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Differential diagnosis of pancytopenia with bone marrow (BM) hypoplasia represented by aplastic anemia (AA) is often challenging for physicians, because no laboratory tests have been established, until recently, to distinguish immune-mediated BM failure, which includes acquired AA (aAA) and a subset of low-risk myelodysplastic syndrome (MDS), from non-immune BM failure, which is primarily caused by genetic abnormalities in hematopoietic stem cells (HSCs). HSCs of healthy individuals often undergo somatic mutations, and some acquire phenotypic changes that allow them to escape immune attack against themselves. Once an immune attack against HSCs occurs, HSCs that undergo somatic mutations survive the immune attack and continue to produce their progenies with the same genetic or phenotypic changes. The presence of mature blood cells derived from mutated HSCs in the peripheral blood serves as evidence of the immune-mediated destruction of HSCs. Glycosylphosphatidylinositol-anchored protein-deficient (GPI[-]) blood cells and HLA class I allele-lacking (HLA[-]) leukocytes are two major aberrant cell types that represent the immune mechanism underlying BM failure. This review focuses on the importance of identifying immune mechanisms using laboratory markers, including GPI(-) cells and HLA(-) leukocytes, in the management of BM failure., (© 2023. Japanese Society of Hematology.)

Acquired aplastic anemia (AA) is a bone marrow failure (BMF) disease, characterized by fatty bone marrow (BM) and BM hypocellularity resulted from auto-immune dysregulated T cells-mediated destruction of BM haemopoietic stem cells (HPSC). The objective of this study was to investigate potential therapeutic effect of irisin, a molecule involved in adipose tissue transition, on AA mouse model. Our results showed that the concentration of irisin in serum was lower in AA patients than in healthy controls, suggesting a role of irisin in the pathogenesis of AA. In the AA mice, irisin administration prolonged the survival rate, prevented or attenuated peripheral pancytopenia, and preserved HPSC in the BM. Moreover, irisin also markedly reduced BM adipogenesis. In vitro results showed that irisin increased both cell proliferation and colony numbers of HPSC. Furthermore, our results demonstrated that irisin upregulated the expression of mitochondrial ATPase Inhibitory Factor 1 (IF1) in HPSC, inhibited the activation of mitochondrial fission protein (DRP1) and enhanced aerobic glycolysis. Taken together, our findings indicate novel roles of irisin in the pathogenesis of AA, and in the protection of HPSC through stimulation of proliferation and regulation of mitochondria function, which provides a proof-of-concept for the application of irisin in AA therapy., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Clinical trials have shown that lentiviral-mediated gene therapy can ameliorate bone marrow failure (BMF) in nonconditioned Fanconi anemia (FA) patients resulting from the proliferative advantage of corrected FA hematopoietic stem and progenitor cells (HSPC). However, it is not yet known if gene therapy can revert affected molecular pathways in diseased HSPC. Single-cell RNA sequencing was performed in chimeric populations of corrected and uncorrected HSPC co-existing in the BM of gene therapy-treated FA patients. Our study demonstrates that gene therapy reverts the transcriptional signature of FA HSPC, which then resemble the transcriptional program of healthy donor HSPC. This includes a down-regulated expression of TGF-β and p21, typically up-regulated in FA HSPC, and upregulation of DNA damage response and telomere maintenance pathways. Our results show for the first time the potential of gene therapy to rescue defects in the HSPC transcriptional program from patients with inherited diseases; in this case, in FA characterized by BMF and cancer predisposition.

Ribosomal protein (Rp) gene haploinsufficiency can result in Diamond-Blackfan Anemia (DBA), characterized by defective erythropoiesis and skeletal defects. Some mouse Rp mutations recapitulate DBA phenotypes, although others lack erythropoietic or skeletal defects. We generated a conditional knockout mouse to partially delete Rps1 2. Homozygous Rps12 deletion resulted in embryonic lethality. Mice inheriting the Rps12 KO/+ genotype had growth and morphological defects, pancytopenia, and impaired erythropoiesis. A striking reduction in hematopoietic stem cells (HSCs) and progenitors in the bone marrow (BM) was associated with decreased ability to repopulate the blood system after competitive and non-competitive BM transplantation. Rps12 KO/+ lost HSC quiescence, experienced ERK and MTOR activation, and increased global translation in HSC and progenitors. Post-natal heterozygous deletion of Rps12 in hematopoietic cells using Tal1-Cre-ERT also resulted in pancytopenia with decreased HSC numbers. However, post-natal Cre-ERT induction led to reduced translation in HSCs and progenitors, suggesting that this is the most direct consequence of Rps12 haploinsufficiency in hematopoietic cells. Thus, RpS12 has a strong requirement in HSC function, in addition to erythropoiesis., Competing Interests: VF, KA, JC, NB No competing interests declared, KG has received research funding from iOnctura, SA, and ADC Therapeutics, (© 2023, Folgado-Marco, Ames et al.)

The Chd8 gene encodes a member of the chromodomain helicase DNA-binding (CHD) family of SNF2H-like adenosine triphosphate (ATP)-dependent chromatin remodeler, the mutations of which define a subtype of autism spectrum disorders. Increasing evidence from recent studies indicates that ATP-dependent chromatin-remodeling genes are involved in the control of crucial gene-expression programs in hematopoietic stem/progenitor cell (HSPC) regulation. In this study, we identified CHD8 as a specific and essential regulator of normal hematopoiesis. Loss of Chd8 leads to severe anemia, pancytopenia, bone marrow failure, and engraftment failure related to a drastic depletion of HSPCs. CHD8 forms a complex with ATM and its deficiency increases chromatin accessibility and drives genomic instability in HSPCs causing an activation of ATM kinase that further stabilizes P53 protein by phosphorylation and leads to increased HSPC apoptosis. Deletion of P53 rescues the apoptotic defects of HSPCs and restores overall hematopoiesis in Chd8-/- mice. Our findings demonstrate that chromatin organization by CHD8 is uniquely necessary for the maintenance of hematopoiesis by integrating the ATM-P53-mediated survival of HSPCs., (© 2021 by The American Society of Hematology.)

