Hypoxia Sensing and Persistence Genes Are Expressed during the Intragranulomatous Survival of Mycobacterium tuberculosis.

Although it is accepted that the environment within the granuloma profoundly affects Mycobacterium tuberculosis (Mtb) and infection outcome, our ability to understand Mtb gene expression in these niches has been limited. We determined intragranulomatous gene expression in human-like lung lesions derived from nonhuman primates with both active tuberculosis (ATB) and latent TB infection (LTBI). We employed a non-laser-based approach to microdissect individual lung lesions and interrogate the global transcriptome of Mtb within granulomas. Mtb genes expressed in classical granulomas with central, caseous necrosis, as well as within the caseum itself, were identified and compared with other Mtb lesions in animals with ATB (n = 7) or LTBI (n = 7). Results were validated using both an oligonucleotide approach and RT-PCR on macaque samples and by using human TB samples. We detected approximately 2,900 and 1,850 statistically significant genes in ATB and LTBI lesions, respectively (linear models for microarray analysis, Bonferroni corrected, P < 0.05). Of these genes, the expression of approximately 1,300 (ATB) and 900 (LTBI) was positively induced. We identified the induction of key regulons and compared our results to genes previously determined to be required for Mtb growth. Our results indicate pathways that Mtb uses to ensure its survival in a highly stressful environment in vivo. A large number of genes is commonly expressed in granulomas with ATB and LTBI. In addition, the enhanced expression of the dormancy survival regulon was a key feature of lesions in animals with LTBI, stressing its importance in the persistence of Mtb during the chronic phase of infection.

The DosR Regulon Modulates Adaptive Immunity and Is Essential for Mycobacterium tuberculosis Persistence.

RATIONALE: Hypoxia promotes dormancy by causing physiologic changes to actively replicating Mycobacterium tuberculosis. DosR controls the response of M. tuberculosis to hypoxia. OBJECTIVES: To understand DosR's contribution in the persistence of M. tuberculosis, we compared the phenotype of various DosR regulon mutants and a complemented strain to M. tuberculosis in macaques, which faithfully model M. tuberculosis infection. METHODS: We measured clinical and microbiologic correlates of infection with M. tuberculosis relative to mutant/complemented strains in the DosR regulon, studied lung pathology and hypoxia, and compared immune responses in lung using transcriptomics and flow cytometry. MEASUREMENTS AND MAIN RESULTS: Despite being able to replicate initially, mutants in DosR regulon failed to persist or cause disease. On the contrary, M. tuberculosis and a complemented strain were able to establish infection and tuberculosis. The attenuation of pathogenesis in animals infected with the mutants coincided with the appearance of a Th1 response and organization of hypoxic lesions wherein M. tuberculosis expressed dosR. The lungs of animals infected with the mutants (but not the complemented strain) exhibited early transcriptional signatures of T-cell recruitment, activation, and proliferation associated with an increase of T cells expressing homing and proliferation markers. CONCLUSIONS: Delayed adaptive responses, a hallmark of M. tuberculosis infection, not only lead to persistence but also interfere with the development of effective antituberculosis vaccines. The DosR regulon therefore modulates both the magnitude and the timing of adaptive immune responses in response to hypoxia in vivo, resulting in persistent infection. Hence, DosR regulates key aspects of the M. tuberculosis life cycle and limits lung pathology.

Microdissection approaches in tuberculosis research.

Mycobacterium tuberculosis, a globally significant pathogen, results in active or latent tuberculosis. The granuloma is the characteristic lesion that offers insight into host-pathogen interactions in these distinct states. Microdissection provides a way to isolate and consequently investigate specific tissue sections. We review various techniques available and in use.

Use of Protein Pegylation to Prolong the Antiviral Effect of IFN Against FMDV.

Interferons (IFNs) are considered the first line of defense against viral diseases. Due to their ability to modulate immune responses, they have become an attractive therapeutic option to control virus infections. In fact, like many other viruses, foot-and-mouth disease virus (FMDV), the most contagious pathogen of cloven-hoofed animals, is highly sensitive to the action of IFNs. Previous studies demonstrated that type I, II, and III IFNs, expressed using a replication defective human adenovirus 5 (Ad5) vector, can effectively block FMDV replication in vitro and can protect animals when challenged 1 day after Ad5-IFN treatment, in some cases providing sterile immunity. Rapidly spreading foot-and-mouth disease (FMD) is currently controlled with vaccination, although development of a protective adaptive immune response takes 5-7 days. Therefore, an optimal strategy to control FMD outbreaks is to block virus replication and spread through sustained IFN activity while the vaccine-stimulated adaptive immune response is developed. Challenges with methods of delivery and/or with the relative short IFN protein half-life in vivo, have halted the development of such approach to effectively control FMD in the animal host. One strategy to chemically improve drug pharmacodynamics is the use of pegylation. In this proof-of-concept study, we demonstrate that pegylated recombinant porcine (po)IFNα displays strong and long-lasting antiviral activity against FMDV in vitro and in vivo, completely protecting swine against FMD for at least five days after a single dose. These results highlight the potential of this biotherapeutics to use in combination with vaccines to fully control FMD in the field.

A novel microdissection approach to recovering mycobacterium tuberculosis specific transcripts from formalin fixed paraffin embedded lung granulomas.

Microdissection has been used for the examination of tissues at DNA, RNA, and protein levels for over a decade. Laser capture microscopy (LCM) is the most common microdissection technique used today. In this technique, a laser is used to focally melt a thermoplastic membrane that overlies a dehydrated tissue section(1). The tissue section composite is then lifted and separated from the membrane. Although this technique can be used successfully for tissue examination, it is time consuming and expensive. Furthermore, the successful completion of procedures using this technique requires the use of a laser, thus limiting its use. A new more affordable and practical microdissection approach called mesodissection is a possible solution to the pitfalls of LCM. This technique employs the MESO-1/MeSectr system to mill the desired tissue from a slide mounted tissue sample while concurrently dispensing and aspirating fluid to recover the desired tissue sample into a consumable mill bit. Before the dissection process begins, the user aligns the formalin fixed paraffin embedded (FFPE) slide with a hematoxylin and eosin stained (H&E) reference slide. Thereafter, the operator annotates the desired dissection area and proceeds to dissect the appropriate segment. The program generates an archived image of the dissection. The main advantage of mesodissection is the short duration needed to dissect a slide, taking an average of ten minutes from set up to sample generation in this experiment. Additionally, the system is significantly more cost effective and user friendly. A slight disadvantage is that it is not as precise as laser capture microscopy. In this article we demonstrate how mesodissection can be used to extract RNA from slides from FFPE granulomas caused by Mycobacterium tuberculosis (Mtb).