Genetic control of murine immune responses to larval Dirofilaria immitis

Previous studies have demonstrated that BALB/c mice, immunized against infection with Dirofilaria immitis, were capable of killing a significant percentage of challenge larvae found within diffusion chambers. The percentage of larvae killed by immunized mice was, however, less than in immunized dogs and unlike immunized dogs, mice were unable to retard the development of the surviving larvae. The objective of the present study was to test 3 inbred strains of mice to determine whether a higher level of protective immunity would develop in these hosts and if larval growth retardation would occur. DBA/2J and C57BL/6J mice and their F1 hybrids B6D2F1/J were used in these studies; it was determined that there were differences in susceptibility among the 3 strains but no difference in ability to eliminate larvae from challenge infections. Growth retardation was seen in larvae recovered from immunized DBA/2J and C57BL/6J mice but not in B6D2F1/J. No difference was noted between immune and control mice in the cell types found in the diffusion chambers. The predominant cell types seen were mononuclear macrophages, multinucleate syncytial cells, and neutrophils. Antibody responses to soluble third- and fourth-stage larval antigens and larval excretory/secretory antigens were measured. Although antibodies to all 3 antigen groups were found in higher concentrations in immunized mice than in their respective controls, only antibody responses to soluble L-3 antigens provided a clear correlation with protective immunity.     

Active and passive immunization of mice against larval Dirofilaria immitis

The objective of this study was to determine if Dirofilaria immitis larvae would survive in diffusion chambers implanted in dogs and mice and secondly to determine if mice could be immunized against infection with D. immitis. Dirofilaria immitis third-stage larvae (L3) survived and grew in diffusion chambers implanted in dogs and mice for at least 3 wk. BALB/c mice, which were repeatedly infected with live L3, showed resistance to challenge infections. Dead L3, with or without adjuvants elicited no protective immunity. A correlation was found between the degree of immune protection seen in mice and antibody levels to soluble larval antigen but not to antibody levels to surface antigens. A monoclonal antibody was prepared that reacted with the surface of D. immitis and Onchocerca lienalis L3, but not to the surfaces of other stages and species of various filarial worms. When this antibody was administered to mice prior to challenge no significant reduction in larval survival was observed.     

The negative systemic effect of BCGcw inoculated intraperitoneally

Summary The present studies describe a systemic effect of BCGcw inoculated intraperitoneally by observing its influence on the development of Morris hepatomas inoculated intramuscularly. In none of our studies did we observe an inhibition of tumor growth. Instead, an enhancement of tumor growth was seen with four antigenically distinct Morris hepatomas (3924a, 9098, 7777, and 5123tc) in two strains of inbred rats (Buffalo and ACI). Extensive studies with Morris hepatoma 3924a showed consistent enhancement of intramuscular tumor growth in eight of eight experiments with a tumor cell dose of 1×10⁵ and a BCGcw dose of 900 g. In BCGcw-inoculated animals, palpable tumors (1–2 g) were detected approximately 1 week earlier than in controls, and significantly larger tumor masses were noted on the day of sacrifice. With the threshold dose of cells, 1×10⁴, an increase in tumor incidence was observed. It was not necessary for the host to be immunized with BCGcw prior to tumor inoculation as enhancement of tumor growth occurred when the BCGcw were inoculated the same day or 7 days after tumor inoculation. Splenectomized animals also demonstrated BCGcw-mediated enhancement of tumor growth. BCGcw immunization blocks the induction of tumor-specific immunity. When 3924a ascites tumor cells were inoculated intradermally into normal animals, no tumor growth was observed and tumor-specific immunity resulted. When 3924a ascites tumor cells were inoculated intradermally into BCGcw-immunized animals, progressive intradermal tumors grew in all the animals, implying that tumor-specific immunity was not induced. The administration of BCGcw did not effect established tumor-specific immunity to line 3924a as assessed by tumor-specific rechallenge resistance. Studies with an allograft system showed that the ACI tumor 3924a would not grow in normal Buffalo rats, but transient tumor growth was observed when the Buffalo rats were immunized with BCGcw. The abbreviations used are: BCGcw, bacillus Calmette-Guérin cell walls attached to oil droplets; MLC, mixed lymphocyte culture; MER, methanol extract residue; IM, intramuscular; IP, intraperitoneal; ID, intradermal; Con-A, concanavalin-A     

