Stimulatory effect of staphylococcal leukocidin on granulopoiesis disturbed by cytostatic agents.

Functional tests of granulopoiesis (leukocytosis, bone marrow picture, incubation in vitro of bone marrow with [3H]thymidine followed by radioautography and counting of labeled promyelocytes and myelocytes, serum muramidase level, liberation of granulocyte bone marrow reserve, Nitro-BT reduction in blood granulocytes, enzyme cytochemistry, and phagocytosis) were performed in rabbits given bubulphan (10 mg/kg) or 5-fluorouracil (200 mg/kg). Determinations were carried on serially during treatment with cytostatics. Some of the cytostatic-treated animals received intravenous (i.v.) injections of purified staphylococcal leukocidin in daily doses of 0.1 mg/kg. In control animals, theleukocidin resulted in stimulation of granulopoiesis (leukocytosis, increased number of [3H]thymidine-labeled myelocytes, elevated serum muramidase level). Animals receiving cytostatics suffered from marked inhibition of granulopoiesis accompanied by decrease of bone marrow granulocyte reserve. Injection of staphylococcal leukocidin during the period of myelosuppression evoked by cytostatics resulted in partial protection of granylopoiesis and faster regeneration of the leukocyte system.

Suppression of the activity of natural killer-like cells by peritoneal macrophages obtained from Lewis lung tumor-bearing, Propionibacterium granulosum KP-45-treated, or normal, untreated mice.

Peritoneal adherent cells (PAC) obtained from Propionibacterium granulosum KP-45-treated or Lewis lung carcinoma-bearing BDF1 mice suppressed in vitro the NK-like cytotoxic activity of murine splenocytes against YAC-1 tumor target cells. Maximum inhibition occurred when suppressor and effector cells were preincubated together for 18 h, but the effect was demonstrable also when the two groups of cells were mixed only at the onset of the 4-h cytotoxic assay (i.e. without previous contact). Inhibitory cells appeared to be mostly macrophages, as judged by adherence to plastic and morphologic features, and as little as 5 to 20% of PAC, relative to the total number of co-incubated cells, were required for the clear demonstration of the effect. In addition to activated also normal, resident PAC obtained from untreated animals inhibited the NK cell-mediated cytotoxicity, but the effect was significantly pronounced only when 20% of suppressor cells were incubated overnight with effector splenocytes. The results favor the hypothesis that both functionally activated as well as resting macrophages operate as important regulators of the activity of NK cells in vivo.

Effect of hyperthermia on rabbit macrophages.

The effect of water-bath hyperthermia on rabbit peritoneal macrophages was studied in vitro. The cells were exposed to hyperthermia for 30 min to 4 hours and membrane transport of ions as measured by total and ouabain-inhibited 86Rb influx as well as membrane permeability for 86Rb and 51Cr-labelled intracellular proteins were investigated. Heat-treated macrophages were tested for their ability to phagocyte staphylococci and for reduction of nitroblue-tetrazolium. Moreover the effect of microwave whole-body hyperthermia on rabbit phogocytic cells was studied in vivo. Ion transport to macrophages was stimulated by both intensive (43 degrees C) and moderate (40 degrees C) hyperthermia. On the other hand exposition of the cells to 43 degrees C led to pronounced release of 86Rb and 51Cr from prelabelled cells. NBT reduction was generally decreased in macrophages exposed to 43 degrees C and increased in macrophages kept at 40 degrees C. Clearance of 32P-labelled staphylococci from peripheral blood of microwave-irradiated rabbits diminished when animals were exposed to microwave hyperthermia for f or 7 days (2 hours daily).

Stimulatory effect of Propionibacterium granulosum KP-45, glucan and pyran copolymer on the activity of natural killer (NK) cells in murine lungs.

