A quantitative PCR method for assessing the presence of Pasteurella testudinis DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii).

Pasteurella testudinis has been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Our goal was to develop a sensitive and specific qPCR method for detecting DNA from P. testudinis in nasal lavage fluid collected from desert tortoises in the field. Probes for 16S ribosomal RNA and RNA polymerase ß-subunit (rpoB) genes were designed. A standard curve generated with DNA extracted from known numbers of bacterial cells determined by flow cytometry revealed a lower detection limit of 50 fg/ml (10 bacteria/ml). The nasal lavage fluid contained no interfering substances, and the qPCR method did not recognize normal flora DNA. The nasal lavage samples from 20 desert tortoises captured in Clark County, Nevada, USA in 2007 and housed at the Desert Tortoise Conservation Center, were all positive for P. testudinis DNA by qPCR. Another set of 19 lavage samples collected in 2010 from wild desert tortoises in the Mojave Desert were tested and 84% were positive for P. testudinis DNA. Fully validated, this qPCR method will provide a means of determining colonization rate. When used in conjunction with serological methods and clinical evaluations, both infection rate and disease rate can be determined for this potential URTD pathogen. This new assay provides an important tool for managing the threatened populations of the Mojave Desert tortoise.

Quantitative PCR method for detection of mycoplasma spp. DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii).

Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6pg ml(-1) to a high of 72,962pg ml(-1). Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise.

Flow cytometric method for quantifying viable Mycoplasma agassizii, an agent of upper respiratory tract disease in the desert tortoise (Gopherus agassizii).

AIMS: Mycoplasma agassizii can cause upper respiratory tract disease in the threatened desert tortoise of the Southwestern United States. Two technical challenges have impeded critical microbiological studies of this microorganism: (i) its small size limits the use of light microscopy for cell counting and (ii) its extremely slow growth in broth and agar cultures impedes colony counting. Our aim was to develop a rapid and sensitive flow cytometric method using a vital fluorescent dye to enumerate viable M. agassizii cells. METHODS AND RESULTS: Here, we demonstrate that the nonfluorescent molecule 5-carboxyfluorescein (5-CF) diacetate acetoxymethyl ester penetrates M. agassizii cell membranes and it is converted in the cytoplasm to the fluorescent molecule 5-CF by the action of intracellular esterases. Labelled mycoplasma cells can be easily detected by flow cytometry, and cultures with as few as 100 viable mycoplasma cells ml(-1) can be labelled and counted in less than 1 h. Experiments using temperature-induced cell death demonstrated that only viable M. agassizii cells are labelled with this procedure. CONCLUSIONS: A rapid and sensitive flow cytometric technique has been developed for enumerating viable M. agassizii cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique should facilitate basic immunological, biochemical and pharmacological studies of this important pathogen which may lead to new diagnostic and therapeutic methods.

Germline polymorphisms are potential metastasis risk and prognosis markers in breast cancer.

A number of theories have been proposed to account for the origins of metastasis, although none as yet have adequately explained all of its characteristics. With approximately 90% of cancer-related deaths due to the effects of disseminated tumors, improved understanding of this process is critical for reducing cancer-associated morbidity and mortality. Extensive research to investigate the molecular basis of this process has been conducted, and our lab has focused on the role of germline polymorphism in this complex process. Simple breeding experiments using a highly metastatic mouse model showed that germline polymorphisms significantly contribute to metastasis susceptibility. Genetic mapping studies revealed that a number of genomic regions are linked to metastasis susceptibility, including a metastasis modifier on mouse chromosome 19. Subsequent analysis identified Sipa1 as the most likely candidate for the observed linkage on Chr 19. Evaluation of SNPs in SIPA1 in a pilot association study in a human breast cancer cohort supported this possibility and demonstrated that SIPA1 polymorphisms are associated with various markers of poor prognosis including differential sentinel lymph node status. Taken together, these data suggest that germline polymorphism is an important modulating component in metastatic progression that needs to be investigated if we are to fully understand the metastatic process.

Host genetics and tumour metastasis.