Cellular Immunity of Mice Infected with Listeria monocytogenes in Diffusion Chambers

The importance of bringing live bacteria into intimate contact with macrophages as a prerequisite for establishing cellular immunity was investigated. The bacterium Listeria monocytogenes was shown to replicate and survive in diffusion chambers implanted in the peritoneal cavities of mice. Humoral substances accruing from host responses to diffusing soluble antigens of the microorganism were unable to inactivate the bacteria. The resistance of mice immunized by subcutaneous inoculation of the live organism always exceeded the resistance of mice with Listeria diffusion chamber implants. Animals with sham diffusion chambers were more resistant to a challenge by L. monocytogenes than were normal mice. Host resistance was not significantly different between Listeria diffusion chamber implant groups and sham diffusion chamber implant groups. The results suggested that direct involvement of macrophages with the parasite is necessary to achieve cellular immunity.     

Active specific immunotherapy of guinea pigs with visceral tumor implants

Summary Guinea pigs, each with established, 7-day-old, syngeneic visceral micrometastases of line 10 tumor implanted intravenously, were immunized by intradermal inoculations into several sites of a mixture of irradiated line 10 cells and an emulsion containing heat-killed BCG or Mycobacterium phlei bacilli. This treatment led to survival of 72 of 80 treated animals (90%). Therapeutic effectiveness depended on the dose of mycobacteria and on that of irradiated tumor cells. Animals treated by intradermal injection of mycobacteria attached to oil droplets alone or with irradiated tumor cells alone, all died with multiple foci of pulmonary tumor.     

Recombinant BCG Expressing Mycobacterium ulcerans Ag85A Imparts Enhanced Protection against Experimental Buruli ulcer

Buruli ulcer, an emerging tropical disease caused by Mycobacterium ulcerans (MU), is characterized by disfiguring skin necrosis and high morbidity. Relatively little is understood about the mode of transmission, pathogenesis, or host immune responses to MU infection. Due to significant reduction in quality of life for patients with extensive tissue scarring, and that a disproportionately high percentage of those affected are disadvantaged children, a Buruli ulcer vaccine would be greatly beneficial to the worldwide community. Previous studies have shown that mice inoculated with either M. bovis bacille Calmette–Guérin (BCG) or a DNA vaccine encoding the M. ulcerans mycolyl transferase, Ag85A (MU-Ag85A), are transiently protected against pathology caused by intradermal challenge with MU. Building upon this principle, we have generated quality-controlled, live-recombinant strains of BCG and M. smegmatis which express the immunodominant MU Ag85A. Priming with rBCG MU-Ag85A followed by an M. smegmatis MU-Ag85A boost strongly induced murine antigen-specific CD4⁺ T cells and elicited functional IFNγ-producing splenocytes which recognized MU-Ag85A peptide and whole M. ulcerans better than a BCG prime-boost vaccination. Strikingly, mice vaccinated with a single subcutaneous dose of BCG MU-Ag85A or prime-boost displayed significantly enhanced survival, reduced tissue pathology, and lower bacterial load compared to mice vaccinated with BCG. Importantly, this level of superior protection against experimental Buruli ulcer compared to BCG has not previously been achieved. These results suggest that use of BCG as a recombinant vehicle expressing MU antigens represents an effective Buruli ulcer vaccine strategy and warrants further antigen discovery to improve vaccine efficacy.     

Inoculation of plasmids encoding Japanese encephalitis virus PrM-E proteins with colloidal gold elicits a protective immune response in BALB/c mice

We established a simple and effective method for DNA immunization against Japanese encephalitis virus (JEV) infection with plasmids encoding the viral PrM and E proteins and colloidal gold. Inoculation of plasmids mixed with colloidal gold induced the production of specific anti-JEV antibodies and a protective response against JEV challenge in BALB/c mice. When we compared the efficacy of different inoculation routes, the intravenous and intradermal inoculation routes were found to elicit stronger and more sustained neutralizing immune responses than intramuscular or intraperitoneal injection. After being inoculated twice, mice were found to resist challenge with 100,000 times the 50% lethal dose (LD(50)) of JEV (Beijing-1 strain) even when immunized with a relatively small dose of 0.5 micro g of plasmid DNA. Protective passive immunity was also observed in SCID mice following transfer of splenocytes or serum from plasmid DNA- and colloidal gold-immunized BALB/c mice. The SCID mice resisted challenge with 100 times the LD(50) of JEV. Analysis of histological sections detected expression of proteins encoded by plasmid DNA in the tissues of intravenously, intradermally, and intramuscularly inoculated mice 3 days after inoculation. DNA immunization with colloidal gold elicited encoded protein expression in splenocytes and might enhance immune responses in intravenously inoculated mice. This approach could be exploited to develop a novel DNA vaccine.     

