bluuebunny/crossref_metadata_2025 huggingface dataset:
Treatment of Severe Aplastic Anaemia (SAA) with Antilymphocyte Globulin (ALG), Cyclosporine (CyA) and Granulocyte Colony Stimulating Factor (G-CSF) 5 μG vs 10 μG/Kg : A Gitmo Prospective Randomized Study.
Authors: Locasciulli Anna , Bruno Barbara , Rambaldi Alessandro , Saracco Paola , Dufour Carlo , Finelli Carlo , Sica Simona , Varotto Stefania , Arcese William , Locatelli Franco , Soligo Davide , Bacigalupo Andrea
Published: October 2019

Background and objective. In a previous study we showed encouraging outcome in severe aplastic anemia (SAA) patients treated with anti-lymphocyte globulin (ALG), cyclosporin (CyA) and G-CSF 5mg/Kg/day (Blood2000; 95: 1931–4.). However failure to respond, delayed responses, partial responses, relapses and early deaths remain signifcant problems. The aim of the present study was to test whether an increased dose of G-CSF (10 mg/Kg/day) would reduce these complications.

Design and methods. This is a multicenter prospective trial in 77 SAA patients treated with horse ALG (15 mg/kg/day day1–5) and CyA (5 mg/kg/day day 1–180). Patients were randomized to receive G-CSF 5 mg/Kg/day (n=38, group A) or 10 mg/Kg/day (n=39, group B) from day +1 to day +30. All patients then received G-CSF 5 mg/Kg/day from day +31 to day +90. Primary end point was response at day +120. Secondary end points were early deaths , blood counts at day +120, and survival.

Results. At day +120 responses were classified as absent, partial, complete in 12, 22, 4 patients in group A and in 23, 7, 9 patients in group B (p=0.001). At last follow up these figures were respectively 9,12,17 vs 19,2,18 (p=0.004). Thirteen patients (5 in group A and 8 in group B) died before day 120 (p=0.3). Median peripheral blood counts an day 120 were comparable in the two groups: Hb 10.5 vs 9.5 gr% (p=0.6), Neutrophils 2.4 vs 1.9x10^9/l (p=0.4) and platelets 42 vs 36 x10^9/l (p=0.3). The actuarial survival at 4 years is 72% in group A vs 67% in group B (p=0.3).

An additional finding of this study is a strong age effect, with an actuarial 4 year survival of 81% in patients aged 0–20 , 80% in patients aged 21–40 and 34% in patients over 40 (p=0.0002). This correlated with the inability of older patients to increase their white blood cell (WBC) counts above 5x10^9/L, during G-CSF treatment. In a multivariate COX analysis patient age and highest WBC counts during G-CSF, were both significant predictors of survival.

Interpretation and conclusions. Increasing the dose of G-CSF does not appear to reduce early deaths, does not improve peripheral blood counts nor survival, and may reduce the response rate in patients with SAA receiving ALG and CyA. Older age and failure to improve WBC are negative predictors of survival.
Abnormal Erythropoiesis in Microgravity.
Authors: Xu Kun , Holubec Keith V. , Love John E. , Goodwin Thomas J. , Sytkowski Arthur J.
Published: October 2019

Humans and experimental animals subjected to microgravity, such as experienced during space flight, exhibit alterations in erythropoiesis, including changes in red blood cell morphology, survival and a reduction in red blood cell mass. Some of these alterations have been attributed to a disruption of normal in vivo erythropoietin physiology. However, human bone marrow cells grown on orbit showed a profound reduction in the number of erythroid cells, suggesting a cellular component. We now report the results of a study carried out on orbit on the International Space Station (ISS UF-1) in which an erythroid cell line was induced to differentiate. Rauscher murine erythroleukemia cells, a continuous cell line that can undergo erythropoietin (Epo)- or chemical-induced differentiation similar to normal erythropoiesis, were cultured for 6 days either in microgravity on board the ISS or on earth and then for 3 days in the absence or presence of 50 U Epo/ml or 0.7% dimethyl sulfoxide (DMSO). The cells were fixed, stored on orbit and returned to earth for study. Compared to ground-based controls, cells cultured in microgravity exhibited a greater degree of differentiation (hemoglobinization) (p<0.01). However, TER-119 antigen, a specific marker of the late stages of murine erythroid differentiation, was not detected on the surface of cells grown in microgravity. A significantly higher percentage (p<0.05) of cell clusters formed on orbit, whereas actin content appeared reduced. Furthermore, there was a more profound loss of actin stress fibers in microgravity following Epo or DMSO treatment. These results demonstrate abnormal erythropoiesis in vitro in microgravity and are consistent with the hypothesis that erythropoiesis is affected by gravitational forces at the cellular level.(Supported by NASA Grants NAG9-1368 and NAG2-1592 to AJS)
Pathogenesis and Therapy of a New Model of SOD2 Deficiency-Induced Sideroblastic Anemia.
Authors: Martin Florent M. , Welsh Megan L. , Friedman Jeff
Published: October 2019

