Suppression of murine NK activity induced by Corynebacterium parvum

Summary Formalin-killed Corynebacterium parvum (CP), given at a dose of 0.4–0.7 mg/mouse IV or IP, induced suppressor cells for NK activity in B6C3F₁ mice. The suppressor cells belong to at least two different populations, plastic adherent and nonadherent, and were not depleted by antibodies specific for Thy-1.2, Ia^k, or NK-1.2 surface markers. Administration of p-I:C, an interferon-inducer, to animals 18 h before the assay did not affect the suppressor activity. Hypotonic shock treatment of splenocytes abrogated the in vitro suppressive activity, and subsequent reconstitution of the shock-treated cells with RBC failed to restore the suppressive activity. SJL/J mice, which have low NK activity, exhibited suppressor activity comparable to B6C3F₁ mice following CP treatment, whereas CP-treated BALB/c athymic and euthymic mice showed a lower ability to generate suppressors for NK as compared to B6C3F₁ mice.     

Suppressor T cells and suppressor macrophages induced by Corynebacterium parvum

Corynebacterium parvum, injected intravenously into C57B1/6 mice (H-2^b) previously alloimmunized with P815 (H-2^d) mastocytoma cells, generated splenic suppressor cells that inhibited the development of primary cytotoxic lymphocytes in vitro. These suppressor cells differed from those generated by intravenous C. parvum injection of naive C57B1/6 mice. The former suppressor cells were effectively induced by administration of 700 μg of C. parvum whereas the latter suppressor cells were dependent upon higher doses (1400 μg) of adjuvant for their activation. Furthermore, suppressor cells generated in alloimmunized mice could only suppress C57B1/6 anti-P815 in vitro cytotoxic responses whereas suppressor cells generated in naive mice could suppress C57B1/6 anti-CBA (H-2^k) responses as well. Suppressor cells were not H-2 restricted in their action. Fractionation of spleens from alloimmunized, C. parvum-treated mice revealed the presence of suppressor T cells and suppressor macrophages. We were unable, however, to determine which cell was responsible for “antigen specificity” of suppression since the fractionation procedures seemed to trigger both suppressor cell types prior to adding them to the primary culture.     

Corynebacterium parvum: Effect on radiocurability of murine tumors

Summary In continuation of our earlier studies (Milas et al., 1975c) we have investigated whether treatment of mice with Corynebacterium parvum augments the curative response of a fairly immunogenic fibrosarcoma (FSa) and a weakly immunogenic mammary carcinoma (MDAH-MCa-4) to fractionated v-irradiation. Tumors were 8 mm in diameter at the commencement of radiation treatment. The FSa was exposed to 500 and the MDAH-MCa-4 to 750 rads daily for 3, 6, or 10 consecutive days. The dose of CP was 0.25 mg and was given intravenously (IV), in most experiments within 2 h after the first irradiation dose. In one experiment involving FSa, CP administration varied from 4 days before to 14 days after the start of irradiation. CP greatly augmented FSa radiocurability, especially when applied before irradiation. The effect was evident from the increase in the cure rate, and, in mice that were not cured, from tumor growth retardation, which resulted in prolonged survival of mice. CP also reduced the incidence of pulmonary metastases in mice in which the combination of treatments failed to produce local tumor control. CP was less effective in augmenting radiation response of MDAH-MCa-4. In this case, CP slowed the growth of irradiated tumors and prolonged the survival of mice. The effect of IV CP on FSa radiocurability was not further increased by including intralesional (IL) injection of CP, intraperitoneal (IP) injection of vitamin A, or the radiosensitizer of hypoxic tumor cells misonidazole (Ro-07-0582). Misonidazole, however, significantly improved the effect of 2000 rads on the growth retardation of mammary carcinoma, but CP did not further enhance this effect.     

Splenic suppressor macrophages induced in mice by injection of Corynebacterium parvum.

