Lentivector transduction improves outcomes over transplantation of human HSCs alone in NOD/SCID/Fabry mice.

Fabry disease is a lysosomal storage disorder caused by a deficiency of α-galactosidase A (α-gal A) activity that results in progressive globotriaosylceramide (Gb(3)) deposition. We created a fully congenic nonobese diabetic (NOD)/severe combined immunodeficiency (SCID)/Fabry murine line to facilitate the in vivo assessment of human cell-directed therapies for Fabry disease. This pure line was generated after 11 generations of backcrosses and was found, as expected, to have a reduced immune compartment and background α-gal A activity. Next, we transplanted normal human CD34(+) cells transduced with a control (lentiviral vector-enhanced green fluorescent protein (LV-eGFP)) or a therapeutic bicistronic LV (LV-α-gal A/internal ribosome entry site (IRES)/hCD25). While both experimental groups showed similar engraftment levels, only the therapeutic group displayed a significant increase in plasma α-gal A activity. Gb(3) quantification at 12 weeks revealed metabolic correction in the spleen, lung, and liver for both groups. Importantly, only in the therapeutically-transduced cohort was a significant Gb(3) reduction found in the heart and kidney, key target organs for the amelioration of Fabry disease in humans.

Extensive gene deletions in Japanese patients with Diamond-Blackfan anemia.

Fifty percent of Diamond-Blackfan anemia (DBA) patients possess mutations in genes coding for ribosomal proteins (RPs). To identify new mutations, we investigated large deletions in the RP genes RPL5, RPL11, RPL35A, RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26. We developed an easy method based on quantitative-PCR in which the threshold cycle correlates to gene copy number. Using this approach, we were able to diagnose 7 of 27 Japanese patients (25.9%) possessing mutations that were not detected by sequencing. Among these large deletions, similar results were obtained with 6 of 7 patients screened with a single nucleotide polymorphism array. We found an extensive intragenic deletion in RPS19, including exons 1-3. We also found 1 proband with an RPL5 deletion, 1 patient with an RPL35A deletion, 3 with RPS17 deletions, and 1 with an RPS19 deletion. In particular, the large deletions in the RPL5 and RPS17 alleles are novel. All patients with a large deletion had a growth retardation phenotype. Our data suggest that large deletions in RP genes comprise a sizable fraction of DBA patients in Japan. In addition, our novel approach may become a useful tool for screening gene copy numbers of known DBA genes.

An evaluation of peripherally inserted central venous catheters for children with cancer requiring long-term venous access.

Long-term venous access is essential when treating malignant diseases. We reviewed our experience with peripherally inserted central venous catheters (PICC) in children suffering from various malignancies with regard to catheter life, reasons for removal, and complications. Ninety-three PICCs were inserted in 78 children. Median catheter life was 162 days (range 6-575 days) with a total of 16,266 catheter days. Seventy-five PICCs (80.6%) had been placed until the elective removal or patients' death, whereas 18 PICCs (19.4%) were removed due to PICC-related complications; a rate of 1.11 per 1,000 catheter days. Complications requiring removal of PICCs included infection (n = 12), occlusion (n = 3), dislodgement (n = 2), and phlebitis (n = 1) with rates of 0.74, 0.18, 0.12 and 0.06 per 1,000 catheter days, respectively. We conclude that PICC provides reliable long-term intravenous access in children suffering from malignancies.

Survival and cardiac remodeling after myocardial infarction are critically dependent on the host innate immune interleukin-1 receptor-associated kinase-4 signaling: a regulator of bone marrow-derived dendritic cells.

BACKGROUND: The innate immune system greatly contributes to the inflammatory process after myocardial infarction (MI). Interleukin-1 receptor-associated kinase-4 (IRAK-4), downstream of Toll/interleukin-1 receptor signaling, has an essential role in regulating the innate immune response. The present study was designed to determine the mechanism by which IRAK-4 is responsible for the cardiac inflammatory process, which consequently affects left ventricular remodeling after MI. METHODS AND RESULTS: Experimental MI was created in IRAK-4(-/-) and wild-type mice by left coronary ligation. Mice with a targeted deletion of IRAK-4 had an improved survival rate at 4 weeks after MI. IRAK-4(-/-) mice also demonstrated attenuated cardiac dilation and decreased inflammation in the infarcted myocardium, which was associated with less proinflammatory and Th1 cytokine expression mediated by suppression of nuclear factor-kappaB and c-Jun N-terminal kinase activation. IRAK-4(-/-) mice had fewer infiltrations of CD45+ leukocytes and CD11c+ dendritic cells, inhibition of apoptosis, and reduced fibrosis and nitric oxide production. Cardiac dendritic cells in IRAK-4(-/-) mice were relatively immature or functionally naïve after MI in that they demonstrated less cytokine and costimulatory molecule gene expression. Furthermore, IRAK-4(-/-) dendritic cells have less mobilization capacity. Transfer of wild type-derived bone marrow dendritic cells into IRAK-4(-/-) mice for functional dendritic cell reconstitution negated the survival advantage and reduced the cardiac dilation observed with IRAK-4(-/-) mice at 28 days after MI. CONCLUSIONS: Deletion of IRAK-4 has favorable effects on survival and left ventricular remodeling after MI through modification of the host inflammatory process by blunting the detrimental bone marrow dendritic cells mobilization after myocardial ischemia.

