bluuebunny/crossref_metadata_2025 huggingface dataset:
Variability of the Iron Specific Magnetic Susceptibility of Spleen Tissue in β-Thalassemia and β-Thalassemia/Hb E Patients.
Authors: Hackett Sara L. , Chua-anusorn Wanida , St Pierre Timothy G. , Pootrakul Pensri
Published: April 2020

Patients with β-Thalassemia major are transfusion dependent and become iron loaded owing to the large number of blood transfusions they receive. Patients with β-Thalassemia/Hb E become iron loaded even in the absence of regular blood transfusions because of increased erythropoeisis and stimulated dietary iron absorption. In both cases, iron accumulates in tissues such as the liver, spleen, and heart in the form of insoluble iron(III) oxyhydroxide nanoparticles found in ferritin and hemosiderin. The body iron burden is usually estimated by measurement of the liver iron concentration (LIC). Non-invasive techniques for measuring LIC often exploit the large magnetic susceptibility of tissue iron, taken to be 1600 x 10−9 m3/kgFe at body temperature. The current study examined spleen tissue samples from Australian β-Thalassemia major patients (n = 7) who had undergone chelation and transfusion therapy and Thai β-Thalassemia/Hemoglobin E disease patients (n = 11) who had received very few transfusions and no chelation therapy. Magnetometry measurements of the iron specific magnetic susceptibility of the spleen tissue samples ranged from 800 x 10−9 m3/kgFe to 1800 x 10−9 m3/kgFe. The mean iron specific magnetic susceptibility for the β-Thal major spleen tissue was approximately 25% lower than that for the β-Thal/Hb E samples (p = 0.007 Student’s t test). To investigate the underlying cause of this variability the physico-chemical properties of the tissue iron deposits were probed using cryogenic 57Fe Mössbauer spectroscopy. Spectra recorded at 78K showed that the β-Thal major tissues contained iron with a greater fraction in the form of a more crystalline chemical species of iron oxyhydroxide than the β-Thal/Hb E tissues (p =0.01 Wilcoxon-Mann-Whitney test). Significant correlations (Spearman rank correlation tests) were identified between the iron specific magnetic susceptibility measured at body temperature and the Mössbauer spectral parameters recorded at both 5 K (p = 0.02) and at 78 K (p = 0.03). This relationship between the iron specific magnetic susceptibility and the chemical speciation of iron oxyhydroxide tissue deposits implies that the magnetic behaviour of tissue iron deposits is determined by the physical and chemical nature of the iron deposits. Variability in the iron specific magnetic susceptibility of tissues could impact on the precision of non-invasive techniques of tissue iron measurement based on magnetic susceptibility measurements.
Filamin Binding to Glycoprotein Ibα Signals Talin-Directed α IIbβ 3 Activation.
Authors: Feng Shuju , Lu Xin , Kroll Michael H.
Published: April 2020

von Willebrand factor (VWF) binding to platelet glycoprotein (Gp) Ib-IX-V triggers platelet activation. Under conditions of pathologically elevated arterial wall shear stress, VWF-dependent platelet adhesion is coupled to aggregation and thrombus formation principally through GpIbα-induced signaling to α IIbβ 3. To elucidate the mechanism of GpIbα signaling to α IIbβ 3, we have examined molecular interactions involving structural proteins that bind to the cytoplasmic domains of GpIbα and β 3. In CHO cells co-expressing human GpIb-IX and α IIbβ 3, the activation of α IIbβ 3 as reported by monoclonal antibody PAC-1 binding is stimulated by ristocetin (1 mg/ml) + purified human VWF(5 μg/ml). When filamin binding to the cytoplasmic domain of GpIbα is eliminated by deleting GpIbα residues 560-570, PAC-1 binding is eliminated. When human platelets in reconstituted whole blood are treated with a peptide that interferes with filamin binding to GpIbα (as reported in Blood2003;102:2122–2129), shear-dependent (1500 sec−1 shear rate or 60 dynes/cm2 shear stress) platelet deposition onto bovine type I collagen is inhibited. In washed resting platelets and platelets activated by 120 dynes/cm2 shear stress, filamin co-immunoprecipitates with both GpIbα and α IIbβ 3; only its association with β 3 is eliminated by DNaseI (1 mg/ml), demonstrating that filamin binds indirectly to α IIbβ 3 through other cytoskeletal elements. One such element is observed to be talin, which co-immunoprecipitates with filamin, α IIbβ 3 and small amounts of GpIbα in resting platelets. When platelets are sheared for two minutes at 120 dynes/cm2, talin’s DNaseI-resistant association with filamin is decreased and its DNaseI-sensitive association with α IIbβ 3 is increased. These changes are prevented when shear-dependent VWF binding to GpIb-IX-V is blocked by monoclonal antibody AK2. Shear-dependent VWF binding to GpIb-IX-V also results in the proteolysis of talin, which is considered to be one mechanism by which the N-terminal head domain of talin regulates α IIbβ 3 activation. Shear-dependent talin proteolysis is not affected by blocking VWF binding to α IIbβ 3 with a RGD peptide. These results demonstrate that the cytoplasmic domain of GpIbα transduces signals to activate α IIbβ 3 through its interactions with filamin. These signals depend only on VWF binding to GpIb-IX-V. Under pathologically elevated wall shear stress in vitro, the mechanism of signaling may be the release of talin by filamin, thus permitting the proteolysis of talin and enhancing talin’s interaction with α IIbβ 3.
Elevated Hemoglobin F in an African American Girl with HbS/β Thalassemia: Unraveling the Mechanism.
Authors: Fond Aaron M. , Prchal Josef T. , Ou Ching-Nan , Rognerud Cheryl L. , Mahoney Donald , Mims Martha P.
Published: April 2020

Factors controlling hemoglobin F production beyond the neonatal period are poorly understood; however, unraveling the mechanisms underlying hereditary persistence of fetal hemoglobin (HPFH) could lead to therapies for β-thalassemia and sickle cell disease. We identified a non-anemic 5 year old girl with Hbs S, F, and A. Investigation revealed elevated HbF in the mother and paternal sickle trait. More than 90% of the gamma globin chains in the daughter were Gγ. Mother and daughter share the common -29 β thalassemia mutation, but have normal MCVs.

