Missing self by heterogeneous natural killer cells

Some murine (YAC, P815 and SP20) and human (Molt4, Raji and HR7) tumour cell lines were (i) treated with IFN-γ for inducing enhanced expression of MHC class I antigen, or (ii) given a brief treatment with citrate buffer (pH 3.0), which resulted in denaturation of class I MHC antigens on these tumour cells. IFL-γ or acid treated tumour cells were used as unlabelled competing targets in cold target inhibition assays. The results indicated that the competing ability of acid-treated tumour cells remained unaltered, whereas IFN-γ treated tumour cells competed with significantly less efficiency. These results have been evaluated in light of the current view of NK cell development and the expression of inhibitory receptors for MHC class I molecules (IRMs), on NK cells. A modified view on NK cell heterogeneity based upon IRM expression has been proposed which reconciles several apparently discordant observations about the activity and role of NK cells. Two classes of NK cells have been proposed. Type I NK ceils have target recognition receptors which do not recognize autologous normal cells, lack IRMs, and may participate in first line of defence against transformed cells in vivo. Type II NK cells have target recognition receptors for autologous normal cells and express at least one self-reactive IRM in order to prevent auto-killing. Type II NK cells participate in killing those transformed cells which down-regulate their MHC class I expression in order to escape cytotoxic T-cell surveillance. It is also postulated that mechanism of inverse correlation of target cell MHC class I expression levels and their susceptibility to NK cells, involves interference model of missing self hypothesis for type I NK cells and inhibitory signal model of missing self hypothesis for type II NE cells. Finally, it is proposed that acid treatment of tumour cells enhances their lysis susceptibility by making them additionally susceptible to type II NK cells, rather than enhancing their killing by type I NK cells. This proposition would explain the lack of effect of acid treatment on the competing ability of tumour cells, when target cells are only lysed by type I NE cells

Effect of diesel exhaust particulate on bacillus Calmette-Guerin lung infection in mice and attendant changes in lung interstitial lymphoid subpopulations and IFNγ response

The effect of exposure to diesel exhaust particulate (DEP) on bacillus Calmette-Guerin (BCG) lung infection in mice was studied. C57Bl/6J female mice were infected with BCG (2.5×104 bacteria/mouse) by intrapulmonary instillation, with or without coadministration of DEP (100 µ g/mouse). Five weeks later, mice exposed to DEP + BCG had about a four-fold higher BCG load in the lungs than mice exposed only to BCG (p < 0.05). DEP treatment alone had no effect on the total number of lung lymphocytes or numbers of T, B, or NK cells recovered from lungs. In contrast, BCG infection significantly increased (p < 0.05) recovery levels of all types of lymphocytes from lungs. Coexposure to DEP + BCG further increased the recovery of lymphocytes from lungs of BCG-infected mice. The pulmonary lymphocyte subpopulation expressing the greatest levels of mRNA for IFNγ after BCG infection was CD4+ T cells. Expression levels were similar in mice exposed to BCG or BCG + DEP and were elevated as compared to noninfected mice and mice treated with DEP alone. Recovery of IFNγ-secreting lymphocytes and IFNγ-secreting T cells was significantly higher (p < 0.05) from lungs of BCG-infected mice as compared to control or DEP-exposed mice. BCG and BCG+DEP groups of mice did not differ significantly in the numbers of IFNγ-secreting lymphocytes in lungs. Taken together, these results indicated that coexposure to DEP+BCG did not significantly affect the level of IFNγ response of mice to BCG infection. However, DEP treatment was found to inhibit IFNγ-induced nitric oxide (NO) production by mouse alveolar macrophages in vitro. Our results indicate that DEP exposure did not alter the IFNγ response to BCG infection, but reduced responsiveness of alveolar macrophages to IFNγ . Reduced sensitivity of DEP-exposed alveolar macrophages to IFNγ may contribute to a greater load of BCG in the lungs of BCG-infected mice given DEP

Natural Killer Cells Interaction with Carbon Nanoparticles

The increased use of nanomaterials for biomedical purposes has warranted the need to introspect their toxicological properties and assess their utility to human health, particularly the immune system. Natural killer (NK) cells hold a pivotal position in innate immunity and serve as first line of defense against foreign bodies. Acid functionalized Carbon nanotubes (CNTs) that easily polydisperse in aqueous solution and could be coupled with fluorescent molecules were used to study the effect of carbon nanoparticles on NK cells in vitro and in vivo. Flow cytometry-based assays were used to study the effect of CNTs on various physiological parameters of NK cells, such as cell recovery, apoptosis, cell cycle, and generation of reactive oxygen species. A downregulation of the cytotoxicity of IL-2-activated murine NK cells was observed in the presence of acid-functionalized CNTs. The mechanistic basis of this downregulation was studied by assessing markers of NK cell activation (CD69), generation (NLK1.1), degranulation (CD107a) and apoptosis (annexin V assay). This chapter provides a blueprint for assessing the effect of carbon nanoparticles on NK cells. The assays mentioned in this chapter can be extrapolated to study the effect of other nanoparticles on different cell types as well