Propionibacterium granulosum KP-45, glucan and pyran copolymer stimulated the elimination of 75Selenomethionine-labelled 3LL tumor cells from murine lungs, as measured 4 hr after intravenous injection of these cells into 16- to 25-week-old B6DF1 mice. This effect was most pronounced 4 to 6 days following intravenous administration of the above biological response modifiers and disappeared 6 to 8 days later. Intraperitoneal injection of all three agents produced only insignificant stimulation results. Spontaneous clearance of 3LL cells from lungs of 8-week-old B6DF1 mice was significantly more effective than in animals over 16 weeks old. Cyclophosphamide suppressed the elimination of tumor cells from lungs in both young and older mice and neutralized the stimulatory effect of P. granulosum KP-45 and glucan. The results suggest that the effector cells responsible for the clearance of radiolabelled 3LL cells from lungs of B6DF1 mice are at least similar to natural killer (NK) lymphocytes.

The search for an influence of whole-body microwave hyperthermia on anti-tumor immunity.

The effect of microwave whole-body hyperthermia was examined on the takes of tumor after i.v. administration of the tumor single cell suspension. It was shown that the longer the exposure to hyperthermia treatment the higher the number of lung nodules obtained by injection of the same dose of tumor cells. Also, the expression of contact hypersenstivity to oxazolone was strongly inhibited after hyperthermia treatment. It was documented that it is possible to transfer the diminished resistance to tumor with bone marrow, whereas impaired responsiveness to oxazolone was transferred with spleen cells or thymocytes.

Effect of whole-body microwave hyperthermia on delayed cutaneous hypersensitivity in tumor-bearing mice.

Tumor-bearing and normal BALB/c mice were examined for their responsiveness to contact-sensitizing agent oxazolone after whole-body microwave hyperthermia treatment. The effective therapy with hyperthermia prolonging mean survival time of the animals and causing tumors regression resulted in strongly pronounced hyporeactivity to oxazolone. The impaired responsiveness remained unchanged until death of the tumor-bearing animals. In the case of tumor-free animals delayed cutaneous response returned to normal values within 8-12 days after hyperthermia treatment. It is documented that the longer exposure to hyperthermia sessions the more pronounced depression of reactivity to oxazolone occurs.

Beneficial response of local immunotherapy with Propionibacterium granulosum KP-45 in combined treatment of inflammatory breast carcinoma.

Nine 44- to 67-year-old patients with inflammatory breast carcinoma were treated over 2 weeks with intratumoral injections of Propionibacterium granulosum KP-45 (KP). This period of immunotherapy was succeeded by four courses of chemoimmunotherapy (FAC: 5-fluorouracil, adriamycin, and cyclophosphamide + intratumoral KP). Inflammatory symptoms disappeared in three patients during immunotherapy and in the remaining six patients during the following chemoimmunotherapy. Finally, 3 to 4 months after starting the therapy, all nine patients were free from inflammatory symptoms and it became possible to perform radical (seven cases) or simple (two cases) surgery. Thereafter routine therapy (radiotherapy, fractionated dose of 5500 R, followed by 10 FAC courses + single injections of KP for each FAC course) was used. After 19 to 32 months observation time all patients are still in complete remission with no local recurrences. Only one patient showed distant metastases during the observation period.

A 2.5-year follow-up of local immunotherapy of advanced stomach and intestinal adenocarcinoma with Propionibacterium granulosum.

Intratumoral injections of 10 mg cells walls from Propionibacterium granulosum strain KP-45 (PG) were applied in 14 patients with advanced, metastatic stomach (five cases) and colorectal (nine cases) adenocarcinoma. Each patient had his own "twin" control. All patients received no other anticancer treatment, except analgetics and/or palliative surgery. Treatment with PG resulted in partial regression of tumors accompanied by improvement of the clinical state of these patients as well as the reappearance of normal values in blood count biochemical parameters. In each pair of twin cases, survival of the PG-treated patient was longer than the untreated control. The mean survival of PG-treated patients was 23.5 months (4 of 14 patients being still alive after 2.5 years follow-up), while all control patients died with a mean survival period of 6.4 months. The difference between these two patient groups of about 17 months is significant (p greater than 0.01).

