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Biomedical subjects

C H Contag

Publications and source records attributed to C H Contag.

33 records · Page 2Linked to original sources
PubMed1999

Noninvasive assessment of tumor cell proliferation in animal models.

Revealing the mechanisms of neoplastic disease and enhancing our ability to intervene in these processes requires an increased understanding of cellular and molecular changes as they occur in intact living animal models. We have begun to address these needs by developing a method of labeling tumor cells through constitutive expression of an optical reporter gene, and noninvasively monitoring cellular proliferation in vivo using a sensitive photon detection system. A stable line of HeLa cells that expressed a modified firefly luciferase gene was generated, and proliferation of these cells in irradiated severe combined immunodeficiency (SCID) mice was monitored. Tumor cells were introduced into animals via subcutaneous, intraperitoneal and intravenous inoculation and whole body images, that revealed tumor location and growth kinetics, were obtained. The number of photons that were emitted from the labeled tumor cells and transmitted through murine tissues was sufficient to detect 1x10(3) cells in the peritoneal cavity, 1x10(4) cells at subcutaneous sites and 1x10(6) circulating cells immediately following injection. The kinetics of cell proliferation, as measured by photon emission, was exponential in the peritoneal cavity and at subcutaneous sites. Intravenous inoculation resulted in detectable colonies of tumor cells in animals receiving more than 1x10(6) cells. Our demonstrated ability to detect small numbers of tumor cells in living animals noninvasively suggests that therapies designed to treat minimal disease states, as occur early in the disease course and after elimination of the tumor mass, may be monitored using this approach. Moreover, it may be possible to monitor micrometastases and evaluate the molecular steps in the metastatic process. Spatiotemporal analyses of neoplasia will improve the predictability of animal models of human disease as study groups can be followed over time, and this method will accelerate development of novel therapeutic strategies.

Animals↗
PubMed1999

Real-time monitoring of Escherichia coli O157:H7 adherence to beef carcass surface tissues with a bioluminescent reporter.

A method for studying bacteria that are attached to carcass surfaces would eliminate the need for exogenous sampling and would facilitate understanding the interaction of potential human food-borne pathogens with food animal tissue surfaces. We describe such a method in which we used a bioluminescent reporter strain of Escherichia coli O157:H7 that was constructed by transformation with plasmid pCGLS1, an expression vector that contains a complete bacterial luciferase (lux) operon. Beef carcass surface tissues were inoculated with the bioluminescent strain, and adherent bacteria were visualized in real time by using a sensitive photon-counting camera to obtain in situ images. The reporter strain was found to luminesce from the tissue surfaces whether it was inoculated as a suspension in buffer or as a suspension in a bovine fecal slurry. With this method, areas of tissues inoculated with the reporter strain could be studied without obtaining, excising, homogenizing, and culturing multiple samples from the tissue surface. Use of the complete lux operon as the bioluminescent reporter eliminated the need to add exogenous substrate. This allowed detection and quantitation of bacterial inocula and rapid evaluation of adherence of a potential human pathogen to tissue surfaces. Following simple water rinses of inoculated carcass tissues, the attachment duration varied with different carcass surface types. On average, the percent retention of bioluminescent signal from the reporter strain was higher on lean fascia-covered tissue (54%) than on adipose fascia-covered tissue (18%) following water washing of the tissues. Bioluminescence and culture-derived viable bacterial counts were highly correlated (r2 = 0.98). Real-time assessment of microbial attachment to this complex menstruum should facilitate evaluation of carcass decontamination procedures and mechanistic studies of microbial contamination of beef carcass tissues.

Abattoirs↗
PubMed1997

Imaging brain structure and function, infection and gene expression in the body using light.

Light can be used to probe the function and structure of human tissues. We have been exploring two distinct methods: (i) externally emitting light into tissue and measuring the transmitted light to characterize a region through which the light has passed, and (ii) internally generating light within tissue and using the radiated light as a quantitative homing beacon. The emitted-light approach falls within the domain of spectroscopy, and has allowed for imaging of intracranial haemorrhage in newborns and of brain functions in adults. The generated-light approach is conceptually parallel to positron emission tomography (PET) or nuclear medicine scanning, and has allowed for real-time, non-invasive monitoring and imaging of infection and gene expression in vivo using low-light cameras and ordinary lenses. In this paper, we discuss recent results and speculate on the applications of such techniques.

Adult↗
PubMed1997

Visualizing gene expression in living mammals using a bioluminescent reporter.