A Double In Vivo Biotinylation Technique to Assess Erythrocyte Turnover in Blood Circulation

We have developed a new double in vivo biotinylation (DIB) technique that may be used for assessing turnover patterns of erythrocytes in circulation. This technique involves two successive in vivo biotinylation steps, interspersed by a period of 5–30 days, which would enable us to tag with biotin a population of erythrocytes entering blood circulation over a defined period of time, between the two biotinylation steps. As such we can track the age-related changes in a lifetime of the circulating erythrocytes, or we can simultaneously study two defined age cohorts of aged as well as young erythrocytes in circulation. We have extensively used this technique to look at erythrocyte loss in mouse models of anemia induced by (a) heavy metal cadmium (Cd), (b) herbicide Paraquat (PQ), (c) carbon nanotubes (CNTs), and (d) autoantibody in autoimmune hemolytic anemia (AIHA). We have found that the pattern of erythrocyte removal is distinctly different in different models of murine anemia. In certain types of anemia (CNT and AIHA), younger erythrocytes in blood circulation are preferentially removed, whereas in other cases (Cd and PQ), old erythrocytes are specifically eliminated

Audit of caesarian deliveries in a tertiary care center, in rural Bangalore, India

Background: The Robson’s Ten-Group Classification System allows critical analysis of caesarean deliveries according to characteristics of pregnancy. The objective was to analyze caesarean section rates in a rural tertiary care teaching hospital in Bangalore, using Robson’s ten groups classification.Methods: This study was done in MVJ Medical College and Research Hospital, a rural tertiary care teaching hospital. All patients who underwent caesarean delivery, between November 2017 and October 2018, were included in the study. Women were classified in 10 groups according to Robson’s classification. For each group, authors calculated its relative contribution to the overall caesarean rate.Results: The overall caesarean section rate was 46.7%. The main contributors to this high caesarean rate were primiparous women in spontaneous labour (group 1) and women with previous caesarean section (group 5).  52.1% of CS were conducted on women who were unbooked or booked at a peripheral health facility and referred to present institution due to complications in labor. Strategies to lower CS rates would include encouraging women with previous CS, to undergo trial of labor to reduce CS rates for group 5C. Sensitization of staff in peripheral medical facilities for early referral of high-risk pregnancies to a tertiary care center for better control of medical complications like hypertensive disorders of diabetes mellitus. Other strategies include offering external cephalic version to eligible women with breech presentation and consider offering vaginal breech delivery to suitable women in groups 6 and 7.Conclusions: The Robson’s classification is easy to use. It is time to implement obstetric audit to lower the overall CS rates

Age-dependent increase in green autofluorescence of blood erythrocytes

Green auto-fluorescence (GAF) of different age groups of mouse blood erythrocytes was determined by using a double in vivo biotinylation (DIB) technique that enables delineation of circulating erythrocytes of different age groups. A significant increase in GAF was seen for erythrocytes of old age group (age in circulation >40 days) as compared to young erythrocytes (age <15 days). Erythrocytes are removed from blood circulation by macrophages in the reticulo-endothelial system and depletion of macrophages results in an increased proportion of aged erythrocytes in the blood. When mice were depleted of macrophages for 7 days by administration of clodronate loaded liposomes, the overall GAF of erythrocytes increased significantly and this increase could be ascribed to an increase in GAF of the oldest population of erythrocytes. Using the DIB technique, the GAF of a cohort of blood erythrocyte generated during a 5 day window was tracked in vivo. GAF of the defined cohort of erythrocytes remained low till 40 days of age in circulation and then increased steeply till the end of the life span of erythrocytes. Taken together our results provide evidence for an age dependent increase in the GAF of blood erythrocytes that is accentuated by depletion of macrophages. Kinetics of changes in GAF of circulating erythrocytes with age has also been defined

Murine model of BCG lung infection: Dynamics of lymphocyte subpopulations in lung interstitium and tracheal lymph nodes

