Resveratrol-loaded sulfated Hericium erinaceus ß-glucan-chitosan nanoparticles: Preparation, characterization and synergistic anti-inflammatory effects.

Resveratrol (RES) is a natural polyphenol with excellent biological activity. But the poor stability and bioavailability of RES severely limit its application. Thus, the resveratrol-loaded sulfated Hericium erinaceus ß-glucan-chitosan nanoparticles (DS-CS-RES NPs) were prepared using electrostatic self-assembly to solve these problems in this study. The structure of DS-CS-RES NPs was spherical or sub spherical shape with small average particle size (191.07 nm), which was characterized by FT-IR, FS, XRD and TEM. DS-CS-RES NPs exhibited good stability and RES had a sustainable release from the nanoparticles in gastrointestinal digestion. Meanwhile, DS-CS-RES NPs could improve the inflammatory injury of LPS stimulated RAW264.7 macrophages by inhibiting the production of NO, IL-1ß, IL-6 and TNF-α. Furthermore, DS-CS-RES NPs had strong anti-inflammatory activity by regulating protein levels of NF-κB p65, STAT1 and TLR4 through NF-κB and JAK-STAT1 signaling pathway in vitro, and sulfated H. erinaceus ß-glucan-chitosan nanoparticle (DS-CS NPs) and RES had synergistic anti-inflammatory effect. Overall, DS-CS NPs can serve as a potential green and safe functional carrier for encapsulating resveratrol, which can improve its anti-inflammatory activity. This work may be conducive to the development of functional carrier for encapsulating RES and applications of hydrophobic active molecules in functional foods or medicines.

Predictive value of thyroid function in severe aplastic anemia patients treated with immunosuppressive therapy.

To explore the predictive value of thyroid function in severe aplastic anemia (SAA) patients treated with immunosuppressive therapy (IST), 149 SAA patients in our center were enrolled between February 2015 and June 2020 in this study. We assessed the thyroid function of 134 patients without primary thyroid diseases, and discovered that 89 patients were accompanied by abnormal thyroid hormone, especially low triiodothyronine (T3). Patients with higher pretreatment-free T3 (FT3) levels (>5 pmol/L) demonstrated superior response rates at 3 and 6 months after IST compared to those with lower FT3 levels (54.5% vs 35.4%, P = .020; 67.3% vs 46.9%, P = .020). Multivariate analysis indicated that shorter disease duration (≤56 days) and response at 6 months were independent favorable factors of overall survival (relative risk [RR] = 2.66, 95% confidence interval [CI] = 1.03-6.90, P = .040; RR = 30.10, 95% CI = 4.02-225.66, P = .001). The 6-year failure-free survival (FFS) was 53.8% (95% CI = 40.9%-65.1%). Multivariate analysis revealed that patients with a response at 6 months, shorter duration (≤56 days) and receiving rabbit antithymocyte globulin (ATG) had better FFS outcomes than those without a response at 6 months, with a longer duration and receiving porcine ATG (RR = 22.6, 95% CI = 7.9-64.9, P < .001; RR = 2.4, 95% CI = 1.3-4.5, P = .006; RR = 2.5, 95% CI = 1.1-5.8, P = .030). In conclusion, FT3 levels reflect the severity of SAA, and patients with higher FT3 levels (>5 pmol/L) had superior response rates than those with lower ones.

Distinct use of super-enhancer elements controls cell type-specific CD25 transcription and function.

The IL-2 receptor α chain (IL-2Rα/CD25) is constitutively expressed on double-negative (DN2/DN3 thymocytes and regulatory T cells (Tregs) but induced by IL-2 on T and natural killer (NK) cells, with Il2ra expression regulated by a STAT5-dependent super-enhancer. We investigated CD25 regulation and function using a series of mice with deletions spanning STAT5-binding elements. Deleting the upstream super-enhancer region mainly affected constitutive CD25 expression on DN2/DN3 thymocytes and Tregs, with these mice developing autoimmune alopecia, whereas deleting an intronic region decreased IL-2-induced CD25 on peripheral T and NK cells. Thus, distinct super-enhancer elements preferentially control constitutive versus inducible expression in a cell type-specific manner. The mediator-1 coactivator colocalized with specific STAT5-binding sites. Moreover, both upstream and intronic regions had extensive chromatin interactions, and deletion of either region altered the super-enhancer structure in mature T cells. These results demonstrate differential functions for distinct super-enhancer elements, thereby indicating previously unknown ways to manipulate CD25 expression in a cell type-specific fashion.

