Development of Indian Council of Medical Research (ICMR) Standard Treatment Workflows for Skin Diseases: A Step Toward Universal Health Coverage.

Background: Skin conditions form a major bulk of diseases in the community. With a disproportionately low number of dermatologists in the country, and a greatly unequal distribution between urban versus rural areas, ineffective treatment and mismanagement of skin conditions are, however, commonplace. Objective: To develop standard treatment workflows (STWs) for certain skin diseases for use by clinicians at primary, secondary, and tertiary care centers. Methodology: Seven members, from various academic institutes across the country, were selected for formulation of the STWs. They were provided logistic and technical support by the ICMR, Department of Health Research (DHR), and WHO India office. Multiple rounds of online and physical discussions were performed to identify topics that would be most useful in the management of skin diseases for the health care personnel (HCP) and subsequently to frame the STW content. Results: The selected diseases included acne and rosacea, alopecia, bacterial skin infections, cutaneous adverse drug reactions, dermatophytosis, eczema/dermatitis, immunobullous dermatoses, psoriasis, scabies, varicella and herpes, vitiligo, and urticaria. There was one separate module on rational use of topical corticosteroids. The STWs for these conditions have been finalised and are available as physical posters in health centers and can also be accessed online and through mobile applications. Conclusion: Thirteen STW modules have been prepared with a view to optimize management of skin diseases at various levels of health care system of the country.

Clinical Reasoning: A 32-Year-Old Man With Painless Bilateral Shoulder Girdle Weakness and Atrophy.

We report a case of 32-year-old man with progressive, asymmetric, proximal weakness of both upper limbs for 14 months. On examination, he had gynecomastia and wasting and weakness of his deltoid, supraspinatus, infraspinatus, pectoralis, biceps, and triceps muscles, along with sensory loss of his left C5-C8 dermatomes. Deep tendon reflexes were depressed in the upper limbs and normal in the lower limbs. There was a history of a road traffic accident 2 years ago without any neurologic deficits. We discuss the clinical approach, differential diagnosis, investigations, and treatment options for bibrachial weakness.

Optimizing alkaline hydrothermal treatment for biomimetic smart metallic orthopedic and dental implants.

Orthopedic and dental implant failure continues to be a significant concern due to localized bacterial infections. Previous studies have attempted to improve implant surfaces by modifying their texture and roughness or coating them with antibiotics to enhance antibacterial properties for implant longevity. However, these approaches have demonstrated limited effectiveness. In this study, we attempted to engineer the titanium (Ti) alloy surface biomimetically at the nanometer scale, inspired by the cicada wing nanostructure using alkaline hydrothermal treatment (AHT) to simultaneously confer antibacterial properties and support the adhesion and proliferation of mammalian cells. The two modified Ti surfaces were developed using a 4 h and 8 h AHT process in 1 N NaOH at 230 °C, followed by a 2-hour post-calcination at 600 °C. We found that the control plates showed a relatively smooth surface, while the treatment groups (4 h & 8 h AHT) displayed nanoflower structures containing randomly distributed nano-spikes. The results demonstrated a statistically significant decrease in the contact angle of the treatment groups, which increased wettability characteristics. The 8 h AHT group exhibited the highest wettability and significant increase in roughness 0.72 ± 0.08 µm (P < 0.05), leading to more osteoblast cell attachment, reduced cytotoxicity effects, and enhanced relative survivability. The alkaline phosphatase activity measured in all different groups indicated that the 8 h AHT group exhibited the highest activity, suggesting that the surface roughness and wettability of the treatment groups may have facilitated cell adhesion and attachment and subsequently increased secretion of extracellular matrix. Overall, the findings indicate that biomimetic nanotextured surfaces created by the AHT process have the potential to be translated as implant coatings to enhance bone regeneration and implant integration.

Antimony susceptible Leishmania donovani: evidence from in vitro drug susceptibility of parasites isolated from patients of post-kala-azar dermal leishmaniasis in pre- and post-miltefosine era.

