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Identifying genes that regulate bone marrow (BM) engraftment may reveal molecular targets for overcoming engraftment barriers. To achieve this aim, we applied a forward genetic approach in a mouse model of nonmyeloablative BM transplantation. We evaluated engraftment of allogeneic and syngeneic BM in BALB.K and B10.BR recipients. This allowed us to partition engraftment resistance into its intermediate phenotypes, which are firstly the immune-mediated resistance and secondly the nonimmune rejection of donor BM cells. We observed that BALB.K and B10.BR mice differed with regard to each of these resistance mechanisms, thereby providing evidence that both are under genetic control. We then generated a segregating backcross (n = 200) between the BALB.K and B10.BR strains to analyze for genetic linkage to the allogeneic BM engraftment phenotype using a 127-marker genome scan. This analysis identified a novel quantitative trait locus (QTL) on chromosome 16, termed Bmgr5(logarithm of odds 6.4, at 11.1 cM). The QTL encodes susceptibility alleles, from the BALB.K strain, that are permissive for allogeneic BM engraftment. Further identification of Bmgr5genes by positional cloning may reveal new and effective approaches for overcoming BM engraftment obstacles. more...

In a search for new genes involved in the regulation of erythropoiesis, we identified murine Penumbra cDNA from a multipotent hematopoietic cell line based on its predominant expression in erythroblasts. Subsequently, we identified the human PENUMBRA from a bone marrow cDNA library. Penumbra is a new member of the tetraspanin superfamily of membrane proteins, many of which are thought to function as organizers of supramolecular signaling complexes. Human and murine Penumbras contain 283 amino acids and are 97% identical. The human PENUMBRA gene is mapped to chromosome 7q32, a hot spot for deletions in myelodysplastic syndromes and acute myelogenous leukemias. Penumbra is targeted to the cell surface and forms disulfide-bonded homodimers. To study the effects of Penumbra deletions, we created a knockout mouse model by gene targeting. Penumbra−/− mice develop massive splenomegaly, basophilic macrocytic red blood cells, and anemia as they age. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra−/− mouse. EMX exhibits ineffective erythropoiesis in the presence of erythropoietin, a defect that is reversed by reexpression of Penumbra. These findings indicate that Penumbra has a positive function in erythropoiesis and its deletion or mutation may result in anemia. more...

In a search for new genes involved in the regulation of erythropoiesis, we identified murine PenumbracDNA from a multipotent hematopoietic cell line based on its predominant expression in erythroblasts. Subsequently, we identified the human PENUMBRAfrom a bone marrow cDNA library. Penumbra is a new member of the tetraspanin superfamily of membrane proteins, many of which are thought to function as organizers of supramolecular signaling complexes. Human and murine Penumbras contain 283 amino acids and are 97% identical. The human PENUMBRAgene is mapped to chromosome 7q32, a hot spot for deletions in myelodysplastic syndromes and acute myelogenous leukemias. Penumbra is targeted to the cell surface and forms disulfide-bonded homodimers. To study the effects of Penumbradeletions, we created a knockout mouse model by gene targeting. Penumbra−/−mice develop massive splenomegaly, basophilic macrocytic red blood cells, and anemia as they age. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra−/−mouse. EMX exhibits ineffective erythropoiesis in the presence of erythropoietin, a defect that is reversed by reexpression of Penumbra.These findings indicate that Penumbrahas a positive function in erythropoiesis and its deletion or mutation may result in anemia. more...

The influence of graft composition on clinical outcomes after reduced-intensity conditioning is not well-characterized. In this report we prospectively enumerated CD34+, CD3+, CD4+, and CD8+ cell doses in granulocyte colony-stimulating factor–mobilized peripheral blood mononuclear cell (G-PBMC) allografts in 63 patients who received transplants following non-myeloablative conditioning with total body irradiation 200 cGy plus fludarabine as treatment for malignant diseases. Donors were HLA-identical siblings (n = 38) or HLA-matched unrelated individuals (n = 25). By univariate analyses G-PBMC CD8+ T-cell dose in at least the 50th percentile favorably correlated with full donor blood T-cell chimerism (P = .03), freedom from progression (P = .001), and overall survival (P = .01). No G-PBMC cell dose influenced grade II to IV acute or extensive chronic graftversus-host disease. In multivariate analysis only G-PBMC CD8+ T-cell dose (P = .003; RR = 0.2, 95% CI = 0.1-0.6) was associated with improved freedom from progression. Infusion of low G-PBMC CD8+ T-cell dose for reduced-intensity allografting may adversely affect T-cell engraftment and survival outcome. more...

The influence of graft composition on clinical outcomes after reduced-intensity conditioning is not well-characterized. In this report we prospectively enumerated CD34+, CD3+, CD4+, and CD8+cell doses in granulocyte colony-stimulating factor–mobilized peripheral blood mononuclear cell (G-PBMC) allografts in 63 patients who received transplants following non-myeloablative conditioning with total body irradiation 200 cGy plus fludarabine as treatment for malignant diseases. Donors were HLA-identical siblings (n = 38) or HLA-matched unrelated individuals (n = 25). By univariate analyses G-PBMC CD8+T-cell dose in at least the 50th percentile favorably correlated with full donor blood T-cell chimerism (P= .03), freedom from progression (P= .001), and overall survival (P= .01). No G-PBMC cell dose influenced grade II to IV acute or extensive chronic graftversus-host disease. In multivariate analysis only G-PBMC CD8+T-cell dose (P= .003; RR = 0.2, 95% CI = 0.1-0.6) was associated with improved freedom from progression. Infusion of low G-PBMC CD8+T-cell dose for reduced-intensity allografting may adversely affect T-cell engraftment and survival outcome. more...

