BACKGROUND:Lilac-colored aggregates (LicAGs) are often observed on bone marrow (BM) aspirate smears stained with May-Grünwald-Giemsa (MG) in patients with amyloid light-chain (AL) amyloidosis. However, the clinicopathological features of LicAGs observed on MG-stained BM smears remain poorly understood. METHODS:We examined MG-stained BM aspirate smears and clinical data from 103 patients with AL amyloidosis. To determine whether LicAGs represented amyloid, LicAG-positive specimens were decolorized, re-stained with Congo red, and examined under polarized light. Patients were grouped based on the presence or absence of LicAGs, and their clinical characteristics were compared. Serial changes in LicAG counts following therapy were assessed in two cases. RESULTS:Of the 103 patients, 20 showed LicAGs (BM[+] group), confirmed as amyloid fibrils using Congo red staining. Compared to the BM(-) group, the BM(+) group had higher proportions of κ-type amyloid, liver and tongue involvement, and negative monoclonal immunoglobulin on immunofixation electrophoresis. LicAG counts decreased on serial BM examinations in two patients after treatment. CONCLUSIONS:LicAGs are amyloid fibrils, indicating their potential significance in AL amyloidosis, especially when monoclonal immunoglobulins are undetectable. They may also serve as indicators of organ involvement and as surrogate markers for treatment response.

T-cell abnormalities have been implicated in the pathogenesis of acquired pure red cell aplasia (PRCA), particularly in its major subtypes such as idiopathic PRCA, thymoma-associated PRCA and large granular lymphocytic leukaemia (LGLL)-associated PRCA, and the precise details remain unclear. Furthermore, signal transducer and activator of transcription 3 (STAT3) mutations are frequently detected in PRCA patients, but their association with cellular immune abnormalities is not well understood. In order to elucidate the immunogenetic backgrounds of PRCA, we conducted human leucocyte antigen (HLA) typing in 39 PRCA patients. The results showed a significantly higher allele frequency of HLA-B*44:03:01, -C*14:03, -DRB1*13:02, compared to HLA database. Additionally, analysis of inferred HLA haplotypes revealed significantly increased frequencies of two haplotypes: HLA-A*24:02-C*07:02-B*07:02-DRB1*01:01 and -A*33:03-C*14:03-B*44:03:01-DRB1*13:02. Clonotypic analyses of T-cell receptor beta (TCRβ) chain revealed that 80% of the PRCA cases possessed mono- or oligoclonally expanded T cells. Particularly among those with STAT3 mutations, the 'QGXG' motif in 15 AA sequences of TCRβ complementarity-determining regions 3 (CDR3) regions was specifically recognized. These findings suggest that a specific immunogenetic background defined by particular HLA alleles, the expansion of somewhat limited T-cell clones and STAT3-mutated T cells are involved in the pathogenesis of chronic acquired PRCA.

Dysregulation of T cell-mediated immunity is considered a major pathophysiological mechanism in acquired pure red cell aplasia (PRCA), including idiopathic PRCA, large granular lymphocytic leukemia-associated PRCA, and thymoma-associated PRCA. Although STAT3 mutations are frequently detected in PRCA patients, the roles of other mutational profiles and their impact on clinical characteristics remain unclear. In this study, whole-exome sequencing and targeted sequencing using a custom-designed panel were performed on 53 PRCA patients. The most frequently mutated genes were STAT3 (36%), PCLO (9%), TET2 (9%), NEB (6%), DNMT3A (6%), and POT1 (6%). Based on genetic profiles, patients were classified into three groups: those with STAT3 variants (group S), those without STAT3 variants but with variants in clonal hematopoiesis (CH)-related genes (group C), and those without variants in either STAT3 or CH-related genes (group O). Patients in group O had a higher median age compared to group S, while group S exhibited milder anemia severity than group C. Additionally, POT1 variants were associated with the idiopathic subtype of PRCA in females, often co-occurring with STAT3 variants. Variants in CH-related genes and other genes, including STAT3 and POT1, may play crucial roles in the pathophysiology of PRCA.

