Mycobacterium tuberculosis infection drives differential responses in the bone marrow hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) play a vital role in the host response to infection through the rapid and robust production of mature immune cells. These HSPC responses can be influenced, directly and indirectly, by pathogens as well. Infection with Mycobacterium tuberculosis (Mtb) can drive lymphopoiesis through modulation of type I interferon (IFN) signaling. We have previously found that the presence of a drug resistance (DR)-conferring mutation in Mtb drives altered host-pathogen interactions and heightened type I IFN production in vitro. But the impacts of this DR mutation on in vivo host responses to Mtb infection, particularly the hematopoietic compartment, remain unexplored. Using a mouse model, we show that, while drug-sensitive Mtb infection induces expansion of HSPC subsets and a skew toward lymphopoiesis, DR Mtb infection fails to induce an expansion of these subsets and an accumulation of mature granulocytes in the bone marrow. Using single-cell RNA sequencing, we show that the HSCs from DR Mtb-infected mice fail to upregulate pathways related to cytokine signaling across all profiled HSC subsets. Collectively, our studies report a novel finding of a chronic infection that fails to induce a potent hematopoietic response that can be further investigated to understand pathogen-host interaction at the level of hematopoiesis.

A single centre experience of local perforator flaps in oncoplastic breast surgery; a cross-sectional study.

Background & objectives: Various oncoplastic techniques have emerged over the years to preserve breast cosmesis and symmetry without compromising the principles of tumor excision. One of the newer techniques for breast volume replacement to achieve symmetry and cosmesis is the use of fasciocutaneous pedicled chest wall perforator flaps or local perforator flaps (LPF). The objectives of this study were to document the details of the surgical procedure as well as patient-reported satisfaction and well-being following the procedure using a validated BREAST-Q tool among Pakistani women. Materials & methods: This cross-sectional study was conducted from March 2019 to February 2021 enrolling 25 female patients who underwent breast conservative surgery using LPF for breast tumors at The Aga Khan University Hospital, Karachi. Data related to the procedure was collected on a pre-designed proforma. Cosmetic outcomes and patient satisfaction were evaluated using 2 scales from BREAST-Q BCT domain version 2.0. The questionnaire was self-administered by the patients during their routine follow-up in the clinic. Data was analyzed using the Statistical Package for Social Sciences (SPSS) version 23. Mean (SD)/median (IQR) were computed for quantitative variables and frequency and percentages were calculated for qualitative variables. 2 sample t-test was applied. P-value ≤0.05 was considered significant. Results: 25 patients underwent LPF with a mean age of 47 ± 13.1 years. 8 LICAP, 7 AICAP, and 10 LTAP flaps were performed. Two postoperative complications of wound site erythema were encountered. 23 women were eligible for the BREAST-Q survey. Median (IQR) postoperative satisfaction with breasts and physical well-being chest (equivalent Rasch transformed score) was 100 (41) and 76 [18] respectively. We found high satisfaction with breasts and comparable physical well-being among Pakistani women after LPF surgeries. Conclusion: Local perforator flaps in oncoplastic breast-conserving surgery are a good option showing high satisfaction with breasts and physical well-being in Pakistani women.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Male Breast Cancer: The Three Decades' Experience of a Tertiary Care Hospital in a Lower-Middle Income Country.

