Influence of immunotherapy (IL2 + LAK + inhibition of prostaglandin synthesis) on peripheral blood immune parameters and in vitro cytokine production in metastatic renal cell carcinoma.

Previous results on the peripheral blood immune status of renal cell carcinoma had indicated immunosuppression in metastatic disease, possibly mediated by prostaglandin E2 (PGE2). In the present study the immunologic effects of inhibition of PG synthesis by piroxicam in combination with interleukin 2 (IL 2) + lymphokine-activated killer (LAK) cell therapy were tested by immunomonitoring. In addition to peripheral blood parameters (lymphocyte subpopulations, neopterin, beta 2-microglobulin, TNF, IL 1, IFN gamma) we recorded in vitro cellular activity by incubating the patients' peripheral blood mononuclear cells (PBMC) in media containing fetal calf serum (FCS) or autologous serum, and either IL 2 or buffer. After 24 h of incubation we measured PGE2 and cytokine levels in supernatants. Systemic application of IL 2 induced in vivo lymphocyte proliferation and clearly influenced the serum levels of neopterin, beta 2-microglobulin and TNF. There was minor affection of IFN gamma and none of IL 1. PBMC in vitro produced high amounts of PGE2, IL 1 and TNF pretherapeutically, during therapy in vitro synthesis of these parameters decreased. Consistent production of IFN gamma was detected in supernatants only when FCS and IL 2 were added to the medium. Lack of affection of IFN gamma production in the autologous system during therapy indicated impaired cellular activity, which could neither be improved by therapy of the patient using IL 2 nor by adding IL 2 to the culture medium. Immunosuppression seems to interfere in a complex way with immunotherapy. Therapeutical influence of immunosuppression based on the results of immunomonitoring, however, seems to be a promising strategy for improving the still limited clinical results of immunotherapy.

Immune modulating effects of low doses of cyclophosphamide and keyhole limpet hemocyanin on peripheral blood immune parameters in patients with metastatic renal cell carcinoma.

Assessing the peripheral blood immune status, we had found evidence of immunosuppression in metastatic renal cell carcinoma. Since immunosuppression might interfere with the outcome of immunotherapy, it was submitted to further investigation. Immunosuppression can be mediated by T suppressor cells, which for their part are inhibited by low-dose cyclophosphamide (Cy). We tested whether the immunomodulating effects of the biological response modifier keyhole limpet hemocyanin (KLH) on cellular and humoral immune parameters would be intensified by low-dose Cy. 10 patients were given 300 mg/m2 Cy i.v. 3 days before application of 1 mg KLH i.m. up to 8 times in 4-weekly intervals. Immune parameters were assayed twice pre-therapeutically, and prior to and 1 day and 1 week after each KLH injection. In contrast to the results obtained with KLH-mono-application, lymphocyte subsets-with the exception of T4 cell counts and the T4/T8 ratio-remained stable during the first 4 months of observation. There were increases in immunoglobulins and in the immunoactivation markers tumor necrosis factor, neopterin and beta 2-microglobulin. The tendencies found here differed from those found in a previous study on patients who were given KLH alone, and were similar to those found in patients with nonmetastatic disease who received KLH alone. However, the increases of the activation markers during KLH+Cy application were at best half as much as in patients with nonmetastatic disease. In conclusion, immunosuppression was influenced, but not counterbalanced by low-dose Cy. Most probably other mediators of immunosuppression than T suppressor cells, for example prostaglandin E2, must be considered as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Postoperative long-term course of peripheral blood immune parameters and immunomodulating effects of keyhole limpet hemocyanin in patients with nonmetastatic renal cell carcinoma.