Abstract Dysregulation of T cell-mediated immunity is considered a major pathophysiological mechanism of acquired pure red cell aplasia (PRCA), such as idiopathic PRCA, large granular lymphocytic leukemia-associated PRCA, and thymoma-associated PRCA. Although STAT3 mutations are frequently detected in PRCA patients, other mutational profiles and their involvement in the clinical characteristics are yet to be clarified. Whole-exome sequencing and targeted sequencing were performed using a custom-designed panel for PRCA (n = 53). The frequently mutated genes were NEB (40%), STAT3 (36%), PCLO (30%), TET2 (23%), and KMT2D (15%). Four of the 12 patients with mutations in TET2 had germline TET2 variants. Patients positive for TET2 variants had significantly more variants of lymphoid clonal hematopoiesis-related genes than those without TET2 variants (11/12 vs. 23/41, P = 0.038). Patients with TET2 variants relapsed after immunosuppressive therapy more frequently than those without TET2 variant (55% [6/11] vs. 11% [4/35], P = 0.0065). These data suggest that variants of clonal hematopoiesis-related genes, including TET2, in addition to STAT3, play important roles in the pathophysiology of PRCA.

Primary tumor cells metastasize to a distant preferred organ. However, the most decisive host factors that determine the precise locations of metastases in cancer patients remain unknown. We have demonstrated that post-translational citrullination of fibrinogen creates a metastatic niche in the vulnerable spots. Pulmonary endothelial cells mediate the citrullination of fibrinogen, changing its conformation, surface charge, and binding properties with serum amyloid A proteins (SAAs), to make it a host tissue-derived metastatic pathogen. The human-specific SAAs-citrullinated fibrinogen (CitFbg) complex recruits cancer cells to form a protein-metastatic cell aggregation in humanized SAA cluster mice. Furthermore, a CitFbg peptide works as a competitive inhibitor to block the homing of metastatic cells into the SAAs-CitFbg sites. The potential metastatic sites in the lungs of patients are clearly visualized by our specific antibody for CitFbg. Thus, CitFbg deposition displays metastatic risks for cancer patients, and the citrullinated peptide is a new type of metastasis inhibitor.

Activated neutrophils release neutrophil extracellular traps (NETs) composed of chromatin filaments containing bactericidal proteins and enzymes. This process, known as NETosis, is an innate host defense mechanism. However, NET accumulation can lead to uncontrolled inflammation and organ damage. Therefore, NET detection provides clinically important information for the assessment of inflammatory conditions. We investigated whether quantification of citrullinated fibrinogen (C-Fbg), which is catalyzed by peptidylarginine deiminase (PAD) released during NETosis, can be used to detect NETs. Human neutrophils were stimulated with fibrinogen using phorbol 12-myristate 13-acetate (PMA). The myeloperoxidase (MPO)-DNA complex and C-Fbg concentrations in the culture supernatants were quantified using an enzyme-linked immunosorbent assay. The protein levels of peptidylarginine deiminase 2 and 4 in culture supernatants and mRNA levels in PMA-stimulated neutrophils were also assessed. The levels of the MPO-DNA complex in the supernatants of PMA-stimulated neutrophils increased, indicating NETosis. C-Fbg level also increased, which was suppressed by both NETosis and PAD inhibitors. PAD2 was detected in the culture supernatant; however, PAD4 , but not PAD2 , mRNA levels increased in PMA-stimulated neutrophils. This study quantitatively demonstrates that fibrinogen is citrullinated by PAD derived from PMA-stimulated neutrophils upon NETosis. Although further studies are needed for clinical application, quantification of C-Fbg in blood may help detect the presence of NETs.

Thrombocytopenia is a common abnormality encountered in the neonatal period, and immature platelet fraction (IPF) may be an informative indicator of thrombopoiesis; however, data on IPF in neonates are scarce. To define reference intervals (RIs) and factors affecting IPF in neonates, we measured the IPF of 533 consecutive neonates. With a multiple regression analysis of 330 newborns with normal platelet counts at birth, premature delivery, neonatal asphyxia, intrauterine infection, chromosomal abnormalities, and respiratory disorders were identified as independent factors for IPF%. The RIs of IPF% and absolute IPF value in neonates were determined to be 1.3% to 5.7% and 3.2 to 14.5×109/L, respectively. On day 14 after birth, IPF% increased to twice the value at birth and thereafter returned to the previous value on day 28. Reticulocyte counts, in contrast, were the lowest at day 14. IPF% was increased in 16 thrombocytopenic patients with various clinical conditions, especially those with immune-mediated thrombocytopenia. IPF in neonates may be evaluated essentially based on the same RIs as in adults, although some precautions must be taken when evaluating IPF in neonates in the first 2 weeks of life. IPF may be useful for evaluating thrombopoiesis and thrombocytopenia in neonates.