[Melatonin-Mediated Inhibitory Effect on Hyperimmune Status of Acquired Aplastic Anemia].

OBJECTIVE: To evaluate the expression level of melatonin and its effects on immune function in aplastic anemia (AA) patients. METHODS: The enzyme-linked immunosorbent assay (ELISA) was used to detect the plasma levels of melatonin in AA patients, and the correlation between melatonin levels and laboratory indexs was analyzed. The activation, proliferation, and apoptosis of T cells from AA patients were analyzed by flow cytometry with or without melatonin in vitro. RESULTS: The plasma levels of melatonin in AA patients were significantly lower compared with healthy controls (HC) (12.23 pg/ml vs 20.04 pg/ml, P < 0.01), while the plasma melatonin levels of AA patients in remission group after immunosuppressive therapy (IST) were significantly higher than those in non-remission group (29.16 pg/ml vs 11.73 pg/ml, P =0.04). Moreover, the melatonin levels were positively correlated with platelets (r =0.49), the absolute reticulocyte count (r =0.45), and the percentage of neutrophils (r =0.43). Meanwhile, there was a negative correlation between melatonin levels and the percentages of lymphocytes (r =-0.45). The expressions of CD25 and CD69 in both CD4+ and CD8+ T cells from AA patients were remarkably inhibited by melatonin in vitro (all P < 0.05). When cultured with melatonin, the proliferation rates of both CD4+ and CD8+ T cells from AA patients were markedly suppressed (P =0.01 andP < 0.01). CONCLUSION: The plasma levels of melatonin were decreased in AA patients, which might play an important role in the mechanism of immunological abnormalities. The hyperimmune status of AA patients could be partially ameliorated by melatonin in vitro.

Impaired immunosuppressive effect of bone marrow mesenchymal stem cell-derived exosomes on T cells in aplastic anemia.

BACKGROUND: Previous studies have verified the dysfunction of mesenchymal stem cells (MSCs) for immunoregulation in acquired aplastic anemia (AA) patients. Exosomes derived from MSCs can partially substitute MSCs acting as immune regulator. Dysfunction of exosomes (Exos) derived from AA-MSC (AA-Exos) may play a key role in immunologic dissonance. METHOD: In this study, CD3 + T cells were collected and cocultured with AA-Exos and exosomes derived from HD-MSC (HD-Exos). The proliferation, differentiation and activation of CD3 + T cells were detected to compare the immunosuppressive effects between AA-Exos and HD-Exos. An immune-mediated murine model of AA was structured to compare the therapeutic effect of AA-Exos and HD-Exos. Furthermore, total RNA including miRNA from exosomes we purified and total RNA of CD3 + T cells were extracted for RNA-seq in order to construct the miRNA-mRNA network for interactions and functional analysis. RESULTS: AA-Exos had impaired inhibition effects on CD3 + T cells in terms of cell proliferation, activation and differentiation compared with exosomes from HD-Exos. HD-Exos showed a more effective rescue of AA mice compared to AA-Exos. Importantly, we found some differentially expressed miRNA involved in immune response, such as miR-199, miR-128 and miR-486. The Gene Ontology analysis of differentially expressed genes (DEGs) revealed involvement of various cellular processes, such as lymphocyte chemotaxis, lymphocyte migration and response to interferon-gamma. The Kyoto Encyclopedia of Genes and Genomes analysis illustrated upregulation of critical pathways associated with T cell function after coculturing with AA-Exos compared with HD-Exos, such as graft-versus-host disease, Th17 cell differentiation and JAK-STAT signaling pathway. A miRNA-mRNA network was established to visualize the interaction between them. CONCLUSION: In summary, AA-Exos had impaired immunosuppressive effect on T cells, less ability to rescue AA mice and differently expressed miRNA profile, which might partly account for the pathogenesis of AA as well as provide a new target of AA treatment.

[Deciphering Hypoplastic Myelodysplastic Syndrome and Aplastic Anemia via In-Depth Analysis of Lymphocyte Subsets].