Manganese superoxide dismutase (SOD2) is a major antioxidant protein which protects cells against the toxicity of superoxide anion, a by-product of mitochondrial respiration. While knockout of SOD2 results in late embryonic or early neonatal death, SOD2 deficient hematopoeitic stem cells (HSC) are capable of rescuing lethally irradiated normal hosts. The resulting hematopoeitic chimeric mice show a persistent hemolytic anemia similar to human sideroblastic anemia. Loss of SOD2 in erythroid progenitor cells results in mitochondrial proliferation and excess iron deposition within mitochondria of reticulocytes. Mature red blood cell (RBC) show abundant iron inclusions with evidence of a defect in iron incorporation into heme and altered survival. SOD2 deficient cells show both enhanced reactive oxygen species production and protein oxidative damage. We recently published a comparative RBC proteome analysis. Our results show that the expression of several proteins involved in folding/chaperone function, redox regulation, ATP synthesis and RBC metabolism is altered in SOD2 deficient cells. To define early events in the pathogenesis, we are currently performing a gene expression array strategy. The overall goal of this procedure is to characterize differential gene expression at the level of TER-119+ CD71+ erythroid precursor cells from the bone marrow of SOD2 deficient HSC mice versus wild-type and antioxidant-treated SOD2 deficient recipients. Preliminary analysis generated a list of 718 genes as significantly discriminating between wild-type and SOD2 deficient RBC progenitors. Taken together, our results and subsequent validation of candidate genes in in vivo experiments will identify genes involved in response of erythroid progenitors to oxidative stress as well as determinants of RBC lifespan in other hemolytic disorders.
Is EPO Receptor Expressed in Human Tumor Cells?.
Authors: Sinclair Angus M. , Busse Leigh , Rogers Norma , Sarosi Ildiko , Van Gwyneth , Elliott Steve
Published: October 2019

EPO stimulates formation of red blood cells by binding to and activating the EPO receptor (EPOR) expressed on the surface of erythroid progenitor cells. Recently EPOR was reportedly expressed in other, non-hematopoietic tissues including brain, kidney, heart and endothelium. EPOR expression has also been reported in tumor tissues and tumor cell lines and many investigators have suggested that EPO may promote tumor growth. This has raised concerns about the use of rHuEPO to treat anemia associated with chemotherapy in cancer patients. To evaluate this, we have examined EPOR expression and binding in tissues and cell lines by quantitative PCR, Western blot analysis and EPO binding assays. Quantitative RT-PCR analysis revealed that the EPOR gene is transcribed in most tissues and cell lines. However non-hematopoietic EPOR transcript levels were lower than that found in hematopoietic cells. No evidence of elevated EPOR gene transcription in tumor vs. non-tumor tissues or in tumor cell lines analyzed was observed. Using Western analysis, we found that commercial anti-EPOR antibodies commonly used to examine EPOR protein expression in published tumor studies, cross-reacted with multiple proteins and/or reacted with proteins of sizes different from that predicted for EPOR. This suggests that in many publications where these antibodies were used, claims of expression of EPOR protein in tumors are of questionable significance. A rabbit polyclonal antibody was developed that specifically reacted with a protein band on Western blots of the size predicted for EPOR. The antibody reacted specifically with a band in COS cells transfected with a human EPOR expressing construct but not in transfection controls. This antibody also reacted with the same sized protein in non-hematopoietic cells. We concluded that EPOR mRNA and EPOR protein were present in most normal cell types examined. Thus the detection of EPOR in tumors and tumor cell lines likely represents the continued expression reflecting the tissue of tumor origin. Since expression does not equate to the presence of surface receptor, binding assays with 125I EPO to non-hematopoietic tumor cell lines were performed. Tumor cell lines showed weak to no detectable EPO binding. These results suggest that although EPOR protein is synthesized in these cells, little or no receptor is expressed on their cell surface. Furthermore, if limited amounts of EPO bound EPOR in tumor lines any biological effect may be different or muted compared to classical responses in hematopoietic cells.
Placental Growth Factor Concentrations in Bone Marrow of Sickle Cell Patients.
Authors: Means Robert T. , Fleury Thomas W. , Dallalio Gail A. , Brunson Chris Y.
Published: October 2019

