An Ocular Commensal Protects against Corneal Infection by Driving an Interleukin-17 Response from Mucosal γδ T Cells.

Mucosal sites such as the intestine, oral cavity, nasopharynx, and vagina all have associated commensal flora. The surface of the eye is also a mucosal site, but proof of a living, resident ocular microbiome remains elusive. Here, we used a mouse model of ocular surface disease to reveal that commensals were present in the ocular mucosa and had functional immunological consequences. We isolated one such candidate commensal, Corynebacterium mastitidis, and showed that this organism elicited a commensal-specific interleukin-17 response from γδ T cells in the ocular mucosa that was central to local immunity. The commensal-specific response drove neutrophil recruitment and the release of antimicrobials into the tears and protected the eye from pathogenic Candida albicans or Pseudomonas aeruginosa infection. Our findings provide direct evidence that a resident commensal microbiome exists on the ocular surface and identify the cellular mechanisms underlying its effects on ocular immune homeostasis and host defense.

Dysbiosis and Staphylococcus aureus Colonization Drives Inflammation in Atopic Dermatitis.

Staphylococcus aureus skin colonization is universal in atopic dermatitis and common in cancer patients treated with epidermal growth factor receptor inhibitors. However, the causal relationship of dysbiosis and eczema has yet to be clarified. Herein, we demonstrate that Adam17(fl/fl)Sox9-(Cre) mice, generated to model ADAM17-deficiency in human, developed eczematous dermatitis with naturally occurring dysbiosis, similar to that observed in atopic dermatitis. Corynebacterium mastitidis, S. aureus, and Corynebacterium bovis sequentially emerged during the onset of eczematous dermatitis, and antibiotics specific for these bacterial species almost completely reversed dysbiosis and eliminated skin inflammation. Whereas S. aureus prominently drove eczema formation, C. bovis induced robust T helper 2 cell responses. Langerhans cells were required for eliciting immune responses against S. aureus inoculation. These results characterize differential contributions of dysbiotic flora during eczema formation, and highlight the microbiota-host immunity axis as a possible target for future therapeutics in eczematous dermatitis.

Psoriatic lesions are characterized by higher bacterial load and imbalance between Cutibacterium and Corynebacterium.

BACKGROUND: Several studies have found that the microbiota of psoriatic lesions is different from that of healthy skin. OBJECTIVE: To characterize the microbiota of lesional and unaffected skin in patients with psoriasis and controls and investigate the correlation between cutaneous microbiota and clinical features of psoriasis. METHODS: Using quantitative polymerase chain reaction and 16S rRNA sequencing, we assayed the profiles of cutaneous microbiota in controls, unaffected skin, and psoriatic lesions. We also investigated the correlation of psoriasis-associated taxa with clinical characteristics. RESULTS: High bacterial load was identified in the psoriatic lesions compared with unaffected skin and controls. There was an imbalance between Cutibacterium (also known as Propionibacterium) and Corynebacterium in psoriatic skin. Lesions showed a higher proportion of Corynebacterium and a lower proportion of Cutibacterium compared with unaffected skin and controls. Corynebacterium was correlated with the severity of local lesions, whereas Cutibacterium showed correlation with the abnormity of skin capacitance. LIMITATIONS: We did not conduct a longitudinal study. CONCLUSIONS: Psoriatic lesions are characterized by higher bacterial load and imbalance between Cutibacterium and Corynebacterium.

Corynebacterium kroppenstedtii in granulomatous mastitis: Analysis of formalin-fixed, paraffin-embedded biopsy specimens by immunostaining using low-specificity bacterial antisera and real-time polymerase chain reaction.