Post-kala-azar dermal leishmaniasis (PKDL) patients are a key source of Leishmania donovani parasites, hindering the goal of eliminating visceral leishmaniasis (VL). Monitoring treatment response and parasite susceptibility is essential due to increasing drug resistance. We assessed the drug susceptibility of PKDL isolates (n = 18) from pre-miltefosine (MIL) era (1997-2004) with isolates (n = 16) from the post-miltefosine era (2010-2019) and post-miltefosine treatment relapse isolates (n = 5) towards miltefosine and amphotericin B (AmB) at promastigote stage and towards sodium antimony gluconate (SAG) at amastigote stage. PKDL isolates were examined for mutation in gene-encoding AQP1 transporter, C26882T mutation on chromosome 24, and miltefosine-transporter (MT). PKDL isolates from the post-miltefosine era were significantly more susceptible to SAG than SAG-resistant isolates from the pre-miltefosine era (P = 0.0002). There was no significant difference in the susceptibility of parasites to miltefosine between pre- and post-miltefosine era isolates. The susceptibility of PKDL isolates towards AmB remained unchanged between the pre- and post-miltefosine era. However, the post-miltefosine era isolates had a higher IC50 value towards AmB compared with PKDL relapse isolates. We did not find any association between AQP1 gene sequence variation and susceptibility to SAG, or between miltefosine susceptibility and single nucleotide polymorphisms (SNPs in the MT gene. This study demonstrates that recent isolates of Leishmania have resumed susceptibility to antimonials in vitro. The study also offers significant insights into the intrinsic drug susceptibility of Leishmania parasites over the past two decades, covering the period before the introduction of miltefosine and after its extensive use. IMPORTANCE: Post-kala-azar dermal leishmaniasis (PKDL) patients, a key source of Leishmania donovani parasites, hinder eliminating visceral-leishmaniasis. Assessment of the susceptibility of PKDL isolates to antimony, miltefosine (MIL), and amphotericin-B indicated that recent isolates remain susceptible to antimony, enabling its use with other drugs for treating PKDL.

Actionable mechanisms of drug tolerance and resistance in Mycobacterium tuberculosis.

The emergence of antimicrobial resistance (AMR) across bacterial pathogens presents a serious threat to global health. This threat is further exacerbated in tuberculosis (TB), mainly due to a protracted treatment regimen involving a combination of drugs. A diversity of factors contributes to the emergence of drug resistance in TB, which is caused by the pathogen Mycobacterium tuberculosis (Mtb). While the traditional genetic mutation-driven drug resistance mechanisms operate in Mtb, there are also several additional unique features of drug resistance in this pathogen. Research in the past decade has enriched our understanding of such unconventional factors as efflux pumps, bacterial heterogeneity, metabolic states, and host microenvironment. Given that the discovery of new antibiotics is outpaced by the emergence of drug resistance patterns displayed by the pathogen, newer strategies for combating drug resistance are desperately needed. In the context of TB, such approaches include targeting the efflux capability of the pathogen, modulating the host environment to prevent bacterial drug tolerance, and activating the host anti-mycobacterial pathways. In this review, we discuss the traditional mechanisms of drug resistance in Mtb, newer understandings and the shaping of a set of unconventional approaches to target both the emergence and treatment of drug resistance in TB.

Advances and challenges in organ-on-chip technology: toward mimicking human physiology and disease in vitro.

Organs-on-chips have been tissues or three-dimensional (3D) mini-organs that comprise numerous cell types and have been produced on microfluidic chips to imitate the complicated structures and interactions of diverse cell types and organs under controlled circumstances. Several morphological and physiological distinctions exist between traditional 2D cultures, animal models, and the growing popular 3D cultures. On the other hand, animal models might not accurately simulate human toxicity because of physiological variations and interspecies metabolic capability. The on-chip technique allows for observing and understanding the process and alterations occurring in metastases. The present study aimed to briefly overview single and multi-organ-on-chip techniques. The current study addresses each platform's essential benefits and characteristics and highlights recent developments in developing and utilizing technologies for single and multi-organs-on-chips. The study also discusses the drawbacks and constraints associated with these models, which include the requirement for standardized procedures and the difficulties of adding immune cells and other intricate biological elements. Finally, a comprehensive review demonstrated that the organs-on-chips approach has a potential way of investigating organ function and disease. The advancements in single and multi-organ-on-chip structures can potentially increase drug discovery and minimize dependency on animal models, resulting in improved therapies for human diseases.

