Rv0646c, an esterase from M. tuberculosis, up-regulates the host immune response in THP-1 macrophages cells.

The genome sequence of Mycobacterium tuberculosis revealed the presence of several hydrolases involved in lipid metabolism including the members of Lip gene family. Rv0646c (LipG) is one of them. It is annotated as putative esterase/lipase because of the presence of consensus sequence 'GXSXG.' The gene was cloned, expressed, and purified in E. coli. It showed 22 U/mg specific activity with pNP-butyrate as a preferred substrate. However, it actively worked on substrates with short chain. The enzyme was optimally active at 50 °C/pH 8.0 and also stable up to 50 °C and in a lower pH range (pH 6-8). The Km, Vmax, and catalytic efficiency of the enzyme were calculated to be 500 µM, 58.82 µmoles/min/ml, and 3.92 µM/min, respectively. Homology modeling of Rv0646c revealed the presence of a canonical putative catalytic triad (Ser123, His279, and Asp251). The esterase activity was abolished in the presence of serine hydrolase inhibitors, THL and PMSF. Various antigenic epitopes were predicted in Rv0646c. The protein mounted significantly high antibody response against the sera of extrapulmonary and MDR-TB patients. Rv0646c up-regulated the production of various pro-inflammatory cytokines (TNF-α and IFN-γ), chemokine (IL-8), and nitric oxide in THP-1-derived macrophages. The secretion of IL-6 from macrophages was also found to be elevated in response to Rv0646c. The treatment resulted in the increased level of reactive oxygen species. Conclusively, Rv0646c could be classified as esterase having vast immunogenic property by eliciting strong humoral response as well as cell-mediated immunity.

Dual Inhibition of Rip2 and IRAK1/4 Regulates IL-1ß and IL-6 in Sarcoidosis Alveolar Macrophages and Peripheral Blood Mononuclear Cells.

Sarcoidosis is a multisystem granulomatous disease of unknown etiology that primarily affects the lungs. Our previous work indicates that activation of p38 plays a pivotal role in sarcoidosis inflammatory response. Therefore, we investigated the upstream kinase responsible for activation of p38 in sarcoidosis alveolar macrophages (AMs) and PBMCs. We identified that sustained p38 phosphorylation in sarcoidosis AMs and PBMCs is associated with active MAPK kinase 4 but not with MAPK kinase 3/6. Additionally, we found that sarcoidosis AMs exhibit a higher expression of IRAK1, IRAK-M, and receptor interacting protein 2 (Rip2). Surprisingly, ex vivo treatment of sarcoidosis AMs or PBMCs with IRAK1/4 inhibitor led to a significant increase in IL-1ß mRNA expression both spontaneously and in response to TLR2 ligand. However, a combination of Rip2 and IRAK-1/4 inhibitors significantly decreased both IL-1ß and IL-6 production in sarcoidosis PBMCs and moderately in AMs. Importantly, a combination of Rip2 and IRAK-1/4 inhibitors led to decreased IFN-γ and IL-6 and decreased percentage of activated CD4(+)CD25(+) cells in PBMCs. These data suggest that in sarcoidosis, both pathways, namely IRAK and Rip2, are deregulated. Targeted modulation of Rip2 and IRAK pathways may prove to be a novel treatment for sarcoidosis.

Findings at brain MRI in children with dengue fever and neurological symptoms.

Dengue is a flavivirus of the genus arbovirus with four serotypes, from DEN 1 to DEN 4. There has been an increase in incidence of dengue infection in children in the tropics and subtropics. Dengue has a variable clinical presentation, with many patients being asymptomatic. Its clinical manifestations in children vary from fever and arthralgia to life-threatening dengue hemorrhagic fever and dengue shock syndrome. We describe MRI findings in children with neurological involvement including dengue encephalopathy, acute hypoxic injury and dengue encephalitis. Dengue encephalopathy is usually secondary to multisystem derangement such as shock, hepatitis, coagulopathy and concurrent bacterial infection and is relatively common. Dengue encephalitis from direct neuronal invasion is rare. Nonspecific changes are seen on brain MRI in dengue infection. Clinical and laboratory findings as well as outcome do not necessarily correspond with brain MRI findings.

Rapamycin induces mitogen-activated protein (MAP) kinase phosphatase-1 (MKP-1) expression through activation of protein kinase B and mitogen-activated protein kinase kinase pathways.

Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

TLR4-mediated AKT activation is MyD88/TRIF dependent and critical for induction of oxidative phosphorylation and mitochondrial transcription factor A in murine macrophages.