Immunotherapy with allogeneic immune peritoneal cells activated by BCG or pyran copolymer

Summary Both pyran copolymer and BCG augmented specific macrophage-mediated cytotoxicity against allogeneic MBL-2 leukemia cells in a synergistic fashion. Similarly, the local passive transfer of peritoneal exudates (PE) from MBL-2 alloimmune mice treated with pyran or BCG protected against MBL-2 tumor development in the syngeneic host, whereas exudates from immunized or adjuvant-treated animals alone were ineffective. Tumor neutralization by alloimmune-activated PE was specific for the immunizing cell type and required intimate cell contact with target cells. Both adherent and nonadherent PE cells were required for optimal therapeutic effect. Long-term survivors demonstrated resistance to subsequent tumor challenge.     

Annexin-V promotes anti-tumor immunity and inhibits neuroblastoma growth in vivo

The goal of the current study is to determine the effects of blocking phosphatidylserine (PS) on the growth of neuroblastoma in mice. PS, an anionic phospholipid restricted to the cytoplasmic surface of plasma membranes in most cells, is externalized to the surface of apoptotic cells. PS has been shown to induce immune tolerance to self-antigens. PS can also be found on the surface of live cells and in particular tumor cells. Annexin-V (AnV) is a protein that specifically binds and blocks PS. To determine the effects of blocking PS with AnV on tumor growth and immunogenicity, mice were inoculated with AGN2a, a poorly immunogenic murine neuroblastoma that expresses high level of PS on the cell surface. Survival and anti-tumor T cell response were determined. AGN2a were engineered to secrete AnV. Secreted protein effectively blocked tumor PS. 40?% of mice inoculated with AnV-expressing AGN2a cells survived free of tumor, whereas none of the mice inoculated with control cells survived (p?=?0.0062). The benefits of AnV were lost when mice were depleted of T cells. The findings suggest that AnV could protect mice from tumor challenge through an immune mediated mechanism. Mice were then immunized with irradiated AnV-secreting or control cells, and challenged with wild-type AGN2a cells. AnV-secreting cell vaccine protected 80?% of mice from AGN2a challenge, while control cell vaccine prevented tumor growth in only 30?% of animals (p?=?0.012). ELISPOT analysis demonstrated that AnV-secreting cell vaccine induced a greater frequency of interferon-gamma producing splenic T cells. T cells isolated from mice immunized with AnV-secreting but not control vaccine lysed AGN2a. In summary, AnV blocked PS, enhanced T cell mediated tumor immunity, and inhibited tumor growth.     

Experimental Salmonellosis VIII. Postinfective Immunity and Its Significance for Conferring Cellular Immunity

In the process of live-vaccine immunization of Salmonella enteritidis infection in mice, the relation between the number of bacteria in the organs of mice and their protecting effect was studied. Treatment with antibiotics was used to control the number of immunizing bacteria in the tissues. Mice, which were infected with 10(-5) mg (1,000 mouse MLD) of virulent S. enteritidis and treated with kanamycin simultaneously, acquired high antilethal resistance against infection with the same organisms. However, the administration of large amounts of kanamycin, which caused a rapid decrease in bacterial numbers in the organs of infected mice, was incapable of conferring immunity. This indicated the necessity of persistence of live bacteria in the host for the production of immunity. A large number of microorganisms were maintained for 53 weeks in a diffusion chamber inserted into the mouse abdominal cavity. The mice implanted with diffusion chambers containing large numbers of virulent S. enteritidis did not acquire antilethal resistance against infection with the same organisms, although agglutinins against S. enteritidis were observed in these mice. Agglutinin was also found in the fluid contained in diffusion chambers inserted into mice immunized with a killed vaccine of S. enteritidis. This indicated that antibody penetrated the membrane filter of diffusion chambers from outside to inside and vice versa. From these results, it is suggested that contact of live microorganisms with the host cell is necessary for conferring postinfective immunity in salmonellosis.     