Granulomatous mastitis (GM) is a rare inflammatory disease of the post-lactation breast, clinically mimicking breast cancer. GM is microscopically characterized by formation of epithelioid granulomas and abscess (suppurative granulomas) with lipid droplet-centered inflammation. Corynebacterium kroppenstedtii (Ck) is known as a causative bacterium of GM, and identification of Ck infection within the lesion should thus be essential for confirming the diagnosis. In the present study, we analyzed formalin-fixed, paraffin-embedded (FFPE) biopsy specimens of a total of 18 GM lesions with immunostaining and real-time PCR for Ck genome. Widely cross-reactive rabbit antisera against Bacillus Calmette-Guerin (BCG), Bacillus cereus, Treponema pallidum and Escherichia coli were chosen. With real-time PCR, Ck genome was demonstrated in 7 of 18 GM lesions. Immunohistochemically, the low-specificity antisera reacted with the cytoplasm of phagocytes and/or granuloma-engulfed lipid droplets in 12 of 18 GM lesions. Antigenic positivity was observed in the following order: BCG > B. cereus > T. pallidum > E. coli. Real-time PCR using DNA extracted from FFPE sections was useful but not consistent for identifying the Ck genome in GM, while immunostaining using cross-reactive antisera against four kinds of bacteria was not Ck-specific but was applicable to visualizing bacterial infection within the GM lesions.

Contemporary microbiology and identification of Corynebacteria spp. causing infections in human.

The Corynebacterium is a genus of bacteria of growing clinical importance. Progress in medicine results in growing population of immunocompromised patients and growing number of infections caused by opportunistic pathogens. A new infections caused by new Corynebacterium species and species previously regarded as commensal micro-organisms have been described. Parallel with changes in Corynebacteria infections, the microbiological laboratory diagnostic possibilities are changing. But identification of this group of bacteria to the species level remains difficult. In the paper, we present various manual, semi-automated and automated assays used in clinical laboratories for Corynebacterium identification, such as API Coryne, RapID CB Plus, BBL Crystal Gram Positive ID System, MICRONAUT-RPO, VITEK 2, BD Phoenix System, Sherlock Microbial ID System, MicroSeq Microbial Identification System, Biolog Microbial Identification Systems, MALDI-TOF MS systems, polymerase chain reaction (PCR)-based and sequencing-based assays. The presented assays are based on various properties, like biochemical tests, specific DNA sequences, composition of cellular fatty acids, protein profiles and have specific limitations. SIGNIFICANCE AND IMPACT OF THE STUDY: The number of opportunistic infections caused by Corynebacteria is increasing due to increase in number of immunocompromised patients. New Corynebacterium species and new human infections, caused by this group of bacteria, has been described recently. However, identification of Corynebacteria is still a challenge despite application of sophisticated laboratory methods. In the study we present possibilities and limitations of various commercial systems for identification of Corynebacteria.

Toll-Like Receptor 2 and Mincle Cooperatively Sense Corynebacterial Cell Wall Glycolipids.

Nontoxigenic Corynebacterium diphtheriae and Corynebacterium ulcerans cause invasive disease in humans and animals. Host sensing of corynebacteria is largely uncharacterized, albeit the recognition of lipoglycans by Toll-like receptor 2 (TLR2) appears to be important for macrophage activation by corynebacteria. The members of the order Corynebacterineae (e.g., mycobacteria, nocardia, and rhodococci) share a glycolipid-rich cell wall dominated by mycolic acids (termed corynomycolic acids in corynebacteria). The mycolic acid-containing cord factor of mycobacteria, trehalose dimycolate, activates the C-type lectin receptor (CLR) Mincle. Here, we show that glycolipid extracts from the cell walls of several pathogenic and nonpathogenic Corynebacterium strains directly bound to recombinant Mincle in vitro Macrophages deficient in Mincle or its adapter protein Fc receptor gamma chain (FcRγ) produced severely reduced amounts of granulocyte colony-stimulating factor (G-CSF) and of nitric oxide (NO) upon challenge with corynebacterial glycolipids. Consistently, cell wall extracts of a particular C. diphtheriae strain (DSM43989) lacking mycolic acid esters neither bound Mincle nor activated macrophages. Furthermore, TLR2 but not TLR4 was critical for sensing of cell wall extracts and whole corynebacteria. The upregulation of Mincle expression upon encountering corynebacteria required TLR2. Thus, macrophage activation by the corynebacterial cell wall relies on TLR2-driven robust Mincle expression and the cooperative action of both receptors.