Introduction Male breast cancer is uncommon and managed on the guidelines of female breast cancer due to tumor rarity. We sought to identify the incidence, clinicopathological features, and survival of all male breast cancer patients managed in our hospital. Methods A retrospective cross-sectional study was conducted at Aga Khan University Hospital (AKUH), Karachi, Pakistan, from January 1986 to December 2018. Demographic data, treatment records, and follow-up data of all male breast cancer patients who were treated at AKUH was reviewed. Results Thirty-eight out of 42 patients who presented over a period of 32 years were included. The mean age was 63 years. The most common tumor type and subtype were invasive ductal carcinoma (89.5%) and luminal A (73.7%), respectively. The majority (36.8%) of the patients presented at stage III. Among 30 (78.9%) patients who underwent surgery, mastectomy was performed in 30 (78.9%), upfront axillary clearance in 24 (63.2%), axillary sampling in five (15.1%) cases, and sentinel lymph node biopsy in one (2.6%) case. Neoadjuvant chemotherapy was given to 10 (26.3%) patients, and adjuvant chemotherapy to eight (21.1%) patients. Adjuvant hormonal treatment was administered to 22 (57.9%) patients, and 13 (34%) patients received adjuvant radiation to the chest wall. The five-year overall survival was 38.2% and the median survival was 36 months. The five-year disease-free survival (DFS) was found to be 33.7%. Conclusion Breast cancer in males presents at an advanced stage with poor survival. Multicenter studies are required to accurately identify incidence, prognostic factors, and outcomes in order to have a better understanding of its management.

BCG vaccination provides protection against IAV but not SARS-CoV-2.

Since the vast majority of species solely rely on innate immunity for host defense, it stands to reason that a critical evolutionary trait like immunological memory evolved in this primitive branch of our immune system. There is ample evidence that vaccines such as bacillus Calmette-Guérin (BCG) induce protective innate immune memory responses (trained immunity) against heterologous pathogens. Here we show that while BCG vaccination significantly reduces morbidity and mortality against influenza A virus (IAV), it fails to provide protection against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In contrast to IAV, SARS-CoV-2 infection leads to unique pulmonary vasculature damage facilitating viral dissemination to other organs, including the bone marrow (BM), a central site for BCG-mediated trained immunity. Finally, monocytes from BCG-vaccinated individuals mount an efficient cytokine response to IAV infection, while this response is minimal following SARS-CoV-2. Collectively, our data suggest that the protective capacity of BCG vaccination is contingent on viral pathogenesis and tissue tropism.

Analysing the trends in breast surgery practice during COVID-19 pandemic: A comparative study with the Pre-COVID era.

BACKGROUND: The emergence of coronavirus disease 2019 (COVID-19) pandemic has crippled the healthcare systems all over the world. Cancer treatment is indispensable and disruption in its provision can lead to unanticipated consequences. No local data exists that has quantified the impact of COVID-19 pandemic on breast cancer surgery in a lower middle-income country (LMIC), therefore, the present retrospective comparative cohort study is directed to determine the trends in breast surgery operative volumes and its outcomes at our institution in Pakistan. MATERIALS AND METHODS: Data was collected retrospectively from Pre-COVID-19 and COVID-19 era to determine impact of the current pandemic on breast cancer management practices and outcomes. RESULTS: Cohort results showed a decline in the number of surgeries during COVID-19 era. A total 149 cases were operated during study period vs. 231 during same Pre-COVID-19 i.e. a 35.5% drop in cancer surgeries. In early COVID-19 time frame, only 4 patients had breast reconstruction, 12 out of 149 (8.05%) surgical candidates were identified having positive COVID-19 status preoperatively and one ASA class 3 patient caught COVID-19 post-surgery and succumbed to virus. CONCLUSION: Pandemic has a negative effect on cancer management in a LMIC with compromised access and care of cancer patients.

Human alveolar macrophage metabolism is compromised during Mycobacterium tuberculosis infection.

Pulmonary macrophages have two distinct ontogenies: long-lived embryonically-seeded alveolar macrophages (AM) and bone marrow-derived macrophages (BMDM). Here, we show that after infection with a virulent strain of Mycobacterium tuberculosis (H37Rv), primary murine AM exhibit a unique transcriptomic signature characterized by metabolic reprogramming distinct from conventional BMDM. In contrast to BMDM, AM failed to shift from oxidative phosphorylation (OXPHOS) to glycolysis and consequently were unable to control infection with an avirulent strain (H37Ra). Importantly, healthy human AM infected with H37Ra equally demonstrated diminished energetics, recapitulating our observation in the murine model system. However, the results from seahorse showed that the shift towards glycolysis in both AM and BMDM was inhibited by H37Rv. We further demonstrated that pharmacological (e.g. metformin or the iron chelator desferrioxamine) reprogramming of AM towards glycolysis reduced necrosis and enhanced AM capacity to control H37Rv growth. Together, our results indicate that the unique bioenergetics of AM renders these cells a perfect target for Mtb survival and that metabolic reprogramming may be a viable host targeted therapy against TB.

