Liposome-mediated cytosolic delivery of macromolecules and its possible use in vaccine development

In the majority of bacterial and viral infections the generation of cytotoxic T cells is of particular interest because such pathogens are able to escape the host defence mechanisms by surviving intracellularly within the phagocytic cells. To generate a CD8+ T lymphocyte response against exogenous antigens, the prerequisite is their delivery into the cytosol followed by processing and presentation along with class I major histocompatibility complex (MHC-I) molecules. In the present study we describe the method of liposome-based delivery of antigens and other macromolecules into the cytosol of target cells. To develop safe and effective methods for generating CD8+ T lymphocytes, we exploited the fusogenic character of lipids derived from lower organisms, that is baker's yeast (Saccharomyces cerevisiae). The degree of fusion with model membrane systems using yeast lipid liposomes varied from 40-70%, as opposed to 1-8% observed with egg PtdCho liposomes, depending on the assay system used. The fusion of yeast lipid liposomes with macrophages resulted in effective delivery of the entrapped solutes into the cytoplasmic compartment. This was further supported by the inhibition of cellular protein synthesis in J774 A1 cells by ricin A, encapsulated in the yeast lipid liposomes. Interestingly, the model antigen ovalbumin, when entrapped in the yeast lipid liposomes, successfully elicited antigen reactive CD8+ T cell responses. It may be concluded that the liposomes made of lipids derived from S. cerevisiae can spontaneously fuse with macrophages, delivering a significant portion of their contents into the cytoplasmic compartment of the cells

Chloroquine encapsulated in malaria-infected erythrocyte-specific antibody-bearing liposomes effectively controls chloroquine-resistant Plasmodium berghei infections in mice

The suitability of liposomes as drug carriers in the treatment of drug-resistant rodent malaria was examined after covalently attaching F(ab')2 fragments of a mouse monoclonal antibody (MAb), MAb F10, raised against the host cell membranes isolated from the Plasmodium berghei-infected mouse erythrocytes, to the liposome surface. The antibody-bearing liposomes thus formed specifically recognized the P. berghei-infected mouse erythrocytes under both in vitro and in vivo conditions. No such specific binding of the liposomes with the infected cells was observed when MAb F10 was replaced by another mouse monoclonal antibody, MAb D2. Upon loading with the antimalarial drug chloroquine, the MAb F10-bearing liposomes effectively controlled not only the chloroquine-susceptible but also the chloroquine-resistant P. berghei infections in mice. The chloroquine delivered in these liposomes intravenously at a dosage of 5 mg/kg of body weight per day on days 4 and 6 postinfection completely cured the animals (75 to 90%) of chloroquine-resistant P. berghei infections. These results indicate that selective homing of chloroquine to malaria-infected erythrocytes may help to cure the chloroquine-resistant malarial infections with low doses of chloroquine

Use of liposomes as an immunopotentiating delivery system: in perspective of vaccine development

Liposomes have been widely used to deliver antigens to the antigen-presenting cells (APCs) and also to modify their immunological behaviour in model animals. We recently demonstrated the potential of yeast lipid liposomes to undergo membrane-membrane fusion with cytoplasmic membrane of the target cells. Interestingly, studies in the present report revealed that antigen encapsulated in yeast lipid liposomes could be successfully delivered simultaneously into the cytosolic as well as endosomal processing pathways of APCs, leading to the generation of both CD4+ T helper and CD8+ cytotoxic T cells. In contrast, encapsulation of same antigen in egg phosphatidyl-choline (PC) liposomes, just like its free form, has inefficient access to the cytosolic pathway of major histocompatibility complex (MHC) I dependent antigen presentation and failed to generate antigen specific CD8+ cytotoxic T-cell response. However, both egg PC as well as yeast lipid liposomes have elicited strong antigen specific antibody responses in immunized animals. These results imply usage of liposome encapsulated antigen as potential candidate vaccine capable of eliciting both cell mediated as well as humoral immune responses