Inborn errors of immunity (IEI) are a genetically heterogeneous group of disorders with a broad clinical spectrum. Identification of molecular and functional bases of these disorders is important for diagnosis, treatment, and an understanding of the human immune response. We identified 6 unrelated males with neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases bone marrow failure associated with 3 different variants in the X-linked gene TLR8, encoding the endosomal Toll-like receptor 8 (TLR8). Interestingly, 5 patients had somatic variants in TLR8 with <30% mosaicism, suggesting a dominant mechanism responsible for the clinical phenotype. Mosaicism was also detected in skin-derived fibroblasts in 3 patients, demonstrating that mutations were not limited to the hematopoietic compartment. All patients had refractory chronic neutropenia, and 3 patients underwent allogeneic hematopoietic cell transplantation. All variants conferred gain of function to TLR8 protein, and immune phenotyping demonstrated a proinflammatory phenotype with activated T cells and elevated serum cytokines associated with impaired B-cell maturation. Differentiation of myeloid cells from patient-derived induced pluripotent stem cells demonstrated increased responsiveness to TLR8. Together, these findings demonstrate that gain-of-function variants in TLR8 lead to a novel childhood-onset IEI with lymphoproliferation, neutropenia, infectious susceptibility, B- and T-cell defects, and in some cases, bone marrow failure. Somatic mosaicism is a prominent molecular mechanism of this new disease.

The only available option to treat radiation-induced hematopoietic syndrome is allogeneic hematopoietic cell transplantation, a therapy unavailable to many patients undergoing treatment for malignancy, which would also be infeasible in a radiological disaster. Stromal cells serve as critical components of the hematopoietic stem cell niche and are thought to protect hematopoietic cells under stress. Prior studies that have transplanted mesenchymal stromal cells (MSCs) without co-administration of a hematopoietic graft have shown underwhelming rescue of endogenous hematopoiesis and have delivered the cells within 24 h of radiation exposure. Herein, we examine the efficacy of a human bone marrow-derived MSC therapy delivered at 3 h or 30 h in ameliorating radiation-induced hematopoietic syndrome and show that pancytopenia persists despite MSC therapy. Animals exposed to radiation had poorer survival and experienced loss of leukocytes, platelets, and red blood cells. Importantly, mice that received a therapeutic dose of MSCs were significantly less likely to die but experienced equivalent collapse of the hematopoietic system. The cause of the improved survival was unclear, as complete blood counts, splenic and marrow cellularity, numbers and function of hematopoietic stem and progenitor cells, and frequency of niche cells were not significantly improved by MSC therapy. Moreover, human MSCs were not detected in the bone marrow. MSC therapy reduced crypt dropout in the small intestine and promoted elevated expression of growth factors with established roles in gut development and regeneration, including PDGF-A, IGFBP-3, IGFBP-2, and IGF-1. We conclude that MSC therapy improves survival not through overt hematopoietic rescue but by positive impact on other radiosensitive tissues, such as the intestinal mucosa. Collectively, these data reveal that MSCs could be an effective countermeasure in cancer patients and victims of nuclear accidents but that MSCs alone do not significantly accelerate or contribute to recovery of the blood system.

Alterations in copper homeostasis is an uncommon cause for hematologic alterations frequently presenting with dysplastic features in the bone marrow. Most of these alterations have been documented in adult patients with copper deficiency. Rare cases show hematogone hyperplasia in these patients. Effects of mild copper excess have not been documented in literature. We are describing a pediatric patient who presented with pancytopenia associated with hypercupraemia (excess of copper). Bone marrow examination showed hematogone hyperplasia. Interestingly, correction of serum copper levels with zinc therapy lead to complete improvement in pancytopenia. Hematogones had also reduced in subsequent marrow biopsy after therapy.

Background: Endothelial cells (ECs) function as an instructive platform to support haematopoietic stem cell (HSC) homeostasis. Our recent studies found that impaired bone marrow (BM) ECs are responsible for the defective haematopoiesis in patients with poor graft function (PGF), which is characterised by pancytopenia post-allotransplant. Although activated autophagy was reported to benefit ECs, whether EC autophagy plays a critical role in supporting HSCs and its effect on PGF patients post-allotransplant remain unclear., Methods: To evaluate whether the autophagy status of ECs modulates their ability to support haematopoiesis, human umbilical vein endothelial cells (HUVECs) and primary BM ECs derived from healthy donors were subjected to knockdown or overexpression of Beclin-1 (an autophagy-related protein). Moreover, BM ECs derived from PGF patients were studied., Findings: Beclin-1 knockdown significantly reduced the haematopoiesis-supporting ability of ECs by suppressing autophagy, which could be restored by activating autophagy via Beclin-1 upregulation. Moreover, autophagy positively regulated haematopoiesis-related genes in HUVECs. Subsequently, a prospective case-control study demonstrated that defective autophagy reduced Beclin-1 expression and the colony-forming unit (CFU) plating efficiency in BM ECs from PGF patients compared to matched patients with good graft function. Rapamycin, an autophagy activator, quantitatively and functionally improved BM ECs from PGF patients in vitro and enhanced their ability to support HSCs by activating the Beclin-1 pathway., Interpretation: Our results suggest that the autophagy status of ECs modulates their ability to support haematopoiesis by regulating the Beclin-1 pathway. Defective autophagy in BM ECs may be involved in the pathogenesis of PGF post-allotransplant. Rapamycin provides a promising therapeutic approach for PGF patients., Funding: Please see funding sources., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interests., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To explore effects of Epstein-Barr virus (EBV) infection on CD19+ B lymphocytes in patients with immunorelated pancytopenia (IRP)., Methods: An enzyme-linked immunosorbent assay (ELISA) in vitro diagnostic kit was used to detect EBV capsid antigen- (CA-) IgG and VCA-IgM antibodies in the serum. We analyzed the EBV-DNA copies of CD19+ B lymphocyte by using real-time quantitative polymerase chain reaction (RT-qPCR). CD21, CD23, CD5, CD80, and CD86 receptors on the surfaces of CD19+ B cells were detected by flow cytometry (FCM). The correlation between these receptors and EBV-DNA copies were evaluated., Results: The results revealed that the positive rate of EBVCA-IgM and CD19+ B lymphocyte EBV-DNA copy in the IRP group were significantly higher than those in the control group ( P < 0.05). CD19+ B lymphocyte EBV-DNA copies were also more abundant in IRP patients than in control subjects ( P < 0.05). CD19+ B lymphocyte EBV-DNA copies were also more abundant in IRP patients than in control subjects ( P < 0.05). CD19+ B lymphocyte EBV-DNA copies were also more abundant in IRP patients than in control subjects (., Conclusions: EBV infection may activate CD19+ B lymphocytes and further disrupt bone marrow hematopoiesis in IRP patients., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2020 Yang Zhao et al.)