Potent induction of B- and T-cell immunity against human carcinoembryonic antigen-expressing tumors in human carcinoembryonic antigen transgenic mice mediated by direct lentivector injection.

The applicability of immunotherapy would be dramatically broadened to a greater number of recipients if direct "off-the-shelf" products could be engineered to engender functionally potent immune responses against true "self"-tumor antigens. This would obviate the need for ex vivo culture of dendritic cells or T cells on a patient-by-patient basis, for example. The carcinoembryonic antigen (CEA) is a glycoprotein expressed in normal gut epithelium that is up-regulated in the majority of colon cancers, non-small cell lung cancers, and half of all breast cancers. Such properties make CEA an excellent and important target for cancer immunotherapy. In this study, we show stabilization of 14-day established s.c. mGC4CEA tumors in human CEA (huCEA) transgenic mice following two direct low-dose injections of 0.15x10(6) transducing units of a lentiviral vector (LV) that directs expression of huCEA (LV-huCEA). This stabilization result was reproducible and detailed analyses including antibody assays, multiplex cytokine analyses on unstimulated splenocytes, lymph node cell characterizations, tetramer staining, and immunofluorescence staining of tumor sections showed that this outcome correlated with both a cellular and humoral immune response. Similar tumor outcomes were not seen when mice were vaccinated with a control LV that engineered expression of enGFP only. The long-term potency of this vaccination strategy was also studied and revealed the requirement for maintenance of tumor antigen-specific immunity for efficient tumor control. These data support the use of direct injections of low doses of LV-huCEA for enhancement of tumor immunotherapy directed against CEA.

Treatment of McLeod phenotype chronic granulomatous disease with reduced-intensity conditioning and unrelated-donor umbilical cord blood transplantation.

Patients with chronic granulomatous disease (CGD) complicated by antimycotics-refractory invasive aspergillosis have an extremely poor prognosis if they cannot undergo allogeneic hematopoietic stem cell transplantation from a suitable related donor while in good clinical condition. We successfully treated a 20-year-old man with very rare McLeod phenotype CGD with reduced-intensity conditioning and unrelated-donor umbilical cord blood transplantation. We postulate that reduced-intensity conditioning-allogeneic hematopoietic stem cell transplantation is a promising therapeutic strategy for patients with CGD even if only unrelated-donor umbilical cord blood is available.

Treatment of stage IV malignant rhabdoid tumor of the kidney (MRTK) with ICE and VDCy: a case report.

The prognosis of stage IV malignant rhabdoid tumor of the kidney (MRTK) has been extremely poor. However, a combination of ICE (ifosfamide, carboplatin, and etoposide) and VDCy (vincristine, doxorubicin, and cyclophosphamide) was recently reported to be effective for metastatic MRTK. We describe a 21-month-old girl with stage IV MRTK who was successfully treated with ICE, VDCy, and radiotherapy. She remained well, without recurrence, 24 months after diagnosis. Alternating therapy with ICE and VDCy might become a standard regimen for stage IV MRTK, although further study is required to confirm its effectiveness.

Induction of autologous CD4- and CD8-mediated T-cell responses against acute lymphocytic leukemia cell line using apoptotic tumor cell-loaded dendritic cells.

OBJECTIVE: Several studies have demonstrated that dendritic cells (DCs) pulsed with tumor lysate or apoptotic tumor cells can elicit effective T-cell responses. This technique does not require the identification of the tumor antigen or HLA haplotype of the patient. We applied this approach to induce HLA class I- and class II-restricted T-cell responses directed against autologous acute lymphocytic leukemia (B-ALL) cell line NH-1. METHODS: Autologous T cells were stimulated by apoptotic tumor cell-loaded DCs generated from a patient with ALL. The stimulated and expanded T cells were isolated into CD8(+) T-cell line and CD4(+) T-cell line, and each of them was examined as to their functions. RESULTS: Both CD8(+) and CD4(+) T-cell lines demonstrated cytotoxicity against NH-1 in an major histocompatibility complex-dependent manner. Finally, we established two independent CD4(+) T-cell clones restricted to HLA-DR. The CD4(+) T-cell line responded strongly to autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (EBV-LCL) but not to autologous normal cells. Furthermore, the T-cell clones also responded to allogeneic EBV-LCLs and B-ALL cell lines in the context of the HLA-DRB1( *)04051 molecule. Interestingly, 293T and COS-7 cells, which had been transfected with the HLA-DRB1( *)04051, were also recognized by T-cell clones. CONCLUSION: These findings indicate that B-ALL has shared and strong immunogenic epitopes expressed on HLA class II molecules, the expression of which is limited to immortalized cells. These data suggest that vaccinations using DCs loaded with apoptotic tumor cells might be a potent strategy in the treatment of B-ALL.

