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Ubiquitin is suggested to play a key role in essential intracellular functions, such as heat shock response, protein breakdown, and regulation of immune responses. Ubiquitin has also been detected in the extracellular space, but the function and biologic significance is unclear. We describe a new function of extracellular ubiquitin and show that extracellular ubiquitin specifically inhibits ex vivo secretion of tumor necrosis factor-alpha (TNF-alpha) and TNF-alpha mRNA expression from peripheral blood mononuclear cells (PBMNCs) in response to endotoxin in a dose-dependent manner. In contrast, the TNF-alpha response to zymosan or Staphylococcus aureus as well as the interleukin-6 (IL-6) and IL-8 responses to endotoxin were unaffected by ubiquitin. Measurement of serum ubiquitin levels showed a significant 5- to 7-fold increase in sepsis and trauma patients, to the level required for inhibition of the PBMNC TNF-alpha response to endotoxin by ubiquitin. Elevated ubiquitin levels in serum were significantly correlated with a reduced TNF-alpha production. Antibodies to ubiquitin were able to (1) significantly increase (2- to 5-fold) the TNF-alpha response to endotoxin in whole blood from trauma and sepsis patients, (2) completely neutralize the inhibitory effect of trauma patients' serum on healthy donors' TNF-alpha production, and (3) partially neutralize the inhibitory effect of sepsis patients' serum on healthy donors' TNF-alpha production. Ubiquitin-depleted serum from trauma patients lost the inhibitory activity for TNF-alpha production, whereas extracted endogenous ubiquitin exerts the inhibitory activity. The results demonstrate that extracellular ubiquitin acts as a cytokinelike protein with anti-inflammatory properties and indicate that extracellular ubiquitin is involved in the regulation of immunodepression in critical illness.

Hypogammaglobulinemia is the hallmark of common variable immunodeficiency (CVID) syndrome, a heterogeneous disorder predisposing patients to recurrent bacterial infections. In this study, we investigated the peripheral B-cell compartment of 30 well-characterized CVID patients in comparison to 22 healthy controls. Flow cytometric analysis of peripheral blood lymphocytes revealed a reduction of class-switched CD27+IgM−IgD− memory B cells below 0.4% in 77% of our patients (group I), while this B-cell subpopulation exceeded 0.5% in all healthy donors and in 23% of CVID patients (group II). These results correlate well with the capacity of peripheral blood lymphocytes to produce immunoglobulins in vitro upon stimulation with Staphylococcus aureus Cowan I (SAC) plus interleukin-2 because the production of immunoglobulin G in vitro is entirely dependent on the presence of switched memory B cells. The subdivision of group I into patients with an increased proportion of CD21− peripheral B cells (> 20%; group Ia) and patients with normal percentages of CD21− B cells (

The simultaneous appearance of B-CLL and MM in the same patient (pt) seems to be a rare phenomenon. In those cases - our experience included - 1. the myeloma exhibits an aggressive course, 2. with CLL-treatment, MM seems to emerge, 3. which, if well treated (e.g. high-dose chemotherapy [CTx] with autologous peripheral blood stem cell transplantation [auto-PBSCT]), may lead to a slower myeloma course, improved clinical condition and CLL disapearance. Here we report on the simultaneous (n=2) and successive (n=2) occurrence of CLL and MM in four pts, who were diagnosed at our center during the last year. We suggest that the occurrence of CLL and MM, when carefully monitored, may be more frequent than assumed so far. Case 1. This 64-year-old male was evaluated due to weight loss, leukocytosis, anaemia, foamy urine and increased creatinine. Kappa (k) Bence-Jones (BJ) proteinuria and elevated k-serum free light chains (SFLC) were found. A bone survey was normal, but BM biopsy showed infiltrates of plasma- (PC) and B-CLL-cells. Due to Rai 0 CLL and MM stage IIIB, the pt received 2 cycles of VAD followed by an auto-PBSCT. Thereby, he obtained a PR. Maintenance with thalidomide stabilized both diseases. Case 2. This 77-year-old male was simultaneously diagnosed with RAI 0 CLL and stage IIIB MM. Diagnostics revealed a lambda (l)-paraprotein, BJ-l-proteinuria and lytic bone lesions. The BM showed monoclonal l-LC-expressing PCs, and k-LC-CLL-cells. The pt received melphalan/prednisone (MP) and thereby obtaining SD. Case 3. This 62-year old pt was diagnosed with RAI II B-CLL. Two years later, he showed increased splenomegaly, anemia, elevated l-SFLC and dense CLL-BM-infiltrates. Chlorambucil/prednisone-CTx was initiated, but failed to improve his condition. Further evaluation due to renal impairment revealed multiple lytic lesions and monoclonal l-LC-expressing PCs, coexisting with remaining CLL infiltrates in his BM. Cyclophosphamide-CTx, 2 cycles of VAD and auto-PBSCT were performed and Bortezomib maintenance, improving the pt’s general condition, his PB counts and l-SFLC secretion. Despite these efforts, MM relapse occurred 5 months later with persisting absence of his preceding CLL and the pt eventually died due to myeloma progression. Case 4. This 73-year-old male was diagnosed with B-CLL, showing moderate splenomegaly, lymphocytosis, initially not requiring any therapy. Two years later, he showed a deteriorating clinical condition, multiple osteolytic lesions, anemia, hypercalcemia, monoclonal IgA paraprotein and k-BJ-proteinuria. A BM biopsy confirmed k-LC B-CLL- and k-PC-infiltrates. The diagnosis of stage IIIA IgA k-MM, and RAI II B-CLL led to MP-CTx that induced SD of his MM and CLL. In summary, our 4 pts had a median age of 69 years (range; 62–77) and showed a median time interval of their CLL and MM diagnosis of 11 months (0–31). All pts had stage III MM and renal impairment in 3/4, whereas CLL showed an indolent course. LCs of CLL and MM were different in all except one pt. Cytogenetic and genomic analyses are currently ongoing and will be reported at the meeting. We conclude that the elucidation of the coincidence of CLL and MM will allow to understand why and how often both occur and also, how they can be efficiently treated. The question of their clonal relationship should be answered via genomic analyses that will allow to gain further insight into the origin of both diseases.