Although sickle cell anemia (SCA) is the result of an abnormality in the beta globin chain of hemoglobin, its clinical manifestations cannot be explained solely on that basis. It has become clear over the last several years that inflammation and inflammatory mechanisms contribute to the clinical syndromes associated with SCA, and that sickle cell disease, even in steady-state, is an inflammatory process. Placental growth factor (PlGF) is a member of the vascular endothelial growth factor family and is associated with inflammation and with pathologic angiogenesis. Perelman and colleagues have reported that PlGF is released from marrow erythroid cells and that its circulating concentration is 50% higher in patients with severe SCA than it is in healthy controls, and also that PlGF induces the expression of inflammatory cytokines like interleukin-1 (IL-1) and tumor necrosis factor (TNF) (Blood2003;102:1506–14; Blood2003;102:1514–24). As part of our studies of the regulation of erythropoiesis by inflammation in sickle disease, we collected bone marrow aspirates from homozygous SCA patients not currently in crisis (n=5) and from healthy volunteers (n=10), and evaluated PlGF concentrations in the marrow aspirate plasma. The SCA patients studied were all adults, and were not on chronic transfusion, erythropoietin, or hydroxyurea. PlGF concentrations were significantly higher in the marrow aspirate plasma from SCA patients compared to healthy controls (range 38.7 – 90.2 pg/mL vs. 8.7–21.9 pg/mL; p = 0.002). In contrast, plasma PlGF concentrations were overlapping (range 6.2– 11.3 pg/mL for SCA patients vs. 2.2–27.0 pg/mL; p = 0.59). When PlGF concentration was normalized to marrow CFU-E concentration, mean PlGF protein per CFU-E was substantially higher for SCA patients (0.86 pg/105 CFU-E vs. 0.13 pg/105 CFU-E ), although the difference did not achieve statistical significance (p = 0.07). Since cytokines induced by PlGF, such as TNF and IL-1, are known to inhibit CFU-E colony formation, the effects of PlGF on erythroid colony formation in vitro by marrow from SCA patients and healthy volunteers were studied. At concentrations 10 – 1000 pg/mL, no inhibition of CFU-E colony formation was observed, and there was no difference in the response of progenitors from SCA patients and from normal volunteers. In summary, PlGF concentrations in bone marrow aspirate plasma are significantly greater than those observed in healthy controls, and the degree of this difference is more substantial than has been reported for circulating PlGF concentrations. Erythroid progenitors from SCA patients tend to produce PlGF at a higher level then do progenitors from healthy controls. PlGF does not inhibit erythroid colony formation in vitro, and would not be expected to contribute directly to impairment of erythropoiesis in SCA patients.
L-Carnitine and Apoptosis in Thalassemia Major Patients.
Authors: Taher Ali , Seoud H. , Ibrahim A. , Gabre H. , Ibrahim I. Youssry , ElGawada G. Abd , Aoun E. , El-Beshlawy A.
Published: October 2019

Background and Objectives:

Thalassemia major patients suffer from severe anemia due to intramedullary hemolysis of erythroid progenitors. Depending on the severity of their disease, thalassemic patients exhibit different patterns of apoptosis. In cases of β-thalassemia major, apoptosis appears to be greatly enhanced in the early-stage erythroid precursors in the bone marrow and it is considered to be the machinery via which ineffective erythropoiesis develops. L-Carnitine is a butyrate derivative found to strongly reduce apoptosis in different diseases. We investigated the effect of oral L-carnitine therapy on apoptosis in thalassemia major patients.