The altered landscape of the human skin microbiome in patients with primary immunodeficiencies.

While landmark studies have shown that microbiota activate and educate host immunity, how immune systems shape microbiomes and contribute to disease is incompletely characterized. Primary immunodeficiency (PID) patients suffer recurrent microbial infections, providing a unique opportunity to address this issue. To investigate the potential influence of host immunity on the skin microbiome, we examined skin microbiomes in patients with rare monogenic PIDs: hyper-IgE (STAT3-deficient), Wiskott-Aldrich, and dedicator of cytokinesis 8 syndromes. While specific immunologic defects differ, a shared hallmark is atopic dermatitis (AD)-like eczema. We compared bacterial and fungal skin microbiomes (41 PID, 13 AD, 49 healthy controls) at four clinically relevant sites representing the major skin microenvironments. PID skin displayed increased ecological permissiveness with altered population structures, decreased site specificity and temporal stability, and colonization with microbial species not observed in controls, including Clostridium species and Serratia marcescens. Elevated fungal diversity and increased representation of opportunistic fungi (Candida, Aspergillus) supported increased PID skin permissiveness, suggesting that skin may serve as a reservoir for the recurrent fungal infections observed in these patients. The overarching theme of increased ecological permissiveness in PID skin was counterbalanced by the maintenance of a phylum barrier in which colonization remained restricted to typical human-associated phyla. Clinical parameters, including markers of disease severity, were positively correlated with prevalence of Staphylococcus, Corynebacterium, and other less abundant taxa. This study examines differences in microbial colonization and community stability in PID skin and informs our understanding of host-microbiome interactions, suggesting a bidirectional dialogue between skin commensals and the host organism.

Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

Function of milk polymorphonuclear neutrophil leukocytes in bovine mammary glands infected with Corynebacterium bovis.

Corynebacterium bovis is one of the most commonly isolated bacteria from aseptically collected bovine milk samples. The objective of the current study was to characterize the bovine innate immune response by evaluating milk polymorphonuclear neutrophilic leukocytes (PMNL) in mammary glands infected with C. bovis. Twenty quarters infected with C. bovis and 28 culture-negative quarters (with milk somatic cell count <1×10(5) cells/mL) were used. The percentages of milk PMNL and the PMNL expression of L-selectin (CD62L), ß2-integrin (CD11b), and one of the endothelial-selectin ligands (CD44), as well as the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus, were evaluated by flow cytometry. The apoptosis and necrosis rates of the PMNL were quantified using dual-color flow cytometry with fluorescein-labeled annexin and propidium iodide. The present study revealed a higher percentage of PMNL in the milk from C. bovis-infected quarters, although no significant differences were found in levels of CD44, CD62L, or CD11b expression among the PMNL. A lower percentage of apoptotic PMNL was observed in C. bovis-infected quarters, as well as higher percentages of viable PMNL and of PMNL that produced intracellular ROS. However, no alterations were observed in phagocytosis of Staph. aureus by the PMNL or in intensity of intracellular ROS production by PMNL. Thus, results from this investigation of the PMNL function support, at least in part, the fact that intramammary infections by C. bovis may offer protection against intramammary infections by other bacteria.

Targeting IL-23 by employing a p40 peptide-based vaccine ameliorates murine allergic skin and airway inflammation.

