Automatic generation of alloreactivity-reduced donor lymphocytes and hematopoietic stem cells from the same mobilized apheresis product.

INTRODUCTION: In vitro or in vivo depletion of alloreactive T cells can facilitate haplo-identical hematopoietic stem cell transplantation (HSCT). Very satisfactory transplant outcomes were thus reported for TCRαß/CD19-depleted hematopoietic stem/progenitor cell (HSPC) grafts. The current semi-automatic manufacturing process on the CliniMACS Plus, although robust, still requires a significant amount of manual labor to be completed. Towards advancing and further facilitating large scale cell processing, a new TCRαß/CD19 depletion module combined with the previously described CD45RA depletion module (to serve as allo-reactivity attenuated donor lymphocyte infusion) was established on the CliniMACS Prodigy. METHODS: We evaluated six apheresis products from G-CSF-mobilized volunteer donors which were split automatically by the Prodigy, one portion each depleted of CD45RA+ or of TCRαß+ and CD19+ cells. We investigated critical quality attributes for both products. Products were assessed for recovery of HSPCs and mature subsets, as well as depletion efficiency of targeted cells using flow cytometry. Effects of apheresis and product age post 48 h storage at 2-6 °C as well as freeze-thawing on product viability and recovery of WBC and HPSCs were assessed by flow cytometry. RESULTS: Ten sequential automatic processes were completed with minimal hands-on time beyond tubing set installation. Depletion efficiency of CD45RA+ resp. TCRαß+ and CD19+ cells was equivalent to previous reports, achieving mean depletions of 4 log of targeted cells for both products. HSPC products retained TCRγδ+ and NK cells. 48 h storage of apheresis product was associated with the expected modest loss of HSPCs, but depletions remained efficient. Depleted products were stable until at least 72 h after apheresis with stem cell viabilities > 90%. Freeze-thawing resulted in loss of NK cells; post-thaw recovery of viable CD45+ and HSPCs was > 70% and in line with expectation. CONCLUSION: The closed, GMP-compatible process generates two separate medicinal products from the same mobilized apheresis product. The CD45RA-depleted products contained functional memory T cells, whereas the TCRαß/CD19-depleted products included HSPCs, TCRγδ+ and NK cells. Both products are predicted to be effectively depleted of GVH-reactivity while providing immunological surveillance, in support of haplo-identical HSCT.

Automated isolation of primary antigen-specific T cells from donor lymphocyte concentrates: results of a feasibility exercise.

BACKGROUND: The safety and clinical efficacy of adoptive transfer of prospectively isolated antigen-specific T cells are well established. Several competing selection methods are available, one of which is based on immunomagnetic enrichment of T cells secreting IFNγ after incubation with the relevant antigen. The proprietary, GMP-conforming selection technology, called 'cytokine capture system' (CCS) is established in many laboratories for the CliniMACS Plus system. It is robust and efficient, but labour-intensive and incompatible with a single-shift working schedule. An automatic immunomagnetic cell processing system, CliniMACS Prodigy ('Prodigy'), including a protocol for fully automatic CCS execution was recently released. MATERIAL AND METHODS: Feasibility of clinical-scale CMV-specific T-cell selection using Prodigy was evaluated using leukoapheresis products from five healthy CMV sero-positive volunteers. Clinical reagents and consumables were used throughout. RESULTS: The process required no operator input beyond set-up and QC-sample collection, that is, feasibility was given. An IFNγ-secreting target T-cell population was detectable after stimulation, and >2 log-scale relative depletion of not CMV-reactive T cells in the target population was achieved. Purity, that is the frequency of CMV-reactive T cells among all CD3(+) cells ranged between 64 and 93%. CONCLUSION: The CCS protocol on Prodigy is unrestrictedly functional. It runs fully automatically beyond set-up and thus markedly reduces labour. The quality of the products generated is similar to products generated with CliniMACS Plus. The automatic system is thus suitable for routine clinical application.

Enhancement of the anti-tumor activity of a peripheral blood progenitor cell graft by mobilization with interleukin 2 plus granulocyte colony-stimulating factor in patients with advanced breast cancer.

