Management of chronic spontaneous urticaria in real life--in accordance with the guidelines? A cross-sectional physician-based survey study.

BACKGROUND: Recently, the updated EAACI/GA(2) LEN/EDF/WAO guidelines for urticaria have been published. OBJECTIVE: To examine how chronic spontaneous urticaria (csU) patients in Germany are diagnosed and treated, and to compare the outcome to the guideline recommendations. METHODS: During this cross-sectional survey study, most dermatologists, paediatricians and 5149 general practitioners in private practice in Germany were asked to participate. All physicians who agreed were requested to complete a standardized questionnaire about their diagnostic and therapeutic management of csU. RESULTS: A total of 776 questionnaires were available for analysis. Most physicians (82%) were attempting to identify underlying causes in their csU patients, but with only limited success. More than 70% reported to check for total serum IgE and to do skin prick testing (not suggested in first line by guideline). In contrast, only 10% applied the autologous serum skin test. The most common first-line treatments were non-sedating antihistamines in standard or higher doses (as recommended). However, many physicians reported still using first generation sedating antihistamines (23%) (not recommended) or systemic steroids (18%). Experience with alternative options was low. Less than one-third of the participants reported to be familiar with the guidelines. Those who did, were found to be more likely to check for underlying causes, to be more experienced with antihistamine updosing and to be more reluctant to use sedating antihistamines or systemic steroids. CONCLUSION: The diagnostic and therapeutic management of csU by private practice physicians does not sufficiently comply with the guidelines. Awareness of the guidelines can lead to improved care.

Impact of cyclosporine and low-dose steroid therapy on insulin sensitivity and beta-cell function in patients with long-term liver grafts.

To examine whether factors controlling glucose tolerance, i.e., insulin sensitivity (SI) and first-(phi1) and second-phase insulin secretion (phi2), are impaired in after orthotopic liver transplantation (OLT), they were assesssed in patients that had undergone OLT for cirrhosis (n = 10) with cyclosporin A and low-dose steroid therapy (5 mg prednisone per day) and were compared with those of healthy matched control subjects (n = 10). These factors were determined by means of computer-based analysis of frequently sampled intravenous glucose tolerance tests (FSIGTT). Glucose and insulin profiles (posthepatic insulin) did not differ between both groups, whereas C-peptide levels (prehepatic insulin) were elevated in the transplant group after the FSIGTT, indicating an increased hepatic insulin degradation. SI and (phi1 did not differ between both groups. phi2, however, was significantly enhanced (23.94 +/- 2.63 vs 13.88 +/- 1.25 min(-1), P < 0.05). These results indicate that cyclosporine and low-dose steroid therapy do not impair SI and phi1. However, enhanced phi2 compensates the increased hepatic insulin clearance.

A prospective study of positive/negative ex vivo B-cell depletion in patients with chronic lymphocytic leukemia.

OBJECTIVE: Autologous peripheral blood stem cell (PBSC) transplantation is increasingly being used in patients with chronic lymphocytic leukemia (CLL). As the autografts are frequently contaminated with large numbers of tumor cells, we have prospectively investigated the feasibility and efficacy of ex vivo double purging of PBSC grafts in an open, nonrandomized, single-center phase I/II clinical study. MATERIALS AND METHODS: Twenty consecutive patients with poor-risk CLL underwent uniform stem cell mobilization with chemotherapy and granulocyte colony-stimulating factor (G-CSF). Double B-cell depletion of the harvested PBSC products was performed using immunomagnetic CD34(+) cell selection (Isolex300i Nexell, Irvine, CA) followed by a negative step with anti-CD19/20/23/37-labeled immunomagnetic beads. The purified PBSC were reinfused after myeloablative treatment with TBI/CY. RESULTS: A total of 25 separation runs was accomplished using collection products containing 3.4% (1.1-8.1) CD34(+) cells and 1.2% (0.1-42) CD19(+)CD5(+) CLL cells. After double selection, 33% (15-67) CD34(+) cells were recovered with a purity of 98.8% (89.1-99. 8). CLL cells were undetectable by high-resolution flow cytometry in 15 of 25 final products; median purging efficacy was 5 (4.1-6) log. The CD34(+) content of the 20 final grafts was 4.6 (2.2-6.5) x 10(6)/kg. Rapid and durable engraftment developed in all cases. With a median follow-up of 20 (6-29) months, 17 patients live in complete clinical remission, two have recurrent disease, and one patient died due to pulmonary embolism five months after transplant. Persistence of the leukemic clone on the molecular level was demonstrated by dot blotting with clone-specific CDR3 probes in an additional five patients. Serious or unexpected infectious complications did not occur. CONCLUSIONS: Positive/negative purging with the Isolex system allows preparation of highly purified CD34(+) fractions and up to six log of tumor cell depletion in patients with B-CLL and can be safety reinfused after myeloablative therapy without affecting hematopoietic engraftment.