Loss-of-function studies have determined that Notch signaling is essential for hematopoietic and endothelial development. By deleting a single allele of the Notch1 transcriptional activation domain we generated viable, post-natal mice exhibiting hypomorphic Notch signaling. These heterozygous mice, which lack only one copy of the transcriptional activation domain, appear normal and have no endothelial or hematopoietic phenotype, apart from an inherent, cell-autonomous defect in T-cell lineage development. Following chemotherapy, these hypomorphs exhibited severe pancytopenia, weight loss and morbidity. This phenotype was confirmed in an endothelial-specific, loss-of-function Notch1 model system. Ang1, secreted by hematopoietic progenitors after damage, activated endothelial Tie2 signaling, which in turn enhanced expression of Notch ligands and potentiated Notch1 receptor activation. In our heterozygous, hypomorphic model system, the mutant protein that lacks the Notch1 transcriptional activation domain accumulated in endothelial cells and interfered with optimal activity of the wildtype Notch1 transcriptional complex. Failure of the hypomorphic mutant to efficiently drive transcription of key gene targets such as Hes1 and Myc prolonged apoptosis and limited regeneration of the bone marrow niche. Thus, basal Notch1 signaling is sufficient for niche development, but robust Notch activity is required for regeneration of the bone marrow endothelial niche and hematopoietic recovery., (Copyright© 2019 Ferrata Storti Foundation.)

In the past decade, a group of cases with persisting haemocytopenia were separated from those with idiopathic cytopenia of undetermined significance due to the optimal response of these patients to immunosuppression therapy and due to the detection of autoantibodies in the bone marrow of haemopoietic cells. This condition was termed immune‑related haemocytopenia (IRH). However, the quantity of T lymphocytes remained unknown. In the present study, the percentage of CD4+ T‑cell subsets and related cytokines was measured using flow cytometry and an enzyme‑linked immunosorbent assay. An abnormal number of CD4+ T cell subsets was found, including increased percentages of T helper (Th)2, Th9 and Th17 cells and a decreased number of regulatory T (Treg) cells. In addition, the results showed downregulation in the levels of interleukin (IL)‑2, transforming growth factor‑β and IL‑35, and upregulation in the levels of IL‑4, IL‑6, IL‑17, IL‑23 and interferon‑γ in patients who did not receive therapy (untreated patients). These levels were significantly associated with the number of peripheral blood cells and were recovered following treatment. In conclusion, an abnormal number of CD4+ T cell subsets and corresponding abnormal levels of regulatory cytokines resulted in the stimulation of B1 lymphocytes to produce autoantibodies in IRH, which may be considered as markers to evaluate disease prognosis and treatment strategies.

Idiopathic portal hypertension (IPH) and extrahepatic portal venous obstruction (EHPVO) are prototype noncirrhotic causes of portal hypertension (PHT), characterized by normal hepatic venous pressure gradient, variceal bleeds, and moderate to massive splenomegaly with preserved liver synthetic functions. Infections, toxins, and immunologic, prothrombotic and genetic disorders are possible causes in IPH, whereas prothrombotic and local factors around the portal vein lead to EHPVO. Growth failure, portal biliopathy, and minimal hepatic encephalopathy are long-term concerns in EHPVO. Surgical shunts and transjugular intrahepatic portosystemic shunt resolve the complications secondary to PHT. Meso-Rex shunt is now the standard-of-care surgery in children with EHPVO., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Kidney proximal tubular cells (PTCs) are highly specialized for ultrafiltrate reabsorption and serve as paradigm of apical epithelial differentiation. Vps34/PI3-kinase type III (PI3KC3) regulates endosomal dynamics, macroautophagy and lysosomal function. However, its in vivo role in PTCs has not been evaluated. Conditional deletion of Vps34/PI3KC3 in PTCs by Pax8-Cre resulted in early (P7) PTC dysfunction, manifested by Fanconi-like syndrome, followed by kidney failure (P14) and death. By confocal microscopy, Vps34 ∆/∆ PTCs showed preserved apico-basal specification (brush border, NHERF-1 versus Na + /K + -ATPase, ankyrin-G) but basal redistribution of late-endosomes/lysosomes (LAMP-1) and mis-localization to lysosomes of apical recycling endocytic receptors (megalin, cubilin) and apical non-recycling solute carriers (NaPi-IIa, SGLT-2). Defective endocytosis was confirmed by Texas-red-ovalbumin tracing and reduced albumin content. Disruption of Rab-11 and perinuclear galectin-3 compartments suggested mechanistic clues for defective receptor recycling and apical biosynthetic trafficking. p62-dependent autophagy was triggered yet abortive (p62 co-localization with LC3 but not LAMP-1) and PTCs became vacuolated. Impaired lysosomal positioning and blocked autophagy are known causes of cell stress. Thus, early trafficking defects show that Vps34 is a key in vivo component of molecular machineries governing apical vesicular trafficking, thus absorptive function in PTCs. Functional defects underline the essential role of Vps34 for PTC homeostasis and kidney survival.

Background: Immuno-related hemocytopenia (IRH) is defined as idiopathic cytopenia of undetermined significance (ICUS) patients with autoantibodies. In our previous studies, we found that IgG1 levels were increased in IRH patients and might cause the destruction of hematopoietic cells., Methods: In this study, we analyzed IgG subclasses in 30 IRH patients (male:female = 13:17, median age 32 years, range 18 - 56), 15 IRH remission patients (IRH-R) (male:female = 6:9, median age 34, range 20 - 52) and 20 normal controls (male:female = 8:12, median age 27, range 24 - 36) by Cytometric Bead Array, Flow Cytometry and Immunohistochemical staining., Results: Levels of IgG1/IgG3 in the bone marrow supernatant of IRH patents, as well as the proportion of CD5+ B lymphocytes and Th2 cells (CD3+CD8-IL-4+) were higher than those of IRH-R patients and normal controls, and IgG1 levels had a positive correlation with the proportion of Th2 cells. In IRH patients, IgG1 and IgG3 were positive on nucleated erythrocytes and granulocytes, which were negative in IRH-R patients and healthy controls and had inverse correlations with hematopoietic function. Using immunohistochemical staining, IgG1 were also detected on bone marrow biopsies of IRH patients., Conclusions: The results indicated that IgG1 and IgG3 autoantibodies in IRH patients might play a key role in the IRH pathogenesis and in the abnormal immune function of IRH patients.

