Plasma radio-metabolite analysis of PET tracers for dynamic PET imaging: TLC and autoradiography.

Background: In molecular imaging with dynamic PET, the binding and dissociation of a targeted tracer is characterized by kinetics modeling which requires the arterial concentration of the tracer to be measured accurately. Once in the body the radiolabeled parent tracer may be subjected to hydrolysis, demethylation/dealkylation and other biochemical processes, resulting in the production and accumulation of different metabolites in blood which can be labeled with the same PET radionuclide as the parent. Since these radio-metabolites cannot be distinguished by PET scanning from the parent tracer, their contribution to the arterial concentration curve has to be removed for the accurate estimation of kinetic parameters from kinetic analysis of dynamic PET.

Candidate modifier genes for immune function in 22q11.2 deletion syndrome.

Background: The 22q11.2 deletion syndrome (22q11.2DS) is the most common contiguous microdeletion affecting humans and exhibits extreme phenotypic heterogeneity.

Comparative analytical evaluation of the respiratory TaqMan Array Card with real-time PCR and commercial multi-pathogen assays.

In this study, a multicenter evaluation of the Life Technologies TaqMan(®) Array Card (TAC) with 21 custom viral and bacterial respiratory assays was performed on the Applied Biosystems ViiA™ 7 Real-Time PCR System. The goal of the study was to demonstrate the analytical performance of this platform when compared to identical individual pathogen specific laboratory developed tests (LDTs) designed at the Centers for Disease Control and Prevention (CDC), equivalent LDTs provided by state public health laboratories, or to three different commercial multi-respiratory panels. CDC and Association of Public Health Laboratories (APHL) LDTs had similar analytical sensitivities for viral pathogens, while several of the bacterial pathogen APHL LDTs demonstrated sensitivities one log higher than the corresponding CDC LDT.

Optimization of a real-time RT-PCR assay reveals an increase of genogroup I norovirus in the clinical setting.

Although norovirus has been identified as the most common cause of gastroenteritis, the majority of cases have no etiologic agent identified. In this study, we describe the optimization of a real-time RT-PCR assay for the improved detection of genogroup I norovirus in patient specimens based upon sequence data from a collection of representative clinical norovirus sequences. The redesigned assay demonstrated a 64 fold increase in sensitivity, a 2 log decrease in the limit of detection, and an 18% increase in amplification efficiency, when compared to the standard assay.

Inactivation of Mycobacterium tuberculosis mannosyltransferase pimB reduces the cell wall lipoarabinomannan and lipomannan content and increases the rate of bacterial-induced human macrophage cell death.

The Mycobacterium tuberculosis (M.tb) cell wall contains an important group of structurally related mannosylated lipoglycans called phosphatidyl-myo-inositol mannosides (PIMs), lipomannan (LM), and mannose-capped lipoarabinomannan (ManLAM), where the terminal alpha-[1-->2] mannosyl structures on higher order PIMs and ManLAM have been shown to engage C-type lectins such as the macrophage mannose receptor directing M.tb phagosome maturation arrest.

The duration of mumps virus shedding after the onset of symptoms.

To determine how long people shed virus after the onset of mumps, we used logistic regression modeling to analyze data from the 2006 outbreak of mumps in Iowa. Our model establishes that the probability of mumps virus shedding decreases rapidly after the onset of symptoms. However, we estimate that 8%-15% of patients will still be shedding the virus 5 days after the onset of symptoms and, thus, may still be contagious during this period.

Real-time reverse transcription-PCR assay for detection of mumps virus RNA in clinical specimens.

The mumps virus is a negative-strand RNA virus in the family Paramyxoviridae. Mumps infection results in an acute illness with symptoms including fever, headache, and myalgia, followed by swelling of the salivary glands. Complications of mumps can include meningitis, deafness, pancreatitis, orchitis, and first-trimester abortion.

Overexpression of Mycobacterium tuberculosis manB, a phosphomannomutase that increases phosphatidylinositol mannoside biosynthesis in Mycobacterium smegmatis and mycobacterial association with human macrophages.

Mycobacterium tuberculosis (M. tb) pathogenesis involves the interaction between the mycobacterial cell envelope and host macrophage, a process mediated, in part, by binding of the mannose caps of M. tb lipoarabinomannan (ManLAM) to the macrophage mannose receptor (MR).

The human macrophage mannose receptor directs Mycobacterium tuberculosis lipoarabinomannan-mediated phagosome biogenesis.

Mycobacterium tuberculosis (M.tb) survives in macrophages in part by limiting phagosome-lysosome (P-L) fusion. M.

Emergent strain of human adenovirus endemic in Iowa.

We evaluated 76 adenovirus type 7 (Ad7) isolates collected in Iowa from 1992 to 2002 and found that genome type Ad7d2 became increasingly prevalent. By 2002, it had supplanted all other Ad7 genome types. The association of Ad7d2 with severe illness and death calls for heightened public health concern.

A fatal case of West Nile virus infection in a bone marrow transplant recipient.

West Nile virus (WNV) can cause severe, potentially fatal neurological illnesses, which include encephalitis, meningitis, Guillain-Barré syndrome, and anterior myelitis. Because of the short viremic phase, WNV infection is most commonly diagnosed by detection of immunoglobulin M antibody to WNV in serum or cerebrospinal fluid (CSF). We describe a patient with T cell lymphoma who had undergone a T cell-depleted bone marrow transplantation and developed fatal WNV infection.

Mycobacterium tuberculosis-infected human macrophages exhibit enhanced cellular adhesion with increased expression of LFA-1 and ICAM-1 and reduced expression and/or function of complement receptors, FcgammaRII and the mannose receptor.

