A novel strategy to elicit enduring anti-morphine immunity and relief from addiction by targeting Acr1 protein nano vaccine through TLR-2 to dendritic cells.

Morphine addiction poses a significant challenge to global healthcare. Current opioid substitution therapies, such as buprenorphine, naloxone and methadone are effective but often lead to dependence. Thus, exploring alternative treatments for opioid addiction is crucial. We have developed a novel vaccine that presents morphine and Pam3Cys (a TLR-2 agonist) on the surface of Acr1 nanoparticles. This vaccine has self-adjuvant properties and targets TLR-2 receptors on antigen-presenting cells, particularly dendritic cells. Our vaccination strategy promotes the proliferation and differentiation of morphine-specific B-cells and Acr1-reactive CD4 T-cells. Additionally, the vaccine elicited the production of high-affinity anti-morphine antibodies, effectively eliminating morphine from the bloodstream and brain in mice. It also reduced the expression of addiction-associated µ-opioid receptor and dopamine genes. The significant increase in memory CD4 T-cells and B-cells indicates the vaccine's ability to induce long-lasting immunity against morphine. This vaccine holds promise as a prophylactic measure against morphine addiction.

Immunosuppressive effects of morphine on macrophage polarization and function.

Macrophages play a pivotal role in safeguarding against a broad spectrum of infections, from viral, bacterial, fungal to parasitic threats and contributing to the immune defense against cancer. While morphine's immunosuppressive effects on immune cells are extensively documented, a significant knowledge gap exists regarding its influence on macrophage polarization and differentiation. Hence, we conducted a study that unveils that prior exposure to morphine significantly impedes the differentiation of bone marrow cells into macrophages. Furthermore, the polarization of macrophages toward the M1 phenotype under M1-inducing conditions experiences substantial impairment, as evidenced by the diminished expression of CD80, CD86, CD40, iNOS, and MHCII. This correlates with reduced expression of M1 phenotypical markers such as iNOS, IL-1ß, and IL-6, accompanied by noticeable morphological, size, and phagocytic alterations. Further, we also observed that morphine affected M2 macrophages. These findings emphasize the necessity for a more comprehensive understanding of the impact of morphine on compromising macrophage function and its potential ramifications for therapeutic approaches.

Holocene surface-rupturing paleo-earthquakes along the Kachchh Mainland Fault: shaping the seismic landscape of Kachchh, Western India.

This study explores the seismotectonics of Kachchh in western India, a region with a low-to-moderate strain rate and a history of significant earthquakes, notably the 1819, Mw 7.8 Allah Bund, and the 2001, Mw 7.6 Bhuj. Despite its substantial seismic risk, comprehensive studies on Kachchh's seismogenic sources are scarce. This is attributed to the concealed nature of active structures, hindering definitive age constraints in paleoseismological research. Our research comprises a detailed paleoseismic analysis of the north-verging, reverse Jhura Fault underlying the Jhura anticline, a segment of the Kachchh Mainland Fault. This fault segment shows evidence of surface-rupturing earthquakes in the area south of the Great Rann of Kachchh. The investigation reveals three paleoseismic events: Event I before 9.72 ka B.P., Event II between 8.63-8.20 ka B.P., and Event III between 6.20-6.09 ka B.P. The elapsed time since the last event on this fault is > 8000 years, suggesting that the area is exposed to a significant earthquake hazard. This highlights the need for more precise characterization of individual seismogenic sources for future earthquake preparedness.

Revolutionizing medicine with toll-like receptors: A path to strengthening cellular immunity.

Toll-like receptors play a vital role in cell-mediated immunity, which is crucial for the immune system's defense against pathogens and maintenance of homeostasis. The interaction between toll-like-receptor response and cell-mediated immunity is complex and essential for effectively eliminating pathogens and maintaining immune surveillance. In addition to pathogen recognition, toll-like receptors serve as adjuvants in vaccines, as molecular sensors, and recognize specific patterns associated with pathogens and danger signals. Incorporating toll-like receptor ligands into vaccines can enhance the immune response to antigens, making them potent adjuvants. Furthermore, they bridge the innate and adaptive immune systems and improve antigen-presenting cells' capacity to process and present antigens to T cells. The intricate signaling pathways and cross-talk between toll-like-receptor and T cell receptor (TCR) signaling emphasize their pivotal role in orchestrating effective immune responses against pathogens, thus facilitating the development of innovative vaccine strategies. This article provides an overview of the current understanding of toll-like receptor response and explores their potential clinical applications. By unraveling the complex mechanisms of toll-like-receptor signaling, we can gain novel insights into immune responses and potentially develop innovative therapeutic approaches. Ongoing investigations into the toll-like-receptor response hold promise in the future in enhancing our ability to combat infections, design effective vaccines, and improve clinical outcomes.