The fundamental structure of eukaryotic cell plasma membrane is the phospholipid bilayer, which contains four major phospholipids. These phospholipids are asymmetrically distributed between the outer and inner leaflets. P4-ATPase flippase complexes play essential roles in ensuring this asymmetry. We found that conditional deletion of Tmem30a, the β subunit of P4-ATPase flippase complex, caused pancytopenia in mice. Tmem30a deficiency resulted in depletion of lineage-committed blood cells in the peripheral blood, spleen, and bone marrow. Ablation of Tmem30a also caused the depletion of hematopoietic stem cells (HSCs). HSC RNA sequencing results revealed that multiple biological processes and signal pathways were involved in the event, including mammalian target of rapamycin signaling, genes for HSC stemness, and genes responding to interferons. Our results also revealed that targeting Tmem30a signaling had therapeutic utility in BCR/ABL1-induced chronic myeloid leukemia., (Copyright © 2018 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

A morphometric analysis was performed on aspirated clots of bone marrow to identify the presence of micromegakaryocytes after immune staining with a monoclonal antibody raised against CD41. Quantitative and qualitative abnormalities of micromegakaryocytes were assessed based on both standard staining and CD41 immune staining. Micromegakaryocytes were largely detected in bone marrow from patients with myelodysplastic syndrome (MDS), while almost no micromegakaryocytes were present in aplastic anemia. CD41 immune staining clearly improved the efficiency of micromegakaryocyte detection under any conditions, showing strong potential as a tool for the auxiliary diagnosis of MDS and differentiation of MDS from pancytopenia, particularly aplastic anemia(AA)., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Acute leukemia, myelodysplastic syndromes (MDS), myeloproliferative neoplasms and lymphomas are the most prevalent diagnoses in adults presenting with new onset cytopenia. Here, we describe two 10-color panels of surface markers (screening and comprehensive panel) applied at the Flow Cytometry Laboratory, University Health Network, Toronto, ON, Canada. A 10-color flow cytometry is applied using the stain-lyse-wash sample preparation method. In patients with <10% blasts and no clear involvement by hematological malignancy based on cytomorphological evaluation of bone marrow (BM) smear, the recently published one-tube 10-color 14-antibody screening panel is applied. This panel allows detection of major B- and T-cell abnormalities, enumeration of cells in blast region (CD45 dim), and gives insight into myeloid BM compartment, including calculation of four-parameter score for MDS-related abnormalities. In patients who present with ≥10 - <20% blasts in blood or BM smears, a comprehensive three-tube panel of surface markers is used up front. The analysis is focused on the detection of abnormal antigen expression patterns not seen in normal/reactive BM, according to the guidelines developed by International/European LeukemiaNet Working Group for Flow Cytometry in MDS. In patients with ≥20% blasts, an additional tube is added to allow the detection of cytoplasmic markers necessary to diagnose mixed phenotype acute leukemia., (© 2015 John Wiley & Sons Ltd.)

Introduction: Carboplatin is a congener of cisplatin used in the treatment of ovarian, head and neck and small-cell lung cancer. However, the clinical efficacy of carboplatin is marred by the development of ROS-dependent nephrotoxicity. The pathophysiological damage inflicted upon the kidney by carboplatin closely resembles to that of Fanconi syndrome., Aims and Objectives: The present study aimed at inducing Fanconi-like syndrome in rats by administration of carboplatin. Objectives of the study involved evaluation of biochemical parameters coherent to Fanconi-like syndrome. Further, an attempt was made to evaluate the potential therapeutic effect of pentoxifylline in this condition., Results: The results of the study demonstrated that the urinary excretion profile of carboplatin treated rats closely resembled to that of patients suffering from Fanconi-like condition. Pentoxifylline was able to ameliorate this nephrotoxic condition as suggested by the change in levels of membrane bound ATPases, MDA and GSH. The urinary levels of tyrosine and cysteine correlate well with that of Fanconi-like condition in animals and humans., Conclusion: In lieu of these observations, our study suggested that carboplatin-induced renovascular damage resembles to Fanconi-like condition which can be mitigated by pentoxifylline., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Context: Collagen vascular diseases are frequently included in the differential diagnosis for unexplained cytopenias and often prompt a bone marrow biopsy in this patient population to exclude malignancy. Few large-scale studies have characterized the bone marrow morphology in patients with collagen vascular disease, and most are limited to systemic lupus erythematosus or rheumatoid arthritis., Objective: To identify morphologic and immunohistochemical abnormalities specific to each of a wide range of collagen vascular disease cases., Design: We examined 102 cases of collagen vascular disease and 38 controls and evaluated the complete blood count, peripheral blood morphology, bone marrow morphology, as well as immunohistochemical staining, for numerous cell lineages., Results: Bone marrow findings, including abnormalities such as lymphoid aggregates, lipogranulomas, or abnormal localization of immature precursors, were not significantly different as compared to the control group., Conclusions: Bone marrow examination in patients with collagen vascular disease with cytopenias seldom provides new information. Caution should be exercised in interpreting morphologic findings suggestive of myelodysplasia since these are of a reactive nature in up to 27% of patients with collagen vascular disease. In a cost-effective diagnostic strategy, successful utilization may favor postponing a bone marrow biopsy while a more standardized autoimmune diagnostic panel is being performed.

We made an attempt to evaluate/compare the cardioprotective activity of two different doses (50 and 100 mg/kg body weight, given orally for 30 consecutive days) of Egyptian sweet marjoram leaf powder (MLP) and marjoram leaf aqueous extract (MLE) against isoproterenol (ISO)-induced myocardial infarcted rats (150 mg/kg body weight, twice at an interval of 24 h on days 29 and 30). The present study showed (probably for the first time) that both MLP and MLE (especially the high dose) significantly alleviated (P < 0.05-0.001) erythrocytosis, granulocytosis, thrombocytosis, shortened clotting time, the increase in relative heart weight, myocardial oxidative stress and the leakage of heart enzymes (creatine phosphokinase (CPK), CPK-MB isoenzyme, lactate dehydrogenase and aminotransferase) in ISO-treated rats through reactivating non-enzymic (reduced glutathione) and enzymic (catalase, glutathione peroxidase, glutathione S-transferase, superoxide dismutase) antioxidant defence system and inhibiting the production of nitric oxide and lipid peroxidation in heart tissues. The modulatory effects of marjoram leaves shown in the present study were dose-dependent in most cases and much higher in MLE (4.3-20.3 % for all parameters taken together). In addition, the doses used in the present study were considered safe. In conclusion, this study may have a significant impact on myocardial infarcted patients.

