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Proliferation and migration of lung epithelial cells following the injury to the epithelial lining of alveoli and airways in the lung are pivotal for remodeling and repair of the wound to restore normal lung function. In the present study, we examined the modulatory effect of carboxylated nanodiamonds (cNDs) on the cell division, migration, and adhesion of epithelial cells in the well-established in vitro model of wound repair and cell migration. Flow cytometry and confocal microscopy results indicated that both LA4 and A549 cells effectively internalized fluorescent carboxylated nanodiamonds (cFNDs) and the internalized nanodiamonds were essentially localized in the cytoplasmic region. Treatment with cNDs blocked the division and migration of cells to fill the scratch wound. Live cell imaging and time-lapse videography of the wound healing process indicated a significant inhibition of cell proliferation activity in cND-treated cells and blocked the wound repair process. Trans-well cell-migration assay results further support the inhibitory effect of cNDs on the cell migration process. Western blotting and immunofluorescence staining indicated that the crucial proteins involved in epithelial-mesenchymal transition (EMT) and cell migration i.e. β-catenin, Vimentin, NM-myosin, and Focal Adhesion Kinase (FAK) were downregulated after treatment with cNDs, while the expression of E-cadherin and Claudin-1, major cell adhesion markers remained unaltered. Taken together, our results indicate that the decline in cell proliferation activity, downregulation in the expression of various crucial protein like β-Catenin, NM-myosin, FAK, and Vimentin involved in the cell migration and unaltered expression of cell adhesion molecules E-cadherin and Claudin-1, may be the factors that contribute to the cND-mediated inhibition of EMT during the wound repair process in the monolayers of lung epithelial cells.

The present study is focused on the modulation of Mycobacterium bovis BCG-induced inflammatory response by poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) in macrophages. Flow cytometric and confocal microscopy studies indicated that both BCG and AF-SWCNTs were efficiently internalized by RAW 264.7 and MH-S macrophage cell lines and were essentially localized in the cytoplasmic area. BCG-induced production of reactive oxygen species (ROS) and nitric oxide by the two cell lines was significantly inhibited by AF-SWCNTs. Using RT-PCR technique, a marked decline was observed in the expression of BCG-induced pro-inflammatory genes COX-2, iNOS, TNF-α, IL-6, and IL-1β upon treatment with AF-SWCNTs. Results of gelatin zymography indicated that the AF-SWCNTs treatment also induced a marked decline in BCG-induced release of matrix metalloproteinases MMP-2 and MMP-9 by the two macrophage cell lines. The anti-inflammatory effect of AF-SWCNTs in downregulating BCG-induced inflammatory response was further validated in murine peritoneal macrophages. Treatment with AF-SWCNTs led to a steep decline in BCG-induced NO production in murine peritoneal macrophages in vitro as well as in vivo. Peritoneal macrophages isolated from mice treated with BCG and AF-SWCNTs had a significantly lower intracellular expression of COX-2 as compared to the peritoneal macrophages derived from mice treated with BCG alone. Taken together, our results demonstrate a potent anti-inflammatory effect of AF-SWCNTs in alleviating BCG-induced inflammatory responses in macrophages in vitro and in vivo.

Background The severe acute respiratory syndrome coronavirus-2 was initially believed to be an infection of only the respiratory system. It is now known to have many extrapulmonary affections including gastrointestinal symptoms associated with high mortality rates. An unusual presentation of bowel perforation in a patient who had recently undergone cesarean delivery prompted us to report this mini review of bowel perforations attributed to coronavirus. Case Our patient, a 19-year-old primigravida, underwent an emergency cesarean section under spinal anesthesia. Intraoperative events and postoperative period were uneventful, and the patient was discharged on postoperative day 5. On the 10th postoperative day, the patient presented in septic shock to the emergency department with complaints of acute epigastric pain, vomiting, loose stools, and bilious discharge from the wound site. Oral contrast computed tomography was done, which revealed a proximal jejunal perforation. The patient was taken up for laparotomy followed by bowel repair with placement of intraperitoneal drains. Immediate postoperatively, the patient developed high-grade fever for which reverse transcription polymerase chain reaction (RT-PCR)_ for COVID-19 was sent. The result for the same turned out to be positive. Twenty-four hours after the laparotomy, the intraperitoneal drains again started draining copious bilious discharge along with the deterioration of general condition of the patient. She expired on the third postoperative day. This was followed by a thorough research of the literature into possible etiologies for bowel perforation. In this mini review, we shall be discussing various case reports to determine the cause of such unusual presentation in our patient. How to cite this article Ramani MV, Saxena RK, Indrani C. Bowel Perforation due to COVID-19: A Mini Review. J South Asian Feder Obst Gynae 2020;12(6):427–431.