Control of gene expression often involves an interwoven set of regulatory processes. As information regarding regulatory pathways may be lost in ex vivo analyses, we used bioluminescence to monitor gene expression in living mammals. Viral promoters fused to firefly luciferase as transgenes in mice allowed external monitoring of gene expression both superficially and in deep tissues. In vivo bioluminescence was detectable using either intensified or cooled charge-coupled device cameras, and could be detected following both topical and systemic delivery of substrate. In vivo control of the promoter from the human immunodeficiency virus was demonstrated. As a model for DNA-based therapies and vaccines, in vivo transfection of a luciferase expression vector (SV-40 promoter and enhancer controlling expression) was detected. We conclude that gene regulation, DNA delivery and expression can now be noninvasively monitored in living mammals using a luciferase reporter. Thus, real-time, noninvasive study of gene expression in living animal models for human development and disease is possible.

Animals↗
PubMed1997

Mother-to-infant transmission of human immunodeficiency virus type 1 involving five envelope sequence subtypes.

Genetic analysis of human immunodeficiency virus type 1 (HIV-1) from cases of mother-to-infant transmission were analyzed in an effort to provide insights into the viral selection that may occur during transmission, as well as the timing and source of transmitted viruses. HIV-1 env genes obtained from seven mothers and their perinatally infected infants in Sweden were studied. Five envelope sequence clades (A to E) were found to be represented. We used a heteroduplex tracking assay (HTA) to assess the genetic relatedness between early viral isolates from the infants and serial maternal virus populations taken during pregnancy and at delivery. HTA findings were used to select for DNA sequence analysis maternal virus populations that were either closely or more distantly related to the infant virus. In each case, nucleotide sequence analysis confirmed the genetic relationships inferred by the HTA. Only maternal peripheral blood was sampled, and large sets of maternal specimens throughout pregnancy were generally not available. However, no consistent correlation was found to support the hypothesis that infant viruses should match blood-derived maternal virus genotypes found early in pregnancy if infants were found to be infected at birth or, conversely, that infant viruses should match blood-derived maternal virus genotypes found at delivery if infants were found to be infected only some time later.

Amino Acid Sequence↗
PubMed1995

Photonic detection of bacterial pathogens in living hosts.

The study of pathogenic processes is often limited to ex vivo assays and cell-culture correlates. A greater understanding of infectious diseases would be facilitated by in vivo analyses. Therefore, we have developed a method for detecting bacterial pathogens in a living host and used this method to evaluate disease processes for strains of Salmonella typhimurlum that differ in their virulence for mice. Three strains of Salmonella were marked with bioluminescence through transformation with a plasmid conferring constitutive expression of bacterial luciferase. Detection of photons transmitted through tissues of animals infected with bioluminescent Salmonella allowed localization of the bacteria to specific tissues. In this manner progressive infections were distinguished from those that were persistent or abortive. We observed patterns of bioluminescence that suggested the caecum may play a pivotal role in Salmonella pathogenesis. In vivo efficacy of an antibiotic was monitored using this optical method. This study demonstrates that real time non-invasive analyses of pathogenic events and pharmacological monitoring can be performed in vivo.

Animals↗
PubMed1989

Age-dependent poliomyelitis of mice: expression of endogenous retrovirus correlates with cytocidal replication of lactate dehydrogenase-elevating virus in motor neurons.

The widespread presence of endogenous retroviruses in the genomes of animals and humans has suggested that these viruses may be involved in both normal and abnormal developmental processes. Previous studies have indicated the involvement of endogenous ecotropic murine leukemia virus (MuLV) in the development of age-dependent poliomyelitis caused by infection of old C58 or AKR mice by lactate dehydrogenase-elevating virus (LDV). The only genetic components which segregate with susceptibility to LDV-induced paralytic disease are multiple proviral copies of ecotropic MuLV and the permissive allele, at the Fv-1 locus, for N-tropic, ecotropic virus replication (Fv-1n/n). Using in situ hybridization and Northern (RNA) blot hybridization, we have correlated the expression of the endogenous MuLV, both temporally and spatially, with LDV infection of anterior horn motor neurons and the development of paralysis. Our data indicate that treatment of 6- to 7-month-old C58/M mice with cyclophosphamide, which renders these mice susceptible to LDV-induced paralytic disease, results in transient increases in ecotropic MuLV RNA levels in motor neurons throughout the spinal cord. Peripheral inoculation of C58/M mice with LDV, at the time of elevated MuLV RNA levels, results in a rapid spread of LDV to some spinal cord motor neurons. LDV infections then spread slowly but progressively throughout the spinal cord, involving an increasing number of motor neurons. LDV replication is cytocidal and results in neuron destruction and paralysis of the infected animals 2 to 3 weeks postinfection. The slow replication of LDV in the spinal cord contrasts sharply with the rapid replication of LDV in macrophages, the normal host cells for LDV, during the acute phase of infection. The data indicate that the interaction between the endogenous MuLV with the generally nonpathogenic murine togavirus LDV occurs at the level of the motor neuron. We discuss potential mechanisms for the novel dual-virus etiology of age-dependent poliomyelitis of mice.