Mother Father Child HbA 77.6% 54% 8.7% HbA2 3.6% 3% 2.7% HbF 18.8% 0% 32.4% HbS 0% 43% 56.2% Hemoglobin 11.4 15.3 13.3 MCV 85.7 82.4 78.2

We set out to determine:

Whether a thalassemia trait explains the MCVs in the mother and child. Whether increased HbF is due to γ globin promoter mutations linked to the −29 β thalassemia mutation.

A duplex PCR strategy was used to genotype family members for the common African −3.7 kb α-thalassemia deletion. Our studies show that the mother is heterozygous for the a globin −3.7 kb deletion while the father and child are homozygous for the wild type α globin locus. The upstream region (−627 to −5) of the Gγ-globin genes of each family member were amplified and the PCR products were digested with the Xmn1 restriction enzyme to determine whether the Xmn1 polymorphism ( C→T at position −158) contributed to the increased fetal hemoglobin. None of the family members had this polymorphism. Sequencing of the upstream region of the Gγ gene revealed that the patient is homozygous for a novel mutation (A →G) at position −309 for which both parents are heterozygous. In summary we describe a novel Gγ gene −309 A →G mutation in association with elevated HbF; further research is in progress to determine if this polymorphism contributes to the patient’s elevated fetal hemoglobin.
The Effect of Combined Chelation Therapy in the Treatment of Severe Myocardial Iron Loading in Beta Thalassaemia Major.
Authors: Tanner Mark A. , Galanello Renzo , Dessi Carlo , Westwood Mark A. , Pennell Dudley J.
Published: April 2020

Introduction: Thalassaemia major ™ is an inherited haemoglobinopathy resulting from mutations of the β-globin gene. Survival is dependent upon lifelong blood transfusions and results in progressive total body iron overload. Cardiac failure secondary to myocardial iron loading is by far the commonest cause of death with only 50% of patients in the UK in recent cohorts surviving beyond the age of 35 years. Conventional treatment with the parenteral iron chelator deferoxamine improves morbidity and mortality but fails to clear the heart of iron in approximately two-thirds of patients. More recently, the oral iron chelator deferiprone has been demonstrated to remove myocardial iron and it has been proposed that in combination with deferoxamine it may have an additive effect. Through the T2* multi-echo cardiac magnetic resonance (CMR) technique, myocardial iron can now be non-invasively and reproducibly quantified, thereby providing a means to assess myocardial iron loading in response to chelation treatment.

Purpose: To report the changes in myocardial T2* and cardiac function in patients with severe myocardial iron loading (myocardial T2* <8ms) following treatment with the un-blinded addition of deferiprone to continued deferoxamine chelation therapy.

Methods: A mobile CMR scanner (1.5T Siemens Sonata) was transported to Cagliari, Italy. The myocardial T2* was assessed in 167 patients with TM aged 18v42 (30 +/− 5.3years). Of these, 22 patients were identified as having severe myocardial iron loading (T2* <8ms), 15 of whom (at their clinician’s discretion) were subsequently given deferiprone in addition to continued deferoxamine therapy. Patients underwent further CMR assessment at baseline, 6 and 12 months.

Results: Over a period of 12 months myocardial T2* improved significantly (6.1+/− 1.0ms to 8.2ms +/−2.5ms, p=0.007) in association with an improvement in cardiac function (LV ejection fraction 52 +/−12% to 65% +/−6.6%, p<0.0001). See Figures 1 and 2.

Conclusion: This prospective study shows that patients with severe myocardial iron loading show significant improvement in iron loading and myocardial function with the addition of deferiprone to continued parenteral deferoxamine.

Figure 1 Figure 1. Figure 2 Figure 2.
Genotype Phenotype Correlation in Thalassemia Syndromes and Their Correlation with Xmn-1 Polymorphism.
Authors: Sachdeva Anupam , Raina Aditya , Khanna Varinder Kumar , Arya Subhash Chander , Yadav Satya Prakash , Verma Ishwar
Published: April 2020

Among the inherited disorders of blood, thalassemia constitutes a major bulk of genetic diseases in India. It causes a high degree of morbidity. In a study conducted in India it has been estimated that there is a frequency of 1:2700 at the time of birth. Thus on an average 9000 new thalassemics are born every year in India.. The present study has been aimed at investigating the clinical and hematological spectrum in the above syndrome. It also assessed the prevalence of Xmn -1 polymorphism its relationship with various mutations and its role in modifying the clinical manifestations. The study was conducted on fifty patients representing 49 families and consisting of 33 males and 17 females who were homozygous for beta thalassemia and ranged in age from 3 months to 32 years. The patients were screened for common Indian mutations and their Xmn polymorphism status and this was correlated with their clinical parameters. Apositive correlation in presence of Xmn-1 polymorphism and IVS1-1 mutation was noted. There was also a correlation between age at diagnosis and also the age at first transfusion. There was a correlation between Xmn polymorphism and IVS1-1 mutation. This was found most commonly in the Punjabi Khatri community. None of the patients with blood group O had positivity of Xmn polymorphism. There was no correlation between Xmn and hemoglobin at diagnosis, HbF at diagnosis, MCV at diagnosis, amount of blood in mL/Kg/year and sex and religion.
Atypical Hemoglobin H Disease Due to Compound Heterozygosity for -α3.7 and --SEA Deletions and Heterozygosity for the β-Chain Variant Hemoglobin Raleigh (β1 VAL→ALA).
Authors: Kutlar Ferdane , Knovich Mary Ann , Elam Dedrey , Jobe Daniel B. , Buss David H. , Harbin Jeanette , Davis Deborah H. , Sabio Hernon , Kutlar Abdullah
Published: April 2020