Campath-1H is effective therapy for patients with relapsed and refractory chronic lymphocytic leukemia (CLL) and prolymphocytic leukemia (PLL), but it is associated with profound lymphopenia and deficiencies in cell-mediated immunity. We report the incidence of cytomegalovirus (CMV) viremia in 34 patients treated with Campath-1H for relapsed or refractory CLL and PLL. All patients received infection prophylaxis during therapy and continuing for at least 2 months following Campath-1H. Five patients (15%) developed CMV viremia at a median of 28 days (range, 20–30 days) after the first dose of Campath-1H. The median CMV viral load was 860/mL (range, 420–2100/mL), as determined by quantitative plasma polymerase chain reaction (PCR). All 5 patients had a temperature 38.5°C, normal chest radiographs, normal liver function tests, and negative bacterial blood cultures with no clinical evidence of CMV disease at the time of presentation with CMV viremia. The median absolute neutrophil count (ANC) was 740/µL (range, 340-1600/µL), and the median absolute lymphocyte count (ALC) was 16/µL (range, 11–169/µL) for the 5 patients at the time of CMV viremia. All 5 patients received ganciclovir therapy followed by prompt fever resolution and clearance of CMV viremia by plasma PCR. By univariate regression analysis, the following were not risk factors for CMV viremia: age, number of prior regimens, prior rituximab therapy, prior splenectomy, modified Rai stage at Campath-1H therapy (low/intermediate vs. high), ANC, and ALC; although, there was a trend towards significance for prior rituximab therapy (P= 0.07). Cytomegalovirus viremia may be a significant infectious complication during Campath-1H therapy and should be investigated further in future studies. more...

A retrospective analysis was performed to investigate the outcome of high-dose therapy (HDT) and autologous hematopoietic cell transplantation in patients with follicular lymphomas beyond first remission. Ninety-two patients with primary induction failure or relapsed follicular low-grade lymphoma (FLGL), follicular large cell lymphoma (FLCL), and transformed follicular lymphoma (TFL) were treated with myeloablative therapy consisting of etoposide (60 mg/kg), cyclophosphamide (100 mg/kg), and either carmustine (BCNU;15 mg/kg) or fractionated total body irradiation (FTBI; 1200 cGy) followed by transplantation of purged autologous bone marrow or peripheral blood hematopoietic cells. For the 49 patients with relapsed FLGL, the median age was 49 years and the median interval from diagnosis to HDT was 30 months. The 4-year estimate of overall survival (OS) was 60% (95% confidence interval [CI], 45%-75%) and of disease-free survival (DFS) was 44% (95% CI, 29%-59%). Treatment with the FTBI-containing HDT regimen was associated with significantly longer DFS (P = .04) and OS (P = .04) in our multivariate analysis. OS was also significantly longer among those treated with 3 or fewer chemotherapy regimens. For the 26 FLCL patients, the median age was 51 years and in 31% the indication for HDT was primary induction failure. For FLCL patients, the 4-year estimate of OS was 58% (95% CI, 37%-79%) and of DFS was 51% (95% CI, 30%-72%). Among the 17 patients with TFL, 13 (76%) transformed at first relapse, and only 6 patients (35%) achieved complete remission with salvage therapy prior to HDT. For TFL patients, the 4-year estimate of OS was 50% (95% CI, 24%-76%) and of DFS 49% (95% CI, 20%-78%). There were 3 occurrences of myelodysplasia (1 after treatment with TBI, 2 after BCNU treatment), yielding an estimated incidence of 7% (95% CI, 0%-16%) at 56 months. This analysis shows that relapsed FLGL patients treated with 3 or fewer different chemotherapy regimens show inferior survival. The HDT regimen containing FTBI appears to be superior to the BCNU-based regimen for relapsed FLGL, although longer follow-up is needed to evaluate late effects. Lastly, patients with TFL or induction failure and relapsed FLCL can achieve survival outcome comparable to those observed with the indolent follicular lymphomas. more...

We performed a retrospective review to investigate pulmonary toxicity syndrome (PTS) in a cohort of breast cancer patients undergoing BCNU-containing high-dose chemotherapy (HDC). Our aim was to characterize presentation, identify risk factors, determine outcome following therapy, and find any association with differences in survival. We reviewed the data of 152 patients with stage II or III or metastatic breast cancer treated with cyclophosphamide 5625 mg/m2, cisplatin 165 mg/m2, and BCNU 600 mg/m2 followed by autologous peripheral blood hematopoietic cell transplantation. During follow-up, PTS was diagnosed when the following criteria were met: (1) presentation with typical clinical symptoms of PTS, (2) an absolute carbon monoxide diffusion capacity (DLCO) decline of 10% compared with pre-HDC DLCO, and (3) no clinical evidence of active pulmonary infection. Patients were then treated with a course of corticosteroid therapy. The incidence of PTS for all 152 patients was 59%, with a median onset at 45 days (range, 21-149 days) post-HDC. The median absolute DLCO decrement was 26% (range, 10%-73%) at diagnosis of PTS. There was no significant correlation between patient age, stage of breast cancer, pre-HDC chemotherapy regimen, pre-HDC chest wall radiotherapy, tobacco use, prior lung disease, or baseline pulmonary function test results and the development of PTS. We did observe an interesting association between PTS and the development of a noncholestatic elevation of transaminases. Of PTS patients treated with prednisone therapy for a median of 105.5 days (range, 44-300 days), 91% achieved resolution of their PTS without pulmonary sequelae. At 3 years, the overall survival (OS) of stage II or III patients who developed PTS was 84% (95% confidence interval [CI], 73%-95%); of metastatic breast cancer patients with PTS, the OS was 58% (95% CI, 38%-78%). These values were not significantly different from those of patients who did not develop PTS (91% [95% confidence interval [CI], 81%-100%] and 53% [95% CI, 32%-74%], respectively). No significant differences in disease-free or event-free survival were observed between patients with and without PTS. The incidence of PTS in breast cancer patients treated with a BCNU-containing HDC regimen can be remarkably high. Treatment with a course of corticosteroid therapy is successful in the vast majority. more...

