Dual targeting macrophages and microglia is a therapeutic vulnerability in models of PTEN-deficient glioblastoma.

Tumor-associated macrophages and microglia (TAMs) are critical for tumor progression and therapy resistance in glioblastoma (GBM), a type of incurable brain cancer. We previously identified lysyl oxidase (LOX) and olfactomedin like-3 (OLFML3) as essential macrophage and microglia chemokines, respectively, in GBM. Here, single-cell transcriptomics and multiplex sequential immunofluorescence followed by functional studies demonstrate that macrophages negatively correlate with microglia in the GBM tumor microenvironment. LOX inhibition in PTEN-deficient GBM cells upregulates OLFML3 expression via the NF-κB-PATZ1 signaling pathway, inducing a compensatory increase of microglia infiltration. Dual targeting macrophages and microglia via inhibition of LOX and the CLOCK-OLFML3 axis generates potent anti-tumor effects and offers a complete tumor regression in more than 60% of animals when combined with anti-PD1 therapy in PTEN-deficient GBM mouse models. Thus, our findings provide a translational triple therapeutic strategy for this lethal disease.

Chronic Myeloid Leukemia Unveils Its Dark Side: A Rare Case of Megakaryocytic Blast Crisis.

Chronic myeloid leukemia (CML) can progress from a chronic phase (CP) to an accelerated phase (AP) or an acute leukemia-like blastic phase (BP). However, transformation into a megakaryoblastic phase is very rare, and such a progression is clinically significant due to its poor prognosis and resistance to standard tyrosine kinase inhibitors (TKIs). This report discusses a case of CML that progressed to a megakaryoblastic phase and the patient's death within a month despite receiving one cycle of daunorubicin, cytarabine, and TKI chemotherapy. A 39-year-old female with CML (CP) initially achieved hematological remission with nilotinib but later presented with B symptoms and cytopenias indicative of disease progression. A complete diagnostic workup was performed, including blood counts, bone marrow examination, flow cytometry, fluorescence in-situ hybridization (FISH), and cytogenetic testing. Peripheral blood and bone marrow evaluation confirmed blast crisis with 84% medium to large-sized blasts with basophilic cytoplasm and cytoplasmic blebs. The blasts were positive for CD41 and CD61 by immunohistochemistry (IHC). The blasts also expressed CD45 (dim), CD34, CD33, CD117, CD41, and CD61 by flow cytometry. While BCR-ABL1 positivity is typically associated with CML (90-95%), the additional findings point towards a transformation to acute megakaryoblastic leukemia (AMKL or AML-M7). The rare instance of CML's transformation to AMKL highlights the need for megakaryocytic markers in diagnostic panels to ensure accurate diagnosis and timely, tailored therapies for improved outcomes.

From Practice to Proficiency: Evaluation of a Novel Workplace-Based Assessment (WBA) in a Postgraduate Chemical Pathology Residency Program in Pakistan.

Introduction: A workplace-based assessment (WBA) model was implemented in the postgraduate (PG) residency program of Chemical Pathology at the Department of Pathology & Laboratory Medicine, Aga Khan University (AKU). PGs were assessed using direct observation of practical skills (DOPS), evaluation of clinical events (ECE) and case-based discussion (CBD) on a virtual learning environment (VLE) platform. Objectives: To evaluate WBA frequency, case mix, feedback, and satisfaction levels of faculty and PGs of Chemical Pathology at AKU. Methods: Data from January 2019 to June 2023 was assessed. Tool utilization and case mix frequencies were calculated. PG and faculty satisfaction levels, as well as feedback and discussion time, were averaged. A thematic analysis was conducted on descriptive comments. Results: Out of 911 WBAs attempted, 79.1% (n=730) were CBDs, 10.8% (n=98) were DOPS, and 9.1% (n=83) were ECEs, showing a well-distributed case mix. Average satisfaction levels for CBD, ECE, and DOPS among both PGs and faculty were 8.38, 8.48, and 8.59, and 8.20, 8.36, and 8.46, respectively. Faculty feedback averaged 8.40, 8.65, and 7.85 minutes for CBD, ECE, and DOPS, respectively. Discussion times averaged 9.37, 9.52, and 13.36 minutes for CBD, ECE, and DOPS, respectively. Suggestions for development were noted in 20.82% (n=225) of CBDs, 21.69% (n=18) of ECEs, and 16.32% (n=16) of DOPS. Positives were documented in 40% (n=292) of CBDs, 28.92% (n=24) of ECEs, and 7.14% (n=7) of DOPS. Conclusion: This study evaluated a web-based WBA model in chemical pathology training, suggesting its applicability in diverse pathology specialties and regional training programs.

