Gene expression-based prediction of myeloma cell sensitivity to histone deacetylase inhibitors.

BACKGROUND: Multiple myeloma (MM) is still a fatal plasma cell cancer. Novel compounds are currently clinically tested as a single agent in relapsing patients, but in best cases with partial response of a fraction of patients, emphasising the need to design tools predicting drug efficacy. Histone deacetylase inhibitors (HDACi) are anticancer agents targeting epigenetic regulation of gene expression and are in clinical development in MM. METHODS: To create a score predicting HDACi efficacy, five MM cell lines were treated with trichostatin A (TSA) and gene expression profiles were determined. RESULTS: The expression of 95 genes was found to be upregulated by TSA, using paired supervised analysis with Significance Analysis of Microarrays software. Thirty-seven of these 95 genes had prognostic value for overall survival in a cohort of 206 newly diagnosed MM patients and their prognostic information was summed up in a histone acetylation score (HA Score); patients with the highest HA Score had the shorter overall survival. It is worth noting that MM cell lines or patients' primary MM cells with a high HA Score had a significant higher sensitivity to TSA, valproic acid, panobinostat or vorinostat. CONCLUSION: In conclusion, the HA Score allows identification of MM patients with poor survival, who could benefit from HDACi treatment.

Exclusive involvement of H-2Db or H-2Kd product in the interaction between T-killer lymphocytes and syngeneic H-2b or H-2d viral lymphomas.

It was demonstrated previously that the cytolysis of murine viral lymphoma cells by anti-murine sarcoma virus (MSV) syngeneic T-killer lymphocytes was restricted by some products of the H-2 complex. The respective role of the products of different regions of the H-2 complex were studied with six H-2(b) and three H-2(d) lymphomas induced by five different type C viruses. They were tested in a classical chromium release test against anti-MSV T-killer cells obtained from different inbred strains of mice, including several H-2 recombinants. Tumors o pound the H-2(b) haplotype were lysed only when effectors and target cells have in common the D(b) region. On the contrary an identity limited to the K end of the H-2 complex is necessary and sufficient in the H-2(d) haplotype. An in vitro restimulation of the spleen cells with concanavalin A strongly increased the activity of in vivo-primed T lymphocytes but did not provide any response for in vivo-primed but nonresponder cells. Preincubation of the tumor cells with anti-H-2 sera abolished the lysis by syngeneic anti-MSV effector lymphocytes. The same results were obtained by preincubating the H-2(b) targets with anti-H-2D(b), or the H-2(d) target with anti-H-2K(d). Preincubation with anti-H-2K(b) or anti- H-2D(d) were ineffective. These results show that the T-killer/target cells interaction in the MSV system involved some products of the H-2 complex which might be different with the various H-2 haplotypes and could possibly vary according to the antigenic specificity. A specific association of a viral product with a normal cellular structure, directed by the H-2 region during the viral budding could explain the observed results.

Clonal V alpha 12.1+ T cell expansions in the peripheral blood of rheumatoid arthritis patients.

Rheumatoid arthritis (RA) represents a heterogenous disease characterized by chronic polyarthritis. Most patients with adult RA inherit HLA-DR4 or -DR1 major histocompatibility complex (MHC) genes. While the molecular basis for this genetic predisposition is unknown, the major function of these MHC-encoded molecules is to present peptides to T lymphocytes. It is hypothesized that an endogenous or environmental antigen initiates a MHC-restricted immune response mediated by T lymphocytes, which is followed by a chronic inflammatory reaction involving many cell types. In chronic RA, previous or ongoing antigenic activation might result in detectable skewing of the peripheral alpha/beta T cell receptor (TCR) repertoire. Here we demonstrate a marked expansion of V alpha 12.1-bearing CD8+ T cells in the peripheral blood (mean, 22%; range, 10-43%) of > 15% of RA patients. A major proportion of these patients shared HLA-DQ2 in addition to the expected high frequency DR1 and DR4 alleles. Detailed molecular analysis in three of the RA patients with elevated V alpha 12.1+ T cells identified repeated TCR alpha chain sequences consistent with clonal V alpha 12.1+,CD8+ T cell expansion. In addition to shared TCR V alpha 12.1 germline gene usage among unrelated subjects, a conserved J alpha motif was also detected. Together, these results suggest an antigen-driven mechanism of T cell expansion in these patients and may offer a new approach in examining specific antigen that stimulate T cells in RA.