Activated mouse B cells as compared to the resting B cells are known to internalize substantially more acid-functionalized single walled carbon nanotubes (AF-SWCNTs). It was hypothesized that the antigens coupled to AF-SWCNTs would also be taken up more efficiently by B cells. Further the enhanced uptake of the antigen by B cells may facilitate antigen presentation by B cells resulting in a better antibody response. Aim of this study was to test this hypothesis. Ovalbumin was chemically coupled to AF-SWCNTs that yielded a coupled product that had 0.08% of all carbon atoms in AF-SWCNTs occupied by ovalbumin. Coupling of ovalbumin to AF-SWCNTs was confirmed by staining the product with anti-ovalbumin antibodies. B cells incubated with ovalbumin-AF-SWCNT internalized more ovalbumin than the B cells incubated with free ovalbumin. Groups of mice were immunized subcutaneously with (a) free ovalbumin, (b) free ovalbumin and AF-SWCNTs at two different subcutaneous sites respectively on mice, and (c) ovalbumin-AF-SWCNT coupled product. In each case a primary immunization was followed by three weekly booster doses. It was found that the anti-ovalbumin antibody response assessed by ELISA, was highest in the group where ovalbumin coupled to AF-SWCNTs was used for immunization (p 0.001). Antibody response in ovalbumin-AF-SWCNT group was comparable to the group where ovalbumin was used for immunization using complete and incomplete Freund's adjuvant (primary and secondary immunizations respectively). We propose that AF-SWCNTs could be explored as an adjuvant to improve the antibody response especially in vaccine development.

BackgroundThe surgical transversus abdominis plane (TAP) block is an established technique for postoperative analgesia following abdominal surgery. However, it has not been practiced frequently by obstetricians. This work was done to study the efficacy of the surgical TAP block after cesarean delivery (CD).Materials and methodsThe study included 100 women undergoing CD in MVJ Medical College and Research Hospital, Hoskote, Bengaluru, Karnataka. The operating obstetrician performed a surgical TAP block for 50 participants in the “Study” group. Twenty milliliters of 0.25% bupivacaine was injected in the transversus abdominis neurofascial plane, on either side. Another 50 participants were included in the “Control” group. Postoperatively, visual analog scores (VAS) for pain were monitored for 24 hours. Whenever the patient demanded rescue analgesia or VAS was recorded as 4 or more, rescue analgesia was administered with intravenous injection tramadol 50 mg. The time of the first rescue analgesia and the total dose of rescue analgesics required in the first 24 hours were recorded. The patients in the “Control” group routinely received intravenous injection tramadol 50 mg, 8th hourly for pain relief.ResultsThe difference in the mean VAS score at all time intervals between the two groups was statistically significant (p< 0.05). The mean time to first rescue analgesia in the “Study” group and “Control” group was 11.7 ± 7.344 and 2.1 ± 1.125 hours, respectively, and the total tramadol consumption was 50 ± 28.571 and 180 ± 26.726 mg, respectively. This difference was statistically highly significant (p< 0.001).ConclusionSurgical TAP block is an important adjunct in the multimodal pain management strategy and should be practiced more often in obstetric practice.How to cite this articleSravani P, Indrani C, Rajanna SP,et al. Efficacy of Surgical Transversus Abdominis Plane Block in Patients Undergoing Cesarean Delivery. J South Asian Feder Obst Gynae 2020;12(5):302–306.

Breath alcohol analyser is used to detect alcohol content in end-expiratory breaths in order to enforce driving regulations under the influence of alcohol legislation. The accuracy and reliability of the routine measurements of alcohol content performed with breath alcohol analyser can be achieved by the calibration of the breath alcohol analyser using standards traceable to SI reference material. Proper calibration is essential for transparency in legal verification for which reference material is needed. At international level, a number of NMIs are active to address this important measurement issue of providing accurate measurements. Several international key comparison programs have been organized so far for the determination of ethanol content in aqueous and in nitrogen/air matrix. NIST, USA; BAM, Germany; IRMM, Belgium, Portugal, INMETRO, Brazil, LGC, UK etc. have developed certain reference materials of ethanol in water solution/air with different concentration ranges. However, no such reference material is introduced in India as an indigenous standard, rather, being procured from abroad or using high purity alcohol for calibration purposes. CSIR-NPL, India, being the NMI is now focusing on establishing the calibration facility and development of SI traceable aqueous alcohol standard to provide test reliability for the testing in breath alcohol analyser. This program has a societal impact which contributes to human health and regulatory needs for the nation.

The present study is focused on the modulation of Mycobacterium bovis BCG-induced inflammatory response by poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) in macrophages. Flow cytometric and confocal microscopy studies indicated that both BCG and AF-SWCNTs were efficiently internalized by RAW 264.7 and MH-S macrophage cell lines and were essentially localized in the cytoplasmic area. BCG-induced production of reactive oxygen species (ROS) and nitric oxide by the two cell lines was significantly inhibited by AF-SWCNTs. Using RT-PCR technique, a marked decline was observed in the expression of BCG-induced pro-inflammatory genes COX-2, iNOS, TNF-α, IL-6, and IL-1β upon treatment with AF-SWCNTs. Results of gelatin zymography indicated that the AF-SWCNTs treatment also induced a marked decline in BCG-induced release of matrix metalloproteinases MMP-2 and MMP-9 by the two macrophage cell lines. The anti-inflammatory effect of AF-SWCNTs in downregulating BCG-induced inflammatory response was further validated in murine peritoneal macrophages. Treatment with AF-SWCNTs led to a steep decline in BCG-induced NO production in murine peritoneal macrophages in vitro as well as in vivo. Peritoneal macrophages isolated from mice treated with BCG and AF-SWCNTs had a significantly lower intracellular expression of COX-2 as compared to the peritoneal macrophages derived from mice treated with BCG alone. Taken together, our results demonstrate a potent anti-inflammatory effect of AF-SWCNTs in alleviating BCG-induced inflammatory responses in macrophages in vitro and in vivo.