Features of Cu and TiO2 in the flow of engine oil subject to thermal jump conditions.

The recent work investigates the heat transfer attributes in the flow of engine oil which comprises of nano-particles such as Cu and TiO2. The performance of Copper and Titanium oxide is over looked in the flow of engine oil. The energy equation is amended by the features of thermal radiation, viscous dissipation, and heat generation. The mathematical model signifies the porosity, entropy generation and moving flat horizontal surface with the non-uniform stretching velocity. Quasi-linearization, which is a persuasive numerical technique to solve the complex coupled differential equations, is used to acquire the numerical solution of the problem. Flow and heat transfer aspects of Cu-TiO2 in the flow are examined against the preeminent parameters. The flow is significantly affected by the thermal jump conditions and porous media. It is observed here that the temperature as well as heat transport rate is reduced with the effect of involved preeminent parameters. However, such fluids must be used with caution in applications where a control on the heat transfer is required. We may conclude that the recent study will provide assistance in thermal cooling systems such as engine and generator cooling, nuclear system cooling, aircraft refrigeration system, and so forth.

Oncoplastic surgery: A suitable alternative to conventional breast conserving surgery in low - Middle income countries; a retrospective cohort study.

INTRODUCTION: Breast Conserving Surgery (BCS) with whole breast radiation is now standard of care as a safer alternative to Mastectomy in terms of loco-regional recurrence and long-term survival. Despite this, a frequent pitfall of conventional BCS is positive surgical margins and need for second surgery with a reported frequency of 12-59 % in literature. Oncoplastic Surgery can be a safer, more cost effective alternate to conventional BCS owing to its higher rate of negative surgical margins (4-6% vs 12-59 %) and better cosmetic results. We aim to prove utility of Oncoplastic surgery for Low-Middle income countries. OBJECTIVE: The aim of this study was to determine Oncoplastic Surgery as a more appropriate alternative to Conventional Breast Conserving Surgery for Low-Middle Income countries in terms of its lower positive margins and re-excision rates. METHODOLOGY: A retrospective comparative single center study by reviewing patient's medical records from August 2016 to June 2020 was conducted. Rate of positive margins and re-excisions along with mean volume of resection specimen, mean tumor size and quadrant dealt by both surgical procedures were compared. RESULTS: Out of 421 patients 249 patients underwent oncoplastic surgery and were compared with 173 patients who had conventional breast conserving surgery. Positive margins were seen in 5 patients (2 %) in OPS group whereas in 31 (17.9 %) patients in BCS group (p value < 0.001). Therefore, 2 from OPS group and 17 from BCS group underwent re-excision (p value < 0.002).None in OPS group while 7 out of 17 patients in BCS group underwent mastectomy as second procedure. Mean tumor size in OPS group was 2.26 cm ± SD 1.66 and in BCS group was 1.94 cm ± SD 1.28. Majority of Lobular carcinoma and Ductal carcinoma in-situ, multifocal, upper inner and central quadrant tumors and those unresponsive to neo-adjuvant therapy were treated by Oncoplastic techniques. CONCLUSION: Oncoplastic surgery has shown promising results as a safer tool to deal with large, complex tumors, lesions in difficult anatomical locations, multifocal or progressing on neo-adjuvant therapy. With its low Re-excision rates, it is a better alternative to traditional Breast Conserving approach for overburdened and resource limited health care system of Low-Middle Income countries. Multi-center, prospective trials are needed to determine its feasibility.

Utility of post mastectomy radiotherapy among patients with T1/ T2 N1 disease: A retrospective cohort study.