Metastasis, the spread and growth of tumours at secondary sites, is an extremely important clinical event, since a majority of cancer mortality is associated with the metastatic tumours, rather than the primary tumour. In spite of the importance of metastasis in the clinical setting, the actual process is extremely inefficient. Millions of tumour cells can be shed into the vasculature daily; yet, few secondary tumours are formed. The classical hypothesis explaining the inefficiency was a series of secondary events occurring in the tumour, resulting in a small subpopulation of cells capable of completing all of the steps required to successfully colonise a distant site. However, recent discoveries demonstrating the ability to predict metastatic propensity from gene expression profiles in bulk tumour tissue are not consistent with only a small subpopulation of cells in the primary tumour acquiring metastatic ability, suggesting that metastatic ability might be pre-programmed in tumours by the initiating oncogenic mutations. Data supporting both of these seemingly incompatible theories exist. Therefore, to reconcile the observed results, additional variables need to be added to the model of metastatic inefficiency. One possible variable that might explain the discrepancies is genetic background effects. Studies have demonstrated that the genetic background on which a tumour arises on can have significant affects on the ability of the tumour to metastasise and on gene expression profiles. Thus, the observations could be reconciled by combining the theories, with genetic background influencing both metastatic efficiency and predictive gene expression profiles, upon which, subsequently, metastasis-promoting mutational and epigenetic events occur. If the genetic background is an important determinant of metastatic efficiency, it would have significant implications for the clinical prediction and treatment of metastatic disease, as well as for the design of potential prevention strategies.

Preparation of microparticulate beta-glucan from Saccharomyces cerevisiae for use in immune potentiation.

AIMS: To develop a method for the preparation of an immunologically active, homogeneous, nonaggregated, microparticulate beta-glucan-containing material from the budding yeast Saccharomyces cerevisiae. METHODS AND RESULTS: Using a combination of sonication and spray-drying, a homogeneous preparation of 1-2-mu diameter beta-glucan-containing particles was made from alkali- and acid-insoluble yeast cell wall material. This microparticulate beta-glucan remained in suspension longer and, following oral administration at 0.1 mg kg(-1) for 14 d, enhanced phagocytosis of mouse peritoneal macrophages significantly better than did aggregated beta-glucan particles. CONCLUSIONS: A new sonication and spray-drying method can be employed to overcome the problem of aggregation of beta-glucan microparticles in aqueous media. SIGNIFICANCE AND IMPACT OF THE STUDY: A microparticulate form of beta-glucan that remains in suspension longer for pharmaceutical applications and has superior immune potentiation characteristics has been developed.

Predisposition to efficient mammary tumor metastatic progression is linked to the breast cancer metastasis suppressor gene Brms1.

Tumor metastasis is one of the most important clinical aspects of neoplastic disease because patient mortality is frequently attributable to disseminated rather than primary tumors. However, it still is not possible to definitively distinguish those individuals at high risk for disseminated disease, who would benefit from aggressive adjuvant therapy, from the low-risk patients who might be spared the side effects of additional anticancer therapy. To identify factors that predispose toward metastatic disease, we have used a genetic approach. Using a highly metastatic model of mammary cancer, we identified previously inbred mouse strains (DBA/2J, NZB/B1NJ, and I/LnJ) that harbor genetic factors that significantly suppress metastatic efficiency. In this study, we report the results of four experiments to localize the genetic map locations of the metastasis efficiency modifier genes. One statistically significant locus was identified on proximal Chr 19 designated Mtes1. Secondary candidate intervals were detected on Chrs 6, 9, 13, and 17. Interestingly, Mtes1 colocalizes with the murine orthologue of the human breast cancer metastasis suppressor gene Brms1, suggesting that allelic variants of Brms1 might be responsible for the metastasis suppression observed.

Three loci modify growth of a transgene-induced mammary tumor: suppression of proliferation associated with decreased microvessel density.

In earlier studies it was observed that the genetic background significantly affected the phenotype of a transgene-induced mammary tumor. Tumors arising in an (I/LnJ x PyMT) F1 hybrid background appeared earlier than in the FVB/N-TgN(MMTV-PyVT)(634Mul) parent, but accumulated less tumor mass, indicating a net decrease in tumor growth. Quantitative genetic mapping in a backcross identified three loci that were associated with the decreased proliferative capacity of the I/LnJ F1 tumors. Molecular analysis of the tumors suggests that these loci may act by restricting the tumor's ability to recruit microvessels. The three loci, designated Mmtg1-3, are unlinked to the angiogenic genes Fgf2, Flt1, Flk4, Flk1, Vegf, and Vegfc, as well as the precursors of the endogenous antiangiogenic molecules angiostatin and endostatin. The Mmtg loci may therefore provide novel targets for antiangiogenic therapeutic strategies.

Identification of inbred mouse strains harboring genetic modifiers of mammary tumor age of onset and metastatic progression.