The in vivo cytostatic effect induced by Propionibacterium granulosum on murine tumor cells.

The cytostatic action of Propionibacterium granulosum was studied in a mouse sarcoma in vivo. Kinetic analysis of tumor cells 28 days after tumor implantation and systemic immunotherapy showed that the cell cycle time was identical in both treated and untreated tumors. P. granulosum treatment resulted in a marked prolongation of the S phase and shortening of the G1 phase of the cell cycle. A pronounced drop in the number of labeled interphases and the reduction of the growth fraction were observed in tumors obtained from mice given injection of P. granulosum. Cloning efficiency of tumor cells from P. granulosum treated animals was quantitatively similar to that of control animals and only differences in the size of lung colonies were observed.

Transrectal hyperthermia as palliative treatment for advanced adenocarcinoma of prostate and studies of cell-mediated immunity.

During a fifteen-month period, 15 patients with progressive adenocarcinoma of the prostate (CaP) were treated with transrectal microwave hyperthermia (TRHT). There were 5 Stage T4 and 11 Stage T3 patients including 6 patients with skeletal metastases. Nine of the patients had severe and 6 had moderately severe signs and symptoms of CaP. TRHT was given six times at 2,450 MHz with temperature controlled at 43.5 degrees for thirty minutes. Cell-mediated immunity tests were performed before TRHT and at two, four, and six months post-therapy. The results of these tests were compared with those of 15 patients with benign prostatic hyperplasia (BPH) treated with the same TRHT and with 30 untreated normal volunteers. TRHT was well tolerated with mild acute toxicity noted in 3 patients (20%). Of the 15 patients treated, 2 (13%) showed scintigraphic evidence of regression of bone metastases. Five patients survived more than five years since treatment, and in 3 patients there was no evidence of CaP. A decrease of marked or moderate degree in signs and symptoms of CaP was noted in 8 patients (53%). The results of cell-mediated immunity tests were of interest. The 15 CaP patients prior to TRHT had lower OKT4/OKT8 ratio, lower PHA transformation index, and lower Con-A induced T-cell suppressor activity as compared with the 15 BPH patients and 30 healthy volunteers, who had normal immune parameters (p < 0.01). Following TRHT there was a significant increase in the monitored immune parameters noted in the 15 CaP patients (p < 0.01). This immune stimulation peaked at two months and gradually decreased to near pretreatment levels at six months. In the 15 BPH patients post-TRHT changes in immune tests were less apparent. The results of this small study, if confirmed, could be of clinical importance in patients with advanced prostate cancer.

Susceptibility of Pseudomonas aeruginosa to a photodynamic effect of the arginine hematoporphyrin derivative.

The photodynamic effect of the arginine hematoporphyrin derivative (HpD-Arg(2)) on the growth of selected Pseudomonas aeruginosa clinical isolates is described as compared to the effect of hematoporphyrin derivative (HpD). Growth inhibition of HpD-Arg(2) of three Gram-positive strains (Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus) and five laboratory strains of Gram-negative bacteria (Escherichia coli, Proteus vulgaris, P. aeruginosa, Klebsiella pneumoniae and Enterobacter cloacae) was observed as well. Serratia marcescens strain was resistant to light-activated HpD-Arg(2) or HpD. The antimicrobial activity of light-activated HpD-Arg(2) against P. aeruginosa may offer an additional option in the anti-pseudomonal therapy of wounds.

Effects of peptides cleaved from human fibrinogen by plasmin on rabbit kidney cells in culture.