Present study is focussed on the modulation ofMycobacterium bovisBCG induced inflammatory response by poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) in macrophages. Flow cytometric and confocal microscopy studies indicated that both BCG and AF-SWCNTs were efficiently internalized by RAW 264.7 and MH-S macrophage cell lines and were essentially localized in the cytoplasmic area. The results indicated strong antioxidant activity of AF-SWCNTs in mitigating BCG induced oxidative and nitrosative stress. We also found a marked decline in expression of BCG induced pro-inflammatory genes like COX-2, iNOS, TNF-α, IL-6 and IL-1β on treatment with AF-SWCNTs at transcriptional level. Decline in expression of BCG induced COX-2 by AF-SWCNTs was also confirmed at protein level using Western blotting. Anti-inflammatory activity of AF-SWCNTs was further validated by our results showing that AF-SWCNTs treatment induced a precipitous decline in BCG induced release of Matrix Metalloproteinases MMP-2 and MMP-9 by macrophage cell lines, by using Gelatin zymography. Taken together, our results demonstrate potent anti-inflammatory role of AF-SWCNTs in alleviating BCG induced inflammation.

Epithelial cell lining of the lung alveoli is under constant onslaught of airborne pathogens and pollutants that may cause injury and disruption of the epithelial lining. Repair mechanisms involve proliferation and migration of nearby healthy epithelial cells to the site of injury. Using murine LA4 and human A549 lung epithelial cell lines, and in vitro models of cell migration we have examined the modulation of cellular adhesion and migration by poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs). Flow cytometric and confocal microscopy studies indicated that AF-SWCNTs were efficiently internalized by both cell lines and were localized essentially in the cytoplasmic area. In the scratch wound repair model, exposure to AF-SWCNTs blocked the filling of the scratched area of the cellular monolayers in both cells. Behaviour of the cells around the scratch area was examined in by live-cell imaging time-lapse micrography. The results indicated active cell proliferation around the scratch area that was totally blocked by AF-SWCNTs in LA4 cells and significantly inhibited in A549 cells. Cell migration across a porous membrane in transwell assay system also indicated a marked inhibition of migration of both cells across the membrane. Effect of AF-SWCNTs on the expression levels of important cell proteins involved in cell migration and adhesion were examined by western blotting and immunofluorescence staining. Expressions of proteins like β-Catenin, NM-Myosin and Vimentin that play crucial role in cell migration were suppressed in AF-SWCNTs-exposed cells whereas the expression levels of E-cadherin and Claudin-1, involved in cell-cell adhesion remained unaltered. Our results provide an insight into the mechanism of repair of lung epithelial cell layers.

Graft versus host disease (GVHD) results from hyper-activation of transplanted lymphocytes against the host antigens. Bone marrow transplantation in humans as well as some cases of blood transfusion and organ transplantation are associated with a strong GVH reaction resulting in GVHD that in many cases may be fatal. We had previously shown that poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) specifically target activated T and B lymphocytes and kill them. In the present study, efficacy of AF-SWCNTs to suppress the GVH reaction was tested in the mouse model. Acute GVHD was induced in mice by administering intravenously 30 or 60 million spleen cells from a parental strain (C57bl/6 mouse, MHC haplotype H-2b) to host (C57bl/6 x Balb/c) F1 mice (MHC haplotype H-2b/d)and waiting for 8-10 days. Chronic GVHD was similarly induced by administration of 30 million parent spleen cells to F1 mice and waiting for a period of 60 days. Our results demonstrate a marked decline in splenomegaly and recovery of spleen T (both CD4 and CD8) and B cells in GVHD mice treated with AF-SWCNTs. AF-SWCNTs treatment also limited T and B cell proliferation by restricting S-phage of cell cycle. Generation of anti-host cytotoxic T cells (CTLs) was also markedly suppressed by AF-SWCNT treatment of acute GVHD mice, and a significant reduction in the generation of anti-host antibodies could also be demonstrated. Taken together, our results suggest that the AF-SWCNTs can be considered as a potential therapeutic agent for treating GVHD.

Graft versus host disease (GVHD) results from hyper-activation of transplanted lymphocytes against the host antigens. Bone marrow transplantation in humans as well as some cases of blood transfusion and organ transplantation are associated with a strong GVH reaction resulting in GVHD that in many cases may be fatal. We had previously shown that poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) specifically target activated T and B lymphocytes and kill them. In the present study, efficacy of AF-SWCNTs to suppress the GVH reaction was tested in the mouse model. Acute GVHD was induced in mice by administering intravenously 30 or 60 million spleen cells from a parental strain (C57bl/6 mouse, MHC haplotype H-2b) to host (C57bl/6 × Balb/c) F1 mice (MHC haplotype H-2b/d) and waiting for 8-10 days. Chronic GVHD was similarly induced by administration of 30 million parent spleen cells to F1 mice and waiting for a period of 60 days. Our results demonstrate a marked decline in splenomegaly and recovery of spleen T (both CD4 and CD8) and B cells in GVHD mice treated with AF-SWCNTs. AF-SWCNTs treatment also limited T and B cell proliferation by restricting S-phage of cell cycle. Generation of anti-host cytotoxic T cells (CTLs) was also markedly suppressed by AF-SWCNT treatment of acute GVHD mice, and a significant reduction in the generation of anti-host antibodies could also be demonstrated. Taken together, our results suggest that the AF-SWCNTs can be considered as a potential therapeutic agent for treating GVHD.