OBJECTIVE: To explore the difference of lymphocyte subsets in peripheral blood (PB) between aplastic anemia (AA) and hypoplastic myelodysplastic syndrome (hypo-MDS) patients, meanwhile to compare the clinical parameters obtained from PB and bone marrow (BM). METHODS: The lymphocyte subsets in hypo-MDS (n=25) and AA (n=33) patients were investigated by flow cytometry. Meanwhile, the differences in PB cell counts, biochemical indicators, BM cell counts and abnormal chromosomes between the two groups were analyzed. RESULTS: The percentage of CD8+T cells in AA group was significantly higher than that in hypo-MDS group (P=0.001), while the percentage of CD4+ T cells and the CD4+/CD8+ ratio in AA group were obviously lower than those in hypo-MDS group (P=0.015 and 0.001, respectively). Furthermore, the proportion of CD4+ and CD8+ activated T (TA) cells, and memory Tregs in AA group was distinctly lower than those in hypo-MDS group (P=0.043, 0.015 and 0.024, respectively). Nevertheless, the percentage of CD8+ naive T (TN) cells in AA patients was remarkably higher (P=0.044). And hypo-MDS patients had declined lymphocyte counts (P=0.025), increased levels of total bilirubin (TBil), lactate dehydrogenase (LDH), vitamin B12 and proportion of BM blasts than AA patients (P=0.019, 0.023, 0.027 and 0.045, respectively). CONCLUSION: In this study it was confirmed that the percentages of CD4+ and CD8+ TA cells, memory Tregs and CD8+ TN cells were significantly different between AA and hypo-MDS patients, which provide an essential basis for the identification of these two diseases.

Acacetin attenuates the pancreatic and hepatorenal dysfunction in type 2 diabetic rats induced by high-fat diet combined with streptozotocin.

Acacetin is a natural flavonoid compound with multiple therapeutic potential in oxidative stress, inflammation, cancers, cardiovascular disease and infections. The present study aimed to detect the effect of acacetin on pancreatic and hepatorenal dysfunction in type 2 diabetic rats. The diabetic rats were induced by high-fat diet (HFD) followed by intraperitoneal injection of streptozotocin (STZ) at a dose of 45 mg/kg. Different doses of acacetin were orally administrated once a day for 8 weeks after the diabetic model was successfully established. The experimental results revealed that acacetin and acarbose displayed obvious attenuation in the levels of fasting blood glucose (FBG) and lipids compared to the untreated diabetic rats. In addition, the physiological function of liver and kidney was impaired in the persistent environment of hyperglycemia, while acacetin improved the damage of liver and kidney. Furthermore, hematoxylin-eosin (H&E) staining indicated that acacetin alleviated the pathological alterations of the pancreas, liver and kidney tissues. Besides, the increased levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-8 and malondialdehyde (MDA) were recused by acacetin treatment, while the reduction of superoxide dismutase (SOD) levels were suppressed by acacetin treatment. In conclusion, the experimental results demonstrated that acacetin improved the lipids and glucose levels, and hepatorenal antioxidant capacity, as well as ameliorated hepatorenal dysfunction in type 2 diabetic rats, and the potential mechanism might be associated with its antioxidant and anti-inflammatory activities.

Association between human leukocyte antigen and immunosuppressive treatment outcomes in Chinese patients with aplastic anemia.

Background: Activated cytotoxic T cells (CTLs) recognize the auto-antigens presented on hematopoietic stem/progenitor cells (HSPCs) through class I human leukocyte antigen (HLA) molecules and play an important role in the immune pathogenesis of aplastic anemia (AA). Previous reports demonstrated that HLA was related to the disease susceptibility and response to immunosuppressive therapy (IST) in AA patients. Recent studies have indicated that specific HLA allele deletions, which helped AA patients to evade CTL-driven autoimmune responses and escape from immune surveillance, may lead to high-risk clonal evolution. Therefore, HLA genotyping has a particular predictive value for the response to IST and the risk of clonal evolution. However, there are limited studies on this topic in the Chinese population. Methods: To explore the value of HLA genotyping in Chinese patients with AA, 95 AA patients treated with IST were retrospectively investigated. Results: The alleles HLA-B*15:18 and HLA-C*04:01 were associated with a superior long-term response to IST (P = 0.025; P = 0.027, respectively), while the allele HLA-B*40:01 indicated an inferior result (P = 0.02). The allele HLA-A*01:01 and HLA-B*54:01 were associated with high-risk clonal evolution (P = 0.032; P = 0.01, respectively), and the former had a higher frequency in very severe AA (VSAA) patients than that in severe AA (SAA) patients (12.7% vs 0%, P = 0.02). The HLA-DQ*03:03 and HLA-DR*09:01 alleles were associated with high-risk clonal evolution and poor long-term survival in patients aged ≥40 years. Such patients may be recommended for early allogeneic hematopoietic stem cell transplantation rather than the routine IST treatment. Conclusion: HLA genotype has crucial value in predicting the outcome of IST and long-term survival in AA patients, and thus may assist an individualized treatment strategy.