Low molecular weight fibrinogen degradation products (LMW-FDP) containing a mixture of dialysable peptides cleaved from human fibrinogen by plasmin are cytotoxic to an established line of rabbit kidney cells and to primary cultures of rabbit kidney cells. The presence of LMW-FDP in a concentration of 50 micrograms/ml during the cell cultivation caused a considerable release of 51Cr from prelabelled cells and inhibited 3H-thymidine and 86Rb uptake. Among three isolated peptides of established primary structure only one, 6D: Ser-Gln-Leu-Gln-Lys-Val-Pro-Pro-Glu-Trp-Lys, induced a significant effect, i.e. it enhanced 3H-thymidine incorporation. Two others, 6A: Ala-Arg-Pro-Ala-Lys and 6E: Thr-Ser-Glu-Val-Lys, did not influence the examined parameters. Hence other components of LMW-FDP must be assumed to be responsible for the cytotoxic effect on kidney cell cultures.

Prevention of hepatic metastases by liver lectin blocking with D-galactose in colon cancer patients. A prospectively randomized clinical trial.

76 colon adenocarcinoma patients (stages 1-3, NO, MO) were enrolled into a prospectively randomized clinical trial, 39 patients were perioperatively treated with D-galactose-(therapy group: 1.9 g/kg BW and day) or D-glucose-containing electrolyte infusions (control group: n = 37). There were no cases of perioperative mortality. The complication rate was 17.1% (therapy group: 15.3%; control group: 18.9%). Since tumor staging and grading were equally distributed for therapy and control groups, a non-stratified statistical analysis yielded a) significantly reduced hepatic metastases and b) improved overall survival for patients of the therapy group. Stage-dependent analysis demonstrated that stage 3 carcinoma patients of the treatment group developed significantly less hepatic metastases as compared to patients of the control group.

Prevention of hepatic metastases by liver lectin blocking with D-galactose in stomach cancer patients. A prospectively randomized clinical trial.

80 stomach adenocarcinoma patients (T1-3, NO, MO) were enrolled into a prospectively randomized clinical study. 40 patients were perioperatively treated with D-galactose (treatment group: 1.9 g/kg BW and per day) or D-glucose-containing electrolyte infusions (control group: n = 40). Perioperative mortality was low (3.7%), complication rate was 11.2% (treatment group: 12.5%; control group: 10%). Since tumor staging and grading were similarly distributed for treatment and control groups, a non-stratified statistical analysis yielded a) significantly reduced hepatic metastases b) significantly improved overall survival for patients of the treatment group. A statistically significant effect on survival free of hepatic metastases and overall survival could be demonstrated for stage T3 patients of the verum group, as compared to patients of the control group.

Local immunotherapy with propionibacterium granulosum KP-45 in advanced breast cancer.

50 patients with advanced metastatic breast cancer were matched into 25 "twin" pairs. In each pair, one "twin" received chemotherapy (FAC) and the other received chemoimmunotherapy (FAC + intratumorally Propionibacterium granulosum KP-45 (PG)). The therapeutic effects of this 2-year follow-up study were carefully documented and analysed. The mean survival time of FAC + PG-treated patients was about 17 months as compared to 8.5 months in FAC controls. PG-therapy responders showed increasing values of T-lymphocytes in peripheral blood, as well as higher blast transformation indices than nonresponders and controls. The skin reactivity to PHA, Distreptase, and Tuberculin was markedly enhanced in the FAC + PG-group. The incidence rate of hematological and/or infectious complications was significantly lower in PG-treated patients than in the controls. Local PG-immunotherapy was proven beneficial in advanced breast cancer when combined with FAC-chemotherapy, providing better toleration of chemotherapy and lower risk of myelosuppression and infections.

Thermoimmunotherapy of advanced neoplasms. A concept and preliminary results.