Advancing medical laboratory practice in Pakistan: insights from a focus group study on technologists' training needs.

BACKGROUND: The realm of medical laboratory technology (MLT) training and education is unstructured in Pakistan. The primary challenge currently confronting the workforce in MLT is the absence of standardized curricula and assessments in education and training. This was an exploratory study aiming to inquire experiences of trainees, alumni and teaching coordinators regarding the technologist training program in a single institute at Pakistan. METHODS: To gain an in depth understanding of MLT program, three focus group discussions (FGDs) were held at Department of Pathology and Laboratory Medicine, Aga Khan University, Pakistan during Feb-April 2024. A team of pathologists and educationists developed interview guides for FGDs in English. Interviews were bilingual, transcribed verbatim and coded using thematic analysis. Participants included current trainees, alumni, teaching and learning coordinators and moderators for the interview. RESULTS: A total of 29 participants were engaged; these included current MLT trainees (n = 10), alumni (n = 10), and teaching/learning coordinators and sectional supervisors (n = 9). Five main themes emerged from the analysis of FGDs: (Scott MG, Rifai N, Smith B, Oellerich M, Panteghini M, Apple F et al. The changing face of laboratory medicine: a more service and less academically oriented profession? 2015;61(2):322-9.) Recognition of key features of the MTT program, (Ferraro S, Braga F, Panteghini MJCC, Medicine L. Lab Med new Healthc Environ. 2016;54(4):523-33.) Evaluating curriculum design, (Waheed U, Ahmad M, Wazeer A, Saeed M, Saba N, Rasheed FJMJMS. Medical laboratory science education; shaping competent and skilled healthcare professionals. 2023;1(1):58-63.) Teaching and learning strategies, (Ned-Sykes R, Johnson C, Ridderhof JC, Perlman E, Pollock A, DeBoy JM. Competency guidelines for public health laboratory professionals. 2015.) Addressing the need to improve assessment methods, and (Linder RJJM, Education B. Educating medical laboratory technologists: revisiting our assumptions in the current economic and health-care environment. 2012;13(2):150-4.) Navigating the transition from a trainee to a competent technologist. CONCLUSION: Our investigation demonstrated its potential as a valuable needs assessment study, highlighting key strengths, drawbacks, and challenges of the existing MTT program. Importantly, these findings at our institute can inform further research efforts to design competency-based MLT education and training programs in Pakistan.

Epigenetic regulation of tumor immunity.

Although cancer has long been considered a genetic disease, increasing evidence shows that epigenetic aberrations play a crucial role in affecting tumor biology and therapeutic response. The dysregulated epigenome in cancer cells reprograms the immune landscape within the tumor microenvironment, thereby hindering antitumor immunity, promoting tumor progression, and inducing immunotherapy resistance. Targeting epigenetically mediated tumor-immune crosstalk is an emerging strategy to inhibit tumor progression and circumvent the limitations of current immunotherapies, including immune checkpoint inhibitors. In this Review, we discuss the mechanisms by which epigenetic aberrations regulate tumor-immune interactions and how epigenetically targeted therapies inhibit tumor progression and synergize with immunotherapy.