Acquired chronic pure red cell aplasia (PRCA) develops idiopathically or in association with other medical conditions, including T cell large granular lymphocytic leukemia (T-LGLL) and thymoma. T cell dysregulation is considered a cardinal pathogenesis of PRCA, but genetic–phenotypic associations in T cell abnormalities are largely unclear. We evaluated an extended cohort of 90 patients with acquired PRCA, including 26 with idiopathic, 36 with T-LGLL-associated and 15 with thymoma-associated PRCA, for their T cell immuno-phenotypes, clonalities and STAT3 mutations. TCR repertoire skewing of CD8+ T cells was detected in 37.5% of idiopathic, 66.7% of T-LGLL-associated and 25% of thymoma-associated PRCA patients, and restriction to Vβ1 was most prominent (41%). Clonalities of TCRβ or γ chain and STAT3 mutational status were statistically associated (P = 0.0398), and they were detected in all three subtypes. The overall response rate to cyclosporin A was 73.9%, without significant difference by subtypes nor STAT3 mutational status. The T cell dysregulations, such as TCR repertoire skewing with predominant Vβ1 usage, clonality and STAT3 mutations, were frequently found across the subtypes, and the close associations between them suggest that these T cell derangements reflect a common pathophysiological mechanism among these PRCA subtypes.

We identified a patient with a novel heterozygous variant fibrinogen, γp.C352R (Niigata II; N-II), who had a bleeding episode and failed infertility treatment and was suspected to have hypodysfibrinogenemia based on low and discordant fibrinogen levels (functional assay 0.33 g/L, immunological assay 0.91 g/L). We analyzed the mechanism of this rare phenotype of a congenital fibrinogen disorder. Patient plasma fibrinogen was purified and protein characterization and thrombin-catalyzed fibrin polymerization performed. Recombinant fibrinogen-producing Chinese hamster ovary (CHO) cells were established and the assembly and secretion of variant fibrinogen analyzed by ELISA and western blotting. Purified N-II plasma fibrinogen had a small lower molecular weight band below the normal γ-chain and slightly reduced fibrin polymerization. A limited proportion of p.C352R fibrinogen was secreted into the culture medium of established CHO cell lines, but the γ-chain of p.C352R was synthesized and variant fibrinogen was assembled inside the cells. We demonstrated that fibrinogen N-II, γp.C352R was associated with markedly reduced secretion of variant fibrinogen from CHO cells, that fibrin polymerization of purified plasma fibrinogen was only slightly affected, and that fibrinogen N-II produces hypodysfibrinogenemia in plasma.

Background: Citrullinated fibrinogen (C-Fbg) has been detected in rheumatoid arthritis; however, few studies have reported the role of C-Fbg in other inflammatory diseases. This study aimed to clarify the changes in serum C-Fbg associated with the bacteremia phase. Methods: We measured serum C-Fbg concentration in bacteremia patients. C-Fbg levels at each phase of bacteremia, classified by white blood cell (WBC) count and neutrophil left shift change, were compared with those of healthy control (HC). The correlation between C-Fbg concentration and certain inflammatory markers, or citrullinated histone H3 concentration was assessed. Multiple linear regression (MLR) analysis was used to examine the association of log C-Fbg with certain inflammatory markers. Result: Serum C-Fbg levels were significantly higher in bacteremia patients than in HC (p< 0.001) and positively correlated with WBC and neutrophil count. Further, C-Fbg levels were significantly higher in phases III and IV of bacteremia than in HC (p< 0.001). MLR analysis indicated that log C-Fbg had a stronger relationship with log neutrophil counts than other certain inflammatory markers (p < 0.01). Conclusion: Serum C-Fbg levels increased in bacteremia patients, and this was consistent with an influx of neutrophils into the blood stream in accordance with the bacteremia phase.

