SummaryThe RD1 genomic region is present in virulent strains of Mycobacterium tuberculosis (MTB), missing from the vaccine strain M. bovis BCG, and its importance to virulence has been established experimentally. Based on in silico analysis, it has been suggested that RD1 may encode a novel secretion system, but the mechanism by which this region affects virulence is unknown. Here we examined mutants disrupted in five individual RD1 genes. Both in vitro and in vivo , each mutant displayed an attenuated phenotype very similar to a mutant missing the entire RD1 region. Genetic complementation of individual genes restored virulence. Attenuated mutants could multiply within THP-1 cells, but they were unable to spread to uninfected macrophages. We also examined export of two immunodominant RD1 proteins, CFP-10 and ESAT-6. Export of these proteins was greatly reduced or abolished in each attenuated mutant. Again, genetic complementation restored a wild-type phenotype. Our results indicate that RD1 genes work together to form a single virulence determinant, and argue that RD1 encodes a novel specialized secretion system that is required for pathogenesis of MTB.

Nuclear imaging is an established clinical molecular imaging modality that offers good sensitivity deep in tissue. However, nuclear imaging is limited by several factors such as timeconsuming data acquisition, expensive equipment, exposure to radioactivity, the need for highly skilled personnel, and relatively poor spatial resolution. 1 Optical imaging is a relatively new imaging modality that offers real-time, non-radioactive, and, depending on the technique, high-resolution imaging of fluorophores embedded in diseased tissues. 2 Of the various optical imaging techniques investigated to date, near-infrared (NIR, 700−900 nm wavelength) fluorescence-based imaging is of particular interest for noninvasive in vivo imaging because of the relatively low tissue absorption, scatter, and minimal autofluorescence of NIR light. 3 NIR fluorescence has the potential to provide rapid, inexpensive, and non-radioactive population-based screening for breast cancer. 4-6 However, it is unclear whether currently available optical imaging systems have adequate sensitivity and/or resolution to identify breast pathology such as microcalcifications. In this study, we developed a critical reagent for exploring the limits of NIR fluorescence-based breast cancer diagnosis, namely, a simultaneous optical and nuclear contrast agent.Bisphosphonates (BPs) bind avidly to hydroxyapatite (HA) bone mineral surfaces, 7 and have many uses. BP-based radiotracers are used to diagnose osteoblastic bone lesions and to treat bone metastasis associated with breast cancer. 8 In addition, contrast agents (CAs) with BPs and phosphonates as the targeting group have been developed for use with nuclear imaging. 9, 10 Although, our group 11-13 and others 14 have explored NIR imaging with BPs, to the best of our knowledge, no BP-based dual modality nuclear-NIR contrast agents have been reported. Dual-labeled targeting imaging agents, such as the one described herein, allow cross validation and direct comparison between nuclear (the gold-standard) and fluorescence optical imaging.The tri-functional diagnostic agent Pam-Tc/Re-800 was synthesized in 5 chemical steps (Scheme 1), with an overall yield of 53%, from N-ε-t.-Boc-L-lysine 1, Me-Pam, 13 MAS 3 , and IRDye800CW. Primary amine of compound 1 was conjugated with N-hydroxy succinimide ester of IRDye800CW to obtain Lys(t.-Boc)-800CW-carboxylic acid 2. Me-Pam reacted with activated compound 2 in presence of HCTU and NMM to generate Lys(t.-Boc)-800CW-PamMe 3. Compound 3 was treated with trifluoroacetic acid and trimethylsilyl bromide to deprotect E-mail: jfrangio@bidmc.harvard.edu. Supporting Information Available: Experimental procedures and spectroscopic data. This material is available free of charge via the Internet at http://pubs.acs.org. Pam-Tc/Re-800 6 was fully characterized for its radioactivity, spectral properties (Supporting Information), and calcium salt specificity (Figure 1). Specific activity of Pam-Tc-800 6a was greater than or equal to 6,250 Ci/mmol and radiochemical purity was greater than or eq...