Influence of chronic administration of morphine and its withdrawal on the behaviour of zebrafish.

Morphine is a potent analgesic opiate used to treat chronic pain, mostly in cancer patients. In addition, it is widely used as a drug of abuse. Due to the continuous rise of morphine-associated addiction, there is an urgent need to develop pre-clinical animal models to understand the behavioural pattern of drug dependence and its withdrawal. Recently, the experimental use of zebrafish has attained significance in behavioural neuroscience studies. The literature on zebrafish is conflicting with regard to morphine withdrawal symptoms. Unfortunately, no single model provides comprehensive details to evaluate zebrafish behaviour on opiate exposure. Further, the current models have various limitations, such as short duration, complexity of phenotypes, intricate quantification, and difficulty in studying withdrawal symptoms. Consequently, a firm standardization of the protocol to understand the influence of opiates on physiological and psychological behaviours is required. In this study, we have tried to overcome the shortcomings associated with the existing models and to optimize the protocols involving an array of parameters. We observed that the administration of morphine caused a significant increase in zebrafish behavioural patterns of spiral movements, circular movements, erratic movements, upper transitions, water surface transitions, wall licking, wall licking with upper transitions, wall licking with lower transitions, absolute angle changes, and time spent in the upper compartment. A decline in the freezing bouts and time spent in the lower compartment were noticed. In essence, this study offers a zebrafish model to comprehensively examine changes in behaviour of animals on opiate dependence and its withdrawal. The present study also reported that in zebrafish, the influence of chronic exposure of morphine modulates key gene targets involved in behaviour, neuroinflammation, and autophagy, which directly or indirectly are associated with morphine addiction in a chronic morphine model.

Diagnostic efficacy of chest CT imaging in diagnosis of COVID-19 cases based on duration of symptoms.

Background: Chest computed tomography (CT) imaging provides results more rapidly and with higher sensitivity than reverse transcription polymerase chain reaction in diagnosis of COVID-19. Aim: To evaluate diagnostic efficacy of chest CT imaging in diagnosis of COVID-19 cases based on age and duration of symptoms. Materials and Methods: A retrospective study conducted during December 2020 to June 2021 in a tertiary care hospital, India. Total 495 patients with typical clinical symptoms of COVID-19, reverse transcription polymerase chain reaction positive for COVID-19 and had undergone chest CT imaging were included. Descriptive statistical analysis was performed for all the variables. Receiver operating characteristic curve analysis was used to determine threshold value of chest CT severity score (CT_SS) based on duration of symptoms and age to diagnose COVID-19. Results: Mean age of patients was 61.86 ± 10.77 years and 367 (71.4%) patients were male. Ground glass opacities were observed in 456 (92.1%) patients and in 332 (67.1%) patients, multilobes were affected. Total CT_SS showed positive correlation with age (r = 0.257) and duration of symptoms (r = 0.625). Total CT_SS >6 after a duration of 2 days of symptoms identified COVID-19 cases with sensitivity 90.8% (95% confidence interval [CI]: 87.5%-93.5%) and specificity 84.6% (95% CI: 76.2%-90.9%). Total CT_SS >11 in patients aged more than 60 years identified COVID-19 cases with sensitivity 47.4% (95% CI: 41.2%-53.6%) and specificity 87.3% (95% CI: 82.3%-91.4%). Conclusion: Threshold value of CT_SS determined will help to expedite diagnosis of COVID-19 patients by the clinicians in an early stage especially in India and other developing countries which have a high patient volume and limited health resources.

Age-mediated gut microbiota dysbiosis promotes the loss of dendritic cells tolerance.

The old age-related loss of immune tolerance inflicts a person with a wide range of autoimmune and inflammatory diseases. Dendritic cells (DCs) are the sentinels of the immune system that maintain immune tolerance through cytokines and regulatory T-cells generation. Aging disturbs the microbial composition of the gut, causing immune system dysregulation. However, the vis-à-vis role of gut dysbiosis on DCs tolerance remains highly elusive. Consequently, we studied the influence of aging on gut dysbiosis and its impact on the loss of DC tolerance. We show that DCs generated from either the aged (DCOld ) or gut-dysbiotic young (DCDysbiotic ) but not young (DCYoung ) mice exhibited loss of tolerance, as evidenced by their failure to optimally induce the generation of Tregs and control the overactivation of CD4+ T cells. The mechanism deciphered for the loss of DCOld and DCDysbiotic tolerance was chiefly through the overactivation of NF-κB, impaired frequency of Tregs, upregulation in the level of pro-inflammatory molecules (IL-6, IL-1ß, TNF-α, IL-12, IFN-γ), and decline in the anti-inflammatory moieties (IL-10, TGF-ß, IL-4, IDO, arginase, NO, IRF-4, IRF-8, PDL1, BTLA4, ALDH2). Importantly, a significant decline in the frequency of the Lactobacillus genus was noticed in the gut. Replenishing the gut of old mice with the Lactobacillus plantarum reinvigorated the tolerogenic function of DCs through the rewiring of inflammatory and metabolic pathways. Thus, for the first time, we demonstrate the impact of age-related gut dysbiosis on the loss of DC tolerance. This finding may open avenues for therapeutic intervention for treating age-associated disorders with the Lactobacillus plantarum.