The entry of Mycobacterium tuberculosis (Mtb) into the host macrophage and its survival in this environment are key components of tuberculosis pathogenesis. Following intracellular replication of the bacterium within alveolar macrophages, there is spread of bacilli to regional lymph nodes in the lungs and subsequent presentation of antigens to the host immune system. How this process occurs remains poorly understood, but one mechanism may involve the migration of macrophages containing Mtb across the alveoli to lymph nodes, where there is development of a protective host response with formation of granulomas composed in part of aggregated and fused, apoptotic, infected macrophages.

Priming reverse transcription with oligo(dT) does not yield representative samples of Mycobacterium tuberculosis cDNA.

Several recent publications have suggested that oligo(dT) can prime reverse transcription of several mycobacterial mRNAs. To determine if this is the case for most Mycobacterium tuberculosis mRNA species, reverse transcription reactions of M. tuberculosis RNA were primed with oligo(dT) or with other primers that did not target polyadenylylated sequences, and the resultant cDNA product was evaluated.

Microaerophilic induction of the alpha-crystallin chaperone protein homologue (hspX) mRNA of Mycobacterium tuberculosis.

Among the products that are expressed when Mycobacterium tuberculosis undergoes hypoxic shiftdown to nonreplicating persistence (NRP) is the alpha-crystallin chaperone protein homologue (Acr). This expression coincides with the previously reported appearance of a respiratory type of nitrate reductase activity, the increase in glycine dehydrogenase activity, and the production of a unique antigen, URB-1. In a timed sampling study, using a slowly stirred oxygen depletion culture model, we have demonstrated that the hspX mRNA that codes for Acr protein as well as the protein itself is induced just as the bacilli enter the microaerophilic NRP stage 1 (NRP-1).

An increase in expression of a Mycobacterium tuberculosis mycolyl transferase gene (fbpB) occurs early after infection of human monocytes.

Changes in the mRNA levels of two Mycobacterium tuberculosis genes (fbpB known as antigen 85B, and hspX known as Acr) were studied in infected human monocytes. Antigen 85B is an enzyme involved in cell wall biosynthesis and is also a major target of the immune response. Acr is a stress protein believed to be involved in the bacillary response to adverse conditions and in non-replicating persistence.

Isolation of M. tuberculosis RNA from Sputum.

Analysis of RNA has many applications and has become increasingly important both in basic research and clinical application. The type and number of particular mRNA transcripts expressed in M. tuberculosis (MTB) bacilli under various conditions can provide insight into models of pathogenesis.

Quantification of M. tuberculosis DNA in Sputum During the Treatment of Pulmonary Tuberculosis.

The most common laboratory measures of response to therapy for patients with pulmonary tuberculosis are conversion of acid-fast bacilli (AFB) sputum smear to negative, or culture positive sputum to negative. AFB enumeration lacks sensitivity and specificity and the culture of M. tuberculosis (MTB) bacteria can take weeks.

Measurement of sputum Mycobacterium tuberculosis messenger RNA as a surrogate for response to chemotherapy.

Effective treatment regimens for pulmonary tuberculosis are difficult to assess because of the slow growth rate of Mycobacterium tuberculosis in culture and its protracted clearance from sputum. A rapid method that reflects effective antimicrobial activity would markedly advance evaluation of treatment and promote the assessment of new antituberculosis drugs. Conventional methods measure the progressive reduction of numbers of acid-fast bacilli in the sputum smear and the clearance of organisms in sputum culture.

Detection of viable Mycobacterium tuberculosis by reverse transcriptase-strand displacement amplification of mRNA.

Numerous assays have been described for the detection of DNA and rRNA sequences that are specific for the Mycobacterium tuberculosis complex. Although beneficial to initial diagnosis, such assays have proven unsuitable for monitoring therapeutic efficacy owing to the persistence of these nucleic acid targets long after conversion of smears and cultures to negative. However, prokaryotic mRNA has a typical half-life of only a few minutes and we have previously shown that the presence of mRNA is a good indicator of bacterial viability.

Quantitative analysis of mRNA as a marker for viability of Mycobacterium tuberculosis.

Numerous assays which use conserved DNA or rRNA sequences as targets for amplification have been described for the diagnosis of tuberculosis. However, these techniques have not been applied successfully to the monitoring of therapeutic efficacy owing to the persistence of amplifiable nucleic acid beyond the point at which smears and cultures become negative. Semiquantitative analysis of rRNA has been used to reduce the time required for antimicrobial susceptibility testing of Mycobacterium tuberculosis, although growth for up to 5 days in the presence of some drugs is still required to discriminate resistant strains.

Comparison of the ABI 7700 system (TaqMan) and competitive PCR for quantification of IS6110 DNA in sputum during treatment of tuberculosis.

Mycobacterium tuberculosis can persist in sputum for long periods of time after the initiation of antituberculosis chemotherapy. The purpose of this study was to determine whether quantitative estimates of M. tuberculosis DNA in sputum correlate with the numbers of viable bacilli and thus measure the therapeutic response of patients during treatment.

Hyperproliferation of BXSB B cells is linked to the Yaa allele.

Systemic lupus erythematosus is characterized by polyclonal B cell activation, the production of autoantibodies, and often by renal disease. Previous studies demonstrated that unfractionated B cells from several strains of mice with lupus hyperproliferate in culture when stimulated with lipopolysaccharide (LPS) or anti-IgM. We wished to further examine proliferation of resting B cells from the BXSB mouse model of lupus and mice with the Yaa allele, when activated with a number of stimuli.