We have previously demonstrated that important regulatory elements responsible for regulated expression of the human GLUT4 promoter are located between -1154 and -412 relative to transcription initiation (Olson, A. L., and Pessin, J. E. (1995) J. Biol. Chem. 270, 23491-23495). Through further analysis of this promoter regulatory region, we have identified a perfectly conserved myocyte enhancer factor 2 (MEF2)-binding domain (-CTAAAAATAG-) that is necessary, but not sufficient, to support tissue-specific expression of a chloramphenicol acetyltransferase reporter gene in transgenic mice. Biochemical analysis of this DNA element demonstrated the formation of a specific DNA-protein complex using nuclear extracts isolated from heart, hindquarter skeletal muscle, and adipose tissue but not from liver. DNA binding studies indicated that this element functionally interacted with the MEF2A and/or MEF2C MADS family of DNA binding transcription factors. MEF2 DNA binding activity was substantially reduced in nuclear extracts isolated from both heart and skeletal muscle of diabetic mice, which correlated with decreased transcription rate of the GLUT4 gene. MEF2 binding activity completely recovered to control levels following insulin treatment. Together these data demonstrated that MEF2 binding activity is necessary for regulation of the GLUT4 gene promoter in muscle and adipose tissue. more...

Hematopoietic progenitors committed to the myeloid lineage, the common myeloid and granulocyte-monocyte progenitors (CMP/GMP), have been shown to protect against opportunistic pathogens following myeloablative radiation; however, the efficacy of this approach has not been studied in the setting of chemotherapy-induced neutropenia. In this mouse model, the infusion of CMP/GMP on the day after 5-fluorouracil (5-FU) administration (D+1) resulted in a significant increase in the number of splenic neutrophils by D+8 when compared with 5-FU–only controls (P = .02), the majority of which were CMP/GMP-derived (54%). Moreover, 19% and 28% of neutrophils in the blood and bone marrow, respectively, were CMP/GMP-derived. Survival following intranasal challenge with the fungus Aspergillus fumigatus was significantly higher in CMP/GMP-infused mice than the controls (56% and 33% respectively; P = .019). Thus, a single infusion of CMP/GMP enhances tissue neutrophil content and increases survival against a lethal challenge with A fumigatus in the setting of chemotherapy-induced neutropenia. more...

Hematopoietic progenitors committed to the myeloid lineage, the common myeloid and granulocyte-monocyte progenitors (CMP/GMP), have been shown to protect against opportunistic pathogens following myeloablative radiation; however, the efficacy of this approach has not been studied in the setting of chemotherapy-induced neutropenia. In this mouse model, the infusion of CMP/GMP on the day after 5-fluorouracil (5-FU) administration (D+1) resulted in a significant increase in the number of splenic neutrophils by D+8 when compared with 5-FU–only controls (P= .02), the majority of which were CMP/GMP-derived (54%). Moreover, 19% and 28% of neutrophils in the blood and bone marrow, respectively, were CMP/GMP-derived. Survival following intranasal challenge with the fungus Aspergillus fumigatuswas significantly higher in CMP/GMP-infused mice than the controls (56% and 33% respectively; P= .019). Thus, a single infusion of CMP/GMP enhances tissue neutrophil content and increases survival against a lethal challenge with A fumigatusin the setting of chemotherapy-induced neutropenia. more...

Hybrid spin-mechanical systems have great potential in sensing, macroscopic quantum mechanics, and quantum information science. In order to induce strong coupling between an electron spin and the center-of-mass motion of a mechanical oscillator, a large magnetic gradient usually is required, which is difficult to achieve. Here we show that strong coupling between the electron spin of a nitrogen-vacancy (NV) center and the torsional vibration of an optically levitated nanodiamond can be achieved in a uniform magnetic field. Thanks to the uniform magnetic field, multiple spins can strongly couple to the torsional vibration at the same time. We propose utilizing this coupling mechanism to realize the Lipkin-Meshkov-Glick (LMG) model by an ensemble of NV centers in a levitated nanodiamond. The quantum phase transition in the LMG model and finite number effects can be observed with this system. We also propose generating torsional superposition states and realizing torsional matter-wave interferometry with spin-torsional coupling. [ABSTRACT FROM AUTHOR] more...

No relevant conflicts of interest to declare.