STAT3 protects hematopoietic stem cells by preventing activation of a deleterious autocrine type-I interferon response.

Hematopoietic stem and progenitor cells (HSPCs) maintain blood-forming and immune activity, yet intrinsic regulators of HSPCs remain elusive. STAT3 function in HSPCs has been difficult to dissect as Stat3-deficiency in the hematopoietic compartment induces systemic inflammation, which can impact HSPC activity. Here, we developed mixed bone marrow (BM) chimeric mice with inducible Stat3 deletion in 20% of the hematopoietic compartment to avoid systemic inflammation. Stat3-deficient HSPCs were significantly impaired in reconstitution ability following primary or secondary bone marrow transplantation, indicating hematopoietic stem cell (HSC) defects. Single-cell RNA sequencing of Lin-ckit+Sca1+ BM cells (LSKs) revealed aberrant activation of cell cycle, p53, and interferon (IFN) pathways in Stat3-deficient HSPCs. Stat3-deficient LSKs accumulated γH2AX and showed increased expression of DNA sensors and type-I IFN (IFN-I), while treatment with A151-ODN inhibited expression of IFN-I and IFN-responsive genes. Further, the blockade of IFN-I receptor signaling suppressed aberrant cell cycling, STAT1 activation, and nuclear p53 accumulation. Collectively, our results show that STAT3 inhibits a deleterious autocrine IFN response in HSCs to maintain long-term HSC function. These data signify the importance of ensuring therapeutic STAT3 inhibitors are targeted specifically to diseased cells to avoid off-target loss of healthy HSPCs.

Multiparatopic antibodies induce targeted downregulation of programmed death-ligand 1.

Programmed death-ligand 1 (PD-L1) drives inhibition of antigen-specific T cell responses through engagement of its receptor programmed death-1 (PD-1) on activated T cells. Overexpression of these immune checkpoint proteins in the tumor microenvironment has motivated the design of targeted antibodies that disrupt this interaction. Despite clinical success of these antibodies, response rates remain low, necessitating novel approaches to enhance performance. Here, we report the development of antibody fusion proteins that block immune checkpoint pathways through a distinct mechanism targeting molecular trafficking. By engaging multiple receptor epitopes on PD-L1, our engineered multiparatopic antibodies induce rapid clustering, internalization, and degradation in an epitope- and topology-dependent manner. The complementary mechanisms of ligand blockade and receptor downregulation led to more durable immune cell activation and dramatically reduced PD-L1 availability in mouse tumors. Collectively, these multiparatopic antibodies offer mechanistic insight into immune checkpoint protein trafficking and how it may be manipulated to reprogram immune outcomes.

CD38-Specific Gallium-68 Labeled Peptide Radiotracer Enables Pharmacodynamic Monitoring in Multiple Myeloma with PET.

