Bortezomib retreatment in relapsed multiple myeloma - results from a retrospective multicentre survey in Germany and Switzerland.

OBJECTIVES: This multicenter, retrospective survey evaluated the efficacy and safety of bortezomib retreatment in patients with relapsed multiple myeloma who had responded to initial bortezomib treatment. METHODS: Clinical records of 94 patients receiving bortezomib retreatment in Germany and Switzerland were reviewed. RESULTS: Sixty patients were included according to prespecified criteria. Patients had received a mean 3.7 ± 2.3 therapies prior to initial bortezomib. Overall response rate to bortezomib retreatment was 63.3%; 8 (13.3%), 3 (5.0%) and 27 (45.0%) patients achieved complete response (CR), near-CR and partial response, respectively. Response to retreatment was associated with response to initial treatment (75.0% of patients with CR to initial treatment responded to retreatment) and treatment-free interval (TFI) after initial treatment (76.9 vs. 38.1% overall response rate for patients with TFI >6 vs. ≤ 6 months). After retreatment, median time to progression was 9.3 months. Median TFI was 5.7 months; 31.7, 25.0 and 15.0% of patients experienced a TFI longer than 6, 9 and 12 months, respectively. Reported adverse drug reactions were consistent with the known safety profile of bortezomib and most resolved completely. CONCLUSIONS: These results demonstrate that relapsed multiple myeloma patients who respond to initial bortezomib treatment have a sustained susceptibility to bortezomib and do not experience uncommon toxicity to retreatment.

DNA typing for natural killer cell inhibiting HLA-Cw groups NK1 and NK2 by PCR-SSP.

Over the last few years, natural killer (NK) cells have been shown to express MHC molecule recognizing receptors which are thought to function primarily as negative signaling receptors. HLA-Cw seems to play a key role as the corresponding ligand. Two distinct HLA-Cw groups which differ in amino acid residues 77 and 80 inhibit separate subsets of NK cells. In order to classify target cells with respect to their expression of HLA-Cw groups we established a group specific PCR-SSP which directly amplifies the relevant epitope coding sequences. The PCR protocol was validated by retyping cell lines obtained from the International Histocompatibility Workshop and by comparing those results with those acquired from allele-specific genotyping and serotyping on 80 donor-recipient pairs from our kidney transplantation unit. In the context of inhibitory HLA-Cw receptors, our protocol which definitively discriminates the two alternative epitopes is the more direct and thus more reliable approach, and is less labor intensive compared to an allele specific PCR or serotyping. In addition serotyping does not detect at all certain alleles. Basic NK cell research and clinical transplantation immunology may benefit from this newly established PCR SSP technique.

Kinetics and organ distribution of allogeneic natural killer lymphocytes transfused into patients suffering from renal cell carcinoma.

The transfusion of natural killer (NK) lymphocytes into patients suffering from malignant diseases is an approach of current interest in the field of immunotherapy. Little is known about the organ distribution, survival, and clearance of donor immune effector cells in cellular therapy, and no reports exist on these important parameters considering NK cells in particular or any other type of allogeneic lymphocytes in humans. In the context of a clinical Phase I/II study we examined the distribution of transfused allogeneic NK cells in patients suffering from renal cell carcinoma. The NK cells were ex vivo cultivated and activated before transfusion. To assess the circulation of the transfused cells in the peripheral blood, we used a nested PCR technique to detect HLA DRB1 alleles of the NK cell donors. Post-transfusion, all patients showed evidence of circulating donor cells for up to 3 days. After 7 days, all donor cells were cleared from the blood to undetectable levels. To assess organ distribution, (111)In-labeled NK cells were injected and monitored by whole-body scintiscans. A distribution to the whole body, with preference for liver, spleen, and bone marrow, was observed after a short initial uptake in the lungs. No activity was observed in lymphatic nodes. A total of 2/4 evaluable metastases showed a clear accumulation of transfused NK cells. The half-life corrected activity in all body compartments remained almost constant over the 6-day observation period in concordance with the absence of any excretion of radioactivity. This may indicate an extended survival of the transfused cells, despite their foreign nature, in the host organism.

Association between inflammation and nigral neuronal damage following striatal excitotoxic lesion.

