Characterization of Argonaute-containing protein complexes in Leishmania-infected human macrophages.

The intracellular protozoan parasite Leishmania causes leishmaniasis in humans, leading to serious illness and death in tropical and subtropical areas worldwide. Unfortunately, due to the unavailability of approved vaccines for humans and the limited efficacy of available drugs, leishmaniasis is on the rise. A comprehensive understanding of host-pathogen interactions at the molecular level could pave the way to counter leishmaniasis. There is growing evidence that several intracellular pathogens target RNA interference (RNAi) pathways in host cells to facilitate their persistence. The core elements of the RNAi system are complexes of Argonaute (Ago) proteins with small non-coding RNAs, also known as RNA-induced silencing complexes (RISCs). Recently, we have shown that Leishmania modulates Ago1 protein of host macrophages for its survival. In this study, we biochemically characterize the Ago proteins' interactome in Leishmania-infected macrophages compared to non-infected cells. For this, a quantitative proteomic approach using stable isotope labelling by amino acids in cell culture (SILAC) was employed, followed by purification of host Ago-complexes using a short TNRC6 protein-derived peptide fused to glutathione S-transferase beads as an affinity matrix. Proteomic-based detailed biochemical analysis revealed Leishmania modulated host macrophage RISC composition during infection. This analysis identified 51 Ago-interacting proteins with a broad range of biological activities. Strikingly, Leishmania proteins were detected as part of host Ago-containing complexes in infected cells. Our results present the first report of comprehensive quantitative proteomics of Ago-containing complexes isolated from Leishmania-infected macrophages and suggest targeting the effector complex of host RNAi machinery. Additionally, these results expand knowledge of RISC in the context of host-pathogen interactions in parasitology in general.

Leishmania infection upregulates and engages host macrophage Argonaute 1, and system-wide proteomics reveals Argonaute 1-dependent host response.

Leishmania donovani, an intracellular protozoan parasite, is the causative agent of visceral leishmaniasis, the most severe form of leishmaniasis in humans. It is becoming increasingly clear that several intracellular pathogens target host cell RNA interference (RNAi) pathways to promote their survival. Complexes of Argonaute proteins with small RNAs are core components of the RNAi. In this study, we investigated the potential role of host macrophage Argonautes in Leishmania pathogenesis. Using Western blot analysis of Leishmania donovani-infected macrophages, we show here that Leishmania infection selectively increased the abundance of host Argonaute 1 (Ago1). This increased abundance of Ago1 in infected cells also resulted in higher levels of Ago1 in active Ago-complexes, suggesting the preferred use of Ago1 in RNAi in Leishmania-infected cells. This analysis used a short trinucleotide repeat containing 6 (TNRC6)/glycine-tryptophan repeat protein (GW182) protein-derived peptide fused to Glutathione S-transferase as an affinity matrix to capture mature Ago-small RNAs complexes from the cytosol of non-infected and Leishmania-infected cells. Furthermore, Ago1 silencing significantly reduced intracellular survival of Leishmania, demonstrating that Ago1 is essential for Leishmania pathogenesis. To investigate the role of host Ago1 in Leishmania pathogenesis, a quantitative whole proteome approach was employed, which showed that expression of several previously reported Leishmania pathogenesis-related proteins was dependent on the level of macrophage Ago1. Together, these findings identify Ago1 as the preferred Argonaute of RNAi machinery in infected cells and a novel and essential virulence factor by proxy that promotes Leishmania survival.

Effect of long-term medium to high-dose inhaled budesonide on bone mineral density in children with asthma: a cross-sectional study.

OBJECTIVE: The objective of this study was to examine the impact of long-term medium to high-dose inhaled budesonide on bone mineral density in children with asthma. METHODS: We conducted a cross-sectional study in children aged 7-17 years with asthma, who received long-term (≥2 years), medium to high-dose inhaled budesonide (≥400µg/day in 6-11 years old; ≥800 µg/day in >11 years old). We measured bone mineral density (BMD) using dual-energy X-ray absorptiometry and compared it with reference Indian normative values. RESULTS: Thirty-five children with moderate to severe asthma receiving long-term medium to high-dose inhaled budesonide, were included in the study. We found a significantly low lumbar-spine BMD in the study population compared to reference Indian values (p-value 0.002). Eight cases had short stature. Despite the adjustment for height-age in these short-stature cases, lumbar-spine BMD remained significantly low in the study population (p-value 0.020). No significant difference was found in 25-hydroxy vitamin D levels between subjects with "low BMD" and "BMD z-score > -2". CONCLUSIONS: The findings of this study suggest that long-term medium to high-dose inhaled budesonide treatment in children with asthma is associated with decreased BMD. However, further investigation with a larger sample size is necessary to confirm this relationship.