BACKGROUND: Pakistan has the highest incidence of breast cancer among Asian Countries but there is insufficient representation of local data addressing breast cancer treatment and outcome. We sought to determine the role of post-mastectomy radiotherapy (PMRT) in T1- T2 breast cancer with 1-3 positive axillary lymph nodes. METHODS: Data was reviewed retrospectively of total 755 patients out of which 291 received PMRT and 464 did not from two large breast cancer centres. RESULTS: With a median follow up of 78 months, 4 (4.5%) patients developed loco regional recurrence (LRR) in the PMRT group while a substantial number 74 (24.4%) recurred in the non PMRT group (p = 0.000). Loco regional free survival rate (LRFS) and overall survival rate (OS) was significantly better for PMRT patients than non-PMRT patients (P = <0.000). Multivariate analysis identified young age, lymphovascular invasion, extra capsular extension, triple negative and ER/PR negative were independent prognostic factors affecting loco regional free survival (LRFS). CONCLUSION: Disease recurrence is a substantial issue in 1-3 node group despite early stage, PMRT has an instrumental effect in improving LRFS and OS.

M. tuberculosis Reprograms Hematopoietic Stem Cells to Limit Myelopoiesis and Impair Trained Immunity.

A greater understanding of hematopoietic stem cell (HSC) regulation is required for dissecting protective versus detrimental immunity to pathogens that cause chronic infections such as Mycobacterium tuberculosis (Mtb). We have shown that systemic administration of Bacille Calmette-Guérin (BCG) or ß-glucan reprograms HSCs in the bone marrow (BM) via a type II interferon (IFN-II) or interleukin-1 (IL1) response, respectively, which confers protective trained immunity against Mtb. Here, we demonstrate that, unlike BCG or ß-glucan, Mtb reprograms HSCs via an IFN-I response that suppresses myelopoiesis and impairs development of protective trained immunity to Mtb. Mechanistically, IFN-I signaling dysregulates iron metabolism, depolarizes mitochondrial membrane potential, and induces cell death specifically in myeloid progenitors. Additionally, activation of the IFN-I/iron axis in HSCs impairs trained immunity to Mtb infection. These results identify an unanticipated immune evasion strategy of Mtb in the BM that controls the magnitude and intrinsic anti-microbial capacity of innate immunity to infection.

ß-Glucan Induces Protective Trained Immunity against Mycobacterium tuberculosis Infection: A Key Role for IL-1.

ß-glucan is a potent inducer of epigenetic and functional reprogramming of innate immune cells, a process called "trained immunity," resulting in an enhanced host response against secondary infections. We investigate whether ß-glucan exposure confers protection against pulmonary Mycobacterium tuberculosis (Mtb) infection. ß-glucan induces trained immunity via histone modifications at gene promoters in human monocytes, which is accompanied by the enhanced production of proinflammatory cytokines upon secondary Mtb challenge and inhibition of Mtb growth. Mice treated with ß-glucan are significantly protected against pulmonary Mtb infection, which is associated with the expansion of hematopoietic stem and progenitor cells in the bone marrow and increased myelopoiesis. The protective signature of ß-glucan is mediated via IL-1 signaling, as ß-glucan shows no protection in mice lacking a functional IL-1 receptor (IL1R-/-). The administration of ß-glucan may be used as a novel strategy in the treatment of mycobacterial infections and possibly as an adjuvant to improve anti-tuberculosis vaccines.

NK cell recruitment limits tissue damage during an enteric helminth infection.