Mitochondria play a critical role in cell survival and death. Mitochondrial recovery during inflammatory processes such as sepsis is associated with cell survival. Recovery of cellular respiration, mitochondrial biogenesis, and function requires coordinated expression of transcription factors encoded by nuclear and mitochondrial genes, including mitochondrial transcription factor A (T-fam) and cytochrome c oxidase (COX, complex IV). LPS elicits strong host defenses in mammals with pronounced inflammatory responses, but also triggers activation of survival pathways such as AKT pathway. AKT/PKB is a serine/threonine protein kinase that plays an important role in cell survival, protein synthesis, and controlled inflammation in response to TLRs. Hence we investigated the role of LPS-mediated AKT activation in mitochondrial bioenergetics and function in cultured murine macrophages (B6-MCL) and bone marrow-derived macrophages. We show that LPS challenge led to increased expression of T-fam and COX subunits I and IV in a time-dependent manner through early phosphorylation of the PI3K/AKT pathway. PI3K/AKT pathway inhibitors abrogated LPS-mediated T-fam and COX induction. Lack of induction was associated with decreased ATP production, increased proinflammatory cytokines (TNF-α), NO production, and cell death. The TLR4-mediated AKT activation and mitochondrial biogenesis required activation of adaptor protein MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-ß. Importantly, using a genetic approach, we show that the AKT1 isoform is pivotal in regulating mitochondrial biogenesis in response to TLR4 agonist.

Inflammatory Myofibroblastic Tumor With Rapid Recurrence and Distant Metastasis: Report of a Rare Case.

Inflammatory myofibroblastic tumors (IMTs) are rare spindle cell tumors clinically, morphologically, and genetically heterogeneous, mimicking many other reactive and neoplastic lesions and creating great diagnostic problems. Although it is generally characterized by oncogene-derived proliferation of myofibroblasts in a background of polyclonal inflammatory cell infiltrates, morphological variations do occur requiring immunohistochemistry and molecular genetics to confirm the diagnosis. It encompasses a wide age range, and locations, mostly said to be of intermediate grade having a low risk of recurrence and metastasis. However, its biological behavior and course are variable and unpredictable. Here, we report a case of thoracic IMT in a 32-year-old adult female presenting with a history of fever, cough, and chest pain associated with neutrophilic leukocytosis. Radiological investigations revealed a large mass in the thoracic region with possibilities of hydatid cyst and neurogenic tumor. Initial core needle biopsy specimen and subsequent local resection specimen revealed the diagnosis of IMT on histopathology and immunohistochemistry, having conventional morphology with expression of Anaplastic lymphoma kinase (ALK) protein. The patient developed rapid local recurrence and was started with first-generation ALK inhibitor Crizotinib. After a brief period of response, she developed vertebral and brain metastasis within a short span of time and was switched to a third-generation ALK inhibitor, Lorlatinib. The patient is on regular follow-up, has stable disease, and maintains a good quality of life after two years of diagnosis.

Successful Surgical Management of Giant Mucinous Cystic Neoplasm of Liver (MCN-L) Presenting With Peritoneal Rupture and Biliary Prolapse: Case Report and Review of Literature.

Mucinous cystic neoplasms of liver (MCN-L) are generally considered benign indolent cystic liver lesions, not associated with significant clinical symptoms in majority of patients. However, rarely these benign-appearing lesions may have a complicated clinical course, presenting with jaundice, acute abdomen, or malignant transformation. We report one such rare clinical presentation of MCN-L presenting with obstructive jaundice and abdominal pain due to prolapse of cystic component in biliary system and peritoneal rupture occurring simultaneously. Despite the complex nature of presentation, it was successfully managed surgically with normal follow-up imaging.

Dysregulation of p38 and MKP-1 in response to NOD1/TLR4 stimulation in sarcoid bronchoalveolar cells.