We analyzed data from 38 patients (median age = 56, range: 8 – 68 years) with acute leukemia (n=15), chronic idiopathic myelofibrosis (n=6), myelodysplastic syndrome with or without myeloproliferative disorder (n=5), chronic myeloid leukemia (n=4), non- Hodgkin lymphoma (n=4), aplastic anemia (n=2), multiple myeloma (n=1) and renal cell carcinoma (n=1), who underwent salvage allogeneic hematopoietic cell transplantation (HCT) for allograft failure. In 14 cases the original donors were used for second HCT, while in 24 cases different donors were identified (Table 1). Conditioning regimens for first HCTs included total body irradiation (TBI; 2 Gy) with or without fludarabine (Flu; n=28), myeloablative regimens (busulfan-cyclophosphamide, n=6; cyclophosphamide-TBI, n=2); and other, cyclophosphamide-anti-thymocyte globulin-based regimens (n=3). Conditioning for salvage HCT consisted of Flu 30 mg/m2/day on days -4 to -2 followed by TBI of 3 (n=24) or 4 (n=14) Gy on day 0. Cyclosporine and mycophenolate mofetil were used for postgrafting immunosuppression. The median time between first and salvage HCTs was 91 (range, 29 to 1004) days. Sustained second grafts were achieved in 34 patients (89%), while grafts failed in 4 patients (11%), all of whom had idiopathic myelofibrosis. With a median follow-up among surviving patients of 2.0 (range, 0.3 to 7.8) years, the 2 and 4 year Kaplan-Meier survival estimates were 49% (95% CI: 31%, 66%) and 42% (95% CI: 23%, 61%), respectively. The 2 year relapse-rate and non-relapse mortality were 36% (95% CI: 20%, 52%) and 25% (95% CI: 11%, 41%), respectively. The cumulative incidences of grades 2–4 acute and moderate-severe chronic graft-versus-host disease (GVHD) at 2 years were 42% and 41%, respectively. Four patients with chronic GVHD discontinued systemic immunosuppressive therapy at a median of 2.5 years. Within the limitations of the small patient numbers studied, TBI dose (3 vs. 4 Gy), same vs. different donors for salvage HCT, donor type (related, unrelated, HLA-haploidentical related vs. double umbilical cord), and HCT comorbidity scores did not appear to affect outcomes. Based on this retrospective multicenter analysis, we conclude that graft failure following allogeneic HCT can be effectively overcome by second transplantation using conditioning with Flu and low dose TBI (3 or 4 Gy), which should be further investigated in a prospective manner. more...

Comprehensive analyses of unrelated donor (URD) and recipient HLA-matching for allogeneic hematopoietic cell transplantation (AHCT) have demonstrated better outcomes when allele typing is performed using high-resolution nucleotide sequence-based techniques. To evaluate survival following myeloablative AHCT using allele-level HLA-matched URD as compared with HLA-identical sibling donors, we analyzed outcomes for 430 patients treated at our center between March 1991 and April 2005. Sequence-based allele typing was retrospectively performed for HLA-A, B, C, DR and DQ when not done at time of AHCT for URD (n = 124; 29%) and non-sibling related donors (n = 19; 4%). Donors were HLA-identical siblings (n = 276; 64%), HLA allele-matched URD (n = 52; 12%), single HLA-locus mismatched donors (n = 52; 12%), or > 1 locus mismatched donors (n = 50; 12%). The median age at transplant was 23.4 years (range: 0.2 – 61). The most common diagnoses were AML (n = 107; 25%), CML (n = 90; 21%), ALL (n = 86; 20%) and MDS (n = 50; 12%). Total body irradiation-based preparative regimens were used for 283 patients (66%). Bone marrow (BM) was the graft for 388 patients (90%) and GCSF-mobilized peripheral blood stem cells (PBSC) for the remaining 42 (10%). Graft-versus-host disease (GVHD) prophylaxes were cyclosporine and methotrexate (n = 327; 76%), long methotrexate (n = 42; 10%), T-cell depletion (n = 19; 4%), or other regimens (n = 42; 10%). With a median follow-up of 4.8 years (range: 0.2 – 12.1), the 5-year estimate of overall survival (OS) for the entire group was 48.2% (95% CI: 45.7 – 50.7) and transplant-related mortality (TRM) was 31.4% (95% CI: 28.8 – 34). As shown in the Table, OS and TRM were indistinguishable between AHCT performed with HLA-identical siblings compared with HLA allele-matched URD. There was also no difference in grade III – IV acute GVHD (P = .46) between these two groups whereas there was a trend towards more extensive chronic GVHD (HR 1.8; 95% CI: 0.9 – 3.6; P = 0.12) for the URD recipients. Using a multivariate analysis to adjust for advanced disease, age (> vs ≤ 30 years), graft (BM vs PBSC) and female-to-male gender mismatch, there remained no difference in OS between HLA-identical siblings and HLA allele-matched URD (P = 0.67). These results demonstrate that key outcomes (OS, TRM, and severe acute GVHD) are equivalent in recipients of grafts from either allele-level 10/10 HLA-matched URD or HLA-identical siblings. Overall Survival TRM Number Hazard Ratio 95% CI P value Hazard Ratio95% CI P value HLA-ID Sibling 276 1 - - 1 - - HLA-ID URD 52 1.1 0.7 – 1.7 0.67 0.8 0.4 – 1.6 0.58 1 Locus MM 52 1.3 0.9 – 2.0 0.19 1.4 0.8 – 2.4 0.25 > 1 Locus MM 50 2.0 1.4 – 2.9 < 0.001 2.6 1.7 – 4.1 < 0.001 more...