OBJECTIVE: Autologous interleukin 2 (IL-2)-activated natural killer (NK) cells kill a broad spectrum of tumor targets, including breast cancer. We hypothesized that mobilization with IL-2 and granulocyte colony-stimulating factor (G-CSF) for collection of peripheral blood progenitor cells (PBPC) may enhance the anti-tumor activity of the graft in autograft recipients. We determined the dose-limiting toxicity and maximum tolerated dose of subcutaneous IL-2 given with G-CSF for PBPC mobilization, the ability of IL-2 + G-CSF mobilized stem cells to reconstitute hematopoiesis, and the in vitro immunologic function of the graft in patients with advanced breast cancer. MATERIALS AID METHODS: Forty-three women with stage IIIA/B or metastatic breast cancer underwent mobilization of PBPC with IL-2 administered subcutaneously for 14 days along with G-CSF for the latter 7 days. IL-2 was given in a dose-escalated manner, with the maximum tolerated dose determined to be 1.75 x 10(6) IU/m(2)/day. Fifteen women with stage IIIA/B or metastatic breast cancer underwent G-CSF mobilization alone and served as a control group. RESULTS: [corrected] Fifty-two percent of the patients mobilized with 1L-2 at the maximum tolerated dose reached the target number of CD34(+) cells for transplantation with three aphereses compared to 93% of control patients who were mobilized with G-CSF alone. [corrected] There was no significant impact on time to engraftment of neutrophils or platelets using either mobilization regimen. The addition of subcutaneous IL-2 to mobilization increased the cytotoxicity of IL-2-activated mononuclear cells from the PBPC product against the breast cancer cell target, MCF-7, and increased the percentage of NK cells and activated T cells in the PBPC product. The enhanced NK cell number was sustained in the early posttransplant period. CONCLUSIONS: [corrected] IL-2 + G-CSF mobilization is safe, may lead to a more immunologically functional graft without impairing hematologic recovery, and thus merits further exploration to evaluate the clinical anti-tumor efficacy of these immunocompetent grafts. [corrected] Limitations of this combined approach to stem cell mobilization include a decrease in the number of CD34(+) cells mobilized with the combined cytokines and the short duration of the increased number of anti-tumor effector cells after transplant.

[2D-SPLASH spectroscopy to determine the fat/water ratio in the muscle of the rotator cuff]. / 2D-SPLASH-Spektroskopie zur Bestimmung des Fett/Wasser-Verhältnisses in der Rotatorenmanschette.

AIM: The degree of fatty infiltration of the rotator cuff is an important factor for the prognosis of an operative reconstruction afterrotator cuff tear. The aim of this work was to develop a method using a clinical MR scanner that allows the quantification of the fat/water ratio with the necessary spatial resolution. METHOD: A SPLASH sequence consisting of 19 complex 2D-FLASH images was implemented on a clinical 1.5 T MR scanner. The echo time was gradually increased from 5.0 ms to 50.0 ms. A spatial in plane resolution of 1.17 mm, a spectral resolution of 0.33 ppm and a spectral width of 6.25 ppm were achieved in a total acquisition time of about 3 min. The quantitative evaluation of the spectra in arbitrarily shaped regions of interest (ROIs) was obtained using a home-built reconstruction program and the time domain fit program AMARES. RESULTS: Phantom studies show a linear relation of the concentration determined by SPLASH spectroscopy (r = 0.997). Because of the high spatial resolution and the possibility to evaluate arbitrarily shaped ROIs, the determination of the fat/water ratio in single muscles in the shoulder has been possible. CONCLUSIONS: By the use of the 2D-SPLASH sequence the degree of fatty infiltration in the rotator cuff can now be determined quantitatively for the first time.

Avascular necrosis of the capital femoral epiphysis in a child receiving corticosteroids: a case report.

Exogenous steroids are known to induce aseptic necrosis in the femoral head of adults. This case report of a 9-year-old girl with corticosteroid induced femoral head necrosis demonstrates effective treatment with an abduction device in a manner similar to that prescribed for the treatment of Legg-Calve-Perthes disease.

