Quantitative Comparison of Active and Latent Tuberculosis in the Cynomolgus Macaque Model

We previously described that low-dose Mycobacterium tuberculosis infection in cynomolgus macaques results in a spectrum of disease similar to that of human infection: primary disease, latent infection, and reactivation tuberculosis (S. V. Capuano III, D. A. Croix, S. Pawar, A. Zinovik, A. Myers, P. L. Lin, S. Bissel, C. Fuhrman, E. Klein, and J. L. Flynn, Infect. Immun. 71:5831-5844, 2003). This is the only established model of latent infection, and it provides a unique opportunity to understand host and pathogen differences across of range of disease states. Here, we provide a more extensive and detailed characterization of the gross pathology, microscopic histopathology, and immunologic characteristics of monkeys in each clinical disease category. The data underscore the similarities between human and nonhuman primate M. tuberculosis infection. Furthermore, we describe novel methods of quantifying gross pathology and bacterial burden that distinguish between active disease and latent infection, and we extend the usefulness of this model for comparative studies. Early in infection, an abnormal chest X ray, M. tuberculosis growth by gastric aspirate, and increased mycobacterium-specific gamma interferon (IFN-γ) in peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage (BAL) cells were associated with the development of active disease. At necropsy, disease was quantified with respect to pathology and bacterial numbers. Microscopically, a spectrum of granuloma types are seen and differ with disease type. At necropsy, monkeys with active disease had more lung T cells and more IFN-γ from PBMC, BAL, and mediastinal lymph nodes than monkeys with latent infection. Finally, we have observed a spectrum of disease not only in monkeys with active disease but also in those with latent infection that provides insight into human latent tuberculosis.Mycobacterium tuberculosis is a remarkably successful human pathogen. Infection, usually via the respiratory route, results in primary tuberculosis or latent infection. Primary tuberculosis, defined as active disease within 2 years of the initial infection, is likely the result of a failure of the host immune response to control the infection. In this case, the initial infection can progress rapidly or slowly but causes contagious, active tuberculosis. Primary disease occurs in ∼5 to 10% of infected individuals (30). In contrast, most persons control the initial infection but apparently do not eliminate the bacteria completely. This asymptomatic condition is termed latent infection and is not considered to be contagious. A small percentage (5 to 10%) of latently infected persons reactivate during the course of a lifetime, resulting in active disease (reactivation). In humans, reactivation rates are increased in the setting of immune suppression, including human immunodeficiency virus (HIV) coinfection (30), aging (34), and tumor necrosis factor (TNF) neutralization (15, 24).The factors that dictate the outcome of M. tuberculosis infection in humans are not well understood. Studies of murine models have identified T cells, particularly CD4 T cells, as important in the control of initial and chronic infection. The cytokines gamma interferon (IFN-γ), interleukin-12 (IL-12), and TNF also have been shown to be crucial for the control of infection. IFN-γ is necessary for the activation of macrophages, and TNF has multiple functions in the immune response to M. tuberculosis (reviewed in reference 6). This is recapitulated in human studies, in which TNF neutralization (15, 24) and HIV infection (either as the direct loss of CD4 T cells or another immunologic perturbation) (30) has been associated with increased susceptibility to tuberculosis.Although the mouse model has been instrumental in defining several important aspects of the immune response to M. tuberculosis, infection in the mouse is progressive and ultimately leads to death. Mice control initial infection and a state of chronic infection ensues that is characterized by slowly advancing pathology, although the progression of disease is determined in part by the mouse strain used and the inoculating dose. Regardless, the mouse is not a model of latent infection. Mice also lack the organizational characteristics of human granulomas and do not have hypoxic granulomas, which are seen in humans and our model (32). Human granulomas have much more structured architecture, often with caseous necrosis in the center, and can be surrounded by a peripheral rim of fibrosis. Other animal models can display more human-like granulomas, such as the guinea pig and the rabbit, but latent infection has not been demonstrated in either of these animal models and immunology tools are limited.We previously reported that cynomolgus macaques infected with a low dose of M. tuberculosis either develop primary tuberculosis or have latent infection (3). To validate our clinical classification of disease states and quantify the outcome of infection, here we perform an in-depth comparison of monkeys with active and latent disease, and we demonstrate significant differences between these two clinically determined infection states with respect to gross and microscopic histopathology, bacterial numbers, and immune responses. There is a spectrum of disease in active tuberculosis and also in the subclinical state that is classified as latent infection. This is an effective model for understanding the events that predict disease outcome and for investigating therapies for active and latent tuberculosis and reactivation from latent infection. The close similarities between human and nonhuman primates with M. tuberculosis infection provide an excellent model for the study of tuberculosis. We demonstrate here that the outcomes of infection and amount of disease now can be quantified, allowing an analysis of monkey groups that will be useful when using this model to study immune interventions or drugs.     