Introduction Congenital fibrinogen disorders are classified as afibrinogenemia, hypofibrinogenemia, dysfibrinogenemia, and hypodysfibrinogenemia. However, difficulties are associated with discriminating between dysfibrinogenemia, hypofibrinogenemia, and hypodysfibrinogenemia using routine analyses. We previously reported a heterozygous variant fibrinogen (gamma A289V; Kanazawa III) as hypodysfibrinogenemia; however, the same variant had previously been described as hypofibrinogenemia. To clarify the production of gamma A289V fibrinogen, we expressed recombinant gamma A289V (r-gamma A289V) fibrinogen and compared it with wild-type (WT) and adjacent recombinant variant fibrinogens. Methods Target mutations were introduced into a fibrinogen gamma-chain expression vector by site-directed mutagenesis, and the vector was then transfected into Chinese hamster ovary cells to produce recombinant fibrinogen. Fibrinogen was purified from the plasma of the proposita, and culture media and fibrinogen functions were analyzed using fibrin polymerization, plasmin protection, and FXIIIa-catalyzed fibrinogen cross-linking. Results The fibrinogen concentration ratio of the culture media to cell lysates was markedly lower for r-gamma A289V fibrinogen than for WT. Because the secretion of recombinant gamma F290L (r-gamma F290L) fibrinogen was similar to WT, we compared r-gamma F290L fibrinogen functions with WT. The fibrin polymerization of Kanazawa III plasma (K-III) fibrinogen was significantly weaker than normal plasma fibrinogen. Moreover, K-III fibrinogen showed a markedly reduced "D:D" interaction. However, all functions of r-gamma F290L fibrinogen were similar to WT. An in silico analysis confirmed the above results. Conclusion The present results demonstrated that gamma A289 is crucial for the gamma-module structure, and the gamma A289V substitution markedly reduced fibrinogen secretion. Moreover, K-III fibrinogen showed markedly reduced fibrin polymerization and "D:D" interactions. gamma A289V fibrinogen was confirmed as hypodysfibrinogenemia.