Nonoperative treatment for painful adolescent spondylolysis or spondylolisthesis.

Eighty-two adolescent patients with symptomatic spondylolysis or spondylolisthesis were treated by nonoperative modalities. Patients' ages ranged from 6.5 to 21 years, and follow-up ranged from 1 to 14.3 years. Thirty-one patients became asymptomatic, 17 had significant but incomplete relief of pain, nine had no relief, and 25 required surgical treatment for pain. Of 12 patients with grade 3 or 4 slip, only one had significant relief of pain, whereas 48 of 70 patients with lesser degrees of slip had significant relief of pain. Nonoperative treatment of degrees of spondylolisthesis of less than or equal to grade 2 can reliably relieve pain in two-thirds of patients.

Gly429 is the major determinant of uncompetitive inhibition of human germ cell alkaline phosphatase by L-leucine.

The catalytic activity of human placental alkaline phosphatase (PLAP) and germ cell alkaline phosphatase (GCAP) can be inhibited, through an uncompetitive mechanism, by L-Phe. GCAP is also selectively inhibited by L-Leu. Site-directed mutagenesis of five of the 12 residues which are different in PLAP and GCAP revealed that Gly429 is the primary determinant of GCAP inhibition by L-Leu, and Ser84 and Leu297 play a modulatory role in the inhibition.

Catastrophic failure of modular zirconia-ceramic femoral head components after total hip arthroplasty.

Catastrophic failure of two zirconia-ceramic modular femoral heads occurred, despite the theoretical improved toughness of zirconia-ceramic relative to alumina-ceramic. This experience led the authors to return to cobalt-chromium as the metal of choice for articulation against polyethylene in total hip arthroplasty.

AtSUC3, a gene encoding a new Arabidopsis sucrose transporter, is expressed in cells adjacent to the vascular tissue and in a carpel cell layer.

The cDNA corresponding to the open reading frame T17M13.3 from Arabidopsis chromosome II was isolated and the encoded protein was characterized as a member of a subgroup of higher plant sucrose transporters. The AtSUC3 (Arabidopsis thaliana sucrose transporter 3) open reading frame encodes a protein with 594 amino acid residues, being 81 and 82 residues longer than the previously described Arabidopsis sucrose carriers AtSUC1 and AtSUC2. About 50 of these additional amino acids are part of an extended cytoplasmic loop separating the N-terminal from the C-terminal half of the protein. For functional characterization the AtSUC3 cDNA was expressed in baker's yeast. Substrate specificities, energy dependence and K(m) values of the recombinant protein were determined. Removal of the enlarged cytoplasmic loop and expression of the truncated cDNA caused no detectable change in the kinetic properties of the protein, suggesting a transport-independent function for this cytoplasmic domain. Immunolocalization with an AtSUC3-specific antiserum identified the protein in a cell layer separating the phloem from the mesophyll and in a single, subepidermal cell layer of the carpels that is important for pod dehiscence. These localizations suggest a possible role of AtSUC3 in the funnelling of sucrose from the mesophyll towards the phloem, and possibly in pod shatter.

Meniscal repair: outcomes and clinical follow-up.

A retrospective study of arthroscopic meniscal repair in 101 consecutive patients was conducted. Sixty-three patients constituted our study group. The arthroscopic technique used for meniscal repair was the inside-out method using malleable cannulas. Forty-five patients were available for clinical examination, with a mean follow-up of 27 months. Tegner and Lysholm scores were comparable to those previously reported for arthroscopic meniscal repair. The HSQ (similar to the SF-36) scores were equal to those from an age- and sex-matched normal population, indicating that individuals with repaired menisci do not have any residual negative effects on global health at mean 26.9 months' follow-up. The physical functioning subscale of the HSQ was found to be sensitive to patient perception of results. Complications included one case of restricted knee range of motion requiring arthroscopy and lysis of adhesions. Overall clinical results were 64% excellent, 27% good, and 9% failure. Age, sex, and length of the meniscal tear had no affect on clinical outcome.