Gene expression profile of human endometrial receptivity: comparison between natural and stimulated cycles for the same patients.

BACKGROUND: The adjunction of exogenous hormones for controlled ovarian stimulation (COS) may alter endometrial receptiveness. In order to identify the genes misregulated under COS, we compared the endometrium gene expression profiles, from the same patients, in a natural cycle and in a subsequent COS cycle. METHODS: For the same normal-responder patients (n = 21), endometrial biopsies (n = 84) were collected during the pre-receptive (LH + 2) and receptive stages (LH + 7) of a natural cycle and, subsequently, on oocyte retrieval day (hCG + 2) and on transfer day (hCG + 5) of a stimulated cycle. Samples were analyzed using DNA microarrays. Gene expression profiles and biological pathways involved in endometrial receptivity were analyzed. RESULTS: Although endometrium transition profiles from pre-receptive to receptive phases are similar between patients, COS regimens alter endometrial receptivity in comparison with natural cycle. Under COS conditions, two endometrial profiles were identified and were associated either with a moderately altered receptivity profile for the majority of the patients or a strongly altered profile for a sub-category of patients. The receptive endometrium transcription profile under COS was defective for biological functions such as TGFbeta signaling, leukocyte transendothelial migration and the cell cycle. CONCLUSIONS: Gonadotrophin treatments in COS cycles led to disruptions of the transcriptional activation of genes involved in normal endometrial receptivity. We propose that when the receptiveness of the endometrium is seriously compromised by the COS protocol, fresh embryo replacement should be cancelled, the embryo frozen and thawed embryo replacement should be performed under natural cycles.

Identification of new biomarkers of human endometrial receptivity in the natural cycle.

BACKGROUND: Identification of new markers assessing endometrial receptivity may help in improving the clinical outcome of IVF. This study aimed at identifying genes expressed in human endometrium during the implantation window that could be used as such markers. METHODS: A series of normoresponder patients (n = 31) underwent endometrial biopsies (n = 62, 2 per patient) during the early secretory phase, 2 days after the LH surge (LH + 2) and the mid-secretory phase (LH + 7) of the same natural cycle that preceded a new ICSI attempt for male infertility factor. Samples were analyzed using DNA microarrays and gene expression profiles at the time of the implantation window were computed. Systems biology analysis allowed the identification of biological pathways that were over-represented in this signature. A new approach for class prediction applied to microarray experiments was then used to identify biomarkers putatively involved in endometrial receptiveness. RESULTS: Five genes expressed during the implantation window were all up-regulated in the LH + 7 samples compared with LH + 2 [laminin beta3 (P = 0.002), microfibril-associated protein 5 (P = 0.009), angiopoietin-like 1 (P = 0.005), endocrine gland-derived vascular endothelial growth factor (P = 0.049) and nuclear localized factor 2 (P = 0.007)]. Increased expression was validated by quantitative RT-PCR. CONCLUSIONS: Five genes have been identified for the first time as being up-regulated during the implantation window and are proposed as new biomarkers for exploration of endometrial receptiveness. As the endometrial biopsy procedure can be performed during a natural cycle, it would be worth testing this approach as a novel strategy in patients with poor implantation after IVF or ICSI.

A non-invasive test for assessing embryo potential by gene expression profiles of human cumulus cells: a proof of concept study.

Identification of new criteria for embryo quality is required to improve the clinical outcome of in vitro fertilization. The aim of this study was to determine the gene expression profile of cumulus cells (CC) surrounding the oocyte as biomarkers for embryo potential and to identify genes to be used as prognostic indicators of successful pregnancy. CC from single oocytes were analysed using DNA microarrays. Gene expression profiles of CC surrounding the oocyte associated with good embryonic quality and pregnancy outcome were computed. We observed that CC issued from oocytes that developed into embryos with a good morphology had differing gene expression profile according to the pregnancy outcome of the embryo. We demonstrated that the expression of BCL2L11, PCK1 and NFIB in CC is significantly correlated with embryo potential and successful pregnancy. These results were confirmed by quantitative RT-PCR. The gene expression profiling of human CC correlates with embryo potential and pregnancy outcome. BCL2L11, PCK1 and NFIB genes are proposed as biomarkers for predicting pregnancy. Our findings suggest a non-invasive approach, offering a new potential strategy for competent embryo selection. This approach should be validated in single-embryo transfer programmes.

Bone marrow mesenchymal stem cells are abnormal in multiple myeloma.

