Dose Of BCG Research Articles

Socio-biological factors in underfive deaths in a rural area.

In 1985-86, 286 underfive deaths occurred among a population of 30,000 in a rural area of Haryana. Two hundred and eighty one were analysed for socio-biological factors related to under five mortality. Females had a higher mortality. About 2/3 of the deaths were in infants, and 90% in first 3 years. Most of the deaths (94%) occurred in the village itself, 58.4% did not seek any medical care during the terminal illness, 80-90% did not receive even a single dose of BCG, DPT or O.P.V., and 36.7% died in the first attack of illness. Though 68% had at least one episode earlier, 31.0% had been admitted in hospitals for an earlier episode of illness. In 42.8% of deaths, the birth order was IV or above. Deaths in socially and economically disadvantaged cases constituted 77.6%. The triad of diarrhea, ARI and malnutrition claimed 56% deaths. In 93% of the deaths, the mothers were illiterate and 96.4% were house wives. There was a sibling death earlier in the family in 78.3%, and 60.1% deaths were of those living in poor housing conditions. About 50% had radio for communication, 85.8% had bicycle for conveyance, and in 66.9% the family had piped water supply. All these findings have been discussed in the study.

Immunoprophylactic trial with combined Mycobacterium Leprae/BCG vaccine against leprosy: preliminary results

In an attempt to find a vaccine that gives greater and more consistent protection against leprosy than BCG vaccine, we compared BCG with and without killed Mycobacterium leprae in Venezuela. Close contacts of prevalent leprosy cases were selected as the trial population since they are at greatest risk of leprosy. Since 1983, 29 113 contacts have been randomly allocated vaccination with BCG alone or BCG plus 6 x 108 irradiated, autoclaved M leprae purified from the tissues of infected armadillos. We excluded contacts with signs of leprosy at screening and a proportion of those whose skin-test responses to M leprae soluble antigen (MLSA) were 10 mm or more (positive reactions). By July, 1991, 59 postvaccination cases of leprosy had been confirmed in 150 026 person-years of follow-up through annual clinical examinations of the trial population (31 BCG, 28 BCG/M leprae). In the subgroup for which we thought an effect of vaccination was most likely (onset more than a year after vaccination, negative MLSA skin-test response before vaccination), leprosy developed in 11 BCG recipients and 9 BCG/Mleprae recipients; there were 18% fewer cases (upper 95% confidence limit [CL] 70%) in the BCG/M leprae than in the BCG alone group. For all cases with onset more than a year after vaccination irrespective of MLSA reaction the relative efficacy was 0% (upper 95% CL 54%; 15 cases in each vaccine group). Retrospective analysis of data on the number of BCG scars found on each contact screened suggested that BCG alone confers substantial protection against leprosy (vaccine efficacy 56%, 95% CL 27-74%) and there was a suggestion that several doses of BCG offered additional protection. There is no evidence in the first 5 years of follow-up of this trial that BCG plus M leprae offers substantially better protection against leprosy than does BCG alone, but the confidence interval on the relative efficacy estimate is wide.

Comparative trial of administration of half (0.05 mg) and quarter (0.025 mg) dose of intradermal Pasteur BCG on 291 infants from birth to 1 year in French Guyana

The study concerns 291 newborns and infants aged 0 to 1 year placed randomly in two groups which respectively received 0.025 and 0.05 mg Pasteur BCG by intradermal injection, between 1 December 1988 and 28 February 1989. This random test aimed to determine if the administration of one quarter dose (0.025 mg) of intradermal BCG conferred immunity comparable to that of one half (0.05 mg) while diminishing the risk of complications, in particular suppurative adenopathy, in infants of ages 0–1 year. Statistical analysis of the results showed that the 0.025 mg dose of intradermal BCG entails an immunoresponse as satisfactory as that entailed by the 0.05 mg dose, while the rate of suppurative adenitis is significantly higher in the group that received the 0.05 mg dose. It is therefore well justified to recommend a dose of 0.025 mg of intradermal Pasteur BCG for infants aged 1 year and less.

High-dose inhibition and low-dose enhancement of murine sarcoma growth exhibited by BCG vaccine

An in vivo assay for BCG anti-cancer efficacy was developed, utilizing subcutaneous injection into CFW Swiss-Webster mice of cultured S180 sarcoma cells and mixed with freeze-dried Tice ™ BCG vaccine. A BCG dose of 0.156–1.56 mg dry weight significantly inhibited tumor formation, but a BCG dose of 0.156–1.56 μg significantly enhanced tumor growth, evidenced by increased tumor incidence, volume and initial growth rate. These antagonistic activities may contribute to the high variability of BCG anti-cancer efficacy seen in animals and humans and indicate the need to exercise caution when employing even low doses of BCG for cancer immunotherapy.