Spleen cells from C57BL/6N mice injected with killed Corynebacterium parvum (CP) had a marked growth inhibitory effect on the in vitro proliferation of RBL-5 murine lymphoma cells. It was most marked 12 to 14 days after injection and was usually no longer detectable later than 21 days. It could be demonstrated at effector cell to target ratios between 20:1 and 5:1 at which normal spleen cells had a growth-promoting effect. Addition of CP to an in vitro mixture of spleen cells and tumor cells augmented the inhibitory effect of spleen cells from CP-injected mice although it conferred no inhibitory potential on normal spleen cells. Growth inhibiton by CP spleen cells was not mediated by T cells and various depletion experiments suggested that the effector cells of the phenomenon were macrophages. Spleen cells of CP-injected mice also showed strongly depressed responses to the T cell mitogens PHA and Con A and suppressed the mitogen responses of syngeneic normal spleen cells. The characteristics of the suppressor cells mediating this effect appeared to be very similar to those inhibiting lymphoma cell growth. The responses to LPS were also strongly suppressed in mice injected with 2.1 mg of CP. However, after injection of one-tenth of the dose a relative sparing of the LPS response was noted, whereas the PHA response was still suppressed.     

Inhibition of cell-mediated cytotoxicity by Corynebacterium parvum

Summary We investigated the effect of altering dose and route of Corynebacterium parvum (C. parvum) administration on the adjuvant's inhibition of cell-mediated cytotoxicity (CMC). Primary in vivo and secondary in vitro CMC of C57B1/6 mice alloimmunized to P815 were depressed if C. parvum was administered systemically (IV or IP) but not when it was given SC. Similarly, only systemic C. parvum generated cells capable of suppressing in vitro CMC. Primary and secondary CMC in spleen was equally inhibited by 700 and 70 g, whereas suppressor cell activity was marked with 700 g and minimal with 70 g. Administration of C. parvum SC admixed with alloantigen resulted in early enhancement and late depression of primary CMC. Secondary CMC was depressed but suppressor activity was absent. Dissociation of CMC depression from suppressor cell generation indicates that these phenomena can be separated under certain conditions.     

Production of an antiviral factor by murine spleen cells after treatment with Corynebacterium parvum.

We have previously shown that injection of Corynebacterium parvum (CP) in mice protected them against lethal encephalitis induced by herpes simplex virus, (HSV). It is shown here that spleen cells of CP-injected mice in vitro produce a factor capable of inhibiting the replication of HSV in mouse embryo fibroblasts (MEF). A similar activity was produced after in vitro exposure of spleen cells from untreated mice to CP. CP was only slightly mitogenic in contrast to phytohemagglutinin (PHA), concanavalin A (Con A), and bacterial lipopolysaccharide (LPS) which were strongly mitogenic but did not induce antiviral activity high enough to be detected in the HSV-MEF system. The activity produced by CP-treated spleen cells appeared to be interferon since it was trypsin sensitive and species specific and not virus specific, and since preincubation of the cells was required to demonstrate an antiviral effect. However, the identity of CP-induced interferon with any of the previously described subclasses of interferon remains to be determined.     

Immunomodulation by Corynebacterium parvum in normal humans

Four normal human donors were immunized with 2 mg of heat-killed Corynebacterium parvum by subcutaneous and intracutaneous injections, and lymphocyte surface markers, antibody-dependent (ADCC), and spontaneous cell-mediated (SCMC) cytotoxicities followed for a 28-day period. Although no changes were observed in the relative proportions of B, T, and Fc receptor-carrying lymphocytes, two T cell subpopulations, namely, the autologous rosette-forming cells and active rosette-forming cells, both exhibited significant increases. Significant increases were also demonstrated in the proportion of monocytes carrying Fc receptors and in the proportion of monocytes phagocytizing Latex beads. Although no consistent changes could be found in ADCC against the P 815 mastocytoma cell line, the SCMC against both the myeloid leukaemia line K 562 and the lymphoma line RAJI was found to be elevated as early as 6 hr post-vaccination. The possibility that the enhanced SCMC activity was induced in vivo by interferon was supported thus: 1) Enhancement of SCMC in vitro by interferon was abrogated by the vaccination. 2) Serum interferon determinations showed significant increases in parallel with the lack of in vitro SCMC enhancement.     

