Emerging role of exosomes as a liquid biopsy tool for diagnosis, prognosis & monitoring treatment response of communicable & non-communicable diseases.

ABSTRACT: From an initial thought of being used as a cellular garbage bin to a promising target for liquid biopsies, the role of exosomes has drastically evolved in just a few years of their discovery in 1983. Exosomes are naturally secreted nano-sized vesicles, abundant in all types of body fluids and can be isolated intact even from the stored biological samples. Being stable carriers of genetic material (cellular DNA, mRNA and miRNA) and having specific cargo (signature content of originating cells), exosomes play a crucial role in pathogenesis and have been identified as a novel source of biomarkers in a variety of disease conditions. Recently exosomes have emerged as a promising 'liquid biopsy tool'and have shown great potential in the field of non-invasive disease diagnostics, prognostics and treatment response monitoring in both communicable as well as non-communicable diseases. However, there are certain limitations to overcome which restrict the use of exosome-based liquid biopsy as a gold standard testing procedure in routine clinical practices. The present review summarizes the current knowledge on the role of exosomes as the liquid biopsy tool in diagnosis, prognosis and treatment response monitoring in communicable and non-communicable diseases and highlights the major limitations, technical advancements and future prospects of the utilization of exosome-based liquid biopsy in clinical interventions.

Stimulated expression of ELR+ chemokines, VEGFA and TNF-AIP3 promote mycobacterial dissemination in extrapulmonary tuberculosis patients and Cavia porcellus model of tuberculosis.

Pathogenic mycobacteria induce and accelerate blood vessel formation driven by extensive inflammation during granuloma formation, which is a central feature of mycobacterial pathogenesis. Tumor necrosis factor-alpha (TNF-α) enhances the expression of vascular endothelial growth factor (VEGF) and glutamic acid-leucine-arginine (ELR+) chemokines, which are potent inducers of vascularization. Most of the reported research work contends that VEGF growth factor induces neovascularization in human tuberculosis (TB) patients, but the evidence is inconclusive. Considerable ambiguity exists concerning the factors responsible for miliary tuberculosis. To identify such factors, we proposed an alternative explanation that could be found in miliary tuberculosis (MTB) cases. We performed a comparative analysis of angiogenic factors TNF-α, VEGF, and angiogenic ELR+ CXC and CC chemokine ligands in extrapulmonary tuberculosis (EPTB) and pulmonary tuberculosis (PTB) patients. To observe the relationship of these factors with the severity of bacterial burden, guinea pigs were infected with Mycobacterium tuberculosis (M.tb) and levels of the angiogenic factors were examined at different time intervals. Expression of these factors also exhibited a significant positive correlation with bacterial burden in other organs like the spleen, liver, and lymph nodes. We demonstrated statistical data on bacterial burden at different time points following the dissemination of infection in guinea pigs. In this study, we observed that there was a stimulated increase in the expression of ELR+ chemokines and VEGF in EPTB patients as compared to PTB patients. Following increased dissemination, the host immune response clears bacteria from the lungs during disease progression in guinea pigs.

Genetic variability in multidrug-resistant Mycobacterium tuberculosis isolates from patients with pulmonary tuberculosis in North India.

BACKGROUND: Information on the genetic variability of drug resistant isolates of Mycobacterium tuberculosis is of paramount importance to understand transmission dynamics of disease and to improve TB control strategies. Despite of largest number of multidrug-resistant (MDR) tuberculosis cases (1, 30,000; 27% of the global burden), strains responsible for the expansion or development of drug-resistant Mycobacterium tuberculosis infections have been poorly characterized in India. Present study was aimed to investigate the genetic diversity in MDR isolates of Mycobacterium tuberculosis in North India. RESULTS: Spacer oligonucleotide typing (spoligotyping) was performed on 293 clinical MDR isolates of Mycobacterium tuberculosis recovered from cases of pulmonary tuberculosis from North India. Spoligotyping identified 74 distinct spoligotype patterns. Comparison with an international spoligotype database (spoldb4 database) showed that 240 (81.91%) and 32 (10.92%) strains displayed known and shared type patterns, while 21 (7.16%) strains displayed unique spoligotype patterns. Among the phylogeographic lineages, lineage 3 (East African-Indian) was found most predominant lineage (n = 159, 66.25%), followed by lineage 2 (East Asian; n = 34, 14.16%), lineage 1 (Indo-Oceanic; n = 30, 12.50%) and lineage 4 (Euro American; n = 17, 7.08%). Overall, CAS1_DEL (60.41%; SITs 2585, 26, 2694, 309, 381, 428, 1401, 141, 25, 1327) was found most pre-dominant spoligotype pattern followed by Beijing (14.16%; SITs255, 260, 1941, 269) and EAI3_IND (5.00%; SITs 298, 338, 11). The demographic and clinical characteristics were not found significantly associated with genotypic lineages of MDR-M.tuberculosis isolates recovered from pulmonary TB patients of North India. CONCLUSIONS: Present study reveals high genetic diversity among the Mycobacterium tuberculosis isolates and highlights that SIT141/CAS1_Del followed by SIT26/ Beijing lineage is the most common spoligotype responsible for the development and transmission of MDR-TB in North India. The high presence of shared type and unique spoligotype patterns of MDR strains indicates epidemiological significance of locally evolved strains in ongoing transmission of MDR-TB within this community which needs to be further monitored using robust molecular tools with high discriminatory power.

