Immunogenicity of PE18, PE31, and PPE26 proteins from Mycobacterium tuberculosis in humans and mice.

Introduction: The large family of PE and PPE proteins accounts for as much as 10% of the genome of Mycobacterium tuberculosis. In this study, we explored the immunogenicity of three proteins from this family, PE18, PE31, and PPE26, in humans and mice. Methods: The investigation involved analyzing the immunoreactivity of the selected proteins using sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy donors from the TB endemic country Mozambique. Antigen-recall responses were examined in PBMC from these groups, including the evaluation of cellular responses in healthy unexposed individuals. Moreover, systemic priming and intranasal boosting with each protein, combined with the Quil-A adjuvant, were conducted in mice. Results: We found that all three proteins are immunoreactive with sera from TB patients, IGRA-positive household contacts, and IGRA-negative BCG vaccinated healthy controls. Likewise, antigen-recall responses were induced in PBMC from all groups, and the proteins stimulated proliferation of peripheral blood mononuclear cells from healthy unexposed individuals. In mice, all three antigens induced IgG antibody responses in sera and predominantly IgG, rather than IgA, responses in bronchoalveolar lavage. Additionally, CD4+ and CD8+ effector memory T cell responses were observed in the spleen, with PE18 demonstrating the ability to induce tissue-resident memory T cells in the lungs. Discussion: Having demonstrated immunogenicity in both humans and mice, the protective capacity of these antigens was evaluated by challenging immunized mice with low-dose aerosol of Mycobacterium tuberculosis H37Rv. The in vitro Mycobacterial Growth Inhibition Assay (MGIA) and assessment of viable bacteria in the lung did not demonstrate any ability of the vaccination protocol to reduce bacterial growth. We therefore concluded that these three specific PE/PPE proteins, while immunogenic in both humans and mice, were unable to confer protective immunity under these conditions.

Serological analysis reveals differential antibody responses between TB patients and latently infected individuals from the TB endemic country of Mozambique.

Serological antibody profiling of tuberculosis (TB) patients and household contacts with latent TB infection (LTBI) could identify risk indicators of disease progression, and potentially also serve as an easily accessible diagnostic tool to discriminate between these two stages of Mycobacterium tuberculosis (Mtb) infection. Yet, despite significant efforts over many decades, neither application has yet fully materialised, and this is at least in part due to inconsistent and varying antibody profiles from different TB endemic regions. In this study, we conducted a retrospective exploratory analysis of serum antibodies in a cohort of active TB patients (ATB) and their interferon-gamma release assay (IGRA) positive household contacts (LTBI), as well as healthy controls (HC) from Mozambique, a country with a high TB burden from the Sub-Saharan region. Using several Mtb antigens as well as crude preparations of culture filtrate proteins (CFP) from Mtb and Bacille Calmette Guérin (BCG), we report that the most discriminatory response for TB and LTBI was observed for serum IgA antibodies to the MPT64 antigen, followed by IgG antibodies to Ag85B and CFP, with ATB patients having significantly higher levels than LTBI or BCG-vaccinated healthy controls. Conversely, sera from LTBI individuals had higher levels of IgG antibodies to the HBHA antigen than ATB. While our sample size (n = 21 for ATB, 18 for LTBI and 17 for HC) was too small to fully evaluate the diagnostic potential of these differing serological profiles, our study however preliminarily indicated high level of sensitivity (95%) and specificity (97%) of an ELISA MPT64-IgA test for discriminating TB from LTBI and healthy controls, supporting the notion that it alone, or possibly in combination with other antigens such as Ag85B or CFP could lead to development of an easily accessible diagnostic tool for TB.

Mycobacterium tuberculosis PE/PPE proteins enhance the production of reactive oxygen species and formation of neutrophil extracellular traps.