Age Factors↗
PubMed1988

Susceptibility of C58 mice to paralytic disease induced by lactate dehydrogenase-elevating virus correlates with increased expression of endogenous retrovirus in motor neurons.

The induction of poliomyelitis by lactate dehydrogenase-elevating virus (LDV) in C58 mice is dependent upon several host factors including old age, loss of immune competence and genetic predisposition. Two genetic components segregate with susceptibility to this neurological disease: the presence of multiple proviral copies of N-tropic endogenous murine leukemia viruses (MuLV) and homozygosity of the permissive allele for N-tropic viral replication (Fv-1n/n). We have quantified the levels of RNA for several endogenous retroviruses, using virus specific oligonucleotide probes, in various tissues of C58 mice in relation to age and immunosuppression. A tissue specific increase in expression of 3.0 kb AKR MuLV RNA in the spinal cords of mice occurred with increasing age of the mice and was enhanced several-fold by immunosuppression in old mice. Susceptibility to LDV-induced poliomyelitis occurs in the same age dependent manner as AKR MuLV expression and is also enhanced by immunosuppression. In contrast, the mink cell focus forming virus (MCF) RNA levels in the spinal cord remained constant despite apparent variations in MCF RNA expression in other tissues, and no xenotropic retrovirus RNA was detectable in spinal cords or brains of the C58 mice. The increased AKR MuLV RNA in the spinal cord was shown by in situ hybridization to be mainly located in the same motor neurons that become infected with LDV in these mice and are destroyed as paralysis develops. These results support a novel dual virus virus hypothesis for LDV-induced poliomyelitis in which increased endogenous retroviral expression in motor neurons renders these cells susceptible to cytocidal replication of LDV and hence to the development of LDV-induced poliomyelitis.

Animals↗
PubMed1987

Protection of C58 mice from lactate dehydrogenase-elevating virus-induced motor neuron disease by non-neutralizing antiviral antibodies without interference with virus replication.

The paralytic poliomyelitis induced in old, immunosuppressed C58 mice by a primary infection with the lactate dehydrogenase-elevating virus (LDV) is prevented by the presence of anti-LDV antibodies in the virus inoculum or by passive transfer of LDV-free plasma from chronically LDV-infected mice one day before infection. Non-neutralizing antibodies were protective and specifically directed to the lowest molecular weight form of the envelope glycoprotein of LDV (VP-3), which seems to exist in virions in at least ten molecular forms ranging from 24 to 44 kDa. The antibodies did not prevent the productive infection of the subpopulation of macrophages that represents the primary permissive cell type in the mouse as evidenced by normal plasma LDV levels nor the spread of LDV to the central nervous system. Many non-neuronal cells containing LDV RNA were detected by in situ hybridization in the spinal cords of mice that had been infected with LDV in the presence of protective antibodies. However, no LDV RNA-positive neurons were detected, which are normally found coincidental with the development of paralytic symptoms in LDV-infected C58 mice. We propose that an early event after infection is critical for the infection of neurons and is inhibited by the presence of non-neutralizing antibodies to the LDV glycoprotein.

Animals↗
PubMed1986

Correlation between presence of lactate dehydrogenase-elevating virus RNA and antigens in motor neurons and paralysis in infected C58 mice.