T-cell receptor (TCR)γδ-type large granular lymphocytic leukaemia (LGLL) is a rare T-cell LGLL that is often complicated with cytopenia and autoimmune abnormalities.1-3 In CD8+ TCRαβ-type LGLL, a major type of T-cell LGLL, dysregulation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling system with an interleukin 6 (IL-6)-dependent and/or independent manner, including activation of STAT molecules, has been recognised, and STAT3 is the most frequently mutated gene.4, 5 In CD4+ TCRαβ-type LGLL, STAT5B is frequently mutated.6 Mutations of the above-mentioned genes were located mainly in the src-homology (SH)-2 domain, and are considered to be activating mutations.7 On the other hand, the molecular basis of TCRγδ-type LGLL is still uncertain. We analysed the mutational profiles of the STAT3, STAT5B and tumour necrosis factor alpha-induced protein 3 (TNFAIP3) genes in TCRγδ-type LGLL. The present study was conducted in accordance with the Declaration of Helsinki and was approved by the Institutional Review Boards of Shinshu University School of Medicine. Written informed consent was obtained from the patients. Six patients with TCRγδ-type LGLL were recruited Table I. The median age was 48 years and five patients were male. The median haemoglobin value was 58 g/l. Neutropenia and pure red cell aplasia were observed in four cases each. The platelet count was within the reference range in all cases. The TCRγδ cells were positive for CD2 and CD3, and negative for CD4 and CD56. CD8 and CD16 positivity was detected in three cases each. The analysis of the TCRγ and TCRδ chain genes by the BioMed-2 protocol showed monoclonal patterns in all cases. Vγ9 and Vγ5 were utilised in two cases, Vγ2 in one case, Vδ1 in four cases and Vδ3 in two cases. Most of the TCRγδ T cells in the peripheral blood of normal subjects utilised a combination of the Vγ9 and Vδ2 chains, which were not found among the patient samples. We analysed the STAT3, STAT5B and TNFAIP3 coding regions of genomic DNA extracted from the mononuclear cells of the patients by deep amplicon sequencing as previously described.8 Briefly, amplicon sequencing was performed using Ion AmpliSeq technology on Ion PGM according to the standard protocol using the Ion 314 or 318 Chip Kit v2 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The data were analysed using the Torrent Suite software program (version 5.2.2). The main variant calling settings were as follows: variant frequency filter 0·005, base quality Q-value ≥20; minimum coverage of depth 1000; and maximum strand bias 0·95 (SNP), 0·9 (INDEL). Allele-specific polymerase chain reaction (PCR) for Y640F and D661Y of STAT3 and N642H and Y665F of STAT5B was performed as previously reported8 and the primers used are described in Table SI. The mean coverage of amplicon sequencing was 3,425x. All six cases were positive for STAT3 mutations Table I and no STAT5B mutations were recognised. The STAT3 mutation sites were H410R, Q448E, Y640F, D661Y and D661V Fig 1. The median (range) variant allele frequency (VAF) of STAT3 was 5·8 (1·3–22·6)%. In case 3, a STAT3 D661Y mutation was detected only by allele-specific PCR. Among the detected STAT3 mutations, four mutations (67%) were within the SH-2 domain. Case 3 was also positive for TNFAIP3 gene mutations at D117fs (VAF, 17·4%) and D119N (VAF, 17·3%). The other five patients were negative for TNFAIP3 mutations. These mutations were detected in TCRγδ T-cell fractions obtained by cell sorting in all four cases that were examined, but not in TCRαβ T cells. When compared with 50 other LGLL cases, which included 25 CD8+TCRαβ-type LGLLs, eight CD4+TCRαβ-type LGLLs and 17 chronic lymphoproliferative disorder of NK cells (CLPD-NK), the patients with TCRγδ-type LGLL were younger (P = 0·0498), had lower white blood cell counts and lymphocyte counts (P = 0·038, P = 0·024 respectively), and had lower haemoglobin values (P = 0·0033). The frequency of STAT3 mutations in TCRγδ-type LGLL was significantly higher than that in other LGLLs (6/6 vs. 22/50, P = 0·023) in an analysis using the system that was used in the present study Table SII. We detected STAT3 mutations in all the six patients by deep amplicon sequencing. In previous reports on TCRγδ-type LGLL, Sanger sequencing was applied for the analysis of the STAT3 gene, mainly the SH-2 domain, revealing that the rates of positivity for STAT3 mutations were between 25% and 67%.9, 10 The STAT3 mutated clones of TCRγδ-type LGLL were relatively small in size, with a median VAF of 5·8%, which implies that STAT3 mutation might occur as a late event in leukaemogenesis. However, the high incidence of STAT3 mutations in TCRγδ-type LGLL would suggest that the STAT3 mutations identified with high throughput sequencing could be diagnostic for and a biomarker of TCRγδ-type LGLL. In case 3, mutations in two genes, STAT3 and TNFAIP3, were recognised, which suggests that multiple mutational processes might play roles in the development of TCRγδ-type LGLL. Further analyses should be performed to clarify the entire mutational landscape of TCRγδ-type LGLL. In addition, TCRγδ-type lymphomas include hepatosplenic T-cell lymphoma, primary cutaneous γδT-cell lymphoma, and monomorphic epitheliotrophic intestinal T-cell lymphoma. Most patients with such lymphomas follow an aggressive clinical course, show resistance to various treatments, and have a poor prognosis. STAT5B is frequently mutated in these lymphomas,11, 12 in contrast to TCRγδ-type LGLL, which is usually clinically indolent. These different genetic features might be attributed to their distinct pathophysiology or clinical behaviour. In conclusion, STAT3 mutations occur with high frequency in TCRγδ-type LGLL, and their diagnostic implications should be confirmed in a larger cohort. The genetic-phenotypic relationship of the mutations in TCRγδ-type LGLL warrants further analyses. This research was supported in part by AMED under Grant Number JP18ek0109272. Fumihiro Ishida, Hideyuki Nakazawa and Taku Yamane designed the study and wrote the paper. Taku Yamane, Toru Kawakami, Jun Kobayashi, Fumihiro Kawakami and Yumiko Higuchi performed the experiments and analysed the data. Nodoka Sekiguchi, Toshimitsu Ueki, Hikaru Kobayashi, Sayaka Nishina, Hitoshi Sakai and Kazuo Oshimi collected the patients’ samples and the data. All the authors read and approved the final version of the manuscript. Table SI. Primers for allele-specific PCR to detect STAT3 and STAT5B. Table SII. Comparison of TCRγδ-type T-cell large granular lymphocytic leukaemia with other types of large granular lymphocytic leukaemia. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Introduction: Congenital fibrinogen disorders result from genetic mutations in FGA, FGB, or FGG resulting in quantitative fibrinogen deficiencies (afibrinogenemia or hypofibrinogenemia) or qualitative fibrinogen deficiencies (dysfibrinogenemia). Hypodysfibrinogenemia sharing features with hypoand dysfibrinogenemia is rare. We performed genetic and functional analyses of a 31-year-old woman with suspected hypodysfibrinogenemia. Materials and methods: Functional and antigenic fibrinogen values of patient were 1.05 and 1.24 g/L, respectively. DNA sequence and western blotting analyses for plasma fibrinogen were performed. A minigene incorporating the mutational region was transfected into a Chinese hamster ovary cell line (CHO), and reverse transcription products were analyzed. Assembly and secretion were examined using the recombinant variant fibrinogen. We purified the patient's plasma fibrinogen and analyzed thrombin-catalyzed fibrin polymerization (TCFP). Results and conclusions: DNA sequencing revealed compound heterozygous nucleotide mutations with FGB 35 bp c.1245-17_1262 or-16_1263 del and FGB c.510T > A (resulting in B beta p.N170K substitution) on different alleles. We did not detect shortened B beta-chain peptides in the plasma using western blotting analysis. A minigene incorporating the deletion DNA showed two aberrant mRNA products. The secretion of B beta p.N170K-fibrinogenCHO was almost same as normal B beta-fibrinogen-CHO. TCFP of plasma B beta p.N170K fibrinogen was slightly lower than that of normal plasma fibrinogen. Aberrant splicing products derived from the 35 bp deletion caused hypofibrinogenemia due to nonsense-mediated mRNA decay and suggested the presence of only B beta p.N170K fibrinogen in patient's plasma. B beta p.N170K caused dysfibrinogenemia due to a delay in lateral aggregation. These findings demonstrated that these mutations respectively affected the fibrinogen quality and quantity, resulting in hypodysfibrinogenemia.

