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Radiotherapy is inevitably intertwined with various side effects impairing the quality of life of cancer patients. Here, we report the possibility that alterations of the oral microbiota influence the therapeutic efficacy and prognosis of radiotherapy for primary rectal cancer and colorectal cancer (CRC) liver metastases that pathologically disrupt gastrointestinal integrity and function. 16S rRNA sequencing shows that oral microbiota alterations change the gut bacterial composition within tumors but not in adjacent peritumor tissues in CRC mouse models. Specifically, buccal Fusobacterium nucleatum migrates to the CRC locus and impairs the therapeutic efficacy and prognosis of radiotherapy. Administration of a specific antibiotic, metronidazole, abrogates the adverse effects of oral microbiome fluctuation on radiotherapy for CRC. The oral microbiota were also associated with radiation-induced intestinal injury via intestinal microbes. Our findings demonstrate that the oral microbiome in synergy with its intestinal counterparts impinges on the efficacy and prognosis of radiotherapy for CRC., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

The availability of L-arginine in tumours is a key determinant of an efficient anti-tumour T cell response 1-4 . Consequently, increases of typically low L-arginine concentrations within the tumour may greatly potentiate the anti-tumour responses of immune checkpoint inhibitors, such as programmed death-ligand 1 (PD-L1)-blocking antibodies 5 . However, currently no means are available to locally increase intratumoural L-arginine levels. Here we used a synthetic biology approach to develop an engineered probiotic Escherichia coli Nissle 1917 strain that colonizes tumours and continuously converts ammonia, a metabolic waste product that accumulates in tumours 6 , to L-arginine. Colonization of tumours with these bacteria increased intratumoural L-arginine concentrations, increased the number of tumour-infiltrating T cells and had marked synergistic effects with PD-L1 blocking antibodies in the clearance of tumours. The anti-tumour effect of these bacteria was mediated by L-arginine and was dependent on T cells. These results show that engineered microbial therapies enable metabolic modulation of the tumour microenvironment leading to enhanced efficacy of immunotherapies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background & Aims: Streptococcus thermophilus was identified to be depleted in patients with colorectal cancer (CRC) by shotgun metagenomic sequencing of 526 multicohort fecal samples. Here, we aim to investigate whether this bacterium could act as a prophylactic for CRC prevention., Methods: The antitumor effects of S thermophilus were assessed in cultured colonic epithelial cells and in 2 murine models of intestinal tumorigenesis. The tumor-suppressive protein produced by S thermophilus was identified by mass spectrometry and followed by β-galactosidase activity assay. The mutant strain of S thermophilus was constructed by homologous recombination. The effect of S thermophilus on the gut microbiota composition was assessed by shotgun metagenomic sequencing., Results: Oral gavage of S thermophilus significantly reduced tumor formation in both Apc min/+ and azoxymethane-injected mice. Coincubation with S thermophilus or its conditioned medium decreased the proliferation of cultured CRC cells. β-Galactosidase was identified as the critical protein produced by S thermophilus by mass spectrometry screening and β-galactosidase activity assay. β-Galactosidase secreted by S thermophilus inhibited cell proliferation, lowered colony formation, induced cell cycle arrest, and promoted apoptosis of cultured CRC cells and retarded the growth of CRC xenograft. The mutant S thermophilus without functional β-galactosidase lost its tumor-suppressive effect. Also, S thermophilus increased the gut abundance of known probiotics, including Bifidobacterium and Lactobacillus via β-galactosidase. β-Galactosidase-dependent production of galactose interfered with energy homeostasis to activate oxidative phosphorylation and downregulate the Hippo pathway kinases, which partially mediated the anticancer effects of S thermophilus., Conclusion: S thermophilus is a novel prophylactic for CRC prevention in mice. The tumor-suppressive effect of S thermophilus is mediated at least by the secretion of β-galactosidase., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Administration of probiotics to regulate the immune system is a potential anti-tumor strategy. However, oral administration of probiotics is ineffective because of the poor inhabitation of exogenous bacteria in host intestines. Here we report that smectite, a type of mineral clay and established anti-diarrhea drug, promotes expansion of probiotics (especially Lactobacillus) in the murine gut and subsequently elicits anti-tumor immune responses. The ion-exchangeable microstructure of smectite preferentially promotes lactic acid bacteria (LABs) to form biofilms on smectite in vitro and in vivo. In mouse models, smectite laden with LAB biofilms (Lactobacillus and Bifidobacterium) inhibits tumor growth (when used alone) and enhances the efficacy of chemotherapy or immunotherapy (when used in combination with either of them) by activating dendritic cells (DCs) via Toll-like receptor 2 (TLR2) signaling. Our findings suggest oral administration of smectite as a promising strategy to enrich probiotics in vivo for cancer immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background & Aims: Immune checkpoint inhibitors have limited efficacy in many tumors. We investigated mechanisms of tumor resistance to inhibitors of programmed cell death-1 (PDCD1, also called PD-1) in mice with gastric cancer, and the role of its ligand, PD-L1., Methods: Gastrin-deficient mice were given N-methyl-N-nitrosourea (MNU) in drinking water along with Helicobacter felis to induce gastric tumor formation; we also performed studies with H/K-ATPase-hIL1B mice, which develop spontaneous gastric tumors at the antral-corpus junction and have parietal cells that constitutively secrete interleukin 1B. Mice were given injections of an antibody against PD-1 or an isotype control before tumors developed, or anti-PD-1 and 5-fluorouracil and oxaliplatin, or an antibody against lymphocyte antigen 6 complex locus G (also called Gr-1), which depletes myeloid-derived suppressor cells [MDSCs]), after tumors developed. We generated knock-in mice that express PD-L1 specifically in the gastric epithelium or myeloid lineage., Results: When given to gastrin-deficient mice before tumors grew, anti-PD-1 significantly reduced tumor size and increased tumor infiltration by T cells. However, anti-PD-1 alone did not have significant effects on established tumors in these mice. Neither early nor late anti-PD-1 administration reduced tumor growth in the presence of MDSCs in H/K-ATPase-hIL-1β mice. The combination of 5-fluorouracil and oxaliplatin reduced MDSCs, increased numbers of intra-tumor CD8 + T cells, and increased the response of tumors to anti-PD-1; however, this resulted in increased tumor expression of PD-L1. Expression of PD-L1 by tumor or immune cells increased gastric tumorigenesis in mice given MNU. Mice with gastric epithelial cells that expressed PD-L1 did not develop spontaneous tumors, but they developed more and larger tumors after administration of MNU and H felis, with accumulation of MDSCs., Conclusions: In mouse models of gastric cancer, 5-fluorouracil and oxaliplatin reduce numbers of MDSCs to increase the effects of anti-PD-1, which promotes tumor infiltration by CD8 + T cells. However, these chemotherapeutic agents also induce expression of PD-L1 by tumor cells. Expression of PD-L1 by gastric epithelial cells increases tumorigenesis in response to MNU and H felis, and accumulation of MDSCs, which promote tumor progression. The timing and site of PD-L1 expression is therefore important in gastric tumorigenesis and should be considered in design of therapeutic regimens., (Copyright © 2021 AGA Institute. Published by Elsevier Inc. All rights reserved.)