Methods:

eighteen Thalassemia major patients were included. Detection of apoptosis was done by Photometric enzyme immunoassay (Elisa) and agarose gel electrophoresis before and after 6 months oral therapy with L-Carnitine (50mg/kg/day).

Results:

The mean age was 12.2±6.6 years. All patients tolerated L-carnitine with no side effects. A significant decrease of apoptosis rates in the erythroid precursors in the bone marrow of studied cases was noted after therapy by. The enrichment factor measured by the photometric enzyme immunoassay dropped from 3.65±1.338 before therapy to 1.60±0.65 after therapy (p=0.005). A positive ladder pattern reflecting apoptosis on agarose gel electrophoresis was detected in 88.9% of cases prior to treatment (figure 1) versus 11.1% after therapy (figure 2) (p=0.006). Patients also had significant decrease in the frequency of transfusions and increase in the pre-transfusion hemoglobin levels after therapy. These results were found to significantly correlate with the reduction in apoptosis rates (p=0.006, r = 0.543 and p=0.027 &r = −0.7762 respectively).

Conclusion:

Apoptosis plays a major role in the underlying pathophysiology of anemia in thalassemic patients. L-Carnitine seems to be a good modulator of apoptotic processes in such patients leading to decreased rates of programmed erythroblast death and general improvement of the disease condition. Further large-scale studies are needed to evaluate the overall role of L-Carnitine in the management of these patients.

Figure Figure
Hydroxyurea to Increase Hemoglobin Level in Thalassemia Intermedia.
Authors: Baronciani Donatella , Vaquer Stefania , Carta Paola , Lai Eliana
Published: October 2019

Hydroxyurea (HU) has been shown to increase fetal hemoglobin (HbF) levels in patients with Sickle Cell Disease and Thalassemia Intermedia. Because there are no defined protocols for the use of this drug, we studied the effect of HU in a group of thalassemia intermedia (genetically defined) patients with at least one of the following features:

Low hemoglobin level ( hemoglobin < 7 gr/dl) Bulky extramedullary erythroid masses

We started the enrollment in February 2003 and 8 patients with a minimum follow up of 8 months have been evaluated ( range 8–16 months ) ( mean 13,6 months; median 14 months ). Mean age of the patients was 39.8 years ( range 28–49 years); all were not transfused at the moment of the study entry. HU was administered orally at a mean daily dose of 8.8 mg/kg (range 4–12 mg/kg). The drug was well tolerated; only one patient discontinued the treament for 2 months for pleural effusion. Table summarizes the pre and post treatment characteristics of this group of patients. Data reported are median (range), the differences between pre and post treatment values were analyzed with the the Wilcoxon test for non parametric observations. In this group of patients a mean increase of hemoglobin level of 1.2 g/dl was registered. Extramedullary erythroid expansion, studied by computed tomography scan did not show any change in this follow-up period.

These results confirm that HU may improve Hb level in thalassemia intermedia patients even if other aspects must be studied and longer follow-up is necessary.

Treatment results

Pre treatment Post treatment Two tailed P value Values are expressed as median (range). P value = Wilcoxon test Hemoglobin level (g/dl) 6.6 (4.9–7.9) 7.8 (7.1–9.8) 0.05 Erythropoietin value (mU/ml) 679 (98–2855) 191 (71–1690) 0.2 Erythroblasts % leucocytes 191 (80–567) 153 (2–426) 0.2 Mean Corpuscolar volume (micron3) 77 (66–87) 82 (69–91) 0.08 Mean Hemoglobin Concentration (pg/rbc) 22 (21–25) 25 (22–27) 0.02 Hemoglobin F (%) 94.7 (52.9–97.7) 96.8 (58.7–98.4) 0.06
dHPLC as a Rapid and Reliable Screening Method for A-Thalassemia Screening.
Authors: Bernasconi Luca , Herklotz Roberto , Hergersberg Martin , Huber Andreas
Published: October 2019