BACKGROUND: Studies have found that the IL-23/Th17 pathway plays an important role in the pathogenesis of atopic dermatitis (AD) and severe and steroid-resistant asthma. Targeting IL-23/Th17 pathway with monoclonal antibodies (mAb) has been successful in the reduction of skin and airway inflammation in animal models. However, the mAb has a short half-life, requiring repeated administrations. For the long-term suppression of IL-23/Th17 pathway, we have previously developed an IL-23p40 peptide-based virus-like particle vaccine, which induces long-lasting autoantibodies to IL-23. OBJECTIVE: We sought to evaluate the effects of this IL-23p40 peptide-based vaccine on the down-regulation of allergic skin and airway inflammation in mice. METHODS: Mice were subcutaneously injected three times with the IL-23p40 vaccine, or the vaccine carrier protein or saline as controls. Two weeks later, mice were epicutaneously sensitized with ovalbumin four times at a 2-week interval. One week after the final sensitization, mice were nasally administrated with ovalbumin daily for 3 days. One day later, bronchoalveolar lavage fluids (BALF), sera, lung and skin tissues were obtained and analysed. RESULTS: Mice immunized with the vaccine produced high levels of IgG antibodies to IL-23, p40 and IL-12 that in vitro inhibited IL-23-dependent IL-17 production. The numbers of total cells, neutrophils, and eosinophils in BALF were significantly reduced in the vaccine group, compared with controls. The levels of IL-13, IL-5, IL-23 and, IL-17 in BALF and levels of serum ovalbumin-specific IgE, IgG1, and total IgE were also significantly decreased. Histological analysis showed less inflammation of the lung and skin tissues in the vaccine group, compared with controls. CONCLUSION AND CLINICAL RELEVANCE: Administration of an IL-23p40 peptide-based vaccine down-regulates allergic skin and airway inflammation, suggesting that this strategy may be a potential therapeutic approach in the treatment of AD and asthma.

[Production of cytokines by vaginal epitheliocytes in the process of interaction with dominant and associative microsymbionts].

AIM: Study the expression of cytokines by vaginal epitheliocytes in the process of interaction with dominant and associative microsymbionts. MATERIALS AND METHODS: IL-8, IL-6, IL-1beta and TNFalpha expression in response to interaction with heat inactivated Lactobacillus spp., Staphylococcus aureus, Escherichia coli, Corynebacterium spp. or their secretory products in comparison with basal expression of cytokines by vaginal epitheliocytes was studied. Results. Lactobacilli secretory products were shown not to influence the expression of IL-8 and IL-1beta and moderately stimulated IL-6 and TNFalpha expression. Contact of epitheliocytes with heat inactivated lactobacilli increased secretion of IL-8, IL-6 and IL-1beta and reduced TNFalpha production. Secretory products of S. aureus and E. coli caused stimulation of IL-6, IL-1beta production and practically did not change the expression of IL-8 and TNFalpha. Contact of epitheliocytes with heat inactivated S. aureus sup pressed TNFalpha production and had no influence on IL-8, IL-6 and IL-1beta expression, contact with E. colistimulated TNFalpha and IL-1beta expression and suppressed IL-6 expression. Changes in cytokine expression during interaction of epitheliocytes with corynebacteria were largely similar to the results of interaction with lactobacilli except IL-6 production that was markedly stimulated by corynebacteria secretory products. Conclusion. In epithelial-bacterial interactions dominant and associative microorganisms have a differential effect on functional status of mucosal epitheliocytes manifesting in production of cytokines that could be the basis of mucosal immunity regulation.

Peculiar immunobiology of bone marrow allografts. I. Graft rejection by irradiated responder mice.

