Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

Trial of BCG immunotherapy in the treatment of resectable squamous cell carcinoma of the bronchus (stages I and II)

Summary Thirty-nine patients with squamous cell carcinoma of the bronchus were randomized into two groups after radical surgery. The first group of 18 patients received 75 mg of living BCG on a scratched area once a week; the second group of 21 patients received no further treatment. After 2 years 3 patients of the group treated with BCG are dead and 1 is in relapse; 8 patients of the control group are dead and 2 are in relapse.In this study the hope of survival for more than 2 years for patients with squamous cell carcinoma of the bronchus is 66% in the group of patients treated with BCG, and 38% in the control group. From these results we can see the beneficial effect of BCG as a complementary treatment of surgery for patients with squamous cell carcinoma of the bronchus.     

Equine sarcoid: BCG immunotherapy compared to cryosurgery in a prospective randomised clinical trial

Summary A total of 30 horses with single or multiple sarcoid tumors of the skin were randomly divided into three treatment groups: (i) cryosurgical treatment, (ii) intralesional immunotherapy with a live BCG vaccine, (iii) intralesional immunotherapy with a BCG cell wall preparation. Complete tumour regression was obtained in all 10 crysurgically treated horses, in 6 of 10 live BCG treated horses, and in 7 of 10 BCG cell wall treated horses. One live BCG and 2 BCG cell wall treated horses showed partial tumour regression of more than 50% of the tumour area. Eleven horses with sarcoid tumours were not eligible for random allocation in the trial because unfavourable site or size of the tumour precluded cryosurgical treatment. These animals were treated with BCG cell wall vaccine except for 1 animal, which was treated with live BCG. In 4 cases this treatment was combined with cytoreductive surgery of the tumour. In this prognostically unfavourable group 8 animals showed complete tumour regression and 3 animals did not respond.Regression after BCG immunotherapy appeared to correlate with size (larger tumours worse response) and localization of the sarcoid (less favourable results in the limb), and increase in peripheral blood leucocytes after the first injection. Horses with a positive delayed type hypersensitivity reaction to PPD before the start of treatment showed a tendency to more favourable prognosis than PPD negative horses. No correlation was present between regression and single or multiple presence of sarcoids, increase in body temperature after injection of BCG and the formation of specific antibodies to BCG. None of the cured animals have shown tumour recurrence 3 to 40 months following treatment.Animals were maintained under the guidelines prescribed by the Faculty of Veterinary Medicine, State University Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation)     

Regional immunotherapy in resectable squamous cell lung carcinoma

Summary Patients with resectable squamous cell carcinoma were randomly allocated after surgery to receive either no further treatment (57 patients) or a single intrapleural injection of BCG (61 patients). No significant improvement in survival was observed in patients treated with BCG, even when their disease was staged as N₀. These was a slight trend for the recurrence rate to be lower in patients classed as N₀, but this was not significant.     

Immunotherapy of bovine ocular squamous cell carcinoma by repeated intralesional injections of live bacillus Calmette-Guérin (BCG) or BCG cell walls

Summary Thirty cows of the Dutch Friesian and the Maas-Rijn-Ijssel breed with histologically confirmed ocular squamous cell carcinoma were treated by repeated intralesional injection of live bacillus Calmette-Guérin (BCG) (n = 14) or a BCG cell-wall vaccine (n = 16). Complete regression of the primary tumour was observed in 64% and 57% of the animals respectively. In the 2-year follow-up period there was no recurrence of primary tumours. This sharply contrasts with the recurrence frequency (40%–50%) after complete remission induced by a single intralesional injection with BCG, observed in an earlier study. In 1 animal a new primary tumour developed. At necropsy metastases were present in 33% of the treated animals: in 3 of 17 animals that showed complete regression of the primary tumour and in 7 of 13 animals with partial regression or progressive disease. This did not differ significantly from results obtained after a single treatment (27%). Delayed-type hypersensitivity toM. bovis purified protein derivative (PPD) was more persistent in animals showing regression of the primary tumour than in non-responding animals. Of the animals with a positive PPD response 6 months after treatment, 79% showed tumour regression. Regression was observed in only 28% of the animals not responding to PPD after the same period of time. In conclusion: (a) recurrence of the primary tumour was not observed after repeated BCG treatment; (b) the frequency of metastases was not decreased compared to results obtained with a single treatment; (c) regression was correlated with a positive delayed-type hypersensitivity reaction to PPD (P <0.05) 6 months after treatment; (d) no significant differences were observed when the clinical results of treatment with live BCG and the BCG cell wall vaccine were compared.     