Identification and functional characterization of sorbitol-6-phosphate dehydrogenase protein from rice and structural elucidation by in silico approach.

The sorbitol-6-phosphate dehydrogenase (S6PDH) is a key enzyme for sorbitol synthesis and plays an important role in the alleviation of salinity stress in plants. Despite the huge significance, the structure and the mode of action of this enzyme are still not known. In the present study, sequence analysis, cloning, expression, activity assays and enzyme kinetics using various substrates (glucose-6-phosphate, sorbitol-6-phosphate and mannose-6-phosphate) were performed to establish the functional role of S6PDH protein from rice (Oryza sativa). For the structural analysis of the protein, a comparative homology model was prepared on the basis of percentage sequence identity and substrate similarity using the crystal structure of human aldose reductase in complex with glucose-6-phosphate and NADP(+) (PDB ID: 2ACQ) as a template. Molecular docking was performed for studying the structural details of substrate binding and possible enzyme mechanism. The cloned sequence resulted into an active recombinant protein when expressed into a bacterial expression system. The purified recombinant protein was found to be active with glucose-6-phosphate and sorbitol-6-phosphate; however, activity against mannose-6-phosphate was not found. The K m values for glucose-6-phosphate and sorbitol-6-phosphate were found to be 15.9 ± 0.2 and 7.21 ± 0.5 mM, respectively. A molecular-level analysis of the active site of OsS6PDH provides valuable information about the enzyme mechanism and requisite enantioselectivity for its physiological substrates. Thus, the fundamental studies of structure and function of OsS6PDH could serve as the basis for the future studies of bio-catalytic applications of this enzyme.

Hemoglobin e syndromes: emerging diagnostic challenge in north India.

Hemoglobin E (HbE) is one of the world's most common and important mutations. HbE disorders may be found in heterozygous (AE), homozygous (EE) and compound heterozygous state. It is important to distinguish HbE disorders diagnostically because of marked differences in clinical course among different genotypes. To find out whether RBC indices as obtained from automated cell counter can provide a clue to the diagnosis of HbE disease. This study was carried out in the Department of Clinical Pathology, PGIMER, Dr. Ram Manohar Lohia Hospital, New Delhi. It included antenatal pregnant females brought for routine check-up as well as referred patients suspected of having hemoglobinopathies. High Performance liquid chromatography was used as a confirmatory test for identification of hemoglobinopathy. Total 20 cases of subtype homozygous HbE (3), HbE trait (12) and Eß-thalassemia (5) were identified. Statistical analysis was done to find out correlation between levels of HBA2, HBF with RBC indices. (a) There was negative correlation between HbA2/E peak values and RBC indices (Mean corpuscular volume (MCV) and Mean corpuscular hemoglobin) among all the three groups taken together. (b) There was positive correlation between HbA2/E and Red cell distribution width (RDW). (c) There was positive correlation between HbF values with MCV. The finding of positive correlation between HbA2/E and RDW may help in differentiating ßthal (RDW normal) from HbE/ßthal. In a patient with microcytic hypochromic blood picture and increased RDW, diagnosis of HbE/ßthal should also be considered along with the more common Iron deficiency anemia. Thus, new insights into the knowledge of these diseases are important because they impart diagnostic challenges to all the experts involved in the treatment of anemic patients.

Prevalence of non-alcoholic fatty liver disease and its correlation with coronary risk factors in patients with type 2 diabetes.