Parasitic helminths cause significant damage as they migrate through host tissues to complete their life cycle. While chronic helminth infections are characterized by a well-described Type 2 immune response, the early, tissue-invasive stages are not well understood. Here we investigate the immune pathways activated during the early stages of Heligmosomoides polygyrus bakeri (Hpb), a natural parasitic roundworm of mice. In contrast to the Type 2 immune response present at later stages of infection, a robust Type 1 immune signature including IFNg production was dominant at the time of parasite invasion and granuloma formation. This early response was associated with an accumulation of activated Natural Killer (NK) cells, with no increase of other innate lymphoid cell populations. Parabiosis and confocal microscopy studies indicated that NK cells were recruited from circulation to the small intestine, where they surrounded parasitic larvae. NK cell recruitment required IFNγ receptor signaling, but was independent of CXCR3 expression. The depletion of tissue-infiltrating NK cells altered neither worm burden nor parasite fitness, but increased vascular injury, suggesting a role for NK cells in mediating tissue protection. Together, these data identify an unexpected role for NK cells in promoting disease tolerance during the invasive stage of an enteric helminth infection.

Predominance of M2 macrophages in gliomas leads to the suppression of local and systemic immunity.

Glioblastoma is a highly prevalent and aggressive form of primary brain tumor. It represents approximately 56% of all the newly diagnosed gliomas. Macrophages are one of the major constituents of tumor-infiltrating immune cells in the human gliomas. The role of immunosuppressive macrophages is very well documented in correlation with the poor prognosis of patients suffering from breast, prostate, bladder and cervical cancers. The current study highlights the correlation between the tumor-associated macrophage phenotypes and glioma progression. We observed an increase in the pool of M2 macrophages in high-grade gliomas, as confirmed by their CD68 and CD163 double-positive phenotype. In contrast, less M1 macrophages were noticed in high-grade gliomas, as evidenced by the down-regulation in the expression of CCL3 marker. In addition, we observed that higher gene expression ratio of CD163/CCL3 is associated with glioma progression. The Kaplan-Meier survival plots indicate that glioma patients with lower expression of M2c marker (CD163), and higher expression of M1 marker (CCL3) had better survival. Furthermore, we examined the systemic immune response in the peripheral blood and noted a predominance of M2 macrophages, myeloid-derived suppressor cells and PD-1+ CD4 T cells in glioma patients. Thus, the study indicates a high gene expression ratio of CD163/CCL3 in high-grade gliomas as compared to low-grade gliomas and significantly elevated frequency of M2 macrophages and PD-1+ CD4 T cells in the blood of tumor patients. These parameters could be used as an indicator of the early diagnosis and prognosis of the disease.

CD109 Restrains Activation of Cutaneous IL-17-Producing γδ T Cells by Commensal Microbiota.

Interleukin-17-producing γδ T (γδ17) cells play a central role in protective and pathogenic immune responses. However, the tissue-specific mechanisms that control the activation of these innate lymphocytes are not known. Here, we demonstrate that CD109, a glycosylphosphatidylinositol (GPI)-anchored protein highly expressed by keratinocytes, is an important regulator of skin homeostasis and γδ17 cell activation. Genetic deletion of CD109 results in spontaneous epidermal hyperplasia, aberrant accumulation of dermal-derived γδ17 cells, and enhanced susceptibility to psoriasiform inflammation. In this context, γδ17 activation requires interleukin (IL)-23 signals and is reversed by transient depletion of the skin microbiota. Mechanistically, CD109 restrains γδ17 cell activation in a cell-extrinsic manner by fortifying skin barrier integrity. Collectively, our data provide insight into the regulation of the skin IL-23/IL-17 immune axis and how homeostasis is maintained at this important barrier site.

TLR-3 Stimulation Skews M2 Macrophages to M1 Through IFN-αß Signaling and Restricts Tumor Progression.