Future perspectives of emerging novel drug targets and immunotherapies to control drug addiction.

Substance Use Disorder (SUD) is one of the major mental illnesses that is terrifically intensifying worldwide. It is becoming overwhelming due to limited options for treatment. The complexity of addiction disorders is the main impediment to understanding the pathophysiology of the illness. Hence, unveiling the complexity of the brain through basic research, identification of novel signaling pathways, the discovery of new drug targets, and advancement in cutting-edge technologies will help control this disorder. Additionally, there is a great hope of controlling the SUDs through immunotherapeutic measures like therapeutic antibodies and vaccines. Vaccines have played a cardinal role in eliminating many diseases like polio, measles, and smallpox. Further, vaccines have controlled many diseases like cholera, dengue, diphtheria, Haemophilus influenza type b (Hib), human papillomavirus, influenza, Japanese encephalitis, etc. Recently, COVID-19 was controlled in many countries by vaccination. Currently, continuous effort is done to develop vaccines against nicotine, cocaine, morphine, methamphetamine, and heroin. Antibody therapy against SUDs is another important area where serious attention is required. Antibodies have contributed substantially against many serious diseases like diphtheria, rabies, Crohn's disease, asthma, rheumatoid arthritis, and bladder cancer. Antibody therapy is gaining immense momentum due to its success rate in cancer treatment. Furthermore, enormous advancement has been made in antibody therapy due to the generation of high-efficiency humanized antibodies with a long half-life. The advantage of antibody therapy is its instant outcome. This article's main highlight is discussing the drug targets of SUDs and their associated mechanisms. Importantly, we have also discussed the scope of prophylactic measures to eliminate drug dependence.

Molecular analysis of Panton-Valentine Leucocidin (pvl) gene among MRSA and MSSA isolates.

The present study was conducted in order to determine the frequency of pvl gene among the pathogenic and healthy population isolates of Methicillin Resistant Staphylococcus aureus (MRSA) and Methicillin Sensitive Staphylococcus aureus (MSSA). For this purpose, nasal swab samples were collected from the healthy individuals (to be used as controls, all the samples were collected irrespective of the sex and age factors), the pathogenic samples were collected from different patients suffering from skin &soft tissue infections caused by S. aureus (to be used as test samples).Both of these population samples were analyzed for the presence of pvl gene. S.aureus were identified through conventional microbiological identification procedures. In the case of normal samples, 70 nasal swabs were collected and only 33 (47%) proved to be S. aureus while 20 pathogenic samples were collected and all (100%) were cleared as S. aureus. For further distribution of samples into MRSA and MSSA, antibiotic susceptibility pattern was checked by using the standard protocols of Kirby-Bauer disc diffusion method. Two antibiotic discs Oxacillin (OX: 1ug) and cefoxitin (FOX: 30ug) were used. Among healthy population, MRSA was found to be 79% (n=26) and MSSA were present as 21% (n= 7). Among pathogenic strains 100% MRSA was detected where n= 20. Detection of pvl gene among the MRSA and MSSA isolates was done by using the uniplex PCR followed by gel electrophoresis. MRSA and MSSA of normal healthy population carried 49% and 7% pvl gene respectively. While, pathogenic MRSA samples carried 46% pvl gene among them. Potentially alarming percentage of pvl gene is present among the normal healthy individuals which indicates a future threat and a major health concern.

Mycobacterium tuberculosis exploits MPT64 to generate myeloid-derived suppressor cells to evade the immune system.