OBJECTIVES: To estimate the prevalence of non-alcoholic fatty liver disease (NAFLD) by ultrasonography, and to correlate NAFLD with coronary artery disease (CAD) and coronary risk factors in a group of Indian type 2 diabetics. METHODS: Consecutive patients of type 2 diabetes were recruited. History and physical examination were recorded. Laboratory investigations included fasting and 2-hour post-prandial blood glucose, blood urea, serum creatinine, liver function tests, lipid profile, glycated haemoglobin, microalbuminuria, and ultrasonographic measurement of carotid intimal-medial thickness (CIMT). NAFLD was diagnosed on the basis of ultrasound assessment of the liver. RESULTS: The study group (n=124) was divided into a NAFLD group (n=71) and a non-NAFLD group (n=53). The prevalence of NAFLD was 57.2%. CAD was more prevalent in the NAFLD subgroup (60.5%) compared to the non-NAFLD subgroup (45.2%). The NAFLD subgroup had higher prevalence of hypertension, smoking, obesity (measured by BMI), central obesity (measured by waist circumference and waist hip ratio), higher HbAlc, higher triglyceride levels and lower HDL levels, and higher mean CIMT. Using binary logistic regression analysis, it was found that hypertension (p=0.013), LDL cholesterol (p=0.049), microalbuminuria (p=0.034) and NAFLD (p=0.016) were significantly correlated with CAD. CONCLUSION: Among type 2 diabetics, NAFLD clusters with traditional coronary risk factors. It is a surrogate and fairly reliable marker of risk for CAD amongst type 2 diabetic patients. Ultrasonographically detected NAFLD is a simple, cheap, and safely assessable parameter for coronary risk stratification in type 2 diabetics.

JAK2-positive Philadelphia-negative myeloproliferative neoplasms.

The recent discovery of the JAK2 mutations has rekindled interest in the approach to classic BCR/ABL-negative myeloproliferative neoplasms (MPNs) in terms of both diagnostic evaluation and treatment. However, additional clinical, laboratory and histological parameters play a key role to allow diagnosis and subclassification, regardless of whether JAK2 V617F mutation is present or not. Here are two cases which incidentally presented with splenomegaly and moderate leukocytosis, and were diagnosed as MPN-primary myelofibrosis (PMF) in prefibrotic phase and polycythemia vera (PV), respectively, using revised World Health Organization (WHO) 2008 criteria.

Ataxia telangiectasia: A report of two cousins and review of literature.

Ataxia telangiectasia (AT) is a rare multisystem, neurodegenerative genetic disorder. Due to its wide clinical heterogeneity, it often leads physicians to an incorrect or missed diagnosis, and insight into this rare disease is important. Here is a case report of two cousins from the same family who showed salient characteristic features of AT along with the incidental finding of co-inheritance of hemoglobin E trait. Though both of them were from the same family, they showed differences in the type of humoral immune deficiencies, laboratory findings, and their susceptibility to develop different types of malignancies. One of them developed T cell acute lymphoblastic leukemia, isolated immunoglobulin A deficiency, and normal serum carcinoembryonic antigen (CEA) and carbohydrate antigen 19.9 (CA 19.9) levels. He expired at the age of nine years. The other, though a year older, has still got normal blood counts, normal immunoglobulin levels, and elevated serum CEA and CA 19.9 levels. Thus, insight into this disease is very important as AT patients require protection from unnecessary exposure to ionizing radiation to prevent malignancies. Diagnosis of AT allows appropriate genetic counseling for the family.

Prevalence of coronary risk factors in type 2 diabetics without manifestations of overt coronary heart disease.

AIMS: To find the prevalence of cardiovascular risk factors in type-2 diabetics without manifestations of overt coronary heart disease and to estimate the prevalence of silent myocardial ischaemia in these patients. METHODS: Seventy seven patients of type 2 diabetes were recruited for the study (one patient lost after recruitment; 76 completed the study). History and physical examination were recorded. Laboratory investigations included fasting and 2-hour post-prandial blood sugar, blood urea, serum creatinine, lipid profile, glycated haemoglobin, and microalbuminuria. Ultrasonographic scanning of the carotid arteries was performed to measure the carotid IMT. For identification of cases of silent ischaemia, treadmill test (TMT) was performed. RESULTS: The study group was divided into a non-CAD group (n=54), and a silent CAD group (n=22). Twenty-two diabetics were found to have silent CAD as evidenced by a positive TMT result (28.9%). The prevalence of silent myocardial ischaemia was almost similar in both males and females. Serum LDL levels more than 140 mg% had a significant correlation with the prevalence of silent CAD (p = 0.04). The difference in CCA-IMT values was found to be statistically significant between the silent CAD and non-CAD groups (p = 0.019). CONCLUSION: High LDL level and greater carotid intima-media thickness are particularly important parameters that can predict if a patient of type 2 diabetes is at risk for silent ischaemia. A high carotid IMT is a surrogate and reliable marker of higher risk of CAD amongst type 2 diabetic patients, even in those without overt CAD. The study also underlines the utility of carotid IMT as a simple, non-invasive, safe, and cheap screening test for the assessment of risk of CAD in type 2 diabetics.