A rationale for the use of local hyperthermia (LH) in cases of advanced neoplasms as well as indications for its therapeutic application are discussed. LH applied alone is of limited use, only about 50% of patients with advanced neoplasms benefit from this therapy, with a complete remission (CR) rate of 10-15%. Thus LH should be considered as a supplementary form of cytoreductive therapy and it should be combined with other antineoplastic modalities. Clinical studies on thermoimmunotherapy (a combination of LH with a potent immunomodulator--Propionibacterium granulosum KP-45) were undertaken in advanced prostatic and breast cancer. The results now available after 1-2 years observation periods indicate that the number of responders is higher in groups treated with thermoimmunotherapy than in those with LH only. Although the observations are still in progress, the authors suggest that thermoimmunotherapy offers a real alternative for advanced neoplasms and opens a new field for experimental and clinical investigations.

Acceleration of the development of benzopyrene-induced skin cancer in mice by microwave radiation.

Development and growth of skin cancer may be affected by various physical and chemical factors present in human environment. Of these factors electromagnetic radiation of radio- and microwave spectra are among the most common. In the present study Balb/c mice were exposed to chemical carcinogen, 3,4-benzopyrene, painted on the skin every 2nd day for a total of 6 months, and simultaneously irradiated with athermal (5 mW/cm2) or subthermal (15 mW/cm2) doses of 2,450 MHz microwaves. The other group of animals was preirradiated with microwaves at 10 mW/cm2 power level for 1, 2, or 3 months and then treated with benzopyrene, as above. Control mice were exposed for 6 months to benzopyrene, resulting in the development of baso- or spinocellular skin carcinoma within approximately 9 months, and sham-irradiated with microwaves. The growth of the tumour was assessed according to a self-designed 7-range macroscopic scale, supported by microscopical examinations of skin sections. All protocols of microwave irradiations resulted in a significant acceleration of the development of benzopyrene-induced skin cancer and in shortening of life span of the tumour-bearing hosts. This effect seemed to be dose-dependent since subthermal doses (15 mV/cm2) and longer (3 months) expositions to microwaves were more efficient as compared to athermal doses (5 mW/cm2) and shorter preirradiations. In addition, low-level, long-lasting exposure to microwaves led to a marked suppression of delayed hypersensitivity of mice treated with benzopyrene, as assessed by their reactivity to dinitrofluorbenzene (DNFB). It is suggested that the observed co-carcinogenic effect of microwave radiation may, at least in part, result from the inhibitory action of microwaves on cellular immune reactions of exposed animals.

Autoradiographic analysis of protein synthesis and measurements of nuclear volume in WISH cell cultures irradiated with 3 GHz electromagnetic radiation.

WISH cell cultures were irradiated 24 h after passage with 3 GHz microwaves for 30 min at intensites of 5 or 20 mW cm-2. After times from exposure of 1, 4, 8, 12 and 24 h the cells were incubated with 5 muCi ml-2 of 3H-glycine and autoradiographs were made using stripping film. The incorporation of the amino acid was measured for four groups, 0-10, 10-20, 20-40 and above 40 grains per cell. Karyometric analysis of irradiated cell cultures was performed 1, 4, 8, 12, 24 and 48 h after exposure. In cultures irradiated at an intensity of 20 mW cm-2, inhibition of protein synthesis with lowering of nuclear volume and enlargement of nucleoli was found one hour after irradiation. This was followed by increase in glycine incorporation 12 and 24 h after irradiation, with enlargement of nuclei. In cultures irradiated at an intensity of 5mW cm-2 stimulation of protein synthesis and enlargement of nuclei were seen 24 h after irradiation. These results suggests that low intensity microwaves stimulate metabolism of cells grown in vitro, although at higher intensities the phase of stimulation is preceded by a short period of inhibition in the culture viability.

Comparison of preoperative embolization followed by radical nephrectomy with radical nephrectomy alone for renal cell carcinoma.