Due to their unique properties, carbon nanotubes (CNTs) are being widely explored for industrial and medical applications. This has necessitated a thorough assessment of the effect of CNTs on human and animal physiology and health. Impact of CNTs on epithelial tight junctions has not been evaluated in the context of their toxic effects in many biological systems. In the present study, we examined the effect of acid functionalized single-walled carbon nanotubes (AF-SWCNTs) on the function and expression of two tight junction proteins (ZO-1 and occludin) in the Madin-Darby canine kidney (MDCK) cell line. Treatment of MDCK cells with AF-SWCNT resulted in a downregulation of tight junction proteins, decreased trans-epithelial electrical resistance (TER), increased paracellular permeability, and disruption of tight junctions. Taken together, our data demonstrate that AF-SWCNT disrupts tight junction barrier by downregulating tight junction proteins in MDCK epithelial cells.

Due to their unique properties, carbon nanotubes (CNTs) are being widely explored for industrial and medical applications. This has necessitated a thorough assessment of the effect of CNTs on human and animal physiology and health. Impact of CNTs on epithelial tight junctions has not been evaluated in the context of their toxic effects in many biological systems. In the present study, we examined the effect of acid functionalized single-walled carbon nanotubes (AF-SWCNTs) on the function and expression of two tight junction proteins (ZO-1 and occludin) in the Madin-Darby canine kidney (MDCK) cell line. Treatment of MDCK cells with AF-SWCNT resulted in a downregulation of tight junction proteins, decreased trans-epithelial electrical resistance (TER), increased paracellular permeability, and disruption of tight junctions. Taken together, our data demonstrate that AF-SWCNT disrupts tight junction barrier by downregulating tight junction proteins in MDCK epithelial cells.

In the present study a simple, sensitive and accurate method has been developed for the speciation analysis of chromium(III) and chromium(VI) in aqueous solution using ion chromatography separation followed by detection through a UV detector. Chromium(III) was detected as its pyridine-2,6-dicarboxylic acid (PDCA) complex at 365 nm by pre-derivatizing it with eluent containing PDCA. Chromium(VI) was detected by post column derivatization with diphenylcarbazide at 540 nm. This paper discusses in details about the method validation and established each parameter according to the IUPAC and EURACHEM approaches. Method is validated for linearity, accuracy, precision, limit of detection and limit of quantification as per the guide lines. The evaluation of expanded uncertainty in measurement of Cr(III) and Cr(VI) was done at 95 % confidence level (k = 2) according to EURACHEM GUM guide. The method can be applied to determine Cr(III) and Cr(VI) up to 2 mg/kg and 40 µg/kg respectively with associated measurement uncertainty value of about 5 %. The major contribution of uncertainty is due to recovery. The method described in the present study is sensitive, selective and capable of quantifying trace level of Cr(VI) and Cr(III).

The interaction of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNT) with NK cells undergoing activation was examined. Exposure to AF-SWCNT during NK activation in vitro by interleukin (IL)-2, and in vivo by Poly(I:C) significantly lowered cytotoxic activity generated against YAC-1 tumor cells. Recoveries of spleen NK1.1(+) cells as well as the activated subset of NK cells (NK1.1(+)CD69(+) cells) were significantly reduced by the AF-SWCNT exposure. The proportion of apoptotic NK cells (NK1.1(+) phosphatidylserine(+)) in the spleen cell preparations activated in vitro was also significantly elevated. Expression levels of CD107a [for assessing NK cell degranulation] as well as of FasL marker [mediating non-secretory pathway of NK cell killing] were significantly lower in cells exposed to AF-SWCNT during the activation phase. Intracellular levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the cells were also significantly reduced. Fluorescent AF-SWCNT (FAF-SWCNT) were internalized by the NK cells and uptake was significantly greater in activated cells. Confocal microscopy indicated the internalized FAF-SWCNT were localized to the cytoplasm of the NK cells. These results indicated that AF-SWCNT were internalized by NK cells and caused a general down-regulation of a variety of parameters associated with NK cell cytotoxicity and other cellular functions.