Endometrial intraepithelial carcinoma: a case report and brief review.

This case report describes the precursor lesion of uterine papillary serous carcinoma (UPSC). A 65-year-old post-menopausal female presented with prolapse and vaginal discharge and underwent a hysterectomy revealing an atrophic endometrium, highly atypical endometrial glands, the lining cells of which showed pseudostratification, hobnailing, a high nuclear to cytoplasmic ratio, and prominent nucleoli. A p53 immunoreactivity score of 8 and a MIB-1 index of 80% was obtained leading to a diagnosis of endometrial intraepithelial carcinoma (EIC). Since serous EIC is commonly associated with extra-uterine serous carcinoma, it is a uniquely aggressive precursor lesion. Molecular studies support the hypothesis that EIC is a precursor of both uterine and extra-uterine invasive serous carcinomas. This is why the treatment protocol for EIC cases is total abdominal hysterectomy (TAH), accompanied by a staging procedure. In our patient, EIC was limited to the endometrium; associated with an excellent clinical outcome.

Pleomorphic adenoma with squamous and appendageal metaplasia mimicking mucoepidermoid carcinoma on cytology.

BACKGROUND: Histological diversity is the hallmark of pleomorphic adenoma, the most common salivary gland tumor. It may cause difficulty in cytological interpretation, due to limited and selective sampling. CASE PRESENTATION: A 16-year-old female patient presented with right cheek swelling. Fine needle aspiration cytology showed squamous cells, basaloid cells, and foamy cells, along with extracellular keratin and foreign body giant cells. Characteristic metachromatic fibrillary chondromyxoid stroma, which is usually seen in pleomorphic adenoma, was not seen in the aspirate. A diagnosis of mucoepidermoid carcinoma was given on cytology. Subsequent resection revealed an encapsulated pleomorphic adenoma, with extensive squamous metaplasia and appendageal differentiation on histology. CONCLUSION: This case illustrates that pleomorphic adenoma with squamous metaplasia presents a potential for misinterpretation as mucoepidermoid carcinoma on cytology. We discuss the various pitfalls and the features that are helpful in distinguishing these two lesions.

Myofibroblastoma of male breast: a case report.

Myofibroblastoma of breast is an extremely rare, benign mesenchymal lesion composed ofmyofibroblasts. Less than seventy cases of this entity are reported in the literature. In spite of the widely varying morphological patterns, there are few characteristic cytological, histopathological, immunohistochemical, and ultrastructural features that permit its accurate identification among a vast group of mammary spindle cell tumours. We present the case ofa 60 year male presenting with a left breast nodule, interpreted as gynaecomastia on cytology. Histopathology however revealed characteristicfeatures of a myofibroblastoma, confirmed by immunohistochemistry.

Cytodiagnosis of multiple myeloma presenting as orbital involvement: a case report.

BACKGROUND: Plasma cell neoplasms represent autonomous proliferations of plasma cells and can manifest as diffuse myeloma with systemic involvement (plasma cell myeloma or multiple myeloma), monoclonal gammopathy of undetermined significance (MGUS), or as variants of plasma cell myeloma such as indolent myeloma, smoldering myeloma, osteosclerotic myeloma, plasma cell leukaemia and non-secretory myeloma. Localized neoplastic proliferation of plasma cells presents as solitary plasmacytoma of bone or extramedullary plasmacytoma. Involvement of orbit can occur as a solitary plasmacytoma, or as part of systemic involvement in multiple myeloma, the clinical outcome being significantly worse in the latter setting. Orbital involvement in multiple myeloma is very rare with less than 50 cases reported in the literature. Early cytological diagnosis of such lesions is vital for timely institution of appropriate therapy. As far as we are aware only six previous cases of cytological diagnosis of multiple myeloma involving the orbit are on record. CASE PRESENTATION: A 37 year old male presented with low grade fever showing evening rise, headache, diplopia and swelling in the right periorbital and temporal region. Imaging studies revealed destructive lesion of sphenoid, frontal bone and zygomatic arch with soft tissue component extending to infratemporal fossa and orbit. A fine needle aspirate from the temporal region swelling showed features of a plasmacytoma, and subsequent workup confirmed the presence of systemic disease. A final diagnosis of multiple myeloma with orbital involvement at presentation was made. CONCLUSION: Present case describes the extremely rare presentation of multiple myeloma with orbital involvement and highlights the utility of cytology in such lesions. Fine needle aspiration diagnosis of plasmacytoma at extramedullary sites offers an opportunity for non-invasive verification of systemic involvement, and thus plays a major role in early diagnosis and management of these patients.