The co-existence of Hemoglobin H (Hb H) disease and heterozygosity for β-chain structural variants is a rare occurrence. Hb H disease has been reported in conjunction with Hb E, Hb C, Hb S, and Hb Hamilton. The combination of Hb H disease with Hb C and Hb S reportedly results in a mild hemolytic anemia without detectable Hb H. We present a new case of atypical Hb H disease that was also heterozygous for the rare β-chain variant Hb Raleigh. The patient is a 27-year-old Cambodian female referred for the evaluation of microcytic anemia unresponsive to iron. She had a lifelong history of generalized fatigue, exertional dyspnea, and weakness in her legs. Physical exam was unremarkable except for pallor of mucuous membranes. There was no hepatosplenomegaly. She had a Hb of 9.7, HCT 30.8, MCV 56, MCH 17.6, MCHC 31.5, ferritin 92. Hb analysis on IEF revealed Hb A, Hb A2, and an abnormal band slightly more anodic to Hb A. No Hb H was observed. On cation exchange HPLC, she had 49.7% Hb A, 48.1% Hb X, and 2.2% Hb A2. Reverse phase HPLC revealed a βx chain eluting immediately before βA. Oxygen affinity was slightly reduced. PCR amplification and sequencing of the β-globin gene revealed heterozygosity for Hb Raleigh (Exon 1, codon 1, GTG→GCG, VAL→ALA). The patient was also found to be a compound heterozygote for -α3.7 and --SEA deletions. This case represents a novel interaction of a structural β-chain variant with Hb H disease. Hb Raleigh has previously been reported in Caucasians and in two Swedish families. It has decreased oxygen affinity. This is the first report of this variant in a Cambodian population. The absence of any detectable Hb H likely results from the inability of variant β-chains to form a viable tetramer with a resultant decrease in βA. The low oxygen affinity did not negatively impact on the degree of anemia. This case, like some others reported previously, shows that the accurate diagnosis of Hb H disease in association with structural β-chain variants can be established by molecular methods, and the detection of Hb H on electrophoretic and chromatographic analyses may not always be reliable.
How To Optimize the Detection of the Left Shift with the Instrument COULTER LH750®.
Authors: Capel-Casbas Maria J. , Duran Jose J. , Naranjo Dolores , Pastor Isabel , Berlanga Rosalia , Masset Maria J. , Piqueras Josep , Pujol-Moix Nuria
Published: April 2020

The Coulter LH750® is a last generation automated hematology analyzer. In 1999 we evaluated the optimization of immature granulocytes/bands (IG/B) detection in the Coulter GEN*S (Blood Vol.94 n° 10 sup.1, 1999 page 45 b), in which we observed that the Mean Volume of the Neutrophil population (M NE V) and the standard deviation of the neutrophil volume (SD NE V) were the most useful parameters to improve the detection of the presence of IG/B. Recently the utility of SD NE V has been proposed in the diagnosis of septic patients.(ISLH 05). The purpose of this study is to confirm that the NE morphometric parameters in the LH750 have the same values and cut-off as the GEN*S to discriminate the cases with lefts shift and if it is possible to create rules/flags that use these parameters to allow the automated detection of IG/B with cut-off levels specific to the origin (emergency room, outpatients, intensive care etc.) of the patient sample.

In addition we investigated if the parameters are useful when taking into account not only the % of IG/B, but also their absolute number as recommended by the NCCLS. The overall intent being to optimize the efficiency of our laboratory in the detection of IG/B by decreasing the number of smears, but without increasing the number of false negatives. We have selected from the routine samples of a 2000 beds hospital, 340 consecutive cases on the LH750® that triggered the bands /Immature granulocytes flags, Immature NE 1 (NE1) or Immature NE 2 (NE2). We considered the samples with more than 0,9 IG/B (103/mL) as positive. Blood smears were performed on all the positive samples (340) and a WBC differential was performed according the NCCLS protocol (H 20A). The percentage and absolute number of IG/B were collected. The statistical analysis was performed using Medcalc® version 8.1.0.0. A ROC Curve and the cut-off for the detection of positive samples with immature IG/B using the SD NE V and M NE V was calculated.

RESULTS: For the flag NE1 the best parameter to select the postive cases with left shift was the SD NE V with a cut-off of >24,2. (although this flag is actually defined for a moderate left shift). For the flag NE2 the best parameter to select the postive cases with left shift was the M NE V with a cut-off of > 161.