Background: a-thalassemia, the most prevalent of all thalassemias, is an inherited single gene disorder of the hemoglobin synthesis characterized by the absence or the reduced expression of a-globin genes resulting in an imbalanced ratio between a- and b-gobin chain synthesis. While some DNA alterations lead to microcytic anemia, the most others cause severe anemia and intrauterine death if co-inherited in a homozygous constellation. The most common genetic defects leading to the a-thalassemia phenotype are deletions of one or more of the two highly homologous a-globin genes. Less frequently, a-thalassemia derives from nondeletional mutations located in parts of the a-globin gene that are critical for its normal expression. So far the identification of the genetic background of a-thalassemic patients has included specific amplification of each a-globin gene followed by laborious and expensive mutation analysis methods such as denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP) or direct sequencing of the entire a-genes.

Method: We attempted to evaluate the performance of denaturing high performance liquid chromatography (dHPLC) technique to identify nondeletional mutations located in the a1- or a2-globin gene. Due to the high sequence homology of a1- and a2-globin genes, separate amplification of the two genes followed by nested PCR were performed. This generated four overlapping amplicons (a-dHPLC1 to 4) covering the whole a1/a2-globin locus including the 3′- and 5′-untranslated regions. The formation of DNA hetero-/homoduplexes was ensured by denaturing and slow reannealing of crude PCR products. Through a DNA separation column under partial-denaturing conditions, DNA homoduplexes were retained longer than their corresponding heteroduplexes generating distinguishable chromatographic profiles and hence allowing for the differentiation between wildtype and mutant DNA. Samples which differed from the wildtype in their elution profile were sequenced in both directions.

Results: 50 patients showing a presumptive a-thalassemic hematological profile not due to deletional mutations (excluded by gap PCR technique) were tested. 10 carriers of a-thalassemia nondeletional alleles (previously identified by endonuclease restriction analysis) and 10 healthy patients were also included in the evaluation.

Conclusion: Our results show that dHPLC is a reliable, sensitive, and specific screening method to detect pointmutations and small deletions located in the a-globin genes. Furthermore, the low costs of analysis and the rapidity of the screening (about 6 min/sample) make the dHPLC technique an appropriate alternative to technically demanding and time consuming mutation screening methods such as DGGE or SSCP analysis.
Comparative Benefits of the Non-Human Primate in Long-Term Toxicity Studies with Iron Chelators: 12-Month Studies with Deferiprone.
Authors: Connelly John , Spino Michael , Tricta Fernando
Published: October 2019

Cynomolgus monkeys and rats have been the species of choice for evaluation of general toxicity during the non-clinical development of iron chelators. Each model has its advantages in safety assessment studies, although the relatively poor conservation of iron in rats and other differences in their iron handling compared with primates, including man, have raised questions about their appropriateness. It was reported that in a study in cynomolgus monkeys conducted during the early development of deferiprone, doses greater than 150 mg/kg caused deaths when given orally once daily for c. 20 days of a scheduled 3-month treatment period. We present results from a 12-month oral toxicity study of deferiprone in naive and iron-loaded cynomolgus monkeys, using twice daily dosing and compare them with findings in a similar study in rats. Cynomolgus monkeys (4–6/sex/group) and rats (20/sex/group), iron loaded by intraperitoneal injection of iron dextran, were given deferiprone as two equal daily oral doses totalling 0, 75, 150 and 200 (250 after 3 months in monkeys) mg/kg/day; naive monkeys were given 0 and 150 mg/kg/day for 12 months. Measurement of all standard parameters, including toxicokinetics, was included. No mortalities, no adverse clinical signs, and no effects on body weight gain, food intake, cardiovascular function or eye morphology, were observed in either iron-loaded or naive monkeys. Haematological and plasma chemistry parameters were largely unaffected by treatment with deferiprone; intermittent increases in serum activities of some hepatic enzymes were related to iron loading. Similarly, histopathological changes were limited to those associated with iron deposition in tissues. Plasma deferiprone concentrations achieved were significantly in excess of clinical exposures. Both naive and iron-loaded rats were less tolerant of long-term administration of deferiprone.