We examined the expression of TNF-alpha within the substantia nigra pars reticulata (SNR) following intrastriatal injection of quinolinic acid (QA) and studied the effect of rolipram, a TNF-alpha-inhibitor, on the secondary neuronal damage. QA (240 nmol in 1 microl) was injected stereotactically into the striatum of male Wistar rats. After survival of 1, 3 or 10 days, the animals were sacrificed and immunohistochemical staining with an antibody against TNF-alpha was performed. From day 1 to day 10 after striatal QA injection TNF-alpha positive cells were observed within ipsilateral substantia nigra which were neither present on the contralateral side nor in sham-operated controls. Double labeling with antibodies against TNF-alpha and NeuN, keratan sulfate proteoglycan or GFAP displayed a good overlap between TNF-alpha and NeuN, which suggests that TNF-alpha positive cells are neurons. For the pharmacological approach, three groups of QA rats were treated intraperitoneally with either solvent (n=5), the NMDA receptor antagonist MK 801 (4 mg/kg, n=6) or the TNF-alpha inhibitor rolipram (0.3 mg/kg, n=6), which was started 24 h after QA-injection and continued with daily applications for 14 days. The amount of striatal damage did not differ between the three groups. The number of intact neurons within the ipsilateral substantia nigra of the solvent treated group was reduced by approximately 30% compared to the contralateral side. Both MK 801 and rolipram ameliorated this secondary damage and reduced the number of TNF-alpha positive cells. The observed association between expression of TNF-alpha and secondary neuronal damage within the substantia nigra induced by intrastriatal QA application might hint towards an involvement of this cytokine in transneuronal degeneration.

[Eye burns caused by wolf's milk]. / Wolfsmilchverätzung.

If the sap of Euphorbia inadvertently gets in the eye, it can cause conjunctivitis, keratitis or iritis. Therapy consists of rinsing, antibiotics, steroids and mydriatics when the anterior chamber is inflamed. It may also be necessary to protect the damaged eye from light, because photoallergic reactions are possible.

The repertoire of HLA-Cw-specific NK cell receptors CD158 a/b (EB6 and GL183) in individuals with different HLA phenotypes.

Over the last few years, natural killer (NK) cells have been shown to express major histocompatibility complex (MHC) molecules recognizing receptors that are thought to function primarily as negative signalling receptors. Much attention has been focused on the NK cell receptors CD158a (EB6) and CD158b (GI 183), which recognize two alternative epitopes on the HLA-Cw locus. In order to investigate whether HLA type affects the CD158a/b repertoire, expressed as percentage positive cells for a particular receptor and mean expression on this population of NK cells, peripheral blood lymphocytes of 47 HLA-typed donors were examined. Peripheral blood samples were examined by flow cytometric analysis to investigate the expression of CD158a and CD158b receptors on the surface of NK cells. In parallel, we determined each individual's HLA phenotype. There was a great heterogeneity in CD158 expression; nevertheless all individuals had NK cells belonging either to the CD158 a+b-, a-b+ or a-b- populations. No positive or inverse correlations could be shown between either receptor expression intensity or proportion of positive cells, and presence of the appropriate ligand. Thus no association between an individual's NK receptor repertoire and HLA serotype could be demonstrated. It is concluded that CD158 is expressed on NK cells in a highly redundant fashion. Our data do not support either a positive selection mechanism or the receptor calibration model.

Intraocular pressure and anterior chamber depth before and after extracapsular cataract extraction with posterior chamber lens implantation.

Long-term intraocular pressure (IOP) was evaluated in 41 glaucoma patients after extracapsular cataract extraction (ECCE) with posterior chamber lens (PC-IOL) implantation. All patients were initially monitored for a mean of 19 days. Eight failed to return for reexamination, but follow up of the other 33 continued for a mean of 12 months. IOP dropped significantly and the need for medication was reduced in all patients (particularly in those with open-angle glaucoma and prior iridotomy and iris suturing). The reduction in pressure remained significant in patients with simple or exfoliation glaucoma even after long-term observation. Pressure also significantly dropped in patients who had undergone previous ophthalmic surgery. The pressure drop was possibly due to a surgical deepening of the chamber angle. (Using the laser tomographic scanner, we found the same phenomenon in 50 patients without glaucoma: following ECCE/PC-IOL, the anterior chamber angle widened 9.3 +/- 4.3 degrees.)

Hemolytic anemia after kidney transplantation: case report and differential diagnosis.