Objective: To evaluate the possible mechanism of transcription factors B cell lymphoma 6 (Bcl-6) , forkhead/winged helix transcription factor 3 (Foxp3) and retinoic acid related orphan receptor (RORγt) in CD4(+) T cells for immuno-related hematocytopenia (IRH)., Methods: CD4(+) T cells were harvested from 40 IRH patients, 38 aplastic anemia subjects and 25 normal controls and separated by magnetic activated cell sorting (MACS). Then the expressions of transcription factors of Foxp3, RORγ and Bcl-6 in CD4(+) T cells were measured by real time fluorescent quantitative-polymerase chain reaction (QRT-PCR)., Results: Auto-antibody was detected on CD34(+) cells (67.5% (27/40) ), CD15(+) cells (65.0% (26/40)), GlyA(+) cells (75.0% (30/40) ), auto-antibody involving three, two or one myeloid cell were detected in 27.5% (11/40), 52.5% (21/40), 20.0% (8/40) of IRH patients. Compensatory increase of Foxp3 mRNA was found in IRH (0.124 (0.073-0.198) vs 0.071 (0.046-0.118), P < 0.05). The expression of Bcl-6 was higher (2.243 (0.854-4.544) vs 1.211 (0.131-2.816), P < 0.05). Compared to aplastic anemia, the expression of RORγt was lower in IRH (0.133 (0.068-0.189) vs 0.290 (0.138-0.480), P < 0.01) and the ratio of Treg/Th17 shifted to Th17 in patients with aplastic anemia (Foxp3/RORγt ratio,0.500 (0.240-0.795) vs 0.975 (0.483-1.416), P < 0.01)., Conclusion: As one kind of bone marrow failures caused by autoantibody to bone marrow cells, IRH may occur due to a high expression of Bcl-6 in CD4(+) T cells, its immunopathogenesis is different from that of aplastic anemia.

Background: Immune-related hematocytopenia (IRH) is considered to be related with the production of autoantibody, as well as the activation of humoral immunity which is stimulated by B lymphocyte. This study aimed to observe the levels of various cytokines in the blood serum and the in situ active state of macrophage (Mφ) in the medullary hematopoietic microenvironment of IRH patients, and to probe into the immune mechanism and clinical significance of Mφ in hematopoietic cell injury., Methods: ELISA is used to detect the IL-4, IL-6, IL-12, IL-17, and IFN-γ levels in the peripheral blood serum of 376 patients in pre- and post-therapy. Cytochemistry and cell immunochemistry methods are used to observe the peroxidase (POX), nonspecific esterase (NSE), hemosiderin granules, and HLA-DR activity of Mφ in the bone marrow of patients. Immunofluorescence is used to observe the expression of hemocyte antihuman globulin IgG antibody, lymphocytes CD4 molecule, Mφ membrane FcγIIreceptor (FcγIIR), mannitose receptor (MR), IFN-γ, ICAM-1, IL-12, and IL-17A and the formation mechanism of antibody-dependent cell-mediated cytotoxicity (ADCC) hematopoietic cell islands (HI) in the medullary hematopoietic microenvironment of patients. Glucocorticoid is used for treatment on the basis of anti-infection therapy, and gamma globulin stoss therapy is used for the appearance of ADCC-type HI or serious Mφ bloodthirsty phenomenon; if necessary, association of Cyclosporine A (CsA) should be used and chalybeate should be supplemented., Results: In the patient group, the levels of IL-4, IL-6, IL-12, IL-17, and IFN-γ were increased. After treatment, the cytokine levels gradually became normal. The activated Mφ in the marrow highly expressed NSE and POX, and Mφ swallowed more hemosiderin particles, but the iron in the cytoplasm of immature erythrocytes decreased. The activated Mφ expressed HLA-DR, MR, ICAM-1, IFN-γ, and IL-12. For patients with humoral immunity activation and bacterial infection, Mφ weakly expressed IL-17A but highly expressed FcγIIR, and the phenomenon that ADCC-type HI broke pathological blood corpuscles often occurred; for the cellular immune activation along with virus infection, the white blood count (WBC) significantly reduced, Mφ weakly expressed FcγIIR, secretory highly expressed IL-17A, and the phenomena that Mφ adhered to, captured and swallowed blood cell often occurred. After four weeks of anti-infective and immunosuppressive therapy, nuclear apoptosis of Mφ occurred in the bone marrow of patients, HI and bloodthirsty phenomenon disappeared, and the peripheral blood picture started to improve., Conclusions: Mφ is an important antigen presenting cell in the IRH marrow for hematopoiesis destruction and an immune effector cell of hematopoietic injury; infection can promote the activation of Mφ, upregulate the impression of immune molecule and receptors, form ADCC HI, aggravate hematopoietic injury, and accelerate the destruction on hematopoietic cell.

Immune-related pancytopenia (IRP) is one kind of bone marrow failure diseases which is related to autoantibodies. Autoantibodies have been detected on the membrane of various bone marrow (BM) hemopoietic cells by BM mononuclear-cell-Coombs test or flow cytometric analysis. There are autoantibodies in the BM supernatant of IRP patients, which can target several antigens on hematopoietic cells membranes by western blot. T follicular helper (Tfh) cells are the true helper cells for Ab responses, which represent one of the most numerous and important subsets of effector T cells. Dysregulation of Tfh cell function or expression of Tfh cell-associated molecules could contribute to the pathogenesis of autoimmune diseases. Currently, there are no studies regarding the role of Tfh cells in IRP patients. The percentages of Tfh cells, Tfh-related molecules ICOS, CD40L, IL-21, and Bcl-6 in BM were investigated in 90 patients with IRP, and 25 healthy controls. We observed that there exist increased quantity and hyperfunction of Tfh cells in IRP, and the results were correlated with patient characteristics. It was indicated that dysregulated Tfh cells might be involved in the pathogenesis of IRP and that inhibition of Tfh cells effector molecules might provide opportunities for new therapeutic approaches to IRP and even other human autoimmune diseases.