The limited availability of molecularly targeted low-molecular-weight imaging agents for monitoring multiple myeloma (MM)-targeted therapies has been a significant challenge in the field. In response, a first-in-class peptide-based radiotracer, [68Ga]Ga-AJ206, is developed that can be seamlessly integrated into the standard clinical workflow and is specifically designed to noninvasively quantify CD38 levels and pharmacodynamics by positron emission tomography (PET). A bicyclic peptide, AJ206, is synthesized and exhibits high affinity to CD38 (KD: 19.1 ± 0.99 × 10-9 m) by surface plasmon resonance. Further, [68Ga]Ga-AJ206-PET shows high contrast within 60 min and suitable absorbed dose estimates for clinical use. Additionally, [68Ga]Ga-AJ206 detects CD38 expression in cell line-derived xenografts, patient-derived xenografts (PDXs), and disseminated disease models in a manner consistent with flow cytometry and immunohistochemistry findings. Moreover, [68Ga]Ga-AJ206-PET successfully quantifies CD38 pharmacodynamics in PDXs, revealing increased CD38 expression in the tumor following all-trans retinoic acid (ATRA) therapy. In conclusion, [68Ga]Ga-AJ206 exhibits the salient features required for clinical translation, providing CD38-specific high-contrast images in multiple models of MM. [68Ga]Ga-AJ206-PET could be useful for quantifying total CD38 levels and pharmacodynamics during therapy to evaluate approved and new therapies in MM and other diseases with CD38 involvement.

Platypnea-orthodeoxia syndrome (POS) in a patient who had undergone partial liver resection.

A South Asian male in his early 60s presented with acute-onset dyspnoea on postoperative day 4 after undergoing middle hepatic vein sacrificing partial liver resection for epithelioid angiomyolipoma. The patient's SpO2 on presentation was 65% in standing position which improved to 90% in left lateral decubitus. He was suspected of having platypnea-orthodeoxia syndrome (POS) which was confirmed on echocardiogram with microbubble contrast showing a large intracardiac right-to-left shunt. The patient was taken up for transcatheter closure of patent foramen ovale (PFO). A 30 mm Amplatzer PFO Occluder was deployed across the PFO which reduced the intracardiac shunt resulting in an improved arterial saturation as well as immediate relief of patient's symptoms. This case illustrates the importance of suspecting and recognising POS clinically as well as the efficacy of transcatheter closure of PFO in such cases leading to resolution of hypoxaemia in a short span of time.

The 'qRBBB myocardial infarction': Unwrapping an old enigma.

This single-center, prospective, observational study was conducted at a tertiary-care center over a span of two years. Patients presenting with acute Anterior-Wall STEMI were included as a study population. The subgroups included qRBBB pattern on ECG and non-qRBBB group. Among 1128 patients included in the study, 100 (11.28 %) patients presented with qRBBB pattern. Increased risk of cardiogenic shock, increased hospital-stay, a higher Killip class on presentation, high incidence of recanalized IRA, remarkably depressed LVEF were significantly associated with qRBBB-MI, which is a menacing form of ACS that leads not only to a high mortality but also to a long-term morbidity.

LncRNA Malat1 suppresses pyroptosis and T cell-mediated killing of incipient metastatic cells.

The contribution of antitumor immunity to metastatic dormancy is poorly understood. Here we show that the long noncoding RNA Malat1 is required for tumor initiation and metastatic reactivation in mouse models of breast cancer and other tumor types. Malat1 localizes to nuclear speckles to couple transcription, splicing and mRNA maturation. In metastatic cells, Malat1 induces WNT ligands, autocrine loops to promote self-renewal and the expression of Serpin protease inhibitors. Through inhibition of caspase-1 and cathepsin G, SERPINB6B prevents gasdermin D-mediated induction of pyroptosis. In this way, SERPINB6B suppresses immunogenic cell death and confers evasion of T cell-mediated tumor lysis of incipient metastatic cells. On-target inhibition of Malat1 using therapeutic antisense nucleotides suppresses metastasis in a SERPINB6B-dependent manner. These results suggest that Malat1-induced expression of SERPINB6B can titrate pyroptosis and immune recognition at metastatic sites. Thus, Malat1 is at the nexus of tumor initiation, reactivation and immune evasion and represents a tractable and clinically relevant drug target.

Noncanonical Activity of Med4 as a Gatekeeper of Metastasis through Epigenetic Control of Integrin Signaling.

