Gamma-interferon reduces expression of the protooncogene c-erbB-2 in human ovarian carcinoma cells.

The overexpression of the protooncogene c-erbB-2 (HER-2/neu) in ovarian and mammary carcinoma is an important indicator for a bad prognosis. In this study we demonstrate that, in three of four ovarian carcinoma cell lines, there is a gamma-interferon-mediated reduction in c-erbB-2 specific protein, and this effect was found to correlate with the antiproliferative action. It is interesting to note that there is no relation between the absolute amount of c-erbB-2 protein expressed and the sensitivity of the ovarian carcinoma cells for an antiproliferative activity of gamma-interferon. Other chemotherapeutic agents did not affect c-erbB-2 expression, although they inhibited the proliferation. The oncogene expression was lowered only in the ovarian carcinoma cell lines and not in three gamma-interferon-sensitive human breast cancer cell lines. Expression of the oncogene c-erbB-2 is the leading prognostic factor in ovarian cancer. Its modulation might represent a mechanism by which gamma-interferon inhibits cell proliferation.

Generation of mature dendritic cells from human blood. An improved method with special regard to clinical applicability.

Two methods to generate human dendritic cells from hematopoietic precursor cells in peripheral blood have recently been published. One approach utilizes the rare CD34+ precursors and GM-CSF plus TNF-alpha. The other method makes use of the more abundant CD34- precursor population and GM-CSF plus IL-4. Here we report a method that is based on the latter approach. However, the GM-CSF and IL-4 treated cells are not stable mature dendritic cells, e.g., the characteristic morphology and nonadherence of dendritic cells is lost if the cytokines are removed. We describe the need for a monocyte-conditioned medium to generate fully mature and stable dendritic cells. This is achieved by adding a 3 day 'maturation culture' to the initial 6-7 day culture in the presence of GM-CSF and IL-4. Macrophage-conditioned medium contains the critical maturation factors. Mature dendritic cells are defined by their pronounced display of motile cytoplasmic processes ('veils'), their high capacity to induce proliferative responses in resting T cells, particularly in naive umbilical cord T cells, their down-regulated antigen processing ability, and their characteristic phenotype: expression of CD83, high levels of MHC molecules and CD86, lack of CD115 and perinuclear dot-like CD68 staining. These features are stable for at least 3 days upon withdrawal of cytokines and conditioned media. IL-4 can be replaced by IL-13. When CD34+ progenitors are depleted from blood, there is only a minor reduction in the yield of dendritic cells by this method. We have adapted the method to consider several variables that are pertinent to clinical use, including a change from fetal calf serum to human plasma and to media approved for clinical use like X-VIVO or AIM-V. 1% plasma and RPMI 1640 are currently optimal. Additional reagents used for cell culture (Ig. cytokines) and cell separation (immunomagnetic beads) are approved for or already used in clinical applications. For 40 ml blood, the yield is 0.8-3.3 x 10(6) mature dendritic cells as defined by the expression of the new dendritic cell-restricted marker CD83. CD83+ cells constitute between 30 and 80% of all cells recovered at the end of the culture period. Yields can be enhanced up to six-fold if the blood donors are pretreated with G-CSF. Stable, mature dendritic cells generated by this method should be a powerful tool for active immunotherapy.

Immune response modulation by pentoxifylline in vitro.