A critical limitation of Salmonella typhimurium (S. typhimurium) as an anti-cancer agent is the loss of their invasive or replicative activities, which results in no or less delivery of anti-cancer agents inside cancer cells in cancer therapy. Here we developed an oxytolerant attenuated Salmonella strain (KST0650) from the parental KST0649 (ΔptsIΔcrr) strain using radiation mutation technology (RMT). The oxytolerant KST0650 strain possessed 20-times higher replication activity in CT26 cancer cells and was less virulent than KST0649. Furthermore, KST0650 migrated effectively into tumor tissues in mice. KST0650 was further equipped with a plasmid harboring a spliced form of the intracellular pro-apoptotic protein sATF6, and the expression of sATF6 was controlled by the radiation-inducible recN promoter. The new strain was named as KST0652, in which sATF6 protein expression was induced in response to radiation in a dose-dependent manner. This strain was effectively delivered inside cancer cells and tumor tissues via the Salmonella type III secretion system (T3SS). In addition, combination treatment with KST0652 and radiation showed a synergistic anti-tumor effect in murine tumor model with complete inhibition of tumor growth and protection against death. In conclusion, we showed that RMT can be used to effectively develop an anti-tumor Salmonella strain for delivering anti-cancer agents inside tumors.

Tightly regulated immune responses must occur in the intestine to avoid unwanted inflammation, which may cause chronic sequela leading to diseases such as colorectal cancer. Toll-like receptors play an important role in preventing aberrant immune responses in the intestine by sensing endogenous commensal microbiota and delivering important regulatory signals to the tissue. However, the role that specific innate receptors may play in the development of chronic inflammation and their impact on the composition of the colonic microbiota is not well understood. Using a model of inflammation-induced colorectal cancer, we found that Lactobacillus species are lost more quickly in wild-type (WT) mice than TLR6-deficient mice resulting in overall differences in bacterial composition. Despite the longer retention of Lactobacillus , the TLR6-deficient mice presented with more tumors and a worse overall outcome. Restoration of the lost Lactobacillus species suppressed inflammation, reduced tumor number, and prevented change in the abundance of Proteobacteria only when given to WT mice, indicating the effect of these Lactobacillus are TLR6 dependent. We found that the TLR6-dependent effects of Lactobacillus could be dissociated from one another via the involvement of IL10, which was necessary to dampen the inflammatory microenvironment, but had no effect on bacterial composition. Altogether, these data suggest that innate immune signals can shape the composition of the microbiota under chronic inflammatory conditions, bias the cytokine milieu of the tissue microenvironment, and influence the response to microbiota-associated therapies., (©2019 American Association for Cancer Research.)

There is limited understanding of how walnut consumption inhibits the development of colorectal cancer. A possible mechanism may involve alterations to the gut microbiota. In this study, the effects of walnut on gut microbiota were tested in a mouse tumor bioassay using the colonotropic carcinogen, azoxymethane (AOM) added to the total Western diet (TWD). 16S rRNA pyrosequencing identified three enterotype-like clusters (E1, E2, and E3) in this murine model. E1, E2, and E3 are associated with AOM exposure, walnut consumption, and TWD diet, respectively. E2 and E3 showed distinct taxonomic and functional characteristics, while E1 represented an intermediate state. At the family level, E1 and E3 were both enriched with Bacteroidaceae , but driven by two different operational taxonomic units (OTU; OTU-2 for E1, OTU-4 for E3). E2 was overrepresented with Porphyromonadaceae and Lachnospiraceae , with OTU-3 (family Porphyromonadaceae ) as the "driver" OTU for this cluster. Functionally, E3 is overrepresented with genes of glycan biosynthesis and metabolism, xenobiotic metabolism, and lipid metabolism. E2 is enriched with genes associated with cell motility, replication and repair, and amino acid metabolism. Longitudinally, E2 represents the gut microbial status of early life in these mice. In comparison with E1 and E3, E2 is associated with a moderate lower tumor burden ( P = 0.12). Our results suggest that walnuts may reduce the risk of colorectal cancer within a Western diet by altering the gut microbiota. Our findings provide further evidence that colorectal cancer risk is potentially modifiable by diet via alterations to the microbiota., (©2019 American Association for Cancer Research.)

Inulin-rich foods exert a prebiotic effect, as this polysaccharide is able to enhance beneficial colon microbiota populations, giving rise to the in situ production of short-chain fatty acids (SCFAs) such as propionic and butyric acids. These SCFAs are potent preventive agents against colorectal cancer due to their histone deacetylases inhibitory properties, which induce apoptosis in tumor colonocytes. As colorectal cancer is the fourth most common neoplasia in Europe with 28.2 new cases per 100,000 inhabitants, a cost-effective preventive strategy has been tested in this work by redesigning common porcine meat products (chorizo sausages and cooked ham) consumed by a substantial proportion of the population towards potential colorectal cancer preventive functional foods. In order to test the preventive effect of these inulin-rich meat products against colorectal cancer, an animal model (Rattus norvegicus F344) was used, involving two doses of azoxymethane (10 mg/kg) and two treatments with dextran sodium sulfate (DSS) during a 20-week assay period. Control feed, control sausages, functional sausages (15.7% inulin), control cooked ham and functional cooked ham (10% inulin) were used to feed the corresponding animal cohorts. Then, the animals were sacrificed and their digestive tract tissues were analyzed. The results showed a statistically significant 49% reduction in the number of colon polyps in the functional meat products cohorts with respect to the control meat products animals, as well as an increase in the cecum weight (an indicator of a diet rich in prebiotic fiber), a 51.8% increase in colon propionate production, a 39.1% increase in colon butyrate concentrations, and a reduction in the number of hyperplastic Peyer's patches. Metagenomics studies also demonstrated colon microbiota differences, revealing a significant increase in Bacteroidetes populations in the functional meat products (mainly due to an increase in Bacteroidaceae and Prevotellaceae families, which include prominent propionate producers), together with a reduction in Firmicutes (especially due to lower Lachnospiraceae populations). However, functional meat products showed a remarkable increase in the anti-inflammatory and fiber-fermentative Blautia genus, which belongs to this Lachnospiraceae family. The functional meat products cohorts also presented a reduction in important pro-inflammatory bacterial populations, such as those of the genus Desulfovibrio and Bilophila. These results were corroborated in a genetic animal model of CRC (F344/NSlc-Apc 1588/kyo ) that produced similar results. Therefore, processed meat products can be redesigned towards functional prebiotic foods of interest as a cost-effective dietary strategy for preventing colorectal cancer in human populations.