Mice are capable of rejecting H-2-incompatible bone marrow grafts after a single lethal exposure to X-rays. The onset of rejection begins 18-24 hr after transplantation and is completed by 96 hr. Maturation of this type of allograft reactivity does not occur until the 22nd day of life. In adult mice, the resistance to marrow allografts can be weakened by administration of cyclophosphamide or dead cultures of Corynebacterium parvum, but not heterologous anti-thymocyte serum. Sublethal exposures to X-rays 7 or 14 days before transplantation also weaken resistance. There is considerable interstrain variation in the ability of mice to resist allografts, even when H-2 differences between hosts and donor are kept identical. Although H-2 incompatibility is a necessary prerequisite for resistance, additional genetic factors influence the outcome of marrow allografts, presumably by controlling recognition. The regulator genes are determinant specific and the alleles for resistance or responder status appear to be dominant. The responder phenotype is expressed by hemopoietic cells and not by the environment. Accordingly, resistance is conferred to otherwise susceptible mice upon transfer of bone marrow cells but not of serum. The production and differentiation of effector cells for marrow graft rejection are thymus independent. In conclusion, bone marrow allografts elicit a particular transplantation reaction, previously unknown, in irradiated mice. Peculiar features of this reaction are the lack of proliferation of host lymphoid cells, tissue specificity, thymus independence, and regulation by genetic factors which apparently do not affect the fate of other grafts.

Cytokine Levels in the In Vitro Response of T Cells to Planktonic and Biofilm Corynebacterium amycolatum.

Unravelling of the interplay between the immune system and non-diphtheria corynebacteria would contribute to understanding their increasing role as medically important microorganisms. We aimed at the analysis of pro- (TNF, IL-1ß, IL-6, IL-8, and IL-12p70) and anti-inflammatory (IL-10) cytokines produced by Jurkat T cells in response to planktonic and biofilm Corynebacterium amycolatum. Two reference strains: C. amycolatum ATCC 700207 (R-CA), Staphylococcus aureus ATCC 25923 (R-SA), and ten clinical strains of C. amycolatum (C-CA) were used in the study. Jurkat T cells were stimulated in vitro by the planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM) derived from the relevant cultures of the strains tested. The cytokine concentrations were determined in the cell culture supernatants using the flow cytometry. The levels of the cytokines analyzed were lower after stimulation with the BCM when compared to the PCM derived from the cultures of C-CA; statistical significance (p < 0.05) was observed for IL-1ß, IL-12 p70, and IL-10. Similarly, planktonic R-CA and R-SA stimulated a higher cytokine production than their biofilm counterparts. The highest levels of pro-inflammatory IL-8, IL-1ß, and IL-12p70 were observed after stimulation with planktonic R-SA whereas the strongest stimulation of anti-inflammatory IL-10 was noted for the BCM derived from the mixed culture of both reference species. Our results are indicative of weaker immunostimulatory properties of the biofilm C. amycolatum compared to its planktonic form. It may play a role in the persistence of biofilm-related infections. The extent of the cytokine response can be dependent on the inherent virulence of the infecting microorganism.Unravelling of the interplay between the immune system and non-diphtheria corynebacteria would contribute to understanding their increasing role as medically important microorganisms. We aimed at the analysis of pro- (TNF, IL-1ß, IL-6, IL-8, and IL-12p70) and anti-inflammatory (IL-10) cytokines produced by Jurkat T cells in response to planktonic and biofilm Corynebacterium amycolatum. Two reference strains: C. amycolatum ATCC 700207 (R-CA), Staphylococcus aureus ATCC 25923 (R-SA), and ten clinical strains of C. amycolatum (C-CA) were used in the study. Jurkat T cells were stimulated in vitro by the planktonic-conditioned medium (PCM) and biofilm-conditioned medium (BCM) derived from the relevant cultures of the strains tested. The cytokine concentrations were determined in the cell culture supernatants using the flow cytometry. The levels of the cytokines analyzed were lower after stimulation with the BCM when compared to the PCM derived from the cultures of C-CA; statistical significance (p < 0.05) was observed for IL-1ß, IL-12 p70, and IL-10. Similarly, planktonic R-CA and R-SA stimulated a higher cytokine production than their biofilm counterparts. The highest levels of pro-inflammatory IL-8, IL-1ß, and IL-12p70 were observed after stimulation with planktonic R-SA whereas the strongest stimulation of anti-inflammatory IL-10 was noted for the BCM derived from the mixed culture of both reference species. Our results are indicative of weaker immunostimulatory properties of the biofilm C. amycolatum compared to its planktonic form. It may play a role in the persistence of biofilm-related infections. The extent of the cytokine response can be dependent on the inherent virulence of the infecting microorganism.