Pentoxifylline (PTX) has recently been shown to modulate TNF-alpha production and to reduce the incidence and severity of all major complications after BMT, including mucositis, veno-occlusive disease, renal insufficiency, hypertension, and graft-versus-host disease. To analyze in detail the effect of PTX on immune complications after BMT, we investigated the immunomodulatory effect of PTX on immune responses in vitro. The continuous presence of PTX significantly reduced the proliferative response of PBMC to PHA stimulation and to alloantigens in a dose-dependent manner. Starting at concentrations of 100 micrograms/ml, PTX was able to inhibit and, at 1000 micrograms/ml, completely block mitogen-induced proliferation. Maximal inhibition of more than 90% (91 +/- 4%) was also observed at PTX concentrations of 1000 micrograms/ml in the mixed lymphocyte culture (MLR) and by addition on day 0. However, lower but still significant suppression (13 +/- 7%) was achieved at concentrations of 10 micrograms/ml PTX. The inhibitory capacity of PTX was increased by mAbs against TNF-alpha (34 +/- 5% additional suppression at 100 micrograms/ml PTX) and not reversed by the addition of rTNF-alpha. The effect of PTX on the generation of CTLs in vitro was studied in the cell-mediated lymphotoxicity assay. PTX (100 micrograms/ml) significantly inhibited (P = 0.0178) the in vitro generation of CTLs when PTX was added to the culture on day 0. PTX also showed profound modulatory properties in the NK assay, with a reduction of 23 +/- 3% in specific lysis at 10 micrograms/ml PTX and maximal reductions of 88 +/- 3% at 1000 micrograms/ml PTX. Immunomodulatory properties of PTX were not only associated with blockage of TNF-alpha, as shown by decreased mRNA expression and TNF-alpha values in the culture supernatants, but also with an impaired production of other cytokines and secondary messages such as IFN-gamma and neopterin. PTX treatment, however, did not affect IFN-alpha or IL-1 beta production, and IL-6 release was even increased. PTX, therefore, has profound immunomodulatory properties in vitro, which are associated with selective inhibition of cytokine release and can be enhanced by the addition of mAbs against TNF-alpha, but not reversed by the addition of rTNF-alpha.

Serum levels of interleukin 6, interleukin 8, and C-reactive protein after human allogeneic bone marrow transplantation.

Serum levels of interleukin 6 (IL-6) and C-reactive protein (CRP) were measured every second day from day -6 to day +86 in 24 patients undergoing allogeneic (n = 23) and syngeneic (n = 1) bone marrow transplantation (BMT). Endogenous serum levels of IL-6, IL-8, and CRP were further analyzed during complications after BMT, such as fever of unknown origin (FUO), severe infectious complications and acute graft-versus-host disease (GVHD). In addition, CRP levels were measured in 10 patients with interstitial pneumonitis of various origins (CMV, idiopathic). In all 24 patients IL-6 and CRP levels showed a characteristic monophasic pattern. After a slight decrease in the first days after BMT, a significant increase was observed, starting on day +3/+5 (P < 0.05) and reaching peak levels on day +9/+11 (P < 0.01). CRP had a similar pattern, with an increase in serum levels on day +3/+5 and maximum levels one to three days after the IL-6 peak was reached. The magnitude of the peak was related to the development of complications in the further course of BMT and was high in patients with and low in patients without complications. Serum levels of both molecules returned to baseline after day 14 posttransplant. Increased IL-6 and CRP levels were observed in the further course of BMT during severe infections or FUO either on the day of clinical onset (IL-6) or three days later (CRP), but not during acute GVHD grade III/IV. CMV interstitial pneumonitis (CMV-IP) was accompanied by an increase in CRP levels, while no such elevations were observed in patients with idiopathic interstitial pneumonitis (IIP). Elevated IL-8 serum levels occurred during bacterial infections, but to a lesser amount also during GVHD and CMV-IP. In conclusion, a characteristic pattern of IL-6 and CRP was observed after allogeneic BMT and a further increase associated with infectious complications. Since no significant elevations were seen in patients with GVHD, we conclude that both molecules are not involved in the induction of GVHD and might be useful diagnostic tools for the prediction and diagnosis of infectious complications after BMT. In contrast, assessment of IL-8 serum values does not permit clinical complications to be specified.

Serum levels of cytokines and secondary messages after T-cell-depleted and non-T-cell-depleted bone marrow transplantation: influence of conditioning and hematopoietic reconstitution.

Cytokines are increasingly recognized as important mediators of graft-versus-host disease (GVHD). Measurements of cytokine serum levels in patients with GVHD, and successful prevention and treatment of the disease with the use of cytokine antagonists to either the cytokine or its receptor, are only two of several factors demonstrating the involvement of cytokines in GVHD. To further investigate the role of cytokines in the pathomechanism of acute GVHD, we investigated endogenous serum levels of various cytokines and dependent molecules in sera of 14 patients after T-cell-depleted (TCD) bone marrow transplantation (BMT) and compared the results with those of 12 patients undergoing non-TCD BMT. The effect of various conditioning regimens and of hematopoietic reconstitution on cytokine serum levels was analyzed in detail in these cohorts of patients by measuring interferon (IFN)-gamma, IFN-alpha, tumor necrosis factor-alpha, interleukin-6, neopterin, and beta2-microglobulin. The analyses showed that an increase in IFN-gamma and neopterin serum levels was a specific feature of cyclophosphamide administration and was not observed after other cytostatic drugs or total body irradiation, and that an increase in IFN-gamma, neopterin, beta2-microglobulin, and IFN-alpha release depends on the presence of T cells in the graft. We conclude that significant cytokine serum alterations were noted after TCD BMT as compared with after non-TCD BMT. These alterations, besides depletion of cytotoxic effector cells, might be involved in preventing GVHD after TCD BMT. In addition, more attention should be devoted to the cytokine release-inducing capacity of the conditioning regimen, because such a release might influence the occurrence of transplant-related complications after BMT.