Breast cancer metastatic relapse after a latency period, known as metastatic dormancy. Through genetic screening in mice, we identified the mediator complex subunit 4 (Med4) as a novel tumor-cell intrinsic gatekeeper in metastatic reactivation. Med4 downregulation effectively awakened dormant breast cancer cells, prompting macroscopic metastatic outgrowth in the lungs. Med4 depletion results in profound changes in nuclear size and three-dimensional chromatin architecture from compacted to relaxed states in contrast to the canonical function of the Mediator complex. These changes rewire the expression of extracellular matrix proteins, integrins, and signaling components resulting in integrin-mediated mechano-transduction and activation of YAP and MRTF. The assembly of stress fibers pulls on the nuclear membrane and contributes to reinforcing the overall chromatin modifications by Med4 depletion. MED4 gene deletions were observed in patients with metastatic breast cancer, and reduced MED4 expression correlates with worse prognosis, highlighting its significance as a potential biomarker for recurrence.

A mitochondrial quality control mechanism reverses the phagosome maturation arrest caused by Mycobacterium tuberculosis.

Phagosome maturation arrest (PMA) imposed by Mycobacterium tuberculosis ( Mtb ) is a classic tool that helps Mtb evade macrophage anti-bacterial responses. The exclusion of RAB7, a small GTPase, from Mtb -phagosomes underscores PMA. Here we report an unexpected mechanism that triggers crosstalk between the mitochondrial quality control (MQC) and the phagosome maturation pathways that reverses the PMA. CRISPR-mediated p62/SQSTM1 depletion ( p62 KD ) blocks mitophagy flux without impacting mitochondrial quality. In p62 KD cells, Mtb growth and survival are diminished, mainly through witnessing an increasingly oxidative environment and increased lysosomal targeting. The lysosomal targeting of Mtb is facilitated by enhanced TOM20 + mitochondria-derived vesicles (MDVs) biogenesis, a key MQC mechanism. In p62 KD cells, TOM20 + -MDVs biogenesis is MIRO1/MIRO2-dependent and delivered to lysosomes for degradation in a RAB7-dependent manner. Upon infection in p62 KD cells, TOM20 + -MDVs get extensively targeted to Mtb -phagosomes, inadvertently facilitating RAB7 recruitment, PMA reversal and lysosomal targeting of Mtb . Triggering MQC collapse in p62 KD cells further diminishes Mtb survival signifying cooperation between redox- and lysosome-mediated mechanisms. The MQC-anti-bacterial pathway crosstalk could be exploited for host-directed anti-tuberculosis therapies.

Host-directed therapy against tuberculosis: Concept and recent developments.

Tuberculosis (TB) continues to remain at the forefront of the infectious disease burden globally, albeit with some aberrations during the COVID-19 pandemic. Among many factors, the emergence of drug resistance or antimicrobial resistance (AMR) has necessitated a renewed focus on developing novel and repurposed drugs against TB. Host-directed therapy (HDT) has emerged as an attractive alternative and a complementary strategy to the conventional antibiotic-based therapy of tuberculosis since HDT enjoys the advantage of disarming the pathogen of its ability to develop drug resistance. Considering the imminent threat of AMR across the spectrum of bacterial pathogens, HDT promises to overcome the drug shortage against superbugs. While all these make HDT a very attractive strategy, identifying the right set of host targets to develop HDT remains a challenge, despite remarkable development in the field over the past decade. In this review, we examine the host mechanisms, that either inadvertently or through targeted perturbation by the pathogen, help TB pathogenesis, and we discuss the latest developments in the targeting of some of the key pathways to achieve newer TB therapeutics.

Stabilizing vimentin phosphorylation inhibits stem-like cell properties and metastasis of hybrid epithelial/mesenchymal carcinomas.