Therapeutic approaches to metastatic renal cell carcinoma (RCC) often focus on the application of immune modulators; the success rates, however, are not satisfactory. Up to the time of this study, no diagnostic tool has been available to select those patients who might profit from immunotherapy. Starting from this point, we have been assessing the immune status of patients suffering from RCC, intending to find markers that would characterize the more favorable prognosis. Our interest is focused not only on the metastatic but also on the nonmetastatic disease, i.e., the disease with the better prognosis. In the present study, we have assessed both the postoperative long-term course of several immune parameters of the peripheral blood and the reactivity of the immune system to immunostimulation with keyhole limpet hemocyanin (KLH) in patients with nonmetastatic RCC. In a prospectively randomized study, the verum group (n = 8) got 1 mg KLH per month up to 1 year while the control group (n = 9) got no immunostimulator after tumor nephrectomy. Both patient groups had stable or even increasing cell counts of lymphocyte subpopulations (T, B, natural killer, T4, T8 cells), and the humoral immunoactivation markers neopterin, beta 2-microglobulin and tumor necrosis factor increased considerably after tumor nephrectomy. An effect of KLH is evident 4-8 months postoperatively: here, the neopterin values in the KLH group are more than twice as high as in the control group. Thus, while patients with metastatic disease had turned out to be immunosuppressed (previous study), in patients with nonmetastatic RCC, both the long-term course indicating postoperative immunostimulation and the reactivity to KLH give evidence of immunocompetence.

Immune status and immune therapy of renal cell carcinoma.

At present, no sufficient therapy for metastatic renal cell carcinoma is available. Several immunotherapeutical protocols have been studied, success rates, however, were inconsistent. The purpose of this study was to assess the pretherapeutic immunological status of 13 patients with metastatic and 16 patients with nonmetastatic renal cell carcinoma and of 15 healthy volunteers. Determined were differential blood counts, lymphocyte subpopulations, beta 2-microglobulin, tumor necrosis factor (TNF), neopterin, immunoglobulin, fibronectin and ferritin. Additionally, these parameters were recorded for monitoring an immunotherapeutical approach with the xenogeneic biological response modifier Keyhole limpet hemocyanine (KLH) in 10 patients with metastatic and in 5 patients with nonmetastatic disease. The pretherapeutic immunological status of patients with metastatic disease was characterized by significantly reduced T4-, T8- and B-cell counts. Significantly increased were granulocyte counts, beta 2-microglobulin, neopterin and TNF. In patients who did not suffer from metastases, only beta 2-microglobulin and neopterin were increased significantly. During immunotherapy, in patients with metastases, there was a decline of lymphocyte subsets and of the T4/T8-ratio, which correlated with progress of the disease. Humoral immune parameters showed no changes compared to pretherapeutic values. In patients who did not suffer from metastases, cellular immune parameters showed stable values during immunotherapy; neopterin, beta 2-microglobulin and TNF increased considerably. These findings indicate immunosuppression in patients with metastatic renal cell carcinoma, increasing with progression of the disease and possibly impairing the immunostimulating effects of biological response modifiers during immunotherapy. In conclusion, the clinical response of metastatic renal cell carcinoma to immunotherapy might be improved if the immunostimulant is combined with agents suitable to overcome immunosuppression, i.e. low doses of cyclophosphamide or inhibitors of prostaglandin synthesis. In addition, assessment of immune parameters for monitoring the actual immune status of a patient and the immunological effects of therapy was found to be a necessary part of immunotherapy.

Tumor necrosis factor in benign and malign tissue of the kidney.