Endogenous interleukin 1 receptor antagonist during human bone marrow transplantation: increased levels during graft-versus-host disease, during infectious complications, and after immunoglobulin therapy.

In order to understand in more detail the role of endogenous interleukin 1 receptor antagonist (IL-1ra) during bone marrow transplantation, IL-1ra serum levels of 28 patients undergoing allogeneic (n=25) or autologous (n=3) bone marrow transplantation were measured with a commercially available ELISA. In addition, the impact of intravenous immunoglobulin (IVIG) was evaluated by analyzing IL-1ra serum levels before and 2, 5, and 24 hr after IVIG infusion. IL-1ra measurements revealed a nadir of circulating IL-1ra levels 3-5 days after bone marrow transplantation, with an increase during conditioning and hematological reconstitution. Circulating IL-1ra levels were significantly increased in patients with cytomegalovirus (CMV) disease, CMV reactivation, graft-versus-host disease (GVHD), or fever of unknown origin, when compared with time-matched controls without complications. Highest levels were observed in patients with CMV disease (1922+/-388 pg/ml), followed by patients with CMV reactivation (1575+/-435 pg/ml) and GVHD (1178+/-317 pg/ml). The magnitude of IL-1ra increase in GVHD was related to disease severity. Patients with grade III-IV GVHD developed higher IL-1ra levels than did patients with grade I-II GVHD. Lower but still significantly elevated IL-1ra levels were observed during fever of unknown origin (384+/-87 pg/ml). An increase of IL-1ra serum levels followed the administration of IVIG before transplantation and after hematopoietic reconstitution, but not during aplasia, pointing to the important role of hematopoietic cells in the production of IL-1ra. In conclusion, we show that IL-1ra release is related to conditioning regimen, hematopoietic reconstitution, complications of infectious and alloimmune etiology after bone marrow transplantation, and exogenously administered IVIG.

CD4 monoclonal antibody VIT4 in human alloimmune response in vitro and in vivo.

In the present report the immunosuppressive effects of the murine anti-human CD4 monoclonal antibody (mAb) VIT4 on human alloimmune response in vitro were analyzed. Moreover, the antibody was tested for its activity to prolong allograft survival in seven patients with steroid-refractory allograft rejection. VIT4 inhibited the proliferative response to alloantigens in the mixed lymphocyte reaction (MLR) in a dose-dependent manner. At concentrations of 1 and 10 micrograms/ml VIT4 blocked MLR by 55 +/- 11% and 77 +/- 1%, respectively. Also alloantigen-specific proliferation of in vitro- generated memory T cells was dose-dependently reduced to 23 +/- 1% at a VIT4 concentration of 100 micrograms/ml. Furthermore, at the same dose level VIT4 blocked proliferation of antigen-specific short-term alloreactive CD4+ cell lines and significantly inhibited the in vitro generation of cytotoxic T lymphocytes (CTL). In a pilot study VIT4 (5 mg/d i.v.) was administered to 7 patients with steroid-refractory allograft rejection for 14 days. In 4 of 7 patients graft function transiently improved and graft survival in all patients was prolonged to a mean of 694 days (range 128-2163) from the beginning of the VIT4 treatment. In the light of our in vitro results and the preliminary clinical data, further clinical trials using higher antibody doses are greatly warranted to assess the efficacy of anti-CD4 mAb VIT4 in the treatment of allograft rejection.

[Interferon-gamma suppresses expression of the HER-2 oncogene in ovarian cancer cells]. / Interferon-Gamma supprimiert die Expression des Onkogens HER-2 in Ovarialkarzinomzellen.