Interleukin-10 promoter variability is associated with the susceptibility, severity, and clinical outcomes of aplasitc anemia in Han-Chinese population.

INTRODUCTION: Acquired aplastic anemia (AA), a heterogeneous bone marrow (BM) failure disease, is mainly mediated by the immune destruction of hematopoietic stem cells (HSCs). Given the predominant role of immunosuppressive therapy (IST) in AA, it is sensible to theorize that variants of cytokine genes might affect the outcome of IST. METHODS: In this study, we analyzed three single nucleotide polymorphisms (SNPs) of interleukin (IL)-10 gene in promoter region to clarify their relationship with susceptibility, clinical efficacy and prognosis of AA. RESULTS: We observed that CT genotype of IL-10 rs1800896 was associated with a decreased risk of AA (adjusted OR = 0.541 [95% CI 0.295-0.993], p = .047). Besides, the disease severity differed considerably by IL-10 gene promoter genotypes and alleles. Furthermore, IL-10 SNPs influenced efficacy of IST, with unfavorable response exhibited by rs1800871 and rs1800872 in dominant models (GG + AG vs. AA, adjusted OR = 0.409 [95% CI 0.178-0.943, p = .036] for rs1800871 and GG + GT vs. TT, adjusted OR = 0.396 [95% CI 0.173-0.909, p = .028] for rs1800872, respectively). CONCLUSION: The polymorphisms of IL-10 promoter region were informatively genetic risk factors which might be conducive to the insights into the mechanisms of AA and the design of individual regimens.

Natural killer cells in peripheral blood at diagnosis predict response to immunosuppressive therapy in severe aplastic anemia.

Immunosuppressive therapy (IST) consisting of antihuman thymocyte globulin and cyclosporine A is the first-line therapy for patients with severe aplastic anemia (AA) who are ineligible for undergoing bone marrow transplantation. The aim of the study was to evaluate the correlation between natural killer (NK) cells and response to IST in SAA patients. We retrospectively included 93 AA patients and detected NK cells in peripheral blood by flow cytometry. Both the proportion and absolute number of NK cells in newly diagnosed SAA patients were significantly lower than in controls, while the proportion and absolute number of NK cells in complete remission patients treated with IST were remarkably increased compared with treatment-naïve SAA patients. Additionally, the absolute number of NK cells at diagnosis was positively correlated with initial blood counts. For SAA patients receiving IST, the proportion of NK cells at baseline and 6 months was significantly higher in responders than in non-responders. Unexpectedly, we found that the increase in the proportion of NK cells at 6 months after IST was closely related to the recovery of hematopoiesis. ROC curve identified 7.3% of NK cells proportion at diagnosis as the cutoff value to predict response to IST. The response rate was higher in NK proportion high group than in NK proportion low group. Multivariate logistic regression analysis further confirmed the independent predictive value of NK cells proportion in assessing IST response. The proportion of NK cells at diagnosis may serve as a promising predictor of response to IST in patients with SAA.

Cluster analysis of lymphocyte subset from peripheral blood in newly diagnosed idiopathic aplastic anaemia patients.

INTRODUCTION: Idiopathic aplastic anaemia (IAA) is a heterogeneous autoimmune disease characterised by pancytopenia and bone marrow failure. The objective of the study was to investigate the clusters of lymphocyte subset in newly diagnosed IAA patients and explore their correlation with clinical characteristics. METHODS: A total of 124 newly diagnosed IAA patients were enrolled. Lymphocyte subset was detected by flow cytometry. Cluster analysis was conducted to identify subgroups of patients based on lymphocyte subset. RESULTS: Cluster analysis classified patients into four distinctive subgroups: Cluster 1 (CD4+ T cells dominant), Cluster 2 (CD8+ T cells dominant), Cluster 3 (NK cells dominant) and Cluster 4 (B cells dominant). Patients in Clusters 1 and 4 suffered more severe disease status than ones in Clusters 2 and 3 (p = .013). And with it, patients in Cluster 2 had the highest white blood cell count, haemoglobin level, reticulocyte count and reticulocyte percentage, while patients in Cluster 3 had the lowest lymphocyte percentage and the highest neutrophil count (all p < .05). Unexpectedly, patients in Cluster 3 tended to have superior curative effect than ones in other clusters, an ordinal logistic regression analysis further confirmed the independent correlation between Cluster 3 and good response to treatment. Lymphocyte subset clustering may serve as a biomarker for assessing disease severity and treatment efficacy in newly diagnosed IAA patients.Key MessagesNewly diagnosed IAA patients could be classified into 4 distinctive subgroups with similar immune patterns by using cluster analysis of lymphocyte subset.Clusters of lymphocyte subset were closely correlated with disease severity and treatment response of IAA.Lymphocyte subset clustering may serve as a promising tool for assessing disease severity and treatment efficacy in newly diagnosed IAA patients.