We identified two heterozygous dysfibrinogenemias, Bβp.Gly45Cys (Kyoto VII; K-VII) and Bβp.Arg74Cys (Iida II; I-II). The impairment of polymerization of Bβp.G45C has been well analyzed; however, that of Bβp.R74C has not. Thus, we compared fibrin polymerization between these variants. To determine the structural and functional characterization of purified fibrinogens, we performed immunoblotting analysis, kinetic analyses of fibrinopeptide A and B release, and thrombin- or batroxobin-catalyzed fibrin or fibrin monomer polymerization. Immunoblotting analysis showed that both variant fibrinogens had variant fibrinogen-albumin complexes and variant fibrinogen multimers, and the amounts of fibrinogen-albumin complexes with fibrinogen K-VII was more than with fibrinogen I-II. Moreover, fibrinopeptide B release from fibrinogen K-VII was about 50% of the control, whereas the others were normal. The maximum slopes of polymerization for variant fibrinogens were reduced, but fibrinogen K-VII was reduced more than fibrinogen I-II. The present study demonstrated that both Bβp.G45C and Bβp.R74C variants showed the presence of variant fibrinogen-albumin complexes and variant fibrinogen multimers, and polymerization of Bβp.G45C was impaired more than Bβp.R74C. Our study and several previous reports concerning the clinical phenotype of both variants suggested the risks of bleeding for patients with Bβp.G45C and thrombosis for patients with Bβp.R74C.