Immunological consequences of innate resistance and susceptibility to BCG

The immunological consequences of genetically controlled innate resistance and susceptibility to Mycobacterium bovis (BCG) infection in mice were investigated. The susceptible ( Bcg s) BALB/c and the congenic resistant BALB/c. Bcg r mouse strains were employed to test for differences in the specific immune response to BCG. Antigen-specific lymphocyte proliferation and production of interleukin 2 (IL-2) to purified protein derivative (PPD) in vitro were measured following infection of congenic mice with low (10 4) and high (10 6) doses of BCG. The lymphocyte subsets were identified by testing the ability of spleen cells to respond after separation of T and B cells and after cytotoxic depletion of T cell subsets. In addition, fluorescence activated cell sorter (FACS) analysis with anti-T and -B cell monoclonal reagents was used to enumerate lymphocyte populations in BCG-infected spleens. The results indicated that the innately resistant mice displayed antigen-specific T helper cell function three weeks following the injection of BCG. The susceptible animals were found to be T cell-unresponsive since they lacked both proliferative and IL-2 secreting specific T cells. No evidence for suppression of IL-2 production or proliferation was detected in BALB/c (susceptible) spleen cultures in cell mixing experiments. The results provide evidence for a regulatory role of the Bcg gene on the generation of lymphocyte responses to BCG.

Evidence for a biphasic memory T-cell response to high dose BCG vaccination in mice

Memory immunity in mice to BCG vaccination is mediated by Thy-1.2 + L3T4 + Lyt-2 − cyclophosphamide-resistant T-lymphocytes. The time required for the emergence of acquired memory immunity was inversely proportional to the BCG inoculum size, although the level of memory expressed, once established, was equivalent regardless of the original dose of BCG administered. In mice given a high intravenous dose (10 8) of BCG, an apparently biphasic memory repsonse was observed, initially peaking on day 15–20, then declining for 10 days or so before increasing again to maximal levels between day 30 and 60 of the infection. This trough in resistance was not due to an active immunosuppressive mechanism; instead, it is suggested that it represents an initial loss or consumption of memory T-cells generated early during the infection which are stimulated to give rise to a state of active immunity as a result of the persisting high mycobacterial load.

Immunization coverage of children in a semi-urban village panchayat in Nepal, 1985

This research project, funded by the Yale International Committee and in cooperation with UNICEF, Nepal, used a questionnaire to survey and assess the immunization status of children 3 years or younger in a semi-urban panchayat. Mothers of children ranging in age from less than 1 month to 3 years were questioned both about immunization status of their children and their own use of health care and specific sources of information. 54% (124/228) of the children had received at least one vaccine and only 4% (10/228) had full coverage with the recommended vaccines (3 doses of DPT and TOPV, one dose of measles and BCG). The mean number of vaccinations for males was significantly greater than that for females (2.12 vs 1.39, P = 0.007, t = 2.80). Male children were twice as likely to have received vaccines as females (76/122, 62% vs 48/106, 45%; Odds Ratio = 2.00). Children born at a hospital were more likely than those born at home to have been vaccinated (23/34, 68% vs 101/194, 52%; P = 0.05, x 2 = 12.52, d.f. = 2). Most mothers obtained health information from neighbors (38%), radio (22%), or health workers (18%), and 85% of the children were born at home; the majority (164/228, 72%) of the women received assistance during childbirth, 28% of the total reported self delivery. More than 70% of the women felt that vaccinations were good preventive measures. The study results suggest that health education about immunization efforts should be focused on women and these efforts should be intensified.

The effect of activation of macrophages on the advancement of experimental lung emphysema

Experiments were carried out on 68 Wistar rats of both sexes. Five s.c. injections of BCG vaccine (once a week) were applied as macrophage mobilizing and activating agent. Papain or physiological saline was given once intratracheally after the 3rd dose of BCG. 4 weeks after the latter substances had been administered the animals were sacrificed. The lungs and the pulmonary alveolar macrophages (PAM) were examined using histological and morphometric techniques; SEM and TEM were applied for assessing ultrastructure of PAM and lung sections. In rats given BCG the number of PAM was increased, and their morphological features showed enhanced activity. In these animals the aggravated emphysematous changes induced by papain should be ascribed to the cumulation of PAM and their increased activity in the lungs.