Levamisole Suppresses CD4+ T-Cell Proliferation and Antigen-Presenting Cell Activation in Aplastic Anemia by Regulating the JAK/STAT and TLR Signaling Pathways.

Aplastic anemia (AA) is a life-threatening disease primarily caused by a metabolic disorder and an altered immune response in the bone marrow (BM) microenvironment, where cytotoxic immune cells attack resident cells and lead to hematopoietic failure. We previously reported an efficient strategy by applying cyclosporin (CSA) combined with levamisole (CSA+LMS-based regimen) in the treatment of AA, but the immunoregulatory mechanism of LMS was still unclear. Here, the therapeutic effects of LMS were examined in vivo using the BM failure murine model. Meanwhile, the proportion and related function of T cells were measured by flow cytometry in vivo and in vitro. The involved signaling pathways were screened by RNA-seq and virtual binding analysis, which were further verified by interference experiments using the specific antagonists on the targeting cells by RT-PCR in vitro. In this study, the CSA+LMS-based regimen showed a superior immune-suppressive response and higher recession rate than standard CSA therapy in the clinical retrospective study. LMS improved pancytopenia and extended the survival in an immune-mediated BM failure murine model by suppressing effector T cells and promoting regulatory T-cell expansion, which were also confirmed by in vitro experiments. By screening of binding targets, we found that JAK1/2 and TLR7 showed the highest docking score as LMS targeting molecules. In terms of the underlying molecular mechanisms, LMS could inhibit JAK/STAT and TLR7 signaling activity and downstream involved molecules. In summary, LMS treatment could inhibit T-cell activation and downregulate related molecules by the JAK/STAT and TLR signaling pathways, supporting the valuable clinical utility of LMS in the treatment of AA.

Impaired immunosuppressive role of myeloid-derived suppressor cells in acquired aplastic anemia.

Myeloid-derived suppressor cells (MDSC) are a group of heterogeneous immature myeloid cells and display immunosuppressive function. In this study, MDSC populations were evaluated in acquired aplastic anemia (AA) (n=65) in which aberrant immune mechanisms contributed to bone marrow destruction. Our data demonstrate that both the proportion and immunosuppressive function of MDSC are impaired in AA patients. Decreased percentage of MDSC, especially monocytic MDSC, in the blood of AA patients (n=15) is positively correlated with the frequency of T-regulatory cells, bone marrow level of WT1 and decreased plasma level of arginase-1. RNA sequencing analyses reveal that multiple pathways including DNA damage, interleukin 4, apoptosis, and Jak kinase singnal transducer and activator of transcription are upregulated, whereas transcription, IL-6, IL-18, glycolysis, transforming growth factor and reactive oxygen species are downregulated in MDSC of AA (n=4), compared with that of healthy donors (n=3). These data suggest that AA MDSC are defective. Administration of rapamycin significantly increases the absolute number of MDSC and levels of intracellular enzymes, including arginase-1 and inducible nitric-oxide synthase. Moreover, rapamycin inhibits MDSC from differentiating into mature myeloid cells. These findings reveal that impaired MDSC are involved in the immunopathogenesis of AA. Pharmacologically targeting of MDSC by rapamycin might provide a promising therapeutic strategy for AA.

The novel SLC40A1 (T419I) variant results in a loss-of-function phenotype and may provide insights into the mechanism of large granular lymphocytic leukemia and pure red cell aplasia.