The overexpression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian and mammary carcinoma is an important indicator for a bad prognosis. In this study we demonstrate that in 7 out of 8 ovarian carcinoma cell lines there is an interferon-gamma-mediated reduction in HER-2 specific protein, and this effect was found to correlate with the antiproliferative action. It is interesting to note that there is no relationship between the absolute amount of HER-2 protein expressed and the sensitivity of the ovarian carcinoma cells for an antiproliferative activity of interferon-gamma. Other chemotherapeutic agents did not affect HER-2 expression although they inhibited the proliferation. The oncogene expression was lowered only in the ovarian carcinoma cell lines and not in 3 interferon-gamma sensitive human breast cancer cell lines. Expression of the oncogene HER-2 is the leading prognostic factor in ovarian cancer. Its modulation might represent a mechanism by which interferon-gamma inhibits cell proliferation.

Graft-versus-host disease after autologous bone marrow transplantation: a realistic expectation?

The immunobiological mechanisms involved in the manifestation of an autoaggressive disease after autologous bone marrow transplantation (BMT) and cyclosporine A (CyA) treatment are still unclear. This disease, which in animals shows clinical and histopathological features in the skin, liver and gut similar to those of graft-versus-host disease (GVHD), is called autologous GVHD and associated with the appearance of CD8+ cells with a lytic specificity for a public epitope on MHC class II. 2 steps are supposed to be essential for the induction of autologous GVHD: firstly, the elimination of a host resistance or an autoregulatory mechanism of self tolerance by high dose chemotherapy including total body irradiation, and secondly, the prevention of clonal deletion of MHC class II-restricted auto-reactive T-cells by CyA. Several experiments describe a possible antitumor effect of this disease in vitro as well as in vivo. The CD8+ cells kill tumor cells in vitro and survival of animals with autologous GVHD challenged with low doses of tumor cells is better than that of animals without autologous GVHD. In an attempt to reduce the relapse rate after autologous BMT, induction of GVHD with CyA treatment was investigated in clinical trials. A syndrome similar to the one described in rats was observed also in humans. However, symptoms were confined to the skin, appeared earlier than in experimental transplantation and were always self-limiting. Further investigations aiming at understanding in more detail the immunobiological mechanisms in human autologous GVHD and phase III clinical trials will have to be completed in order to define the role of this disease in clinical transplantation.

Structural polymorphism of the DC1 light chains from DR1-, DR2-, and DRw6-positive cell lines.

The heavy and light chain subunits of the DC1 antigen from several cell lines expressing different DR specificities were compared by two-dimensional (2-D) gel electrophoresis. The DC1 light chains from cell lines typed as DR1, DR2, or DRw6 differed in their isoelectric points. No difference in charge was observed for the DC1 heavy chain from the three cell lines. The DC1 light chains from different DRw6-positive cell lines were also found to be structurally polymorphic. The DRw6 cell lines examined did not all express the same DR-like light chains, indicating that the DRw6 specificity is biochemically complex, which is in agreement with the serologic studies of others.

Effect of an individualised training programme during weight reduction on body composition: a randomised trial.

OBJECTIVE: To study the effect of a standardised training programme focusing on maintenance of fat free mass during weight reduction by energy reduction in obese children. DESIGN: Randomised trial of physical training programme and dietary advice (group A) versus dietary advice alone (group B). SUBJECTS: Thirty obese children and adolescents (14 group A, 16 group B) participated in the 12 week long programme; 20 children (10 group A, 10 group B) were also reassessed after one year. MEASUREMENTS: Fat free mass was estimated from the resistance index, obtained by bioelectrical impedance analysis at baseline, after four, eight, and 12 weeks in all subjects, and after one year in 20 subjects. RESULTS: The mean (SD) change in fat free mass was significantly different between the two groups after 12 weeks (group A, 2.68 (3.74) kg; group B, 0.43 (1.65) kg). The change in body weight after one year was inversely correlated with the change in fat free mass after 12 weeks (r = -0. 44), as assessed in the 20 subjects. CONCLUSIONS: A standardised training programme as used in this study can prevent reduction in fat free mass during weight loss in obese children. Reduction in fat free mass during weight reduction might be a risk factor for regain of weight.

