Adiponectin Deficiency Suppresses Rhabdomyosarcoma Associated with Gut Microbiota Regulation.

The gut microbiota is very important in the initiation, progression, and dissemination of cancer, and the regulation of microbiota has been employed as a novel strategy to enhance the effect of immunotherapy. Adiponectin (APN), an adipocyte-derived hormone, plays a vital role in regulating the immune response of innate immune cells. The deficiency of APN inhibits rhabdomyosarcoma growth. However, whether this function is associated with regulating gut microbiota remains unknown. To investigate, we performed 16S ribosomal RNA (rRNA) gene sequencing on the fecal microbiome of APN gene knockout mice to determine whether APN deletion affects the gut microbiota. We found APN deficiency alters gut microbial functions involved in metabolism, genetic information processing, and cellular processes. In addition, a decreased abundance of Bacteroides and an increased abundance of Prevotella and Helicobacter were observed in rhabdomyosarcoma-bearing APN knockout mice; these bacteria were associated with the inhibition of rhabdomyosarcoma growth. These findings suggest that gut microbiota may be a potential target of APN deficiency against rhabdomyosarcoma.

Clostridium spores as anti-tumour agents.

The successful treatment of cancer remains a huge challenge. Consequently, efforts are being made to develop alternative methods of tumour therapy. One of these is the use of live Clostridium species, based on the observation that obligatory anaerobic bacteria specifically colonize the hypoxic and necrotic regions that are present in solid tumours but normally absent in other parts of the body. Although past results have fuelled scepticism about its clinical use, recent promising findings emphasize the potential of Clostridium-directed tumour therapy. These recent developments are reviewed and the reintroduction of this tumour-targeting protein delivery system into clinical settings is discussed.

Ulcerated rhabdomyosarcoma as portal of entry for tetanus in a previously nonimmunized child.

Infectious diseases represent one of the most important secondary problems related to the treatment of childhood cancer, being the leading cause of death in this population. They are predominantly of bacterial and fungal etiology. The association between tetanus, a bacterial vaccine-preventable disease, and cancer is virtually undescribed. The authors present the case of a previously nonimmunized child, due to his parents' choice, who developed severe tetanus with an ulcerated rhabdomyosarcoma as portal of entry. Due to an unfavorable evolution, the child underwent a hip disarticulation to provide tetanus control. The ulterior tumor management was successful: the child has been off therapy for more than 108 months with no evidence of disease.

Recurrent isolation of an uncommon yeast, Candida pararugosa, from a sarcoma patient.

A yeast was repeatedly isolated from the saliva of a sarcoma patient. A relatively uncommon species, Candida maris, was identified based on the API 20C profile. The yeast species most frequently obtained from the patient's mother and from clinic staff was Candida albicans. A comparison of the yeast obtained from the patient with the type strain of C. maris strongly suggested that the former was not representative of C. maris. Analysis of partial ribosomal DNA sequences of the patient strain and from the type strain of C. maris showed that the two are phylogenetically not closely related. The patient strain was very close to Candida pararugosa, a relatively uncommon asporogenous yeast. DNA reassociation studies among C. pararugosa and patient isolates showed that they were conspecific. We could not determine the source of the yeast infection. This case will alert hospital staff to be aware of the possibility of unexpected environmental microorganisms as causes of infections, colonizations and persistent environmental contamination events in immunocompromised patients.

Optimization of tumor-targeted gene delivery by engineered attenuated Salmonella typhimurium.

BACKGROUND: Attenuated Salmonella typhimurium has been demonstrated as a potential gene delivery vector. Previous findings induce the necessity to optimize tumor selectivity and bacterial dosing in relation to tumor volume and intratumoral therapeutic gene expression. MATERIALS AND METHODS: Attenuated Salmonella VNP20009 and VNP20047 (expressing cytosine deaminase) were systemically administered to tumor-bearing rats. The bacteria were quantified in tumor and normal organs. Conversion of 5-fluorocytosine to 5-fluorouracil was evaluated using thin layer chromatography. RESULTS: Tumor colonization efficiency was dependent on Salmonella density, administration route and tumor volume. Colonization of normal tissues gradually decreased with time, while intratumoral proliferation of bacteria remained high during the follow-up period. The Optimal Therapeutic Dose (OTD) was found to be 5.10(7) cfu/rat. Intratumoral VNP20047-expressed CDase leading to the conversion of 5-FC to 5-FU was detected in vivo. CONCLUSION: Our results indicate the need to define an OTD, probably for each species, when using genetically engineered Salmonella as a tumor- and species-selective vector in cancer therapy.

Improvement of Clostridium tumour targeting vectors evaluated in rat rhabdomyosarcomas.