Chronic inflammation and gut microbiota dysbiosis, in particular the bloom of genotoxin-producing E. coli strains, are risk factors for the development of colorectal cancer. Here, we sought to determine whether precision editing of gut microbiota metabolism and composition could decrease the risk for tumor development in mouse models of colitis-associated colorectal cancer (CAC). Expansion of experimentally introduced E. coli strains in the azoxymethane/dextran sulfate sodium colitis model was driven by molybdoenzyme-dependent metabolic pathways. Oral administration of sodium tungstate inhibited E. coli molybdoenzymes and selectively decreased gut colonization with genotoxin-producing E. coli and other Enterobacteriaceae. Restricting the bloom of Enterobacteriaceae decreased intestinal inflammation and reduced the incidence of colonic tumors in two models of CAC, the azoxymethane/dextran sulfate sodium colitis model and azoxymethane-treated, Il10 -deficient mice. We conclude that metabolic targeting of protumoral Enterobacteriaceae during chronic inflammation is a suitable strategy to prevent the development of malignancies arising from gut microbiota dysbiosis., (© 2019 Zhu et al.)

Mucus-invasive bacterial biofilms are identified on the colon mucosa of approximately 50% of colorectal cancer (CRC) patients and approximately 13% of healthy subjects. Here, we test the hypothesis that human colon biofilms comprise microbial communities that are carcinogenic in CRC mouse models. Homogenates of human biofilm-positive colon mucosa were prepared from tumor patients (tumor and paired normal tissues from surgical resections) or biofilm-positive biopsies from healthy individuals undergoing screening colonoscopy; homogenates of biofilm-negative colon biopsies from healthy individuals undergoing screening colonoscopy served as controls. After 12 weeks, biofilm-positive, but not biofilm-negative, human colon mucosal homogenates induced colon tumor formation in 3 mouse colon tumor models (germ-free ApcMinΔ850/+;Il10-/- or ApcMinΔ850/+ and specific pathogen-free ApcMinΔ716/+ mice). Remarkably, biofilm-positive communities from healthy colonoscopy biopsies induced colon inflammation and tumors similarly to biofilm-positive tumor tissues. By 1 week, biofilm-positive human tumor homogenates, but not healthy biopsies, displayed consistent bacterial mucus invasion and biofilm formation in mouse colons. 16S rRNA gene sequencing and RNA-Seq analyses identified compositional and functional microbiota differences between mice colonized with biofilm-positive and biofilm-negative communities. These results suggest human colon mucosal biofilms, whether from tumor hosts or healthy individuals undergoing screening colonoscopy, are carcinogenic in murine models of CRC.

Over the past decades, Salmonella has been proven capable of inhibiting tumor growth. It can specifically target tumors and due to its facultative anaerobic property, can be more penetrative than other drug therapies. However, the molecular mechanism by which Salmonella inhibits tumor growth is still incompletely known. The antitumor therapeutic effect mediated by Salmonella is associated with an inflammatory immune response at the tumor site and a T cell-dependent immune response. Many tumors have been proven to have a high expression of indoleamine 2, 3-dioxygenase 1 (IDO), which is a rate-limiting enzyme that catalyzes tryptophan to kynurenine, thus causing immune tolerance within the tumor microenvironment. With decreased expression of IDO, increased immune response can be observed, which might be helpful when developing cancer immunotherapy. The expression of IDO was decreased after tumor cells were infected with Salmonella. In addition, Western blot analysis showed that the expression levels of phospho-protein kinase B (P-AKT), phospho-mammalian targets of rapamycin (P-mTOR), and phospho-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased after Salmonella infection. In conclusion, our results indicate that Salmonella inhibits IDO expression and plays a crucial role in anti-tumor therapy, which might be a promising strategy combined with other cancer treatments.

IL-18 is a proinflammatory and immune regulatory cytokine, member of the IL-1 family. IL-18 was initially identified as an IFN-γ-inducing factor in T and NK cells, involved in Th1 responses. IL-18 is produced as an inactive precursor (pro-IL-18) that is enzymatically processed into a mature form by Casp1. Different cells, such as macrophages, DCs, microglial cells, synovial fibroblasts, and epithelial cells, express pro-IL-18, and the production of bioactive IL-18 is mainly regulated at the processing level. PAMP or DAMP molecules activate inflammasomes, which trigger Casp1 activation and IL-18 conversion. The natural inhibitor IL-18BP , whose production is enhanced by IFN-γ and IL-27, further regulates IL-18 activity in the extracellular environment. Inflammasomes and IL-18 represent double-edged swords in cancer, as their activation may promote tumor development and progression or oppositely, enhance anti-tumor immunity and limit tumor growth. IL-18 has shown anti-tumor activity in different preclinical models of cancer immunotherapy through the activation of NK and/or T cell responses and has been tested in clinical studies in cancer patients. However, the dual role of IL-18 in different experimental tumor models and human cancers raises critical issues on its therapeutic use in cancer. This review will summarize the biology of the IL-18/IL-18R/IL-18BP system and will address the role of IL-18 and its inhibitor, IL-18BP, in cancer biology and immunotherapy., (© Society for Leukocyte Biology.)

Bacteria of many species are able to invade and colonize solid tumours in mice. We have focused on Salmonella enterica serovar Typhimurium. Detailed analysis revealed that such tumour-invading Salmonella form biofilms, thus providing a versatile in vivo test system for studying bacterial phenotypes and host-pathogen interactions. It appears that biofilm formation by S. typhimurium is induced as a defence against the immune system of the host, and in particular against neutrophils. Further, we extended our work to the clinically more relevant biofilm infection by Pseudomonas aeruginosa. The induction of P. aeruginosa biofilms in neoplastic tissue appears to be elicited as a reaction against the immune system. Reconstitution experiments reveal that T cells are responsible for biofilm induction. Isogenic mutants that are no longer able to form biofilms can be used for comparison studies to determine antimicrobial resistance, especially therapeutic efficacy against P. aeruginosa located in biofilms., (© 2014 The Association for the Publication of the Journal of Internal Medicine.)