Autografting of highly purified peripheral blood progenitor cells following myeloablative therapy in patients with lymphoma: a prospective study of the long-term effects on tumor eradication, reconstitution of hematopoiesis and immune recovery.

In a prospective study, we have investigated CD34+ selection of peripheral blood progenitor cells (PBPC) for autotransplantation in patients with lymphoma. Twenty-six consecutive patients (10 follicular lymphomas, seven mantle cell lymphomas, seven B-CLL, two immunocytomas) were mobilized using chemotherapy plus G-CSF. Sufficient numbers of PBPC could be collected from 24 patients and were immunoselected with the semiautomated Isolex 300 (n = 17) or the fully integrated Isolex 300i (n = 7) devices. The selection products were assayed by PCR amplification of clonal CDRIII or t(14;18) rearrangements for residual tumor cell content. Residual disease and long-term hematopoietic and immune recovery were studied by assessing the following parameters at 3, 6, and 12 months post-transplant: CDRIII or t(14;18) PCR, platelet count, lymphocyte subsets, serum IgG, serum IgA, and measles titer. With the Isolex 300 device 26% (10-65) of input CD34+ cells were recovered with a median purity of 89.2% (49.4-98.9) after CD34+ selection. The Isolex 300i device allowed significantly better recoveries (46% (22-86)) and purities of CD34+ cells (98.8% (92.2-99.2)). The overall purging efficacy was 3.2 (0.6-5.1) log. Twenty patients have been reinfused with CD34+ selected grafts after myeloablative preparation. Rapid engraftment occurred in all patients. With a median follow-up of 28 (19-42) months, 14 patients are alive without clinical or molecular evidence of disease recurrence, whereas five have relapsed and one additional patient shows persistent presence of the disease-specific molecular marker without clinical progression. Cellular and serological parameters of hematopoietic and immune functions were largely normal at 12 months post-transplant including the measles titer which was present in all patients. Kinetics of immunohematopoietic recovery were similar to those of 12 control patients who had received unmanipulated PBPC during the same time period except for the recovery of CD4+ CD45RA+ T cells which was significantly delayed in the CD34+ group. During the first year post-transplant, transient monoclonal or oligoclonal gammopathies were observed in seven of 16 study patients. We conclude that CD34+ selection with the Isolex system allows preparation of highly purified CD34+ fractions and effective tumor cell depletion. The CD34+ products can be reinfused safely after myeloablative treatment and result in sustained hematopoietic and immune recovery. The fact that all patients retained their specific measles immunity suggests that myeloablative treatment with reinfusion of highly purified CD34+ PBPC is not immunoablative.

Combined positive/negative selection for highly effective purging of PBPC grafts: towards clinical application in patients with B-CLL.

Autologous PBPC transplantation is a potentially curative treatment for patients with chronic lymphocytic leukemia (CLL). As the autografts are frequently contaminated with large numbers of tumor cells, we have studied double purging of PBPC using immunomagnetic CD34+ cell selection (Isolex 300i) followed by negative depletion with anti-CD19/20/23/37-labeled immunomagnetic beads. In four small-scale experiments using PBPC from patients with CLL or leukemic low-grade lymphoma, double purging resulted in CD34+ enrichment from 0.9-4.4% to 95.8-99.4%. Lymphoma cells were always undetectable by FACS and PCR (CDR3 or t(14;18)) after negative depletion. Next, seven heavily contaminated full grafts from five patients with CLL or lymphoplasmocytoid immunocytoma were subjected to the double purging procedure, resulting in a CD34+ enrichment from 1.6% (0.7-5.6) to 98.5% (96-99.8). The CD34+ yield after double purging was 1.3-6.3 x 10(6)/kg, according to a median recovery of 32%. The overall reduction of lymphoma cells was 5.6 (>4.6-6) log. Although CLL cells were completely absent after purging in five cases as assessed by FACS, all grafts remained PCR positive. The first two patients have been reinfused with double selected products after myeloablative radiochemotherapy and showed prompt and uneventful hematopoietic engraftment. We conclude that without significant loss of CD34+ cells, negative depletion adds 2 log of CLL cell depletion to CD34+ selection, resulting in an overall purging efficacy of more than 5 log. This combination of positive and negative selection can be successfully applied even to heavily contaminated PBPC grafts.