CONCLUSION: Before the study performed in 1999 we validated the Imm NE1 and Imm NE2 flags according to the NE% and NE# or the WBC values, that statistically demonstrated limited utility. Parameters which permitted to optimize the detection of IG/B, were the Neutrophile morphological parameters; M NE V and SD NE V. The values of the LH 750 are slightly different to those of the GEN*S. We will subsequently study their utility and advantages compared to the IG/B counting in the detection of bacterial infections and sepsis due to better reproducibility.
Rituximab in the Treatment of Secondary Autoimmune Neutropenia.
Authors: Pivkova Aleksandra A. , Cevreska Lidija L. , Stojanoski Zlate Z. , Panovska Irina I. , Siljanoski Nikola N. , Stavrik Sonja S. Genadieva , Balkanov Svetlana S. Krstevska , Trajkova Sanja S. , Georgievski Borce B.
Published: April 2020

Autoimmune cyclical neutropenia is a rare syndrome with periodically severe but temporary neutropenia with occurrence of 3–4 weeks, but not rarely in 2 or 5 week intervals. Antibodies with specificity for the neutrophils has been demonstrated in a number of different neutropenic disorders and is often associated with systemic autoimmune disease. Since the role of monoclonal antibodies in the treatment of autoimmune disorders is not clearly optimized, we evaluated the effectiveness of Rituximab in the treatment of immune neutropenia.

We present two cases (male 66 years and 42 years female) with immune neutropenia, associated with systemic autoimmune disease, Chron’s disease in male patient and rheumatoid arthritis in female patient. Bone marrow biopsy revealed reduction of granulopoesis and absence of neutrophils in the peripheral smear-agranulocytosis. Immunological investigations revealed increased polyclonal IgG, positive indirect granulocyte immunofluorescence test. Both patients had a clinical course with continuously intermittent neutropenia every 2 weeks, presented with recurrent febrile episodes and serious lower respiratory tract infections. Treatment with G-CSF was immediately started in both patients to reach neutrophil recovery, but after one year of periodically persistent neutropenia it was decided to start treatment with Rituximab, an infusion with standard single agent 375 mg/m2 weekly for 4 weeks. After 4 infusions neutrophils reached normal levels and the treatment continued with every 2 weeks infusion of monoclonal antibody in duration of 2 months and now once a month as maintenance treatment. The patients are one year and six months after beginning treatment with monoclonal antibodies, in stable clinical and laboratory condition. We can conclude that B cells play a central role in the autoimmune process and Rituximab treatment has been used in patients with autoimmune neutropenia. Further randomized studies will define optimal role of Rituximab in the treatment of autoimmune diseases.
Human Cytomegalovirus Inhibits Granulopoiesis Via Direct Infection and Inducing Apoptosis.
Authors: Wang Qing Wen , He Zheng Xian , Li Xiao Feng , Xiong Wei , Chen Mei Lian , Li Karen Kwai Har , Yang Mo
Published: April 2020

Human cytomegalovirus (HCMV) infection can cause delayed leukocyte recovery after bone marrow transplantation and often associated with suppression of granulocyte/macrophage progenitor. Leukocyte lineage may be one of the major sites of HCMV infection. However, whether HCMV can interfere with CFU-GM formation and induce apoptosis in granulocyte/macrophage progenitor have not been well investigated. Human bone marrow mononuclear cells (Ficoll), promyelocyte cell line HL-60 and HCMV AD169 strain were co-cultured. Each bone marrow specimen was HCMV DNA and HCMV IgM negative by PCR and ELISA test. Our results showed that HCMV significantly inhibited the formation of CFU-GM as shown in two different concentrations of viral infection groups: 139.26 ± 5.42 (2×105pfu/ml), and 124.19 ± 8.82 (2×106pfu/ml) (colonies/2×105cells/ml, n=26). These were significant differences compared with the blank control group (P<0.01, P<0.05) respectively. HCMV also significantly inhibited the growth of HL-60 cells in three different concentrations of viral infection groups (101pfu/ml, 102pfu/ml, 103pfu/ml). After incubation for 7 days, viability cells in control group and each infection groups (n=20) were 96%, 83%, 73%, and 64%, showing that HCMV infection decreased the viability of HL-60 cells in a dose-dependent manner. The apoptotic effect was also investigated by Annexin V assay using flow cytometry. The percentage of Annexin V -positive cells were 19.60 ± 1.63 (in group of 101pfu/ml) (P<0.05), 28.70 ± 2.61 (102pfu/ml) (P<0.05), and 36.3 ± 2.57 (103pfu/ml) (P<0.01), compared with control group 3.96 ± 1.75 (n=8). The apoptotic cells were further confirmed by morphologic observation and DNA ladder formation. Furthermore, HCMV DNA was positive in HL-60 cells and cells of CFU-GM measured by PCR and IS-PCR analysis respectively. These were negative in blank and mock control groups. Our data demonstrated that: (1) HCMV AD169 strain inhibited the growth of HL-60 and CFU-GM formation in a dose-dependent manner; (2) HCMV transcription occurred in HL-60 and CFU-GM cells; and (3) This virus induced apoptosis in HL-60 cells. HCMV may have an inhibiting effect on granulopoiesis via direct infection and inducing apoptosis on myeloid progenitors.
An Indispensable Role of GM-CSF in the Differentiation of Human Blood Monocytes toward Langerhans Cells.
Authors: Shibasaki Tetsunori , Katayama Naoyuki , Ohishi Kohshi , Monma Fumihiko , Yu Zhaocai , Nishii Kazuhiro , Masuya Masahiro , Shiku Hiroshi
Published: April 2020