Comprehensive proteomic analysis of autophagosomes derived from Leishmania-infected macrophages.

Recently, autophagy has been implicated as a host defense mechanism against intracellular pathogens. On the other hand, certain intracellular pathogens such as Leishmania can manipulate the host's autophagy to promote their survival. Our recent findings regarding the regulation of autophagy by Leishmania donovani indicate that this pathogen induces non-classical autophagy in infected macrophages, independent of regulation by the mammalian target of rapamycin complex 1. This suggests the fine-tuning of autophagy to optimally promote parasite survival, possibly by the sequestration or modulation of specific autophagosome-associated proteins. To investigate how Leishmania potentially manipulates the composition of host-cell autophagosomes, we undertook a quantitative proteomic study of the human monocytic cell line THP-1 following infection with L. donovani. We used stable isotope labeling by amino acid in cell culture and liquid chromatography-tandem mass spectrometry to compare expression profiles between autophagosomes isolated from THP-1 cells infected with L. donovani or treated with known autophagy inducers. Selected proteomic results were validated by Western blotting. In this study, we showed that L. donovani modulates the composition of macrophage autophagosomes during infection when compared to autophagosomes induced by either rapamycin (selective autophagy) or starvation (non-selective autophagy). Among 1787 proteins detected in Leishmania-induced autophagosomes, 146 were significantly modulated compared to the proteome of rapamycin-induced autophagosomes, while 57 were significantly modulated compared to starvation-induced autophagosomes. Strikingly, 23 Leishmania proteins were also detected in the proteome of Leishmania-induced autophagosomes. Together, our data provide the first comprehensive insight into the proteome dynamics of host autophagosomes in response to Leishmania infection and demonstrate the complex relations between the host and pathogen at the molecular level. A comprehensive analysis of the Leishmania-induced autophagosome proteome will be instrumental in the advancement of understanding leishmaniasis.

Thinking beyond infections in children with recurrent/persistent pneumonia.

Children with recurrent or persistent pneumonia often have underlying chronic cardiopulmonary disease, but few reports on this subject have been published. Children with isolated common cardiac diseases, uncomplicated bronchial asthma or with incomplete records were excluded. Of 4361 children followed during a five-year period, 107 were included in our study. Underlying causes were identified in 99.0%: immunodeficiency disorders (20.2%), cardiothoracic malformations (18.3%), syndromic conditions (14.4%), infections (10.6%) bronchiectasis (10.6%), gastro-oesophageal reflux disease (6.6%), interstitial lung disease (3.8%) and other miscellaneous conditions (15.4%). Thus, children with recurrent or persistent pneumonia should be carefully evaluated for an underlying aetiology, as early diagnosis and appropriate management will decrease morbidity and mortality in most of these children.

Predictors of severe dengue amongst children as per the revised WHO classification.

BACKGROUND & OBJECTIVES: World Health Organization (WHO) revised its guidelines for classification and management of dengue in 2009. This revised system was found out to have good sensitivity and negative predictive value but poor specificity as well as positive predictive value. METHODS: This retrospective study was carried out in a tertiary care hospital of Delhi, India to assess factors predicting the occurrence of severe dengue in children as per the revised classification. A total of 647 suspected dengue cases were admitted in the hospital in the year 2015. Detailed clinical and epidemiological data of 170 patients who were confirmed as dengue either by NS1 antigen test or by serology (Ig M positive) were recorded and statistically analyzed. RESULTS: The number of laboratory-confirmed cases was 170 and included thirty (17.65%) dengue fever (DF), 106 (62.35%) dengue with warning signs (DWS) and 34 (20.0%) severe dengue (SD) patients. Regression analysis revealed that presence of vomiting, altered sensorium, shock, peri-orbital edema, hepatomegaly, splenomegaly, severe anemia, thrombocytopenia, elevated urea and creatinine, decreased total protein and globulin were significantly associated with occurrence of severe disease. INTERPRETATION & CONCLUSION: The addition of clinical features (peri-orbital edema and splenomegaly); and laboratory findings (elevated urea and creatinine, decreased serum protein and globulin) might help improve the sensitivity and specificity of the revised WHO dengue classification in predicting severe dengue.

Leishmania regulates host macrophage miRNAs expression by engaging transcription factor c-Myc.