Crystalline cell surface layer of Mycobacterium bovis BCG.

A paracrystalline surface layer (S layer) was found as the outermost layer of the cell wall of five Mycobacterium bovis BCG strains. An oblique arrangement of the subunits in the S layer was only clearly seen in thin-sectioned and shadowed preparations, and the unit constant was about 5.5 nm.     

Intralymphatic administration of BCG in melanoma patients

Summary From November 1973 to December 1974, 20 patients with advanced malignant melanoma were treated with BCG given by intralymphatic route at the Cancer Institute of Milan. The lyophilized Pasteur BCG was used. Patients were treated with a single dose ranging from 0.2–80 mg. Patients' performance status was never severely impaired.The most frequent side effects were fever, lymphangitis, and lymph node enlargement.Variations were observed in white cell count, ERS and immunoglobulins; in no case did we find evidence of liver toxicity or tumor growth enhancement. It is concluded that the intralymphatic route is a safe way of administrating BCG.     

Chemoimmunotherapy of stage III breast carcinoma with BCG and a live allogeneic tumor cell vaccine

Summary Ten women with metastatic breast carcinoma were treated with CMF, BCG, and a live TCV in a phase I study to determine whether viable allogeneic tumor cells can safely be given to women receiving adjuvant chemotherapy. Side effects of fatigue, nausea, vomiting, alopecia, and myelosuppression were attributable to chemotherapy. Side effects of BCG therapy were malaise, fever, and pruritis at the injection site, as previously described. Four patients developed hepatitis B due to viral contamination of the human agamma serum in which the tumor cells were grown. This complication has now been eliminated by omitting human sera, and hence the possibility of hepatitis B virus, from tumor cell growth media. No local tumor growth was observed in any patient. Results of this study show that live TCV is safe for patients receiving chemotherapy, and could be tested in combination with BCG and chemotherapy for control of micrometastases from breast cancer following mastectomy. Abbreviations used in this paper: BCG, bacillus Calmette-Guérin; CMF, cyclophosphamide, methotrexate, and 5-fluorouracil; DNCB, dinitrochlorobenzene; PPD, purified protein derivative; TCV, tumor cell vaccine(s).     

Intratumoral BCG and Corynebacterium parvum therapy of canine mammary tumours before radical mastectomy

Summary In two parallel studies, bitches with mammary tumour received single intralesional injections of BCG (1 mg: 10⁷ living bacteria) and Corybacterium parvum (10⁹ killed bacteria) (53 bitches) or C. parvum alone (129 bitches) at the same dosage. Control groups received injections, following the same protocol, of 1 ml BCG suspension medium diluted in saline in the first study (51 bitches) or no injections at all (120 bitches in the second study).A block dissection, including mammary tumours, adjacent mammary glands, and regional lymph nodes, was performed 2 weeks later in all animals. On the basis of histologically confirmed malignant tumours, 48 bitches (25 treated by immunotherapy and 23 controls) in the first study and 67 bitches (30 treated by immunotherapy and 37 controls) in the second study remained for postsurgical follow-up.The clinical tolerance of the treatment was generally good. No significant differences were found in cumulative survival rates between treated and control group in either studies.     

Post-operative intrapleural BCG in lung cancer: A 5-year follow-up report

Summary Of a total study group of 99 patients 56 received intrapleural BCG in three different dosages (5 patients 16×10⁶, 25 patients 32×10⁶, and 26 patients 64×10⁶ culturable particles) following resection treatment for lung cancer. This study group was compared with an historical control population, very closely matched with regard to age, sex, stage of disease, histology and type of operation.Although the clinical condition of the patients selected for BCG treatment was above average, no survival benefit ensued from the intrapleural BCG. On the contrary, disease-free survival in BCG receivers with stage I and II squamous cell carcinoma was shortened significantly at 2 years follow-up due to the earlier appearance of local recurrences, with the same tendency still present after 5 years. This observation suggests an enhancement of tumour growth by the intrapleural BCG treatment. This alarming phenomenon could be a warning to clinicians when planning clinical immunotherapy trials to be aware of the potentially deleterious effects of such treatment.     