C57B1/6 female mice were infected with an intrapulmonary dose of 2.5×104 BCG(Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium and lung-associated tracheal lymph nodes (LN) were examined at 1,2, 4, 5, 6, 8 and 12 weeks after infection. BCG load in lungs peaked between 4-6 weeks post-infection and declined to very low levels by the 12th week of infection. Lung leukocytes were obtained over the course of infection by enzyme digestion of lung tissue followed by centrifugation over Percoll discontinuous density gradients. By 4 to 6 weeks after infection, numbers of lung leukocytes had more than doubled but the proportions of lymphocytes (about 70%), macrophages (about 18%) and granulocytes (about 12%) remained essentially unaltered. Flow cytometric studies indicated: (i) the total number of CD3+ T cells in lungs increased by 3-fold relative to uninfected controls at 5 to 6 weeks post-infection, but the relative proportions of CD4 and CD8 cells within the T cell compartment remained unaltered; (ii) relative proportion of NK cells in lungs declined by 30% but the total number of NK cells (NK1.1+) per lung increased by about 50%, 5-6 weeks post infection; (iii) tracheal LN underwent marked increase in size and cell recoveries (6-10-fold increase) beginning 4 weeks after infection. While both T and B cells contributed to the increase in cell recoveries from infected tracheal LNs, the T/B ratio declined significantly but CD4/CD8 ratio remained unaltered. In control mice, IFNγ producing non-T cells outnumbered T cells producing IFNγ . However, as the adaptive response to infection evolves, marked increase occur in the number of IFNγ producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNγ response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNγ, suggesting that maturational changes associated with migration to the lungs or residence in the lungs enhance the capability of some T cells to produce this cytokine

ESBL, MBL AND AMP C-β LACTAMASES PRODUCED BY SUPERBUGS: AN EMERGING THREAT TO CLINICAL THERAPEUTICS

Objectives: The present study was undertaken to determine the prevalence of multi drug resistant (MDR) and multiple β-lactamase producing Pseudomonas aeruginosa isolates in lower respiratory tract infection (LRTI) patients at a tertiary care hospital in India.Methods: A total of 80 consecutive, non-duplicate isolates of P. aeruginosa were studied for the presence of class A or B β-lactamase. Antibiotic susceptibility tests and PCR amplification of genes encoding class A (PER-1 and CTX-M 1, 2, 9) and class B β-lactamases (blaVIM-2, blaIMP-1 and blaSIM-1) were performed.Results: Out of 80 P. aeruginosa isolates, 65% (52/80) of the isolates were MDR with 34 being Metallo-β-lactamase (MBL) producers, 23 were extended spectrum β-lactamase (ESBL) producers and 21 were positive for AmpC production. The cross-class resistance rates to other antibiotics was significantly higher in class A and B β-lactamase producers than in non-producers (P<0.05 for fluoroquinolone, aztreonam, ceftazidime and meropenem). Combined disk test (CDT) for MBL highest sensitivity and specificity compared to PCR. Combined disk method (CDM) for ESBL co-related well with PCR (sensitivity and specificity).Conclusion: This study reports the validation of a simple and accurate MBL and ESBL detection method which can be easily integrated into the daily routine of a clinical laboratory.Â

Differential pulmonary retention of diesel exhaust particles in Wistar Kyoto and spontaneously hypertensive rats

Spontaneously hypertensive (SH) and normotensive Wistar Kyoto (WKY) rats have been used for understanding the mechanisms of variations in susceptibility to airborne pollutants. We examined the lung burden of diesel exhaust particles (DEP) following inhalation of diesel engine exhaust (DEE) in both strains. The kinetics of clearance was also examined after single intratracheal (IT) instillation of DEP. Lungs were analyzed for DEP elemental carbon (EC) after exposure to DEE (0, 500, or 2000 μ g/m3 4 h/day, 5 days/week×4 weeks). SH rats had 16% less DEP-EC at 500 and 32% less at 2000 μ g/m3 in the lungs, despite having 50% higher than the average minute volume. No strain-related differences were noted in number of alveolar macrophages or their average DEP load as evident from examining cells in bronchoalveolar lavage fluid (BALF). The kinetics of DEP clearance from lungs of male WKY and SH rats was studied following a single instillation at 0.0 or 8.33 mg/kg of DEP standard reference material (SRM 2975) from the National Institute of Standards Technology. SH rats cleared 60% DEP over 112 days while minimal clearance occurred from the lungs of WKY. The pattern of DEP-induced inflammatory response assessed by BALF analysis was similar in both strains, although the overall protein leak was slightly greater in SH rats. A time-dependent accumulation of DEP occurred in tracheal lymph nodes of both strains (SH > WKY). Thus, SH rats may clear DEP more efficiently from their lungs than normotensive WKY rats, with a small contribution of more effective lymphatic drainage

Type IIA2 urethral duplication: report of an unusual case

This report describes a rare case of type IIA2 sagittal urethral duplication. The presentation, investigation, and management of this rare anomaly are briefly discussed. A 3½-year-old boy presented with urinary obstruction and recurrent urinary tract infection due to a stenosed dorsal urethra and segmental stenosis of the dominant ventral urethra. The child also had left-sided vesicoureteric reflux. Staged surgical management consisted of an initial vesicostomy followed by serial dilatation of the ventral urethral stricture, left ureteric reimplantation, and a 2-cm long distal urethrourethrostomy between the dorsal urethra, opening at the tip of the penis, and the ventral urethra, which had a hypospadic opening at the base of the glans. The functional and cosmetic outcomes were satisfactory. The management needs to be individualized as best suited for the patient.Keywords: urethra, urethral duplication, urethral strictur