Tumor necrosis factor neutralization results in disseminated disease in acute and latent Mycobacterium tuberculosis infection with normal granuloma structure in a cynomolgus macaque model

Objective

An increased risk of tuberculosis has been documented in humans treated with tumor necrosis factor α (TNFα)–neutralizing agents. In murine models, impaired signaling by TNF causes exacerbation of both acute and chronic infection associated with aberrant granuloma formation and maintenance. This study was undertaken to investigate immune modulation in the setting of TNF neutralization in primary and latent tuberculosis in a non‐human primate model.

Methods

Cynomolgus macaques 4 years of age or older were infected with Mycobacterium tuberculosis and subjected to clinical, microbiologic, immunologic, and radiographic examinations. Monkeys were classified as having active or latent disease 6–8 months after infection, based on clinical criteria. Monkeys used in acute infection studies were randomized to receive either adalimumab (prior to and during infection) or no treatment. Monkeys with latent infection that were randomized to receive TNF‐neutralizing agent were given either an inhibitor of soluble TNF, recombinant methionyl human soluble TNF receptor I (p55‐TNFRI), or adalimumab. Control monkeys with latent infection were given no treatment or saline. Data from previously studied monkeys with active or latent disease were also used for comparison.

Results

Administration of TNF‐neutralizing agents prior to M tuberculosis infection resulted in fulminant and disseminated disease by 8 weeks after infection. Neutralization of TNF in latently infected cynomolgus macaques caused reactivation in a majority of animals as determined by gross pathologic examination and bacterial burden. A spectrum of dissemination was noted, including extrapulmonary disease. Surprisingly, monkeys that developed primary and reactivation tuberculosis after TNF neutralization had similar granuloma structure and composition to that of control monkeys with active disease. TNF neutralization was associated with increased levels of interleukin‐12, decreased levels of CCL4, increased chemokine receptor expression, and reduced mycobacteria‐induced interferon‐γ production in blood but not in the affected mediastinal lymph nodes. Finally, the first signs of reactivation often occurred in thoracic lymph nodes.

Conclusion

These findings have important clinical implications for determining the mechanism of TNF neutralization–related tuberculosis.

     

General principles of abdominal wall reconstruction--20 years of experience

After 20 years of reconstructive abdominal wall surgery and almost five thousand surgical interventions for this pathology, most of them resolved with prosthesis, authors present a retrospective study concerning to increased biocompatibility, world dynamics of alloplastic solution and also the technical surgical problems that appears in incisional hernia repair. There are underlined two concepts: first--the maximal transversal diameter--that define the size of musculo-aponeurosis defect and the span of alloplastic substitution, in opposition to the old idea that allude to the size of hernia sack; the second concept--receptor prosthetic layer--define the interface between abdominal viscera and prosthetic material. This is not just a mechanical divider, being also a conjunctive and vascular support, helping prosthetic tissue integration.     

Alcohol-induced S-adenosylhomocysteine accumulation in the liver sensitizes to TNF hepatotoxicity: possible involvement of mitochondrial S-adenosylmethionine transport

Hepatocytes are resistant to tumor necrosis factor-alpha- (TNF) induced killing/apoptosis under normal circumstances, but primary hepatocytes from rats chronically fed alcohol have increased TNF cytotoxicity. Therefore, there must be mechanism(s) by which alcohol exposure "sensitizes" to TNF hepatotoxicity. Abnormal metabolism of methionine and S-adenosylmethionine (SAM) are well-documented acquired metabolic abnormalities in ALD. S-adenosylhomocysteine (SAH) is the product of SAM in hepatic transmethylation reactions, and SAH hydrolase (SAHH) is the only enzyme to metabolize SAH to homocysteine and adenosine. Our previous studies demonstrated that chronic intracellular accumulation of SAH sensitized hepatocytes to TNF cytotoxicity in vitro. In the current study, we extended our previous observations by further characterizing the effects of chronic alcohol intake on mitochondrial SAM levels in liver and examining its possible involvement in SAH sensitization to TNF hepatotoxicity. Chronic alcohol consumption in mice not only increased cytosolic SAH levels, but also decreased mitochondrial SAM concentration, leading to decreased mitochondrial SAM to SAH ratio. Moreover, accumulation of hepatic SAH induced by administration of 3-deaza-adenosine (DZA-a potent inhibitor of SAHH) enhanced lipopolysaccharide (LPS)/TNF hepatotoxicity in mice in vivo. Inhibition of SAHH by DZA resulted not only in accumulation of cytoplasmic SAH, but also in depletion of the mitochondrial SAM pool. Further studies using mitochondrial SAM transporter inhibitors showed that inhibition of SAM transport into mitochondria sensitized HepG2 cells to TNF cytotoxicity. In conclusion, our results demonstrate that depletion of the mitochondrial SAM pool by SAH, which is elevated during chronic alcohol consumption, plays a critical role in SAH induced sensitization to TNF hepatotoxicity.     