During tumor progression, macrophages shift their protective M1-phenotype to pro-tumorigenic M2-subtype. Therefore, conversion of M2 to M1 phenotype may be a potential therapeutic intervention. TLRs are important pathogen recognition receptors expressed by cells of the immune system. Recently, a crucial role of TLR-3 has been suggested in cancer. Consequently, in the current study, we defined the role of TLR-3 in the reversion of M2-macrophages to M1. We analyzed the role of TLR-3 stimulation for skewing M2-macrophages to M1 at mRNA and protein level through qRT-PCR, flow cytometry, western blotting, and ELISA. The effectiveness of TLR-3L stimulation to revert M2-macrophages to M1 was evaluated in the murine tumor model. To determine the role of IFN-αß signaling in vitro and in vivo, we used Ifnar1-/- macrophages and anti-IFN-αß antibodies, respectively. We observed upregulation of M1-specific markers MHC-II and costimulatory molecules like CD86, CD80, and CD40 on M2-macrophages upon TLR-3 stimulation. In contrast, reduced expression of M2-indicators CD206, Tim-3, and pro-inflammatory cytokines was noticed. The administration of TLR-3L in the murine tumor reverted the M2-macrophages to M1-phenotype and regressed the tumor growth. The mechanism deciphered for macrophage reversion and controlling the tumor growth is dependent on IFN-αß signaling pathway. The results indicate that the signaling through TLR-3 is important in protection against tumors by skewing M2-macrophages to protective M1-subtype.

BCG Educates Hematopoietic Stem Cells to Generate Protective Innate Immunity against Tuberculosis.

The dogma that adaptive immunity is the only arm of the immune response with memory capacity has been recently challenged by several studies demonstrating evidence for memory-like innate immune training. However, the underlying mechanisms and location for generating such innate memory responses in vivo remain unknown. Here, we show that access of Bacillus Calmette-Guérin (BCG) to the bone marrow (BM) changes the transcriptional landscape of hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), leading to local cell expansion and enhanced myelopoiesis at the expense of lymphopoiesis. Importantly, BCG-educated HSCs generate epigenetically modified macrophages that provide significantly better protection against virulent M. tuberculosis infection than naïve macrophages. By using parabiotic and chimeric mice, as well as adoptive transfer approaches, we demonstrate that training of the monocyte/macrophage lineage via BCG-induced HSC reprogramming is sustainable in vivo. Our results indicate that targeting the HSC compartment provides a novel approach for vaccine development.

T-cell exhaustion in tuberculosis: pitfalls and prospects.

T-cells play an important role in immunity but when these cells are overexposed to specific antigens, their function may decline. This state is usually referred to as exhaustion and the T-cells show reduced proliferation and functions such as cytokine release. T-cell exhaustion has been observed in several cancers as well as in chronic infections such as tuberculosis (TB). In chronic Mycobacterium tuberculosis (Mtb) infection, T-cells may express the exhaustion phenotype and show a progressive loss of secretion of IL-2, IFN-γ and TNF-α. In some cancers and chronic infection models, blocking the exhaustion phenotype can be achieved with the so-called checkpoint inhibitors. This results in tumor control and more effective immunity. However, in the case of TB, the T-cell exhaustion results are quite ambiguous. Hence, there is a need to investigate and explain the contribution of checkpoint at a molecular level to the outcome of events in chronic TB. Such information could help to guide the success of new therapies against chronic TB. This review highlights the mechanism through which T-cells undergo exhaustion and the approaches that can avert such events. This will help to design immunotherapies that can reinvigorate T-cell potency to protect patients from TB.

Infergen Stimulated Macrophages Restrict Mycobacterium tuberculosis Growth by Autophagy and Release of Nitric Oxide.

IFN alfacon-1 (Infergen) is a synthetic form of Interferon (IFN)-α2b. Infergen has immunomodulatory activity and is effective against hepatitis C virus. However, the effect of Infergen (IFG) on Mycobacterium tuberculosis (Mtb) has not yet been reported. Therefore, for the first time, we have studied the influence of IFG in constraining the survival of Mtb in human macrophages. We observed that IFG significantly enhanced the maturation and activation of macrophages. Further, it substantially augmented the secretion of IL-6, nitric oxide (NO) and antigen uptake. Moreover, macrophages exhibited remarkably higher bactericidal activity, as evidenced by reduction in the Mtb growth. Infergen-mediated mechanism was different from the type-1 interferons; since it worked through the activation of NF-κB, phosphorylation of STAT-3 and Akt-PI3K that improved the bactericidal activity through autophagy and NO release. In future, IFG immunotherapy can be a novel strategy for treating patients and controlling TB.