We recently demonstrated that GM-CSF, TGFβ1, and Notch ligand Delta-1, all of which are synthesized in the milieu of the skin, instruct human blood monocytes to differentiate into Langerhans cells (LCs) that are characterized by the expression of CD1a, Langerin, CLA, CCR6, and E-cadherin and the presence of Birbeck granules. Previous studies have shown similar biologic activities of GM-CSF and IL-3 on human blood monocytes. In the present study, we investigated whether GM-CSF and IL-3 have a similar action on blood monocytes with respect to their differentiation into LCs. In contrast to CD14+ monocytes cultured for 7 days in the presence of GM-CSF, TGF-β1, and Delta-1, the cells obtained from culture with IL-3, TGF-β1, and Delta-1 were negative for CLA, CCR6, Langerin, and E-cadherin. These cells expressed HLA-ABC, HLA-DR, CD80, CD86, CD40, CD54, and CD11c but not DC-SIGN. The expression level of CD1a declined, while CD14 was upregulated. The difference between cells cultured with GM-CSF, TGF-β1, and Delta-1 and those with IL-3, TGF-β1, and Delta-1 was corroborated by microarray analysis of the gene expression profiles. Thus, GM-CSF and IL-3 exert distinct effects on human blood monocytes in the presence of TGF-β1 and Delta-1. When GM-CSF was added to cultures containing IL-3, TGF-β1, and Delta-1, the phenotype of cultured cells closely resembled that observed with GM-CSF, TGF-β1, and Delta-1. As GM-CSF and IL-3 share common receptor β subunits, the signals mediated through GM-CSF receptor α subunits appear to be required for the development of LCs from monocytes. We also compared the action of GM-CSF with that of IL-3 in terms of the differentiation of monocytes into macrophages and dendritic cells (DCs). When CD14+ monocytes were cultured in the presence of GM-CSF or IL-3 for 7 days, the resulting macrophages obtained from both cultures were phenotypically indistinguishable. Next, we examined whether IL-3 as well as GM-CSF induces CD14+ monocytes to differentiate into DCs in the presence of IL-4. The phenotypic profiles in cells cultured with IL-3 plus IL-4 were parallel to those seen with DCs cultured in the presence of GM-CSF plus IL-4. These data suggest that although IL-3 can substitute for GM-CSF in the differentiation pathway of CD14+ monocytes toward macrophages or DCs, GM-CSF is indispensable for their LC development.
Immunopotent Polysaccharides of Different Sources Selectively Activate Human Dendritic Cells.
Authors: Chan Godfrey ChiFung , Chan W.K. , Law H.K. , Lin Z.B. , Lau Y.L.
Published: April 2020

Background: Purified polysaccharides extracted from plants and fungi have been shown to induce immune responses in-vivo and vitro over the past decade. Currently, most of these polysaccharides are found to be glucan but with different branch structure and sizes. Their relative potency and effect on human immune cells remains unknown. This study aims to compare their relative effect on human dendritic cell, the most potent antigen presenting cell.

Materials & Methods: We selected 2 prototypes of purified polysaccharides extracted from: 1) Ganoderma lucidum (GL, Lingzhi, Reishi) mycelium, a widely used herb with long and branching β (1® 3), (1® 6) glucan structure (provided by Prof. Lin ZB, Beijing) and 2) Barley with shorter and different branching β (1® 3), (1® 4) structure (provided by Prof. Cheung VNK, NY). Their characteristics and chemical properties had been reported previously. Human peripheral blood mononuclear cells (PBMCs) proliferation was studied by XTT assay. Human dendritic cells (DCs) were derived from monocytes and maturation of DCs were determined by: a) immunophenotypic shift using flow cytometer; 2) dextran endocytosis assay and 3) mixed lymphocytes reaction. Cytokine secretions were determined by ELISA test. Comparisons between means were by nonparametric Student’s t test (2-tailed).

Results: We found that purified polysaccharides from GL but not barley could induce PBMCs proliferation and maturation of DCs. GL polysaccharides could enhance phenotypic and functional maturation of DCs with significant IL-12 and IL-10 production. DCs were relatively inert to Barley glucans stimulation. However, both polysaccharides did not polarize T cells into the direction of T helper 1, T helper 2 or regulatory T cells.

Conclusions: Our study shown that purified polysaccharides extracted from plants and fungi have different effect on human DCs and their potency and effects are probably affected by their respective sources and structures.
Mast Cell-Specific Gene Targeting.
Authors: Melicoff Ernestina , Thakurdas Shakeel M. , Siddiqi Tanya , Murthy Jayasimha , Sansores-Garcia Leticia , Adachi Roberto
Published: April 2020