A comparative study of the in vitro immunomodulatory activity of human intact immunoglobulin (7S IVIG), F(ab')2 fragments (5S IVIG) and Fc fragments. Evidence for post-transcriptional IL-2 modulation.

During the past few decades intravenous immunoglobulin (IVIG) has been used successfully in the treatment of various immunoregulatory disorders. Treatment results have been attributed to immunomodulation mainly via Fc receptors or by anti-idiotypic antibodies to disease-causing autoantibodies. From the present study it is clearly evident that 7S IVIG (intact immunoglobulin) as well as 5S IVIG [F(ab')2 fragments] and Fc fragments have a potent immunomodulatory capacity. We demonstrate that mainly 7S IVIG inhibits alloantigen-induced T-cell proliferation and generation of cytotoxic T lymphocytes. Reduced interleukin-2 (IL-2) protein levels in culture supernatants of IVIG-supplemented mixed lymphocyte reactions (MLR) but unchanged IL-2 mRNA levels strongly argue in favour of a post-transcriptional interference of IVIG with cytokines and/or cytokine production. Interferon-gamma (IFN-gamma), soluble IL-2 receptor (sIL-2R) and monokines such as IL-1 beta, IL-6, IFN-alpha and tumour necrosis factor (TNF-alpha) were not affected by IVIG supplementation to MLR. Fc fragments were superior to F(ab')2-containing IVIG (5S and 7S IVIG) in inhibiting lectin stimulation of peripheral blood mononuclear cells (PBMC), whereas natural killer (NK) cytotoxicity was primarily inhibited by Fc-bearing IVIG (7S IVIG and Fc fragments), suggesting multiple mechanisms of IVIG immunomodulatory activity.

Survival after bone marrow transplantation from cytomegalovirus seropositive sibling donors.

HLA-A2-restricted T cells show peptide-specific activity against cytomegalovirus and leukaemia cells. We retrospectively analysed the influence of donor cytomegalovirus serostatus on the outcome of 103 consecutive patients who had leukaemia and who received bone-marrow transplants from HLA-identical sibling donors. We found that donor cytomegalovirus seropositivity significantly improved overall survival (p=0.02) as a result of lower relapse incidence (p=0.035) in HLA-A2-positive but not HLA-A2-negative recipients. In HLA-A2-positive recipients donor cytomegalovirus seropositivity was associated with chronic graft-versus-host disease (GVHD), but even in patients without chronic GVHD donor cytomegalovirus seropositivity significantly improved survival (p=0.0483). These preliminary data provide evidence that at least in HLA-A2-positive recipients, transplantation of bone marrow from cytomegalovirus positive, HLA-identical sibling donors seems to be associated with substantial graft-versus-leukaemia activity, and suggests a cross-reactivity of cytomegalovirus-specific donor-derived cytotoxic T cells with HLA-A2-restricted recipient minor histocompatibility antigens.

Dendritic cells generated from blood precursors of chronic myelogenous leukemia patients carry the Philadelphia translocation and can induce a CML-specific primary cytotoxic T-cell response.

Dendritic cells (DC) are professional antigen-presenting cells specialized in the initiation of primary immune responses. We were interested to know whether mature DC can be grown in vitro from peripheral blood mononuclear cells (PBMC) of patients with chronic myelogenous leukemia (CML), and whether they carry the Philadelphia (Ph) translocation. Using a method recently described, DC were generated from PBMC precursors of 12 patients with CML using GM-CSF, IL-4, and monocyte-conditioned medium. DC exhibited the typical morphology with thin cytoplasmatic processes and expressed high levels of MHC class II, CD86, and CD83 typical for mature DC. After sorting with the monoclonal antibody CD83, a cell population of more than 95% CD83 positive cells was obtained. The presence of the Ph translocation was analyzed in these cells, in PBMC, lymphoblastoid cell lines (LCL), and in phytohemagglutinin (PHA)-induced T blasts from the same patients by fluorescence in situ hybridization (FISH). In contrast to all other cells analyzed, the vast majority of DC (95.9 +/- 0.7%) displayed the Ph translocation, irrespective of disease stage or therapy. PBMC were predominantly positive for the Ph chromosome (67.6 +/- 7.3%), whereas only 11.4 +/- 1% of the B cells and 4.4 +/- 1.1% of the PHA blasts carried the Ph translocation. Using such leukemic DC as antigen-presenting cells, a primary CML-directed cytotoxic immune response in vitro was obtained, as shown by the specific recognition of Ph chromosome positive cells. We conclude that DC can be generated from blood progenitors of CML patients in vitro and exhibit, to a large extent, the Ph translocation. Such DC, which in a preliminary experiment have been able to induce a primary CML-directed cytotoxic immune response in vitro, might be ideal candidates for adoptive immunotherapy either by direct transfer of DC for in vivo generation of a T-cell response or by in vitro generation of CML-specific cytotoxic autologous or HLA-matched normal T-cell clones for use in vivo.