Mycobacterium tuberculosis (Mtb) is a smart and successful pathogen since it can persist in the intimidating environment of the host by taming and tuning the immune system. Mtb releases MPT64 (Rv1980c) protein in high amounts in patients with active tuberculosis (TB). Consequently, we were curious to decipher the role of MPT64 on the differentiating dendritic cells (DCs) and its relation to evading the immune system. We observed that pre-exposure of differentiating DCs to MPT64 (DCMPT64) transformed them into a phenotype of myeloid-derived suppressor cells (MDSCs). DCMPT64 expressed a high level of immunosuppressive molecules PD-L1, TIM-3, nitric oxide (NO), arginase 1, IDO-1, IL-10 and TGF-ß, but inhibited the production of pro-inflammatory cytokines TNF-α, IL-6 and IL-12. DCMPT64 chemotaxis function was diminished due to the reduced expression of CCR7. DCMPT64 promoted the generation of regulatory T cells (Tregs) but inhibited the differentiation of Th1 cells and Th17 cells. Further, high lipid and methylglyoxal content, and reduced glucose consumption by DCMPT64, rendered them metabolically quiescent and consequently, reduced DCMPT64 ability to phagocytose Mtb and provided a safer shelter for the intracellular survival of the mycobacterium. The mechanism identified in impairing the function of DCMPT64 was through the increased production and accumulation of methylglyoxal. Hence, for the first time, we demonstrate the novel role of MPT64 in promoting the generation of MDSCs to favor Mtb survival and escape its destruction by the immune system.

Corrigendum: Curdlan Limits Mycobacterium tuberculosis Survival Through STAT-1 Regulated Nitric Oxide Production.

[This corrects the article DOI: 10.3389/fmicb.2019.01173.].

Outcomes of normothermic machine perfusion of liver grafts in repeat liver transplantation (NAPLES initiative).

BACKGROUND: Retransplantation candidates are disadvantaged owing to lack of good-quality liver grafts. Strategies that can facilitate transplantation of suboptimal grafts into retransplant candidates require investigation. The aim was to determine whether late liver retransplantation can be performed safely with suboptimal grafts, following normothermic machine perfusion. METHODS: A prospectively enrolled group of patients who required liver retransplantation received a suboptimal graft preserved via normothermic machine perfusion. This group was compared with both historical and contemporaneous cohorts of patient who received grafts preserved by cold storage. The primary outcome was 6-month graft and patient survival. RESULTS: The normothermic machine perfusion group comprised 26 patients. The historical (cold storage 1) and contemporaneous (cold storage 2) groups comprised 31 and 25 patients respectively. The 6-month graft survival rate did not differ between groups (cold storage 1, 27 of 31, cold storage 2, 22 of 25; normothermic machine perfusion, 22 of 26; P = 0.934). This was despite the normothermic machine perfusion group having significantly more steatotic grafts (8 of 31, 7 of 25, and 14 of 26 respectively; P = 0.006) and grafts previously declined by at least one other transplant centre (5 of 31, 9 of 25, and 21 of 26; P < 0.001). CONCLUSION: In liver retransplantation, normothermic machine perfusion can safely expand graft options without compromising short-term outcomes.

Liver transplantation is a life-saving procedure for many different diseases. In the UK, one in 10 patients awaiting transplant have had a previous liver transplant. These retransplant operations are complex, and the general belief is that a good-quality donor liver graft is required for best outcomes. However, there is a significant shortage of good-quality organs for liver transplantation, so many patients awaiting retransplantation spend longer on the waiting list. This study investigated whether a new technology, called normothermic machine perfusion, could be used to preserve lower-quality donor livers and have successful outcomes for patients undergoing retransplantation. Traditionally, good-quality livers are preserved in an ice box and the study compared the outcomes of these two different approaches. The aim was to prove that normothermic machine perfusion improves access to transplantation for this group of patients, without compromising outcomes. A group of patients who underwent retransplantation and received a lesser-quality liver preserved with normothermic machine perfusion was compared with two groups of patients who had received a transplant with traditional ice-box preservation. The complications, graft, and patient survival of the former group was compared with those in the latter two groups who underwent liver retransplantation with better-quality liver grafts. The rate of survival and adverse surgical outcomes were comparable between the groups of patients who received a liver preserved via traditional ice-box preservation, and those who received a lesser-quality liver preserved via normothermic machine perfusion. Normothermic machine perfusion can potentially expand the number of suitable donor livers available for retransplant candidates.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Endocrine dysfunction in psychology during social distancing measures.