Both divided daily dosing and iron loading may have contributed to the markedly better survival of monkeys in the 12-month study compared with the earlier investigation, and the results of this work confirm the value of the model in the evaluation of the safety of iron chelators.
Clinical Application of Matrix Metalloproteinase-8 and Matrix Metalloproteinase-9 Expression in Patients with CML.
Authors: Lin Liang-In , Lee Cheng-Yeh , Lin Dong-Tsamn , Wen Hwei-Wen , Tien Hwei-Fang
Published: October 2019

Neutrophils are the first cells to arrive at the site of infection where they play a critical role in host defense against invading microbes. To perform this process, neutrophils produce and secrete a lot of molecules to destroy the infectious agent. Among proteolytic enzymes identified in the neutrophils, matrix metalloproteinases (MMPs) have attracted considerable attention for their proposed role in the destruction of extracellular matrix under physiological and pathological conditions. The first MMP identified in neutrophils is MMP-8 or neutrophil collagenase that is stored in the specific granules and is capable of cleaving type I collagen. MMP-9, neutrophil gelatinase, are stored in the gelatinase granules and is capable of cleaving type VI collagen. Chronic myeloid leukemia (CML) is a malignant clonal disorder that originates from a transformed stem cell and involves myeloid lineage. The affected cells have both proliferative and functional impairment. Leukemoid reaction (LR) often encountered in association with severe infections or malignancy, shows extreme elevations of the leukocyte count with left shift including myeocytes, promyelocytes and blasts that would be confused with CML. The laboratory differential diagnosis of CML and LR are based on the leukocyte alkaline phosphatase (LAP) score, philadelphia chromosome analysis and bone marrow analysis. Our previous study revealed that the level of marrow MMP-9 may be a useful surrogate marker for monitoring disease status in AML and propose it as a potential prognostic factor. In this study, CML patients at diagnosis, various patients with leukemoid reaction and several normal individuals were assessed, to evaluate the potentiality of MMP-9 or/and MMP-8 to be differential diagnostic markers between CML and LR. We determined the MMP-8 and MMP-9 concentrations in peripheral blood by ELISA method, and the MMP-8 and MMP-9 contents in neutrophils by immunocytochemical staining and flow cytometry. We found that the plasma MMP-8 levels were less than 10ng/ml in all normal individuals studied, those were 10 – 100ng/ml in CML patients and those were more than 100ng/ml in LR patients. The same trend was shown in MMP-9 levels, which were less than 50ng/ml, 50 – 450ng/ml and more than 450ng/ml, respectively. Immunocytochemical staining revealed MMP-8 and MMP-9 contents in cytoplasm of neutrophils were abundant in patients with LR, compared with those in patients with CML and normal individuals; further flow cytometric analysis also showed the same trend. These results suggest that the MMP-8 and MMP-9 concentrations and individual neutrophil MMP-8 and MMP-9 contents might contribute to discriminate between CML and LR, and to be useful surrogate markers for differential diagnosis.
Changing Patterns of Mortality in Gaucher Disease Prior to and Following the Advent of Enzyme Replacement Therapy.
Authors: Weinreb Neal J. , Lee Robert E.
Published: October 2019

Enzyme replacement therapy (ERT) with exogenous glucosylceramide β-glucosidase (alglucerase, [Ceredase®] or imiglucerase, [Cerezyme®]) has been shown to improve anemia, thrombocytopenia, hepatosplenomegaly, bone symptoms and quality of life in patients with Gaucher disease. However, the impact of ERT on mortality has not been assessed since the inception of ERT in 1991. Data from the epoch prior to the availability of ERT were obtained from the University of Pittsburgh Gaucher Disease Registry. Data from the time period following the advent of ERT were obtained from the International Collaborative Gaucher Group (ICGG) Gaucher Registry and the Ceredase/Cerezyme pharmacovigilance database (Genzyme Corporation). Age of death was obtained for patients with reported Type 1 (non-neuronopathic) Gaucher disease. Only patients with a known cause of death were included in this analysis.