Recent literature suggested that cells of the microenvironment of tumors could be abnormal as well. To address this hypothesis in multiple myeloma (MM), we studied bone marrow mesenchymal stem cells (BMMSCs), the only long-lived cells of the bone marrow microenvironment, by gene expression profiling and phenotypic and functional studies in three groups of individuals: patients with MM, patients with monoclonal gamopathy of undefined significance (MGUS) and healthy age-matched subjects. Gene expression profile independently classified the BMMSCs of these individuals in a normal and in an MM group. MGUS BMMSCs were interspersed between these two groups. Among the 145 distinct genes differentially expressed in MM and normal BMMSCs, 46% may account for a tumor-microenvironment cross-talk. Known soluble factors implicated in MM pathophysiologic features (i.e. IL (interleukin)-6, DKK1) were revealed and new ones were found which are involved in angiogenesis, osteogenic differentiation or tumor growth. In particular, GDF15 was found to induce dose-dependent growth of MOLP-6, a stromal cell-dependent myeloma cell line. Functionally, MM BMMSCs induced an overgrowth of MOLP-6, and their capacity to differentiate into an osteoblastic lineage was impaired. Thus, MM BMMSCs are abnormal and could create a very efficient niche to support the survival and proliferation of the myeloma cells.

[Risk management of special infections]. / Risikomanagement besonderer Infektionssituationen.

The WHO considers that there is considerable danger of an influenza pandemic. One result of globalisation is that other highly contagious infectious diseases, such as SARS or Ebola, which are potentially endemic, can also be brought into Germany. Bioterorrism must also be considered. As well as all other medical disciplines, hospital surgical departments must be armed against this now. National pandemic planning will be taken as an example to illustrate the planning needed in hospitals to prepare for emergencies arising from infections in such specific situations.

Heparan sulphate proteoglycans are essential for the myeloma cell growth activity of EGF-family ligands in multiple myeloma.

The epidermal growth factor (EGF)/EGF-receptor (ErbB1-4) family is involved in the biology of multiple myeloma (MM). In particular, ErbB-specific inhibitors induce strong apoptosis of myeloma cells (MMC) in vitro. To delineate the contribution of the 10 EGF-family ligands to the pathogenesis of MM, we have assessed their expression and biological activity. Comparing Affymetrix DNA-microarray-expression-profiles of CD138-purified plasma-cells from 65 MM-patients and 7 normal individuals to those of plasmablasts and B-cells, we found 5/10 EGF-family genes to be expressed in MMC. Neuregulin-2 and neuregulin-3 were expressed by MMC only, while neuregulin-1, amphiregulin and transforming growth factor-alpha were expressed by both MMC and normal plasma-cells. Using real-time polymerase chain reaction, we found HB-EGF, amphiregulin, neuregulin-1 and epiregulin to be expressed by cells from the bone marrow-environment. Only the EGF-members able to bind heparan-sulphate proteoglycans (HSPGs) - neuregulin-1, amphiregulin, HB-EGF - promote the growth of MMC. Those ligands strongly bind MMC through HSPGs. The binding and the MMC growth activity was abrogated by heparitinase, heparin or deletion of the HS-binding domain. The number of HS-binding EGF ligand molecules bound to MMC was higher than 10(5) molecules/cell and paralleled that of syndecan-1. Syndecan-1, the main HSPG present on MM cells, likely concentrates high levels of HS-binding-EGF-ligands at the cell membrane and facilitates ErbB-activation. Altogether, our data further identify EGF-signalling as promising target for MM-therapy.

Peripheral neuropathy after autologous blood stem cell transplantation for multiple myeloma.

We report a case of peripheral neuropathy occurring after autologous blood stem cell transplantation (ABSCT) for multiple myeloma. The patient, free of neurological symptoms, was transplanted in partial remission, and achieved a complete remission after transplantation. A severe peripheral, symmetric, distal sensori-motor polyneuropathy appeared at day 25 and worsened progressively until commencement of corticosteroid therapy. A peripheral nerve biopsy showed endoneurial cellular infiltrates which were predominantly composed of T cells identified by immunocytochemistry. Ultrastructural examination showed acute axonal damage. Electrophysiologic studies performed before and during the treatment were consistent with a severe axonal degeneration and showed a marked improvement, concomitant with the favorable clinical outcome. This is the first report of peripheral neuropathy after ABSCT.

In vivo migration of tonsil lymphocytes in rheumatoid synovial tissue engrafted in SCID mice: involvement of LFA-1.