Introduction: Neutrophil granulocytes predominate in the lungs of patients infected with Mycobacterium tuberculosis (Mtb) in earlier stages of the disease. During infection, neutrophils release neutrophil extracellular traps (NETs), an antimicrobial mechanism by which a DNA-backbone spiked with antimicrobial components traps the mycobacteria. However, the specific mycobacterial factors driving NET formation remain unclear. Proteins from the proline-glutamic acid (PE)/proline-proline-glutamic acid (PPE) family are critical to Mtb pathophysiology and virulence. Methods: Here, we investigated NET induction by PE18, PPE26, and PE31 in primary human blood-derived neutrophils. Neutrophils were stimulated with the respective proteins for 3h, and NET formation was subsequently assessed using confocal fluorescence microscopy. Intracellular ROS levels and cell necrosis were estimated by flow cytometry. Additionally, the influence of phorbol-12-myristate-13-acetate (PMA), a known NADPH oxidase enhancer, on NET formation was examined. Neutrophil integrity following incubation with the PE/PPE proteins was evaluated using transmission electron microscopy. Results: For the first time, we report that stimulation of primary human blood-derived neutrophils with Mtb proteins PE18, PPE26, and PE31 resulted in the formation of NETs, which correlated with an increase in intracellular ROS levels. Notably, the presence of PMA further amplified this effect. Following incubation with the PE/PPE proteins, neutrophils were found to remain viable and structurally intact, as verified through transmission electron microscopy, indicating the occurrence of vital NET formation. Discussion: These findings offer valuable insights that contribute to a better understanding of host-pathogen interactions during Mtb infection. Moreover, they underscore the significance of these particular Mtb antigens in triggering NET formation, representing a distinctive and previously unrecognized function of PE/PPE antigens.

Role of phagocyte extracellular traps during Mycobacterium tuberculosis infections and tuberculosis disease processes.

Mycobacterium tuberculosis (M.tb) infections remain one of the most significant causes of mortality worldwide. The current situation shows an emergence of new antibiotic-resistant strains making it difficult to control the tuberculosis (TB) disease. A large part of its success as a pathogen is due to its ability to persist for years or even decades without causing evident clinical manifestations. M.tb is highly successful in evading the host-defense by manipulating host-signalling pathways. Although macrophages are generally viewed as the key cell type involved in harboring M.tb, growing evidence shows that neutrophils also play a fundamental role. Both cells are known to act in multiple ways when encountering an invading pathogen, including phagocytosis, release of cytokines and chemokines, and oxidative burst. In addition, the formation of neutrophil extracellular traps (NETs) and macrophage extracellular traps (METs) has been described to contribute to M.tb infections. NETs/METs are extracellular DNA fibers with associated granule components, which are released upon activation of the cells by the pathogen or by pro-inflammatory mediators. On one hand, they can lead to a protective immune response by entrapment and killing of pathogens. However, on the other hand, they can also play a severe pathological role by inducing tissue damage. Extracellular traps (ETs) produced in the pulmonary alveoli can expand easily and expose tissue-damaging factors with detrimental effects. Since host-directed therapies offer a complementary strategy in TB, the knowledge of NET/MET formation is important for understanding potential protective versus detrimental pathways during innate immune signaling. In this review, we summarize the progress made in understanding the role of NETs/METs in the pathogenesis of TB.

Cellular and Molecular Alterations Underlying Abnormal Bone Growth in X-Linked Hypophosphatemia.

X-linked hypophosphatemia (XLH), the most common form of hereditary hypophosphatemic rickets, is caused by inactivating mutations of the phosphate-regulating endopeptidase gene (PHEX). XLH is mainly characterized by short stature, bone deformities and rickets, while in hypophosphatemia, normal or low vitamin D levels and low renal phosphate reabsorption are the principal biochemical aspects. The cause of growth impairment in patients with XLH is not completely understood yet, thus making the study of the growth plate (GP) alterations necessary. New treatment strategies targeting FGF23 have shown promising results in normalizing the growth velocity and improving the skeletal effects of XLH patients. However, further studies are necessary to evaluate how this treatment affects the GP as well as its long-term effects and the impact on adult height.

Innovative Three-Dimensional Microscopic Analysis of Uremic Growth Plate Discloses Alterations in the Process of Chondrocyte Hypertrophy: Effects of Growth Hormone Treatment.

Chronic kidney disease (CKD) alters the morphology and function of the growth plate (GP) of long bones by disturbing chondrocyte maturation. GP chondrocytes were analyzed in growth-retarded young rats with CKD induced by adenine intake (AD), control rats fed ad libitum (C) or pair-fed with the AD group (PF), and CKD rats treated with growth hormone (ADGH). In order to study the alterations in the process of GP maturation, we applied a procedure recently described by our group to obtain high-quality three-dimensional images of whole chondrocytes that can be used to analyze quantitative parameters like cytoplasm density, cell volume, and shape. The final chondrocyte volume was found to be decreased in AD rats, but GH treatment was able to normalize it. The pattern of variation in the cell cytoplasm density suggests that uremia could be causing a delay to the beginning of the chondrocyte hypertrophy process. Growth hormone treatment appears to be able to compensate for this disturbance by triggering an early chondrocyte enlargement that may be mediated by Nkcc1 action, an important membrane cotransporter in the GP chondrocyte enlargement.

A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes.