We identified a novel heterozygous variant, Bβp.Pro234Leu (fibrinogen Tokorozawa), which was suspected to be associated with hypofibrinogenemia. Therefore, we analyzed the assembly and secretion of this fibrinogen using Chinese hamster ovary (CHO) cells. To determine the impact on the synthesis and secretion of fibrinogen of the Bβp.P234L and γp.G242E substitutions, we established recombinant variant fibrinogen-producing CHO cell lines. Synthesis and secretion analyses were performed using an enzyme-linked immunosorbent assay (ELISA) and immunoblotting analysis with the established cell lines. In addition, we performed fibrin polymerization using purified plasma fibrinogen and in-silico analysis. Both Bβp.P234L and γp.G242E impaired the secretion and synthesis of fibrinogen. Moreover, immunoblotting analysis elucidated the mobility migration of the Bβγ complex in Bβp.P234L. On the other hand, the fibrin polymerization of fibrinogen Tokorozawa was similar to that of normal fibrinogen. In-silico analysis revealed that the Bβp.P234 residue is located in the contact region between the Bβ and γ chains and contacts γp.G242 residue. The present study demonstrated that the Bβp.P234L substitution resulted in hypofibrinogenemia by decreasing the assembly and secretion of fibrinogen. Therefore, there is a possibility that substitutions in the contact region between the Bβ and γ chains impact the assembly and secretion of fibrinogen.

Background: The fibrinogen gamma-module has several functional sites and plays a role in dysfibrinogenemia, which is characterized by impaired fibrin polymerization. Variants, including gamma D318Y and gamma Delta N319D320, have been reported at the high affinity Ca2+-binding site, and analyses using recombinant fibrinogen revealed the importance of this site for fibrinogen functions and secretion. We examined the polymerization abilities of the recombinant fibrinogen variants, gamma D318Y and gamma K321E. Materials and methods: gamma D318Y and gamma K321E were produced using CHO cells and fibrinogen functions were examined using thrombin- or batroxobin-catalyzed polymerization, gel chromatography, protection against plasmin degradation, and factor XIIIa cross-linking. Results: gamma D318Y did not show any polymerization by thrombin or batroxobin, similar to gamma Delta N319D320, whereas gamma K321E had slightly impaired polymerization. The functions of Ca2+ binding, hole 'a', and the "D-D" interaction were markedly reduced in gamma D318Y, and gel chromatography suggested altered protofibril formation. In silico analyses revealed that structural changes in the gamma-module of these variants were inconsistent with polymerization results. The degree of structural changes in gamma D318Y was moderate relative to those in gamma D318A and gamma D320A, which had markedly impaired polymerization, and gamma K321E, which showed slightly impaired polymerization. Conclusion: Our results suggest that no polymerization of gamma D318Y or gamma Delta N319D320 was due to the loss of both "A-a" and "B-b" interactions. Previous studies demonstrated that "B-b" interaction alone causes polymerization of neighboring gamma D318A and gamma D320A fibrinogen, which is subsequently decreased. Marked changes in the tertiary structure of the gamma D318Y gamma-module influenced the location and/or orientation of the adjacent beta-module, which led to impaired "B-b" interactions.

Significant recent advances in cancer immunotherapeutics include the vaccination of cancer patients with tumor antigen-associated peptide-pulsed dendritic cells (DCs). DC vaccines with homogeneous, mature, and functional activities are required to achieve effective acquired immunity; however, the yield of autologous monocyte-derived DCs varies in each patient. Priming with a low dose of recombinant human granulocyte colony-stimulating factor (rhG-CSF) 16–18 h prior to apheresis resulted in 50% more harvested monocytes, with a significant increase in the ratio of CD11c+CD80+ DCs/apheresed monocytes. The detection of antigen-specific cytotoxic T lymphocytes after Wilms’ tumor 1-pulsed DC vaccination was higher in patients treated with rhG-CSF than those who were not, based on immune monitoring using tetramer analysis. Our study is the first to report that DC vaccines for cancer immunotherapy primed with low-dose rhG-CSF are expected to achieve higher acquired immunogenicity.