Additional evidence for the augmented induction of tumor-specific resistance in vaccinia virus-primed mice by immunization with vaccinia virus-modulated syngeneic tumor cells

The augmenting effect of vaccinia virus infection of tumor cells on induction of tumor-specific resistance was examined in mice. C3H/HeN mice were primed intraperitoneally (ip) with live vaccinia virus after whole-body irradiation with 250 rad of X-rays. Three weeks later the mice were immunized ip 3 times at weekly intervals with syngeneic murine hepatoma MH134 or spontaneous myeloma X5563 which had been infected in vitro with vaccinia virus and subsequently irradiated with 7000 rad of X-rays. One week after the third immunization, the mice were challenged with 1 X 10(5) viable cells of MH134 or X5563 ip or 1 X 10(6) tumor cells intradermally (id). On ip challenge with viable MH134 cells all mice that had not been pretreated died within 3 weeks due to ascites tumor out-growth, whereas all mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected MH134 cells survived. On ip challenge with X5563 cells, the percentage survival of vaccinia virus-primed and vaccinia virus-modified tumor-immunized mice was 80%. On id challenge with MH134 and X5563 tumor cells, in un-treated mice tumors grew to more than 5 mm in diameter within 3 weeks, whereas 90% and 60%, respectively, of the mice that had been vaccinia virus-primed and immunized with vaccinia virus-infected tumor cells showed no tumor out-growth. Pretreatment by only immunization with vaccinia virus-infected cells or vaccinia virus-priming and immunization with virus non-infected tumor cells were not effective for preventing induction of tumor-resistance to either ip or id challenge with MH134 or X5563 tumor cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Splenectomy before tumor inoculation prolongs the survival time of cachectic mice

The effects of splenectomy on the development of cachexia, tumor growth and animal survival were studied in tumor-bearing CDF1 mice. Mice were inoculated with two subclones of colon 26 adenocarcinoma, clone 20 (with a potent capacity to induce cachexia) and clone 5 (without such activity), and underwent splenectomy before or after tumor inoculation. Splenectomy significantly prolonged the survival of mice bearing clone 20 when it was performed prior to tumor inoculation, although the progression of cachexia and tumor growth were not affected. The survival rate was higher in splenectomized than it was in nonsplenectomized mice 20–40 days after tumor inoculation. Such effects on survival were not observed, however, in mice splenectomized after inoculation with clone 20 or in mice that underwent splenectomy either before or after inoculation with clone 5. The decrease of peripheral blood lymphocyte count observed in mice bearing clone 20 was magnified when splenectomy was performed before tumor inoculation, but the serum levels of tumor necrosis factor and interleukin-6 were comparable. These results indicate that cancer death from cachexia is not directly attributable to enhanced catabolism. The mechanism by which splenectomy ameliorates the survival of cachectic mice remains to be studied, although several changes observed in the splenectomized mice after inoculation, including decreases in the peripheral blood L3T4⁺ cells and Lyt-2⁺ cells on the 9th day and 15th day respectively, and increase in the L3T4⁺/Lyt-2⁺ cell ratio on the 15th day suggest the involvement of the modified host's immune response.     

Plasmodium berghei: The spleen in sporozoite-induced immunity to mouse malaria

A/J mice were splenectomized (Splx) or sham-splenectomized (SSplx) prior to administration of a single injection of irradiated sporozoites. Following challenge 7 days after immunization, it was found that none of the splenectomized mice were protected whereas 50% of the sham-splenectomized and intact animals were resistant to challenge. In another series of experiments similar groups, along with mice splenectomized just prior to challenge, were injected with 1.5 × 10⁵ irradiated sporozoites over a 5 week period. This resulted in protection of (1) 60–100% of the animals splenectomized before immunization, and (2) 90–100% protection of the animals splenectomized prior to challenge, as well as the intact and sham-splenectomized mice. Serum levels of antisporozoite antibodies (CSP and SNA) increased during immunization of the intact animals. Only 15–20% of the animals splenectomized prior to immunization presented positive CSP reactions and little if any sporozoite neutralizing activity (SNA) was detected. Serum from intact animals immunized and found resistant to sporozoite challenge was used for passive transfer studies. Immune serum recipients were challenged with small numbers of sporozoites. Only one out of 18 recipients was protected against sporozoite challenge.     