Integrin-adressin binding is a critical step in lymphocte attachment to target tissues. The mucosal recognition systems (alpha E beta 7, alpha 4 beta 7, MADcam-1) have been implicated in the autoimmune process in rheumatoid arthritis. We developed a model for in vivo study of radio-labelled lymphocyte circulation and their attachment to human rheumatoid synovium. We studied the homing of tonsil lymphocytes, considered as mucosal lymphocytes, and the involvement of alpha E beta 7 integrin and LFA1 in the homing of tonsil lymphocytes. We engrafted human rheumatoid synovium subcutaneously in 6 week old SCID CB17 mice. Three weeks later, we injected intraperitoneally 20 IO6 human peripheral blood or tonsil mononuclear cells, previously labelled with 3 mCFi HMPAO-99mTc. A mouse total body scintigram was obtained 20 h postinjection. The same protocol was performed after treatment of the MNC and mAb against LFA-1 (CD11a) or alpha E beta 7 (CD103). Tonsil MNC retention in the rheumatoid synovial graft 20 h post-injection was enhanced compared to blood MNC (12731 +/- 8297cpm/200 pixel) versus 5982 +/- 4713cpm/200 pixel, p < 0.05). A monoclonal antibody against LFA 1 decreased the activity in the graft (4152 +/- 1287 cpm/200 pixel), p < 0.05. No significant difference in tonsil MNC attachment to rheumatoid synovial tissue was observed with a mAb against alpha E beta 7 (8057 +/- 5009 cpm/200 pixel). Our results showed an increase in radiolabelled mucosal MNC migration in synovial tissue engrafted in SCID mice compared with blood MNC. Moreover, the date suggest that LFA-1 but not the alpha E beta 7 integrin is involved in tonsil MNC binding to synovial tissue in RA.

Rheumatoid pericarditis: new immunopathological aspects.

Rheumatoid pericarditis (RP) is a well known extraarticular manifestation of rheumatoid arthritis (RA). It is not frequently diagnosed despite its high reported prevalence in post-mortem studies. There have been no immunohistological studies of its presence in pericardial membranes. Here we report a complete immunohistological study of two RA patients with RP complications, using a panel of monoclonal antibodies (mAbs) for the recognition of B, T, and NK cells. Both cases showed strong and almost exclusive pericardial membrane infiltration of CD8+ T-cells which was correlated with a higher than expected similar increase in the subset of peripheral blood lymphocytes (PBL). These findings suggest an important role for CD8+ T-cells in chronic RA, especially in this extraarticular manifestation of the disease.

Serum levels of secretory IgA and in vitro production of IgA in rheumatoid arthritis.

Twenty-three per cent of rheumatoid arthritis (RA) patients show an increase of serum IgA concentrations. To determine the role of mucous-associated lymphoid tissue (MALT) in the elevation of serum IgA in RA, we studied the serum secretory-IgA (s-IgA) in 63 RA patients and in 30 healthy controls. We also analysed the secretion of circulating B cells producing IgA, which is known to reflect mucous tissue activity, in a subgroup of 15 patients with increased serum IgA concentrations, and in control patients. The mean s-IgA in the RA patients was 0.046 mg/ml +/- 0.064, versus 0.002 +/- 0.004 mg/ml in controls (not significant). Active disease defined by clinical criteria was associated with an increase in serum s-IgA (p < 0.001). Furthermore, in a subgroup of RA patients with high serum IgA levels, we found an increase in in vitro IgA production by circulating blood lymphocytes (17.39 +/- 15.2 micrograms/ml), versus RA patients with normal serum IgA levels or controls (p < 0.001). These results were not modified by LPS or PWM. Our results further support the hypothesis of primary MALT activation following environmental antigenic stimulation in RA patients.

Growth factor activity of IL-6 in the synovial fluid of patients with rheumatoid arthritis.