The use of tumor necrosis factor (TNF) in immunotherapy of tumor diseases has attracted increasing interest. Since the direct antitumor effect of the TNF is mediated by receptor-bound TNF, we immunohistologically stained both benign and malignant tissue from 35 tumor-bearing human kidneys for TNF. Using a polyclonal anti-TNF-antiserum, paraffin sections were tested in the presence and absence of in vitro preincubation with TNF. Furthermore, all specimens were stained immunohistologically for Tamm-Horsfall protein (THP) because this renospecific glycoprotein can bind TNF in a lectin-like manner. In the absence of TNF preincubation, malignant tissue was TNF-positive in 34 specimens, as was benign tissue from the same tumor-bearing kidneys in 35 cases. In several specimens the staining was so intense that preincubation with TNF did not enhance the reaction. Whereas TNF staining in tumor tissue was relatively homogenous, that in benign tissue was intensive in distal tubuli, moderate in proximal tubuli, and negative in glomeruli. THP staining was negative in malignant kidney tissue but positive in the distal tubuli of benign tissue, i.e., in the regions in which TNF staining was most intense. These results indicate that TNF binds not only to membrane, most likely in a receptor-mediated manner, but also to THP both in vivo and in vitro. In vivo binding of TNF to THP was confirmed in animal experiments in which pigs were given injections of TNF. Immunohistological staining of the animals' kidneys revealed positive reactions for both TNF and THP at the distal tubuli, indicating TNF binding to THP after in vivo TNF administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Investigations on bone metabolism of urological tumors forming metastases.

Bone metastases of urological tumors occur in nearly 40% of all primary tumors of the prostate, the kidney and the bladder. The quality of metastases may be described as osteolytic, osteoplastic or mixed lesion. Whereas prostate cancers produce mainly osteoplastic lesions, renal cell carcinomas predominantly generate osteolytic lesions. In bladder cancer both forms of metastases occur in tantamount numbers. However, diagnostics still presents many difficulties, since it is not feasible to identify very small metastases until symptoms have manifested themselves. The purpose of our study was to evaluate measurement technique and classification of significant serum markers for monitoring the course of disease. Patients with primary urological tumors and metastases in the skeleton were investigated and compared with healthy volunteers. Osteodensitometry was used to confirm and to replace radiological diagnosis of bone metastases. Thus it was possible to locate the extent and obtain information on the maximum charge and the stability of metastases. Our examinations revealed that distinct serum markers describe the changes in bone evoked by metastases. In comparison with healthy volunteers, patients with osteoplastic lesions and osteolytic lesions showed increases in hydroxproline and pyridinium crosslinks (significance at least p < 0.005). Osteocalcin was elevated only in osteoplastic lesions versus healthy volunteers (p < 0.01). For diagnostics of osteoplastic and osteolytic metastases, either alkaline phosphatase or the skeleton-specific phosphatase (ostase) can be measured serologically. Both parameters showed significant elevation in the patient groups when set against the healthy controls (both p < 0.0001). Compared with lytic lesions osteoplastic carcinomas revealed significant increase of alkaline phosphatase (p < 0.0001) and osteocalcin (p < 0.005). In examination of bone metabolism in patients with skeletal metastases the following parameters are of eminent interest: osteocalcin, hydroxyproline or pyridinium crosslinks, alkaline phosphatase or ostase. These serological parameters could be helpful even with regard to early diagnosis of bone metastases. Evaluation of measuring techniques suggests quantifying pyridinium crosslinks instead of hydroxyproline, because they may be assessed without taking the patient's diet into account. Determination of bone density may be helpful in diagnostics or control of therapy modalities.

Damaging effects of high energy shock waves on cultured Madin Darby canine kidney (MDCK) cells.

Shock wave lithotripsy (ESWL) has become an almost non-invasive standard treatment modality for urolithiasis. Several investigations, however, demonstrated that ESWL is not completely free of side effects. Among others alteration of renal tubular function has been reported. To study the effect of shock waves on tubular cells directly an in-vitro model with cultured Madin Darby Canine Kidney (MDCK) cells was established. Suspensions of MDCK cells (7 groups of 6 containers each) were exposed to 0, 16, 32, 64, 128, 256 shock waves (Dornier HM4, 18 kV). Before and 0, 1, 3, 6, 9, 12, 24 h after ESWL the following parameters were measured in the nutrient medium: lactate dehydroxygenase (LDH), glutamate oxalacetate transaminase (GOT), electrolytes. LDH and GOT increased depending on the number of shock waves indicating a membrane damage of MDCK cells. The MDCK model seems suitable for further studies on the effect of shock waves on renal tubular cells.