Purging peripheral blood progenitor cell grafts from lymphoma cells: quantitative comparison of immunomagnetic CD34+ selection systems.

Autologous peripheral blood progenitor cell (PBPC) transplantation is increasingly being used for treatment of indolent lymphomas. Since involvement of bone marrow and peripheral blood is frequent and methods to reduce the lymphoma cell load of PBPC grafts are thus highly desirable, we have studied purging of PBPC comparing two immunomagnetic CD34+ selection systems (VarioMACS, Miltenyi Biotech; Bergisch Gladbach, Germany, and Isolex50 System, Baxter; Irvine, CA). Samples of freshly collected mobilized PBPCs were contaminated with BALM-3 or KARPAS422 lymphoma cells that had been labeled with the fluorescent DNA stain Hoechst 33342. The mixture was subjected to separation with the two devices and the resulting "CD34+" fractions were screened for lymphoma cells by limiting dilution using fluorescence microscopy and by polymerase chain reaction amplification of t(14;18) or CDRIII-rearrangements. Both devices yielded comparable purities (MACS 97% [87%-99%]; Isolex 97% [84%-99%]) and recoveries of CD34+ cells (MACS 56% [30%-81%]; Isolex 45% [24%-63%]). The overall depletion of lymphoma cells was 3.9 log (2.6-5.9), however, residual contaminating cells were seen in every single experiment. The purging efficacy was dependent on the type of contaminating lymphoma cell (BALM-3: 4.4 log [3.7-4.8]; KARPAS422: 3.2 log [2.6-4.2]; p = 0.018), whereas the type of selection system used or the percentage of CD34+ cells in the starting material had no influence. We conclude that excellent purification of CD34+ cells leading to a vigorous depletion of lymphoma cells can be achieved with both CD34+ selection systems investigated. However, the efficacy of purging may greatly differ between individual lymphomas, and complete eradication of contaminating cells from PBPC grafts may rarely be achieved with CD34+ selection alone.

G-CSF-mobilized peripheral blood progenitor cells for allogeneic transplantation: comparison of T cell depletion strategies using different CD34+ selection systems or CAMPATH-1.

Allogeneic transplantation of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) appears to be an attractive alternative to allogeneic bone marrow transplantation (BMT). However, because vast amounts of potentially graft-vs.-host-reactive T cells are transfused with PBPC grafts, the use of PBPC in the allogeneic setting may be associated with an increased incidence or severity of graft-vs.-host disease (GVHD). To evaluate strategies for prevention of GVHD after PBPC allografting, we have studied T cell depletion (TCD) of G-CSF-mobilized PBPC samples harvested from six healthy donors and from five patients scheduled for autologous PBPC transplantation. Three approaches (CAMPATH-1 plus autologous complement [C], immunomagnetic CD34+ cell selection, and biotin-avidin-mediated CD34+ cell selection) were compared. TCD of PBPC samples with the monoclonal antibody (MAb) CAMPATH-1 plus autologous C resulted in a median elimination of 2.16 log CD3+ T cells, whereas 39% of CD56+ natural killer (NK) cells and 56% of CD34+ progenitor cells were recovered. TCD by CD34+ cell selection with the Isolex (Baxter, Munich, Germany) or Ceprate (CellPro, Bothell, WA) devices achieved median depletions (Isolex vs. Ceprate) of 4.04 vs. 3.12 log T cells and > 5 vs. 3.27 log NK cells while allowing the recovery of 36 vs. 27% CD34+ cells. The median purity of CD34+ cells in the final product was 1.7 (CAMPATH-1), 94 (Isolex), and 65% (Ceprate). We conclude that all methods tested effectively deplete T cells from PBPC preparations harvested from healthy donors. Whereas immunomagnetic CD34+ selection is most effective in terms of elimination of T cells, the less intensive T and NK cell depletions achieved with CAMPATH-1 might be advantageous with regard to retaining engraftment potential and graft-vs.-leukemia (GVL) activity of PBPC allografts.