OBJECTIVE: We set out to determine whether the ability of synovial fluids (SF) in patients with rheumatoid arthritis (RA) to facilitate the proliferation of synovial tissue-derived fibroblastic cell lines was related to the presence of growth factors and/or cytokines. METHODS: The growth factor activity of 20 RA SF was measured by their ability to induce anchorage-independent growth of the rat NRK-49F (49F) fibroblastic strain. The presence of transforming growth factor-beta (TGF-beta) and platelet-derived growth factor (PDGF) was also assessed using neutralising anti-TGF-beta or anti-PDGF-AB mAbs. Cytokines were measured by functional assays or ELISA: RESULTS: We observed a correlation between growth factor activity and the IL-6 levels in SF. Both were correlated to the erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) levels in SF and serum. IL-6 (at concentrations above 10(4) U/ml), synergized with growth factors in the induction of the anchorage independent (AI) growth of 49F cells. Pretreatment of SF with a neutralising anti-IL-6 mAb substantially reduced the capacity of these SF to induce AI growth of 49F cells, confirming the growth factor activity of IL-6 in this test. In contrast, IL-6 alone or in association with PDGF, epidermal growth factor (EGF) or TGF-beta had no effect on the anchored growth of synovial tissue-derived fibroblasts, and treatment of SF with a neutralising anti-IL-6 mAb did not affect their ability to increase the growth rate of synovial tissue-derived fibroblasts. CONCLUSIONS: These results strongly suggest that IL-6 is responsible for the observed correlation between the growth factor activity of SF and inflammatory indexes such as ESR and CRP. However, neither IL-6 nor PDGF were responsible for the observed positive effect of SF on synovial fibroblastic cell lines.

Bone morphogenic protein 6: a member of a novel class of prognostic factors expressed by normal and malignant plasma cells inhibiting proliferation and angiogenesis.

Pathogenesis of multiple myeloma is associated with an aberrant expression of pro-proliferative, pro-angiogenic and bone-metabolism-modifying factors by malignant plasma cells. Given the frequently long time span from diagnosis of early-stage plasma cell dyscrasias to overt myeloma and the mostly low proliferation rate of malignant plasma cells, we hypothesize these to similarly express a novel class of inhibitory factors of potential prognostic relevance. Bone morphogenic proteins (BMPs) represent possible candidates as they inhibit proliferation, stimulate bone formation and have an effect on the survival of cancer patients. We assessed the expression of BMPs and their receptors by Affymetrix DNA microarrays (n=779) including CD138-purified primary myeloma cell samples (n=635) of previously untreated patients. BMP6 is the only BMP expressed by malignant and normal plasma cells. Its expression is significantly lower in proliferating myeloma cells, myeloma cell lines or plasmablasts. BMP6 significantly inhibits the proliferation of myeloma cell lines, survival of primary myeloma cells and in vitro angiogenesis. A high BMP6 expression in primary myeloma cell samples delineates significantly superior overall survival for patients undergoing high-dose chemotherapy independent of conventional prognostic factors (International Staging System (ISS) stage, beta(2) microglobulin).

[Immunosuppressive phenomena in experimental tumor systems (author's transl)]. / Phénomènes d'immunosuppression dans les systèmes tumoraux expérimentaux.

During the growth of spontaneous or chemically-induced tumors in animals, it has been reported that T lymphocytes are present in the spleen and/or the thymus which are able (i) to specifically block tumor rejection by syngeneic immunized animals in in vivo transfer experiments, and (ii) to specifically inhibit the in vitro cell-mediated immune reactions. The relationship between both types of suppressor cells is unclear. On the other hand, mitogen insensitive cells, acting in vitro in a non specific way on normal cell mitosis and cell-mediated cytotoxicity induction, and having macrophage characteristics, are described in viral and non viral tumor bearing hosts as well as in non tumoral animals after hematopoietic hypoplasia. The various suppressor cells types and their possible mechanisms of action are discussed along with soluble factors when present in experimental tumor systems, but the role of these cells in tumor pathogenesis is not yet clear.

In vitro primary allo-CTL generation by enucleated antigen-presenting cells.

It is generally thought that only viable cells can elicit a primary cytotoxic T-lymphocyte (CTL) response. We present evidence that this is not so, since enucleated tumor cells can generate a strong cytolytic response of unprimed allogeneic human T lymphocytes. Cytoplasts (enucleated cells) were obtained by incubation with cytochalasin B and subsequent isopycnic centrifugation. Their purity was assessed by electron microscopy and flow cytometry. Membrane fractions were prepared by nitrogen cavitation, and used in parallel with cytoplasts and intact cells as stimulators in primary allo-CTL generation; although all cell fractions expressed high amounts of class I and II histocompatibility antigens, as assessed by flow cytometry and ELISA technique, only the cytoplasts generated a strong cytotoxic response of naive peripheral T cells, like that induced by intact cells. The dogma that an intact and metabolically active stimulator cell is required for the primary generation of CTLs is questioned.