Evidence for a graft-versus-tumor effect in a patient treated with marrow ablative chemotherapy and allogeneic bone marrow transplantation for breast cancer.

Graft-versus-leukemia (GvL) has been shown to be an important immune-mediated antitumor effect in hematologic malignancies. It is still unknown whether such an immunemediated antitumor effect has clinical implications in patients with solid tumors. A 32-year-old woman with inflammatory breast cancer received a bone marrow transplant (BMT) from her HLA-identical sibling. During graft-versus-host disease (GvHD) cytotoxic T lymphocytes were grown and tested in a chromium-release assay against B and T lymphocytes of the patient and donor and against a panel of breast cancer cell lines. Resolution of liver metastases was observed simultaneously with clinical GvHD in the first weeks after transplant. In addition, minor histocompatibility antigen (MiHA)-specific and major histocompatibility complex (MHC) class I antigen-restricted cytotoxic T lymphocytes recognizing breast carcinoma target cells were isolated from the blood of the patient. Pretreatment of such target cells with tumor necrosis factor (TNF)-alpha but not with interferon (IFN)-alpha or IFN-gamma increased susceptibility of these cells to lysis by cytotoxic T lymphocytes. Clinical course and in vitro results suggest that a graft-versus-tumor (GvT) effect might exist after allogeneic BMT for breast cancer. However, clinical experience on a larger scale would be required to determine the clinical efficacy of GvT effects in patients with solid tumors.

LST1: a gene with extensive alternative splicing and immunomodulatory function.

The gene of the leukocyte-specific transcript (LST1) is encoded within the TNF region of the human MHC. The LST1 gene is constitutively expressed in leukocytes and dendritic cells, and it is characterized by extensive alternative splicing. We identified 7 different LST1 splice variants in PBMC; thus, 14 LST1 splice variants (LST1/A-LST1/N) have been detected in various cell types. These isoforms code for transmembrane as well as soluble LST1 proteins characterized by two alternative open reading frames at their 3' end. We demonstrate the presence of the transmembrane variant LST1/C on the cell surface of the monocytic cell lines U937 and THP1. Recombinant expression of LST1/C permitted its profound inhibitory effect on lymphocyte proliferation to be observed. In contrast, the alternative transmembrane variant LST1/A, the extracellular domain of which shows no amino acid sequence homology to LST1/C exerted a weaker but similar inhibitory effect on PBMC. These data demonstrate the protein expression of LST1 on the cell surface of mononuclear cells, and they show an inhibitory effect on lymphocyte proliferation of two LST1 proteins although they have only a very short amino acid homology.

Expression of c-fos correlates with IFN-alpha responsiveness in Philadelphia chromosome positive chronic myelogenous leukemia.

This study evaluates (i) constitutive levels of oncogene and p53 transcripts in chronic phase CML patients and (ii) their modulations subsequent to in vivo therapy with rIFN-alpha 2c. Peripheral blood mononuclear cells (pbmc) and bone marrow cells of 26 patients were examined for c-fos, c-myc, p53 and the hybrid bcr/abl mRNA levels. Results indicated that (i) constitutive c-fos transcript levels are significantly higher in patients subsequently responding to IFN-alpha therapy (p < 0.01) and positively correlated with the proportion of lymphocytes (r = 0.6895, p < 0.01) and negatively with the proportion of immature cells (r = -0.568, p < 0.01) contained in the pbmc preparations tested, (ii) constitutive mRNA levels of the hybrid bcr/abl, c-myc and p53 are positively correlated with each other, but failed to relate to disease parameters, and (iii) acute and chronic in vivo exposure to IFN-alpha is accompanied by upregulation of c-fos and downregulation of c-myc mRNA levels in responder patients.