Parasites of Leishmania genus have developed sophisticated strategies allowing them to deactivate their host macrophage to promote their survival. It has become clear that miRNAs play important roles in shaping innate and adaptive immune responses toward pathogens. It is not surprising that several pathogens including Leishmania have evolved the ability to regulate host macrophage miRNA expression in order to manipulate host cell phenotypes to their advantage. However, very little is known about the mechanisms used by intracellular pathogens to drive changes in host cell miRNA abundance. In this review, Leishmania exploitation of macrophage transcription factor c-Myc as a critical proxy virulence factor to regulate abundance of macrophage miRNAs influencing macrophage physiology to promote its survival will be discussed.

Diaphragmatic palsy in a 10-month-old boy with infantile tremor syndrome causing respiratory failure with full response to vitamin B12 therapy.

Infantile tremor syndrome (ITS) owing to vitamin B12 deficiency usually presents with tremors, anaemia, pigmentary skin changes, neuro-regression and hypotonia. A 10-month-old boy with ITS and respiratory failure owing to bilateral diaphragmatic palsy who responded to high parenteral doses of vitamin B12 is presented. As far as we are aware, this is the first report of diaphragmatic palsy associated with ITS and vitamin B12 deficiency.

Therapeutic Plasma Exchange in Parvovirus B19-induced Acute Hepatic Failure.

Parvovirus B19 has rarely been associated with acute liver failure (ALF), which has a high mortality. Plasma exchange that usually acts as a bridge to liver transplantation removes toxins, antibodies, cytokines, and can correct coagulopathy while maintaining a euvolemic state. Pediatric data regarding its use are scarce. We report a case of 16-year-old girl with acute liver failure in stage 4 encephalopathy with coagulopathy due to parvovirus B19 who was successfully managed with high-volume therapeutic plasma exchange (TPE). We tried to use it as a treatment modality due to nonavailability of in-hospital transplant facilities. Parvovirus B19 may be an underdiagnosed cause of acute viral hepatitis. Therapeutic plasma exchange can act as a bridge to liver transplant (LT) or bridge to recovery especially in self-limiting illnesses such as viral hepatitis. HOW TO CITE THIS ARTICLE: Singh DP, Agarwal S, Singh R, Nandan D, Gupta A. Therapeutic Plasma Exchange in Parvovirus B19-induced Acute Hepatic Failure. Indian J Crit Care Med 2020;24(5):361-362.

Role of Intrapleural Urokinase in Empyema Thoracis.

OBJECTIVE: To study the role of fibrinolytic therapy in pediatric empyema in relation to duration of hospital stay, need for surgical intervention and survival to discharge. METHODS: Retrospective analysis of case records of children <16 y of age admitted in a tertiary care hospital during January 2013 - December 2017 with diagnosis as empyema thoracis was done. Clinico-laboratory characteristics and the primary and secondary outcomes between the group which received intrapleural urokinase (IPU) and the group which did not (non IPU), were compared. RESULTS: Of the 84 cases, 40 children received IPU. Mean duration of hospital stay in IPU group (17.51 + 4.57 d) was significantly less than non IPU group (24.32 + 10.18 d, CI -10.19 to -3.64, p < 0.001), so was the duration of intercostal drain (ICD) insertion (9.08 + 3.12 d - IPU group vs. 11.20 + 3.95 d - non IPU group, CI -3.68 to -0.50, p < 0.01). No statistically significant difference was found between the groups with regard to need for surgical intervention [IPU - 4 (10%), non IPU - 9 (20.4%), p = 0.23]. There was no mortality or adverse reaction to urokinase in either group. CONCLUSIONS: IPU holds promising results in terms of reduction of hospital stay and duration of ICD insertion. It may be the initial choice of treatment in septated empyema where surgical options are not easily available or cost-effective especially in resource limited settings.

Successful Ventilation of Acute Respiratory Distress Syndrome Complicated by Pneumothorax Using Airway Pressure Release Ventilation: A Case Report.

Pneumothorax can develop in children being mechanically ventilated for 'severe acute respiratory distress syndrome' making the situation worse and challenging for the treating intensivist. There is no evidence on use of 'airway pressure release ventilation' mode in children with acute respiratory distress syndrome complicated by pneumothorax. We present a case of a girl who had severe acute respiratory distress syndrome and developed pneumothorax on pressure control ventilation mode. We had to use 'airway pressure release ventilation' mode in view of severe refractory hypoxemia. Fortunately, the child responded well and weaned off the ventilator over few days. We suggest that 'airway pressure release ventilation' mode may be used successfully in patients with 'acute respiratory distress syndrome' complicated by pneumothorax if intensive and close monitoring is done. KEY MESSAGES: APRV may be a useful mode of ventilation in severe ARDS with pneumothorax. HOW TO CITE THIS ARTICLE: Chandelia S, Kishore S, Nandan D. Successful Ventilation of Acute Respiratory Distress Syndrome Complicated by Pneumothorax Using Airway Pressure Release Ventilation: A Case Report. Indian J Crit Care Med 2019;23(9):437-438.