An audit of trauma related mortality in Government Medical College, Jammu

Background: Surgical audit is defined as a systematic, critical analysis of the quality of surgical care that is reviewed by peers against explicit criteria or recognized standards, and then used to further inform and improve surgical practice with the ultimate goal of improving the quality of care for patients. Aims and objectives were to study the profile of patients who died due to trauma and to identify factors involved in both pre-hospital and hospital care.Methods: This study was conducted in the department of surgery, GMC Jammu during over a period of one year. The profile of all traumatic deaths was studied to evaluate the various causes of trauma related mortality, age and sex relationship, mortality rate, prognostic indicators and also to identify factors involved in both pre-hospital and hospital care.Results: A total of 414 deaths occurred due to trauma. Out of these 317 patients were males and 97 patients were females comprising male to female ratio of 3.3:1. Road traffic accidents accounted for majority of the deaths (57%) followed by falls (35.5%), assaults (5.07%) and miscellaneous causes (2.43%). In children less than 15 years of age, traumatic deaths were more due to falls, whereas in adults it was more due to road traffic accidents. In our study there was steady rise of fatalities during weekend days with a peak on Saturday. Cranio-cerebral injuries were responsible for majority of the traumatic deaths (85.9%) followed by limb injuries including fractures (38.4%), thoracic injuries (27.7%), abdominal injuries (24.6%), pelvic injuries (20.7%) and spine injuries (14.2%).Conclusions: This study can be the impetus to motivate committed professionals in the trauma specialty to help in the organization of available facilities and to upgrade existing facilities for a better response to injury, which should not be different from other public health responses

Comparative study of transabdominal preperitoneal versus open Lichtenstein hernia repair in primary inguinal hernia

Background: Inguinal hernia repair is one of the most commonly performed surgery in surgical practice and has evolved through various techniques. However, which technique is gold standard is still a topic of debate and the clinical studies are not adequate to show clear benefits of one technique over another. Objective was to compare the outcome of transabdominal preperitoneal repair (TAPP) versus open Lichtenstein tension free mesh repair in primary inguinal hernia.Methods: This retrospective cohort study was conducted in a tertiary care hospital with sample size of 80 patients (40 cases in each group) and these patients were compared in terms of operative time, complications, duration of hospital stay, postoperative recovery, postoperative pain and timing of return to normal activity and work.Results: On comparing the results of our study we found that in unilateral cases the operating time was greater in the TAPP group than the Lichtenstein group; however, in the bilateral cases, the operating time was significantly greater in the Lichtenstein repair group than the laparoscopic TAPP group. The incidence of post operative complications was lower in TAPP group (8.2%) then in open hernia repair group (21.6%). The time to return to normal activity was also lower for laparoscopic group in both unilateral and bilateral cases.Conclusions: It can be concluded that laparoscopic TAPP repair offers significant advantage over open tension free mesh hernioplasty in terms of lesser post operative pain, lesser complications and early return to normal activity, better cosmetic outcomes, and less persisting pain but it is associated with a higher operative time depending on surgeon’s expertise, more costly for the patient and there is no significant difference in early post operative complications

Process evaluation of integrated early child development care at private clinics in poor urban Pakistan: a mixed methods study

Background: In poor urban Pakistan, private GP clinics lack adequate services to promote early child development (ECD) care. A clinic-based contextualised ECD intervention was developed for quarterly tool-assisted counselling of mothers. Aim: To explore the experience and implementation of ECD intervention by the private care providers and clients, for further adaptation for scaling of quality ECD care, at primary level private healthcare facilities in Pakistan. Design & setting: A mixed methods approach using quantitative records review and qualitative interviews at poor urban clinics in Rawalpindi and Lahore, Pakistan. Method: Quantitative data from study-specific records were reviewed for 1242 mother–child pairs registered in the intervention. A total of 18 semi-structured interviews with clinic staff, mothers, and research staff were conducted at four clinics. The interviews were audiorecorded and transcribed verbatim. Results: District Health Office (DHO) support allowed transparent and effective selection and training of clinic providers. Public endorsement of ECD care at private clinics and the addition of community advocates promoted ECD care uptake. Clinic settings were found feasible for clinic assistants, and acceptable to mothers, for counselling sessions. Mothers found ECD counselling methods more engaging compared to the usual care provided. Conclusion: In poor urban settings where public health care is scarce, minimal programme investment on staff training and provision of minor equipment can engage private clinics effectively in delivering ECD care

Delivering integrated child development care in Pakistan: protocol for a clustered randomised trial