Evidence for apoptosis in non-small cell lung carcinoma. Relationship with cell kinetics and prognosis

OBJECTIVE: To investigate cell death via apoptosis in non-small cell lung carcinoma (NSCLC) and its correlation with proliferative indices and follow-up. STUDY DESIGN: In 38 cases of NSCLC (21 squamous cell carcinomas and 17 adenocarcinomas) we analyzed apoptosis by nuclear morphology and in situ DNA fragmentation end labeling and the cell kinetics by an autoradiographic method with tritiated thymidine and by immunohistochemistry with anti-proliferating cell nuclear antigen (anti-PCNA) antibodies. We also evaluated mitotic frequency. Apoptotic index (AI) was correlated with the thymidine and PCNA labeling indices (T-LI and PCNA-LI, respectively) and with the mitotic index. RESULTS: The percentage of proliferating cells (T-LI range, 0.1-20.1%; PCNA-LI range, 0-14.7%) was generally considerably higher than that of apoptotic cells (range, 0-8%) and of mitotic cells (range, 0.1-1%). Survival at six years was significantly higher in patients with high levels of apoptosis and low T-LI values. CONCLUSION: AI and T-LI are independent and very useful prognostic factors in NSCLC. A high percentage of proliferating cells in terms of T-LI correlates with poor prognosis, whereas a high AI indicates a favorable outcome.     

Localization and expression of inducible nitric oxide synthase in patients after BCG treatment for bladder cancer

Treatment with Bacillus Calmette Guerin (BCG) bladder instillations is an established treatment modality for superficial urinary bladder cancer and carcinoma in situ (CIS), but the anti-tumor mechanisms following BCG instillations remain largely unknown. Previous data show increased nitric oxide (NO) concentrations in the urinary bladder from patients treated with BCG suggesting that NO-formation may be involved in the BCG mediated effect. In the present study we evaluated 11 patients with urinary bladder cancer who had received BCG treatment and 11 tumor free control subjects. We performed immunohistochemistry, Western blot and real-time polymerase chain reaction (PCR) on bladder biopsies to establish inducible nitric oxide synthase (iNOS) protein levels and localization as well as iNOS mRNA expression. Endogenous NO formation in the bladder was also measured. In patients with bladder cancer who had received BCG treatment iNOS-like immunoreactivity was found in the urothelial cells but also in macrophages in the submucosa. Furthermore, endogenously formed NO was significantly increased (p<0.001) in the BCG treated patients and they had a ten-fold increase in mRNA expression for iNOS compared to healthy controls (p=0.003). In conclusion iNOS was found to be localized to the urothelium and macrophages underlying it. Our study also confirms elevated levels of endogenously formed NO and increased mRNA expression and protein levels for iNOS in patients with BCG treated bladder cancer. These data further support the notion that NO may be involved in the anti-tumor mechanism that BCG exerts on bladder cancer cells.     

Mechanisms of BCG action

Summary Donor mice were treated IV with BCG and after various time intervals the spleens from these animals were injected into syngeneic recipients which were simultaneously challenged with an allogeneic tumour. The spleen cells from the BCG-treated donors, but not untreated donors, conferred on the recipients an ability to induce a potentiated CMC reaction against the tumour. The transference of BCG-induced potentiating activity could not be explained by the transference of viable BCG organisms, but was mediated by a cell that was anti-Thy.1-sensitive, silica-resistant, plastic-nonadherent, and nylon wool-adherent, and was sensitive in vivo to anti-thymocyte serum but resistant to hydrocortisone. By the use of congenic strains of mice that differed at the Thy.1 allele, it was shown that the cells responsible were not precursors of the cytotoxic lymphocytes but were cells that produced an amplification of the response of the recipient host's precursor cytotoxic T cells.     

