Dendritic cell biology and the application of dendritic cells to immunotherapy of multiple myeloma

Dendritic cells (DCs) are extremely efficient antigen-presenting cells that are potent stimulators of both B and T cell immune responses. Although DCs are normally present in extremely small numbers in the circulation, recent advances in DC biology have made it possible to generate DCs in culture. DCs can be generatedin vitro from various cellular sources including bone marrow, cord blood and peripheral blood. Although culture conditions are extremely diverse, the majority of protocols grow DCs in GM-CSF and either TNF-alpha and/or IL-4. The addition of other growth factors such as SCF and Flt-3 ligand can dramatically enhance DC recovery. It is important to appreciate that DC subsets have been identified. Thus, DC at different stages of maturation, based on phenotype and capacity to capture antigen, can be obtained depending on culture conditions. For clinical applications, DCs can be generated in serum-free media and cryopreserved for future clinical applications. The ability to obtain DCs in numbers suitable for manipulating immune responses has pushed DC-based immunotherapies into the spotlight for treatment of various malignancies, including multiple myeloma, a B cell malignancy that is presently incurable. Although high-dose chemotherapy and transplantation have improved complete remission rates and overall survival in myeloma, immunotherapeutic strategies are needed for the additional cytoreduction needed to achieve a cure. Because DCs specialize in antigen capture and are extremely potent at stimulating T cell responses, they are ideally suited for generating anti-myeloma T cell responsesin vivo. Several studies have demonstrated that myeloma protein, also called idiotype (Id), is sufficiently immunogenic and can be used to generatein vivo T cell responses in myeloma patients. Clinical trials using Id-pulsed DCs as a vaccine to treat minimal residual disease or relapsed myeloma are currently underway. Feasibility studies indicate that antigen-pulsed autologous DCs can be used to elicitin vivo Id-specific T cell responses. Additional studies are needed to optimize current DC vaccination protocols and determine clinical benefits associated with this approach. It is hoped that, following conventional therapies, a combination of adoptive immunotherapeutic modalities such as DCs together with myeloma-specific T cells may lead to improved clinical responses in multiple myeloma, and ultimately lead to complete remission and cure.     

Hemoglobin switching in sheep: a comparison of the erythropoietin- induced switch to HbC and the fetal to adult hemoglobin switch

Stimulation of sheep erythropoietic progenitor cells by erythropoietin (epo) has been studied with regard to its effect on the pattern of hemoglobin production. An analysis of hemoglobin (Hb) synthesis in BFU- E- and CFU-E-derived colonies from fetuses either homozygous for HbA (AA) (homozygous also for the beta c gene responsible for HbC production) or HbB (BB) (lacking the beta c gene) indicated the following. Colonies derived from precursor cells from 51- and 89-day fetuses exhibited small but detectable increments of HbB synthesis with prolonged incubation in vitro. This response was not dependent on the epo concentration. Erythropoietic precursor cells from a 124-day BB fetus were already committed to HbB synthesis, since HbF production was replaced by HbB on successive days in vitro as erythroid colonies matured; this switch was not affected by varying the epo concentration. In contrast, progenitor cells from a 124-day AA fetus responded to higher doses of epo by forming colonies in which more HbC was made at the expense of both HbF and HbA. Erythropoietic stress did not result in induction of HbF in vivo or in erythroid colonies derived from CFU-E in young adult BB sheep, whereas our prior studies had shown induction of HbC synthesis under analogous conditions in colonies derived from young adult AA sheep. We conclude that the epo-induced HbF (or HbA) to HbC switch and the fetal to adult hemoglobin switch are regulated by different mechanisms.     

Effect of tricuspid annuloplasty in mitral/aortic valve replacement on the clinical aspects and global function of the right heart chamber

The clinical improvement after mitral or aortic valve surgery is primarily due to the correction of the aortic/mitral valve function and the subsequent decrease of pulmonary artery pressure. The hemodynamic effect of an additional tricuspid annuloplasty, however, is still unclear. To assess the influence of a tricuspid annuloplasty using DeVega- or Carpentier-technique on the clinical outcome, hemodynamics, and right ventricular function in patients with moderate to severe tricuspid insufficiency, 38 patients were studied pre- and 11 +/- 4 months postoperatively. The clinical degree of left heart failure was graded according to the criteria of the NYHA. The extent of right heart failure (RHF) was determined using a clinical score from 0 (no signs) to 3 (severe RHF with pleural effusion/ascites). Mean pulmonary artery pressure (PAPm), end-diastolic volume index (RVEDVI), and ejection fraction (RVEF) of the right ventricle using biplane cineventriculography, as well as the angiographic and dopplerechocardiographic degree of tricuspid insufficiency were determined. The patients were assigned to three groups: gr.I (n = 12): preoperatively no tricuspid insufficiency (TI), gr. II (n = 12): with preop. TI and without tricuspid annuloplasty (TA), gr. III (n = 14): with preop. TI and TA. The patients of all three groups improved postoperatively from NYHA functional class III to class II (p less than 0.001). The clinical score of RHF decreased from 0.8 +/- 0.5 to 0.3 +/- 0.5 in gr. I, from 1.4 +/- 1.1 to 0.6 +/- 0.7 in gr. II, and from 1.7 +/- 1.0 to 0.8 +/- 0.8 in gr. III (p less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Recombinant adeno-associated virus-mediated gene transfer into hematopoietic progenitor cells [published erratum appears in Blood 1995 Feb 1;85(3):862]