For years mast cell (MC) studies have relied on MC lines or bone marrow-derived MC. Regular knockout mice have some disadvantages such as early lethality and developmental defects. Also, the phenotype in MCs from these animals could not be a primary defect but secondary to other cell lines affected by the mutation. The Cre/loxP system allows us to target genetic deletions to specific tissues or cells. Cre, a recombinase from bacteriophage P1, recognizes a DNA sequence motif of 34 bases (loxP). If a DNA segment is flanked by two loxP sites in the same orientation, Cre excises that segment activating or silencing a gene. Many genes have been “floxed” (flanked by two loxP sites), but no MC-specific Cre mouse has been created. We are using promoter regions of MC-specific proteins to drive expression of Cre-recombinase. We produced transgenic mice using the promoter for human alpha-subunit and murine beta-subunit of the high-affinity receptor for IgE (hFcεRIα and mFcεRIβ), both expressed in MCs and basophils. We also used the promoter of the murine mast cell protease mMCP-5, a member of a family of cargo proteins expressed only in MCs. For even more controlled expression, we knocked-in Cre cDNA into the mMCP-5, mMCP-6 and Ras guanine releasing protein 4 (RasGRP4) loci, all of them proteins expressed exclusively in MCs, while knocking-out the respective gene. Heterozygotes will express Cre only in MCs, and homozygotes for mMCP-5, mMCP-6 or RasGRP4 deletions will be regular knockouts that will allow us to study the role of these proteins in mast cells. These three transgenic and three knock-out/knock-in lines have been created and are being crossed with reporter mice. We decided to use a double reporter mouse, the Bgeo/GFP (Jackson Laboratory). This mouse has “floxed” lacZ that is constitutively expressed under the control of the CMV enhancer/chicken actin promoter, and when crossed with our Cre recombinase-expressing mouse, lacZ is excised and enhanced green fluorescent protein (EGFP) is expressed in cells expressing Cre. EGFP can be easily detected in MCs from peritoneal lavage by flow-cytometry, using antibodies against c-kit (CD117) and FcεRI to label the MCs. Results of this screening will be presented at the meeting. After documenting the presence of functional Cre-recombinase in our reporter mice, we will cross them with our “floxed” mice to obtain conditional mast cell-specific knockouts.
Intradermal RNA-Vaccination of Patients with Metastatic Renal Cell Carcinoma.
Authors: Wierecky Jan , Müller Martin R. , Häntschel Maik , Horger Marius S. , Pascolo Steve , Brugger Wolfram , Kanz Lothar , Brossart Peter
Published: April 2020

Background: The use of in vitro transcribed RNA isolated from tumor cells or coding for defined tumor associated antigens was shown to be a very powerfull method to generate antigen specific T cells upon transfection into dendritic cells (DC). More recently it was demonstrated in a mouse model that application of RNA intra dermaly can elicit both, CD8 and CD4 mediated immune responses. Furthermore, in Her-2-neu transgenic mice vaccinations with naked RNA were able to elicit a specific immune response in vivo and delay the development of breast cancer in treated animals. Based on these results we analyzed the clinical and immunological responses in patients with metastatic renal cell carcinoma (RCC) vaccinated with mRNA encoding for tumor associated antigens (TAA) using two different treatment arms.

Methods: In vitro transcribed RNA was generated using plasmids coding for the tumor antigens MUC1, CEA, Her-2/neu, telomerase, survivin and MAGE-1. RNA coding for HbsAg and Influenza matrix protein were included as controls. Vaccinations were performed intradermally on day 1,14, 28 and 42 in a first group of patients and on day 0–3, 7–10, 28 and 42 in a second group (intensified arm). Vaccinations were repeated afterwards monthly until tumor progression. One day after each RNA injection patients additionally received 1 injection of GM-CSF (250 μg) sc. The enhancement of T cell precursor was monitored using IFN-g ELISPOT and tetramer staining..

Results: 25 patients were included in this study. In 3 patients regression of metastases was induced and 6 patients had a stabilization of the disease. Specific CD8 and CD4 T cell responses in vivo were detected in the first analyzed patients. The treatment was well tolerated with no severe side effects. In most cases erythema and induration were observed after injections of GM-CSF. One patient developed an allergic exanthema after GM-CSF application.

Conclusions: This study demonstrates that intradermal RNA-vaccination can be effective in the treatment of metastatic RCC and induces clinical and immunological responses.
Clinical Evaluation of CCL18/PARC and Chitotriosidase Biomarkers in Gaucher Disease.
Authors: Alfonso Pilar , Latre Paz , de Blas Ignacio , Giraldo Pilar , Giralt Manuel , Pocovi Miguel
Published: April 2020

Gaucher’s disease (GD) is an autosomal recessive lysosomal storage disorder, characterized by accumulation of glycosphingolipid in so-called Gaucher cells. The clinical manifestations of Gaucher disease are highly variable, and although certain genotypes are often associated with mild or severe symtomps, a defined correlation between genotype and phenotype does not exist. Identification of serum biochemical markers characteristic of disease may be useful in the diagnosis and monitoring of GD, nevertheless about 6% of the population does not express the chitotriosidase (CT) gene. In this study, we analyzed using currently available enzyme analysis the relationship among two known surrogate markers: CT, which utility in initiation and optimization of costly therapeutic interventions has been highly demonstrated, and a newly-described chemokine, pulmonary and activation-regulated chemokine (CCL18/PARC) as associated to Gaucher disease.

Patients and methods: We have analysed 21 control samples, 149 samples of GD patients on enzyme replacement therapy (ERT), and 52 samples of GD patients on substrate reduction therapy (SRT), 4 samples of GD on combined therapy (ERT+SRT) and 7 samples of GD patients without therapy. The samples were stored at −80°C in the biobank of Biochemistry and Molecular and Cellular Biology Department of Zaragoza University. The CT activity and CCL18/PARC quantification were performed simultaneously in the samples obtained at baseline and yearly during 7 years under ERT.

Results: our results concluded that both markers levels similarly fell with time and their variations correlated strongly each other, mainly in patients under ERT. The poor correlation of both variables in the case of SRT might be due to small number of samples. These findings demonstrated that CCL18 is a good biomarker of monitoring GD, comparable to CT and very useful in patients without expression of CT gene.

