Manfred Herold

CELLULAR AND HUMORAL IMMUNE RESPONSES IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA AFTER VACCINATION WITH ANTIGEN PULSED DENDRITIC CELLS

PURPOSE Dendritic cells are the most potent stimulators of immune responses including antitumor responses. We performed a pilot study of cultured antigen loaded dendritic cells in patients with metastatic renal cell carcinoma. MATERIALS AND METHODS Dendritic cells were obtained by culturing plastic adherent mononuclear cells from peripheral blood for 5 days in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. Day 5 dendritic cells were loaded with cell lysate from cultured autologous tumor cells and with the immunogenic protein keyhole-limpet hemocyanin (KLH) which serves as a helper antigen and as a tracer molecule. During the antigen pulse dendritic cells were activated with a combination of tumor necrosis factor-alpha and prostaglandin E2. Dendritic cells were administered by 3 intravenous infusions at monthly intervals. Cellular and humoral immune responses to KLH and cell lysate were measured in vitro before and after the vaccinations. RESULTS Preparation of 12 dendritic cell vaccines from patients with advanced renal cell carcinoma was successful. Treatment with fully activated CD83+ dendritic cells was well tolerated with moderate fever as the only side effect. Potent immunological responses to KLH and, most importantly, against cell lysate could be measured in vitro after the vaccinations. CONCLUSIONS Our data demonstrate that a dendritic cell based vaccine can induce antigen specific immunity in patients with metastatic renal cell carcinoma. Dendritic cell based immunotherapy represents a feasible, well tolerated and promising new approach for the treatment of advanced renal cell carcinoma.

International recommendations for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies

Anti-nuclear antibodies (ANA) are fundamental for the diagnosis of autoimmune diseases, and have been determined by indirect immunofluorescence assay (IIFA) for decades. As the demand for ANA testing increased, alternative techniques were developed challenging the classic IIFA. These alternative platforms differ in their antigen profiles, sensitivity and specificity, raising uncertainties regarding standardisation and interpretation of incongruent results. Therefore, an international group of experts has created recommendations for ANA testing by different methods. Two groups of experts participated in this initiative. The European autoimmunity standardization initiative representing 15 European countries and the International Union of Immunologic Societies/World Health Organization/Arthritis Foundation/Centers for Disease Control and Prevention autoantibody standardising committee. A three-step process followed by a Delphi exercise with closed voting was applied. Twenty-five recommendations for determining ANA (1–13), anti-double stranded DNA antibodies (14–18), specific antibodies (19–23) and validation of methods (24–25) were created. Significant differences between experts were observed regarding recommendations 24–25 (p<0.03). Here, we formulated recommendations for the assessment and interpretation of ANA and associated antibodies. Notably, the roles of IIFA as a reference method, and the importance of defining nuclear and cytoplasmic staining, were emphasised, while the need to incorporate alternative automated methods was acknowledged. Various approaches to overcome discrepancies between methods were suggested of which an improved bench-to-bedside communication is of the utmost importance. These recommendations are based on current knowledge and can enable harmonisation of local algorithms for testing and evaluation of ANA and related autoantibodies. Last but not least, new more appropriate terminologies have been suggested.

Impaired nocturnal secretion of melatonin in coronary heart disease

Patients with coronary heart disease have increased nocturnal urinary noradrenaline. Because melatonin suppresses sympathetic activity, we measured serum melatonin concentrations at night (0200 h) in 15 patients with coronary heart disease. Melatonin was significantly lower in the patients than in 10 healthy controls (median 7.8 [interquartile range 6.5-11.8] vs 36.2 [32.2-42.5] pg/mL, p < 0.0001). Thus, impaired nocturnal secretion of melatonin is associated with coronary heart disease.

Production of IL-12 by Human Monocyte-Derived Dendritic Cells Is Optimal When the Stimulus Is Given at the Onset of Maturation, and Is Further Enhanced by IL-4