Absorption of Mycobacterium bovis BCG administered by the oral route.

The action of gastric and duodenal juices on BCG as well as on its absorption and its distribution in the organs after intragastric administration in mice were studied. A significant decrease in BCG oxygen uptake and a moderate loss of viability were found after 2 h treatment with gastric juice. Using duodenal juice, a marked decrease of respiration and a notable fall in viability were observed. Labelling of BCG with carbon-14 was accomplished using [14C]glycerol as a precursor of mycobacterial lipids. Similar levels of radioactivity were recovered in organs of mice 24 h after intragastric administration of 14C-BCG, sonicated 14C-BCG and [14C]glycerol. The level of 14C-BCG remained stable from 6 to 24 days, while sonicated 14C-BCG and [14C]glycerol defined a biological decay process. Studies of biological decay from the small intestine and liver indicated that the absorptive process started rapidly and reached its highest level at 24 h, declining thereafter according to the complexity of the material given to mice. However, living bacilli were not cultured from organs of mice given single doses of unlabelled BCG. Therefore, judging from the above data it may be concluded that the majority of BCG bacilli absorbed intact were not alive.

Effect of oral exposure ofMycobacterium avium intracellular on the protective immunity induced by BCG

The relative protective efficacy of oral administration of mycobacteria as compared to the conventional intradermal route of vaccination has been assessed in guinea pigs. Skin test reactivity to partially purified protein derivative and protective immunity to challenge with virulent Mycobacterium tuberculosis were used as parameters of protective immunity. Oral immunisation of guinea pigs either with BCG or with Mycobacterium avium intracellulare induces skin test reactivity and protective immunity comparable to that induced by intradermal route of vaccination. Oral exposure of Mycobacterium avium intracellulare prior to oral or intradermal dose of BCG did not interfere with the protective immunity induced by BCG in guinea pigs challenged with Mycobacterium tuberculosis H37Rv.

Migration of natural suppressor cells from bone marrow to peritoneal cavity by live BCG.

Delayed-type hypersensitivity (DTH) responses were suppressed in mice inoculated with bone marrow cells from mice that had been injected with 10(8) colony-forming units (CFU) of live BCG. Upon analysis of this DTH-suppression by the use of a macrophage migration inhibition (MI) assay, the in vitro correlate of DTH, suppressor macrophages in the peritoneal cavity were found to play an important role in DTH suppression. However, neither suppression of DTH nor production of suppressor macrophages was observed in mice inoculated with bone marrow cells from mice that had been injected with methotrexate (MTX), a folic acid antagonist, and 10(8) CFU of live BCG. Moreover, suppressor cells against the MI activity of peritoneal exudate cells from BCG cell wall-immunized mice existed in bone marrow cells from normal mice, natural suppressor (NS) cells, and they were sensitive to MTX. In addition, these NS cells phagocytized carbonyl iron particles, were adherent to Sephadex G-10, and had Fc receptors, but they had no B or T cell markers, suggesting that these cells belonged to a macrophage compartment. From this evidence, we hypothesized that the origin of suppressor macrophages in the peritoneal cavity induced by live BCG injection was MTX-sensitive NS cells in bone marrow, and that these NS cells were stimulated by a small dose of live BCG trapped in bone marrow after i.v. injection of a high dose of live BCG and migrated from bone marrow to the peritoneal cavity.

Dissociation between interleukin-1 and interleukin-2 production in proliferative response to microbial antigens: restorative effect of exogenous interleukin-2.

The relationship between the production of interleukin-1 (IL-1) and interleukin-2 (IL-2) after stimulation of human mononuclear cells within an antigenic extract from Candida albicans was analyzed in both responder and nonresponder donors. Culture supernatants from responders contained both IL-1 and IL-2 activity, whereas the supernatants from nonresponders contained only IL-1 and no appreciable IL-2. However, the addition of exogenous IL-2 to nonresponder cultures restored the normal proliferative response. Similar observations were made when cells from mice infected intravenously with high doses of Mycobacterium bovis BCG were cultured; these cells showed a marked impairment of the proliferative response to purified protein derivative. Spleen cells from BCG-induced unresponsive mice failed to produce IL-2 despite the fact that normal IL-1 activity was present in the culture. Again, the addition of exogenous IL-2 fully reversed the proliferative unresponsiveness. Thus, the presence of IL-1 does not necessarily induce production of IL-2, and the proliferative unresponsiveness is therefore due to a primary lack of IL-2.

Evolution of cell types and T-cell subsets in the spleens of Mycobacterium bovis BCG-resistant and M. bovis BCG-susceptible strains of mice after infection with M. bovis BCG.