RATIONALE: Sarcoidosis is a systemic inflammatory disorder characterized by distinct up-regulation of Th1 cytokines, such as tumor necrosis factor (TNF)-α and IL-12. The mechanism underlying this up-regulation remains unclear. Recognition of microbial moieties through Toll-like or Nod-like receptors evokes sequential activation of mitogen-activated protein kinases (MAPKs), which plays a role in Th1-immune response. OBJECTIVES: To test the hypothesis that dysregulation in MAPK signaling in response to microbial stimulation is important in mediating Th1 response in sarcoidosis. METHODS: Ex vivo cultured bronchoalveolar lavage (BAL) cells isolated from patients with sarcoidosis and control subjects were stimulated with low-dose Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 1 (NOD1) ligands as a model of microbial stimulation, and MAPK signaling and inflammatory response were analyzed. MEASUREMENTS AND MAIN RESULTS: BAL cells from patients with sarcoidosis exhibited higher basal p38 activity, greater p38 phosphorylation, and more robust production of TNF-α and IL-12/IL-23p40 on stimulation with NOD1 and TLR4 agonists than cells isolated from control subjects. In contrast, control BAL cells had greater basal extracellular signal-regulated kinase (ERK) activity and NOD1 and TLR4 agonists preferentially activated the ERK pathway. Inhibition of p38, but not ERK, attenuated production of both IL12/IL23p40 and TNF-α. Interestingly, stimulation of cells from patients with sarcoidosis with either NOD1 or TLR4 ligand failed to induce MAPK phosphatase 1 (MKP-1). Adenovirus-mediated overexpression of MKP-1 attenuated p38 activation and decreased the production of IL12/IL23p40 and TNF-α in sarcoid BAL cells. CONCLUSIONS: Our results suggest that enhanced p38 signaling in response to microbial products is caused by abnormal regulation of MKP-1 and contributes to heightened inflammation in sarcoidosis.

Portal vein fenestration: a case report of an unusual portal vein developmental anomaly.

Portal vein anatomic variations are common in living donor liver transplantation. Portal vein fenestration, in which a segment of a vessel divides into at least two channels that reunite into a single distal lumen, has not yet been reported in the literature. Failure to identify this anomaly can lead to catastrophic events in donor liver hepatectomy. Herein, we report an unusual portal vein anomaly that was detected intraoperatively in a living liver donor.

Is Fatty Liver Associated with Increased Mortality and Morbidity in Coronavirus Disease 2019 (COVID-19) Pneumonia?

Background: Fatty liver has been shown to be associated with severe COVID-19 disease without any impact on mortality. This is based on heterogenous criteria for defining both fatty liver as well as the severity parameters. This study aimed to study the impact of fatty liver on the mortality and severity of disease in patients with COVID-19 pneumonia. Methods: In a case control study design, patients with COVID-19 pneumonia (COVID-19 computed tomography severity index [CTSI] on high-resolution computed tomography chest of ≥1) with fatty liver (defined as liver to spleen attenuation index ≤5 on noncontrast computed tomography cuts of upper abdomen) were compared with those without fatty liver. The primary outcome measure was in-hospital mortality, and the secondary outcome measures were CTSI score, need for intensive care unit (ICU) care, need for ventilatory support, duration of ICU stay, and duration of hospital stay. Results: Of 446 patients with COVID-19 pneumonia, 289 (64.7%)admitted to Max Hospital, Saket, India, between January 1, 2021, and October 30, 2021, had fatty liver. Fifty-nine of 446 patients died during the index admission. In-hospital mortality was not different between patients with fatty liver (38 [13.24%]) or without fatty liver (21 [13.81%]). COVID-19 CTSI score was found to be significantly higher among patients who had fatty liver (13.40 [5.16] vs 11.81 [5.50]; P = 0.003). There was no difference in the requirement of ICU (94 [32%] vs 62 [39.49%]; P = 0.752), requirement of ventilatory support (27 [9.34%] vs 14 [8.91%]; P = 0.385), duration of ICU stay (8.29 [6.87] vs 7.07 [5.71] days; P = 0.208), and duration of hospital stay (10.10 [7.14] vs 10.69 [8.13] days; P = 0.430) between the groups with fatty liver or no fatty liver. Similarly, no difference was found in primary or secondary outcomes measure between the group with severe fatty liver vs mild/moderate or no fatty liver. High total leucocyte count and Fibrosis-4 (FIB-4) index were independently associated with mortality. Conclusions: Fatty liver may not be associated with increased mortality or clinical morbidity in patients who have COVID-19 pneumonia.

Normative cutoffs of muscle mass, muscle strength, and frailty for healthy Indian population.