Salmonella enterica is a facultative anaerobic bacteria, whose ability to colonize antigen-presenting cells (APCs) such as dendritic cells and macrophages, has allowed its successful use as an alive, attenuated bacterial vector for vaccination. Salmonella enterica elicits efficient cellular, humoral and mucosal immune responses, against heterologous antigens including viruses, parasites, other bacterial species and tumor-associated antigens, since it is capable of delivering these antigens to cells of the immune system. The extracellular expression of heterologous antigens on the surface of Salmonella enterica via its type I, III and V secretion systems, and their delivery into infected cells is essential for its stimulation of immune responses against these antigens. Moreover, Salmonella enterica is a promising therapeutic agent against cancer, as demonstrated by reports of pre-clinical and clinical studies indicating that, after systemic administration, Salmonella enterica preferentially localizes in solid tumors and metastases as compared to normal tissues. In this review, we focus on novel prophylactic and therapeutic anti-cancer approaches using Salmonella enterica as a delivery system of heterologous molecules with the aim of inhibiting tumor growth.

The ability of opportunistic bacterial pathogens to grow in biofilms is decisive in the pathogenesis of chronic infectious diseases. Growth within biofilms does not only protect the bacteria against the host immune system but also from the killing by antimicrobial agents. Here, we introduce a mouse model in which intravenously administered planktonic Pseudomonas aeruginosa bacteria are enriched in transplantable subcutaneous mouse tumors. Electron microscopy images provide evidence that such bacteria reside in the tumor tissue within biofilm structures. Immunohistology furthermore demonstrated that infection of the tumor tissue elicits a host response characterized by strong neutrophilic influx. Interestingly, the biofilm defective PA14 pqsA transposon mutant formed less biofilm in vivo and was more susceptible to clearance by intravenous ciprofloxacin treatment as compared to the wild-type control. In conclusion, we have established an experimentally tractable model that may serve to identify novel bacterial and host factors important for in vivo biofilm formation and to re-evaluate bactericidal and anti-biofilm effects of currently used and novel antibacterial compounds., (Copyright © 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Conventional cancer therapies are often limited in effectiveness and exhibit strong side effects. Therefore, alternative therapeutic strategies are demanded. The employment of tumor-colonizing bacteria that exert anticancer effects is such a novel approach that attracts increasing attention. For instance, Salmonella enterica serovar Typhimurium has been used in many animal tumor models as well as in first clinical studies. These bacteria exhibit inherent tumoricidal effects. In addition, they can be used to deliver therapeutic agents. However, bacterial expression has to be restricted to the tumor to prevent toxic substances from harming healthy tissue. Therefore, we screened an S. Typhimurium promoter-trap library to identify promoters that exclusively drive gene expression in the cancerous tissue. Twelve elements could be detected that show reporter gene expression in tumors but not in spleen and liver. In addition, a DNA motif was identified that appears to be necessary for tumor specificity. Now, such tumor-specific promoters can be used to safely express therapeutic proteins by tumor-colonizing S. Typhimurium directly in the neoplasia.

Pseudomonas aeruginosa is a threatening, opportunistic pathogen causing disease in immunocompromised individuals. The hallmark of P. aeruginosa virulence is its multi-factorial and combinatorial nature. It renders such bacteria infectious for many organisms and it is often resistant to antibiotics. To gain insights into the physiology of P. aeruginosa during infection, we assessed the transcriptional programs of three different P. aeruginosa strains directly after isolation from burn wounds of humans. We compared the programs to those of the same strains using two infection models: a plant model, which consisted of the infection of the midrib of lettuce leaves, and a murine tumor model, which was obtained by infection of mice with an induced tumor in the abdomen. All control conditions of P. aeruginosa cells growing in suspension and as a biofilm were added to the analysis. We found that these different P. aeruginosa strains express a pool of distinct genetic traits that are activated under particular infection conditions regardless of their genetic variability. The knowledge herein generated will advance our understanding of P. aeruginosa virulence and provide valuable cues for the definition of prospective targets to develop novel intervention strategies.

The probiotic bacterium Escherichia coli Nissle 1917 (EcN) constitutes a prospective vector for delivering heterologous therapeutic molecules to treat several human disorders. To add versatility to this carrier system, bacteria should be equipped with expression modules that can be regulated deliberately in a temporal and quantitative manner. This approach is called in vivo remote control (IVRC) of bacterial vectors. Here, we have evaluated promoters P(araBAD), P(rhaBAD) and P(tet), which can be induced with L-arabinose, L-rhamnose or anhydrotetracycline, respectively. EcN harboring promoter constructs with luciferase as reporter gene were administered either orally to healthy mice or intravenously to tumor bearing animals. Subsequent to bacterial colonization of tissues, inducer substances were administered via the oral or systemic route. By use of in vivo bioluminescence imaging, the time course of reporter gene expression was analyzed. Each promoter displayed a specific in vivo induction profile depending on the niche of bacterial residence and the route of inducer administration. Importantly, we also observed colonization of gall bladders of mice when EcN was administered systemically at high doses. Bacteria in this anatomical compartment remained accessible to remote control of bacterial gene expression.

Background: Several facultative anaerobic bacteria with potential therapeutic abilities are known to preferentially colonize solid tumors after systemic administration. How they efficiently find and invade the tumors is still unclear. However, this is an important issue to be clarified when bacteria should be tailored for application in cancer therapy., Methodology/principal Findings: We describe the initial events of colonization of an ectopic transplantable tumor by Salmonella enterica serovar Typhimurium. Initially, after intravenous administration, bacteria were found in blood, spleen, and liver. Low numbers were also detected in tumors associated with blood vessels as could be observed by immunohistochemistry. A rapid increase of TNF-alpha in blood was observed at that time, in addition to other pro-inflammatory cytokines. This induced a tremendous influx of blood into the tumors by vascular disruption that could be visualized in H&E stainings and quantified by hemoglobin measurements of tumor homogenate. Most likely, together with the blood, bacteria were flushed into the tumor. In addition, blood influx was followed by necrosis formation, bacterial growth, and infiltration of neutrophilic granulocytes. Depletion of TNF-alpha retarded blood influx and delayed bacterial tumor-colonization., Conclusion: Our findings emphasize similarities between Gram-negative tumor-colonizing bacteria and tumor vascular disrupting agents and show the involvement of TNF-alpha in the initial phase of tumor-colonization by bacteria.