In mice, the early host response to intravenous infection with small doses of dispersed Mycobacterium bovis BCG is controlled by the Bcg gene. After infection with a low dose of M. bovis BCG, Lyt-1+ cells were generated in the spleens of BCG-susceptible mice (Bcgs) in parallel with an increase in the proportion of phagocytic cells. Very few changes occurred in the splenic cell types of BCG-resistant mice (Bcgr).

Opposite effects of cyclophosphamide pretreatment on tuberculin hypersensitivity during the course of sensitization of mice with mycobacterium bovis BCG

The influence of cyclophosphamide (CY) pretreatment upon the development of tuberculin hypersensitivity has been studied during the course of infection of mice with BCG. An enhancement of the DTH response to BCG antigens occurred during the induction phase, whereas a depression of this response occured at the peak and during the decay phase of sensitization. The development of the early DTH-promoting and of the late DTH-depressing effect of CY was favoured by the use of a supra-optimal dose of BCG. Both these effects were cell-dependent since they could be transferred adoptively to syngeneic recipient mice with sensitized lymphoid cells but not with specific immune sera. Pretreatment with CY favoured the emergence of cells capable of responding in vitro to BCG antigens in the draining lymph nodes of BCG-infected mice. No simple association however, exists between this in vitro lymphocyte transformation response and the DTH response.

Role of first stimulating agents in the production of tumor necrosis factor.

The conditions and kinetics of tumor necrosis factor (TNF) production were examined. For TNF production, dual stimulation is necessary. Priming agents such as BCG, Corynebacterium parvum, and zymosan, which can stimulate the reticuloendothelial system (RES), are good substances for TNF production with the aid of lipopolysaccharide. Wide differences are observed in TNF producibility among different priming agents. The producibility of TNF depends on the degree of stimulation of the RES by the priming agents. Those priming agents, e.g., Propionibacterium acnes and Corynebacterium anaerobium, that are able to induce substantial RES hyperplasia are also able to induce high levels of TNF activity. Following administration of large doses of BCG or zymosan, mice were found to produce TNF activity. However, PPD, OK 432, PSK, and Choreito were unable to induce TNF activity.

In vivo and in vitro administration of interleukin 2-containing preparation reverses T-cell unresponsiveness in Mycobacterium bovis BCG-infected mice.

Mice infected with high doses of Mycobacterium bovis BCG (3 X 10(7)) showed a marked impairment of delayed-type hypersensitivity to PPD in vivo, and their splenic T cells failed to proliferate when cultured in vitro with concanavalin A or PPD. However, this state of unresponsiveness could be reversed both in vitro and in vivo by the administration of an interleukin 2 (IL-2)-containing preparation. IL-2 produced spontaneously by the gibbon lymphosarcoma T-cell line MLA-144 and T-cell-conditioned medium from a mixed lymphocyte reaction were able to increase DNA synthesis of splenic T lymphocytes from BCG-immunosuppressed mice cultured with concanavalin A or PPD. Furthermore, BCG-infected mice treated in vivo with at least 100 U of IL-2 showed a positive skin reaction to PPD, and their spleen cells were fully responsive in vitro. The reversal of BCG-induced immunosuppression was not observed when infected mice were injected with IL-2 preparations previously incubated with blast cells, a procedure known to remove IL-2 activity. These results indicate that the basis of BCG-induced unresponsiveness is a deficiency in the production of IL-2 rather than a lack of reactive T cells.

Suppressor T cells in BCG-treated mice interfere with an in vivo specific antitumoral immune response.

The interference by BCG in the induction and expression of a specific antitumoral immune reaction was studied in B6 mice, using the in vivo Winn assay and also active immunization. T cells immunized against MCA-induced fibrosarcoma (MC B6-1) transferred together with the tumour cells protected the syngeneic host against tumour take. Pretreatment of normal B6 mice with moderate or high doses of BCG prevented the development of a protective immune response after immunization. Moreover, a single dose of 1 mg, or 2 doses of 0.01 mg BCG, completely eliminated an established antitumour immunity. Suppressor cells are involved in the BCG-induced inhibitory effect; they interfered (1) with the expression of the antitumour response, since their addition to immune T cells in the Winn test resulted in decreased protection and (2) with the induction of the antitumour response, since injection of spleen cells from BCG-treated mice (BCG SpC) into normal mice before immunization inhibited the development of immunity. Treatment of BCG SpC with anti Thy 1.2 and anti Lyt 1.2 antibodies plus complement before injection into normal mice significantly decreased the suppressive activity, showing that the suppressor cells induced by BCG are T cells expressing the Lyt 1+ phenotype. The partial increase in protection obtained after IL-2 administration to BCG-treated mice suggests that the suppressive action of BCG SpC on the IL-2 producing capacity of helper T cells is only one of a number of possible mechanisms of T-cell-mediated suppression.