Characterization of Demodex musculi Infestation, Associated Comorbidities, and Topographic Distribution in a Mouse Strain with Defective Adaptive Immunity.

A colony of B6.Cg-Rag1tm1Mom Tyrp1B-w Tg(Tcra,Tcrb)9Rest (TRP1/TCR) mice presented with ocular lesions and ulcerative dermatitis. Histopathology, skin scrapes, and fur plucks confirmed the presence of Demodex spp. in all clinically affected and subclinical TRP1/TCR mice examined (n = 48). Pasteurella pneumotropica and Corynebacterium bovis, both opportunistic pathogens, were cultured from the ocular lesions and skin, respectively, and bacteria were observed microscopically in abscesses at various anatomic locations (including retroorbital sites, tympanic bullae, lymph nodes, and reproductive organs) as well as the affected epidermis. The mites were identified as Demodex musculi using the skin fragment digestion technique. Topographic analysis of the skin revealed mites in almost all areas of densely haired skin, indicating a generalized demodecosis. The percentage of infested follicles in 8- to 10-wk-old mice ranged from 0% to 21%, and the number of mites per millimeter of skin ranged from 0 to 3.7. The head, interscapular region, and middorsum had the highest proportions of infested follicles, ranging from 2.3% to 21.1% (median, 4.9%), 2.0% to 16.6% (8.1%), and 0% to 17% (7.6%), respectively. The pinnae and tail skin had few or no mites, with the proportion of follicles infested ranging from 0% to 3.3% (0%) and 0% to 1.4% (0%), respectively. The number of mites per millimeter was strongly correlated with the percentage of infested follicles. After administration of amoxicillin-impregnated feed (0.12%), suppurative infections were eliminated, and the incidence of ulcerative dermatitis was dramatically reduced. We hypothesize that the Rag1-null component of the genotype makes TRP1/TCR mice susceptible to various opportunistic infestations and infections, including Demodex mites, P. pneumotropica, and C. bovis. Therefore, Rag1-null mice may serve as a useful model to study human and canine demodecosis. D. musculi should be ruled out as a contributing factor in immunocompromised mouse strains with dermatologic manifestations.

Nonspecific Immunotherapy of Murine Solid Tumors With Corynebacterium granulosum.

A single intraperitoneal (ip) or intravenous (iv) injection of Corynebacterium granulosum into C3Hf/Bu mice shortly after subcutaneous (sc) injection of cells from a strongly antigenic syngeneic fibrosarcoma induced by 3-methylcholanthrene caused complete and lasting regressions of 100 and 70% of resulting tumors, respectively. Treatment with this bacterium sc only slightly inhibited the growth of some tumors. C. granulosum given iv to mice 3 days after the sc injection of fibrosarcoma cells caused complete regressions of 39 of 45 tumors; two iv injections with this immunostimulant given 1 month apart were no more effective than a single injection. Intralesional treatment of fibrosarcomas 8 mm in diameter induced complete regressions of tumors in 30% of the animals, whereas sc treatment contralateral to the growing tumor only slightly reduced tumor growth. Intraperitoneal growth of a fibrosarcoma was efficiently controlled (58-80% survival of mice) if C. granulosum was given ip, but not iv, 3 days after inoculation with tumor cells. Again, two injections of C. granulosum (given ip 4 days apart) were only as effective as a single injection. Treatment with C. granulosum iv at 3, 7, 14, or 21 days after sc inoculation of a weakly antigenic, spontaneously arising mammary carcinoma (MC-1) strongly inhibited tumor growth. Three complete but temporary tumor regressions were observed. The subcutaneous growth of another spontaneous mammary carcinoma (MC-2), which contained fairly strong tumor-specific antigen(s), was also significantly inhibited if C. granulosum was given 3,7, or 14 days after, but not 7 days before, tumor cell inoculation. However, pretreatment of mice with the immunostimulant significantly protected the mice against artifically induced pulmonary metastases of this tumor.