Pre-ERT Era (R.E.L.) Post-ERT Era Number of patients 31 137 Treated with ERT No Yes Mean age at death (years) 53.2 55.6 Range of age at death (years) 3–85 0.2–89 Cause of death due to: n % n % • Gaucher disease 4 12.9% 1 0.7% • Leukemia 3 9.7% 7 5.1% • Lymphoma 1 3.2% 3 2.2% • Myeloma 3 9.7% 1 0.7% • Solid tumor 12 38.7% 17 12.4% • Hemorrhage 1 3.2% 14 10.2% • Thromboembolism 0 0.0% 4 2.9% • Other cardiovascular disease 2 6.5% 26 19.0% • Infectious disease 3 9.7% 6 10.9% • Other causes 2 6.5% 49 35.8%

These descriptive data collected prior to the advent of ERT and following approval of ERT in 1991 raise intriguing questions about the changing pattern of mortality in Gaucher disease. Any direct comparison of these populations must be qualified by the possibility of detection bias or a cohort effect. Therefore, pending further information, it is difficult to attribute significance to the difference in mean age at death between the two populations. However, there have been shifts in the pattern of the causes of death. Most notably, deaths due to the primary manifestations of Gaucher disease and to hematologic cancers and solid tumors appear substantially less common in the post-ERT era whereas the proportion of deaths due to cardiovascular disease and other causes appears to be increasing. More accurate estimates of the patterns of mortality in Gaucher disease remain to be determined, particularly for the pre-ERT era. Additional studies of the changing patterns of mortality in Gaucher disease are ongoing.
Down-Regulation of Toll-Like Receptor 4 Expression in Transforming Growth Factor β1 Treated Murine Dendritic Cells: Implications of Maturation Resistant to Lipopolysaccharide.
Authors: Lin Maofang , Mou Haibo , Cen Hong
Published: October 2019

Evidences accumulated that immature dendritic cell (iDC) could inhibit alloantigen-specific T cell responses and prolong the survival time of allografts. However, the tolerogenic properties of these iDCs were often unstable or inconsistent because of in vivo maturation, such as lipopolysaccharide (LPS) stimulating. Toll-like receptor 4(TLR4) has been reported to act as a receptor for LPS and LPS can stimulate iDC to mature DC (mDC) via TLR4 signal transduction pathway. In this study, we investigated the effects of transforming growth factor β1 on murine bone marrow derived DCs. Murine bone marrow cells were cultured with GM-CSF and TGF-β1 to generate TGF-β1 treated DCs (TGFβ-DCs). Compared to iDCs cultured by GM-CSF alone, the TGFβ-DCs had no significant alterations in ultrastructure after LPS stimulation. Surface expression of CD80, CD86, CD40, MHC-II were inhibited by addition of TGF-β1, especially in CD80, CD86 (p<0.05). Furthermore, the iDCs were sensitive to further maturation in response to LPS by showing increased levels of MHC class II, CD80, CD86 and CD40. In marked contrast, TGF-β1 prevented this LPS-mediated maturation and maintained the cells in the immature state, with low levels of surface costimulatory molecules expression. Using BrdU incorporation method, after 96 h mix lymphocyte reaction, TGFβ-DCs had weaker allogeneic stimulating capacity than iDCs. Importantly, LPS stimulating strongly promoted the allostimulatory capacity of iDCs, whereas only slightly affected TGFβ-DCs. TGFβ-DCs also showed decreased IL-12p70 production and impaired NF-κB activation after LPS stimulation. We also found the expression of TLR4 mRNA on TGFβ-DCs was weaker than that on iDCs by RT-PCR. Moreover, the results of flow cytometry revealed the positive expression percentages of TLR4/MD2 complex on iDCs and TGFβ-DCs were (51.8±3.89% vs. 15.7±4.13%, p<0.01) and the mean fluorescence intensities (MFIs) were (2.37±0.26 vs. 1.36±0.17, p<0.05). These results agreed with previous findings that TGFβ-DCs responded weakly to LPS. In summary, TGFβ-DC is resistant to maturation stimulus (LPS) and might have some correlation with the down-modulation of TLR4 expression.
Platelet-Derived Growth Factor Receptor Beta (PDGFRb) Expression in Langerhans Cell Histiocytosis (LCH).
Authors: Palmieri Giovannella , Montella Liliana , Strazzullo Viviana , De Vizio Dolores , Catalano Lucio , Rotoli Bruno , Insabato Luigi
Published: October 2019

On the basis of promising results obtained by imatinib mesilate in a patient with cerebral LCH with high expression of PDGFRb in the affected tissue (Case Report in press in the New Engl J Med), we evaluated PDGFRb expression by immunohistochemistry on banked LCH specimens.

