iterative-bleaching-extends-multiplicity-with-use-staining-automation

Iterative Bleaching Extends Multiplicity with Use of Staining Automation for Core Facilities

Iterative Bleaching Extends Multiplicity (IBEX) is a highly sensitive tyramide signal amplification (TSA)-based multi-round fluorescence imaging technique ...

Boston University

Boston University Chobanian and Avedisian School of Medicine

The causative agent of novel coronavirus disease (COVID-19) is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 possesses RNA as a genetic material with 79% of the match with the bat SARS-CoV genome, which became epidemic in 2002. The SARS-CoV-2 peripheral Spike-Fc protein binds specifically to the ACE2 receptors present on bronchial epithelial cells and alveolar pneumocytes to downmodulates its expression which leads to severe acute respiratory failure. The disease is super infectious from human to human and the symptoms are similar to flu. The old aged and immunocompromised population are severely affected, and healthcare providers globally applied various strategies for treatment including the repurposing of drugs including antimalarial drug, hydroxychloroquine and anti-viral drugs.Herein, we described the SARS-CoV-2 pandemic, immune responses, possible drug targets, vaccines under the trials and correlated the possibility of trained immunity induced by BCG vaccination over control of SARS-CoV-2 infection. The countries with constraint BCG vaccination policy are struggling badly compared to countries with BCG vaccination policy. The BCG vaccination policy supports either lowering the total number of COVID-19 cases or the increasing recovery rate.

An update on COVID-19: SARS-CoV-2 life cycle, immunopathology, and BCG vaccination.

Macrophages contribute to host immunity and tissue homeostasis via alternative activation programs. M1-like macrophages control intracellular bacterial pathogens and tumor progression. In contrast, M2-like macrophages shape reparative microenvironments that can be conducive for pathogen survival or tumor growth. An imbalance of these macrophages phenotypes may perpetuate sites of chronic unresolved inflammation, such as infectious granulomas and solid tumors. We have found that plant-derived and synthetic rocaglates sensitize macrophages to low concentrations of the M1-inducing cytokine IFN-gamma and inhibit their responsiveness to IL-4, a prototypical activator of the M2-like phenotype. Treatment of primary macrophages with rocaglates enhanced phagosome-lysosome fusion and control of intracellular mycobacteria. Thus, rocaglates represent a novel class of immunomodulators that can direct macrophage polarization toward the M1-like phenotype in complex microenvironments associated with hypofunction of type 1 and/or hyperactivation of type 2 immunity, e.g., chronic bacterial infections, allergies, and, possibly, certain tumors.

Channeling macrophage polarization by rocaglates increases macrophage resistance to .

Here, we present a streamlined protocol for assessing intracellular Mycobacterium tuberculosis (Mtb) loads in macrophages. This protocol describes the simultaneous assessment of macrophage viability using automated microscopy. Further, we detail the quantification of mycobacterial loads using a rapid, inexpensive, and accurate approach for mycobacterial DNA isolation from paraformaldehyde-fixed macrophages. Simultaneous assessment of the bacterial loads using internal standard and macrophage viability allows for precise quantification of the effects of perturbations on Mtb and host cells while accounting for technical artifacts. For complete details on the use and execution of this protocol, please refer to Chatterjee et al. (2021).

Medium throughput protocol for genome-based quantification of intracellular mycobacterial loads and macrophage survival during  infection.

Pulmonary infections caused by the group of nontuberculosis mycobacteria (NTM), Mycobacterium avium complex (MAC), are a growing public health concern with incidence and mortality steadily increasing globally. Granulomatous inflammation is the hallmark of MAC lung infection, yet reliable correlates of disease progression, susceptibility, and resolution are poorly defined. Unlike widely used inbred mouse strains, mice that carry the mutant allele at the genetic locus sst1 develop human-like pulmonary tuberculosis featuring well-organized caseating granulomas. We characterized pulmonary temporospatial outcomes of intranasal and left intrabronchial  spp. hominissuis (M.av) induced pneumonia in B6.Sst1S mice, which carries the sst1 mutant allele. We utilized traditional semi-quantitative histomorphological evaluation, in combination with fluorescent multiplex immunohistochemistry (fmIHC), whole slide imaging, and quantitative digital image analysis. Followingintrabronchiolar infection with the laboratory M.av strain 101, the B6.Sst1S pulmonary lesions progressed 12-16 weeks post infection (wpi), with plateauing and/or resolving disease by 21 wpi. Caseating granulomas were not observed during the study. Disease progression from 12-16 wpi was associated with increased acid-fast bacilli, area of secondary granulomatous pneumonia lesions, and Arg1+ and double positive iNOS+/Arg1+ macrophages. Compared to B6 WT, at 16 wpi, B6.Sst1S lungs exhibited an increased area of acid-fast bacilli, larger secondary lesions with greater Arg1+ and double positive iNOS+/Arg1+ macrophages, and reduced T cell density. This morphomolecular analysis of histologic correlates of disease progression in B6.Sst1S could serve as a platform for assessment of medical countermeasures against NTM infection.

