Eleven patients with relapsed fludarabine-resistant B-cell chronic lymphocytic leukemia (CLL) or leukemic variants of low-grade B-cell non-Hodgkin’s lymphoma (NHL) were treated with the chimeric monoclonal anti-CD20 antibody rituximab (IDEC-C2B8). Peripheral lymphocyte counts at baseline varied from 0.2 to 294.3 × 109/L. During the first rituximab infusion, patients with lymphocyte counts exceeding 50.0 × 109/L experienced a severe cytokine-release syndrome. Ninety minutes after onset of the infusion, serum levels of tumor necrosis factor- (TNF-) and interleukin-6 (IL-6) peaked in all patients. Elevated cytokine levels during treatment were associated with clinical symptoms, including fever, chills, nausea, vomiting, hypotension, and dyspnea. Lymphocyte and platelet counts dropped to 50% to 75% of baseline values within 12 hours after the onset of the infusion. Simultaneously, there was a 5-fold to 10-fold increase of liver enzymes, d-dimers, and lactate dehydrogenase (LDH), as well as a prolongation of the prothrombin time. Frequency and severity of first-dose adverse events were dependent on the number of circulating tumor cells at baseline: patients with lymphocyte counts greater than 50.0 × 109/L experienced significantly more adverse events of National Cancer Institute (NCI) grade III/IV toxicity than patients with less than 50.0 × 109/L peripheral tumor cells (P= .0017). Due to massive side effects in the first patient treated with 375 mg/m2 in 1 day, a fractionated dosing schedule was used in all subsequent patients with application of 50 mg rituximab on day 1, 150 mg on day 2, and the rest of the 375 mg/m2 dose on day 3. While the patient with the leukemic variant of the mantle-cell NHL achieved a complete remission (9 months+) after treatment with 4 × 375 mg/m2 rituximab, efficacy in patients with relapsed fludarabine-resistant B-CLL was poor: 1 partial remission, 7 cases of stable disease, and 1 progressive disease were observed in 9 evaluable patients with CLL. On the basis of these data, different infusion schedules and/or combination regimens with chemotherapeutic drugs to reduce tumor burden before treatment with rituximab will have to be evaluated.

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions.In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac α-actin promoter. First, fluorescent areas in ES cell–derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1–2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs.Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.

Patients with HD have an increased risk for inadequate semen quality even prior to treatment. Infertility is more frequent in patients with elevated ESR and advanced stage of disease. This association demonstrates the predominant influence of the disease on fertility. Assuming HD is the major initial cause for infertility efforts should be made to identify new non-gonadal toxic chemotherapies to be able to regain fertility after effective therapy. Further investigations have to be performed to clarify mechanisms inducing fertility defects in patients with HD.

Summary. Multiple myeloma (MM) is characterized by a clonal proliferation of malignant plasma cells in the bone marrow secreting a monoclonal immunoglobulin (paraprotein) with specific antigenic determinants, the idiotype (Id), which can be regarded as a tumour-associated antigen (TAA). In order to analyse the impact of a dendritic cell (DC)-based vaccine, 11 patients with advanced MM were treated with CD34 stem cell-derived dendritic cells that were pulsed with Id peptides. Subsequently, the patients received three boost immunizations every other week with a combination of Id and granulocyte±macrophage colonystimulating factor (GM-CSF) (nine patients) or with Id peptide-pulsed dendritic cells again (two patients). The treatment was well tolerated with no side-effects. The present clinical study was a proof of concept analysis of dendritic cell-based vaccines in MM. The capacity of the dendritic cells to activate idiotype-specific T cells was verified by in vitro stimulation experiments before the vaccination therapy. Immunological effects of the Id vaccination were analysed by monitoring changes in anti-idiotype antibody titres and idiotype-specific T-cell activity. After vaccination, three out of 10 analysed patients showed increased antiidiotype antibody serum titres, indicating the induction of an idiotype-specific humoral immune response. The idiotype-specific T-cell response analysed by ELISpot was increased in four out of 10 analysed patients after vaccination, and one patient had a decreased plasma cell infiltration in the bone marrow. In conclusion, five out of 11 patients showed a biological response after vaccination. Thus, our data indicate that immunotherapy with Id-pulsed DCs in MM patients is feasible and safe. DC generated from CD34 1 progenitor cells can serve as a natural adjuvant for the induction of clinically relevant humoral and cellular idiotype-specific immune responses in patients suffering from advanced MM.

The CC thymus and activation-related chemokine (TARC) is a protein, which is highly expressed by Reed-Sternberg cells in Hodgkin's disease and is found in the majority of Hodgkin's disease patients. Within several trials conducted by the German Hodgkin study group, 62 Hodgkin's disease patients were elected based on availability of serum samples post and prior therapy to assess TARC levels by ELISA. TARC levels from 33 patients with continuous complete response (CCR), 20 patients with relapse, and nine patients with progressive disease (PD) were correlated with freedom from treatment failure and survival. As defined in healthy donors (mean value F 2Â SD), a TARC level of >500 pg/mL was considered as elevated. The median TARC levels of all patients at baseline and after completed primary treatment were 5,803 pg/mL (range, 116-73,074 pg/mL) and 663 pg/mL (50-24,709 pg/mL), respectively. TARC levels of patients with PD were higher than those of patients with CCR at baseline and after therapy. Baseline TARC correlated significantly with stage (P = 0.019), erythrocyte sedimentation rate (P = 0.004), leukocyte count (P < 0.001), and lymphocyte count (P = 0.026). A TARC level of >2,000 pg/mL after completed treatment was a significant risk factor for poorer survival (P = 0.02) but not for relapse. In conclusion, monitoring serum TARC levels in Hodgkin's disease patients may add valuable information about therapy success in Hodgkin's disease patients, especially those with PD and should therefore be prospectively evaluated in future trials. (Cancer Res 2005; 65(13): 5516-9)