Epithelial-mesenchymal transition (EMT) empowers epithelial cells with mesenchymal and stem-like attributes, facilitating metastasis, a leading cause of cancer-related mortality. Hybrid epithelial-mesenchymal (E/M) cells, retaining both epithelial and mesenchymal traits, exhibit heightened metastatic potential and stemness. The mesenchymal intermediate filament, vimentin, is upregulated during EMT, enhancing the resilience and invasiveness of carcinoma cells. The phosphorylation of vimentin is critical to its structure and function. Here, we identify that stabilizing vimentin phosphorylation at serine 56 induces multinucleation, specifically in hybrid E/M cells with stemness properties but not epithelial or mesenchymal cells. Cancer stem-like cells are especially susceptible to vimentin-induced multinucleation relative to differentiated cells, leading to a reduction in self-renewal and stemness. As a result, vimentin-induced multinucleation leads to sustained inhibition of stemness properties, tumor initiation, and metastasis. These observations indicate that a single, targetable phosphorylation event in vimentin is critical for stemness and metastasis in carcinomas with hybrid E/M properties.

Phosphorylation of CFP10 modulates Mycobacterium tuberculosis virulence.

IMPORTANCE: Secreted virulence factors play a critical role in bacterial pathogenesis. Virulence effectors not only help bacteria to overcome the host immune system but also aid in establishing infection. Mtb, which causes tuberculosis in humans, encodes various virulence effectors. Triggers that modulate the secretion of virulence effectors in Mtb are yet to be fully understood. To gain mechanistic insight into the secretion of virulence effectors, we performed high-throughput proteomic studies. With the help of system-level protein-protein interaction network analysis and empirical validations, we unravelled a link between phosphorylation and secretion. Taking the example of the well-known virulence factor of CFP10, we show that the dynamics of CFP10 phosphorylation strongly influenced bacterial virulence and survival ex vivo and in vivo. This study presents the role of phosphorylation in modulating the secretion of virulence factors.

Non-invasive PD-L1 quantification using [18F]DK222-PET imaging in cancer immunotherapy.

BACKGROUND: Combination therapies that aim to improve the clinical efficacy to immune checkpoint inhibitors have led to the need for non-invasive and early pharmacodynamic biomarkers. Positron emission tomography (PET) is a promising non-invasive approach to monitoring target dynamics, and programmed death-ligand 1 (PD-L1) expression is a central component in cancer immunotherapy strategies. [18F]DK222, a peptide-based PD-L1 imaging agent, was investigated in this study using humanized mouse models to explore the relationship between PD-L1 expression and therapy-induced changes in cancer. METHODS: Cell lines and xenografts derived from three non-small cell lung cancers (NSCLCs) and three urothelial carcinomas (UCs) were used to validate the specificity of [18F]DK222 for PD-L1. PET was used to quantify anti-programmed cell death protein-1 (PD-1) therapy-induced changes in PD-L1 expression in tumors with and without microsatellite instability (MSI) in humanized mice. Furthermore, [18F]DK222-PET was used to validate PD-L1 pharmacodynamics in the context of monotherapy and combination immunotherapy in humanized mice bearing A375 melanoma xenografts. PET measures of PD-L1 expression were used to establish a relationship between pathological and immunological changes. Lastly, spatial distribution analysis of [18F]DK222-PET was developed to assess the effects of different immunotherapy regimens on tumor heterogeneity. RESULTS: [18F]DK222-PET and biodistribution studies in mice with NSCLC and UC xenografts revealed high but variable tumor uptake at 60 min that correlated with PD-L1 expression. In MSI tumors treated with anti-PD-1, [18F]DK222 uptake was higher than in control tumors. Moreover, [18F]DK222 uptake was higher in A375 tumors treated with combination therapy compared with monotherapy, and negatively correlated with final tumor volumes. In addition, a higher number of PD-L1+ cells and higher CD8+-to-CD4+ cell ratio was observed with combination therapy compared with monotherapy, and positively correlated with PET. Furthermore, spatial distribution analysis showed higher [18F]DK222 uptake towards the core of the tumors in combination therapy, indicating a more robust and distinct pattern of immune cell infiltration. CONCLUSION: [18F]DK222-PET has potential as a non-invasive tool for monitoring the effects of immunotherapy on tumors. It was able to detect variable PD-L1 expression in tumors of different cancer types and quantify therapy-induced changes in tumors. Moreover, [18F]DK222-PET was able to differentiate the impact of different therapies on tumors.