Conclusion: The availability of sensitive plasma surrogate markers may be of great value for monitoring the efficacy of treatment, especially in cases of deficiencies of some marker.
Gene Expression Profiling of Adenosine Signaling in the Human Monocytic THP-1 Cells.
Authors: Chen Chin-Fu , Cheng Chun-Huai
Published: April 2020

Inflammation, a host response to infection or injury, is usually beneficial to the host. However, uncontrolled or prolonged inflammation can cause tissue and organ damage, and possibly lead to diseases like atherosclerosis, rheumatoid arthritis, asthma, and cancers. Much attention has focused on pro-inflammatory signaling but little is known about the mechanisms that resolve inflammation (anti-inflammation). Adenosine, released from leukocytes and endothelial cells, is an endogenous anti-inflammatory mediator that down-regulates acute inflammation in part by modulating macrophage cytokine production. LPS (lipopolysaccharide) is a potent pro-inflammatory reagent of macrophages that acts primarily through Toll-like receptors (TLRs). Studies using murine cells have showed that co-stimulation of both adenosine receptors and TLRs greatly increases the production of vascular endothelial growth factor (VEGF). The mechanisms for this synergistic interaction are currently not known. It is also unknown whether the adenosine-LPS interaction occurs in human macrophages. To gain insights and to generate new hypotheses, we have utilized oligonucleotide microarrays to examine how adenosine alters global gene expression in the human monocytic THP-1 cell line. We analyzed differentially expressed genes induced by adenosine alone, by LPS alone, and by adenosine in the presence of LPS. The genes activated by adenosine are enriched in processes transcription, RNA modification and cellular defense response, but are lacking processes involved in lipid biosynthesis and protein catabolism. Our result shows that at the mRNA level adenosine alone induces a nine-fold increase of VEGF, and adenosine in the presence of LPS elevates VEGF expression three fold when compared to controls. Many genes with the greatest induction by adenosine in the presence of LPS have unknown functions in macrophages. The genes with known functions include transcription regulation (EGR1, HEY1, and CTBP1), nuclear receptors (NR4A2), immune response modulators (IL1A, STAT4, and INHBA), apoptosis facilitator (BCL2L11), signal transducers (PRPF4B, LNK, and ARHGEF7), and glutamate transporter (SLC1A2). These results suggest that the mechanisms by which adenosine mediates its anti-inflammatory signals are far more complex than simply regulating cytokine production. In addition, we found that three TLR signaling genes are uniquely suppressed by adenosine in the presence of LPS: CREB1, TLR1, and IRF2. CREB1 modulates transcription of cAMP signaling; TLR1 is part of the TLR2/TLR1 complex and can regulate TLR2 mediated pathways; IRF2 is a mediator of interferon signaling. We are in the process of verifying these observations using real-time PCR and independent RNA samples.
Intravenous gamma Globulin Does Not Increase RNA Expression of FcgRIIb in Purified Human Monocytes.
Authors: Chan Howard H. , Su Brenda , Li Wei , Kelton John G. , Denomme Greg
Published: April 2020

Introduction

Intravenous gamma globulin (IVIG) has been shown to modulate immune cytopenia through FcγRIIb in a murine model (1). There are conflicting results from the studies on the protein expression of the FcγRIIb. In that murine model, there is increase in FcγRIIb positive splenocytes following IVIG treatment (1). Yet, in patients with Kawasaki disease, flow cytometric analysis of peripheral CD14+ mononuclear cells shows no change in FcγRIIb expression (2). To study the effect of IVIG on the RNA expression of FcγIIB, we performed RT-PCR using monocytes collected from three healthy volunteers. The monocytes were harvested using a magnetic separation method. The purified monocytes were then cultured with IVIG in vitro.

Methods

Peripheral blood mononuclear cells (PBMC) from three healthy donors were isolated from heparin anticoagulated blood by Ficoll-Hypaque density separation. The PBMC were washed and the monocytes were isolated using anti-CD14 magnetic bead positive selection. In the first set of experiments, purified monocytes were cultured at 37°C, 0.5% CO2 in media containing 10% fetal calf serum (FCS) (control) and in the same media containing IVIG (0.5 mg/mL and 5.0 mg/mL). In the second set of experiemnts, purified monocytes were cultured at 37°C, 0.5% CO2 in serum free medium (SFM), in SFM containing IVIG (5.0 mg/mL), and in SFM containing 10% FCS. Monocytes were harvested from the culture flasks after an 18-hour incubation. Isolated monocytes were lyzed and the total RNA was extracted with TRIzol reagent according to the manufacturer’s protocol. One μg of RNA was used to generate first strand complimentary DNA using oligo-dT and Superscript II reverse transcriptase. Amplitaq Gold DNA polymerase was used to PCR-amplify FCGR2B cDNA. PCR amplification was performed using 5 μL of cDNA and 1 μM each of an FCGR2B-specific primer pair (3) in a total volume of 50 μL. The PCR products were evaluated using 10% polyacylamide gel electrophoresis.

Results

There was no change in FCGR2B expression when purified monocytes were incubated with either 0.5 mg/mL or 5mg/mL IVIG for 18 hours (n=3) (Fig1).

However, there was a demonstrable increase in FCGR2B expression when monocytes were cultured in the presence of FCS (Fig2).

Conclusion

IVIG does not directly modulate monocyte FCGR2B gene transcription.

Figure Figure
Hemophagocytic Lymphohistiocytosis in a Extremely Low Birth Weight Infant Presenting with a Neonatal Thrombocytopenia.
Authors: Lee Mee Jeong , An Young Won , Chang Young Pyo , Lee Won Ae , Park Joowan , Lee Young Suk , Ghim Thad T.
Published: April 2020

INTRODUCTION: Hemophagocytic lymphohistiocytosis(HLH) is a potentially fatal disease presenting at an young children. Symptoms and physical findings include fever, rash and pancytopenia associated with histiocytic infiltration of bone marrow, liver and spleen. Its occurrence in neonates is very rare. We describe a female infant diagnosed with HLH, who was born at 24 week 3 days of gestational age(GA) weighing 666 gm. The infant expired due to aggravating thrombocytopenia and hepatosplenomegaly leading to multiple organ failure associated with suspected sepsis. Autopsy findings lead to a confirmation of HLH.