To search for new genes that might be involved in the regulation of erythropoiesis, we performed cDNA representational difference analysis on a multipotent murine hematopoietic cell line, EML C1. Penumbra was identified based on its predominant, high-level expression in TER119+ erythroblasts. It is not expressed or is expressed at very low levels in Gr1+ or CD3+ or B220+ or CD11c or NK1.1+ bone marrow or spleen cells. The full-length murine Penumbra mRNA contains 2017 nucleotides and encodes a new membrane protein characterized by four transmembrane domains, a small extracellular domain between the first and second transmembrane domains and a large extracellular domain between the third and fourth transmembrane domains. It is a member of the evolutionarily conserved tetraspanin membrane protein superfamily that includes CD63, CD81, CD151 and Peripherin. Using the murine Penumbra as a probe, we identified the human Penumbra from a human bone marrow cDNA library. The human Penumbra also encodes a protein of 283 amino acids and is 97% identical to the murine Penumbra. The human Penumbra gene is mapped to chromosome 7q32, a hotspot for interstitial deletions in myelodysplastic syndromes and acute myelogenous leukemias. Immunofluorescence microscopy indicated that Penumbra is targeted to the cell surface. Immunoprecipitation-Western analysis showed that Penumbra formed disulfide bond-linked homodimers. To study the possible effects of Penumbra deletions, a knockout mouse model was created by homologous recombination. To avoid any pathology that might result from the Neo selectable marker, the targeting vector employed a self-excising Cre-Neo cassette driven by a testis-specific promoter. At 3 months of age, Penumbra-/- mice had similar numbers of RBC, hematocrits, WBC and platelets as the WT littermates. At 1 year of age, Penumbra-/- mice as a group had significantly lower RBC numbers and hematocrits than the WT littermates. Furthermore, about 40–60% of the Penumbra-/- mice had increased percentages of basophilic or polychromatophilic macrocytic RBC. As a group, these mice had even lower numbers of RBC (6.24 ± 1.87 vs. 8.88 ± 0.51 x 106/microliter in WT; p = 0.0002) and hematocrits (36.63 ± 8.28 vs. 46.21 ± 3.62% in WT; p = 0.002). Many such mice had massive splenomegaly due to increased extramedullary hematopoiesis. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra-/-- mouse. EMX exhibits ineffective erythropoiesis in response to erythropoietin, a defect that is reversed by re-expression of Penumbra. These findings indicate that Penumbra plays an important role in erythropoiesis and suggest that Penumbra may be one of the genes that are often deleted during the evolution of myelodysplastic syndromes and acute myelogenous leukemias. more...

The efficacy of high dose therapy (HDT) and autologous hematopoietic cell transplantation (AHCT) for patients with diffuse large cell non-Hodgkin’s lymphoma (NHL) who never achieve a first complete remission (CR) with conventional chemotherapy have been addressed by few published studies. We retrospectively analyzed outcomes for 43 consecutive patients with chemotherapy-sensitive primary refractory diffuse large cell NHL treated with HDT and AHCT at our center between November 1988 and January 2002. The median age at transplant was 43 years (range: 18 – 64). At diagnosis 26 patients (60%) had stage IV disease, 28 patients (65%) had extranodal involvement, and bulky disease was present in 17 patients (40%). The age-adjusted NHL international prognostic index (IPI) score was low in 4 patients (9%), low-intermediate in 18 patients (42%), high or high-intermediate in 12 patients (28%), and not available in 9 patients (21%). All patients failed to achieve a first CR following CHOP (n = 31, 72%), R-CHOP (n = 6, 14%), and other anthracycline-containing regimens (n = 6, 14%). Fourteen patients (33%) did not receive additional conventional chemotherapy before proceeding to HDT and AHCT. The remaining 29 patients (67%) received in addition between 1 – 6 cycles of salvage chemotherapy. None of the 43 patients had achieved a CR at any time point before HDT and AHCT and all patients had measurable disease at the start of the HDT regimen. The median interval from diagnosis to HDT was 8.9 months (range: 2.4 – 15.5). The HDT regimen consisted of total body irradiation, etoposide, and cyclophosphamide in 15 patients (35%); BCNU, etoposide, and cyclophosphamide in 26 patients (60%); and CCNU, etoposide, and cyclophosphamide in 2 patients (5%). All but three patients (7%) were rescued with peripheral blood stem cells mobilized with cyclophosphamide 4 gm/m2 plus G-CSF and purged with monoclonal antibodies and complement. With a median follow-up of 5.8 years (range: 0.2 – 15.8) among surviving patients, the 5-year Kaplan-Meier estimates for overall survival was 67.4% (95% CI: 52.4 – 82.4), freedom from progression was 60.1% (95% CI: 43.7 – 76.6), and event-free survival was 55.8% (95% CI: 39.2 – 72.4). Two patients with relapse post-AHCT proceeded to non-myeloablative allogeneic HCT and were censored at the time of second transplant. By univariate analyses the following characteristics were not prognostically significant for overall survival: disease stage at diagnosis (I–II versus III–IV), prior radiotherapy, age-adjusted IPI score (low versus high) in evaluable patients, and the HDT regimen (TBI versus non-TBI). These results demonstrate that HDT and AHCT is effective treatment for chemotherapy-sensitive primary refractory diffuse large cell NHL. Figure Figure more...