Lactate dehydrogenase-elevating virus (LDV) induces poliomyelitis in immunosuppressed C58 mice resulting in fatal paralysis. We have synthesized and cloned cDNA complementary to the LDV genome, and used the cDNA clones as in situ hybridization probes for the detection of LDV RNA in tissue sections. Direct fluorescent antibody staining using IgG from chronically infected mice was used for the detection of LDV antigens. Using these methods, we have detected LDV RNA and antigens in anterior horn neurons of paralyzed mice. The appearance of LDV RNA and antigen positive motor neurons and their location in the spinal cord correlated with the development of paralytic symptoms. No positive neurons were detected in LDV-infected, susceptible mice without signs of paralysis, but some glial cells of the white and gray matter in the spinal cords of these mice were found to contain LDV RNA. These analyses broaden the host cell range of LDV to include neuronal and other cells in the CNS and support the hypothesis of LDV replication in neurons as the cause of poliomyelitis and paralysis.

Animals↗
PubMed1986

Genomic differences between strains of lactate dehydrogenase-elevating virus.

The RNAs of two independently isolated strains of lactate dehydrogenase-elevating virus (LDV), which differ antigenically and in neurovirulence for C58 mice, were isolated and T1 RNase fingerprinted. Of about 30 unique T1 oligonucleotides, 27 seemed to be common for both strains of LDV, whereas 2 or 3 oligonucleotides were unique for each strain. In other physical and biological properties, such as virion density, molecular weights of their structural proteins, interaction with mouse anti-LDV IgG, and replication in primary cultures of peritoneal macrophages from various mouse strains, the two strains of LDV were indistinguishable. The T1 RNase patterns and the affinity of LDV RNA for oligo(dT) indicated that it contained poly-A.

Centrifugation, Isopycnic↗
PubMedDate not supplied

Use of reporter genes for optical measurements of neoplastic disease in vivo.

Revealing the cellular and molecular changes associated with cancer, as they occur in intact living animal models of human neoplastic disease, holds tremendous potential for understanding disease mechanisms and elucidating effective therapies. Since light is transmitted through mammalian tissues, at a low level, optical signatures conferred on tumor cells by expression of reporter genes encoding bioluminescent and fluorescent proteins can be detected externally using sensitive photon detection systems. Expression of reporter genes, such as the bioluminescent enzyme firefly luciferase (Luc) or variants of green fluorescent protein (GFP) in transformed cells, can effectively be used to reveal molecular and cellular features of neoplasia in vivo. Tumor cell growth and regression in response to various therapies have been evaluated non-invasively in living experimental animals using these reporter genes. Detection of Luc-labeled cells in vivo was extremely sensitive with signals over background from as few as 1000 human tumor cells distributed throughout the peritoneal cavity of a mouse with linear relationships between cell number and signal intensity over five logs. GFP offers the strength of high-resolution ex vivo analyses following in vivo localization of the tumor. The dynamic range of Luc detection allows the full disease course to be monitored since disease progression from small numbers of cells to extensive disease can be assessed. As such, therapies that target minimal disease as well as those designed for late stage disease can be readily evaluated in animal models. Real time spatiotemporal analyses of tumor cell growth can reveal the dynamics of neoplastic disease, and facilitate rapid optimization of effective treatment regimens. Thus, these methods improve the predictability of animal models of human disease as study groups can be followed over time, and can accelerate the development of therapeutic strategies.

Animals↗
PubMedDate not supplied

Rapid and quantitative assessment of cancer treatment response using in vivo bioluminescence imaging.

Current assessment of orthotopic tumor models in animals utilizes survival as the primary therapeutic end point. In vivo bioluminescence imaging (BLI) is a sensitive imaging modality that is rapid and accessible, and may comprise an ideal tool for evaluating antineoplastic therapies. Using human tumor cell lines constitutively expressing luciferase, the kinetics of tumor growth and response to therapy have been assessed in intraperitoneal, and subcutaneous, and intravascular cancer models. However, use of this approach for evaluating orthotopic tumor models has not been demonstrated. In this report, the ability of BLI to noninvasively quantitate the growth and therapeutic-induced cell kill of orthotopic rat brain tumors derived from 9L gliosarcoma cells genetically engineered to stably express firefly luciferase (9LLuc) was investigated. Intracerebral tumor burden was monitored over time by quantitation of photon emission and tumor volume using a cryogenically cooled CCD camera and magnetic resonance imaging (MRI), respectively. There was excellent correlation (r=0.91) between detected photons and tumor volume. A quantitative comparison of tumor cell kill determined from serial MRI volume measurements and BLI photon counts following 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment revealed that both imaging modalities yielded statistically similar cell kill values (P=.951). These results provide direct validation of BLI imaging as a powerful and quantitative tool for the assessment of antineoplastic therapies in living animals.

Animals↗