Filarial tropical pulmonary eosinophilia: a condition masquerading asthma, a series of 12 cases.

INTRODUCTION: Tropical pulmonary eosinophilia (TPE) is a form of occult filariasis, clinically characterized by paroxysmal cough, wheezing and dyspnea which is often misdiagnosed and treated as asthma. These manifestations result from a host immune response to trapped antigens of the microfilarial parasites Wuchereria bancrofti or Brugia malayi in the pulmonary microcirculation. CASE STUDY: We describe three rare presentations of TPE (cor pumonale, cystic lung disease and respiratory distress mimicking acute severe asthma) in our series of 12 cases. All cases were from filaria endemic areas and presented with cough, wheezing and dyspnea, either alone or in combination. Subsequent work-up revealed peripheral eosinophilia, raised serum IgE levels and positive serum filarial antibody and/or antigen in all the cases. RESULTS: All patients were treated with diethylcarbamazine (DEC), while few required inhaled/systemic corticosteroid. Prompt improvement in clinical symptoms with a decrease in eosinophil count was seen in all. Two cases relapsed requiring a second course of DEC. Long-term outcome was good, however, there was a persistence of restrictive lung function and echocardiographic feature of pulmonary hypertension in the patients with cystic lung disease and cor pulmonale, respectively. CONCLUSION: TPE should always be considered in patients from filaria endemic areas presenting with cough, dyspnea or wheezing. High eosinophil count (>3 × 109 cells) with raised IgE level (>1000 IU/mL) in such cases should alert the physician to look for TPE. Early diagnosis and treatment can prevent disease progression and complications.

Miransertib (ARQ 092), an orally-available, selective Akt inhibitor is effective against Leishmania.

Leishmaniasis is amongst the most important neglected diseases, afflicting more than 12 million people in 88 countries. There is an urgent need for safe orally bioavailable and cost-effective drugs for the treatment of leishmaniasis. It has recently been shown that Leishmania activates host macrophage serine/threonine kinase Akt, to promote survival of both parasites and infected cells. Here, we sought to evaluate a compound, Miransertib (ARQ 092), an orally bioavailable and selective allosteric Akt inhibitor currently in clinical trials for patients with PI3K/Akt-driven tumors or Proteus syndrome. Miransertib was tested against Leishmania donovani and Leishmania amazonensis, causative agents of visceral and cutaneous leishmaniasis, respectively. Cultured promastigotes were susceptible to Miransertib. In addition, Miransertib was markedly effective against intracellular amastigotes of L. donovani or L. amazonensis-infected macrophages. Miransertib also enhanced mTOR dependent autophagy in Leishmania-infected macrophages, which may represent one mechanism of Miransertib-mediated killing of intracellular Leishmania. Whereas parasite clearance in the spleen of mice infected with L. donovani and treated with Miransertib was comparable to that when treated with miltefosine, Miransertib caused a greater reduction in the parasite load in the liver. In the cutaneous leishmaniasis infection model, lesions were reduced by 40% as compared to mock treated mice. Together, these results provide direct evidence to support the conclusion that Miransertib is an excellent lead compound for the development of a new oral drug therapy for visceral and cutaneous leishmaniasis.

Evaluation of the Protection Provided by Hepatitis B Vaccination in India.

OBJECTIVE: In India, Hepatitis B vaccination is recommended at 6 wk except for hospital-deliveries. The authors examined protection afforded by the birth dose. METHODS: A case-control study was done. HBsAg and HBcAb were tested in 2671 children, 1 to 5 y and HBsAb was evaluated in a subset of 1413 children. Vaccination history was recorded. Cases were HBsAg carriers. In another analysis, children who got infected (HBsAg and/or HBcAb positive) were considered as cases. Exposed were the unvaccinated. In another analysis, exposed were those vaccinated without the birth dose. RESULTS: The odds ratio (OR) for HBsAg positivity with birth vaccination was 0.35 (95% CI 0.19-0.66); while with vaccination at 6 wk was 0.29 (95%CI 0.14-0.61), both compared to unvaccinated. Birth vaccination has no added protection when compared to the unvaccinated. Unvaccinated children in index study had HBsAg positivity of 4.38%. The number needed to treat (NNT) to prevent one case of HBsAg positivity was 32.6 (95% CI, 20.9 to 73.6). The odds of getting HBV infection was 0.42 (CI 0.25-0.68) with birth dose and 0.49 (CI 0.30-0.82) without the birth dose compared to the unvaccinated. Protective antibody (HBsAb) was present in about 70% of the vaccinated. In the unimmunised, in the first 2 y HBsAb protection was present in 40%. The odds ratio (OR) for HBsAb in the fully vaccinated between 4 and 5 y was 1.4 (95%CI 0.9-2.18) compared to the unvaccinated. CONCLUSIONS: The present study lends support to the pragmatic approach of the Government to vaccinate babies born at home starting at 6 wk.