Hypoxia in solid tumors is a major obstacle in conventional treatment because of inefficient delivery of therapeutic agents to the lesions, but offers the potential for anaerobic bacterial colonization that can lead to tumor destruction. We have previously reported a recombinant Clostridium perfringens (Cp) strain constructed by deletion of the superoxide dismutase (sod) gene and insertion of the Panton-Valentine leukocidin (PVL) gene, Cp/sod(-)/PVL, which showed elevated oxygen sensitivity, tumor selectivity, and oncopathic potency in an orthotopic model of pancreatic cancer in immune-competent and syngeneic mice, and that led to substantial prolongation of animal survival. A major limitation to Cp/sod(-)/PVL in clinical applications is that it expresses phospholipase C (plc), the alpha-toxin and the major virulence determinant in Cp that is causative in the development of gas gangrene. In this study, the plc gene in Cp/sod(-)/PVL was knocked out to create Cp/plc(-)/sod(-)/PVL, which was shown to be incapable of inducing gas gangrene in mice. Intravenous injection of Cp/plc(-)/sod(-)/PVL spores led to a significant survival advantage in tumor-bearing mice with the same efficacy as Cp/sod(-)/PVL, indicating that the oncopathic potency of Cp is independent of a functional plc gene. The treatment also did not lead to an attenuated immune response to a subsequent pathogen challenge, indicating that a systemic immune-suppressive effect in the host is absent. Consequently, Cp/plc(-)/sod(-)/PVL is a novel oncopathic bacterial agent for the effective treatment of pancreatic cancer and other poorly vascularized tumors, with a substantially enhanced safety profile, which is essential for the development of translational studies in the future.

Purpose: Bacteria-based tumor-targeted therapy is a modality of growing interest in anticancer strategies. Imaging bacteria specifically targeting and replicating within tumors using radiotracer techniques and optical imaging can provide confirmation of successful colonization of malignant tissue., Experimental Design: The uptake of radiolabeled pyrimidine nucleoside analogues and [18F]FDG by Escherichia coli Nissle 1917 (EcN) was assessed both in vitro and in vivo. The targeting of EcN to 4T1 breast tumors was monitored by positron emission tomography (PET) and optical imaging. The accumulation of radiotracer in the tumors was correlated with the number of bacteria. Optical imaging based on bioluminescence was done using EcN bacteria that encode luciferase genes under the control of an l-arabinose-inducible P(BAD) promoter system., Results: We showed that EcN can be detected using radiolabeled pyrimidine nucleoside analogues, [18F]FDG and PET. Importantly, this imaging paradigm does not require transformation of the bacterium with a reporter gene. Imaging with [18F]FDG provided lower contrast than [18F]FEAU due to high FDG accumulation in control (nontreated) tumors and surrounding tissues. A linear correlation was shown between the number of viable bacteria in tumors and the accumulation of [18F]FEAU, but not [18F]FDG. The presence of EcN was also confirmed by bioluminescence imaging., Conclusion: EcN can be imaged by PET, based on the expression of endogenous E. coli thymidine kinase, and this imaging paradigm could be translated to patient studies for the detection of solid tumors. Bioluminescence imaging provides a low-cost alternative to PET imaging in small animals.

Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8(+) T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8(+) T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8(+) T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.

As a quarantine of biological materials, we tested 96 transplantable tumors and cell lines for contamination with microorganisms in a mouse antibody production (MAP) test, enzymatic assay and microbiological culture. Contamination with lactic dehydrogenase elevating virus (LDV), mycoplasmas and Pasteurella pneumotropica was detected. A considerable difference in the contamination rate was observed between in vivo- and in vitro- propagated tumors. LDV in the tumors could be eliminated by both in vitro subculture and subpassage in nude rats. Mycoplasmas were eliminated by means of the mycoplasma-removal agent and P. pneumotropica by subpassage in mice. These results suggest that there is still a high risk of contamination in transplantable tumors and emphasizes the importance of adequate microbiological quality control.

To quarantine human tumor samples for transplantation into immune deficient mice or tumor xenograft lines established and introduced from other institutions, we performed isolated implantation and passaging of tumors in a vinyl isolator, and microbiological examinations of sentinel mice kept together with tumor bearing mice. We examined 105 pairs of sentinel mice used to quarantine 907 tumors, and found six cases of contamination or infection with Staphylococcus aureus, 20 cases with Pseudomonas aeruginosa and one case with mouse hepatitis virus (MHV). It was, however, possible that Mycoplasma pulmonis contamination was overlooked because the microbe had been isolated from tumors passaged after quarantine, even though the results of the quarantine of these tumors showed no sign of pathogens. Direct culture of tumors for the microbe was recommended to improve the quarantine system.

The 93-bp region downstream of the enhancer (DEN) in the long terminal repeat (LTR) of the mink cell focus-forming virus (MCF13) has been shown to be important for transcriptional activation and viral lymphomagenicity (J. C. Tupper, H. Chen, E. F. Hays, G. C. Bristol, and F. K. Yoshimura, J. Virol. 66:7080-7088, 1992). In this report, we have further explored the role of the DEN region in transcriptional activation. We observed that it has enhancer-like abilities as well as some unique LTR properties. Transcriptional activation by the DEN region involved interactions with enhancer sequences that were either synergistic or additive, depending on the cell type. The most intriguing property of the DEN region is its ability to induce transcription in activated T cells. This activity is unique for the LTR in that no other LTR region can do this. We also examined the role of the DEN region in retroviral lymphomagenesis. We cloned and sequenced proviral LTRs integrated upstream of the cellular c-myc gene from DNA obtained from thymic tumors induced by DEN region deletion mutant viruses in AKR mice. We determined that for transcriptional activation of the c-myc proto-oncogene, enhancer sequences can substitute for the DEN region. This study identifies the significance of non-enhancer sequences in the LTR for the oncogenesis of the MCF13 retrovirus.

Epstein-Barr virus (EBV) recombinants with specifically mutated BCRF1 genes were constructed and compared with wild-type BCRF1 recombinants derived in parallel for the ability to initiate and maintain latent infection and growth transformation in primary human B lymphocytes. A stop codon insertion after codon 116 of the 170-codon BCRF1 open reading frame or deletion of the entire gene had no effect on latent infection, B-lymphocyte proliferation into long-term lymphoblastoid cell lines (LCLs), or virus replication. LCLs infected with the stop codon recombinant were indistinguishable from wild-type recombinant-infected LCLs in tumorigenicity in SCID mice. However, mutant BCRF1 recombinant-infected cells differed from wild-type recombinant-infected cells in their inability to block gamma interferon release in cultures of permissively infected LCLs incubated with autologous human peripheral blood mononuclear cells. This is the first functional assay for BCRF1 expression from the EBV genome. BCRF1 probably plays a key role in modulating the specific and nonspecific host responses to EBV infection.

