Experimental hematology最新文献

The hematopoietic stem cell (HSC) pool is highly heterogeneous, including a subset of HSCs that rarely contribute to homeostatic hematopoiesis but can be recruited to cycle under stress. Such deeply quiescent HSCs with a low division history perform best when transplanted. However, few tools exist to isolate and interrogate mechanisms regulating the balance between quiescence and activation required to maintain HSC integrity. We recently reported Gprasp2 (G-protein Coupled Receptor (GPCR)-associated Sorting Protein 2) as an HSC regulator during transplantation. Involved in post-endosomal sorting to the lysosome, GPRASP2 is HSC-enriched and heterogeneously expressed in single HSCs. Further, low Gprasp2 (Gprasp2low) expressing HSCs are transcriptionally programmed for lineage-specific differentiation and cell cycling relative to Gprasp2high HSCs. Using our Gprasp2-reporter mouse, we serially transplanted Gprasp2high/low HSCs and found that Gprasp2high HSCs have slower repopulation kinetics with balanced reconstitution and increased, prolonged blood output compared to Gprasp2low HSCs. Single Gprasp2high/low HSC transplantation confirms Gprasp2high clones with delayed yet robust, balanced blood output. Proteomic profiling reveals Gprasp2high HSCs are programmed for quiescence, confirmed by assaying in vivo cycling kinetics. Prospectively, elevated GPRASP2 maintains HSC quiescence by limiting GPCR cell-surface availability via targeted lysosomal degradation. Assessed GPCR candidates show decreased cell surface expression on Gprasp2high HSCs and increased expression on Gprasp2low HSCs. Gprasp2 is hierarchically restricted and heterogeneously expressed in human HSCs, and human Gprasp2high/low HSCs are transcriptionally distinct. Cumulatively, GPRASP2 marks a subset of quiescent, durable repopulating HSCs that preserve function by limiting GPCR cell-surface availability.

Using cord blood units (CBU) for hematopoietic stem cell transplants (HSCT) improves match compatibility and reduces graft versus host disease. Limited cell numbers hinder widespread adult use. In vitro expansion overcomes dose limits, but techniques for long-term HSC expansion are underdeveloped, prompting the need for novel molecular targets. Studies in mice have implicated Mitofusin 2 (MFN2) activity as necessary to maintain potent HSCs in vitro. Recent studies have described efficacious small molecule agonists of MFN2 fusion. Thus, we hypothesized that MFN2 agonist treatment during short-term UCB cell expansion could enhance HSC repopulating function. Our study revealed phenotypic HSCs expanded with MFN2 agonists displayed a highly significant increase in long-term chimerism for both primary and secondary xenografts of MFN2 agonist HSC culture recipients compared to vehicle, revealing MFN2 fusion as necessary for human HSC function. MFN2 agonist-treated HSCs displayed upregulation of genes associated with ribosome, stress granules, and autophagy are hallmarks of HSC maintenance mechanisms. HSC cultures exposed to MFN2 agonist exhibited decreased protein translation by OP-Puro assay, heightened lysosome count and acidification using lysotracker dyes, and increased LC3B puncta, indicating heightened autophagy. Network analysis of DGEs suggested upstream regulation via MTOR signaling. HeLa cells treated with MFN2 agonist or rapamycin displayed increased TFEB nuclear levels (lysosomal biogenesis master regulator), indicating enhanced autophagy. Additionally, reduced S6 phosphorylation, a downstream target of MTOR, and direct MFN2-MTOR interaction indicated by immunoprecipitation assays, suggests MFN2 inhibits MTOR signaling. Overall, our findings elucidate a novel mechanism whereby MTOR inhibition via MFN2 induces catabolic programs to maintain HSC function in vitro.

Hematopoietic stem cells (HSCs) persist throughout life to regenerate blood cells lost to physiological turnover, injury and disease. But how stem cells preserve their immense regenerative potential while simultaneously sustaining their remarkable longevity remains a mystery. The regulation of protein homeostasis (proteostasis) has emerged as being fundamentally and preferentially important for HSCs. Proteostasis is maintained by an integrated network of physiological mechanisms and stress response pathways that coordinate protein synthesis, folding, trafficking and degradation to regulate the content and quality of the proteome. Many of these highly-conserved pathways have long been thought of as housekeeping functions, performed similarly by most cells. However, we found that HSCs exhibit unique cell-type-specific configuration of the proteostasis network that is critical for preserving their fitness, health and longevity. Young adult HSCs exhibit unusually low protein synthesis rates to restrict the biogenesis of misfolded proteins in vivo and utilize non-canonical protein trafficking and degradation pathways to limit the accumulation of protein aggregation to preserve their long-term self-renewal potential. Challenges to proteostasis during aging cause HSCs to remodel their proteostasis network to sustain their fitness and regenerative potential. However, adapting to these selective pressures comes at the cost of increasing the risk of premalignant and malignant disease. Overall, unique and dynamic regulation of proteostasis is key for balancing stem cell regeneration and longevity.

Background

Cardiac diseases pose a global health challenge, necessitating the identification of biomarkers for early detection and management. Inflammation plays a role in many cardiac diseases, highlighting the need for inflammatory markers. Mean platelet volume (MPV), is a promising inflammatory biomarker in cardiac diseases.

Methods

Complete Blood Count reports were collected and observed in treatment-naive patients from January 2021 to January 2023, particularly in those having cardiac diseases (Myocardial infarction {MI}, Pericarditis, Myocarditis, and Atrial fibrillation {Afib}), respective changes in MPV counts were observed and tallied. This prospective study investigated the correlation of MPV with various cardiac diseases, considering the age and sex disparities.

Results

The study comprised 87 inpatients with cardiac diseases, the patients with MPV values exceeding 11 fl are as shown on the graph. The incidence of MPV more than 11fl in the present study is as follows: 50% of AFib exhibited the highest MPV 12.1fl (male 75 yrs). 18.8% of Myocarditis had the highest MPV of 11.3fl in both genders, (average 21.5 yrs). 11.9% of Pericarditis showed the highest MPV 11.9 fl (female 18yrs) 12.4% MI recorded the highest MPV of 11.9 fl in both genders (average 47.5 years).

Conclusion

Based on our evidence and the strong association of platelets in the pathophysiology of cardiac diseases we conclude MPV has significance in the following order Afib, Myocarditis, Pericarditis, and MI. Age, sex, and menopausal status also influence the MPV dynamics. Integrating MPV into clinical practice facilitates personalized preventive strategies and risk stratification.

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.