Previous studies have demonstrated the feasibility of using apathogenic clostridia as a promising strategy for hypoxia-specific tumour targeting. The present study shows that the use of the vascular targeting compound combretastatin A-4 phosphate could significantly (P<0.001) increase the number of Clostridium vegetative cells in rat rhabdomyosarcomas with sizes between 0.2 cm(2) and 3 cm(2). Furthermore, this study showed that administration of metronidazole for a 9-day period was sufficient to eliminate systemically administered Clostridium from the tumour. Moreover, previous Clostridium spore administration did not effect tumour colonisation, regardless of the immune response status of the host.

Clostridium as a tumor-specific delivery system of therapeutic proteins.

The feasibility of gene therapy strategies in cancer treatment still has important pitfalls. Transfer of therapeutic proteins to the hypoxic/necrotic 'extracellular' microenvironment of solid tumors, based on the engineering of nonpathogenic clostridia is proposed as an alternative methodology. Using the rat rhabdomyosarcoma R1 in vivo tumor model, we demonstrated that Clostridium species colonized the tumors, whereas proliferation of these bacteria was absent in normal tissues. C. acetobutylicum was genetically engineered to express and secrete either mTNF-alpha or the E. coli cytosine deaminase. Quantitative in vitro data showed stability of the vectors, and significant levels of biologically active therapeutic proteins in lysates and supernatants of recombinant clostridia. Administration of either of these recombinant Clostridium strains to tumor-bearing rats resulted in the presence of active proteins in the tumor tissue. Based on these data and supported by its selective colonization pattern and safety, the Clostridium gene transfer system offers a potential application in anti-cancer therapies.

Evolution of coxsackie B virus during in vitro persistent infection: detection of protein mutations using two-dimensional polyacrylamide gel electrophoresis.

Serotype 5 coxsackie B virus (CBV5) can establish in vitro persistent infections in human rhabdomyosarcoma (RD) cells. This paper describes the characterisation of the virus released from the persistently infected RD cell line designated piRD-3673. Although infectious virus was released for 42 sequential passages of piRD-3673 cells, no gross virus-specific cytopathic effect was detected when the cells were examined by light microscopy. Two-dimensional polyacrylamide gel electrophoresis was used to compare the virus released from piRD-3673 cells with the CBV5 isolate (CBV-3673) used to initiate the persistent virus infection. Two of the virus intracellular proteins (apparent molecular weights 33,000 and 39,000, designated p33 and p39, respectively) increased in their net basic charge for the virus released from piRD-3673 cells compared to CBV-3673; a reduction in the apparent molecular weights of p33 and p39 was also observed. The charge alteration for both p33 and p39 was a two-stage process, the accumulative effect of which resulted in p33 increasing in pI from 6.14 to 6.53 and p39 increasing in pI from 6.29 to 6.63. The first mutation of p33 and p39 occurred between passages 7 and 10 of piRD-3673 cells and affected both the charge and apparent molecular weight of these two proteins. The second mutation at passage 15 of piRD-3673 cells caused only a change in the charge of p33 and p39. Two other virus proteins (p54 and p75) showed no evidence of mutation over the same passage history of piRD-3673 cells. The virus released from piRD-3673 cells also differed from CBV-3673 by two further criteria, a reduction in plaque-forming efficiency in HEp-2 cells and increased virus replication in RD cells. These data on virus evolution are discussed in relation to the maintenance of persistent CBV infections and the presence of naturally occurring CBV variants.

Mapping of the RD phenotype of the Nancy strain of coxsackievirus B3.

The RD variants of group B coxsackieviruses differ from their parental strains in having the ability to replicate in a human rhabdomyosarcoma cell line, RD. The nucleotide sequence of the P1 region of the RD variant of coxsackievirus B3 strain Nancy (CB3NRD) was determined by sequencing cloned cDNAs, obtained by PCR amplification. A comparison between the established nucleotide sequence and that of the P1 region from the parental virus revealed 12 point mutations which corresponded to six amino acid replacements. To identify if the P1 region is responsible for the phenotype of CB3NRD, a chimeric virus was constructed, using an infectious cDNA clone of CB3. The P1 region of the infectious cDNA was replaced by cDNA fragments from CB3N (parental strain Nancy) or CB3NRD and the resulting recombinants were assayed for their ability to infect and replicate in RD cells. The results showed that the RD phenotype of CB3NRD maps in the P1 region. Furthermore, a chimera which only contained the 5' part of the P1 region derived from CB3NRD and the remaining P1 sequence from CB3N was able to replicate in RD cells, suggesting that the VP2 polypeptide contains at least one determinant for the RD phenotype.