Introduction: Transplantation of functional hematopoietic stem cells (HSC) using peripheral blood (PB), bone marrow (BM) or cord blood (CB) cells is widely used to treat malignant and nonmalignant disorders. Because long-term cryopreservation is performed for PB, BM and CB cells, and these are often used years after cell harvests, the implementation of a quality-assurance is a major requirement to ensure graft safety for clinical use. Methods: We assessed the efficiency of recovery of viable HSC from 37 patients (pts; n=20 NHL, n=6 Hodgkin, n=9 MM, n=2 AML) and 6 allogeneic-donors (AD) with stored PBSC samples. All pts had received an auto-PBSCT between 1992–2004. Stored PBSC samples used in this analysis had been cryopreserved for a median of 5.6 years (y; range: 1.3–12). We determined post-thawing recovery, cell viability, ex vivo expansion potential, CD34+ numbers, CFU growth in methylcellulose culture and LTC-ICs. Viable cells were determined by trypan blue and propidium iodide via FACS analysis, CFUs in 0.9% methylcellulose (supplemented with IMDM, 30% FCS and EPO, IL-3+GM-CSF) and LTC-IC as previously described. Pts and AD were analyzed as a total group and within 3 subgroups of: A) ‘long-term’ cryopreservation: n=21 PBSC harvests had a median cryopreservation of 9.5y (8–12), B) ‘short-term’ cryopreservation: n=16 harvests had a 2.9y (1.3–5.6) cryopreservation period, and C) n=6 pts showing delayed engraftment (EG) or early death after auto-PBSCT: the cryopreservation in these 6 pts was 2.7y (2.2–3.5). Cryopreservation results were correlated with clinical results and EG. Results: Hematopoietic EG in group A and B was prompt with WBC>1000/μl and platelets>20,000/μl on d10–11 post PBSC reinfusion. EG in group C was delayed albeit 4.3x106 CD34+ cells/kg bw (2.1–8.6) had been retransfused (WBC>1000/μl + platelets>20,000/μl: d+13 post PBSC infusion, non-platelet-EG >20,000/μl before death: n=5). Primary cause of death in group C was progressive disease in 3 and serious infections in 5 pts. Group A showed 74.3% viable cells post-thawing in PBSC grafts. Median number of CD34+ cells were 2.9%. Median numbers of CFU-C, BFU-E and GEMM were 36, 60 and 7, respectively. This was comparable with results in group B, showing 70% viable cells post-thawing, CD34+ cells of 4.2% and CFUs of 43, 75 and 6, respectively (p>0.05). Proliferative capacity was intact in both groups after 7 days of suspension culture, generating CFU-C, BFU-E and GEMM of 67, 29 and 1, respectively. In group C, viable cells were present in only 58% and median CFU-C, BFU-E and GEMM were 21, 5 and 0, respectively (p Conclusions: We show that human PBSC can be stored for more than a decade without apparent loss of HSC activity and can be efficiently retrieved. These results reinforce that expiration dates cannot be set for safely stored cryopreserved HSC. Assessment of CD34+ cell numbers, clonogenic potential via methylcellulose and LTC-IC assays are clinically relevant, since they may correlate with clinical outcome. Thus, these hematopoietic assays are valuable to assess the quality of cryopreservation and possibly also outcome of PBSCT.

High-dose chemotherapy with autologous peripheral blood stem cell transplantation (auto-PBSCT) offers a valid treatment option in cancer patients (pts) with advanced, relapsed or high-risk (HR) disease. Auto-PBSCT is in general a safe procedure, however, some pts may have a dismal outcome due to treatment related mortality (TMR), early relapse (ER) and/or failure of blood cell reconstitution (FBR). Risk factors predisposing for early complications after PBSCT have not yet been clearly assessed. From a total of 796 pts having been transplanted over a 10 year (y) period (6/93-5/03), we determined 50 pts (group A, m:f=29:21), who had primary FBR or died within 3 months after PBSCT. As a control group, 50 pts who had undergone PBSCT over the same period, were randomly selected (group B; m:f=25:25). Pre-PBSCT-parameters associated with early complications were examined by univariate analysis. Median age at transplant was similar in group A (52y) and group B (54y). Group A vs. B had underlying lymphoma in 31 vs. 17, solid tumors in 13 vs. 21 and leukemias in 6 vs. 12 pts, respectively. Notable differences were a lower performance status, more pretreatment chemotherapy (CX)-cycles, higher LDH levels, lower platelet counts, hypocellular bone marrow (BM) and lower colony forming units (CFU) in group A. Treatment before PBSCT in group A consisted of a median number of 7 CX (group B: 4 CX) cycles. Median number of retransfused CD34+-cells were similar (group A 4.1 vs. group B 3.7x10e6/kg bw). Total CFUs/1.5e5, determined by methylcellulose culture of PBSC grafts in group A were considerably decreased with 128 vs. 187 (group B), and BFU-E, CFU-GM, and CFU-GEMM were 51 vs. 79, 55 vs. 87 and 5 vs. 4, respectively. Hypocellular BM was observed in group A and B in 46% vs. 4% of pts (odds ratio 20.44 [p