We have recently developed a new technique to objectively identify erythrocyte cohorts of defined age in mouse blood. The technique (termed double in vivo biotinylation, DIB) involves an initial biotinylation of all erythrocytes in circulation, followed after a few days by a second biotinylation, at a lower density, that labels the biotin-negative erythrocytes that have entered since the first biotinylation. The proportions of biotin(high), biotin(low), and biotin(negative) erythrocytes are enumerated by flow cytometry. The DIB technique allows us to track age-related changes on erythrocyte cohorts (Protocol A), and to simultaneously identify very young and older erythrocyte populations in the blood (Protocol B). Using this technique, we have reexamined: i) the relationship between age and buoyant density of erythrocytes, ii) erythrocyte destruction through a random removal mechanism, and iii) the expression of phosphatidylserine on aging erythrocytes. We have also used the DIB technique to study age-related changes in the expression of various markers like CD47 and CD147 and green autofluorescence in aging erythrocyte populations.Vor kurzem wurde von uns eine Technik entwickelt, um objektiv Erythrozytengruppen von definiertem Alter im Mäuseblut zu bestimmen. Diese als Doppelte In-vivo-Biotinylierung (DIB) bezeichnete Technik beinhaltet eine erste Biotinylierung aller Erythrozyten in der Blutzirkulation gefolgt von einer zweiten Biotinylierung wenige Tage später mit einer geringeren Dichte, so dass nur biotinnegative Erythrozyten, die seit der ersten Biotinylierung in die Zirkulation gelangt sind, markiert werden. Die numerischen Verhältnisse von Biotin

Presentation of a prototype lipid antigen α-Galactosylceramide (αGC) was examined on primary epithelial cells derived from mouse lungs and on bronchoalveolar lavage (BAL) cells that essentially comprise alveolar macrophages. Presence of CD1d molecules coupled to αGC was demonstrated on both types of cells pre-treated with αGC, suggesting that both cell types are equipped to present lipid antigens. Internalization of Mycobacterium bovis Bacillus Calmette-Guérin (BCG: a prototype pathogen), a pre-requisite to the processing and presentation of protein as well as lipid antigens, was clearly demonstrated in primary lung epithelial (PLE) cells as well as BAL cells. Both PLE and BAL cells expressed CD1d molecule and a significant up-regulation of its expression occurred upon infection of these cells with BCG. Besides CD1d, the expression of other important molecules that participate in lipid antigen presentation pathway (i.e. microsomal triglyceride transfer protein (MTTP), scavenger receptor B1 (SR-B1) and Saposin) was also significantly upregulated in PLE and BAL cells upon BCG infection. In situ up-regulation of CD1d expression on lung epithelial cells was also demonstrated in the lungs of mice exposed intra-tracheally to BCG. Taken together these results suggest that lung epithelial cells may have the ability to present lipid antigens and this pathway seems to get significantly upregulated in response to BCG infection.

Engineered carbon nanotubes are being developed for a wide range of industrial and medical applications. Because of their unique properties, nanotubes can impose potentially toxic effects, particularly if they have been modified to express functionally reactive chemical groups on their surface. The present study was designed to evaluate whether acid functionalization (AF) enhanced the cardiopulmonary toxicity of single-walled carbon nanotubes (SWCNT) as well as control carbon black particles. Mice were exposed by oropharyngeal aspiration to 10 or 40 microg of saline-suspended single-walled carbon nanotubes (SWCNTs), acid-functionalized SWCNTs (AF-SWCNTs), ultrafine carbon black (UFCB), AF-UFCB, or 2 microg LPS. 24 hours later, pulmonary inflammatory responses and cardiac effects were assessed by bronchoalveolar lavage and isolated cardiac perfusion respectively, and compared to saline or LPS-instilled animals. Additional mice were assessed for histological changes in lung and heart. Instillation of 40 microg of AF-SWCNTs, UFCB and AF-UFCB increased percentage of pulmonary neutrophils. No significant effects were observed at the lower particle concentration. Sporadic clumps of particles from each treatment group were observed in the small airways and interstitial areas of the lungs according to particle dose. Patches of cellular infiltration and edema in both the small airways and in the interstitium were also observed in the high dose group. Isolated perfused hearts from mice exposed to 40 microg of AF-SWCNTs had significantly lower cardiac functional recovery, greater infarct size, and higher coronary flow rate than other particle-exposed animals and controls, and also exhibited signs of focal cardiac myofiber degeneration. No particles were detected in heart tissue under light microscopy. This study indicates that while acid functionalization increases the pulmonary toxicity of both UFCB and SWCNTs, this treatment caused cardiac effects only with the AF-carbon nanotubes. Further experiments are needed to understand the physico-chemical processes involved in this phenomenon.

Mouse lung tissue was digested with dispase and collagenase enzymes to dissociate cells and primary lung epithelial (PLE) cells were isolated by removing leukocytes, erythroid cells, endothelial cells and fibroblasts, using a magnetic-bead based negative selection process. Purified PLE cells were cultured on fibronectin coated plastic dishes. Proliferative activity in PLE cell in culture was monitored by using time lapse live imaging technique. Proliferative activity of PLE cells started after 3 days in culture and peaked on day 4 to 5 when about 11% of the cells in culture were seen to divide. Proliferation activity ceased after day 6 to 7. There was a marked change in the morphology of the cells as the cultured cells became flattened and significantly larger in size. Type I and type II PLE cells were enumerated in culture by flow cytometry based upon staining with anti-podoplanin antibody (type I cells) or CD74 antibody (type II cells). In freshly isolated PLE cells, type I and type II epithelial cells accounted for about 20% each, bulk of the remaining cells being double negative. The proportion of type I cells rapidly increased in culture and was above 80% by day 4 of the culture. Proportion of cells expressing B7.1 and ICAM-1 molecules involved in antigen presentation increased but the MHC class II+ cells declined. These results show that PLE cells isolated by negative selection show a transient proliferative activity in culture become predominantly type I epithelial cells and retain the expression of some important antigen presentation markers.