BACKGROUND: Population-derived cutoffs for low skeletal muscle mass, skeletal muscle strength, and frailty among Indians are lacking. Studies describing sarcopenia and frailty among patients with chronic liver diseases have used cutoffs derived from Caucasian populations giving erroneous results. AIMS: We aimed to derive gender-specific cutoffs for low skeletal muscle mass and skeletal muscle strength from healthy Indians. METHODS: Healthy Indian population consisted of two groups. Group 1 (Gp I) included 242 healthy liver donors and group 2 (Gp II) 272 healthy health care workers. Skeletal muscle index (SMI) was calculated from computed tomography (CT) abdomen performed prior to donor hepatectomy only in Gp I. Liver frailty index (LFI) was computed using the online calculator, after recording hand grip strength (HGS), chair stand-up test (CSUT), and balance test in both groups. HGS was measured using the Smedley handgrip dynamometer. CSUT was noted as time to complete 5 chair stand-ups with subjects' arms folded across the chest. Gender-specific cutoffs of SMI and HGS were derived as <5th percentile of the distribution values and as >95th percentile for CSUT and LFI values. RESULTS: The SMI was measured from Gp I subjects (n=242; 120 males [mean age 31.13] and 122 females [mean age 36.60]). The HGS, CSUT, and LFI were measured in Gp I and Gp II subjects (n=514; 272 males [mean age 34.30] and 242 females [mean age 37.52]). The cutoffs for SMI, HGS, CSUT, and LFI were <27.72 cm2/m2, <25.63 kg, >10 s, and >3.49, respectively for healthy males. The corresponding cutoffs for healthy females were <24.4 cm2/m2, <16.7 kg, > 10 s, and >3.68, respectively. CONCLUSIONS: We derived gender-specific cutoffs for SMI, HGS, CSUT, and LFI from healthy adult Indian population, which can be used to detect sarcopenia and frailty among patients with liver diseases, as well as other conditions.

Amelioration of sleep-disordered breathing with supplemental oxygen in older adults.

Elderly adults demonstrate increased propensity for breathing instability during sleep compared with younger adults, and this may contribute to increased prevalence of sleep-disordered breathing (SDB) in this population. Hence, in older adults with SDB, we examined whether addition of supplemental oxygen (O2) will stabilize breathing during sleep and alleviate SDB. We hypothesized that exposure to supplemental O2 during non-rapid eye movement (NREM) sleep will stabilize breathing and will alleviate SDB by reducing ventilatory chemoresponsiveness and by widening the carbon dioxide (CO2) reserve. We studied 10 older adults with mild-to-moderate SDB who were randomized to undergo noninvasive bilevel mechanical ventilation with exposure to room air or supplemental O2 (Oxy) to determine the CO2 reserve, apneic threshold (AT), and controller and plant gains. Supplemental O2 was introduced during sleep to achieve a steady-state O2 saturation ≥95% and fraction of inspired O2 at 40%-50%. The CO2 reserve increased significantly during Oxy versus room air (-4.2 ± 0.5 mmHg vs. -3.2 ± 0.5 mmHg, P = 0.03). Compared with room air, Oxy was associated with a significant decline in the controller gain (1.9 ± 0.4 L/min/mmHg vs. 2.5 ± 0.5 L/min/mmHg, P = 0.04), with reductions in the apnea-hypopnea index (11.8 ± 2.0/h vs. 24.4 ± 5.6/h, P = 0.006) and central apnea-hypopnea index (1.7 ± 0.6/h vs. 6.9 ± 3.9/h, P = 0.03). The AT and plant gain were unchanged. Thus, a reduced slope of CO2 response resulted in an increased CO2 reserve. In conclusion, supplemental O2 reduced SDB in older adults during NREM sleep via reduction in chemoresponsiveness and central respiratory events.NEW & NOTEWORTHY This study demonstrates for the first time in elderly adults without heart disease that intervention with supplemental oxygen in the clinical range will ameliorate central apneas and hypopneas by decreasing the propensity to central apnea through decreased chemoreflex sensitivity, even in the absence of a reduction in the plant gain. Thus, the study provides physiological evidence for use of supplemental oxygen as therapy for mild-to-moderate SDB in this vulnerable population.

Molecular characterization and immunogenic function of ML1899 (LipG) of Mycobacterium leprae.

Introduction. ML1899 is conserved in all mycobacterium sp. and is a middle member of mle-ML1898 operon involved in mycolic acid modification.Aim. In the present study attempts were made to characterize ML1899 in detail.Methodology. Bioinformatics tools were used for prediction of active-site residues, antigenic epitopes and a three-dimensional model of protein. The gene was cloned, expressed and purified as His-tagged protein in Escherichia coli for biophysical/biochemical characterization. Recombinant protein was used to treat THP-1 cells to study change in production of nitric oxide (NO), reactive oxygen species (ROS), cytokines and chemokines using flowcytometry/ELISA.Results. In silico analysis predicted ML1899 as a member of α/ß hydrolase family with GXSXG-motif and Ser126, His282, Asp254 as active-site residues that were confirmed by site-directed mutagensis. ML1899 exhibited esterase activity. It hydrolysed pNP-butyrate as optimum substrate at pH 8.0 and 50 °C with 5.56 µM-1 min-1 catalytic efficiency. The enzyme exhibited stability up to 60 °C temperature and between pH 6.0 to 9.0. K m, V max and specific activity of ML1899 were calculated to be 400 µM, 40 µmoles min-1 ml-1 and 27 U mg- 1, respectively. ML1899 also exhibited phospholipase activity. The protein affected the survival of macrophages when treated at higher concentration. ML1899 enhanced ROS/NO production and up-regulated pro-inflammatory cytokines and chemokine including TNF-α, IFN-γ, IL-6 and IL-8 in macrophages. ML1899 was also observed to elicit humoral response in 69 % of leprosy patients.Conclusion. These results suggested that ML1899, an esterase could up-regulate the immune responses in favour of macrophages at a low concentration but kills the THP-1 macrophages cells at a higher concentration.