In vitro destruction of tumor cells by macrophages from mice treated with Corynebacterium granulosum.

Peritoneal macrophages from C3Hf/Bu mice treated with killed Corynebacterium granulosum bacteria were tested for their effect on in vitro growth of syngeneic fibrosarcoma cells, tumorigenic mouse L-P59 cells, human malignant melanoma cells, allogeneic fibroblasts, erythrocytes, and epithelial kidney cells. Only the cell cultures having neoplastic properties were destroyed by stimulated macrophages; the rate of tumor cell destruction was greater as the ratio of effector to target cells was increased. Neither irradiation nor trypsinization of macrophage monolayers altered the cytotoxicity of stimulated macrophages. The results indicated that C. granulosum activated macrophages to destroy tumor cells in an immunologically nonspecific manner but had no cytotoxic effect on normal allogeneic cells.

In vivo cortisone sensitivity of nonspecific antitumor activity of Corynebacterium parvum-activated mouse peritoneal macrophages.

The nonspecific antitumor activity of Corynebacterium parvum-activated mouse peritoneal macrophages in vitro was inhibited by their prior treatment with cortisone acetate in vivo. Inhibition was marked (60 percent) 24 hours after cortisone injection and was still significant at 7 days, but antitumor activity was completely recovered by 9 days. This transient inhibition was sufficient to abolish the non-specific protection afforded by C. parvum in an in vivo pretreatment model.

Intralesional immunotherapy of brain tumors with combined Corynebacterium parvum and recombinant interleukin-2 in mice.

Clinical observations and experimental work suggested that inflammatory cells attracted to the brain exert a nonspecific antineoplastic effect. Intralesional treatment of implanted malignant murine brain tumors (KHT sarcomas) with killed Corynebacterium parvum produced an inflammatory cell infiltrate and increased survival in C3H mice relative to that in untreated control C3H mice. This antitumor effect was enhanced when recombinant interleukin-2 was sequentially added as a second intralesional immunomodifier. A high percentage of mice so treated were cured. Inflammatory cells in the brains of treated mice divided for 1-2 weeks, and metabolic activity of astrocytes increased. These findings form the basis for a recently initiated immunotherapy protocol in patients with recurrent glioblastoma multiforme.

Effect of corynebacterium liquefaciens on a C3Hf mouse squamous cell carcinoma.

The antitumor effect of anaerobic Corynebacterium liquefaciens was compared with that of specific immunization. Experimental tumors were fourth or fifth generation isotransplants of a NR-Sl squamous cell carcinoma that arose spontaneously in a C3Hf/He female mouse. Specific immunization failed to exhibit an antitumor effect, whereas a single administration of the bacterium markedly inhibited the growth of the tumor. This growth inhibition was most effective when C. liquefaciens was administered 2 to 4 days before transplantation of tumor cells, but marked inhibition was also observed when this agent was administered after transplantation. The inhibitory effect was independent of dose within a range of 0.1 to 2.0 mg/mouse; a single dose of less than 0.05 mg/mouse did not exhibit antitumor effect. Multiple administrations of large doses, if given with short treatment intervals, were no more effective than one small dose. Multiple doses given at 14-day intervals resulted in marked growth retardation. The dose of cells that produced 50% tumor takes in C. liquefaciens-treated animals was not significantly different from that in nontreated animals, indicating that this bacterium exhibited no lethal effect on the tumor cells studied.