This manuscript reports a novel procedure to imaging growth plate chondrocytes by using confocal microscopy. The method is based on fixed undecalcified bone samples, in-block staining with eosin, epoxy resin embedding and grinding to obtain thick sections. It is simple, inexpensive and provides three-dimensional images of entire chondrocytes inside their native lacunae. Quantitative analysis of volume, shape and cytoplasm density of chondrocytes at different strata of the growth plate allowed to objectively grade chondrocytes of the growth plate in seven different clusters. These seven categories of chondrocytes were subsequently evaluated by immunohistochemistry of some well-defined molecular landmarks of chondrocyte differentiation. Furthermore, immunohistochemical analysis of proteins responsible for ionic changes and water transport allowing chondrocyte swelling during hypertrophy was also performed. Results obtained indicate that four subphases can be defined in the pre-hypertrophic zone and three subphases in the hypertrophic zone, a fact that raises that chondrocytes of the growth plate are less homogeneous than usually considered when different zones are defined according to subjective cell morphological criteria. Results in the present study provide a technological innovation and gives new insights into the complexity of the process of chondrocyte differentiation in the growth plate.

Epidemiology of Paget's disease of bone: a systematic review and meta-analysis of secular changes.

CONTEXT: Several studies have suggested that the prevalence and severity of PDB have fallen in recent years. The magnitude of this trend and its globalization have not been well established. OBJECTIVE: The objective of this study is to estimate the pooled magnitude of the changes in the prevalence of PDB and as a secondary objective, to make up a world atlas of PDB prevalence. METHODS: A systematic review of English and non-English articles using MEDLINE (1946 to 2013) and EMBASE (1980 to 2013) was the method used. Search terms included epidemiology, incidence, prevalence, cohort studies, osteitis deformans or Paget's disease of bone. Studies with incidence and/or prevalence rate for PDB were included. Two authors independently extracted the data using predefined data fields and quality assessment. A pooled analysis based on random-effects models was carried out for secular trends. RESULTS: Twenty-eight articles documented the prevalence of PDB; four articles the incidence and two articles the rate of new referrals. The prevalence of PDB varied greatly between the different countries, from 0.00028% in Japan to 5.4% in the UK. There were available data on changes in prevalence from two different surveys over two different time frames in Europe and New Zealand. In all but one city (Turin), a drop in the prevalence of PDB was recorded (pooled OR 0.64; 95% CI 0.45-0.91). CONCLUSION: The incidence and prevalence rates of PDB vary widely between populations but both have decreased in most regions over recent years. The changes are heterogeneous however and within countries, the largest changes have been in areas that previously had a high prevalence. The reasons for these changes remain unclear at present but are likely to be due to an interaction between genetic factors and environmental triggers which may differ in different regions.

Periodic Lateralized Epileptiform Discharges (PLEDs) and pneumococcal meningoencephalitis.

Pneumococcal meningoencephalitis (PME) is a life-threatening condition of the central nervous system (CNS), and is often the result of a complicated upper airway infection. Periodic Lateralized Epileptiform Discharges (PLEDs) are a typical electroencephalographic (EEG) pattern found in some acutely acquired brain insults. Within the pediatric population they are frequently seen in association with herpetic encephalitis, a CNS infection with a high morbidity and mortality rate. We report the case of a 3-year-old girl with a bilateral ear infection who developed convulsions and coma. She had early PLEDs lateralized to the right on the EEG and microbiological criteria for Streptococcus pneumoniae infection. Concomitant herpetic encephalitis was ruled out. Intensive antibiotic and antiepileptic treatment resulted in a remarkable improvement, with the patient being able to resume her normal activities within months. To our knowledge, the association of PME and PLEDs has not been previously described in children. On the other hand, EEG has scarcely been used in the management of acute CNS infections. Hence, non-herpetic CNS encephalitis with potentially more favorable outcomes ought to be considered in the differential diagnosis of PLEDs. Continuous EEG monitoring should be considered in children with CNS infections presenting with altered sensorium, independent of the presence of seizures.

Overall survival, renal survival and relapse in patients with microscopic polyangiitis: a systematic review of current evidence.