Cytopenias are key prognostic indicators of life-threatening infection, contributing to immunosuppression and mortality. Here we define a role for Caspase-1-dependent death, known as pyroptosis, in infection-induced cytopenias by studying inflammasome activation in hematopoietic progenitor cells. The NLRP1a inflammasome is expressed in hematopoietic progenitor cells and its activation triggers their pyroptotic death. Active NLRP1a induced a lethal systemic inflammatory disease that was driven by Caspase-1 and IL-1β but was independent of apoptosis-associated speck-like protein containing a CARD (ASC) and ameliorated by IL-18. Surprisingly, in the absence of IL-1β-driven inflammation, active NLRP1a triggered pyroptosis of hematopoietic progenitor cells resulting in leukopenia at steady state. During periods of hematopoietic stress induced by chemotherapy or lymphocytic choriomeningitis virus (LCMV) infection, active NLRP1a caused prolonged cytopenia, bone marrow hypoplasia, and immunosuppression. Conversely, NLRP1-deficient mice showed enhanced recovery from chemotherapy and LCMV infection, demonstrating that NLRP1 acts as a cellular sentinel to alert Caspase-1 to hematopoietic and infectious stress., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Context: In cancer cells, the Warburg effect is defined as the avid consumption of glucose through the glycolytic pathway with concomitant lactate production, even under aerobic conditions., Case: We report a 64-yr-old woman who was referred to our institution for pancytopenia and hypoglycemia. Physical examination demonstrated hepatosplenomegaly and petechiae. She had no clinical manifestation of neuroglycopenia, despite serum glucose of 26 mg/dl (1.4 mmol/liter) and serum lactate of 28.5 mmol/liter (normal range, 0.5-3.4 mmol/liter). Bone marrow biopsy demonstrated diffuse large B-cell lymphoma. Staging (18)F-fluorodeoxyglucose (FDG)-positron emission tomography/computed tomography showed increased FDG avidity in an enlarged spleen and absent FDG uptake in the brain. Despite dextrose infusions up to 30 g/h, there was no increase in serum glucose, but there was a paradoxical increase in serum lactate. Immunochemotherapy improved the hematological and metabolic abnormalities. Follow-up FDG-positron emission tomography/computed tomography showed a decrease in splenic avidity and an increase in brain FDG avidity. The patient refused further chemotherapy and died 1 wk after discharge., Methods: Literature review of cases of lymphoma with lactic acidosis, with and without hypoglycemia, demonstrated that these combinations occurred in multiple categories of B- and T-cell lymphoma. There was no difference in the mortality rate in those with (75%) or without (74%) concomitant hypoglycemia., Conclusion: This case represents an exaggerated Warburg effect, or "hyper-warburgism," characterized by excessive lactate production and overwhelming glucose consumption. We speculate that the decreased brain FDG uptake, despite the lack of neuroglycopenic symptoms, supports the hypothesis that lactate served as a fuel for the brain, thus protecting against hypoglycemia.

Background: Bovine neonatal pancytopenia (BNP) is a syndrome characterised by thrombocytopenia associated with marked bone marrow destruction in calves, widely reported since 2007 in several European countries and since 2011 in New Zealand. The disease is epidemiologically associated with the use of an inactivated bovine virus diarrhoea (BVD) vaccine and is currently considered to be caused by absorption of colostral antibody produced by some vaccinated cows ("BNP dams"). Alloantibodies capable of binding to the leukocyte surface have been detected in BNP dams and antibodies recognising bovine MHC class I and β-2-microglobulin have been detected in vaccinated cattle. In this study, calves were challenged with pooled colostrum collected from BNP dams or from non-BNP dams and their bone marrow hematopoietic progenitor cells (HPC) cultured in vitro from sternal biopsies taken at 24 hours and 6 days post-challenge., Results: Clonogenic assay demonstrated that CFU-GEMM (colony forming unit-granulocyte/erythroid/macrophage/megakaryocyte; pluripotential progenitor cell) colony development was compromised from HPCs harvested as early as 24 hour post-challenge. By 6 days post challenge, HPCs harvested from challenged calves failed to develop CFU-E (erythroid) colonies and the development of both CFU-GEMM and CFU-GM (granulocyte/macrophage) was markedly reduced., Conclusion: This study suggests that the bone marrow pathology and clinical signs associated with BNP are related to an insult which compromises the pluripotential progenitor cell within the first 24 hours of life but that this does not initially include all cell types.

Objective: To observe the relationship between erythropoietin receptor (EPOR) and autoantibodies-IgG/IgM (auto-Ab) on the membrane of erythropoietic cells of the patients with bone marrow mononuclear cells (BMMNC)-Coomb's test(+) hemocytopenia (immunorelated pancytopenia (IRP)) and explore the probable autoantigens of auto-Ab in IRP., Methods: A total of 46 newly diagnosed IRP patients (15 with auto-Ab on erythropoietic cells and 31 without) and 18 healthy controls were enrolled. The EPOR expressions on their nuclear erythrocytes were tested with flow cytometry (FCM) to observe the relationship between EPOR and auto-Ab. EPOR mRNA was detected by reverse transcription (RT)-PCR. Stat5 and P-Stat5 proteins in nucleated erythrocytes were measured by Western blot. EPOR expressions on nucleated erythrocytes membrane were re-tested after stripping autoantibodies with glycine buffer., Results: (1) EPOR of auto-Ab(+) group (1.6% ± 0.9%)was significantly lower than that of auto-Ab(-) group (4.6% ± 4.1%, P < 0.01)and the latter was significantly higher than that of normal controls (2.3% ± 1.8%, P < 0.05). EPOR of IRP patients was inversely correlated with their auto-Ab (r = -0.543, P = 0.000). (2) EPOR mRNA of auto-Ab(+) group (0.68 ± 0.14)was significantly higher than that of auto-Ab(-) group (0.55 ± 0.12, P < 0.01) and normal controls (0.58 ± 0.12, P < 0.05). (3) Protein Stat5 of auto-Ab(+) group (1.45 ± 0.94) was significantly higher than that of normal controls (0.54 ± 0.36, P < 0.05). While P-Stat5 of auto-Ab(+) group (0.42 ± 0.18)was significantly lower than that of normal controls (0.85 ± 0.38, P < 0.05). (4) EPOR expression increased significantly after auto-Ab stripping., Conclusions: The auto-Ab of some IRP patients blocks or competitively inhibits EPOR on the membrane of erythropoietic cells. And EPOR may be one of autoantigens in IRP.

TRF1 is part of the shelterin complex, which binds telomeres and it is essential for their protection. Ablation of TRF1 induces sister telomere fusions and aberrant numbers of telomeric signals associated with telomere fragility. Dyskeratosis congenita is characterized by a mucocutaneous triad, bone marrow failure (BMF), and presence of short telomeres because of mutations in telomerase. A subset of patients, however, show mutations in the shelterin component TIN2, a TRF1-interacting protein, presenting a more severe phenotype and presence of very short telomeres despite normal telomerase activity. Allelic variations in TRF1 have been found associated with BMF. To address a possible role for TRF1 dysfunction in BMF, here we generated a mouse model with conditional TRF1 deletion in the hematopoietic system. Chronic TRF1 deletion results in increased DNA damage and cellular senescence, but not increased apoptosis, in BM progenitor cells, leading to severe aplasia. Importantly, increased compensatory proliferation of BM stem cells is associated with rapid telomere shortening and further increase in senescent cells in vivo, providing a mechanism for the very short telomeres of human patients with mutations in the shelterin TIN2. Together, these results represent proof of principle that mutations in TRF1 lead to the main clinical features of BMF.