Countervailing, time-dependent effects on host autophagy promotes intracellular survival of Leishmania.

Autophagy is essential for cell survival under stress and has also been implicated in host defense. Here, we investigated the interactions between Leishmania donovani, the main etiological agent of visceral leishmaniasis, and the autophagic machinery of human macrophages. Our results revealed that during early infection-and via activation of the Akt pathway-Leishmania actively inhibits the induction of autophagy. However, by 24 h, Leishmania switched from being an inhibitor to an overall inducer of autophagy. These findings of a dynamic, biphasic response were based on the accumulation of lipidated light chain 3 (LC3), an autophagosome marker, by Western blotting and confocal fluorescence microscopy. We also present evidence that Leishmania induces delayed host cell autophagy via a mechanism independent of reduced activity of the mechanistic target of rapamycin (mTOR). Notably, Leishmania actively inhibited mTOR-regulated autophagy even at later stages of infection, whereas there was a clear induction of autophagy via some other mechanism. In this context, we examined host inositol monophosphatase (IMPase), reduced levels of which have been implicated in mTOR-independent autophagy, and we found that IMPase activity is significantly decreased in infected cells. These findings indicate that Leishmania uses an alternative pathway to mTOR to induce autophagy in host macrophages. Finally, RNAi-mediated down-regulation of host autophagy protein 5 (ATG5) or autophagy protein 9A (ATG9A) decreased parasite loads, demonstrating that autophagy is essential for Leishmania survival. We conclude that Leishmania uses an alternative pathway to induce host autophagy while simultaneously inhibiting mTOR-regulated autophagy to fine-tune the timing and magnitude of this process and to optimize parasite survival.

Correlation of induced sputum eosinophil levels with clinical parameters in mild and moderate persistent asthma in children aged 7-18 years.

BACKGROUND: Treatment decisions in asthma are currently based on clinical assessment and spirometry. Sputum eosinophil, being a marker of airway inflammation, can serve as a tool for assessing severity and response to treatment in asthma patients. OBJECTIVES: To measure eosinophil percentage in induced sputum in children with asthma and correlate it with clinical asthma parameters. METHODS: A prospective observational study was performed at tertiary care hospital on 91 children aged 7-18 years with newly diagnosed mild or moderate persistent asthma. Theinduced sputum eosinophil percentage was obtained at the time of enrollment and three months after treatment with inhaled budesonide. Patients were specifically evaluated for five clinical parameters of asthma, i.e., days of acute exacerbations, use of salbutamol as rescue medication, emergency visits, nighttime cough and days of school absence. RESULTS: Sputum eosinophil percentage was high (3.1 ± 0.515%) at the time of enrollment which reduced significantly after three months of inhaled budesonide therapy [0.06 ± 0.164% (p < 0.0005)]. Children with moderate persistent asthma had significantly higher values of sputum eosinophil levels than children with mild persistent asthma at the time of enrollment (3.38 ± 0.64% vs. 2.99 ± 0.42%, p  =  0.001) but the difference was not significant after three months of inhaled steroid therapy (0.07 ± 0.18 vs. 0.04 ± 0.12, p  =  0.5104). A significant negative correlation was found between reduction in sputum eosinophil levels and improvement in FEV1 (r = -0.400, p = 0.0001). All the clinical asthma parameters also correlated significantly with the reduction in sputum eosinophil levels after three months of inhaled steroid therapy. CONCLUSION: Eosinophil levels in induced sputum correlate well with clinical asthma parameters and asthma severity in children. It is a simple, noninvasive and cheap method which can be used for the monitoring of asthma in a resource-limited setting.

Chronic Hypersensitivity Pneumonia due to Pigeon Breeders Disease.

BACKGROUND: Pigeon breeders disease usually affects adults. Children are more likely to be affected when they share living space with a backyard poultry or pigeon breeding. CASE CHARACTERISTICS: A 12-year-old girl with persistent cough for 3 years and dyspnea for 2 years. OBERVATION: She was dignosed to be having allergy to pigeon droppings, based on reports of lung biopsy and allergy testing. MESSAGE: Pigeon breaders disease should be considered in a child who presents with features of chronic hypersensiticity pneumonitis.