We have recently established a continuous natural killer (NK) cell line, KIL (for Killer Lymphocyte), from a coculture of normal murine whole bone marrow (WBM) and the OP-9 stromal cell line expressing the Notch ligand, Jagged2, in the presence of stem cell factor and IL-7 (Dehart et al. Blood2005;105:3521). A clonal prototype, KIL C.2, was derived from a C57BL/6 mouse and has the CD3-NK1.1+CD122+ phenotype, expresses the NKG2D activating receptor, and proliferates extensively upon in vitro stimulation with IL-2 (20ng/ml). We now show that KIL C.2 stimulated with IL-2 for 48 hours produce large quantities of interferon-gamma (10,300 ± 1850 pg/ml) and modest amounts of tumor necrosis factor-alpha (200 ± 60 pg/ml). IL-4 and IL-10 secretion was below background levels. We then evaluated the cytolytic potential of KIL C.2 using a FACS-based flourolysis assay using GFP-labeled target cells. We observed that KIL C.2 potently lyse allogeneic A20 (H2d) lymphoma tumor targets without prior activation. In addition, KIL C.2 is moderately lytic against two syngeneic (H2b) targets: a myeloma cell line, 5T33MM, and BLL C.2, a tumorigenic leukemia/lymphoma cell line that was established from a WBM culture and has the CD19+CD25+CD43+sIg- pre-B-cell phenotype (S.T. and T.M.C., unpublished data). To study the mechanism of cytolysis we performed killing assays in the presence of anti-NKG2D (MI-6) or anti-NK1.1 (PK136) blocking antibodies. Blockade of either NK1.1 or NKG2D reduced cytotoxicity against BLL C.2 targets by 50%. BLL C.2 has the C57BL/6 background, thus does not express H60 but expresses the other NKG2D ligands Rae1 at high levels and Mult1 at moderate levels as determined by quantitative RT-PCR. In contrast, blocking NK1.1 and NKG2D had no effect on cytolysis of A20 suggesting the presence of alternative activation pathways such as those mediated by MHC class I alloantigen receptors. To examine the in vivo tumoricidal potential of KIL C.2, we used a C57BL/6 ? BALB/c T-cell depleted (TCD)-WBM transplantation and A20-luciferase tumor model and monitored tumor progression using the Xenogen bioluminescence imaging system. BALB/c mice lethally irradiated and rescued with 2.5 × 107 TCD-WBM along with A20-luciferase inoculation uniformly died from tumor. Mice given TCD-WBM and 1 × 106 donor splenocytes did not develop tumor but died from lethal graft-versus-host disease (GVHD). In contrast, mice given TCD-WBM and two injections of KIL C.2 at doses of 1 × 107 per mouse were protected from tumor progression without developing signs of GVHD. These results show that KIL C.2 is a highly functional NK cell line with the capacity for proliferation, cytokine secretion, and cytotoxicity against syngeneic as well as allogeneic tumors. As such, KIL C.2 provides a useful cell line model for studies of NK cell biology and suggests a potentially effective strategy for NK cell-based immunotherapy. more...

We have recently established a continuous natural killer (NK) cell line, KIL (for Killer Lymphocyte), from a coculture of normal murine whole bone marrow (WBM) and the OP-9 stromal cell line expressing the Notch ligand, Jagged2, in the presence of stem cell factor and IL-7 (Dehart et al. Blood 2005; 105:3521). A clonal prototype, KIL C.2, was derived from a C57BL/6 mouse and has the CD3−NK1.1+CD122+phenotype, expresses the NKG2D activating receptor, and proliferates extensively upon in vitro stimulation with IL-2 (20ng/ml). We now show that KIL C.2 stimulated with IL-2 for 48 hours produce large quantities of interferon-gamma (10,300 ± 1850 pg/ml) and modest amounts of tumor necrosis factor-alpha (200 ± 60 pg/ml). IL-4 and IL-10 secretion was below background levels. We then evaluated the cytolytic potential of KIL C.2 using a FACS-based flourolysis assay using GFP-labeled target cells. We observed that KIL C.2 potently lyse allogeneic A20 (H2d) lymphoma tumor targets without prior activation. In addition, KIL C.2 is moderately lytic against two syngeneic (H2b) targets: a myeloma cell line, 5T33MM, and BLL C.2, a tumorigenic leukemia/lymphoma cell line that was established from a WBM culture and has the CD19+CD25+CD43+sIg−pre-B-cell phenotype (S.T. and T.M.C., unpublished data). To study the mechanism of cytolysis we performed killing assays in the presence of anti-NKG2D (MI-6) or anti-NK1.1 (PK136) blocking antibodies. Blockade of either NK1.1 or NKG2D reduced cytotoxicity against BLL C.2 targets by 50%. BLL C.2 has the C57BL/6 background, thus does not express H60 but expresses the other NKG2D ligands Rae1 at high levels and Mult1 at moderate levels as determined by quantitative RT-PCR. In contrast, blocking NK1.1 and NKG2D had no effect on cytolysis of A20 suggesting the presence of alternative activation pathways such as those mediated by MHC class I alloantigen receptors. To examine the in vivotumoricidal potential of KIL C.2, we used a C57BL/6 → BALB/c T-cell depleted (TCD)-WBM transplantation and A20-luciferasetumor model and monitored tumor progression using the Xenogen bioluminescence imaging system. BALB/c mice lethally irradiated and rescued with 2.5 × 107TCD-WBM along with A20-luciferaseinoculation uniformly died from tumor. Mice given TCD-WBM and 1 × 106donor splenocytes did not develop tumor but died from lethal graft-versus-host disease (GVHD). In contrast, mice given TCD-WBM and two injections of KIL C.2 at doses of 1 × 107per mouse were protected from tumor progression without developing signs of GVHD. These results show that KIL C.2 is a highly functional NK cell line with the capacity for proliferation, cytokine secretion, and cytotoxicity against syngeneic as well as allogeneic tumors. As such, KIL C.2 provides a useful cell line model for studies of NK cell biology and suggests a potentially effective strategy for NK cell-based immunotherapy. more...

