Experimental hematology最新文献

This case report presents a rare condition of pulmonary infiltrate with eosinophilia syndrome (PIES). Incidence of PIES with bronchial asthma has been reported (5 - 11 %). However, actual incidence rate of idiopathic cases is difficult to ascertain. 82-year-old lady with Type 2 diabetes mellitus, lacunar stroke, paroxysmal atrial tachycardia and pernicious anaemia, presented with occasional dry cough and weight loss. Computed Tomography (CT) thorax showed a dense lesion (diameter of 3.6cm) in the right lung apex with pleural fibrosis. Initially, bronchogenic cancer was suspected, but was not confirmed by biopsy. Two years later, Eosinophilia - eosinophil count of 1328 (40-400) 10E6/L was seen in blood. The lung infiltrate continued to be the same.

PIES is characterized by prominent infiltration of lung parenchyma by eosinophils which release pro-inflammatory cytokines, reactive oxygen species, and cationic proteins, leading to development of pseudo-tumorous lesion and fibrosis. Eosinophilic lung diseases include Loeffler syndrome, Tropical pulmonary eosinophilia, Eosinophilic granulomatous with polyangiitis, and Hypereosinophilic syndrome. Thorough history, physical exam, imaging studies, bronchoscopy, blood and sputum analysis where high levels of an absolute eosinophil count greater than 400 10E6/L is needed to confirm diagnosis.

Elevated C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), interleukin -6 (IL-6), Bronchoalveolar Lavage Fluid Analysis, and plasma cytokines - IL-5, IL-4, IL-13, and tumour necrosis factor-alpha provides valuable insight for diagnosing PIES.

Therefore, in patients with pulmonary lesions and eosinophilia, cytokine tests can facilitate accurate and immediate diagnosis.

However, the cause of PIES in our patient remains idiopathic. The treatment plan established for the patient was symptomatic management and observation.

Background

:Neutrophil-Lymphocyte Ratio (NLR), a simple marker to assess immune response (IR) is derived by dividing absolute neutrophil count (ANC) by absolute lymphocyte count (ALC) obtained through a routine complete blood cell count (CBC). Neutrophils, an integral part of the innate IR, are tumour promoting and immune suppressive role. Neutrophilia is usually accompanied by relative lymphocytopenia representing a significant decline in cell-mediated adaptive IR consequently promoting metastasis.

Methods

CBC and Histopathology reports of carcinomas patients in a 2-year period. The ANC and ALC obtained were used to calculate the NLR. Inclusion Criteria: patients with cancer and NLR calculated from the CBC prior to surgery. Exclusion criteria: Cancer patients with any infection or inflammatory process , febrile patients and those on chemotherapy

Results

58 patients (33 females and 25 males) Mean age patients in males and females were 54.2 and 56.78 respectively. In male and female patients with NLR above 10, the most prevalent cancer was in the rectum and the buccal mucosa, respectively. Male patients with stages T0N0M0- T2N1M0 had lower NLR values in the 3.428 to 9.44 range. Male patients with T3N0M0-T4N1M0 stage had an NLR range of 2.54 to 6.38. In the female patients with NLR above 10, two have tongue cancer with staging from T2N0M0-T4bN2M0 and one buccal mucosal cancer patient with T4N2cM0. The lower values of the NLR is more prevalent in female patients with breast cancer.

Conclusion

Rectosigmoid cancer had the highest NLR. Buccal mucosa and tongue cancers had lower NLR, with few outliers. The lowest NLR was seen in breast cancer patients, showing a possible gender correlation. Regarding prognosis, our data suggests that age and gender of the patient, anatomic location and the TNM of the cancer are significant.

Hematopoietic stem cell (HSC) transplantation is a life-saving treatment for both malignant and inherited hematological diseases. Umbilical cord blood (UCB) serves as a widely available source of HSCs; however, low cell numbers remain a limiting barrier for adult transplantations. A safe and efficient method to expand and maintain the number of engraftable blood stem cells ex vivo could greatly increase the proportion of UCB units that can be used for transplantations. From high throughput genetic screens, we have identified several lentivirally delivered shRNA molecules with profound expanding effects on human hematopoietic stem and progenitor cells (HSPCs). Here, we reasoned that the corresponding synthetic siRNAs to these highly potent shRNAs, if delivered transiently, could serve as chemical inducers of ex vivo HSC expansion without the permanent effects that would accompany lentiviral delivery. However, a key challenge would be to find a safe and non-toxic approach to deliver these siRNAs, as standard transfection methods for siRNA show substantial toxicity in primary cells. To this end, we recently established a nanotechnology-based protocol that enables gentle delivery of biomolecules through tube-like structures directly into the cytoplasm of HSCs in a completely non-toxic manner. We optimized this method for delivery of siRNA molecules into UCB derived HSPCs and tested a number of siRNAs converted from the shRNAs identified in our previous screens. In particular one siRNA molecule (si31) strongly enhanced the propagation of CD34+CD90+ HSPCs over 5 days of in vitro culture (4-fold compared to control). We are currently performing more detailed functional tests of the expanded cells both in vitro and in vivo. If successful, our approach has the potential to become a new method for HSC expansion, which may be relevant for both research and clinical applications.