Recombinant adeno-associated viruses (rAAV) containing only the inverted terminal repeats (ITR) from the wild-type virus are capable of stable integration into the host cell genome, and expression of inserted genes in cultured cells. We have now defined the ability of rAAV to introduce genes into primary hematopoietic progenitors. A vector was constructed containing the coding sequences for beta- galactosidase (beta-gal), including a nuclear localization signal, under the control of a strong viral promotor. Infectious vector particles were prepared by cotransfection of the vector plasmid with a second plasmid that contained the coding sequences for AAV proteins into adenovirus-infected human embryonic kidney cells. These vector preparations transferred and expressed the beta-gal gene in human K562 erythroleukemia and Detroit 6 cells. Positive immunoselection yielded a population of enriched CD34+ cells that were transduced with the rAAV beta-gal vector. Nuclear localized enzyme expression was documented in 60% to 70% of infected cells. Progenitor-derived colonies that developed after 2 weeks in clonogenic cultures were shown to have viral- associated DNA at an estimated copy number of 1 to 2 per cell using a semiquantitative polymerase chain reaction (PCR) method. Integration of AAV into hematopoietic progenitors was documented using wild-type virus, as its genome may integrate at a preferred site on chromosome 19. Our data suggest that rAAV will transfer and express genes in primitive hematopoietic progenitors with high frequency, and support the development of this vector system for therapeutic gene transfer.     

Regulation of expression of a human intercellular adhesion molecule (ICAM-1) during lymphohematopoietic differentiation

The intercellular adhesion molecule (ICAM-1) is a cell-surface molecule which binds to leukocyte function antigen-1 (LFA-1) and regulates both leukocyte adhesion to endothelial cells and immune functions requiring cell-cell contact. Membrane expression of ICAM-1 is highly regulated on all hematopoietic lineages. Cell membrane antigen is significantly expressed on a small subset of bone marrow (BM) progenitors but is weak or absent on all cell lineages once they enter the circulation. However, strong expression on tissue macrophages and germinal center B cells suggested that activated cells may show upregulated expression. When B cells, T cells, macrophages, or granulocytes were activated in vitro by suitable mitogens, ICAM-1 expression was induced in all cases. Parallel studies of hematopoietic tumors demonstrated a heterogeneity of expression which correlated with expression on their normal cellular counterparts. In particular, a striking correlation between expression on B-cell tumors and corresponding stages of B-cell differentiation was noted. The widely varying expression of ICAM-1 contrasts with LFA-1 which, while variable, is nevertheless significantly positive at all stages of differentiation. This suggests that the major regulation of homotypic adhesion mediated by the LFA-1/ICAM-1 linkage occurs through control of ICAM-1 expression. In keeping with this notion, ICAM-1 expression was also correlated with the "adhesiveness" of B-lymphoid tumors. Large solitary lymphoma masses showed intense expression of ICAM-1. Conversely, chronic lymphocytic leukemia (CLL) cells and lymphoma cells from tumors exhibiting diffuse, widespread lymph node disease showed weak expression. These observations are discussed in relation to the role of ICAM-1 in regulation of lymphoid recirculation and the biology of lymphoid tumors.     

Long-term in vivo expression of a murine adenosine deaminase gene in rhesus monkey hematopoietic cells of multiple lineages after retroviral mediated gene transfer into CD34+ bone marrow cells

Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.     

Hairy cell leukemia: a tumor of pre-plasma cells

Monoclonal antibodies defining B-, T-, and myeloid-restricted cell surface antigens were used to characterize the lineage and state of differentiation of tumor cells isolated from 22 patients with hairy cell leukemia (HCL). These tumors were shown to be of B lineage because they strongly expressed the B cell-restricted antigens B1 and B4 and lacked T cell- and monocyte-restricted antigens. Moreover, the strong expression of the plasma cell-associated PCA-1 antigen on the majority of hairy cells suggested that these tumors correspond to later stages of B cell ontogeny. Dual fluorescence experiments further confirmed that HCL splenocytes that coexpressed B1 and PCA-1 demonstrated both the morphology and tartrate-resistant acid phosphatase positivity of hairy cells. The observation that some hairy cells either spontaneously produce immunoglobulin (Ig) or could be induced to proliferate and secrete Ig provides complementary support for the view that HCL is a pre-plasma cell tumor. However, staining of hairy cells with anti-IL2R1 monoclonal antibody, which is directed to the T cell growth factor receptor and/or with the anti-Mo1 reagent, directed to C3bi complement receptor, distinguish these cells from currently identified B cells.     