The nondefective Moloney murine leukemia virus (MuLV) induces clonal or oligoclonal T-cell tumors in mice or rats. The proviruses of these nondefective MuLVs have been shown to act as insertion mutagens most frequently activating an adjacent cellular gene involved in cell growth control. Mutations by provirus insertions, recognized as common provirus integration sites, have been instrumental in identifying novel cellular genes involved in tumor formation. We have searched for new common provirus integration sites in Moloney MuLV-induced thymomas. Using cellular sequences flanking a provirus cloned from one of these tumors, we found one region, designated Mis-2, which was the target of provirus integration in a low (3%) percentage of these tumors. Mis-2 was mapped on mouse chromosome 10, approximately 160 kbp downstream of myb. The Mis-2 region may contain a novel gene involved in tumor development.

Recent reports implicate two DNA tumour viruses, herpes simplex virus type 2 (HSV-2) and human papillomavirus types 16 or 18 (HPV-16 and -18), in the pathogenesis of cervical cancer. Previous studies have indicated that primary human fibroblasts transfected with HPV-16 and HSV-2 morphological transforming region III (mtr III) are more aneuploid than fibroblasts immortalized with HPV-16 and that HSV-2 DNA sequences are retained in transformed cells. Since HSV-2 and HPV typically infect cells of epithelial origin, the interactions of these viruses with respect to morphological transformation were examined in human keratinocytes. HPV-16- or HPV-18-immortalized keratinocytes (FEPL and FEA cells, respectively) were transfected with fragments derived from HSV-2 mtr III. When compared to their normal counterparts, FEPL cells and FEA cells transfected with mtr III fragments grew to higher saturation densities and were morphologically transformed. FEPL cells transformed by HSV-2 were capable of growth in soft agar and, when injected into nu/nu mice, lesions developed at the site of injection. Histological examination of the lesions revealed a benign mass which was composed of squamous epithelial cells that were producing keratin. In contrast, immortalized keratinocytes (FEPL or FEA) or FEA cells transfected with HSV-2 did not produce these lesions. These observations suggest that sequences within mtr III can alter the growth properties of human keratinocytes immortalized by HPV-16 or HPV-18.

Viruses rapidly able to transform cultured chicken bone marrow cells have been isolated from cases of myelocytic myeloid leukosis (MML) induced experimentally by the HPRS-103 strain of avian leukosis virus, and from field cases of MML. HPRS-103 virus itself did not acutely transform cultured bone-marrow cells. These findings suggest that during myeloid leukemogenesis by HPRS-103 virus, recombinant viruses are generated with transduced cellular oncogenes. The transformed cell appeared to be a macrophage precursor cell. Transformed cells in culture lost their proliferative capacity after a few weeks and then tended to resemble more differentiated macrophages. This change could be reversed temporarily by addition of a myelomonocytic growth factor, cMGF, to the culture medium. In oncogenicity tests, a selection of the virus strains induced MML, nephroblastomas, renal adenomas/adenocarcinomas, and other tumors in line 21 meat-type chickens but not in line 0 chickens. This difference may have been related to a propensity for the virus strains to induce persistent tolerant viremic infections in the line 21 chickens following infection at 1 day of age. The oncogenic pattern was not clearly related to the ability of the viruses to transform cultured bone-marrow cells. The generation of acutely transforming viruses during myeloid leukemogenesis may be relevant to the occurrence of MML in the field.

Infection with the helper virus-dependent human parvovirus adeno-associated virus (AAV) is known to interfere with cellular transformation in vitro and oncogenesis in vivo. Here we report on sensitization to gamma irradiation by AAV infection of cells in culture and of tumors established from HeLa cells grafted into immunodeficient (nude) mice: infection of HeLa cells with AAV type 2 enhanced cell killing and reduced plating efficiency after irradiation compared with uninfected cells. Similarly, HeLa cell tumors in nude mice displayed a reduced growth rate and were more sensitive to gamma irradiation when the animals were infected with AAV type 2 prior to or after tumor cell inoculation. Since no pathogenicity is known for AAV, the ability of this virus to render radiotherapy of human tumor cells more efficient may up open novel approaches in cancer treatment.

The activity and epithelial tropism of the human papillomavirus type 18 P105 early promoter, which directs the synthesis of the E6 and E7 transforming genes, are controlled by cis elements included in the viral long control region. To identify potential cellular regulators of this promoter, we mutagenized one or both of the 5'-TGACTAA-3' cis elements capable of interacting with the AP1 transcription factor, which is composed either of homodimers or heterodimers of the Jun products or of heterodimers of Jun and Fos. Mutation of both elements completely abolished P105 promoter activity in human keratinocytes. We show that either AP1 site can interact efficiently in vitro with any of the three different Jun products as heterodimers with c-Fos. However, in nuclear extracts prepared from human keratinocytes, JunB was the predominant Jun component bound to the DNA probe containing this cis element. These results implicate JunB as an important factor in human papillomavirus type 18 transcription in keratinocytes and strongly suggest a potential role of this Jun gene product in the tissue-specific transcription of the genital papillomaviruses.

Transgenic mice carrying the entire polyomavirus (Py) early region consistently develop both vascular and bone tumors. This tumor spectrum represents a subset of the tumors found in mice infected with Py and an expansion of the vascular tumor spectrum seen in Py middle T antigen (MT) transgenic mice (V. L. Bautch, S. Toda, J. A. Hassell, and D. Hanahan, Cell 51:529-538, 1987). Transgenic mice of three independent lineages develop these pathologies, and mice of individual lineages also develop lymphangiomas and fibrosarcomas. All tumors are of mesenchymal origin, and all tumor tissues express the Py transgene. Some unaffected tissues, including the testes of mice of all lineages, also express the Py transgene. The number of transgene expression sites in mice of a given lineage correlates with the severity and latency of the tumor phenotype in these animals. Analysis of transgene transcripts indicates that RNAs for Py large T antigen (LT), MT, and small T antigen (ST) are present in both tumors and testes of transgenic mice. The ratio of LT RNA to MT and ST RNAs, however, is higher in testes than in tumors and other unaffected tissues, indicating that tissue-specific differences in the relative amounts of the alternatively spliced Py RNAs exist. The finding that some sites of Py transgene expression are susceptible to tumor formation while other expressing tissues such as testes are refractory to tumorigenesis suggests that mechanisms of viral pathogenesis are influenced by a tissue specificity in the effects of the Py early region.