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 more than 40 days) as compared to young erythrocytes (age less than 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.

Diesel exhaust particles (DEP) constitute a significant component of airborne particulates in urban environment. Exposure to DEP is known to enhance susceptibility to viral and bacterial infections. We hypothesized that DEP could partially exert its effect on disease susceptibility by interfering with antigen presentation required for the generation of protective adaptive immune responses. MHC class I and class II molecules, ICAM 1 (CD54) and B7.1 (CD80) are intimately involved with the presentation of viral and bacterial antigens to T cells. In the present study we examined the effect of in vitro exposure to DEP on basal and cytokine induced expression levels of these key molecules on mouse lung epithelial cells. LA4 and TC1 mouse epithelial cell lines were activated with a cytokine mixture comprising IFNγ, TNFα and IL1β, in the presence or absence of DEP. CM significantly boosted the expression of MHC I and ICAM 1 antigens on epithelial cells. Exposure to DEP significantly inhibited the basal and CM induced expression of MHC I and ICAM 1 expression, but did not interfere with the constitutive expression of B7.1 on epithelial cells. Significant expression of MHC II antigens, which is needed for presentation of exogenous antigenic peptides, was not detected on either LA4 or TC1 cells. These results suggest that DEP may interfere with the expression of the cellular markers involved in the presentation of viral antigens.

Background and Objective We looked at the two most commonly used methods for prenatal diagnosis, cordocentesis and chorionic villus sampling for prenatal diagnosis of haemophilia A in an Indian setting. Material and Methods The study sample included 16 families which reported to us for prenatal diagnosis of haemophilia A at All India Institute of Medical Science, New Delhi, India. Prenatal tests were done on chorionic villus samples or on cord blood (cordocentesis). Molecular work-up included the use of indirect mutation analysis in the form of linkage markers like CA-13, CA-22, Xba1 and Bcl1 as well as direct mutation analysis in the form of inversion 1 and 22 detection. Non-molecular work-up included primarily factor VIII assays. Result Chorionic villus sampling was performed in eight mothers. Of the other eight mothers, six underwent cord blood factor VIII assays because these had absence of family history and were negative for linkage and inversion 1 and 22. One patient had a female child and another had a fetus that showed congenital abnormalities. Conclusion We found that the choice of either technique, chorionic villus or cordocentesis, is not really an alternative, but rather dependent on the gestational age of presentation.

Many potential applications in nanotechnology envisage the use of better-dispersed and functionalized preparations of carbon nanotubes. Single-walled carbon nanotubes (SWCNTs) were treated with 1:1 mixtures of concentrated nitric and sulfuric acids for 3 min in a microwave oven under 20 psi pressure followed by extensive dialysis to remove the acids. This treatment resulted in acid functionalized SWCNTs (AF-SWCNTs) that had high negative charge (Zeta potential −40 to −60 mV) and were well dispersed (98% of the particles

Multiply transfused patients of severe aplastic anemia are at increased risk of graft rejection. Five such patients underwent peripheral blood stem cell transplantation from HLA-identical siblings with a fludarabine-based protocol. The conditioning consisted of fludarabine 30 mg/m(2)/day x 6 days, cyclophosphamide 60 mg/kg/day x 2 days and horse antithymocyte globulin (ATG) x 4 days. Two different ATG preparations were used: ATGAM (dose 30 mg/kg/day x 4 days) or Thymogam (dose 40 mg/kg/day x 4 days). Engraftment: median time to absolute neutrophil count (ANC)0.5 x 10(9)/l was 11 days (range: 8-17) and median time to platelet count20 x 10(9)/l was 11 days (range: 9-17). At a median follow-up of 171 days (range: 47-389), there has been no graft rejection and all patients are in complete remission. Acute GVHD (grade 1) occurred in one patient only. Chronic GVHD developed in two patients (extensive in one and limited in another). The transplants were performed in non-HEPA filter rooms. In only one patient, systemic antifungal therapy (voriconazole) was used. The use of Thymogam brand of ATG for conditioning is being reported for the first time. Our experience suggests that this fludarabine-based protocol allows rapid sustained engraftment in high-risk patients without significant immediate toxicity.

Murine bone marrow (BM) cell preparations lack mature cytotoxic natural killer (NK) cells, but NK cells may be induced in these cell preparations by culturing with interleukin-2 (IL2). Present study was aimed at studying the role of interactions between Ly49 molecules and major histocompatibility complex (MHC) class I molecules during IL2-induced development of mature NK cells in BM cell cultures. Addition of monoclonal antibodies (mabs) specific to class I MHC molecules of H-2b haplotype, to block any interaction of MHC I molecules with their receptors, was found to inhibit NK cell development. Mouse NK cells express several types of Ly49 molecules including Ly49C, which is an inhibitory receptor specific to MHC I molecules of H-2b haplotype. Blocking Ly49–MHC I interaction by using anti-Ly49C mab inhibited the development of cytotoxic NK cells. Addition of anti-Ly49A (no specificity for H-2b MHC I molecules) or anti-Ly49D (activating receptor specific for MHC I molecules of many H-2 haplotypes including H-2b) mabs, however, had no effect on IL2-induced NK cell development in BM cells. Mabs specific to Ly49C molecule and MHC I molecules of H-2b haplotype inhibited the development of mature NK cells from highly purified NK precursor cell population. These results indicate that specific interaction between inhibitory self-reactive Ly49 molecules and MHC I molecules may be crucial for NK cell development. We propose a model in which Ly49–MHC I interaction may have a permissive role in allowing development of only such NK cell clones that expresses at least one self-reactive inhibitory Ly49 molecule so that lysis of autologous healthy cells by mature NK cells may be avoided.