Human immunodeficiency virus type 2 infection and fusion of CD4-negative human cell lines: induction and enhancement by soluble CD4.

We describe human immunodeficiency type 2 (HIV-2) strains which induce cell-to-cell fusion and infect certain CD4- human cell lines. Soluble CD4 (sCD4) induces or enhances fusion by most HIV-2 strains tested. Soluble CD4-immunoglobulin G chimeras and conjugates of sCD4 and antibody to the third domain of CD4 block HIV-2 fusion of CD4- cells. We conclude that HIV-2 can enter CD4- cells via an alternative cell surface receptor to CD4. While some strains entered efficiently, others retained a dependency on an interaction with sCD4 to initiate changes in the virion envelope required for membrane fusion.

Non-cytopathic infection of rhabdomyosarcoma cells by coxsackie B5 virus.

Infection of rhabdomyosarcoma (RD) cells by coxsackie B5 virus (CBV5) was non-cytopathic, although low titres of infectious virus were produced after 24 h post-infection. The extent of CBV5 replication in RD cells increased after sequential passage of the virus in these cells. The RD cells from the first cycle of CBV5 infection were recovered and maintained in culture for 3 months (equivalent to 21 passages) releasing infectious virus throughout this period; these cells were considered to be persistently infected with CBV5 and were designated piRD cells. Coxsackie virus antigen was demonstrated in a small proportion of piRD cells by immunofluorescence staining. High resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the intracellular proteins prepared from piRD cells, three proteins were detected which were absent in uninfected RD cells. These new proteins were similar in charge to virus proteins induced during CBV5 lytic infection of HEp-2 cells. Quantitative densitometry of 2-dimensional protein profiles of piRD and uninfected cells showed no significant disruption of RD cell protein synthesis by the persistent virus infection. Three cloned cell lines were recovered from piRD cells, none of which showed evidence of infectious virus or virus-induced protein synthesis suggesting that the parental cell line was a carrier culture for CBV5.

Comparison of shell vials and conventional tubes seeded with rhabdomyosarcoma and MRC-5 cells for the rapid detection of herpes simplex virus.

Shell vials (SV) and conventional tubes (CT) were seeded with rhabdomyosarcoma (RD) and MRC-5 cells and inoculated with clinical specimens, and the systems were evaluated for the rapid diagnosis of herpes simplex virus (HSV) infections by detection of cytopathic effects (CPE) (for CT, for 7 days) and by using fluoresceinated monoclonal antibodies (for SV, 16 h postinoculation). Of 245 genital specimens (16 from males and 229 from females) 56 (23%) seeded with MRC-5 cells (14 type 1 and 42 type 2) and 55 (22%) seeded with RD cells were detected in CT; however, CPE were recognized in only 26 (46%) of the total HSV-positive cultures 1 day postinoculation. Forty-eight (86% sensitivity, MRC-5) and 46 (84% sensitivity, RD) HSV strains were detected immunologically in SV 16 h postinoculation. Early CPE in CT or fluorescent foci in SV were easier to detect in MRC-5 than in RD cell cultures. MRC-5 and RD cells were equally sensitive to infection with HSV. CT cell cultures were more sensitive than SV but less rapid for the detection of HSV infection (P less than 0.01). We recommend using SV for the rapid diagnosis of HSV infections, but in addition, CT must be inoculated with MRC-5 or RD to ensure maximum detection of this virus.

Studies on human immunodeficiency virus-induced cytopathic effects: use of human rhabdomyosarcoma (RD) cells.

A full-length molecular clone of human immunodeficiency virus (HIV) proviral DNA was transferred to human rhabdomyosarcoma (RD) cells by gene transfer method. RD cells released infectious virus within 12 hours after transfection and the viral particles present in the culture medium could be quantitated by monitoring reverse transcriptase activity. Chronic low level viral producer cell lines of RD were also established. Southern hybridization analysis revealed the presence of HIV sequences in transfected RD cells. Electron microscopic studies of the transfected cell revealed intracellular budding of HIV and also showed structural abnormalities such as giant cell phenotype and vacuolation. These features qualify RD cells as a useful system for studying the regulation and cytopathic effects of HIV.

Clonal derivatives of the RD-114 cell line differ in their antiviral and gene-inducing responses to interferons.