The preventive capability of interleukin 1 alpha (IL-1) against bacterial infections was estimated in normal and anticancer drug-treated BALB/c mice in comparison with OK432, granulocyte colony-stimulating factor, interferons alpha and gamma, and interleukin 2. Pretreatment with IL-1 (days -4 and -2) resulted in a significantly higher survival rate in normal mice inoculated i.p. with Klebsiella pneumoniae, Pseudomonas aeruginosa or Listeria monocytogenes (day 0). The i.p. and s.c. administrations of IL-1 were equally effective for the induction of antibacterial resistance. Pretreatment with OK432 showed an equal degree of resistance to i.p. infection but was effective only by i.p. administration. Enhanced antibacterial resistance by IL-1 and OK432 was also observed in cyclophosphamide- and aminomethylpyrimidinylmethylchloroethylnitrosourea hydrochloride-pretreated (day -5) normal hosts and in cyclophosphamide-treated tumor-bearing hosts. In the case of granulocyte colony-stimulating factor (i.p. or s.c.) (days -4 to -1), a statistical difference in survival rate between granulocyte colony-stimulating factor and its vehicle-treated groups was observed in cyclophosphamide-pretreated hosts, but not in normal hosts or aminomethylpyrimidinylmethylchloroethylnitrosourea hydrochloride-pretreated hosts. Viable bacteria in the peritoneal cavity and blood at 12 h after i.p. infection of K. pneumoniae correlated well with the survival rate. In IL-1-pretreated hosts, the earlier and increased accumulation of neutrophils into peritoneal cavity after the infection was observed and the number of inflammatory cells in peritoneal cavity correlated well with the survival rate. The enhanced resistance to bacterial infection by IL-1 was suggested to be in part due to the enhanced cellular defense mechanisms. The prophylactic administration of IL-1 would be beneficial for the management of serious infections in cancer patients.

Tumorigenicity in immunocompetent syngeneic mice and H-2 class I antigen expression of BPV1-transformed mouse cell lines had no correlation. H-2 expression was examined using monoclonal anti-(H-2Kb) and anti-(H-2Db) antibodies in immunofluorescence staining for flow cytometry analysis and by determining the sensitivity of the cells to cytolysis by allostimulated spleen cells. Nontumorigenic cell lines were as resistant as tumorigenic cell lines to natural killer activity. The results indicate that in our model defence by natural killer cells is not a decisive factor. The results also show that instead of or in addition to H-2 class I antigens other factors (e.g. the presence or absence of virus-specific antigens) are important in determining the tumorigenicity of BPV1-transformed cell lines.

The establishment of an epithelial cell line from a mouse pancreatic adenocarcinoma is described. The cell line, designated LTPA, was aneuploid and exhibited many transformed growth properties (rapid growth rate, failure to show density-dependent inhibition of growth, ability to grow in defined medium). A type C oncornavirus was isolated from the culture medium, and electron microscopy also revealed the presence of intracisternal type A particles. LTPA cells carried a persistent polyoma infection which produced only low levels of cytopathic effects. A mycoplasmal contamination was also carried. When injected s.c. into Swiss nu/nu mice, LTPA cells formed ductular structures which were destroyed by inflammatory reactions within 3 weeks.

The insertion stability and DNA methylation patterns of integrated adenovirus type 12 (Ad12) genomes were investigated in Ad12-induced tumors and in tumor cell lines established from them as a function of time of passage under culture conditions. Upon subcultivation of cells from some of the tumors, the viral genomes were eliminated, apparently in a stepwise process with segments of the left termini of Ad12 DNAs persisting the longest. Morphological variants of these tumor cells lost all viral DNA and yet retained the oncogenic phenotype. All 13 independently isolated clones from one revertant line were devoid of Ad12 DNA. It could not be ruled out that very short sequence elements of viral DNA, such as promoters or enhancing sequences, could have persisted in these variants. The extent of viral DNA methylation was minimal in Ad12-induced tumors, although the viral genome was not extensively expressed, if at all. Upon passage in culture, the levels of viral DNA methylation increased. It was interesting that establishment of the final methylation pattern of integrated Ad12 DNAs required many cell generations after the fixation of foreign DNA in the host genome. The shift in methylation was nonrandom. The late parts of the inserted viral genomes became methylated more extensively than did the early gene segments.

Four cell lines derived from adenovirus type 12-induced rat brain tumors were studied. The polyploid cells displayed neuroepithelial characteristics and were transplantable into syngeneic rats and nude mice. In tissue culture the cells grew in monolayers and multilayers. A very high saturation density was reached, and the cells plated in agar and were easily agglutinated with low concentrations of concanavalin A. Between 2 and 11 copies of the viral genome per diploid cellular genome were detected by reassociation kinetics analysis in the different lines. The patterns of distribution of viral DNA sequences in these lines, as revealed by blot analysis, suggest colinear integration of the intact viral genome into the cellular DNA. The patterns of integration were stable after more than 15 months of prolonged tissue culture and after animal reimplantation. Integration patterns were identical in three of the tumor lines and different in another line. Viral sequences were transcribed. The extent of homology found toward adenovirus type 12 DNA in polyadenylated polysome-associated mRNA isolated from the tumor lines suggests that the early and some of the late genes of adenovirus type 12 DNA are transcribed in these tumor cells. Infectious virus was not rescuable from these lines.

Cellular genes containing nucleotide sequences homologous to retroviral oncogenes (v-onc) have been identified in the genomes of a variety of species of both the vertebrate and invertebrate phyla. Expression of such genes, termed c-onc genes, has been shown to be tissue-specific in chickens and mice and to be modulated during murine development and differentiation of human haematopoietic cells. We report here that the level of the c-fos gene transcripts is 100-fold greater in human term fetal membranes than in other normal human tissues and cells. These levels of c-fos expression in human amniotic and chorionic cells are close to the level of v-fos expression that results in the induction of osteosarcomas in mice and transformation of fibroblasts in vitro. This observation suggests that the induction of neoplastic transformation by the FBJ murine osteosarcoma virus may require the expression of the fos gene product at high levels in an inappropriate cell type. In contrast, the human c-fms gene is expressed at high levels specifically in term placenta and trophoblastic cells. The tissue and cell type-specific patterns of c-fos and c-fms expression suggest that the physiological function of the c-fos and c-fms encoded proteins may be associated with those embryo-derived cells whose primary functions are protection and nourishment of the human fetus.