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 x 104 bacteria/mouse) by intrapulmonary instillation, with or without coadministration of DEP (100 microg/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 IFNgamma 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 IFNgamma-secreting lymphocytes and IFNgamma-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 IFNgamma-secreting lymphocytes in lungs. Taken together, these results indicated that coexposure to DEP + BCG did not significantly affect the level of IFNgamma response of mice to BCG infection. However, DEP treatment was found to inhibit IFNgamma-induced nitric oxide (NO) production by mouse alveolar macrophages in vitro. Our results indicate that DEP exposure did not alter the IFNgamma response to BCG infection, but reduced responsiveness of alveolar macrophages to IFNgamma. Reduced sensitivity of DEP-exposed alveolar macrophages to IFNgamma may contribute to a greater load of BCG in the lungs of BCG-infected mice given DEP.

The trace impurities in high-purity silver granules were determined by fast scanning single collector sector field high-resolution inductively coupled plasma mass spectrometry (HR-ICPMS) after selective separation of the major matrix and silver as silver metal by hydroxylamine hydrochloride. In the filtrate, 32 major and minor metallic impurities including silver were determined using NIST SRM 1643e, Trace Elements in Water, VHG SM75 and SM90 (VHC Labs, Inc., Manchester, NH), and internal standards 7Li, 9Be, 45Sc, 72Ge, 115ln, 209Bi, and 238U. To minimize the matrix effect, reagent blank was measured and subtracted from the samples. The spectral interferences have been resolved by measuring the isotopes 55Mn, 56Fe, 75As, and 78Se in high-resolution mode (m/Δm = 10,000); 52Cr, 63Cu, and 64Zn in medium-resolution mode (m/Δm = 4,000); and others in low-resolution mode (m/Δm = 300). In the present study, the purity of the tested silver granules was found to be 99.9898 ± 0.00007 % with 95 % confidence level. The purity results obtained from impurity assessment by HR-ICPMS were compared with those of the results of the conventional gravimetric method and found to be comparable. Though the gravimetric process is a primary technique for purity determination and the results obtained are directly traceable to SI units, HR-ICPMS provides more information about the impurities present in the silver granules. The proposed method is convenient, accurate, and precise and can be used for purity as well as for determination of trace impurities in silver metal.

Discussions and debates on how languages around the world are endangered and are becoming extinct have been expanding, and this volume, edited by Alexandre Duchene and Monika Heller, brings these i...

Modulation of NK Cell Activation and Interaction with Target Cells

Administration of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs, 10 µg intravenously on alternate days) induced a sustained anemia in mice. Using a modified double in vivo biotinylation (DIB) technique, blood counts of reticulocytes and erythrocytes of different age groups were simultaneously enumerated in control and AF-SWCNT treated mice. A sustained reticulocytosis was observed in AF-SWCNT treated mice. Young erythrocytes (up to 7 days old) that are normally resistant to elimination in control mice were eliminated at a significant rate in AF-SWCNT treated mice. Old erythrocytes, however, accumulated in circulation indicating that younger erythrocytes were selectively eliminated from the blood circulation of AF-SWCNT treated mice. Cells representing various stages of erythroid differentiation in bone marrow and spleen were enumerated flow cytometrically by double staining with anti-Ter119 and anti-transferrin receptor (CD71) monoclonal antibodies. Proportion of erythroid cells was significantly reduced (up to 27%) in bone marrow (BM) indicating a fall in erythropoietic activity. A concomitant increase in the spleen erythroid population was however observed that could be a compensatory response. Changes in erythroid populations in bone marrow and spleen correlated with changes in erythroblast-A population in these organs that represent an early stage of erythroid differentiation. Uptake of intravenously administered fluorescence tagged AF-SWCNTs (FAF-SWCNTs) was relatively low (3–4%) in erythroid cells in bone marrow and spleen. A significantly higher proportion of pro-erythroblasts and erythroblast-A (early stages of erythroid differentiation) took-up FAF-SWCNTs. Uptake of AF-SWCNTs by early precursors of erythroid differentiation with toxic consequences may be a contributing factor in AF-SWCNT induced anemia.