Metastasis is one of the most important and complex processes in human neoplastic disease. A large number of both positive and negative events must occur to permit a tumor cell to colonize a distant site successfully. To identify mouse strains that harbor dominant genetic modifiers of this process, a strain survey was initiated utilizing a transgenic mouse mammary tumor model that exhibits a high incidence of pulmonary metastases. The transgenic animal was bred to 27 different inbred strains of mice and scored for the metastatic organ tropism and metastatic density. Thirteen strains were identified that had a statistically significant reduction in the numbers of pulmonary metastases. In addition, 10 strains were identified that altered the kinetics of induction of the primary mammary tumor. These strains will likely provide useful model systems for the analysis of genetic interactions in the initiation and progression of mammary adenocarcinomas.

Toward the construction of integrated physical and genetic maps of the mouse genome using interspersed repetitive sequence PCR (IRS-PCR) genomics.

Using two recently developed techniques, IRS-PCR YAC walking and IRS-PCR genotyping, a framework-integrated physical and genetic map of the mouse genome was constructed. The map consists of 821 contigs, containing 7746 YAC clones originating from three different YAC libraries. Three hundred eighty of the contigs have been anchored to the genetic map. Approximately 16% of the physical length of the mouse genome is estimated to be represented.

Rapid and efficient construction of yeast artificial chromosome contigs in the mouse genome with interspersed repetitive sequence PCR (IRS-PCR): generation of a 5-cM, > 5 megabase contig on mouse chromosome 1.

We have developed a new technique for the generation of YAC contigs in the mouse genome that is based on the ability to detect overlapping clones by hybridization of shared IRS-PCR products. As a demonstration of the technique, a 5-cM, > 5 megabase contig was developed on the distal half of mouse Chromosome (Chr) 1, spanning the region from Lamb2 to At3. The contig covers roughly 5% of the genetic distance of the chromosome and is comprised of more than 80 clones; 71 probes were assigned physical order to the chromosome, of which 59 were new markers generated in this study. Eight of the new probes were shown to be polymorphic between C3H/HeJ-gld and M. spretus. Three probes were mapped on a [(C3H/HeJ-gld x M. spretus) x C3H/HeJ-gld] interspecific backcross to integrate the physical map with a high-resolution genetic map of the region.

Single-strand conformational polymorphism (SSCP) mapping of the mouse genome: integration of the SSCP, microsatellite, and gene maps of mouse chromosome 1.

Interspersed repetitive sequence (IRS) PCR and repetitive element-to-bubble (IRS-bubble) PCR have been utilized to rapidly generate large numbers of mouse-specific, chromosome 1-enriched STSs from mouse-hamster somatic cell hybrids. Single-strand conformational polymorphism (SSCP) has been used to localize 39 new repetitive element-linked STSs to the mouse map: 22 to Chr 1, 10 to Chr. 15, 2 each to Chrs 12 and 14, and three to Chr 7. In addition, we have integrated the SSCP, single-strand length polymorphism, and restriction fragment length polymorphism maps of mouse Chr. 1, resulting in a high-density map of the chromosome, containing over 100 loci, all typed on a single interspecific backcross.

Reversal of intracellular toxicity of the trichothecene mycotoxin T-2 with monoclonal antibody.

The trichothecene mycotoxin T-2 is a potent inhibitor of intracellular protein synthesis. We have previously shown that a mouse immunoglobulin G1 monoclonal antibody (15H6) specific for T-2 toxin can neutralize the in vitro protein synthesis inhibitory effect of the toxin in human B-lymphoblastoid cultures, and protect rats from lethal toxemia. We now report that these monoclonal antibodies can induce the net efflux of [3H]-T-2 toxin from poisoned human B-lymphoblastoid cells in vitro, and restore protein synthesis. Administration of the monoclonal antibodies (250 mg/kg) 30 min before infusion of a lethal dose (1 mg/kg) of T-2 toxin causes the sequestration of the toxin in the plasma compartment. When administered 35 min after T-2 toxin, a time when the bulk of toxin is in the tissues, the monoclonal antibodies facilitate the migration of toxin back into the plasma compartment. These data demonstrate that monoclonal antibodies can be of therapeutic value against an intracellular toxin.

Biosensors. A new analytic technology for real-time, on-line biochemical monitoring.

A new technology is evolving that has the potential of improving patient management while substantially reducing the overall cost of health care. This new technology is based on biosensors, analytic microelectronic devices that use biologic detector molecules (e.g., antibodies, enzymes, receptor proteins, lectins, nucleic acids) as the sensing or signal transducing elements. An array of different biosensor configurations are under development, spurred on by recent advances in biotechnology and solid-state electronics. Although not all biosensors can detect their target analytes instantaneously, nor perform continuous measurements, certain biosensors embody both capabilities. Real-time, on-line biochemical monitoring will offer important information heretofore unavailable to the physician. It is also inevitable that biosensor-based instruments will decentralize patient testing, but telemetric systems can maintain the active and necessary involvement of the clinical pathologist.