Delineating cell-intrinsic and -extrinsic drivers of hematopoietic stem cell (HSC) self-renewal is critical to improve efforts in ex vivo HSC expansion and to better understand leukemia cell biology. To characterize molecular changes at the proteome level, we applied miniaturized, multiplexed sample preparation protocols in combination with mass spectrometry (MS)-based quantitative proteomics to compare normal and Tet2-deficient HSCs, and data-independent acquisition (DIA)-MS to characterize the extracellular environment of HSCs in vivo and during ex vivo expansion. We show that both the cellular and secreted proteome accurately stratify HSCs based on functional potency and mutational status and identify novel molecular components not captured in transcriptomic analyses. On the pre-leukemia side, we reveal that Tet2-deficient HSCs have altered expression of extracellular matrix (ECM) proteins and that interaction with these proteins in artificial niches affects cellular function. Extracellular proteomic analysis further reveals that Tet2-deficient cells create a microenvironment is pro-inflammatory and pro-thrombotic even in young, asymptomatic animals. In HSC expansion assays, proteomics identifies the requirement for intact DNA repair pathways, specifically mismatch repair proteins, as key components of HSC clones capable of extensive self-renewal compared to unsuccessful expansion cultures. Analysis of the secretome of unsuccessful cultures further identifies mast cell proteins as predictive of failure to expand engraftable HSCs. Collectively these data highlight novel proteins to which transcriptomic studies are blind, and open new opportunities for HSC expansion and preleukemic biology, paving the way for future ex vivo and in vivo modulation of HSC function via manipulation of the cells and their extracellular environment.

Hematopoiesis changes upon aging, including expansion of hematopoietic stem cells (HSCs) and megakaryocyte progenitors (MkPs), which undergo changes in phenotype and function. Platelets, the progeny of MkPs, also undergo age-related alterations that drive inflammation and cardiovascular and thrombotic disease; major health concerns. We discovered a non-canonical (ncMkP) population that specifically expands upon aging. Compared to their canonical (cMkP) counterparts, ncMkPs exhibit increased expansion and survival with age. Upon transplantation and serial blood assessment, both young and old cMkPs, and young ncMkPs, contributed to similar levels of platelet formation. In contrast, aged ncMkPs specifically produced greater platelet output. The progressive increase in ncMkPs with age appears to be, in part, originating from HSCs as in vitro generation of ncMkPs was significantly enhanced by aged compared to young HSCs. Further, single-cell functional analysis revealed that this was not driven by individual HSC clones, a finding substantiated by single-cell RNA sequencing of young and old bone marrow (BM). Progressively increasing, chronic inflammation is thought to be a driver of aging. Inducing chronic inflammation in young mice partially phenocopies known features of aged mice, including altered platelet output and expanded HSC numbers, implicating its role in influencing age-related platelet production. Direct assessment of inflammatory modulation of young and old HSCs, cMkPs, and ncMkPs via in vitro culture revealed distinct age- and population-specific responses, uncovering surprisingly distinct and differential roles for direct inflammatory reprogramming. These new data provide mechanistic insight to cell-based causes of aging-related adverse thrombotic events and may offer targets for mitigation and rejuvenation.

We study how transcriptional and epigenetic programmes are played out on chromatin spanning the alpha globin cluster as hematopoietic cells undergo lineage fate decisions and differentiation to form erythroid cells. The alpha globin cluster and its regulatory elements are silenced in early progenitors, poised for expression in later progenitors and fully expressed during terminal erythroid differentiation. Using a variety of approaches we have established the order in which silencing factors are removed, activating transcription factors bind and epigenetic modifications occur. In addition, we have shown how chromosomal conformation and nuclear sub-localisation change during hematopoiesis. Detailed experimental analysis of individual elements is providing insight into the fundamental regulatory elements of the genome. Natural cis and trans acting mutations that cause alpha thalassaemia provide additional insight into how the long-range regulatory elements may interact with the promoters of the globin genes and other flanking genes to activate their expression. Together these observations establish some of the general principles by which genes within their natural chromosomal environment are switched on and off during differentiation and development and how these processes are perturbed in human disease.