Background: Early childhood developmental delay is associated with significant disadvantage in adult life. In Pakistan, high prevalence of developmental delay is associated with poverty, under-nutrition, and maternal depression. Aim: To assess the effectiveness of an early child development counselling intervention delivered at private GP clinics, in poor urban communities. Design & setting: A clustered randomised trial in Pakistan. Method: The intervention was developed following a period of formative research, and in consultation with local experts. A total of 2112 mother–child pairs will be recruited at 32 clinics, from within the locality (cluster); 16 clinics per arm. A primary care counselling intervention (promoting child development, nutrition, and maternal mental health) will be delivered at 6 weeks, 3, 6, and 9 months of the child’s age. Monitoring, assessment, and treatment will also be performed at quarterly visits in intervention clinics. Primary outcome is the developmental delay at 12 months (ASQ-3 scores). Secondary outcomes are stunting rate, and maternal depression (PHQ-9 score). In addition, a process evaluation and costing study will be conducted. Discussion: This trial will be the first to assess an early child development intervention, delivered in private GP clinics for poor urban communities in Pakistan. If found to be effective, this public–private model may offer a more sustainable, and feasible option for populations in poor urban settings, where private GP clinics are the most accessible provider of primary health care. There is scope for scale-up at provincial level, should the intervention be effective. Trial registration: The trial has been registered with the Current Controlled Trials ISRCTN48032200

Comparison of clinical safety of minimal access surgery/laparoscopy versus open surgery in terms of patient outcomes and risk to theatre staff during the COVID-19 pandemic

Background: The COVID-19 pandemic has greatly affected surgical practice in all parts of the world because the safety of minimal access surgery (MAS) was questioned during the COVID-19 pandemic due to increased concern with regard to disease spread. This study assessed the available evidence on the safety of laparoscopy as compared to open surgery during the COVID-19, explored the possible precautions to be taken to prevent exposure of the operating team to the viral infection. The objective of this study was to access the clinical safety of laparoscopy as compared to open surgery during the COVID-19 pandemic.Methods: This study was a retrospective study conducted during the COVID-19 pandemic in the Department of Surgery, GMC, India, from January 2020 to January 2021. The various outcomes assessed included: burden of covid-19 infection among the patients, deaths due to COVID-19, infection acquired by staff, length of hospital stay and post-discharge symptomatology among patients.Results: There was no statistically significant difference in terms of median age of patients (p=0.853), gender (p=0.835), American Society of Anesthesiologists (ASA) status (p=0.876), urgency of operation (p=0.074), total time in theatre complex (p=0.163) or total number of theatre staff involved (p=0.831). The length of stay in the hospital was significantly shorter in the laparoscopic as compared the open group (3.5 versus 9 days; p=0.011).Conclusions: Based on our review, we concluded that if recommended guidelines are followed and proper precautions are taken, laparoscopic surgery is safe for patients and theatre staff during the COVID-19 pandemic. Only on the basis of COVID-19, laparoscopy should not be replaced by laparotomy. If laparoscopy is strongly indicated in patients, it can be used with precautions because of its benefits over open surgery

Heme oxygenase-1 protects against Alzheimer’s amyloid-β1-42 induced toxicity via carbon monoxide production

Heme oxygenase-1 (HO-1), an inducible enzyme up-regulated in Alzheimer‟s disease (AD), catabolises heme to biliverdin, Fe2+ and carbon monoxide (CO). CO can protect neurones from oxidative stress-induced apoptosis by inhibiting Kv2.1 channels, which mediate cellular K+ efflux as an early step in the apoptotic cascade. Since apoptosis contributes to the neuronal loss associated with amyloid β peptide (Aβ) toxicity in AD, we investigated the protective effects of HO-1 and CO against Aβ1-42 toxicity in SH-SY5Y cells, employing cells stably transfected with empty vector or expressing the cellular prion protein, PrPc, and rat primary hippocampal neurons. Aβ1-42 (containing protofibrils) caused a concentrationdependent decrease in cell viability, attributable at least in part to induction of apoptosis, with the PrPc expressing cells showing greater susceptibility to Aβ1-42 toxicity. Pharmacological induction or genetic over-expression of HO-1 significantly ameliorated the effects of Aβ1-42. The CO-donor CORM-2 protected cells against Aβ1-42 toxicity in a concentration-dependent manner. Electrophysiological studies revealed no differences in the outward current pre- and post-Aβ1-42 treatment suggesting that K+ channel activity is unaffected in these cells. Instead, Aβ toxicity was reduced by the L-type Ca2+ channel blocker nifedipine, and by the CaMKKII inhibitor, STO-609. Aβ also activated the downstream kinase, AMP-dependent protein kinase (AMPK). CO prevented this activation of AMPK. Our findings indicate that HO-1 protects against Aβ toxicity via production of CO. Protection does not arise from inhibition of apoptosis-associated K+ efflux, but rather by inhibition of AMPK activation, which has been recently implicated in the toxic effects of Aβ. These data provide a novel, beneficial effect of CO which adds to its growing potential as a therapeutic agent