Stimulation of natural killer (NK) and antibody-dependent cellular cytotoxicity (ADCC) activities in murine leukocytes by bombesin-related peptides requires the presence of adherent cells

Bombesin and the two mammalian bombesin-related peptides, gastrin-releasing peptide (GRP) and neuromedin C, at physiological concentrations have been previously shown to stimulate significantly in vitro the antibody-dependent cellular cytotoxicity (ADCC) and natural killer (NK) activities in BALB/c mouse leukocytes from axillary nodes, spleen and thymus. In the present work we have shown that adherent cells are required in leukocyte samples for stimulation of cytotoxicity by the neuropeptides, which suggests that this effect may be mediated by those cells. Here we demonstrate the specificity of the effects by reversing them in the presence of the bombesin-antagonist (Leu¹³-ΨCH₂NH-Leu¹⁴)-BN, and by detecting specific receptors for GRP on macrophages of high and low affinity. Using the same binding technics, no receptors for this neuropeptide were found in non-adherent leukocytes.     

Biological effects of Corynebacterium parvum. IV. Adjuvant and inhibitory activities on B lymphocytes

The PFC response to the thymus-independent antigen SIII (type 3 pneumococcal polysaccharide) was amplified in mice injected 4 days previously with killed Corynebacterium parvum. This adjuvant activity was demonstrable with high (2–50 μg) but not low (0.1–0.5 μg) doses of SIII. Induction of tolerance was unaffected. Depression of the response resulted from simultaneous injection of SIII with either C. parvum or Bordetella pertussis, while prior treatment with the latter was without effect. Responsiveness to SIII was transiently but potently suppressed in spleen cells transferred into lethally irradiated, C. parvum pretreated mice.Although C. parvum is an effective B cell adjuvant, other data imply that it acts indirectly on these lymphocytes. It is argued that both adjuvant and suppressive activities of C. parvum on the B cell response to SIII are most probably mediated by activated macrophages.     

The antineoplastic effect of Corynebacterium parvum modified by lysozyme treatment

Summary The study was conducted to evaluate the possibility that host factors may modulate the antitumor effect of C. parvum (CP). Heat-killed CP after being subjected to limited digestion by lysozyme (L-CP) produced a superior effect on the inhibition of local growth of murine mammary carcinoma (CD ₈ F ₁ ) comparsed to unmodified CP. The median survival time (MST) of tumor-bearing animals that were treated with L-CP was slightly but not significantly increased over that of the immunotherapeutic response to unmodified CP. This modified CP was capable of reducing the incidence of early death in tumor-bearing animals. The modified CP was also more toxic than the unmodified. In contrast, mixed glycosidase had no effect on the antitumor potency of CP. The usefulness of enzymatic modification of whole CP in improving the tumor-therapeutic potency of CP is discussed.     

Immunotherapy of lung cancer with Corynebacterium parvum

Summary Thirty-one patients with inoperable carcinoma of the lung, excluding oat-cell carcinoma, were randomized to receive either chemotherapy alone, with methyl CCNU and vinblastine every 6–8 weeks (15 Pts) or such chemotherapy plus immunotherapy with IV infusions of Corynebacterium parvum (16 Pts). Prior duration of the disease was longer, and more patients had received previous therapy, in the immunotherapy group; these groups were otherwise very similar. In vitro lymphocyte response to phytohemagglutinin did not change significantly in either group, but the weaker response to Varidase declined in both groups after chemotherapy. An increased baseline level of circulating B lymphocytes was sharply reduced in the C. parvum group. There were no differences in -globulins or delayed skin test responses between immunotherapy and control patients at entry into this study or on follow-up. Median survival from entry was longer in the immunotherapy group (6 months) than in the control group (3 months), but this difference was not statistically significant and only two patients in each group lived for more than 11 months. It is conceivable that more benefit from C. parvum might have been recorded had more effective chemotherapy been available.     

