a stromal layer entered into the cell cycle whenever exogenous c-fos was downregulated. However, ectopic c-fos did not perturb colony formation by Lin-Sca-1(+) BM cells after they entered the cell cycle. Furthermore, endogenous c-fos is not essential to cell cycle progression of hematopoietic stem cells because the factor-dependent and the stroma-dependent hematopoiesis by Lin-Sca-1(+) fetal liver cells from c-fos-deficient mice was not impaired. These results suggest that the c-fos induced in primitive hematopoietic stem cells negatively controls cell cycle progression and maintains them in a dormant state.     

High efficiency gene transfer to human hematopoietic SCID-repopulating cells under serum-free conditions

Stable gene transfer to human pluripotent hematopoietic stem cells (PHSCs) is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical trials, the levels of gene transfer to this cell population have generally been low, reflecting deficiencies in both the vector systems and transduction conditions. In this study, we have used a pseudotyped murine retroviral vector to transduce human CD34(+) cells purified from bone marrow (BM) and umbilical cord blood (CB) under optimized conditions. After transduction, 71% to 97% of the hematopoietic cells were found to express a low-affinity nerve growth factor receptor (LNGFR) marker gene. Six weeks after transplantation into immunodeficient NOD/LtSz-scid/scid (NOD/SCID) mice, LNGFR expression was detected in 6% to 57% of CD45(+) cells in eight of nine engrafted animals. Moreover, proviral DNA was detected in 8.3% to 45% of secondary colonies derived from BM cells of engrafted NOD/SCID mice. Our data show consistent transduction of SCID-repopulating cells (SRCs) and suggest that the efficiency of gene transfer to human hematopoietic repopulating cells can be improved using existing retroviral vector systems and carefully optimized transduction conditions.     

Infection of human marrow stroma by human immunodeficiency virus-1 (HIV-1) is both required and sufficient for HIV-1-induced hematopoietic suppression in vitro: demonstration by gene modification of primary human stroma

Patients with human immunodeficiency virus-1 (HIV-1) infection often present with bone marrow (BM) failure that may affect all hematopoietic lineages. It is presently unclear whether this failure reflects a direct viral impairment of the CD34+ hematopoietic progenitor cells or whether the virus affects the BM microenvironment. To study the effects of HIV-1 on the BM microenvironment, we examined the stromal cell monolayers in long-term BM culture (LTBMC), which are the in vitro equivalent of the hematopoietic microenvironment. We assessed the hematopoietic support function (HSF) of human stromal layers by determining the cellular proliferation and colony-forming ability of hematopoietic progenitors from BM cells grown on the stromal layers. We show that the HSF is reduced by in vitro infection of the human stromal cell layer by a monocytotropic isolate of HIV-1 (JR-FL). There is no loss of HSF when the stromal cell layer is resistant to HIV-1 replication, either using murine stromal cell layers that are innately resistant to HIV-1 infection or using human stromal cells genetically modified to express a gene that inhibits HIV-1 replication (an RRE decoy). Decreased HSF was seen using either human or murine hematopoietic cells, if the stromal cells were human cells that were susceptible to HIV-1 infection. These in vitro studies implicate HIV-1 replication in the stroma as the essential component causing decreased hematopoietic cell production in HIV-1 infection.