婴儿双歧杆菌对小鼠肉瘤S180的抑制作用

本文主要观察婴儿双歧杆菌(Bif. 189—3)对小鼠皮下移植肉瘤180(S180)的抑制作用。结果发现,该菌无论在S180移植前或移植后经皮下注射,均能明显抑制皮下移植S180的增长,并使带瘤鼠存活期显著延长。病理检查见实验组肿瘤坏死明显,肿瘤周围有大量炎症细胞浸润。提示该菌能非特异性增强肿瘤局部的免疫反应。     

Rodent malaria: BCG-induced protection and immunosuppression.

One dose of 10(7) viable units of Mycobacterium bovis, strain BCG, protected a significant number of Swiss mice from a primary challenge with 10(4) thoracic sporozoites of Plasmodium berghei. Immunization with irradiated sporozoites induced greater protection than that observed in BCG-treated with BCG and surviving a primary sporozoite challenge were not protected from rechallenge, whereas mice immunized with irradiated sporozoites and surviving initial challenge of sporozoites were solidly immune to further challenge. Immunizing mice with BCG and irradiated sporozoites simultaneously resulted in a synergistic effect of increased protection against a primary challenge of sporozoites only if the two immunogens were administered on the same day and if the mice were challenged 1 to 3 days later. Mice given BCG and irradiated sporozoites and surviving a primary challenge of sporozoites were unable to survive rechallenge. BCG given to mice previously immunized with irradiated sporozoites suppressed their protective immunity against sporozoite challenge.     

Allosensitization induced suppression of various murine tumors: role of non-H-2 antigens in antitumor immunity

Presence of alloantigens on various murine tumors was tested by tumor rejection in allosensitized Swiss mice. The results indicated the presence of alloantigen on immunogenic tumors like chemically induced fibrosarcoma (FS), ascitic sarcoma 180 (S 180) and immunogenic variant of lymphosarcoma (LS-A) in Swiss mice, while these antigens could not be detected by this procedure on spontaneous lymphosarcoma (LS). Allosensitization with skin graft was found to offer quantitatively higher antitumor resistance than the allosensitization achieved by allogeneic lymphocytes. Antitumor effect was not seen when tumor cells were inoculated earlier than day 3 of grafting. Further, host immunosuppression with whole body irradiation up to day of 3 of skin grafting abrogated the antitumor effect. H-2 compatible and non-H-2 incompatible skin graft sensitization of host could offer resistance against both S 180 and LS-A. Further, tumor immune mice rejected H-2 compatible, non-H-2 incompatible skin graft significantly earlier.     

Induction of protective immunity against Schistosoma mansoni by a non living vaccine. I. Partial characterization of antigens recognized by antibodies from mice immunized with soluble schistosome extracts

A single intradermal injection of frozen and thawed schistosomula in conjunction with the bacterial adjuvant Mycobacterium bovis strain Bacille Calmette Guerin, Phipps substrain (BCG) induced significant levels of resistance to challenge Schistosoma mansoni infection in C57BL/6 mice. Immunization with the aqueous fraction remaining after 100,000 X G centrifugation of the larval lysate was also protective under these conditions, suggesting that some immunogenic determinants may not be membrane associated. Frozen-thawed cercariae and soluble components of adult worms also protected against challenge infection in these experiments. These observations indicate that soluble immunogens are present in both early and late developmental stages of the parasite, and therefore may be good candidate antigens for an immunochemically defined vaccine against schistosomiasis. Induction of humoral reactivity against soluble or membrane antigens was examined in mice protected against cercarial challenge by prior exposure to frozen-thawed larvae, soluble larval, or soluble adult antigens plus BCG. Animals that were immunized with frozen-thawed larvae produced low but significant levels of antibodies against larval surface antigens when examined by indirect immunofluorescence or by immunoprecipitation of surface-labeled schistosomula. Mice immunized with soluble antigens, however, showed negligible antibody reactivity against surface membrane antigens. Because mice immunized with soluble antigens were resistant to challenge infection, these results strongly suggest that anti-surface membrane reactivity is not required in the mechanism of protective immunity in this model. Sera from mice immunized with either total freeze-thaw larval lysate or soluble schistosome extracts all showed strong reactivity against soluble antigens, as detected by ELISA. Western blot analysis showed these antisera to react with a restricted number of high m.w. antigens that were present both in schistosomula and in adult worms. These antigens are therefore likely to play a major role in the development of resistance in this model as immunogens and/or as targets of protective immune response.     