Evidence-Based Unani Pharmacotherapeutics for the Treatment of Non-Alcoholic Fatty Liver Disease: A Mechanistic Review.

Background: Non-alcoholic fatty liver disease (NAFLD) has emerged as a global health crisis, affecting a quarter of the world's population, and is anticipated to become a leading cause of liver transplantation and hepatocellular carcinoma by 2030. Conventional pharmacotherapy for NAFLD remains imperfect. In this context, Unani medicine offers a promising alternative for managing NAFLD. Objective: This review aims to compile information on Unani medications used for the treatment of NAFLD, aiming to provide evidence of their efficacy and delve into the mechanisms through which these Unani drugs exert their therapeutic effects in NAFLD. Methods: A comprehensive exploration of classical Unani literature was conducted, referencing well-established texts to extract pertinent information regarding NAFLD and its treatment in Unani Medicine. Electronic databases, including PubMed, Science Direct, and Google Scholar, were systematically searched to gather information on the efficacy of Unani drugs in the treatment of NAFLD. Results: Unani medicine offers a rich repository of single herbs and compound formulations. There is a description of about 32 single herbs and 18 compound formulations for the treatment of NAFLD. These drugs act due to their Musakhkhin (calorific), Mudirr (diuretics), Mufattih (deobstruent), Muqawwῑ-i-Jigar (hepatoprotective), and Muhallilat (anti-inflammatory) action. The bioactive components present in these drugs possess antioxidant, hypolipidemic, anti-inflammatory, hypoglycemic, and hepatoprotective activities. These actions of Unani drugs closely align with the multifaceted nature of NAFLD pathogenesis, and thus effective in the treatment of NAFLD. Conclusion: The findings led us to conclude that the use of Unani medicines can improve clinical outcomes in NAFLD, as demonstrated by various clinical and experimental trials. However, further clinical trials are essential to provide a safe and effective option for addressing this prevalent liver condition.

Factor Xa inhibitors versus low-molecular-weight heparin for preventing coagulopathy following COVID-19: a systematic review and meta-analysis of randomized controlled trials.

Background: Hospitalized patients with COVID-19 have shown a significant occurrence of thromboembolism and a heightened risk of death. It remains unclear whether factor Xa inhibitors are superior to enoxaparin in this context. Hence, there is a need for a direct comparison to assess the preventive effects and safety of factor Xa inhibitors versus enoxaparin in hospitalized COVID-19 patients. Methods: MEDLINE, Embase, and Cochrane Central databases were searched for randomized controlled trials (RCTs) or retrospective studies that compared the effectiveness or safety of factor Xa inhibitors and enoxaparin in preventing thromboembolism in hospitalized patients with COVID-19. Embolic incidence, incidence of bleeding, and all-cause mortality were among the outcomes of interest. Mantel-Haenszel weighted random-effects model was used to calculate relative risks (RRs) with 95 percent CIs. Results: The analysis included six RCTs and two retrospective studies containing 4048 patients. Meta-analysis showed a statistically significant reduction among patients on factor Xa inhibitors compared with low-molecular-weight heparin (LMWH) in the embolic incidence [risk ratio (RR) 0.64 (95%, CI 0.42, 0.98); P=0.04, I2=12%]. Upon subgroup analysis by type of study design, no significant reductions were noted in patients on factor Xa inhibitors in RCTs (RR: 0.62; 95% CI: 0.33-1.17; P=0.14) or observational studies (RR: 0.53; 95% CI: 0.23-1.26; P=0.15) when compared with enoxaparin Factor Xa inhibitors were not significantly associated with incidence of bleeding [RR 0.76 (95% CI 0.36, 1.61); P=0.47, I2=0%] or all-cause mortality (RR: 0.81; 95% CI: 0.48-1.36; P=0.43). Consistent results were obtained upon subgroup analysis by the type of study design. Conclusion: Factor Xa inhibitors are more effective than enoxaparin in preventing thromboembolism among patients with COVID-19 who are not acutely ill and are hospitalized. Additional rigorous RCTs comparing factor Xa inhibitors with enoxaparin are warranted.