A series of 474 patients with renal cell carcinoma (RCC), who had radical nephrectomy during a period of 15 years, was studied to assess the prognostic significance of various pathologic parameters (tumor stage [pT], lymph node status, metastasis, tumor grade, venous involvement) and value of preoperative embolization of renal artery. There were: 20 (4%) pT1, 204 (43%) pT2, 245 (52%) pT3, and 5 (1%) pT4 patients. All 474 patients underwent nephrectomy including a group of 118 (25%) patients (24 pT2, 90 pT3, and 4 pT4) who underwent preoperative embolization of the renal artery. To compare treatment outcomes in embolized patients with RCC, a group of 116 (24%) nonembolized patients with RCC was selected. This group was matched for sex, age, stage, tumor size, and tumor grade, with the embolized patients (p<0.01). All important prognostic factors were studied as to their influence on survival by the treatment group. The overall 5- and 10-year survival was 62% and 47%, respectively. The 5- and 10-year survival rates were significantly better (p<0.01) for patients with pT2 than for those with pT3 tumors (79% vs. 50% and 59% vs. 35%, respectively). Involvement of regional lymph nodes (N+) was an important prognostic factor for survival in patients with pT3 tumors. The 5-year survival for pT3 N+ was 39%, compared with 66% in those with pT3N0 (p<0.01). Preoperative embolization was also an important factor influencing survival. The overall 5- and 10-year survival for 118 patients embolized before nephrectomy was 62% and 47%, respectively, and it was 35% and 23%, respectively, for the matched group of 116 patients treated with surgery alone (p = 0.01). The most important finding of this study was an apparent importance of preoperative embolization in improving patients' survival. This finding needs to be interpreted with caution and confirmed in a prospective randomized trial.

Alteration of diurnal rhythms of blood pressure and heart rate to workers exposed to radiofrequency electromagnetic fields.

BACKGROUND: In previous studies we found measurable effects on variability of heart rate and on blood-pressure parameters of workers exposed to radiofrequency electromagnetic fields (EMF)compared with a control population, but none of the effects could be assigned clinical significance. In general, the obtained results strongly suggested that dysregulation of the autonomic control of the circulatory system was occurring. Therefore, it seemed logical that analysis of diurnal rhythms of blood pressure and heart rate, on the basis of data from 24 h recordings, might further support the above hypothesis. OBJECTIVE: The aim of this study was to determine the course of diurnal rhythms of blood pressure and heart rate in a group of workers exposed to various intensities of radiofrequency electromagnetic fields. METHODS: In the study we used 61 healthy workers (aged 30-50 years) who had been exposed to radiofrequency EMF of 0.738-1.503 Mhz and 42 healthy workers at radio-line stations (aged 28-49 years), who had not been exposed to EMF occupationally. The work patterns of these two groups were identical (12 h day working shift, 24 h interval, 12 h night shift and then 48 h rest). During the second day of the rest period 24 h ambulatory blood pressure (ABP) was recorded. For analysis of diurnal rhythms the group of exposed workers was divided into two subgroups: group A of 38 subjects exposed to low intensities of radiofrequency EMF (20-180 V/m) and group B of 23 subjects exposed to high intensities of radiofrequency EMF (200-550 V/m). Parameters of diurnal rhythms of blood pressure and heart rate (acrophase, amplitude and mean) were calculated by performing a least-square fit of a 24 h cosinor (single cosinor analysis) at P < 0.05. RESULTS: Healthy men aged 28-49 years, working on a pattern of 12-24-12-48 h, exhibited typical, well-preserved diurnal rhythms of blood pressure and heart rate with two maxima (at about 1400 and 1700-1800 h) and one minimum (at about 0200-0400 h). For workers exposed to radiofrequency EMF we noted a significant lowering of the amplitudes of rhythms of blood pressure and heart rate (P < 0.01) and a shift of the acrophase to an earlier time (1100-1200 h; P < 0.05). These changes were more pronounced among workers exposed to high intensities of radiofrequency EMF. CONCLUSIONS: Occupational exposure to radiofrequency EMF can result in changes of the diurnal rhythms of blood pressure and heart rate with lowering of their amplitudes and a shift of the acrophase. The clinical relevance of the present finding needs to be investigated in further studies.