OBJECTIVE: There are limited data on the long-term prognosis of microscopic polyangiitis (MPA). A systematic review was performed to estimate the survival, renal survival and relapse rates in patients with MPA. METHODS: Articles included in MEDLINE and EMBASE databases were reviewed. Randomized or non-randomized trials, cohort, case-control and cases-series studies of patients with MPA diagnosed according to Chapel Hill Consensus Conference definitions, a high rate of biopsy-confirmed diagnosis, follow-up >1 year and follow-up losses <10%. Two independent authors using a predefined questionnaire for evaluating the quality and risk of bias for each study extracted data. RESULTS: Eighteen studies for MPA prognosis (n = 940) and six for MPA outcomes after transplantation (n = 65) were included. Survival rates were 77-100% at 1 year, 46-80% at 5 years and 60-80% at 10 years. Higher mortality density occurred within the first months after diagnosis. Vasculitis was the cause of death in 32-50% of patients. Relapses were detected in 19-39% of cases (median time to relapse 15-43 months). Renal graft survival was 85-94% at 1 year and 51-87% at 5 years. Age, renal involvement and immunosuppressive treatment were related to mortality. Lower relapse rate was achieved with 12 vs 6 CYC pulses. CONCLUSION: Evidence regarding MPA prognosis is weak. MPA mortality is mainly concentrated in the first months after diagnosis. Fewer than 50% of deaths are related to MPA activity. MPA long-term prognosis is less severe, although relapses are frequent. End-stage renal failure is a frequent complication of MPA, and renal transplantation could be an effective therapy in these patients. Early diagnosis, early initiation of a tailored therapy according to risk factors and a longer follow-up of the patients are needed.

Use of noninvasive ventilation on internal wards for elderly patients with limitations to respiratory care: a cohort study.

BACKGROUND: The use of noninvasive positive pressure ventilation (NPPV) outside the intensive wards has been evaluated in patients with no limitation on life-sustaining support. Our aim was to evaluate its usefulness in general wards for patients with NPPV as the ceiling of ventilator care when admission to the intensive care unit (ICU) has been withheld. MATERIALS AND METHODS: Noninvasive positive pressure ventilation was used in 44 patients with acute respiratory failure (ARF) and limitations to respiratory care- 22 with chronic obstructive pulmonary disease (COPD) exacerbations and 22 with acute cardiogenic pulmonary oedema (CPE). Survival at hospital discharge, and survival and readmission rate at 12 months were assessed. RESULTS: Sixty-three per cent of COPD and 55% of CPE patients survived hospital discharge; and 50% and 37% respectively, were alive after 1 year. The cause of the in-hospital mortality was related to the admission diagnosis in 88% of cases. Cancer in COPD patients [P = 0·040, odds ratio (OR) = 15, 95% CI = 1·14-198] and the completion of NPPV treatment in both diseases (P = 0·008, OR = 0·03, 95% CI = 0·00-0·39 for COPD and P = 0·010, OR = 0·04, 95% CI = 0·00-0·45 for CPE) were related to in-hospital mortality. Fifty-six per cent of COPD and 33% of CPE patients that survived hospital admission were readmitted. CONCLUSIONS: Our study suggests that the use of NPPV in general wards could be a safe and effective option, as a last choice treatment, in patients with NPPV as the ceiling of ventilator care when admission to ICU has been withheld.

Long-term follow-up of microscopic polyangiitis, 17-year experience at a single center.

BACKGROUND: Long-term prognoses of Wegener granulomatosis (WG) and Churg-Strauss syndrome (CSS) are known; however, few data exist on long-term prognoses for microscopic polyangiitis (MPA). Our aim was to analyse the prognoses of MPA. METHODS: Cohort study with retrospective selection of patients. Twenty-two patients admitted to our Hospital (1990-2006) with biopsy-proven MPA were studied. The start date for entry into the study was the date of diagnosis. Statistical analysis was performed to look for prognostic factors for survival. RESULTS: MPA patients were followed-up for a median of 78 (5-131) months. MPA patients were treated with cyclophosphamide (Cy) plus corticosteroid (Cs) (59%) or Cs alone (41%). Seven MPA patients died. Cumulative MPA patient survival at 1, 5, and 10 years were 85% (75-95%), 85% (75-95%), and 74% (60-88%) in those treated with Cy plus Cs and 50% (32-68%), 36% (14-58%), and 0% (0-30%) in those treated with Cs alone, respectively (P=0.04). Disease extent index <5 (P=0.02) and age <65 years (P=0.02) were associated with improved survival rates in MPA patients treated with Cy. Five MPA (23%) patients relapsed after a median of 54 months (35-93). No variables were related to relapses. Despite treatment, 11MPA (50%) patients developed end-stage renal disease after a median of 9 months (0-53). CONCLUSIONS: Most MPA patients had life-threatening renal or lung involvement at diagnosis. Patients not treated with immunosuppressants had a poorer prognosis. The long-term prognosis of MPA patients who survived 6 months post diagnosis was good, although renal survival rates are low.