CASE: The baby, weighing 666gm, was born from a 36 year-old mother at GA 24 weeks 3 days, by emergency cesarian section due to vaginal bleeding. The mother came from the Republic of Kyrgystan and didn’t receive any antenatal care. The mother had a history of 8 prior abortions due to incompetence of internal os cervix (0–5–3/0–0). CBC on the first day was normal, but on the third day itg showed thrombocytopenia (Hb 11.2 g/dL, WBC 11,700/mm3, Platelet 78x103/mm3). Antibiotics were administered for suspected sepsis. On the 47th day, abdominal ultrasonography was performed due to abdominal distension and TPN-induced conjugated hyperbilirubinemia. GB wall edema and hepatomegaly were noted. Thrombocytopenia persisted and on the 55th day the patient became hypotensive and develped metabolic acidosis. Laboratory work up for persistent thrombocytopenia included anti-platelet antibody(−), TORCH(−), parvovirus-B19 PCR(-), CMV PCR (−), EBV infection(−). Bone marrow biopsy was not done. The patient expired from sepsis and multiorgan failure on the 61st day. Autopsy findings showed histiocyte infiltration of all tissue including the liver, spleen and brain. Bone marrow biopsy also showed infiltration of abundant hemophagocytic histiocytes consistent with a diagnosis of HLH.

CONCLUSION: A premature newborn with HLH described here presented with thrombocytopenia. Therefore, HLH should be included in the differential diagnosis of neonatal thrombocytopenia.
The Role of Endothelial PI3Kγ Activity in Neutrophil Trafficking.
Authors: Diacovo Thomas G. , Doggett Teresa A. , Huang Ching-Yu , Douangpanya Jason , Hayflick Joel , Turner Martin , Penninger Josef , Puri Kamal
Published: April 2020

Phosphoinositide 3-kinase gamma (PI3Kγ) in neutrophils plays a critical role in the directed-migration of these cells into inflamed tissues. In this study, we demonstrate the importance of the endothelial component of PI3Kγ activity relative to its leukocyte counterpart in supporting neutrophil interactions with the inflamed vessel wall. Despite the reconstitution of class Ib PI3K function in neutrophils of p110γ−/− mice, we observed a 45% reduction in accumulation of these cells in an acute lung injury model. Mechanistically, this appears to result from a perturbation in selectin-mediated adhesion as manifested by a 70% reduction in WT neutrophil attachment to and 17-fold increase in rolling velocities on p110γ−/− microvessels in vivo in response to TNFα. This alteration in adhesion was further augmented by a deficiency in p110δ, suggesting that the activity of both catalytic subunits is required for efficient capture of neutrophils by cytokine-stimulated endothelium. Interestingly, E-selectin-mediated adhesion in p110gamma]−/− mice was impaired by > 95%, but no defect in NF-κB-induced gene expression was observed. These findings suggest a previously unrecognized partnership between class I PI3Ks expressed in leukocytes and endothelium, the combination of which is required for the efficient trafficking of immunocompetent cells to sites of inflammation.
Moribund Blood Monocytes: Apoptosis Limits the Yield of Monocyte Derived Dentritic Cells from Apheresis Samples.
Authors: Macke Lars , Geffers Robert , Rohde Manfred , Garritsen Henk S. , Woermann Bernhard , Lindenmaier Werner , Dittmar Kurt E.
Published: April 2020

Cells of the monocyte/macrophage/dendritic cell (DC) lineage play a central role in inflammation and form the bridge between innate and acquired immunity. In recent years monocytes also found special attention as source of precursor cells for the generation of monocyte derived dendritic cells for immunotherapy. However, monocytes circulate in the blood stream only temporarily with a half life of about 24 h and die spontaneously from apoptosis in the absence of specific survival signals. When we investigated the survival of blood monocytes isolated by CD14 positive selection from apheresis samples and cultivated in the presence of GM-CSF/IL4, we observed a loss of viability of about 66% of the cells within 48h. This would be consistent with the view that about 80% of the monocytes are bound to die at the time of isolation. Detailed flow cytometric analysis revealed that loss of vital cells is accompanied by the appearance of a CD14-negative population, which stained positive for annexin V. To analyse the fate of these cell populations CD14-positive and CD14-negative cells were separated by cell sorting at day 1 and day 2. Flow cytometry and electron microscopy identified predominantly dying cells in the CD14-negative population, whereas a large fraction of the CD14-positive population survived. The CD14-positive cells can be differentiated with an efficiency of 64%–91% to mature DC after cultivation under DC generating conditions; these include the presence of GM-CSF and IL4 and addition of a maturation mix (TNFa, IL1b, IL6 and PGE2) on day 5. DC generated from sorted CD14-positive cell populations were qualitatively and quantitatively comparable to DC from unsorted monocytes. To study the genes involved we analyzed the transcriptome on each cultivation day using Affymetrix HG-U133A DNA-Arrays. During the first 24h, the expressions of about 4700 genes were regulated. About 3000 genes were up regulated and about 1700 genes were down regulated. Especially, we focused on the expression of genes involved in the apoptosis pathway during the early phase of cultivation. Among apoptosis related genes caspase 3, a central player in natural apoptosis of monocytes, was up regulated on day 1. In summary these data suggest that apoptosis limits the yield of monocyte derived DC and that cultivation in the presence of caspase inhibitors or survival signals could improve the efficiency of generation of monocyte derived dendritic cells.