Experimental screening of BCG preparations produced for cancer immunotherapy: Safety and immunostimulating and antitumor activity of four consecutively produced batches

Summary Four consecutively produced batches of Bacillus Calmette-Guérin (BCG) especially intended to be used for cancer immunotherapy were investigated for consistency of the vaccine. Each batch was investigated directly after production of the vaccine, so that the four batches were not tested simultaneously. The activity of the four batches was investigated in general safety assays, immunostimulation assays, and two different tumor models.General safety assays showed dose-dependent growth retardation and increased serum glutamic pyruvic transaminase activity in mice, and a long-lasting temperature rise in rabbits after IV administration of the BCG preparations. In a skin reactivity assay, reactions were found acceptable for all preparations when compared with a reference batch.The results of the immunostimulation and antitumor studies can be summarized as follows. All four batches induced a specific delayed-type hypersensitivity reaction to PPD, indicating the induction of cell-mediated immunity. A stimulating effect on lymphoreticular organs was concluded from increased spleen weight and enhanced cell proliferation in draining lymph nodes. Enhanced macrophage function (phagocytosis and killing of bacteria) was demonstrated by an increased resistance to Listeria monocytogenes. YAC lymphoma target cells were killed nonspecifically by BCG-activated peritoneal exudate cells (PEC), indicating the induction of natural killing activity by BCG.Intralesional injection of BCG induced tumor regression in the guinea pig line 10 hepatocellular carcinoma, followed by immunity to the line 10 tumor. In the murine 5D04 squamous cell carcinoma, BCG had no effect on the primary tumor. However, IV-injected BCG resulted in a decreased number of lung metastases.In general, the four consecutively produced batches showed similar safety and activity in the immunostimulation assays and antitumor activity. Since only minor differences between the batches were found, which can also be attributed to the variation in experimental conditions common to biological assays, it is concluded that the vaccine batches produced show an acceptable consistency.Abbreviations used BCG, Bacillus Calmette-Guérin; C. parvum, Corynebacterium parvum; c. p., culturable particles; IU, international unit; PPD, purified protein derivative; PEC, peritoneal exudate cells     

Chronobiological analysis of thyroxine action on cell proliferation in the hypotetraploid strain of Ehrlich's ascitic tumor

Experiments on white random-bred male mice were made to study the effect of L-thyroxine on cell proliferation of the hypotetraploid strain of Ehrlich's ascites carcinoma. It was shown that prolonged thyroxine administration (during 6 days of carcinoma growth) lead to synchronization of cell proliferation and the maximum values of the mitotic index was found 3 hours earlier then in the control experiments. At the same time thyroxine did not exert any noticeable effect on the average daily magnitudes of the number of DNA-synthesizing cells and did not change the pattern of modulations in the radioactive index. The changes in the mitotic index and radioactive index were asynchronous in control and experimental animals. Analogous results were found for hyperdiploid strain of Ehrlich's ascites tumor. Ploidy of cells did not influence the tipe rhythms of the cell proliferation and its reaction on the action of thyroxine.     

EGF induces cell cycle arrest of A431 human epidermoid carcinoma cells

The human carcinoma cell line A431 is unusual in that physiologic concentrations of epidermal growth factor (EGF) inhibit proliferation. In the presence of 5-10 nM EGF proliferation of A431 cells is abruptly and markedly decreased compared to the untreated control cultures, with little loss of cell viability over a 4-day period. This study was initiated to examine how EGF affects the progression of A431 cells through the cell cycle. Flow cytometric analysis of DNA in EGF-treated cells reveals a marked change in the cell cycle distribution. The percentage of cells in late S/G2 increases and early S phase is nearly depleted. Since addition of the mitotic inhibitor vinblastine causes accumulation of cells in mitosis and prevents reentry of cells into G1, it is possible to distinguish between slow progression through G1 and G2 and blocks in those phases. When control cells, not treated with EGF, are exposed to vinblastine, the cells accumulate mitotic figures, as expected, and show progression into S, thus diminishing the number of cells in G1. In contrast, no mitotic figures are found among the EGF-treated cells in the presence or absence of vinblastine, and progression from G1 into S is not observed, as the number of cells in G1 remains constant. These results suggest that there are two EGF-induced blocks in cell cycle transversal; one is in late S and/or G2, blocking entry into mitosis, and the other is in G1, blocking entry into S phase. After 24 hours of EGF treatment, DNA synthesis is reduced to less than 10% compared to untreated controls as measured by the incorporation of [3H]thymidine or BrdU. In contrast, protein synthesis is inhibited by about twofold. Although inhibition of protein synthesis is less extensive, it occurs 6 hours prior to an equivalent inhibition of DNA synthesis. The rapid decrease in protein synthesis may result in the subsequent cell cycle arrest which occurs several hours later.     