Lmo2 has largely been defined by its oncogenic role in T-cell acute lymphoblastic leukemia; however, Lmo2’s natural role is not fully captured by this overexpression context. We hypothesize that in normal T cell development, Lmo2 contributes to a mechanism that stalls entry into the T cell pathway by initially sequestering E proteins in a TF complex, thus preventing E protein homodimerization which would otherwise push the T cell program forward. To validate, we knocked out Lmo2 in bone marrow-derived progenitor cells and analyzed development in the OP9-Dll1 co-culture system. Previous knockout (KO) experiments were conducted at timepoints where Lmo2 is already downregulated, so we utilized input from the PVA culture system to focus on the earliest developmental stages. Lmo2-KO cells differentiated at least three days faster than controls, measured by cell surface markers, and our initial bulk RNA-seq results confirm this acceleration phenotype: CD25- Lmo2-KO cells upregulate multiple features of the T cell program, including Tcf7, Gata3, Bcl11b, Ets1, Thy1, Rag1, Rag2, Cd3 and significant transcriptional activation of the TCRg and TCRb loci. This could be explained by increased Notch1 and Notch3 expression, thus increasing sensitivity to the Notch ligand-rich environment, though the Notch-response gene Hes1 was not affected. Interestingly, certain progenitor cell program members are Lmo2-activated (Spi1, Mef2c, Bcl11a, Hhex) while other canonical members are not influenced by Lmo2 (Hoxa9, Erg, Flt3), and Lmo2 loss causes specific downregulation of the myeloid signature, including C/EBP family members, Csf3r, Csf1r, Mpo, Elane and Gzma. We explore these findings, along with E protein binding data, to reveal the many roles Lmo2 plays in controlling T cell pathway entry via E protein sequestration, altered Notch signaling and/or the persistence of competing programs.

Intratumor heterogeneity (ITH) is a main cause of therapy resistance and relapse in leukemia. While genetic mutations play an important role in clonal evolution during disease development, accumulating evidence suggests that non-genetic mechanisms are also major drivers of ITH. Here we used single-cell trimodal approaches measuring simultaneously RNA, chromatin and over 150 cell surface proteins as well as gene regulatory network construction to decipher the epigenetic basis of tumor heterogeneity in T cell acute lymphoblastic leukemia (T-ALL), an aggressive cancer of thymocytes with a high relapse rate. Our results reveal an unexpected contribution of non-genetic mechanisms in the tumor-initiating process in T-ALL, and allowed us to identify patient-specific combinations of cell surface proteins with critical implications for future targeted therapies.

Our ability to characterize hematopoietic differentiation has been revolutionized by novel single cell technologies of which scRNAseq is undoubtedly the most influential. While this has led to novel insights into early hematopoietic decision events, e.g. how stem cells decide on their future fates, it is important to remember that mRNA levels are only proxies for the levels of the true cellular workhorses, i.e. the proteins. Since protein levels are regulated by additional cellular events such as translational initiation, elongation and protein decay, there is not necessarily a one-to-one relationship between mRNA and protein levels. Therefore, relying only on mRNA levels for the characterization of complex biological systems comes at a risk of missing important biological information.

Here, we present the first single-cell proteomics by Mass Spectrometry (scp-MS) based map of the human CD34+ hematopoietic stem and progenitor cells (HSPCs) compartment (>2,500 cells averaging approximately 1,000 proteins/cell). We used the GLUE autoencoder to integrate the scp-MS data with corresponding scRNAseq data to generate a common embedding, allowing us to compare mRNA and protein levels from similar computationally inferred cells. Trajectory analysis demonstrated high concordance between mRNA and protein levels along the granulocytic/monocytic and erythroid trajectories, whereas early HSC differentiation events were associated with significant lower concordance levels, highlighting the importance of protein-level data. We leveraged these findings to identify and validate novel regulators of early hematopoietic differentiation. This work demonstrates the feasibility and potential of scp-MS to gain novel insights into normal and, in the future, malignant hematopoiesis.

While cancer cells have been identified to have a metabolism distinct from normal cells for almost a century, the clinical success of targeting metabolic enzymes for cancer therapy remains limited. A key reason for this is the ability of cells to rewire their metabolism and adapt to the blockage of a single pathway. Here, we use acute myeloid leukemia (AML), a highly lethal blood cancer, as a model to investigate and target metabolic plasticity. We treated human AML cell lines with combinations of pharmacological compounds targeting metabolic enzymes across central carbon metabolism. An unexpected synthetic lethality was observed when AML cells were simultaneously treated with BPTES, an inhibitor of glutaminase, the rate-limiting enzyme in glutamine catabolism, and TOFA, an inhibitor of acetyl-CoA carboxylase 1, a key enzyme in de novo lipogenesis. Sensitivity to this metabolic inhibitor combination was equally seen in primary AML patient samples, but healthy hematopoietic stem and progenitor cells were not affected. Stable isotope tracing and lipidomics experiments revealed that AML cells are highly lipogenic and have a distinct lipid profile characterized by a high degree of fatty acid saturation. However, we unexpectedly found that the cytotoxic effects of TOFA are not due to its inhibition of lipogenesis, but because this compound also inhibits protein S-acyltransferases. Protein S-acylation in AML cells specifically requires 18-carbon long fatty acids and is essential to maintain correct mitochondrial function and allow metabolic adaptation to inhibition of glutaminolysis. Extended screening further showed that not only AML, but many different cancer types are sensitive to combined inhibition of protein S-acylation and glutaminolysis, highlighting this as a promising strategy to overcome metabolic plasticity and selectively eliminate cancer cells.