Variants in the solute carrier family 40 member 1 (SLC40A1) gene are the molecular basis of ferroportin disease, which is an autosomal dominant hereditary hemochromatosis. Here, we present a patient with pure red cell aplasia (PRCA) and large granular lymphocytic leukemia (LGLL) associated with an extremely high levels of serum ferritin and iron overload syndrome. Whole exon sequencing revealed a novel heterozygous variant in SLC40A1 (p.T419I), which was found in his daughter as well. A series of functional studies in vitro of the T419I variant in ferroportin were conducted and the results revealed a reduced capacity of iron export from cells without changes in protein localization and its sensitivity to hepcidin. Intracellular iron storage in mutated cells was significantly higher than that of wild-type. These findings suggest that the novel variant p.T419I can cause the classical form of ferroportin disease and an elevated intracellular iron level indicates a potential novel pathogenic mechanism underlying PRCA and LGLL.

Leptin-mediated proinflammatory bone marrow environment in acquired aplastic anemia.

Acquired aplastic anemia (AA), a paradigm of bone marrow failure syndrome, is mainly caused by abnormal immune activation. The enhanced adipogenesis of bone marrow-derived mesenchymal stem cell (BM-MSC) results in a fatty marrow of AA. Leptin, an adipokine mainly generated by adipocytes, has powerful proinflammatory effects on immune cells and is associated with various autoimmune diseases. However, the role of leptin in the hyperimmune status of AA remains unknown. In this study, we firstly discovered the higher leptin concentration in AA-BM than that in healthy donors (HD)-BM and myelodysplastic syndrome (MDS)-BM. Then, we found AA-MSC could express high amounts of leptin during the process of adipogenesis. Compared with HD, the leptin receptor was also highly expressed on T cells in AA-BM. Furthermore, leptin significantly accelerated the proliferation and activation of T cells in AA-BM. And, leptin promoted the production of interferon-γby T cells in AA-BM. However, leptin remarkably inhibited the conversion of CD4+CD25- T cells into CD4+Foxp3+ T cells. Finally, we detected the cell signaling pathway in T cells from AA patients and found leptin could activate the STAT3 pathway. In summary, our data revealed the high expression of adipokine leptin in AA-BM which shaped a proinflammatory environment for T cells in AA-BM by activating the JAK2/STAT3 pathway.

Efficacy of anti-thymocyte globulin for platelet transfusion refractoriness in serious aplastic anemia patients.

Platelet transfusion refractoriness (PTR) is a life threatening, intractable clinical issue suffered by some serious aplastic anemia (SAA) patients. Unlike immune thrombocytopenia, effective treatments for PTR remain largely unknown. In our clinical work, we noted that PTR in some SAA patients could be rapidly relieved with the application of anti-thymocyte globulin (ATG), therefore, we retrospectively analyzed its management and outcomes for PTR in SAA patients. A cohort including 29 SAA with PTR patients who received ATG administration was enrolled in this study. All patients suffered from PTR before ATG administration. Among the 29 PTR patients treated with ATG, 21 (72.4.0 %) patients had response, importantly, 13 (44.8 %) patients had an immediately response following the first dose of ATG administration. Bleeding events of grade 3 or above occurred in 23 patients (79.3 %). With the recovery of effective platelet transfusion, the bleeding events in responders could be quickly relieved. The non-responders suffered from aggravated bleeding, including intracranial bleeding in two non-responders, which appeared on eighth and 29th days after ATG administration. Our study indicated that ATG was an effective and safe intervention in the management of PTR in SAA patients.

Platelet/Lymphocyte ratio independently predicts the outcome of severe aplastic anemia patients treated with antithymocyte globulin.

OBJECTIVE: The aim of this study was to determine the clinical role of platelet/lymphocyte ratio and neutrophil/lymphocyte ratio in severe aplastic anemia patients treated with antithymocyte globulin. METHODS: The outcomes of consecutive severe aplastic anemia patients treated with rabbit or swine antithymocyte globulin plus cyclosporine (n=159, from January 2012 to December 2018) were analyzed retrospectively. RESULTS: In a total of 159 patients, the actuarial 5-year survival rate was 85.6%. Low platelet/lymphocyte ratio (PLR≤55) was significantly associated with less complications at 1 month and 24 months after the antithymocyte globulin treatment (p=0.048 and 0.028, respectively). The univariate and multivariate analyses revealed that low platelet/lymphocyte ratio was an independent predictor of overall survival (p=0.03 and 0.04, respectively). Patients with low neutrophil/lymphocyte ratio (NLR≤0.18) had shorter survival time, but there was no significant difference (p=0.056). PLR was positively correlated with neutrophil/lymphocyte ratio (r=0.38, p<0.0001) and age (r=0.17, p=0.0379), while it was negatively correlated with IgG level (r=-0.18, p=0.0309). The ratio of CD4/CD8 was significantly higher in low platelet/lymphocyte ratio group (p=0.005). CONCLUSION: The platelet/lymphocyte ratio reflects the immune abnormality of SAA. Notably, low platelet/lymphocyte ratio is an independently positive prognostic factor for severe aplastic anemia patients treated with antithymocyte globulin.