Bladder-resident bacteria associated with increased risk of recurrence after electrofulguration in women with antibiotic-recalcitrant urinary tract infection.

Background: Antibiotic-recalcitrant recurrent urinary tract infection (rUTI) is has become increasingly observed in postmenopausal women. Therefore, when standard antibiotic therapies have failed, some elect electrofulguration (EF) of areas of chronic cystitis when detected on office cystoscopy. EF is thought to remove tissue-resident bacteria that have been previously detected in the bladder walls of postmenopausal women with rUTI. We hypothesized that increased bladder bacterial burden may be associated with incomplete rUTI resolution following EF. Methods: Following IRB approval, bladder biopsies were obtained from 34 consenting menopausal women electing EF for the advanced management of rUTI. 16S rRNA FISH was performed using both universal and Escherichia probes and tissue-resident bacterial load was quantified. Time to UTI relapse after EF was recorded during a six-month follow-up period and the association of bladder bacterial burden and clinical covariates with UTI relapse was assessed. Results: We observed bladder-resident Escherichia in 52% of all participants and in 92% of participants with recent E. coli UTI. Time-to-relapse analysis revealed that women with high bladder bacterial burden as detected by the universal probe had a significantly ( p =0.035) higher risk of UTI within six months of EF (HR=3.15, 95% CI: 1.09-9.11). Interestingly, bladder-resident Escherichia was not significantly associated ( p =0.26) with a higher risk of UTI relapse (HR= 2.14, 95% CI: 0.58-7.90). Conclusions: We observed that total bladder bacterial burden was associated with a 3.1x increased risk of rUTI relapse within six months. Continued analysis of the relationship between bladder bacterial burden and rUTI outcomes may provide insight into the management of these challenging patients.

Regional variations in antimicrobial susceptibility of community-acquired uropathogenic Escherichia coli in India: Findings of a multicentric study highlighting the importance of local antibiograms.

Objectives: Evidence-based prescribing is essential to optimize patient outcomes in cystitis. This requires knowledge of local antibiotic resistance rates. Diagnostic and Antimicrobial Stewardship (DASH) to Protect Antibiotics (https://dashuti.com/) is a multicentric mentorship program guiding centers in preparing, analyzing and disseminating local antibiograms to promote antimicrobial stewardship in community urinary tract infection. Here, we mapped the susceptibility profile of Escherichia coli from 22 Indian centers. Methods: These centers spanned 10 Indian states and three union territories. Antibiograms for urinary E. coli from the outpatient departments were collated. Standardization was achieved by regional online training; anomalies were resolved via consultation with study experts. Data were collated and analyzed. Results: Nationally, fosfomycin, with 94% susceptibility (inter-center range 83-97%), and nitrofurantoin, with 85% susceptibility (61-97%), retained the widest activity. The susceptibility rates were lower for co-trimoxazole (49%), fluoroquinolones (31%), and oral cephalosporins (26%). The rates for third- and fourth-generation cephalosporins were 46% and 52%, respectively, with 54% (33-58%) extended-spectrum ß-lactamase prevalence. Piperacillin-tazobactam (81%), amikacin (88%), and meropenem (88%) retained better activity; however, one center in Delhi recorded only 42% meropenem susceptibility. Susceptibility rates were mostly higher in South, West, and Northeast India; centers in the heavily populated Gangetic plains, across north and northwest India, had greater resistance. These findings highlight the importance of local antibiograms in guiding appropriate antimicrobial choices. Conclusions: Fosfomycin and nitrofurantoin are the preferred oral empirical choices for uncomplicated E. coli cystitis in India, although elevated resistance in some areas is concerning. Empiric use of fluoroquinolones and third-generation cephalosporins is discouraged, whereas piperacillin/tazobactam and aminoglycosides remain carbapenem-sparing parenteral agents.

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.