Epidemiology & Prognostication of Acute Myeloid Leukemia (AML) in India – A Brief Review

AML is represented by aggregation of ≥20% myeloid immature cells in the spongy marrow and most generally raise in the peripheral blood. A cytogenetic finding plays a vital role in the risk management and stratification of AML patients. AML is genetically and functionally a heterogenous malignant disease. In the western world leukemia is one of the most common among all cancers. India ranked 3rd in cancer disease after US and China. Management of AML is challenging specially for medium and low-income countries as it causes a huge economic burden to the patient and family. Molecular prognostic biomarkers will help in redefining the risk stratification more efficiently. Targeted drugs in pre-clinical and clinical trial recorded to have promising outcomes in AML. In this review we summarize the prevalence, incidence, and prognostication of AML.

Emerging Roles of Myeloid-Derived Suppressor Cells in Diabetes

Diabetes is a syndrome characterized by hyperglycemia with or without insulin resistance. Its etiology is attributed to the combined action of genes, environment and immune cells. Myeloid-derived suppressor cell (MDSC) is a heterogeneous population of immature cells with immunosuppressive ability. In recent years, different studies have debated the quantity, activity changes and roles of MDSC in the diabetic microenvironment. However, the emerging roles of MDSC have not been fully documented with regard to their interactions with diabetes. Here, the manifestations of MDSC and their subsets are reviewed with regard to the incidence of diabetes and diabetic complications. The possible drugs targeting MDSC are discussed with regard to their potential of treating diabetes. We believe that understanding MDSC will offer opportunities to explain pathological characteristics of different diabetes. MDSC also will be used for personalized immunotherapy of diabetes.

A Durable Anatomy with Local Plasticity Enables Normal and Stress Hematopoiesis

Knowledge of the anatomy of each tissue and the relationships between progenitors and daughter cells is necessary to understand physiology and pathology. The anatomy of hematopoiesis in the marrow remains largely unknown. Here we identify strategies to image all steps of blood cell production in the mouse sternum using confocal microscopy. We show that long-distance migration of multipotent progenitors, lineage-committed progenitor recruitment to vessels, and generation of lineage-specific oligoclonal structures that are the main production sites for immature cells are key features of the anatomy of blood production. This structural organization is extremely durable and resilient to insults as it was maintained after hemorrhage, Listeria monocytogenes infection, and aging (80-weeks). Importantly, this anatomy is also enabled with local plasticity as production sites for each blood lineage selectively expand/contract in respond to insults followed by a return to homeostasis. We used immunophenotyping to identify fifty-six surface markers that can be combined to image any populations of interest. For example, we found that ESAM is selectively expressed in 100% of LT-HSC, 90% of ST-HSC, 70% of MPP2 and MPP3, 30% of MPP4, 10% of CMP, and 90% of MkP and megakaryocytes but absent in more mature cells. Transplantation experiments revealed that all functional LT- and ST-HSC, MPP2, MPP3 and CMP were contained -exclusively- in the ESAM positive fraction (p<0.05 when compared with ESAM- cells n= 7 mice per group). ESAM + MPP4 displayed 5-fold more engraftment than ESAM - MPP4 (p<0.05). Combining ESAM with classical HSPC markers allowed imaging of all LT-HSC, ST-HSC, MkP, Pre-MegE, MPP2 and a mixed population of MPP3, CMP and MPP4. We developed similar strategies to map erythropoiesis (Pre-MegE → Pre-CFU-E → CFU-E → early erythroblast → late erythroblast → reticulocyte → erythrocyte) and lymphopoiesis (CLP→ PreProB → ProB →PreB→ Immature B). All strategies allowed clonal fate-mapping using Ubc-cre ERT2:confetti mice. In this model tamoxifen treatment leads to irreversible expression of one out of four fluorescent proteins. We found that multipotent and oligopotent progenitors are found as single cells, evenly distributed through the marrow (e.g. mean LT-HSC distance to closest ST-HSC, MPP2, MPP3, MkP, Pre-MegE >100 μm, no different from random simulations, n=41 LT-HSC from 5 sternum sections of 4 wild-type mice) and are clonally unrelated between them. Multipotent and oligopotent progenitors reside near sinusoids (mean distance =9.7 μm) but this association is not different from that observed for random cells. In contrast, as progenitors become lineage-restricted, they localize to arterioles (for lymphoid progenitors) or sinusoids (all other progenitors) where they enter oligoclonal structures that are the main production sites for immature cells in each lineage. Each production site has distinct architectures: lymphoid sites are characterized by tight clusters of PreProB cells surrounding CLP; erythroid sites are characterized by strings of 4-21 CFU-E decorating the surface of sinusoids with early erythroblasts differentiating orthogonally to the vessel surface; in megakaryocyte sites one or two megakaryocyte progenitors produce megakaryocytes that decorate blood vessels over large (>200μm 3) marrow regions. We previously showed that production sites for neutrophils contain 1 or 2 granulocyte progenitors tightly clustered with preneutrophils and that sites for monocytes/dendritic cells contain loose clusters of dendritic cells surrounding monocyte dendritic cell progenitors (Zhang Nature 2021). This spatial architecture is durable and resilient and is maintained after acute challenge via phlebotomy, L. monocytogenes infection, or physiological aging (80-week-old mice). However, we also observed plasticity of production sites. Two days after phlebotomy we found increases in erythroid site numbers (368 vs 945 per mm 3, p<0.05). These expansions were reversed by day 8 after phlebotomy. Similarly, infection led to increases in the size of neutrophil and dendritic cell production sites (~2-fold by day 6 post-infection) but these changes are reverted by day 20 post-infection. In summary, we have developed strategies that allow imaging of differentiation in situ and defined a complex - but durable and plastic- anatomy for the hematopoietic tissue. Disclosures No relevant conflicts of interest to declare.