Limitations of flow cytometry in the analysis of CDIalHLADR+ human oral mucosal Langerhans cells

Suspensions of human oral epithetial cells were stained with antibodies to CD la and HLADR conjugated with fluorochromes and analysed by flow cytometry with the aim of purifying double-labetled Langerhans cells, a population comprising approximatety 2% of the cell total. Whole suspensions had high levets of autofluorescence and a wide range of forward and right angle scatter properties. The mean percentage of CDIalHLADR+ cells was 2.I%, though the double-labetled cells did not form a discrete group and the percentages of positive cells using control antibodies were similar. Density gradient centrifugation prior to flow cytometry did not facilitate Langerhanr cell identification within the suspension. The results indicate flow cytometric analysis of minority cell populations (such as Langerhans cells) within oral epithetium is limited by the autofluorescence of physically heterogeneous keratinocytes, and emphasise the importance of controls in studies of oral epithetium which use this method.     

LOC283731 promoter hypermethylation prognosticates survival after radiochemotherapy in IDH1 wild‐type glioblastoma patients

MGMT promoter methylation status is currently the only established molecular prognosticator in IDH wild‐type glioblastoma multiforme (GBM). Therefore, we aimed to discover novel therapy‐associated epigenetic biomarkers. After enrichment for hypermethylated fractions using methyl‐CpG‐immunoprecipitation (MCIp), we performed global DNA methylation profiling for 14 long‐term (LTS; >36 months) and 15 short‐term (STS; 6–10 months) surviving GBM patients. Even after exclusion of the G‐CIMP phenotype, we observed marked differences between the LTS and STS methylome. A total of 1,247 probes in 706 genes were hypermethylated in LTS and 463 probes in 305 genes were found to be hypermethylated in STS patients (p values < 0.05, log2 fold change ± 0.5). We identified 13 differentially methylated regions (DMRs) with a minimum of four differentially methylated probes per gene. Indeed, we were able to validate a subset of these DMRs through a second, independent method (MassARRAY) in our LTS/STS training set (ADCY1, GPC3, LOC283731/ISLR2). These DMRs were further assessed for their prognostic capability in an independent validation cohort (n = 62) of non‐G‐CIMP GBMs from the TCGA. Hypermethylation of multiple CpGs mapping to the promoter region of LOC283731 correlated with improved patient outcome (p = 0.03). The prognostic performance of LOC283731 promoter hypermethylation was confirmed in a third independent study cohort (n = 89), and was independent of gender, performance (KPS) and MGMT status (p = 0.0485, HR = 0.63). Intriguingly, the prediction was most pronounced in younger GBM patients (<60 years). In conclusion, we provide compelling evidence that promoter methylation status of this novel gene is a prognostic biomarker in IDH1 wild‐type/non‐G‐CIMP GBMs.     

Efficacy of balloon valvuloplasty in patients with critical aortic stenosis and cardiogenic shock--the role of shock duration

BACKGROUND: Because of limited long-term success, aortic balloon valvuloplasty is considered to be a palliative procedure, including patients at excessive risk for standard therapy-aortic valve replacement-that is, those in cardiogenic shock. HYPOTHESIS: The study was undertaken to evaluate the outcome of balloon valvuloplasty for critical aortic stenosis complicated by cardiogenic shock. METHODS: Over a 10-year-period, we followed 14 patients (age 74+/-11 years, range 50-91) presenting in cardiogenic shock and critical aortic stenosis, who underwent valvuloplasty, together with 19 patients with critical aortic stenosis requiring urgent major noncardiac surgery. RESULTS: In patients in shock, calculated aortic valve area could be increased successfully by at least 0.3 cm2, from 0.38+/-0.09 to 0.81+/-0.12 cm2, with an insignificant increase in cardiac index from 1.89+/-0.33 to 2.01+/-0.41 l/min * m2. In-hospital mortality was 71% (10 patients). Two patients underwent valve replacement within 16 days and survived after 1 year, as did two patients refusing surgery. By multivariate logistic regression analysis, only an interval between onset of shock symptoms and valvuloplasty of > 48 h was significantly associated with fatal outcome (p < 0.01). In those patients requiring noncardiac surgery, this was possible after valvuloplasty in 95% who survived 1 year after hospital discharge. One patient in this group died of pulmonary embolism the day after the procedure. CONCLUSION: These data support the concept of causal treatment in patients with cardiogenic shock, as well as in the setting of cardiogenic shock and critical aortic stenosis, at the earliest possible convenience.