Dendritic cells produce IL-12 both in response to microbial stimuli and to T cells, and can thus skew T cell reactivity toward a Th1 pattern. We investigated the capacity of dendritic cells to elaborate IL-12 with special regard to their state of maturation, different maturation stimuli, and its regulation by Th1/Th2-influencing cytokines. Monocyte-derived dendritic cells were generated with GM-CSF and IL-4 for 7 days, followed by another 3 days ± monocyte-conditioned media, yielding mature (CD83+/dendritic cell-lysosome-associated membrane glycoprotein+) and immature (CD83−/dendritic cell-lysosome-associated membrane glycoprotein−) dendritic cells. These dendritic cells were stimulated for another 48 h, and IL-12 p70 was measured by ELISA. We found the following: 1) Immature dendritic cells stimulated with CD154/CD40 ligand or bacteria (both of which concurrently also induced maturation) secreted always more IL-12 than already mature dendritic cells. Mature CD154-stimulated dendritic cells still made significant levels (up to 4 ng/ml). 2) Terminally mature skin-derived dendritic cells did not make any IL-12 in response to these stimuli. 3) Appropriate maturation stimuli are required for IL-12 production: CD40 ligation and bacteria are sufficient; monocyte-conditioned media are not. 4) Unexpectedly, IL-4 markedly increased the amount of IL-12 produced by both immature and mature dendritic cells, when present during stimulation. 5) IL-10 inhibited the production of IL-12. Our results, employing a cell culture system that is now being widely used in immunotherapy, extend prior data that IL-12 is produced most abundantly by dendritic cells that are beginning to respond to maturation stimuli. Surprisingly, IL-12 is only elicited by select maturation stimuli, but can be markedly enhanced by the addition of the Th2 cytokine, IL-4.

Bacillus Calmette‐Guérin mycobacteria stimulate human blood dendritic cells

Bacillus Calmette‐Guérin (BCG) mycobacteria have been used as adjuvant in the active immunotherapy of various human cancers. In addition, dendritic cells, which are the most potent antigen‐presenting cells, have been shown to be capable of initiating anti‐tumor immune responses. Here we investigated the effects of BCG on dendritic cells cultured from human blood. Addition of BCG resulted in rapid homotypic adhesion of dendritic cells. Moreover, BCG concentrations ranging from 104 to 106 bacteria/ml enhanced expression of the dendritic‐cell‐maturation antigen CD83 and of the T‐cell co‐stimulator CD86 (B7‐2) in a dose‐dependent manner. Concomitant with the increase of CD83 and CD86 expression, the cells lost the ability to capture soluble antigens, as determined by the exclusion of fluoresceinated Dextran molecules. Strikingly, the same dosages of BCG‐bacteria stimulated TNF‐α‐gene transcription and TNF‐α‐protein release from dendritic cells in a dose‐dependent fashion. BCG infection of dendritic cells in the presence of a neutralizing antibody directed against TNF‐α inhibited CD83 expression by more than 50% indicating that the BCG‐induced maturation of dendritic cells was at least partially mediated by dendritic‐cell‐derived TNF‐α. The finding that BCG activates the most potent antigen‐presenting cells reveals a plausible immunological mechanism of the occasionally observed anti‐tumor activity of BCG.

Successful treatment of metastatic renal cell carcinoma with a biologically active dose of recombinant interferon-gamma.

We tested the clinical efficacy of a biologically active dose (BAD) of interferon (IFN)-gamma for treatment of progressive renal cell carcinoma (RCC). Twenty-two RCC patients with disease progression subsequent to nephrectomy were entered on a phase II clinical trial. During an initial dose-finding phase, biochemical responses to repeated once-weekly subcutaneous injections of 10, 100, or 500 micrograms of recombinant IFN-gamma were tested in 16 patients. Results indicated that 100 micrograms IFN-gamma applied once weekly was biologically active with induction of serum beta 2-microglobulin and neopterin. Such a dose induced a nearly maximum response of both markers lasting more than 4 days. This dose was also associated with minimal side effects. A dose of 100 micrograms IFN-gamma given once weekly was, therefore, subsequently given weekly for long-term treatment. During a median time of therapy of 10 months (range, 2 to 32 months) two complete (CR; 20+, 20+ months) and four partial tumor responses (PR; 6+, 7+, 8+, 24+ months) were seen (30% CR plus PR; 95% confidence limits, 12% to 54%) among 20 patients evaluable for response. Patients with refractory disease had significantly lower IFN-gamma-induced increments of serum beta 2-microglobulin than those who achieved clinical remission or stable disease.

Tumour necrosis factor‐alpha plasma level in patients with type 1 diabetes mellitus and its association with glycaemic control and cardiovascular risk factors

Abstract. Lechleitner M, Koch T, Herold M, Dzien A, Hoppichler F (University of Innsbruck, Medical Centre Hentschelhof, Innsbruck, and Hospital Barmherzige Brüder, Salzburg, Austria). tumour necrosis factor‐alpha plasma level in patients with type 1 diabetes mellitus and its association with glycaemic control and cardiovascular risk factors. J Intern Med 2000: 248: 67–76.