A possible mechanism of intravesical BCG therapy for human bladder carcinoma: involvement of innate effector cells for the inhibition of tumor growth

Intravesical bacillus Calmette-Guerin (BCG) therapy is considered the most successful immunotherapy against solid tumors of human bladder carcinoma. To determine the actual effector cells activated by intravesical BCG therapy to inhibit the growth of bladder carcinoma, T24 human bladder tumor cells, expressing very low levels of class I MHC, were co-cultured with allogeneic peripheral blood mononuclear cells (PBMCs) with live BCG. The proliferation of T24 cells was markedly inhibited when BCG-infected dendritic cells (DCs) were added to the culture although the addition of either BCG or uninfected DCs alone did not result in any inhibition. The inhibitory effect was much stronger when the DCs were infected with live BCG rather than with heat-inactivated BCG. The live BCG-infected DCs secreted TNF-α and IL-12 within a day and this secretion continued for at least a week, while the heat-inactivated BCG-infected DCs secreted no IL-12 and little TNF-α. Such secretion of cytokines may activate innate alert cells, and indeed NKT cells expressing IL-12 receptors apparently proliferated and were activated to produce cytocidal perforin among the PBMCs when live BCG-infected DCs were externally added. Moreover, depletion of γδ T-cells from PBMCs significantly reduced the cytotoxic effect on T24 cells, while depletion of CD8β cells did not affect T24 cell growth. Furthermore, the innate effectors seem to recognize MICA/MICB molecules on T24 via NKG2D receptors. These findings suggest the involvement of innate alert cells activated by the live BCG-infected DCs to inhibit the growth of bladder carcinoma and provide a possible mechanism of intravesical BCG therapy.     

Kinetics of cell replication in the uterine cervix. VI. Loci of DNA-synthesizing cells and arrested mitoses in the basal-cell layer

The mode of proliferation in the basal-cell layer of the squamous cervical epithelium was investigated in C57B1 mice with the aid of 3H-thymidine and vincristine. Six hours after vincristine injection and two hours after thymidine injection, 33% of the basal cells were in DNA synthesis and 12% in mitosis. Of these, only 23% of the cells in DNA synthesis and 45% of those in mitosis were found as single cells. The remaining cells proliferated in clusters of two or more cells. As many as 59% of the cells in DNA synthesis and 30% of those in mitosis occurred in colonies of three or more consecutive cells, indicating that multicell clustering is a rather common pattern of basal cell proliferation. Multicell loci of DNA-synthesizing cells occurred contemporaneously with but independently of multicell loci of mitotic cells (the loci were nonconsecutive). Basal-cell replication in the squamous cervical epithelium thus appears to be an organized process of cell renewal.     

Chlorpromazine inhibits the mitotic index, cell number, and formation of mouse blastocysts, and delays implantation of CBA mouse embryos

Chlorpromazine, administered to pregnant CBA mice 56 h after copulation in single doses of 10 or 15 mg/kg bodyweight, inhibited the compaction of embryos, formation of blastocysts, and reduced the mitotic index and cell number of embryos 86 h after copulation but did not adversely influence their viability or induce structural chromosomal aberrations. Blastocyst formation was more severely affected than embryo compaction. When 86-h embryos were treated with chlorpromazine (10 or 15 mg/kg) and subsequently cultured for 120 h, there was delayed hatching from the zona pellucida, delayed attachment to the culture dish, outgrowth of the trophoblast and expansion of the inner cell mass. Mice treated identically and evaluated on the 18th day of gestation had fewer implanted embryos than did controls, and the fetuses weighed less. No resorptions, malformations or significant differences in intrauterine deaths were found. Chlorpromazine given in the same manner but at 0.5 mg/kg did not affect any of the aforementioned criteria. When 56 h embryos were cultured in vitro in the presence of 50 microM-chlorpromazine for a further 40 h, embryo compaction, blastocyst formation, the mitotic index and the total cell number were significantly reduced compared with controls. Blastocyst formation was again more severely affected than embryo compaction. The inhibition of embryo compaction, blastocyst formation, and reduction in mitotic index and cell number associated in this study with chlorpromazine in vivo and in vitro indicate that the drug inhibits the development of cleavage-stage embryos in the mouse. These effects might be mediated by antagonistic effects of calmodulin.