Genomic damage induced by 1‐MHz ultrasound in vitro

Genotoxic effects of therapeutic ultrasound are poorly documented, when compared with the wide use of this physical agent. The aim of this work was to investigate the clastogenic and aneugenic potential of 1 MHz ultrasound, employing intensities (200 and 300 mW/cm²) above the cavitational threshold, but in the range of those normally used in therapeutics. Both normal fibroblasts (AG01522) and tumoral cells (MCF‐7) were sonicated. While no effects on viability were noted, significant increases of CREST‐negative micronuclei (indicative of clastogenesis) and CREST‐positive micronuclei (indicative of aneuploidy) were detected. Clastogenesis was confirmed by increases of γ‐H2AX foci, while increases of spindle anomalies confirmed the induction of aneuploidy. Our results confirm previous works that showed ultrasound‐induced DNA breakage. Moreover, our experiments show that the known effect of ultrasound‐induced damage to microtubules is also able to damage the mitotic spindle and induce aneuploidy. On the overall, this work highlights the importance to further investigate the potential risks related to therapeutics US. Environ. Mol. Mutagen. 59:60–68, 2018. © 2017 Wiley Periodicals, Inc.     

Cognitive Control: Componential and Yet Emergent

In this commentary, I will argue that the componential and emergent views of cognitive control as defined by Cooper (2010) do not necessarily oppose each other, and I will try to make a case for their interdependence. First, I will use the construct of cognitive inhibition—one of the main componential control functions mentioned in the target articles—to illustrate my line of reasoning. Then, I will comment on how some of the target articles, each from a different perspective, bring arguments in favor of this integrative view.     

Vascular diaphragmatic hernia in a patient with cirrhosis first case report

We report the case of an adult patient recently diagnosed with cirrhosis. The ultrasound evaluation described a multinodular inhomogeneous liver, requiring a magnetic resonance imaging scan for further characterization. The performed magnetic resonance imaging examination confirmed the diagnosis of cirrhosis associated with portal hypertension and detected a vascular left transdiaphragmatic hernia. Although various types of diaphragmatic hernias have been described - congenital or acquired - to the best of our knowledge, this type of pathology has never been reported.     

Long-term genotoxic effects in the hematopoietic system of prenatally X-irradiated mice

Purpose: To investigate the genotoxic effects of prenatal X-irradiation in mice and the possible presence of late genomic instability.

Materials and methods: Pregnant mice were exposed to 0, 1 or 2?Gy at embryonic day 11.5. Blood smears were obtained from pups at birth and on post-natal day 11, 21, 42 and 140. Hematological data (diameter of erythrocytes, percentage of reticulocytes and Granulocyte-to-Lymphocyte ratio [GLR]) and genotoxicity (micronucleated erythrocytes, micronucleated reticulocytes, CREST-positive and negative micronuclei) were assessed.

Results: Prenatal irradiation caused perinatal reticulocytosis (which ended on postnatal day 11) and a dose-dependent increase of GLR (indicative of myeloid skewing) on postnatal days 42 and 140. Two temporally distinct genotoxic effects were observed: an early, acute damage (still detectable at birth and soon after) and a late, long-term damage.

Conclusions: Increases in micronuclei frequencies and GLR observed from day 42 on are both ascribable to DNA damage. Time of appearance of this late effect may be linked to the shift of hematopoiesis from spleen to bone marrow and to cell-extrinsic factor such as the microenvironment. This study confirms that ionizing radiation can induce long-term genotoxic effects in the hematopoietic system and shows that prenatal irradiation determines genomic instability in blood-forming tissues of adult mice.