The human rhabdomyosarcoma cell line RD-114 is partially responsive to interferons (IFNs). In these cells, alpha interferon (IFN-alpha) or gamma interferon (IFN-gamma) inhibits the replication of some viruses but not of others. Similarly, some of the IFN-inducible mRNAs are induced poorly, whereas others are induced well. Here we report the isolation of clonal derivatives of this line which display different spectra of responses to IFNs. Among the eight extensively characterized clonal lines, one, C10, did not respond to IFN-alpha or IFN-gamma at all. Retrovirus production by each of the seven other lines was inhibited by both IFN-alpha and IFN-gamma. Replication of vesicular stomatitis virus was inhibited strongly by IFN-alpha in clone B1 but not in others, whereas it was not appreciably affected by IFN-gamma in any clone. Replication of encephalomyocarditis virus was inhibited strongly by IFN-gamma in clones A1, A2, A3, B3, and B8 and by IFN-alpha in clone A2. Neither IFN inhibited the multiplication of these clones greatly, although their doubling times were slightly increased. Five mRNAs were induced by IFNs to varying degrees in the seven clones. mRNA 2A was most strongly induced by IFN-gamma in clone A3. mRNA 1-8 was strongly induced by IFN-alpha in clone A1 and by either IFN in clones A2 and A3. The highest concentrations of 2',5'-oligoadenylate synthetase mRNA, mRNA 561, and mRNA 6-16 were in IFN-alpha-treated clones A1 and A2. These results demonstrated the existence of clonal heterogeneity in IFN responses in a cell line and strengthened the view that IFN treatment of cells generates multiple signals leading to a variety of IFN-induced phenotypes.

Regulation of viral persistence in human glioblastoma and rhabdomyosarcoma cells infected with coronavirus OC43.

Cultures of human rhabdomyosarcoma (RD) and human glioblastoma (U87-MG) were compared for their ability to sustain a persistent infection with coronavirus OC43. Within 28 days, infectious virus and hemagglutinin were being produced at high levels in both types of cells. Temperature sensitive plaque variants were recovered at 31 degrees C. In both cell types, the virus caused increased antigen synthesis and cell death, if the temperature was lowered to 31 degrees C. Infectious virus was lost if cells were treated with antiserum to whole virus or if the temperature was raised to 39.5 degrees C. Probing the cured cells with OC43-specific 32P-cDNA showed that cured cells contained no detectable viral RNA. The relative ease of establishment and cure of these persistent infectious makes them attractive as models to study coronavirus regulatory processes.

Monoclonal antibody that inhibits infection of HeLa and rhabdomyosarcoma cells by selected enteroviruses through receptor blockade.

BALB/c mice were immunized with HeLa cells, and their spleen cells were fused with myeloma cells to produce hybridomas. Initial screening of culture fluids from 800 fusion products in a cell protection assay against coxsackievirus B3 (CB3) and the CB3-RD virus variant yielded five presumptive monoclonal antibodies with three specificities: protection against CB3 on HeLa, protection against CB3-RD on rhabdomyosarcoma (RD) cells, and protection against both viruses on the respective cells. Only one of the monoclonal antibodies (with dual specificity) survived two subclonings and was studied in detail. The antibody was determined to have an immunoglobulin G2a isotype and protected cells by blockade of cellular receptors, since attachment of [35S]methionine-labeled CB3 was inhibited by greater than 90%. The monoclonal antibody protected HeLa cells against infection by CB1, CB3, CB5, echovirus 6, and coxsackievirus A21 and RD cells against CB1-RD, CB3-RD, and CB5-RD virus variants. The monoclonal antibody did not protect either cell type against 16 other immunotypes of picornaviruses. The monoclonal antibody produced only positive fluorescence on those cells which were protected against infection, and 125I-labeled antibody confirmed the specific binding to HeLa and RD cells. The results suggest that this monoclonal antibody possesses some of the receptor specificity of the group B coxsackieviruses.

Altered receptor specificity of coxsackievirus B3 after growth in rhabdomyosarcoma cells.

Serial "blind" passages in human rhabdomyosarcoma (RD) cells of prototype viruses from each of the six immunotypes of the group B coxsackieviruses (CB) resulted in the isolation of intratypic variants of CB1, CB3, CB5, and CB6. Each variant virus strain acquired the capacity to agglutinate human erythrocytes and produce small plaques on HeLa cells, although their serological specificity remained unchanged. An alteration in VP1 mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was noted for CB3-RD. The CB3-RD variant was plaque purified on RD cells and studied for receptor interactions on both HeLa and RD cells. An attachment restriction appeared to exist for prototype CB3 on RD cells, whereas CB3-RD attached well to both cells. In attachment interference assays, HeLa cells saturated with CB3-RD blocked the attachment of CB3. In contrast, saturation of cells with CB1 (which shares a common receptor with parental CB3) failed to block the attachment of CB3-RD. This unidirectional receptor blockade suggested that a second site for the attachment of virions to receptors was acquired by the CB3-RD variant. Thus, more than one virus receptor specificity may be operative in the selection of host range virus mutants. The implications of this phenomenon as they may relate to pathogenesis are discussed.