Cellular biology of Cryptosporidium parvum

With the emergence of Cryptosporidium parvum as a major pathogen encountered in human and veterinary clinical practice, a need for increased knowledge of the cellular- and immuno-biology of this Apicomplexan parasite has developed. Initial work has used paradigms taken from other Apicomplexans, especially Plasmodium, Toxoplasma and Eimeria, as a starting point. In this article, Carolyn Petersen discusses the observation that in these organisms, molecular targets of antibodies (which have protective value, in vivo, against disease) have frequently been located in the apical complex or on the surface of the invasive stages of the parasite and appear to mediate biologically crucial processes including motility, attachment to the host cell, modification of the host membrane, and entry into the host cell. Molecular-biology approaches to the study of enzymes and of structural proteins which mediate motility are also considered. Invasion mechanisms, biochemical pathways and motility may involve molecules that will prove susceptible to immunotherapeutic or chemotherapeutic interruption of cryptosporidiosis.     

Properties of an Antigenic Polysaccharide from Corynebacterium parvum

Corynebacterium parvum strain 10390 is an antitumor agent and stimulant of the reticuloendothelial system and produces a soluble antigen towards the end of its growth cycle. This material, which is a cell wall component and can also be released from the organism by acid or alkaline hydrolysis, has been purified. It is an acidic polysaccharide of molecular weight 100,000 to 150,000 and contains galactose, glucose, fucose, N-acetylgalactosamine, N-acetylglucosamine, uronic acids, sialic acids, and a small proportion of amino acids. The antigen gives a precipitin reaction with antisera raised against the whole organism and also binds to animal cells. The antigenic determinants are extremely resistant to oxidation, reduction, and enzymatic and chemical hydrolysis, but the single cell-binding site is destroyed by alkali and also by Helix pomatia digestive juice, alginase, and neuraminidase without substantially affecting the molecular weight. This site is inaccessible until the molecule is released from the cell surface. The possibility that the soluble antigen is the biologically active fraction of C. parvum is discussed.     

Cellular proliferation kinetics of the murine sarcoma induced by the Moloney sarcoma virus

Abstract. Cell proliferation kinetics of the sarcoma induced by Moloney virus was studied in newborn Swiss OFl mice.
After in vivo injection of tritiated thymidine, followed by autoradiography, it was shown that the majority of cells were actively proliferating (labelling index; 31%, growth fraction 78%). The mean cell cycle was 16 hr and cell loss was relatively low (cell loss factor 48%). The study of tumour specific activity with time after a single [ in vivo ] injection of [³H]dR or [¹²⁵I]UdR did not demonstrate the same degree of cell loss as that calculated by autoradiography. This result is consistent with a massive reutilization of radioactivity released by normal tissues.     

Role of Corynebacterium parvum in the activation of peritoneal macrophages. II. Identification of distinguishable anti-tumor activities by macrophage subpopulations

Two different mechanisms of murine macrophage (MP) antitumor activity are described in this report. C. parvum-activated peritoneal MPs were tested for cytotoxic and cytostatic activity 4 days after ip immunization. Cytotoxic activity could be distinguished from cytostatic activity using two different assay protocols. When MPs were separated by 1g velocity sedimentation, cytotoxic MPs were confined to high velocity fractions. In contrast, cytostatic MPs were found in cell fractions with velocities as low as 5.2 mm/hr. These two MP activities were also distinguishable by culturing at 37 degrees C for 24 hr. Cytotoxicity was abrogated when MPs were incubated in MEM, or MEM supplemented with lymphokine (LK) or indomethacin. In contrast, cytostasis remained at high levels when the cells were incubated with LK or indomethacin. Cytotoxicity was not retained after overnight culture even if LPS was present, or if various spleen or non-adherent peritoneal exudate cells were cocultured with the cytotoxic effector cells. Assays done to determine the presence of suppressor cells failed to find any inhibitory cell type. The phagocytic index, acid phosphatase activity, and H2O2 secretion were also measured before and after overnight culture. Acid phosphatase and phagocytic activities did not decline whereas H2O2 secretion declined significantly. These data indicate that in response to C. parvum, at least two different effector cell types with distinct antitumor activities are generated. Cytotoxicity, like the ability of cells to secrete H2O2, is found to be a short-lived function of CP stimulated MPs. In contrast, cytostasis is a function retained longer by MPs in culture.