To search for new genes that might be involved in the regulation of erythropoiesis, we performed cDNA representational difference analysis on a multipotent murine hematopoietic cell line, EML C1. Penumbrawas identified based on its predominant, high-level expression in TER119+ erythroblasts. It is not expressed or is expressed at very low levels in Gr1+ or CD3+ or B220+ or CD11c or NK1.1+ bone marrow or spleen cells. The full-length murine PenumbramRNA contains 2017 nucleotides and encodes a new membrane protein characterized by four transmembrane domains, a small extracellular domain between the first and second transmembrane domains and a large extracellular domain between the third and fourth transmembrane domains. It is a member of the evolutionarily conserved tetraspanin membrane protein superfamily that includes CD63, CD81, CD151 and Peripherin. Using the murine Penumbraas a probe, we identified the human Penumbrafrom a human bone marrow cDNA library. The human Penumbraalso encodes a protein of 283 amino acids and is 97% identical to the murine Penumbra. The human Penumbragene is mapped to chromosome 7q32, a hotspot for interstitial deletions in myelodysplastic syndromes and acute myelogenous leukemias. Immunofluorescence microscopy indicated that Penumbra is targeted to the cell surface. Immunoprecipitation-Western analysis showed that Penumbra formed disulfide bond-linked homodimers. To study the possible effects of Penumbradeletions, a knockout mouse model was created by homologous recombination. To avoid any pathology that might result from the Neoselectable marker, the targeting vector employed a self-excising Cre-Neocassette driven by a testis-specific promoter. At 3 months of age, Penumbra−/− mice had similar numbers of RBC, hematocrits, WBC and platelets as the WT littermates. At 1 year of age, Penumbra−/− mice as a group had significantly lower RBC numbers and hematocrits than the WT littermates. Furthermore, about 40–60% of the Penumbra−/− mice had increased percentages of basophilic or polychromatophilic macrocytic RBC. As a group, these mice had even lower numbers of RBC (6.24 ± 1.87 vs. 8.88 ± 0.51 x 106/microliter in WT; p= 0.0002) and hematocrits (36.63 ± 8.28 vs. 46.21 ± 3.62% in WT; p= 0.002). Many such mice had massive splenomegaly due to increased extramedullary hematopoiesis. A multipotent hematopoietic cell line, EMX, was established from the bone marrow of a Penumbra−/−-mouse. EMX exhibits ineffective erythropoiesis in response to erythropoietin, a defect that is reversed by re-expression of Penumbra. These findings indicate that Penumbraplays an important role in erythropoiesis and suggest that Penumbramay be one of the genes that are often deleted during the evolution of myelodysplastic syndromes and acute myelogenous leukemias. more...

The influence of graft composition on clinical outcomes after non-myeloablative allogeneic hematopoietic transplantation is not well characterized. In this report we evaluated the influence of CD34+, CD3+, CD4+, and CD8+ cell doses on donor engraftment, graft-versus-host disease (GVHD), freedom from progression (FFP), and overall survival (OS). Patients (n=63) were given total body irradiation 200 cGy (n=8) or total body irradiation 200 cGy plus fludarabine (n=55) followed by allografting with granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell (G-PBMC). The median age was 53 years, and donors were HLA-identical siblings (n=38) or HLA-matched unrelated individuals (n=25). Almost all patient were treated for a hematologic malignancy (n=60), with 30 patients (48%) characterized as having advanced diseases. G-PBMC cell doses were prospectively enumerated by FACS analysis at the time of collection. Median donor G-PBMC cell doses were 7.4x106 CD34+/kg (range: 1.9–17.7), 3.2x108 CD3+/kg (range: 0.8–6.4), 2.1x108 CD4+/kg (range: 0.4–4.1), and 1.1x108 CD8+/kg (range: 0.2–3.4). By univariate analysis only G-PBMC CD8+ T-cell dose ≥ the 50th percentile favorably correlated with attainment of full donor blood T-cell chimerism (P = .03). The incidences of grade 2–4 acute GVHD was 16.3% (95% CI: 6.9–25.8) and extensive chronic GVHD was 42.9% (95% CI: 27.8–57.9). No G-PBMC cell dose significantly influenced grade 2–4 acute or extensive chronic GVHD. With a median follow-up of 13 months (range: 5–45), estimates for FFP was 53.7% (95% CI: 37.7–69.7) and OS was 33.4% (95% CI: 17.4–49.4). G-PBMC CD8+ T-cell dose ≥ the 50th percentile T-cell dose was favorably prognostic for both FFP (P = .001) and OS (P = .01). Survival curves for FFP and OS are shown in Figure 1. Extensive chronic GVHD also predicted better FFP (P = .02), but in multivariate analysis only G-PBMC CD8 + T-cell dose (P = .004; RR = 0.2, 95% CI = 0.1–0.6) was associated with improved FFP. Infusion of low G-PBMC CD8+ T-cell dose for non-myeloablative allografting with total body irradiation 200 cGy plus fludarabine may adversely affect donor T-cell engraftment, disease progression and survival outcomes. more...