Moderate to severe traumatic brain injury (TBI) in humans and rats induces measurable metabolic changes, including a sustained depression in cerebral glucose uptake. However, the effect of a mild TBI on brain glucose uptake is unclear, particularly in rodent models. This study aimed to determine the glucose uptake pattern in the brain after a mild lateral fluid percussion (LFP) TBI. Briefly, adult male rats were subjected to a mild LFP and positron emission tomography (PET) imaging with (18)F-fluorodeoxyglucose ((18)FDG), which was performed prior to injury and at 3 and 24 h and 5, 9, and 16 days post-injury. Locomotor function was assessed prior to injury and at 1, 3, 7, 14, and 21 days after injury using modified beam walk tasks to confirm injury severity. Histology was performed at either 10 or 21 days post-injury. Analysis of function revealed a transient impairment in locomotor ability, which corresponds to a mild TBI. Using reference region normalization, PET imaging revealed that mild LFP-induced TBI depresses glucose uptake in both the ipsilateral and contralateral hemispheres in comparison with sham-injured and naïve controls from 3 h to 5 days post-injury. Further, areas of depressed glucose uptake were associated with regions of glial activation and axonal damage, but no measurable change in neuronal loss or gross tissue damage was observed. In conclusion, we show that mild TBI, which is characterized by transient impairments in function, axonal damage, and glial activation, results in an observable depression in overall brain glucose uptake using (18)FDG-PET.

Region of difference (RD1) genes are present in virulent Mycobacterium tuberculosis but not the vaccine strain M. bovis bacille Calmette-Guérin (BCG). The deletion of RD1 from M. tuberculosis produces an attenuation strikingly like that of BCG, which suggests the use of RD1 mutant strains for improvement of the tuberculosis (TB) vaccine. We performed long-term murine infection with M. tuberculosis H37Rv: Delta RD1 and BCG. Mice infected with H37Rv: Delta RD1 gained less weight than did BCG-infected control mice, and, after >1 year, their lungs harbored many more bacteria and displayed significant levels of inflammation. This difference in virulence has important implications for the pursuit of strains lacking RD1 in the development of the TB vaccine.

Lung biopsy specimens from children after cavopulmonary anastomosis demonstrate increased expression of vascular endothelial growth factor and its receptor. These data confirm earlier findings that blood vessels forming after cavopulmonary anastomosis may have reduced intercellular junctions (decreased CD31 staining). Despite the increased numbers of pulmonary vessels that are present in these patients, these vessels are not highly proliferative (proliferating cell nuclear antigen staining equivalent to that of control subjects). These results suggest that vascular endothelial growth factor may be a mediator of angiogenesis in the lungs of children after cavopulmonary anastomosis; however, other factors, such as vascular dilation and remodeling, may also be important.

No abstract

Although much is understood regarding the role of B7/CD28 family of costimulatory molecules in regulating host resistance in the context of several pathogens, analogous information with Mycobacterium tuberculosis is lacking. To address the requirements of B7-mediated costimulation in host resistance against tuberculosis, mice deficient in both B7.1 and B7.2 (B7DKO) were aerosol infected with M. tuberculosis Erdman and disease progression was monitored. We report herein that B7DKO mice are initially able to contain the bacterial load in the lung, but exhibit enhanced susceptibility during chronic infection. Despite the early control of bacterial replication, B7DKO mice essentially start off with compromised Th1 immunity and slower granulomatous response in the lung, characterized by markedly reduced lymphocytic infiltration. As the infection progresses from acute phase to the chronic phase, the nascent granulomas in the B7DKO lungs never fully achieve the architecture of granulomas developing in wild-type mice. Instead, lesions spread progressively to involve much of the lung in the B7DKO mice, ultimately leading to necrosis. Thus, early control of M. tuberculosis growth in the lung can occur in the absence of B7 costimulation and is less dependent on Th1 immunity and formation of a granulomatous structure. However, B7 costimulation is critical for long-term containment of infection within lung granulomas. These findings suggest that the use of costimulation-based immunomodulators may have significant repercussions on the induction of host protective immunity against tuberculosis.

No abstract