SUMMARYGamma interferon (IFNγ) plays a key role in host defense against pulmonary mycobacterial infections. A variety of lymphocyte subsets may participate in producing pulmonary IFNγ responses, but their relative contributions after mycobacterial infection have not been clearly elucidated. To address this question, C57Bl/6 female mice were infected by intrapulmonary instillation of 2·5 × 104 BCG (Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium were examined at different time points after the infection. BCG load in lungs peaked between 4 and 6 weeks post-infection and declined to very low levels by the 12th week of infection. Recovery of lung interstitial lymphocytes doubled by 4–6 weeks after infection and declined thereafter. Flow cytometric analysis of the lung-derived lymphocytes revealed that about 5% of the these cells made IFNγ in control mice, and this baseline IFNγ production involved T (CD3+NK1.1−), NK (CD3−NK1.1+) and NKT (CD3+NK1.1+) cells. As the BCG lung infection peaked, the total number of CD3+ T cells in the lungs increased threefold at 5–6 weeks post-infection. There was a marked increase (sixfold) in the number of T cells secreting IFNγ 5–6 weeks post-infection. Some increase was also noted in the NKT cells making IFNγ, but the numbers of NK cells making IFNγ in BCG-infected lungs remained unaltered. Our results suggest that whereas NK and NKT cells contribute to baseline IFNγ secretion in control lungs, expansion in the IFNγ-producing T-cell population was essentially responsible for the augmented response seen in lungs of BCG-infected mice.

C57Bl/6 female mice were infected with an intrapulmonary dose of 2.5 x 10(4) 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, IFNgamma producing non-T cells outnumbered T cells producing IFNgamma. However, as the adaptive response to infection evolves, marked increase occur in the number of IFNgamma producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNgamma response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNgamma, 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.

Effect of poly-dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs) was examined on lipid antigen presentation through CD1d pathway on three cell lines, LA4, MHS, and JAWSII used as prototype antigen presenting cells (APCs). CD1d molecule was expressed on 80-90% MHS (prototype macrophages) and JAWSII (prototype dendritic cells) cells whereas

A sensitive sandwich enzyme-linked immunosorbent assay (ELISA) that can detect up to 0.5 ng of culture filtrate proteins (CFPs) of Mycobacterium tuberculosis strain H37Ra is described. This detection system features several special characteristics: (i) the use of CFPs from the H37Ra strain of Mycobacterium tuberculosis to generate capture and detection antibodies in rabbits and mice, respectively; (ii) affinity purification of CFP-reactive antibodies and selection of the antibody preparations for best performance in the sandwich ELISA system; and (iii) the use of urine-derived protein preparations for antigen detection. The sandwich ELISA could detect up to 0.5 ng of CFPs of the H37Ra strain of Mycobacterium tuberculosis. The assay did not detect antigens of Escherichia coli, Candida albicans or Saccharomyces cerivisiae but efficiently detected CFP preparations from nine different clinical isolates of Mycobacterium tuberculosis. Significant variations, however, were noted in the relative efficacy of the assay to detect CFPs from different clinical isolates of Mycobacterium tuberculosis. The procedure was utilized for detecting tubercular antigens in urine samples from 29 patients with confirmed (sputum-positive) tuberculosis and from 25 healthy controls. Significant levels of antigen could be detected in 22 of the 29 samples tested.

NK activation in C57B1/6 mouse spleen cells was carried out with IL-2 in the presence or absence of paraformaldehyde fixed YAC tumor cells. Generation of anti-YAC cytolytic activity was markedly higher when activation was carried out in the presence of fixed tumor cells. In addition the cytotoxic effector cells generated were resistant to anti-Thy-1 + C treatment, indicating that the effector cells were not T lymphocytes. IL-2 activation of NK cells was compared When fixed YAC or EL4 tumor cells were added during the IL-2 activation phase, it was found that the addition of either of these tumor cells significantly boosted the levels of cytotoxic activity generated against both targets. Significantly higher anti-YAC cytotoxic activity was however generated when fixed YAC cells instead of EL4 cells were present during the activation phase. Similarly, significantly greater cytolytic activity was generated against EL4 cells, when fixed EL4 rather than YAC cells were present during the activation phase. Addition of paraformaldehyde fixed syngeneic or allogenic spleen cells instead of tumor cells, did not boost NK activation in response to IL-2, indicating that the boosting of NK activation did not result from exposure to alloantigens during the activation phase. These results indicate that an exposure to tumor cells during IL-2 activation phase, may boost the activation of NK cells in a tumor specific manner.

A novel factor which augments the expression of major histocompatibility complex I (MHC augmenting factor or MHC-AF) antigens on tumor cell lines, has been isolated from the culture supernatants of human peripheral blood mononuclear cells activated by concanavalin?A. A mouse equivalent of this factor has also been isolated from the culture supernatants of mouse spleen cells activated by mitogens or in a mixed lymphocyte reaction. Mouse MHC?AF enhances the expression of class I MHC antigens on murine tumor cell lines ( EL?4 and BW5147 ) but not on human tumor cell lines (K562 and HR?7). Human MHC?AF on the other hand enhances the MHC I expression on both human as well as murine cell lines. Interferon gamma (IFN-γ), a cytokine also known to enhance the expression of MHC I antigens, acts in a highly species specific manner with mouse IFN-γ augmenting the MHC I on murine tumor cell lines and human IFN-γ augmenting the MHC I on human tumor cell lines only. These results indicate important differences in the cross species biological activities of MHC?AF and IFN-γ , and provide additional evidence for MHC-AF being distinct from IFN-γ.