A 58-year-old woman presented with hemolysis and thrombocytopenia 2 weeks after receiving a kidney graft. Hemolytic uremic syndrome was initially suspected, because in addition to hematological changes the graft function was missing. Unexpectedly, the results of the direct antiglobulin test became positive (4+), which is not normally observed in the hemolytic uremic syndrome. Differentiation of the eluted antibodies revealed anti-rhesus D specificity, which had to be interpreted either as an autoantibody of patient's origin or, hypothetically, as a "graft versus host" antibody of donor origin. Gm- and Km allotyping of these antibodies demonstrated a pattern which differed from the patient's but was identical to that of the kidney donor. Therefore hemolysis could be explained unambiguously by "graft versus host" antibodies. Whether the thrombocytopenia was also due to an immune process was not clear, although some evidence favors this hypothesis. Immunosuppressive treatment remained unchanged and several red blood cell transfusions were necessary before reactivity of the direct antiglobulin test diminished and became negative 7 weeks after kidney transplantation. The occurrence of hemolysis in the early posttransplantation period should thus draw attention to the possibility of "graft versus host" antibodies directed against red cells. Concomitant thrombocytopenia may occur. Donor screening for irregular erythrocyte antibodies should be performed whenever solid organ transplantation is intended.

The effect of HLA-C matching on acute renal transplant rejection.

BACKGROUND: The acute immunological rejection and long time survival of kidney allografts are correlated with the human leukocyte antigen (HLA) match status between donor and recipient. HLA-A, -B and -DR have all turned out to be relevant HLA loci in several studies. The role of HLA-C has not been studied before now. METHODS: In 104 consecutive patient/donor pairs from our transplantation unit, we retrospectively analysed whether acute graft rejection is influenced by HLA-C match status between donor and recipient. For typing HLA-C alleles, we used an allele-specific PCR protocol in combination with serology. RESULTS: By analysing groups of donor/recipient pairs with a homogeneous distribution of HLA-B mismatches in order to exclude an effect of the linkage disequilibrium between HLA-B/C, HLA-C mismatch turned out to be significantly correlated with acute transplant rejection in pairs with one additional mismatch on the B locus (P=0.004). Additional parameters that may hypothetically influence acute rejection episodes (HLA-A or DR mismatch, time of cold and warm ischaemia, previous transplantations, pre-existing HLA antibodies) were also analysed but cannot explain this finding. CONCLUSION: HLA-C matching of all kidney donors and recipients seems to be an option to reduce the probability of acute rejection episodes. Further studies of greater patient cohorts analysing organ rejection and organ survival are warranted.

Clinically relevant hypokalaemia, hypocalcaemia, and loss of hemoglobin and platelets during stem cell apheresis.

Since the introduction of hematopoietic growth factors, the collection of mobilized stem cells via leukapheresis has widely replaced the harvest of bone marrow in both autologous and allogeneic transplantation settings. We investigated the frequency and the extent of anticoagulant-induced electrolyte changes and the cell-separation-related loss of hemoglobin and platelets. In our study a total of 200 leukaphereses were performed on 60 patients with hematological malignancies. The electrolytes (calcium and potassium) were determined photometrically pre- and post-apheresis. Blood counts were analyzed to calculate the relative decline in hemoglobin and platelet counts. Stem cells were collected by processing a mean total blood volume of 11.6+/-3.9 L with a citrate consumption of 1,345+/-126 mL. More than 50% of all patients needed replacement therapy of either potassium or calcium. In non-substituted patients the initial serum potassium concentration dropped by 11.3+/-7.0% to 3.25+/-0.33 mmol/L post apheresis. In 21% of non-substituted patients, clinical relevant hypokalaemia was observed with levels < 3 mmol/L. The mean citrate-induced reduction of the total calcium was 5.5+/-6.0%. In addition the relative loss of hemoglobin and platelet counts amounted to 10.7+/-5.2% and 24.2+/-12.5%, respectively. In addition to the well-documented citrate-induced hypocalcaemia, we observed a considerable reduction in serum potassium during stem cell apheresis. This can result in a clinically relevant, substitution requiring hypokalaemia. The modest decline in hemoglobin and platelet counts suggested that levels of >9 g/dl (Hb) and platelets >30 x 10(9)/L are sufficient for a safe standard leukapheresis.

Feasibility of the adoptive transfusion of allogenic human leukocyte antigen-matched natural killer cells in patients with renal cell carcinoma.