A hemizygous p.R204Q mutation in the ALAS2 gene underlies X-linked sideroblastic anemia in an adult Chinese Han man.

BACKGROUND: X-linked sideroblastic anemia (XLSA) is the most common form of congenital sideroblastic anemia (CSA), and is associated with the mutations in the 5-aminolevulinate synthase 2 (ALAS2). The genetic basis of more than 40% of CSA cases remains unknown. METHODS: A two-generation Chinese family with XLSA was studied by next-generation sequencing to identify the underlying CSA-related mutations. RESULTS: In the study, we identified a missense ALAS2 R204Q mutation in a hemizygous Chinese Han man and in his heterozygous daughter. The male proband presented clinical manifestations at 38 years old and had a good response to pyridoxine. CONCLUSIONS: XLSA, as a hereditary disease, can present clinical manifestations later in lives, for adult male patients with ringed sideroblasts and hypochromic anemia, it should be evaluated with gene analyses to exclude CSA.

Small PNH clones detected by fluorescent aerolysin predict a faster response to immunosuppressive therapy in patients with severe aplastic anaemia.

Objectives: To clear the obscure conclusion on the prediction value of paroxysmal nocturnal haemoglobinuria (PNH) clones in severe aplastic anaemia (SAA) patients treated with immunosuppressive therapy (IST). Methods: We retrospectively analyzed 219 consecutive SAA patients treated with IST from October 2008 to October 2015 and evaluated the haematological responses to IST. Results: The presence of a PNH clone was detected in 55 (25.1%) patients prior to IST [37/88 by flow cytometry (FCM) and 18/131 by fluorescent aerolysin (FLAER)] and 27 disappeared after IST (23/37 in initial FCM group, 4/18 in initial FLAER group, p = 0.005). In patients without an initial clone, 12 (30.0%) cases in FCM and 17 (19.5%) in FLAER groups presented a PNH clone at least once after IST (p < 0.001). In patients with a pre-treatment PNH clone detected by FCM, the 3-, 6- and 12-month response rates were higher than patients without (p = 0.006; 0.002 and 0.002, respectively). And in FLAER group, the 3-month response rate was significantly higher in those with a prior clone (p = 0.017), however, the 6- and 12-month response rates showed no differences (p = 0.105, p = 0.144, respectively). By multivariate analysis, a shorter interval between diagnosis and treatment is associated with a better response and survival. Conclusions: A more reliable FLAER method allows us to draw a conclusion that PNH clone predicts a faster response but not a higher response rate to IST. Once a diagnosis is confirmed, the IST should be initiated as soon as possible.

Long-term follow-up of a novel immunosuppressive strategy of cyclosporine alternatively combined with levamisole for severe aplastic anemia.

Hematopoietic stem cell transplantation (HSCT) and immunosuppressive therapy (IST) with antithymocyte globulin (ATG) and cyclosporine (CsA) have been widely accepted as the standard first-line treatments for severe aplastic anemia (SAA). However, most of the patients with SAA had a slim chance to access these strategies in developing countries. Here, we reported 10-year results in a cohort of 232 patients with SAA who received a novel IST of CsA, levamisole, and danazol (CsA&LMS-based regimen). The cumulative incidence of response was 52.1% at 6 months, 66.4% at 12 months, and 77.1% at 24 months. The 10-year overall survival (OS) and failure-free survival was 60.2% and 48.3%, respectively. Positive predictors of OS in multivariate analysis were higher pretreatment ANC, younger age, higher pretreatment absolute reticulocyte count (ARC), and response within 6 months. The probability of CsA&LMS discontinuation was 50.2% at 10 years. With a slow CsA&LMS taper, the actuarial risk for relapse was only 9.5%. The cumulative incidence of MDS/AML was 8.2% at 10 years. The long-term follow-up information demonstrated that the CsA&LMS regimen could be a promising strategy for patients with SAA in developing countries.