Technological advances in bedside monitoring: biosensors.

The need to monitor certain key biochemical parameters in hospitalized patients is driving the development of biosensors, a new class of medical device for real-time, on-line quantitative analysis. A biosensor is a microelectronic device that utilizes a biological molecule (eg, antibody, enzyme, or receptor) as the sensing or signal-transducing element. Biosensors can be configured into simple, rapid, and cost-effective laboratory devices that will allow the clinical pathologist to become even more responsive to the primary care physician. In those instances where measurements on discrete samples do not provide the required information, continuous monitoring with implantable biosensors could provide real-time data on levels of critical endogenous or exogenous substances. By hybridizing recent advances in transdermal substance collection with the analytical capabilities of biosensors, devices for continuous noninvasive monitoring at the bedside can be envisioned. The clinical pathologist can and should play a key role in the clinical evaluation and implementation of such technological advances.

Early diagnosis of Schistosoma mansoni in mice using assays directed against cercarial antigens isolated by hydrophobic chromatography.

Two diagnostic assays are described for the early diagnosis of acute schistosomiasis, using a defined cercarial antigen preparation obtained by hydrophobic chromatography. Circulating IgM antibodies against this antigen fraction could be detected by ELISA as early as 1 wk after exposure in experimentally infected mice; IgM levels against other antigens and IgG levels against all the preparations examined were not significantly elevated until approximately 4-5 wk postinfection. Circulating antigen was detected as early as 3 days after exposure by a competitive inhibition ELISA using rabbit serum prepared against the cercarial antigen; antigen levels in the serum of mice with a 100-worm infection were found to exceed 100 ng/ml. Studies using sera from infected humans indicate that the assay can also recognize chronic S. mansoni, S. haematobium or S. japonicum infections. In a very limited field study, the specificity of the circulating antigen assay with regard to other helminthic infections was found to be 85%; sensitivity 100%. Preliminary characterization of the relevant antigen indicates that it is a relatively hydrophobic polypeptide with a molecular weight of approximately 41,000 daltons. The implications of these findings with regard to the treatment of travelers or the conduct of seroepidemiological studies in endemic areas are discussed.

Acute titration and chronic follow-up with captopril in hypertension. A one-year safety profile on combination therapy with captopril and a diuretic.

The present study examines acute titration with captopril and chronic follow-up data on captopril and a diuretic in patients with all forms of hypertension. Captopril was initiated in those patients in whom previous antihypertensive agents either failed to control high blood pressure or produced adverse reactions. Acute titration was done in 88 patients in whom average diastolic blood pressure was equal to or more than 95 mm Hg. Initial titration dosage was decided on the basis of initial blood pressure recordings. During initial titration, 5 patients received 12.5 mg, 51 received 25 mg, 28 received 50 mg, and the remaining 4 received 100 mg of captopril. Post-captopril blood pressure data were normalized by using pre-captopril data as 100% for each patient. The blood pressure-lowering effect of captopril on both systolic and diastolic blood pressure in all 88 patients was statistically significant (p less than 0.05), within forty-five minutes of captopril administration irrespective of the doses. No adverse reactions were seen during the acute titration. After the initial titration, in all 88 patients a diuretic was added to obtain a synergistic effect. Eleven patients were dropped from the study, for they could not follow the requirements of the protocol. In 77 patients the data for a one-year safety profile with captopril and diuretic were available. There were no overall significant statistical changes in serial white blood cell count, serum potassium, and serum creatinine values in those 77 patients. In 31 patients the initial and maintenance dosage of captopril and the diuretic remained unaltered for one year. Post-captopril blood pressure and heart rate data were normalized, pre-captopril data being considered as 100% in those 31 patients. The blood pressure data following captopril and a diuretic therapy compared with the pre-captopril data were statistically significant (p less than 0.05) throughout the study period. However, no significant changes in heart rates were observed during the study period. In all other patients, diuretic therapy was continued throughout the study period. In 6 severely hypertensive patients, an additional beta-blocker was needed for further control of high blood pressure. In 3 severe hypertensives with renal failure, besides a diuretic and a beta-blocker, minoxidil was needed to normalize their high blood pressure. In 4 of 77 patients, verapamil was used for treatment of either vasospastic angina or paroxsysmal supraventricular arrhythmia.(ABSTRACT TRUNCATED AT 400 WORDS)