Patients with metastasized renal cell carcinoma have a poor prognosis with conventional therapies. The feasibility and safety of donating purified natural killer (NK) cells without additional cytokines were evaluated. In contrast to all previous studies, the NK cells were derived from allogenic donors. The NK cell donors were HLA-C matched to enable NK cell inhibition via killer cell inhibitory receptors and HLA-C. This should obviate a graft-versus-host reaction against nonmalignant HLA-expressing tissues in the allogenic constellation. The average number of cells applied per transfusion was 1.02 +/- 0.265 x 10(9). The purity of the NK cells was 85% to 95%, and most of the contaminating cells were monocytes. Twenty-six transfusions given to 11 patients did not cause any minor or major adverse effects, with the exception of one episode of transient fever. One patient had an objective regression of his lung metastases that had been progressing continuously before. No cytotoxic HLA antibodies could be detected 3 weeks after the transfusions. The observed tolerance to this therapeutic regimen suggests the need for further studies with increased doses of cytokine-activated NK cells.

Lysyl hydroxylation in collagens from hyperplastic callus and embryonic bones.

Tissue from two patients with osteogenesis imperfecta suffering from a hyperplastic callus was studied. Although collagen type I from the compact bone and the skin and fibroblast cultures of these patients showed normal lysyl hydroxylation, collagen types I, II, III and V from the callus tissue were markedly overhydroxylated. Furthermore, the overhydroxylation of lysine residues covered almost equally the entire alpha 1 (I) collagen chain, as demonstrated by the analysis of individual CNBr-derived peptides. In addition, collagen type I was isolated from femoral compact bone of 33 individuals who died between the 16th week of gestational age and 22 years. Lysyl hydroxylation rapidly decreased in both collagen alpha 1 (I) and alpha 2 (I) chains during fetal development, and only little in the postnatal period. The transient increase in lysyl hydroxylation and the involvement of various collagen types in callus tissue argue for a regulatory mechanism that may operate in bone repair and during fetal development.

Evaluation of a flow cytometric method for simultaneous leukocyte phenotyping and quantification by fluorescent microspheres.

We describe a flow cytometric method using a newly designed product, fluorochrome-containing microspheres (Flow Count fluorospheres), which facilitates the precise quantification of cells in whole blood samples or heterogeneous cell suspensions on a single-cell level. These microparticles are easily distinguishable from other events of interest and can be detected by their light-scattering and fluorescence properties. In contrast to the traditional manual or automated cell-counting techniques, this method offers the opportunity to quantify cells simultaneously with flow cytometric immunophenotyping without additional cell loss or other cell preparation steps. We evaluated the accuracy and reproducibility of this flow cytometric method on the determination of CD45+ leukocyte counts and compared the results with those obtained by conventional techniques. Particular interest was focused on the behavior of cells and fluorospheres regarding their sedimentation rate over the period of analysis. The data from 48 blood samples with low, normal, and high leukocyte counts confirmed the reliability and comparability of the flow cytometric method, permitting the determination of white blood cell concentration at least to a limit of 100 cells/microL. A broad field of applications will benefit from this flow cytometric supplement because it is easy to perform and highly accurate. The results appear to be transferable to clinical decision-making monitoring of CD4+ lymphocytes in patients infected with human immunodeficiency virus or of CD34+ hematopoietic cells, optimizing the harvest for peripheral blood stem cell transplantation.

Collection efficiencies of CD34+ progenitor cells and mononuclear cells in leukapheresis products quantified by flow cytometry and calculated on the basis of a new formula.

BACKGROUND AND OBJECTIVES: Optimal mobilization and harvest of hematopoietic progenitors are essential for peripheral blood stem cell transplantation after myeloablative high-dose chemotherapy. Conflicting data have been published concerning the most useful, cost-effective collection strategy which is also convenient for patients. MATERIALS AND METHODS: A total of 66 leukaphereses in 20 patients were retrospectively evaluated. We assessed the predictive value of the number of white blood cells, mononuclear cells (MNCs) and CD34+ cells in peripheral blood for the yield of CD34+ cells in leukapheresis products. The concentrations of MNCs and CD34+ cells were quantified simultaneously by a flow cytometric procedure using fluorescent microparticles. Their collection efficiencies were calculated based on a newly developed formula. RESULTS: The collected hematopoietic progenitor concentration could be predicted only by the number of peripheral blood CD34+ cells prior to apheresis (r = 0.902; p<0.01). Furthermore, the mobilization of at least 30 CD34+ cells/microl peripheral blood was a good predictor that a single leukapheresis would yield a minimum of 2.0x10(6) CD34+ cells/kg body weight. The collection efficiencies calculated by the new formula were 55.2+/-10.7% and 57.7+/-11.2% for MNCs and CD34+ cells, respectively. CONCLUSION: The precise quantification of MNCs and CD34+ cells by a direct flow cytometric assay, as well as the new formula to determine the collection efficiencies, has an impact on optimizing high-quality stem cell products.