Simultaneous occurrence of posterior reversible leukoencephalopathy syndrome in two cases of childhood acute lymphoblastic leukemia induction chemotherapy.

We describe two cases Posterior Reversible Encephalopathy Syndrome (PRES) occurring in childhood B-precursor acute lymphoblastic leukemia within a week of each other during induction chemotherapy in our ward. We review the literature related to the possible etiology of PRES in these cases.

Desmoplastic Fibroma of Bone: a Case Series and Review of Literature.

Desmoplastic fibroma is an uncommon locally aggressive benign tumor of the bone. Patients usually present with a long-standing history of pain and swelling. Radiologically and histologically, it can mimic a variety of tumors. This article presents a report on three cases of desmoplastic fibroma in the proximal humerus, distal femur, and neck femur region. The varied radiographic features and the management of the three cases are described in detail. All the three patients were treated by extended curettage. Follow-up ranged from 2 to 5 years. There were no local or systemic recurrence until the last follow-up. Tumor resection with adequate margins is the preferred treatment, although extended curettage can be an acceptable alternative treatment modality when resection is not possible or acceptable.

Infraphrenic collaterals in malignant superior vena cava obstruction.

We present three cases of malignant superior vena cava obstruction demonstrating infraphrenic venous collaterals, one of which showed intense focal hepatic enhancement on computed tomographic scan. Infraphrenic venous collaterals and focal liver enhancement are uncommon but specific computed tomographic features of superior vena cava obstruction.

Comparative accuracy of CT, dual-echo MRI and MR spectroscopy for preoperative liver fat quantification in living related liver donors.

BACKGROUND: It is of significant importance to assess the extent of hepatic steatosis in living donor liver transplant (LDLT) surgery to ensure optimum graft regeneration as well as donor safety. AIM: To establish the accuracy of non-invasive imaging methods including computed tomography (CT), dual-echo in- and opposed-phase magnetic resonance imaging (MRI), and MR spectroscopy (MRS) for quantification of liver fat content (FC) in prospective LDLT donors with histopathology as reference standard. SETTINGS AND DESIGN: This retrospective study was conducted at our institution on LDLT donors being assessed for biliary and vascular anatomy depiction by Magnetic Resonance Cholangiopancreatography (MRCP) and CT scan, respectively, between July 2013 and October 2014. MATERIALS AND METHODS: Liver FC was measured in 73 donors by dual-echoT1 MRI and MRS. Of these, CT liver attenuation index (LAI) values were available in 62 patients. STATISTICAL ANALYSIS: CT and MRI FC were correlated with histopathological reference standard using Spearman correlation coefficient. Sensitivity, specificity, positive predictive value, negative predicative value, and positive and negative likelihood ratios with 95% confidence intervals were obtained. RESULTS: CT LAI, dual-echo MRI, and MRS correlated well with the histopathology results (r = 0.713, 0.871, and 0.882, respectively). An accuracy of 95% and 96% was obtained for dual-echo MRI and MRS in FC estimation with their sensitivity being 97% and 94%, respectively. False-positive rate, positive predictive value (PPV), and negative predicative value (NPV) were 0.08, 0.92, and 0.97, respectively, for dual-echo MRI and 0.03, 0.97, and 0.95, respectively, for MRS. CT LAI method of fat estimation has a sensitivity, specificity, PPV, and NPV of 73%, 77.7%, 70.4%, and 80%, respectively. CONCLUSION: Dual-echo MRI, MRS, and CT LAI are accurate measures to quantify the degree of hepatic steatosis in LDLT donors, thus reducing the need for invasive liver biopsy and its associated complications. Dual-echo MRI and MRS results correlate better with histological results in the study, as compared to CT LAI method for fat quantification.