Progression and Dissemination of Pulmonary Mycobacterium Avium Infection in a Susceptible Immunocompetent Mouse Model.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from TB-susceptible and TB-resistant mice. We then apply the cSTAR (cell State Transition Assessment and Regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the. transcriptional state of the TB-susceptible macrophages towards that of TB-resistant cells. Finally, we demonstrate that the compounds identified with this approach enhance resistance of the TB-susceptible mouse macrophages to virulent  .

Cell state transition analysis identifies interventions that improve control of  infection by susceptible macrophages.

Understanding cell state transitions and purposefully controlling them to improve therapies is a longstanding challenge in biological research and medicine. Here, we identify a transcriptional signature that distinguishes activated macrophages from the tuberculosis (TB) susceptible and resistant mice. We then apply the cSTAR (cell state transition assessment and regulation) approach to data from screening-by-RNA sequencing to identify chemical perturbations that shift the transcriptional state of tumor necrosis factor (TNF)-activated TB-susceptible macrophages toward that of TB-resistant cells, i.e., prevents their aberrant activation without suppressing beneficial TNF responses. Last, we demonstrate that the compounds identified with this approach enhance the resistance of the TB-susceptible mouse macrophages to virulent .

In the ongoing battle against antimicrobial resistance, phenotypic drug tolerance poses a formidable challenge. This adaptive ability of microorganisms to withstand drug pressure without genetic alterations further complicating global healthcare challenges. Microbial populations employ an array of persistence mechanisms, including dormancy, biofilm formation, adaptation to intracellular environments, and the adoption of L-forms, to develop drug tolerance. Moreover, molecular mechanisms like toxin-antitoxin modules, oxidative stress responses, energy metabolism, and (p)ppGpp signaling contribute to this phenomenon. Understanding these persistence mechanisms is crucial for predicting drug efficacy, developing strategies for chronic bacterial infections, and exploring innovative therapies for refractory infections. In this comprehensive review, we dissect the intricacies of drug tolerance and persister formation, explore their role in acquired drug resistance, and highlight emerging therapeutic approaches to combat phenotypic drug tolerance. Furthermore, we outline the future landscape of interventions for persistent bacterial infections.

Unlocking the enigma of phenotypic drug tolerance: Mechanisms and emerging therapeutic strategies.

To detect multiple protein markers, here we present a protocol for imaging pulmonary tuberculosis (PTB) lesions from murine lungs using Opal-tyramide signal amplification (TSA) conjugated fluorescent dyes in free-floating formalin-fixed thick lung sections (50-100 μm). We describe steps for preparing tissue sections, permeabilization and antigen retrieval, and multiplexing with antibodies raised in the same species. This protocol has been optimized to preserve tissue integrity and endogenously expressed fluorescent reporter signals and nuclear staining. It enhances the signal-to-background ratio and enables 3D image rendering.

Protocol for 3D multiplexed fluorescent imaging of pulmonary TB lesions using Opal-TSA dyes for signal amplification.

Pulmonary tuberculosis (PTB) represents 85% of the disease burden caused by  (Mtb) and promotes aerosol transmission infecting about a quarter of people globally. Most Mtb infections are effectively limited within primary granulomatous lesions. Containment failures lead to hematogenous spread and the formation of post-primary destructive PTB lesions. Factors that favor Mtb survival and replication in the lungs after hematogenous spread despite systemic immunity represent appealing targets for host-directed TB therapies, but are currently unknown. We developed a novel mouse model that mimics progression of chronic post-primary PTB in humans: wherein PTB lesions form after hematogenous spread from a remote primary lesion in immunocompetent but TB-susceptible B6.Sst1S mice. The B6.Sst1S mice developed PTB lesions featuring granulomatous pneumonia, bronchogenic expansion and broncho-occlusion closely resembling post-primary PTB in humans. Using spatial transcriptomic and fluorescent multiplexed immunochemistry, we demonstrated the expansion of myeloid cell populations with the appearance of alternatively activated macrophages, dissolution of initial lymphoid follicles, and accumulation of de-differentiated lung epithelial cells in the advanced PTB lesions. To determine whether lung parenchymal cells or lung oxygenation were necessary for the pulmonary TB progression, we implanted lung and spleen fragments subcutaneously to serve as potential targets for hematogenous spread. The lung (but not spleen) implants displayed characteristic organized granulomas with necrosis and Mtb replication demonstrating that deleterious interactions of aberrantly activated macrophages with the inflammation-injured lung resident cells, and possibly hypoxia, not oxygenation, are critical determinants of PTB progression in immunocompetent hosts. Necrotic TB lesions also developed in subcutaneous implants of human lung tissue in mice with human immune system after respiratory infection. These animal models may serve to further dissect the lung-specific mechanisms of host susceptibility to virulent Mtb and for testing therapeutic interventions targeting these mechanisms.