Objective: To observe the telomerase activity (TA) in bone marrow mononuclear cell (BMMNC) at pre-therapy and post-therapy in patients with immune related hematocytopenia and immune related pancytopenia (IRH/IRP) so as to explore the relationship between TA and disease process., Methods: TA in BMMNC of 108 IRH/IRP patients was detected with telomeric repeat amplification protocol (TRAP)-PCR-ELISA. The expressions of anti-human immunoglobulin IgG, FcγII receptor (FcγIIR), mannose receptor (MR) and interleukin 17A (IL-17A) located on membrane surface were observed by immunofluorescence (IF). A total of 30 iron deficiency anemia patients were selected as case control., Results: The TA of 108 IRH/IRP patients were 0.251 ± 0.021. And it increased as compared with the control group of 0.062 ± 0.031 at pre-therapy (P < 0.01). The HLA-B27(+) patients among them was 0.270 ± 0.020 while HLA-B27(-) patients 0.181 ± 0.013 (P < 0.05). And HLA-B27(+)IgG elevated IRP was 0.341 ± 0.016 and HLA-B27(-)IgG elevated 0.183 ± 0.010, the difference of TA was significant between two groups (P < 0.01). IgG antibody positive immature hemocyte could be observed in marrow of IRH/IRP patients, and FcγIIR was expressed highly on those activated macrophage (Mφ) in bone marrow. Both IL-17A and MR were expressed on activated dendritic cells and Mφ in marrow of HLA-B27(+) patients. After the therapy of glucocorticoids associated with cyclosporine A and other drugs, the patient's hemogram recovered and the telomerase in BMMNC were re-devitalized., Conclusions: The TA of the BMMNC in marrow of IRH/IRP patients is increased, and it shows a close relation between apoptotic degree of hematopoietic cell and the activation of immunocytes. In IRH/IRP, synchronism and equilibrium exist between BMMNC apoptosis and TA.

In recent years, there have been lots of progresses in the studies on red cell diseases in China, especially bone marrow failure diseases including immuno-related pancytopenia, aplastic anemia, myelodysplastic syndrome, and paroxymal nocturnal hemoglobinuria. Numerous laboratory experiments as well as clinical researches have been carried out by Chinese hematologists, which brought about much clearer pathogenesis, more rational diagnosis methods and more effective therapies for red cell diseases.

Hepatoportal sclerosis accompanied by dense elastic fibre deposition is generally regarded as the primary lesion in the development of idiopathic portal hypertension (IPH). This study was performed to clarify the mechanism of elastic fibre deposition in the peripheral portal tracts of IPH liver in relation to serum anti-endothelial cell antibodies (AECA). In-vitro experiments were performed using human dermal microvascular endothelial cells (HMVEC) and patients' sera. The presence of serum AECA was assayed by a cell-based enzyme-linked immunosorbent assay (ELISA) using HMVEC. Immunohistochemical analysis of elastin was performed using liver tissue sections of IPH patients. IPH sera contained one or more AECA that could bind to the vascular endothelial cells of the peripheral portal tracts of the liver. When the value of AECA greater than the mean ± 2 standard deviations of healthy controls was regarded as positive, the positive detection rate of either immunoglobulin (Ig)G, IgA or IgM AECA in IPH sera was 30% (10 of 33 cases). IPH sera induced the expression of elastin in HMVEC, which appeared to be associated with the presence of AECA. Apoptosis was also induced in HMVEC by the stimulation with IPH sera. In vivo, elastin expression was observed in the endothelial cells of the peripheral portal tracts of IPH livers in a proportion of cases. The disease pathogenesis of IPH seems to be heterogeneous, and this study elucidated a possible contribution of the induction of elastin expression in the portal vessels to hepatoportal sclerosis of IPH, which might be linked to serum AECA as a causative factor., (© 2011 The Authors. Clinical and Experimental Immunology © 2011 British Society for Immunology.)

The human proton coupled folate transporter (PCFT) is involved in low pH-dependent intestinal folate transport. In this report, we describe a new murine model of the hereditary folate malabsorption syndrome that we developed through targeted disruption of the first 3 coding exons of the murine homolog of the PCFT gene. By 4 weeks of age, PCFT-deficient (PCFT(-/-)) mice developed severe macrocytic normochromic anemia and pancytopenia. Immature erythroblasts accumulated in the bone marrow and spleen of PCFT(-/-) mice and failed to differentiate further, showing an increased rate of apoptosis in intermediate erythroblasts and reduced release of reticulocytes. In response to the inefficient hematologic development, the serum of the PCFT(-/-) animals contained elevated concentrations of erythropoietin, soluble transferrin receptor (sCD71), and thrombopoietin. In vivo folate uptake experiments demonstrated a systemic folate deficiency caused by disruption of PCFT-mediated intestinal folate uptake, thus confirming in vivo a critical and nonredundant role of the PCFT protein in intestinal folate transport and erythropoiesis. The PCFT-deficient mouse serves as a model for the hereditary folate malabsorption syndrome and is the most accurate animal model of folate deficiency anemia described to date that closely captures the spectrum of pathology typical of this disease.

The management of myelodysplastic syndromes (MDS) in elderly patients is a significant clinical problem. The therapeutic options range from observation alone for patients with low-risk disease, lenalidomide for patients with 5q-syndrome, to 5-azacytidine (5-AZA) for patients with higher risk of disease. In this paper, we summarize the clinical course of three patients with high-risk MDS treated with 5-AZA as well as the management and supportive care measures for adverse events. As expected, based on available clinical trials data, the agent resulted in clinical and hematological improvement in these patients with acceptable side effects. 5-AZA is an attractive option for elderly patients with high-risk MDS.

Transcobalamin II deficiency (# MIM 275350) is a rare, recessively inherited disorder of cobalamin transport that leads to intracellular cobalamin depletion with secondary impairment of methionine synthetase and methyl-malonyl CoA mutase activities. Affected individuals may suffer from long-term neurological sequelae if therapy with intramuscular hydroxocobalamin is not initiated promptly. We report two sisters with complete absence of transcobalamin due to homozygosity for a novel mutation (c.insC110) in the TCN2 gene that leads to a premature stop codon and non-functional protein. The older sister, now 4.5 years old, presented at 6 weeks of age with pancytopenia, protein losing enteropathy and a rapidly declining clinical course. Prompt therapy with 1mg hydroxocobalamin/day led to full recovery within days. Her now 1.5 year old sister was diagnosed shortly after birth and was started on hydroxocobalamin prior to onset of clinical symptoms. Interestingly, urinary methylmalonic acid excretion was increased significantly during the first days of life suggesting that functional cobalamin deficiency is present also during fetal life, although not giving rise to clinical symptoms until well after birth.