Minor histocompatibility antigens (mHAg) are T-cell peptide antigens derived from polymorphic endogenous gene products encoded throughout the genome. Despite extensive human genetic diversity, recent experimental evidence suggests that a mHAg set may be limited in size because alloimmune responses directed against them can be dominated by only a few specific peptides. To extend upon these observations, we performed a genome-wide scan to identify susceptibility loci for graft-versus-host disease (GVHD) in a murine model. We previously identified B10.BR (H2k) mice to be resistant, and BALB.K (H2k) mice susceptible, to lethal GVHD when transplanted with hematopoietic cells from MHC-matched AKR/J (H2k) donors (Cao et al. PNAS2003:11571). In the present study B10.BR, BALB.K, or (B10.BR x BALB.K)F1 mice were lethally irradiated and injected with AKR/J bone marrow at radioprotective doses plus 2.0 or 3.0 x 106 purified splenic T-cells. Confirming our prior observations, B10.BR mice survived to day 60 whereas all BALB.K mice died prior to day 10 from GVHD. All (B10.BR x BALB.K)F1 mice also died after a median of 10 days, indicating that GVHD susceptibility in BALB.K mice is a dominant trait. (B10.BR x BALB.K)F1 mice were then backcrossed to the GVHD-resistant B10.BR parent. Resulting [B10.BR x (B10.BR x BALB.K)]BC mice (n = 180) were genotyped and similarly irradiated and injected with AKR/J bone marrow plus 3.0 x 106 splenic T-cells. Forty (22%) BC mice died with signs of GVHD prior to day 100. Surprisingly, a 90-marker genome-wide scan for this phenotype, lethal GVHD prior to day 100, revealed highly significant linkage (P <.001) only to chromosome 16. Linkage analysis was performed with MapManager QTX 0.29 using Kosambi’s map function. As shown in Figure 1, interval mapping for chromosome 16 with additional markers revealed a broad and complex linkage pattern encompassing 2 markers, D16Mit4 (27.3 cM) and D16Mit189 (55.2 cM), that had peak LOD scores of 8.8 and 8.0 respectively. Another genetic locus on chromosome 13 flanked by markers D13Mit248 and D13Mit149 with a peak LOD score of 2.3 by interval mapping was suggestively linked. While an evaluation of candidate genes within the support intervals is in progress, these results thus far suggest that the critical gene dose required for inducing lethal GVHD may be remarkably small. If validated as encoding for immunodominant mHAg, these results furthermore suggest that a functionally relevant mHAg set may be definable for clinical genotyping. more...

The influence of graft composition on clinical outcomes after non-myeloablative allogeneic hematopoietic transplantation is not well characterized. In this report we evaluated the influence of CD34+, CD3+, CD4+, and CD8+cell doses on donor engraftment, graft-versus-host disease (GVHD), freedom from progression (FFP), and overall survival (OS). Patients (n=63) were given total body irradiation 200 cGy (n=8) or total body irradiation 200 cGy plus fludarabine (n=55) followed by allografting with granulocyte colony-stimulating factor-mobilized peripheral blood mononuclear cell (G-PBMC). The median age was 53 years, and donors were HLA-identical siblings (n=38) or HLA-matched unrelated individuals (n=25). Almost all patient were treated for a hematologic malignancy (n=60), with 30 patients (48%) characterized as having advanced diseases. G-PBMC cell doses were prospectively enumerated by FACS analysis at the time of collection. Median donor G-PBMC cell doses were 7.4x106CD34+/kg (range: 1.9–17.7), 3.2x108CD3+/kg (range: 0.8–6.4), 2.1x108CD4+/kg (range: 0.4–4.1), and 1.1x108CD8+/kg (range: 0.2–3.4). By univariate analysis only G-PBMC CD8+T-cell dose ≥ the 50thpercentile favorably correlated with attainment of full donor blood T-cell chimerism (P= .03). The incidences of grade 2–4 acute GVHD was 16.3% (95% CI: 6.9–25.8) and extensive chronic GVHD was 42.9% (95% CI: 27.8–57.9). No G-PBMC cell dose significantly influenced grade 2–4 acute or extensive chronic GVHD. With a median follow-up of 13 months (range: 5–45), estimates for FFP was 53.7% (95% CI: 37.7–69.7) and OS was 33.4% (95% CI: 17.4–49.4). G-PBMC CD8+T-cell dose ≥ the 50thpercentile T-cell dose was favorably prognostic for both FFP (P= .001) and OS (P= .01). Survival curves for FFP and OS are shown in Figure 1. Extensive chronic GVHD also predicted better FFP (P= .02), but in multivariate analysis only G-PBMC CD8 +T-cell dose (P= .004; RR = 0.2, 95% CI = 0.1–0.6) was associated with improved FFP. Infusion of low G-PBMC CD8+T-cell dose for non-myeloablative allografting with total body irradiation 200 cGy plus fludarabine may adversely affect donor T-cell engraftment, disease progression and survival outcomes. more...

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option. more...