Local immunotherapy of malignant brain tumors. Evaluation of intratumoral injection of BCG and a Streptococcus pyogenes preparation

The anti-tumor effect of local immunotherapy using BCG and Picibanil, a Streptococcus pyogenes preparation, was evaluated in experimental and human malignant brain tumors. BCG and Picibanil produced an identical inflammatory cell reaction in the C3HHe mouse brain after intracerebral injection. BCG had no direct cytocidal effect on Rous sarcoma virus-induced malignant glioma cells of the mouse in vitro. A mixture of the mouse glioma cells and BCG was inoculated into the subcutaneous tissue and brain of the syngeneic mice. The development and growth of subcutaneous and brain tumors were inhibited remarkably by the addition of 100-1, 000 μg of BCG to 2×105 subcutaneously transplanted tumor cells, and by the addition of 50-500 μg of BCG to 1×104 intracerebrally transplanted tumor cells. Intratumoral injection of 5 mg of BCG showed a significant suppression of the growth of subcutaneous tumors which could not be obtained by the systemic administration of the same dose of BCG. The enhancement of tumor-specific immunity was obtained in this mouse-tumor system by the repeated immunization of a mixture of irradiated tumor cells and BCG. Thirteen patients with malignant brain tumors, 12 anaplastic gliomas and one metastatic tumor, received repeated intratumoral injections of Picibanil through one to two intratumorally implanted tubes. All patients tolerated this therapy, and the morbidity rate was acceptable. Significant tumor regression was noted on CT scans in 6 of 11 patients for whom scanning was performed. Histologic examinations of the post-therapy specimens obtained from 9 patients revealed that inflammatory cell reactions were evoked in all of the tumors, although the extent of the inflammatory reactions varied from tumor to tumor and was mostly localized to an area surrounding the intratumoral tubes. Combination therapy of local Picibanil injection and systemic administration of ACNU as maintenance immunochemotherapy was performed in another series of anaplastic glioma patients. Among the 10 patients who were followed for more than 2 years after treatment, 5 survived more than 2 years and 4 obtained long-term survivals of 71, 58, 44 and 43 months postoperatively. In conclusion, local immunotherapy is worth employing as maintenance immunochemotherapy for anaplastic glioma patients.

The immunologic and immunotherapeutic sequelae of intraperitoneal BCG. II: Serial changes in spleen weight, morphology, cell populations and in vitro mitogen responses.

FOLLOWING the IP administration of an immunotherapeutic dose of BCG, sequential analysis of spleen weight, morphology, and cell subpopulations was performed over a sixty day period.3H-thymidine uptake, by basal and optimally stimulated spleen cell cultures, was also measured over the same period. Despite a prolonged increase in spleen weight, little perturbation of the percentage distribution of the cell subpopulations and no alteration inin vitro responses were found. The results are discussed in relation to previous reports of immunosuppression following the administration of BCG.

Cutaneous unresponsiveness to Mycobacterium bovis BCG in intravenously infected mice.

Mice injected with 1 mg (about 1 X 10(7) CFU) of Mycobacterium bovis BCG in the footpad showed high levels of delayed-type hypersensitivity (DTH) to BCG antigens and a continuous increase in circulating specific anti-immunoglobulin G (IgG) antibodies throughout a 6-week observation period. In contrast, mice injected intravenously with a 1-mg dose failed to mount a DTH and showed a depression in antibody production at week 5 postinfection. A dose-response study revealed that an optimum dose of BCG, when injected intravenously, can induce a small but significant DTH response. The delayed cutaneous unresponsiveness in intravenously infected mice lasted at least 6 weeks and was not antigenically specific, in that it depressed the DTH response to Corynebacterium parvum. No simple relationship existed between levels of DTH and the amount of circulating anti-IgG antibodies. Splenectomy and treatment with a high dose of cyclophosphamide before infection, although greatly reducing the humoral response, did not reverse the BCG-induced unresponsiveness nor enhance levels of DTH in mice infected in the footpad. It is concluded that the intravenous infection of mice with BCG exerts a nonspecific inhibitory effect on delayed-type immune reactions by the induction of some type of suppressor mechanisms that are resistant to cyclophosphamide.