Objective: To investigate the variation of bone marrow complement level in cytopenia patients with positive BMMNC-Coombs test (CBCPC), and probe the role of complement in destroying hematopoietic cells of CBCPC patients., Methods: One hundred and twenty-four patients with CBCPC and twenty-three healthy donors as controls were enrolled in this study. The levels of CH50, C3, C4, C5b-9 were tested with ELISA. The auto-antibodies on bone marrow hematopoietic cells (BMHC) were examined with flow cytometry., Results: The level of C5b-9 in bone marrow (BM) of untreated CBCPC patients [(119.8+/-54.0) microg/L] was significantly higher than that of recovered patients [(100.7+/-33.4) microg/L] or normal controls [(93.9+/-28.8) microg/L] (P<0.05). The levels of CH50 in BM of untreated or recovered CBCPC patients [(33.3+/-11.5) kU/L, (30.8+/-10.3) kU/L] were significantly higher than that of normal controls [(24.1+/-6.4) kU/L] (P<0.05). The level of C3 in BM of untreated or recovered CBCPC patients [(4.9+/-2.2) mg/L], (5.0+/-3.5) mg/L] was significantly lower than that of normal controls [(7.0+/-5.6) mg/L] (P<0.05). The level of complement in peripheral blood was consistent with that in BM. CH50 in BM of CBCPC patients was negatively correlated with their C3 (r=-0.303, P=.0007) and positively correlated with their C5b-9 (r=0.241, P=0.003) levels. The level of C5b-9 in BM of CBCPC patients was higher in the BMHC-IgM positive group [(117.6+/-55.7) microg/L] than in the BMHC- IgM negative group [(99.2+/-26.2) microg/L] (P<0.05). The positive rate of CD34(+)-IgG or CD34(+)-IgM of CBCPC patients was positively correlated with their C5b-9 level (r=0.593, P=0.000, r=0.326, P=0.049). The reticulocyte percentage (r=0.421, P=0.000) and serum indirect bilirubin level (r=0.230, P=0.032) of CBCPC patients were positively correlated with their CH50 level., Conclusions: The hematocytopenia of CBCPC patients might be related to the hematopoietic cells destruction caused by auto-antibody activated complements.

Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Migration of donor-derived T cells into GVHD target organs plays a critical role in the development of GVHD and chemokines and their receptors are important molecules involved in this process. Here, we demonstrate in murine bone marrow transplantation models that the expression of the inflammatory CC chemokine receptor 2 (CCR2) on donor-derived CD8+ T cells is relevant for the control of CD8+ T-cell migration and development of GVHD. Recipients of CCR2-deficient (CCR2-/-) CD8+ T cells developed less damage of gut and liver than recipients of wild-type CD8+ T cells, which correlated with a reduction in overall GVHD morbidity and mortality. Assessment of donor CD8+ T-cell target organ infiltration revealed that CCR2-/- CD8+ T cells have an intrinsic migratory defect to the gut and liver. Other causes for the reduction in GVHD could be excluded, as alloreactive proliferation, activation, IFN-gamma production and cytotoxicity of CCR2-/- CD8+ T cells were intact. Interestingly, the graft-versus-tumor effect mediated by CCR2-/- CD8+ T cells was preserved, which suggests that interference with T-cell migration by blockade of CCR2 signaling can separate GVHD from GVT activity.

The molecular mechanisms involved in the pathogenesis of toxic epidermal necrolysis (TEN) remain not fully understood. We report a unique case of antibiotic-induced TEN developed in a patient who also suffered from prolonged severe methotrexate-induced pancytopenia. The objective of the study was to explore the nature of the cutaneous inflammatory infiltrate and the density in dermal dendrocytes (DD). Immunohistochemistry was used to identify activated T lymphocytes (CD45R0), monocyte-macrophages (Mac 387, CD68), DD (Factor XIIIa), and Langerhans cells (CD1a). The proliferation marker (Ki67) and the antibody to Fas receptor (CD95R) were also used to assess the distribution of the germinative pool of keratinocytes and the FAS-related apoptotic process, respectively. Numerous Factor XIIIa+ DD were present in the papillary dermis with only sparce perivascular CD45RO+ T lymphocytes and scattered CD68+ or Mac 387+ macrophages. Double immunostainings revealed that a minority of Factor XIIIa+ DD co-expressed the CD68 glycoprotein (a marker of phagocytic activity). No cells co-expressed factor XIIIa and Mac 387 immunoreactivities. CD45RO+ T lymphocytes, CD68+ and Mac 387+ macrophages were absent in the epidermis. The expression of CD95R was present although restricted to the basal keratinocytes, while the L1-protein (Mac 387+) was diffusely present in the epidermis. Langerhans cells (CD1a+) were sparce, but normal in distribution. The presence of a great number of Factor XIIIa+ DD without any possible recent recruitment from bone marrow suggests that these cells differentiated from resident cells of the skin. Indeed, there was no co-expression of Factor XIIIa and L1-protein, thus showing the absence of recruitment from monocytes. The simultaneous over-expression of Factor XIIIa and CD68 in some DD indicates some phagocytic activity. In view of the absence of inflammatory cells in the epidermis, keratinocytes appeared responsible for their own destruction through CD95-mediated and/or calcium-dependent apoptotic pathways. This finding entails that TEN treatments should target the keratinocyte metabolism rather than the circulating inflammatory cells which presumably play a limited role, if any, in the epidermal destructive process.

Erythropoiesis in the adult mammal depends critically on erythropoietin, an inducible cytokine with pluripotent effects. Erythropoietin gene expression increases under conditions associated with lowered oxygen content such as anemia and hypoxia. HIF-1alpha, the founding member of the hypoxia-inducible factor (HIF) alpha class, was identified by its ability to bind and activate the hypoxia-responsive enhancer in the erythropoietin regulatory region in vitro. The existence of multiple HIF alpha members raises the question of which HIF alpha member or members regulates erythropoietin expression in vivo. We previously reported that mice lacking wild-type HIF-2alpha, encoded by the EPAS1 gene, exhibit pancytopenia. In this study, we have characterized the etiology of this hematopoietic phenotype. Molecular studies of EPAS1-null kidneys reveal dramatically decreased erythropoietin gene expression. EPAS1-null as well as heterozygous mice have impaired renal erythropoietin induction in response to hypoxia. Treatment of EPAS1-null mice with exogenous erythropoietin reverses the hematopoietic and other defects. We propose that HIF-2alpha is an essential regulator of murine erythropoietin production. Impairments in HIF signaling, involving either HIF-1alpha or HIF-2alpha, may play a prominent role in conditions involving altered hematopoietic or erythropoietin homeostasis.