OBJECTIVES: To determine the psychological impact of the social distancing measures in place due to the coronavirus pandemic. METHODS: We conducted a cross-sectional study on the Pakistani population. Informed consent was taken from all the participants. The data was collected through an online questionnaire. Cronbach's alpha was used to assess the internal consistency of the questionnaire, and it was found to be 0.80. The data obtained was analyzed on IBM's statistical package for the social sciences (SPSS) version 26. RESULTS: Out of 706 participants, 489 (69.26 %) were males and 217 (30.74 %) were females. The mean age of the participants was 35.24 ± 12.08 years. The majority of the participants were from Punjab (66.00 %).The mean time since quarantine measures had been established was 10.35 ± 5.09 days. The mean total score was 9.08 ± 2.38 points. A majority of the participants (25.64 %) were daily wage workers. t-test was significant when the time of quarantine was compared to the psychological impact. Significant results were also found when gender was compared to the impact. CONCLUSIONS: Social distancing measures have an impact on psychology and endocrinology of people in general. The impact can take the shape of long-lasting consequences (Tab. 3, Ref. 19).

Deciphering the Structural Enigma of HLA Class-II Binding Peptides for Enhanced Immunoinformatics-based Prediction of Vaccine Epitopes.

Vaccines remain the most efficacious means to avoid and eliminate morbid diseases associated with high morbidity and mortality. Clinical trials indicate the gaining impetus of peptide vaccines against diseases for which an effective treatment still remains obscure. CD4 T-cell-based peptide vaccines involve immunization with antigenic determinants from pathogens or neoplastic cells that possess the ability to elicit a robust T helper cell response, which subsequently activates other arms of the immune system. The available in silico predictors of human leukocyte antigen II (HLA-II) binding peptides are sequence-based techniques, which ostensibly have balanced sensitivity and specificity. Structural analysis and understanding of the cognate peptide and HLA-II interactions are essential to empirically derive a successful peptide vaccine. However, the availability of structure-based epitope prediction algorithms is inadequate compared with sequence-based prediction methods. The present study is an attempt to understand the structural aspects of HLA-II binders by analyzing the Protein Data Bank (PDB) complexes of pHLA-II. Furthermore, we mimic the peptide exchange mechanism and demonstrate the structural implication of an acidic environment on HLA-II binders. Finally, we discuss a structure-guided approach to decipher potential HLA-II binders within an antigenic protein. This strategy may accurately predict the peptide epitopes and thus aid in designing successful peptide vaccines.

Intestinal microbiota disruption limits the isoniazid mediated clearance of Mycobacterium tuberculosis in mice.

Tuberculosis (TB) continues to remain a global threat due to the emergence of drug-resistant Mycobacterium tuberculosis (Mtb) strains and toxicity associated with TB drugs. Intestinal microbiota has been reported to affect the host response to immunotherapy and drugs. However, how it affects the potency of first-line TB drug isoniazid (INH) is largely unknown. Here, we examined the impact of gut microbial dysbiosis on INH efficiency to kill Mtb. In this study, we employed in vivo mouse model, pretreated with broad-spectrum antibiotics (Abx) cocktail to disrupt their intestinal microbial population prior to Mtb infection and subsequent INH therapy. We demonstrated that microbiota disruption results in the impairment of INH-mediated Mtb clearance, and aggravated TB-associated tissue pathology. Further, it suppressed the innate immunity and reduced CD4 T-cell response against Mtb. Interestingly, a distinct shift of gut microbial profile was noted with abundance of Enterococcus and reduction of Lactobacillus and Bifidobacterium population. Our results show that the intestinal microbiota is crucial determinant in efficacy of INH to kill Mtb and impacts the host immune response against infection. This work provides an intriguing insight into the potential links between host gut microbiota and potency of INH.

Gut Dysbiosis Thwarts the Efficacy of Vaccine Against Mycobacterium tuberculosis.

The generation of enduring protective immunity by vaccines is of utmost importance. Intriguingly, there is considerable variation in the efficacy of vaccines amongst individuals. Various studies have shown that normal flora of gastrointestinal tract plays a vital role in maintaining host homeostasis and immunity. Since gut microbiome is also extremely variable between individuals, we speculate that it might impact individual's response to vaccines. Consequently, we administered broad spectrum antibiotics cocktail to induce gut dysbiosis and monitored its impact on the generation of long-lasting memory T cells and thereby BCG vaccine efficacy. Interestingly, gut dysbiosis significantly decreased the activation of CD4+ T cells and CD8+ T cells. Further, there was decline in the frequency of memory CD4+ T cells and CD8+ T cells in lungs and secondary lymphoid organs of the vaccinated animals. Moreover, it dampened the IFN-γ and TNF-α secretion and proliferation of Mtb-specific T cells. Most importantly, dysbiosis hampered Mtb clearance in vaccinated animals, as evidenced by increase in the colony forming units (CFUs) in lungs and spleen. Our findings indicate that gut dysbiosis can be one of the major factors responsible for variable efficacy of TB vaccines across the world.