Large granular lymphocytic leukemia (LGLL) is a heterogenous group of disorders. The clinical presentations of LGLL are highly variable; it may present with neutropenia or anemia, complicated with recurrent infections, autoimmune diseases such as rheumatoid arthritis, and neurological symptoms, requiring therapeutic interventions. It may present only with indolent laboratory changes without any symptoms. The cytological phenotypes of LGLL also vary and they commonly include CD8+TCR αβ-type, CD4+TCR αβ-type, and NK cell-type. NK cell-type LGLL is designated as chronic lymphoproliferative disorder of NK cells (CLPD-NK). TCR γδ-type LGLL (γδT-LGLL) is even rarer and it often confers cytopenia. The genetic backgrounds of LGLL with CD8+TCRαβ-type, CD4+TCRαβ-type and NK cell-type have been elucidated with the recent sequencing technologies. JAK/STAT signaling genes and chromatin-modifying genes were frequently mutated in the non-leukemic counterpart of the lymphoid malignancies with γδ TCR immunophenotypes, such as hepatosplenic T-cell lymphoma, monomorphic epitheliotropic intestinal T-cell lymphoma and primary cutaneous gamma delta T-cell lymphoma. However, the genetic basis of γδT-LGLL and the genetic-phenotypic associations remain unclear. The aim of this study is to identify clinical and genetic characteristics of γδT-LGLL in comparison with other LGL leukemias. In total, 56 LGLL patients were recruited; six patients with γδT-LGLL, 25 CD8+ TCRαβ type, 8 CD4+TCR αβ type and 17 CLPD-NK, in this study. All cases were negative for EBV. The median age of patients with γδT-LGLL was 48 years old and five of them were male. The median LGL counts and hemoglobin level were 1.81 ×109/L (range; 0.60-4.42) and 5.8 g/dL (range; 3.0-11.2), respectively. All patients with γδT-LGLL had anemia and/or neutropenia, although no patients were thrombocytopenic. 4 patients presented with pure red cell aplasia. Common immunophenotypes of γδT cells were CD2+ CD3+ CD4-CD7+. 3 patients were CD8+. CD16, CD56 and CD57 were positive in 3, 0, and 1 patient, respectively. γδT cells of all patients showed a monoclonal pattern of TCRγ gene rearrangements with BioMed 2 protocol. We performed amplicon sequencing of entire coding regions of STAT3, STAT5B and TNFAIP3 genes and allele-specific PCR for several hot spot regions of STAT3 and STAT5B genes using extracted DNA from mononuclear cells of peripheral blood. Mutations in STAT3 and TNFAIP3 were identified in 100% (6/6) and 17% (1/6), respectively. Among the10 STAT3 mutations identified, 5 were located in the SH-2 domain, while other 3 were in coiled-coiled domain and the other 2 were in the DNA binding domain. The median variant allele frequencies of STAT3 was 2.0% (0.7-22.6) and STAT3 mutations were restricted to sorted γδT cells in all 3 patients examined. No patient was positive for STAT5B mutations, including N642H and Y665F with allele specific PCRs. One of 5 patients who were treated with cyclosporin A was refractory. Two patients were treated with cyclophosphamide, showing clinical improvements. The patients with γδT-LGLL were younger than those with other types of LGLL (P=0.048) and anemia and STAT3 mutations were significantly more frequent in γδT-LGLL (P value; 0.003 and 0.02, respectively). PRCA was a consistent cause of anemia in γδT-LGLL. Low frequency of STAT5B mutations among γδT-LGLL patients was in contrast to other aggressive γδT cell lymphoma in which STAT5B frequently mutated. The clinical characteristics of γδT-LGLL patients may be defined by younger age and the anemia due to pure red cell aplasia, and the frequent STAT3 mutations might be a novel molecular marker for γδT-LGLL. Disclosures Yamane: Pfizer: Research Funding; Celgene: Honoraria; Bristol-Meyers Squibb: Research Funding; Chugai Pharmaceutical: Consultancy, Research Funding; Astellas Pharma: Research Funding; Eli Lily and Company: Research Funding. Ishida:Eli Lily and Company: Research Funding; Astellas Pharma: Research Funding; Pfizer: Research Funding; Bristol-Meyers Squibb: Research Funding; Chugai Pharmaceutical: Consultancy, Research Funding; Celgene: Honoraria.