Effect of nonspecific immune stimulation with BCG and polidin on the Ehrlich ascites tumor growth

Investigations were performed to study: 1) the antitumor effect of BCG pretreatment on the development of Ehrlich ascites tumor in mice; 2) the effect of BCG administration in relation to the period of time before tumor inoculation and the dose levels used, and 3) the antitumor effect of an associated pretreatment of BCG and Polidin on the development of Ehrlich ascites tumor. BCG administered prior to Ehrlich ascites tumor inoculation have a protective effect evidenced by a delay in tumor development, a prolonged survival of the tumor host and, in some cases, even inhibition of tumor growth. The effect of BCG was highly dependent on 1) the dose and the time of administration of BCG and) 2 the combined pretreatment of BCG and Polidin.     

Long-term tumor growth suppression in mice immunized with naked DNA of the human tumor antigen mucin (MUC1)

Naked DNA is an attractive tool for vaccination studies. We have studied naked DNA vaccination against the human tumor antigen mucin, encoded by the gene MUC1. C57/BL6 mice were immunized twice, on day 1 and day 10. with plasmid pCI-MUC1, intramuscularly. Five days after the last immunization tumor challenge experiments were performed using the tumor cell line MC38, expressing human MUC1. In 85% of mice immunized with the mucin plasmid tumor growth inhibition was observed, whereas control mice developed tumors. Re-tumor challenge after three months revealed no tumor growth in mice immunized with the mucin plasmid. These encouraging results, showing long-term protection against tumor growth, indicate the potential usefulness of naked DNA vaccination for clinical immunotherapy.     

Antitumor activity of two BCG vaccine preparations against the lewis lung carcinoma in mice

Summary Two BCG vaccine preparations were prepared following different production methods. Immuno-BCG Pasteur F was produced by surface culture on Sauton medium; BCG-RIV was a homogenous stirred deep culture.The antitumor effects of the two BCG vaccines were investigated on the Lewis lung carcinoma (3LL) in C57Bl/6 mice. A direct relationship exists in this tumor model between the log₁₀ dose of single-cell suspension inoculated subcutaneously in the hind footpad of mice and the onset and the degree of local tumor growth and the time of death, which is directly related to the lung metastases. No significant difference from control mice was observed in the two groups of BCG-immunized mice when 3LL tumor cells were injected 2 weeks after BCG immunization. When varying numbers of viable units of the two BCG vaccines were injected together with 10⁵ tumor cells in separate groups of normal mice, a dose-dependent local reaction was observed with Immuno-BCG Pasteur F, which was associated with a delay in the onset and development of tumor growth and an increase in the mean survival time. The local inflammatory reaction produced with BCG-RIV was of lower magnitude, and only the highest concentration (1.8×10⁶ viable units) led to some delay in tumor occurrence and mortality. The antitumor effect of a specific local delayed-type hypersensitivity (DTH) elicited by varying amounts of the two BCG preparations injected together with 10⁵ tumor cells in separate groups of normal or BCG-immunized mice showed that the challenge injection of Immuno-BCG Pasteur F was in all cases more effective than the BCG-RIV, but these two vaccines were more effective in BCG-RIV-immunized mice than in Immuno-BCG F Pasteur-immunized mice.When the same number of viable units within each BCG vaccine was used as a criterion of comparison, Immuno-BCG Pasteur F produced a higher specific and nonspecific local inflammatory reaction (which was associated with a local antitumor effect) than BCG-RIV. But within 2 weeks, the latter was much better able to sensitize the mice to mycobacterial antigens. This was confirmed by the evaluation of local granuloma formation and tuberculin hypersensitivity. BCG vaccines prepared as surface-grown pellets and mechanically dispersed always sensitized mice to a lesser degree and after a much longer period of time than did the well-dispersed deep-cultured vaccine.