Landscape of Myeloid-derived Suppressor Cell in Tumor Immunotherapy

Myeloid-derived suppressor cells (MDSC) are a group of immature cells that produced by emergency myelopoiesis. Emerging evidences have identified the vital role of MDSC in cancer microenvironment, in which MDSC exerts both immunological and non-immunological activities to assist the progression of cancer. Advances in pre-clinical research have provided us the understanding of MDSC in cancer context from the perspective of molecular mechanism. In clinical scenario, MDSC and its subsets have been discovered to exist in peripheral blood and tumor site of patients from various types of cancers. In this review, we highlight the clinical value of MDSC in predicting prognosis of cancer patients and the responses of immunotherapies, therefore to propose the MDSC-inhibiting strategy in the scenario of cancer immunotherapies. Phenotypes and biological functions of MDSC in cancer microenvironment are comprehensively summarized to provide potential targets of MDSC-inhibiting strategy from the aspect of molecular mechanisms.

Insights into in vivo adipocyte differentiation through cell-specific labeling in zebrafish

White adipose tissue hyperplasia has been shown to be crucial for handling excess energy in healthy ways. Though adipogenesis mechanisms have been underscored in vitro, we lack information on how tissue and systemic factors influence the differentiation of new adipocytes. While this could be studied in zebrafish, adipocyte identification currently relies on neutral lipid labeling, thus precluding access to cells in early stages of differentiation. Here we report the generation and analysis of a zebrafish line with the transgene fabp4a(-2.7):EGFPcaax. In vivo confocal microscopy of the pancreatic and abdominal visceral depots of transgenic larvae, revealed the presence of labeled mature adipocytes as well as immature cells in earlier stages of differentiation. Through co-labeling for blood vessels, we observed a close interaction of differentiating adipocytes with endothelial cells through cell protrusions. Finally, we implemented hyperspectral imaging and spectral phasor analysis in Nile Red labeled transgenic larvae and revealed the lipid metabolic transition towards neutral lipid accumulation of differentiating adipocytes. Altogether our work presents the characterization of a novel adipocyte-specific label in zebrafish and uncovers previously unknown aspects of in vivo adipogenesis.

Serum estrogen and its soluble receptor levels in Egyptian patients with acute leukemia: case-control study

Background Acute leukemias are malignant neoplastic diseases that arise from either lymphoid [ALL] or myeloid [AML] cell lines that are distinguished by the proliferation of BM non-functional immature cells and subsequently released into the bloodstream. ALL is prevalent malignancy in young, while AML in older. Diagnosis is usually routinely performed through peripheral blood count and smear then confirmed by BM aspirate. It is remarkable to notice that leukemia can be manifested at high, low, and even at normal leucocyte count. While treatment results have improved steadily over the last decades in younger and adults, limited changes have been in survival among subjects of age > 60 years. Aim of the work is to measure the serum estrogen [E2] and its soluble receptor [ER] levels in acute leukemia patients and extrapolate its possible clinical significance. This study included 40 [20 females and 20 males] healthy volunteers clinically free from any disease, 40 [20 females and 20 males] AML patients, and 40 [20 females and 20 males] ALL. To all subjects, serum E2 and its soluble ER level were investigated by ELISA. Results Serum E2 [pg/ml] level was lower in AML and ALL female and male patients groups than control group. Serum ER [ng/ml] level was lower in AML and ALL female and male patients groups than control group. Conclusion Estimation of serum E2 and its soluble ER level is of edifying diagnostic value. Determination of serum E2 and its soluble ER level in AML and ALL patients is of value in deciding treatment therapeutic target protocol.

Stem Cells Applications in Therapeutics and Site-Specific Genome Editing Through CRISPR Cas9 System

Stem cells are immature cells that have ability to differentiate into all specific and mature cells in body. The two main characteristics of stem cells are self-renewable and ability to differentiate into all mature, functional and adult cells types. There are the two major classes a) pluripotent stem cells which have potential to differentiate in all adult cell and b) multipotent stem cells which have capacity to differentiate into many adult cells but not in all cell types.