Detection of BK virus and adenovirus in the urine from children after allogeneic stem cell transplantation.

The development of hemorrhagic cystitis (HC) and urinary excretion of polyoma BK virus (BKV) and adenovirus (ADV) was investigated by polymerase chain reaction in 20 children undergoing allogeneic stem cell transplantation. Five children developed HC, and all of them excreted BKV; however, only 1 excreted ADV, suggesting that BKV is more significant cause of HC than ADV in children undergoing stem cell transplantation.

Relapse of T-cell all after stem cell transplant presenting as hypertrophic cardiomyopathy: the value of non-invasive diagnostic imaging in detecting cardiac leukemia.

We describe a 14-year-old female with acute lymphoblastic leukemia (ALL) with a mediastinal mass at diagnosis who developed hypertrophic cardiomyopathy (HC) after stem cell transplantation (SCT). During refractory relapse after SCT using bone marrow from her HLA-matched sibling, she underwent whole thorax irradiation because of pleural effusion and a recurrent mediastinal mass. After a second SCT using peripheral blood from the same donor, she developed HC suspected to be related to tacrolimus. However, isolated cardiac relapse was finally diagnosed by several non-invasive imaging techniques. Cardiac irradiation resolved her cardiac failure, though she eventually developed progressive and fatal hematological disease.

Gastric antral vascular ectasia in 2-yr-old girl undergoing unrelated cord blood stem cell transplantation.

Gastrointestinal bleeding is a common complication after hematopoietic stem cell transplantation (HSCT) and is often related to acute graft-vs.-host disease (aGVHD). Gastric antral vascular ectasia (GAVE), recently recognized as a complication after HSCT, is a rare cause of severe gastrointestinal bleeding, which has only been reported in adult patients so far. We report a 2-yr-old girl who developed GAVE after unrelated cord blood stem cell transplantation (CBSCT) as treatment of intractable Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis (EBV-HLH). Her conditioning regimen for CBSCT consisted of etoposide, busulfan, and cyclophosphamide. She was doing well after CBSCT without recurrence and developed only grade I aGVHD. She suddenly developed coffee ground emesis, tarry stools and severe anemia 76 days after CBSCT. As antacids were ineffective, esophagogastroduodenoscopy was performed and revealed GAVE on day 97. Endoscopic coagulation therapy was performed twice; subsequently, she needed no further transfusions and there was no clinical recurrence of GAVE.

[Prognostic value of serum markers for liver fibrosis in transient abnormal myelopoiesis (TAM)].

Transient abnormal myelopoiesis (TAM) is usually a self-limiting myeloproliferative disorder observed in approximately 10% of newborn infants with Down syndrome. However, progressive liver fibrosis may occur in patients with TAM and is often lethal. We investigated the utility of the serum levels of hyaluronic acid (HA) and N-terminal peptide of III procollagen (P-III-P) as markers of liver fibrosis and indication for chemotherapy. We reviewed 4 cases of TAM retrospectively. HA levels were more than one hundred times as high as the upper limit of the normal range in 2 patients, one of whom died from gastrointestinal bleeding. His HA and P-III-P had increased up to 18,800 U/ml and 26.2 ng/ml, respectively, just before he died. Another patient's serum HA and P-III-P increased to 6,100 U/ml and 12.8 ng/ml, respectively, however his liver fibrosis resolved with low-dose cytosine arabinoside treatment after exchange transfusion during his clinical course. We suggest that serum HA is useful as a marker of liver fibrosis and a prognostic indicator for chemotherapy in patients with TAM. Early treatment including both exchange transfusion and chemotherapy should be considered for patients presenting with extremely high or an elevating tendency of their HA serum levels.

Successful cidofovir treatment of adenovirus-associated hemorrhagic cystitis and renal dysfunction after allogenic bone marrow transplant.

We present a patient who developed hemorrhagic cystitis and renal dysfunction after unrelated bone marrow transplantation. Polyoma BK virus and adenovirus 11 were detected in the urine. Vidarabine was administered without effect. Relatively low dose cidofovir was efficacious. Renal function improved, and the urinary secretion of adenovirus stopped.