Serum levels of interleukin-6 in multiple myeloma and other hematological disorders: correlation with disease activity and other prognostic parameters

SummaryInterleukin-6 (IL-6) is a multifunctional cytokine involved in the regulation of the terminal differentiation pathway of B lymphocytes. Recent reports revealed its potential role in the in vitro and in vivo growth of human multiple myeloma cells. The mechanism, however, by which IL-6 triggers proliferation of malignant plasma cells remains controversial. Using the very sensitive 7TD1 bioassay we quantified endogenous circulating IL-6 levels in serum samples of 104 patients suffering from monoclonal gammopathies and other hematological disorders [47 with multiple myeloma (MM), 24 with monoclonal gammopathy of unknown significance (MGUS), 8 with myeloproliferative disease, and 25 suffering from lowgrade non-Hodgkins lymphoma (NHL)]. Elevated serum levels of IL-6 (>5 pg/ml) were detected in 42% of the patients with MM, in 13% with MGUS, in 15% with low-grade B-NHL, and in 1 patient with T-NHL. In patients suffering from chronic myeloproliferative diseases, IL-6 levels were within the normal range. In patients with myeloma, IL-6 levels were significantly higher at advanced stages (II/III) or with progressive disease than in patients with MM stage I, MGUS, or at the plateau phase (P<0.01). In patients with monoclonal gammopathies including MGUS, serum IL-6 levels correlated with neopterin, tumor necrosis factor alpha and β2-microglobulin. An inverse correlation was found with hemoglobin levels. From these results, we propose that in myeloma patients serum IL-6 levels may reflect disease activity and tumor cell mass. The correlation with serum neopterin, a macrophage product, also suggests its origin in an activated immune system.

Diagnostic imaging of gout: comparison of high-resolution US versus conventional X-ray

The aim was to compare X-ray and ultrasound (US) in diagnosing gout. In a prospective study, 105 consecutive patients with clinical suspicion of gout underwent conventional X-ray und high-resolution US in order to help in arriving at a definite diagnosis. X-ray findings suggestive of gout included soft-tissue opacifications with densities between soft tissue and bone, articular and periarticular bone erosions, and osteophytes at the margins of opacifications or erosions. US findings suggestive of gout included bright stippled foci and hyperechoic soft-tissue areas. Fifty-five patients had a definite diagnosis of gout (102 involved sites), 31 patients were diagnosed as having another disease (59 involved sites), and 19 patients were excluded from the study because a definite diagnosis could not be established. X-ray suggested gout with a sensitivity of 31% (32/102) and a specificity of 93% (55/59), whereas US suggested gout with a sensitivity of 96% (98/102) and a specificity of 73% (43/59). US was much more sensitive than conventional X-ray but less specific. Our data show that US often provided additional diagnostic information in patients with clinical suspicion of gout when laboratory findings and X-ray results were negative or inconclusive and should therefore be used in these cases.

Endogenous IFN-gamma during human bone marrow transplantation : analysis of serum levels of interferon and interferon-dependent secondary messages

Serum levels of interferon-gamma and the IFN-dependent marker molecules neopterin and beta 2-microglobulin were assessed in BMT recipients. Concentrations of the latter two markers were corrected for creatinine levels in order to eliminate the impact of alteration of kidney function. Serum levels were assessed daily using commercially available radioimmunoassays. Twelve patients were studied during the early phase of allogeneic bone marrow transplantation and eleven additional patients during complications of BMT. Results indicated that both the conditioning regimen for BMT as well as major clinical complications such as infection and acute graft-versus-host disease strongly influence the endogenous patterns of the lymphokine and its secondary messages. During allogeneic BMT IFN-gamma and neopterin levels exhibited a biphasic pattern with a first peak during conditioning with high-dose cyclophosphamide and a second still higher peak at the time of hemopoietic regeneration. beta-2-microglobulin ratios increased during conditioning and remained elevated throughout observation. Serious infections of bacterial and viral origin as well as GvHD were accompanied by elevated levels of all three serum parameters studied. The kinetics of enhanced endogenous production, however, differed between infectious complications and GvHD. Increasing concentrations were observed during infections subsequent to clinical manifestation, whereas they preceded disease manifestation in GvHD.