Aberrant macrophage activation and maladaptive lung repair promote tuberculosis progression uniquely in the lung.

Platelets, which have been traditionally associated with hemostasis and thrombosis functions, now receive attention for their role in immune responses that may affect vaccine development and effectiveness. Through their interactions with immune cells and modulation of inflammation alongside their role in antigen presentation, platelets become integral components of both innate and adaptive immune systems. New research shows platelets can improve vaccine effectiveness while reducing adverse side effects. During vaccine administration, platelets release cytokines and chemokines, which attract and stimulate immune cells to the injection site. Platelets work together with dendritic cells and T cells to support antigen processing and presentation, which leads to strong immune activation. Platelets' pro-inflammatory mediators strengthen local immune responses to boost protective immunity generation. Significant attention has been given to platelet involvement in vaccine-related thrombotic events, including vaccine-induced immune thrombotic thrombocytopenia (VITT). The rarity and severity of these events demonstrate the need to investigate the complex interplay between vaccine mechanisms and platelet activation. Exploration of the platelet-immune axis can lead to new methods for improving both the effectiveness and safety of vaccines. Researchers are working on creating innovative approaches for treatments that target platelet receptors and thrombosis pathways without interfering with the regular hemostatic functions of platelets. New vaccine development methods and personalized immunization strategies can emerge from targeting platelets with adjuvants and immune modulators.

Vaccination and Platelet Biology: Unraveling the Immuno-Hemostatic Interplay.

Here, we present a protocol for a mouse model for studying mechanisms of post-primary pulmonary tuberculosis (PTB) caused by virulent Mycobacterium tuberculosis (Mtb) using subcutaneous hock infection and lung tissue implantation. We describe steps for collagen instillation of lungs, lung and spleen implantation, preparation of Mtb for infection, and hock infection of mice. We then detail procedures for the perfusion of the lung and collection of organs, tissue processing, and histopathologic interpretation. For complete details on the use and execution of this protocol, please refer to Yabaji et al..

Article	Year
An update on COVID-19: SARS-CoV-2 life cycle, immunopathology, and BCG vaccination. Preparative biochemistry & biotechnology\| PubMed ID: 33226885	2021
Channeling macrophage polarization by rocaglates increases macrophage resistance to . iScience\| PubMed ID: 34381970	2021
Medium throughput protocol for genome-based quantification of intracellular mycobacterial loads and macrophage survival during infection. STAR protocols\| PubMed ID: 35310069	2022
Progression and Dissemination of Pulmonary Mycobacterium Avium Infection in a Susceptible Immunocompetent Mouse Model. International journal of molecular sciences\| PubMed ID: 35682679	2022
Cell state transition analysis identifies interventions that improve control of infection by susceptible macrophages. bioRxiv : the preprint server for biology\| PubMed ID: 36798271	2023
Cell state transition analysis identifies interventions that improve control of infection by susceptible macrophages. Science advances\| PubMed ID: 37756395	2023
Unlocking the enigma of phenotypic drug tolerance: Mechanisms and emerging therapeutic strategies. Biochimie\| PubMed ID: 38168626	2024
Protocol for 3D multiplexed fluorescent imaging of pulmonary TB lesions using Opal-TSA dyes for signal amplification. STAR protocols\| PubMed ID: 39982826	2025
Aberrant macrophage activation and maladaptive lung repair promote tuberculosis progression uniquely in the lung. bioRxiv : the preprint server for biology\| PubMed ID: 40093068	2025
Vaccination and Platelet Biology: Unraveling the Immuno-Hemostatic Interplay. Vaccines\| PubMed ID: 40333325	2025
Protocol for developing a mouse model of post-primary pulmonary tuberculosis after hematogenous spread in native lungs and lung implants. STAR protocols\| PubMed ID: 40714561	2025

Shivraj Yabaji

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