Identification of a novel subset of alveolar type 2 cells enriched in PD-L1 and expanded following pneumonectomy

Alveolar type 2 (AT2) cells are heterogeneous cells; where specialised AT2 subpopulations within this lineage exhibit stem cell properties. However, the existence of quiescent, immature cells within the AT2 lineage, which are activated during lung regeneration, is unknown.SftpcCreERT2/+; tdTomatoflox/flox mice were used for the labelling of AT2 cells and labeled subpopulations were analysed by flow cytometry, qPCR, ATAC-seq, gene arrays, pneumonectomy, and culture of precision-cut lung slides. ScRNA-seq data from human lungs were analysed.In mice, we detected two distinct AT2 subpopulations with low tdTomato level (TomLow) and high tdTomato level (TomHigh). TomLow express lower level of AT2 differentiation markers, Fgfr2b and Etv5, while TomHigh, as bona fide mature AT2 cells, show higher levels of Sftpc, Sftpb, Sftpa1, Fgfr2b and Etv5 expression. ATAC-seq analysis indicates that TomLow and TomHigh constitute two distinct cell populations with specific silencing of Sftpc, Rosa26 and cell cycle gene loci in TomLow. Upon pneumonectomy, the number of TomLow but not TomHigh cells increases and TomLow upregulate the expression of Fgfr2b, Etv5, Sftpc, Ccnd1 and Ccnd2 compared to sham. TomLow cells overexpress PD-L1, an immune inhibitory membrane receptor ligand, which is used by flow cytometry to differentially isolate these two sub-populations. In the human lung, data mining of a recent scRNA-seq AT2 dataset demonstrates the existence of a PD-L1Pos population. Therefore, we have identified a novel population of AT2 quiescent, immature progenitor cells in mouse that expand upon pneumonectomy and provided evidence for the existence of such cells in human.

Protocol for the Use of a Novel Bioreactor System for Hydrated Mechanical Testing, Strained Sterile Culture, and Force of Contraction Measurement of Tissue Engineered Muscle Constructs

Bioreactor systems are built as controlled environments for biological processes and utilized in the field of tissue engineering to apply mechanical, spatial, and chemical cues to developing tissue grafts. Often the systems are applied to instruct differentiation and maturation of the cells grown inside. Perhaps the most obvious targets for strain and compression-based bioreactors are mechanically active tissues, as it is hypothesized that biomimetic mechanical environments instruct immature cells to form differentiated tissues. One such tissue, skeletal muscle, has been identified as a key candidate for strain application due to the close structure-function relationship of myofibers. Here we detail the multiple uses of a custom-built bioreactor system in combination with electrospun fibrin microfibers for muscle tissue engineering. Outlined below are the methods used in the system to test the mechanical properties of hydrogel-based scaffolds in an aqueous environment, including Young’s modulus and poroelasticity. Additionally, we demonstrate the application of tensile strain to sterile cell cultures grown on electrospun scaffolds and perform end-point testing of tissue contractility with the addition of an electrode.

Corneocytes: Relationship between Structural and Biomechanical Properties

<b><i>Background:</i></b> Skin is the interface between an organism and the external environment, and hence the stratum corneum (SC) is the first to withstand mechanical insults that, in certain conditions, may lead to integrity loss and the development of pressure ulcers. The SC comprises corneocytes, which are vital elements to its barrier function. These cells are differentiated dead keratinocytes, without organelles, composed of a cornified envelope and a keratin-filled interior, and connected by corneodesmosomes (CDs). <b><i>Summary:</i></b> The current review focusses on the relationship between the morphological, structural, and topographical features of corneocytes and their mechanical properties, to understand how they assist the SC in maintaining skin integrity and in responding to mechanical insults. <b><i>Key Messages:</i></b> Corneocytes create distinct regions in the SC: the inner SC is characterized by immature cells with a fragile cornified envelope and a uniform distribution of CDs; the upper SC has resilient cornified envelopes and a honeycomb distribution of CDs, with a greater surface area and a smaller thickness than cells from the inner layer. The literature indicates that this upward maturation process is one of the most important steps in the mechanical resistance and barrier function of the SC. The morphology of these cells is dependent on the body site: the surface area in non-exposed skin is about 1,000–1,200 μm², while for exposed skin, for example, the cheek and forehead, is about 700–800 μm². Corneocytes are stiff cells compared to other cellular types, for example, the Young’s modulus of muscle and fibroblast cells is typically a few kPa, while that of corneocytes is reported to be about hundreds of MPa. Moreover, these skin cells have 2 distinct mechanical regions: the cornified envelope (100–250 MPa) and the keratin matrix (250–500 MPa).