The BALB/3T3 mouse embryo cell line, noted for its marked postconfluence inhibition of proliferation, anchorage dependence, and high serum requirement, and frequently studied as a prototype nontumorigenic "fibroblast" line that is compared with tumorigenic sublines transformed with various agents, produced tumors within 2 to 3 months when an average of 3 X 10(4) cells were implanted s.c. attached to 1- X 5- X 10-mm polycarbonate platelets. Plastic platelets alone produced no tumors after 1 year of observation. The tumors, as well as others arising from implants of BALB/3T3 cells attached to 3-mm glass beads, were given the histological diagnosis of "vasoformative saroma" because the tumor cells frequently formed vascular channels. The vasoformative pattern and the results of specific staining for reticulin and collagen support the likelihood that BALB/3T3 cells originated from endothelial cells rather than from fibroblasts. That the tumors were derived from BALB/3T3 cells and not host cells was proved when tumors arising in BALB/c X C57BL/6 F1 hybrids were shown to be transplantable to BALB/c but not to C57BL/6 mice. The cultured tumor cells showed loss of both postconfluence inhibition of proliferation and anchorage dependence. Evidence of the induction of endogenous oncornaviruses was obtained in only one of four tumors tested. These tumors also exhibited tumor-unique transplantation rejection antigens. We conclude that BALB/3T3 cells are preneoplastic and give rise to different spontaneously transformed clones bearing unique tumor rejection antigens when implanted in vivo attached to a solid substrate.

Adenovirus type 12 (Ad12)-specific proteins were determined in nine well-characeterized Ad12-transformed hamster cell lines and in Ad12-induced hamster and rat tumour lines by immunoprecipitation, gel electrophoresis and autoradiography. All cell lines expressed a 60K mol. wt. polypeptide. In several cell lines, the presence of a 58K mol. wt. protein could also be demonstrated by these techniques. Smaller Ad12-specific proteins could be detected only in the Ad12-transformed hamster cell lines HA12/7 and A2497-3, and in the Ad12-induced hamster tumour line CLAC3. The quality of the immunoprecipitation tests performed depended primarily on the sera used; their antibody titres varied widely. For at least one of the Ad12-transformed hamster cell lines (HA12/7), there was good agreement in the number and mol. wt. of Ad12-specific proteins detected by both immunoprecipitation and in vitro translation of hybrid-selected cytoplasmic RNA. There was no clear-cut correlation between the number and the nature of Ad12-specific proteins and the way in which the cell lines or tumours were obtained. Cell lines with the least number of copies of Ad12 DNA persisting appeared to express the largest number of Ad12-specific proteins.

The ubiquitous nononcogenic lactic dehydrogenase RNA virus (LDV) is one of the most common contaminants of transplantable tumors and oncogenic viral preparations. This association of LDV with a large variety of murine tumors and the known effect of LDV infection on immune competence may suggest the existence of a possible etiological relationship between the virus and murine tumors. We have found that the inoculation of transplantable carcinoma cells to acutely LDV-infected mice is followed by an enhanced rate of tumor growth. To examine the possibility that LDV is involved in the carcinogenic process and may act as a promoter or initiator, we infected mice with LDV at various time intervals prior to, simultaneously with or after their treatment with chemical carcinogens (urethan 3-methylcholanthrene, and 9,10-dimethyl-1,2-benzanthracene) and then followed the development of the malignant disease. No differences in the rate of tumor appearance and in mortality were observed between LDV-infected and uninfected mice. In addition, LDV failed to affect the development of spontaneous reticulum cell sarcoma and lymphatic leukemia in SJL/J, AKR/Cu, and AKR/J mouse strains. It is suggested that, although LDV does not affect the induction of oncogenic processes, this virus may still be capable of promoting the progression of transplantable tumors due to its immunoregulatory properties.

A DNA species with buoyant densities greater than mouse cellular DNA was found associated with intracytoplasmic A particles (CAP) isolated from mouse mammary tumor virus-infected mouse mammary tumors and mouse Leydig cell tumors which produce CAP but no complete mouse mammary tumor virus virions. This DNA species was absent in identically prepared tissue fractions from tumors which did not contain CAP. Treatment of CAP-associated DNA with pancreatic RNase A did not alter the buoyant density although a reduction in apparent molecular weight (broadening of the DNA band at equilibrium) was observed upon analytical equilibrium sedimentation in CsCl. The molecular weight of untreated CAP-associated DNA was estimated to range from 0.8 x 10(6) to 3.1 x 10(6). Base composition analysis showed CAP-DNA to possess an approximate guanine plus cytosine content of 38%. Ninety percent of CAP-associated DNA eluted as single-stranded molecules upon hydroxyapatite column chromatography, a characteristic that accounts in part for its higher buoyant density in neutral CsCl compared to native double-stranded mouse DNA. In two preparations, CAP-DNA had a sedimentation coefficient of 7 to 8S.

A series of febrile illnesses, some severe, among hospital personnel was traced to an outbreak of lymphocytic choriomeningitis in a hospital research colony of Syrian hamsters. By rapid virus isolation and identification techniques, a definitive diagnosis was made 5 days after specimens were received. This outbreak emphasizes the dangers of working with experimental hamsters and tumor cell lines passaged in them.

Repeated injections of urethan into suckling BALB/c mice induced multiple papillary adenocarcinomas in the lungs and kidneys. When the pulmonary tumors were transplanted i.p. by cell graft into 6 suckling BALB/c mice, they induced disseminated carcinosarcomas within the peritoneal cavity in all inoculated animals. Tumors resulting from the transplantation of tumor cells were used for preparation of filtered extracts. The filtrates were inoculated into 6 suckling BALB/c mice and induced generalized malignant lymphomas in all animals. The primary urethan-induced pulmonary and renal tumors, the carcinosarcomas that resulted from i.p. cell transfer, and also the generalized malignant lymphomas induced by inoculation of filtered extracts contained C-type virus particles. Theoretically, it could be assumed that both the primary urethan-induced pulmonary and renal tumors, as well as the cell-graft-induced peritoneal carcinosarcomas, contained the C-type virus particles as passengers, not necessarily related etiologically to the tumors in which they were found. It is quite likely, however, that these virus particles were etiologically related to the filtrate-induced malignant lymphomas in which they were also found.