Methods: A 4 m m section obtained from formalin-fixed, paraffin-embedded specimens was incubated in a microwave oven for 15 min in 10 mmol/L buffered citrate pH 6.0, followed by immunohistochemical procedures to detect PDGFRb (using a rabbit polyclonal antibody, Santa Cruz Biotechnology Inc., CA) and KIT (CD117, Dako, Carpinteria, CA), diluted 1:50 and 1:300, respectively. A conventional avidin-biotin complex procedure was then applied, according to manufacturer’s protocol. Briefly, the sections were washed and incubated with the primary antibody overnight at 4°C, then incubated with the secondary antibody. Positive staining was revealed by the DAB chromogen, followed by counterstaining with Mayer’s hematoxilin. The slide was cover-slipped with a xilene-based mounting medium. The staining was scored as Neg, +1 (low expression), +2 (intermediate expression), or +3 (strong expression).

Results. Up to now, we have analysed specimens from 9 patients (see Table). A strong PDGFRb expression with a predominant cytoplasmic localization was detected in 5 cases; two specimens had intermediate expression and two low expression. KIT expression was never observed. The study is ongoing, to evaluate a larger number of cases; however, in the limited series presented, 70% of cases had a significant PDGFRb expression. This finding may stimulate further clinical investigation on the use of tirosin kinase inhibitors in LCH.

PDGFRß and KIT expression in LCH specimens

Case Gender/Age Disease site KIT PDGFRß C: cytoplasmic; N: nuclear 1 M/10 ocular soft tissue Neg +1C 2 F/9 skin Neg +3C/N 3 M/76 skin Neg +2C 4 M/13 bone Neg +3C 5 M/19 bone Neg +3C 6 M/1 bone Neg +1C 7 M/45 oral mucosa Neg +3C 8 F/23 brain Neg +2C 9 M/45 oral mucosa Neg +3C
Enzyme Replacement Therapy in Gaucher Disease: Beneficial Effects on Bone Crisis and Bone Pain.
Authors: Grabowski Gregory , Charrow Joel , Weinreb Neal J. , Dulisse Brian
Published: October 2019

Bone crises and bone pain are major causes of morbidity in type 1 Gaucher disease (GD), patients. Enzyme replacement therapy (ERT) for GD has proven to be of clinical benefit in resolving hematologic abnormalities and organomegaly. However, the clinical effects of ERT on bone crisis and bone pain have not been fully evaluated. This paper reports the results of the first retrospective data analysis undertaken to examine the association between ERT in this population and the reduction of bone crisis and bone pain.

Data were analyzed from the International Collaborative Gaucher Group Registry, established in 1991 as a longitudinal database to track the clinical outcomes of patients with GD, irrespective of treatment status. Inclusion criteria were GD type 1, treatment by ERT, and data available on the occurrence of bone crises and/or bone pain for 1 year prior to and for each of the 3 years following the initiation of ERT. A bone crisis was defined as pain with acute onset requiring immobilization, narcotics for pain, and periosteal elevation, leucocytosis, fever or debilitation. For bone crises and bone pain, 219 and 244 patients, respectively, met the criteria.

TYPE 1 GAUCHER DISEASE PATIENTS WITH BONE CRISES AND BONE PAIN 1 YEAR PRE- AND 1, 2 AND 3 YEARS POST-ERT

The numbers of patients with reports of bone crises and bone pain in each of the time periods are presented in the table. Bone crisis frequency, at 17% prior to ERT, declined to 5% or less in each of the 3 years following the initiation of ERT (t-test, p <0.0001). Similarly, bone pain, reported in 49% of patients prior to ERT, declined to approximately 30% in each of the 3 years following the initiation of ERT (t-test, p <0.0001)

Yr from ERT initial infusion −1 YEAR 1 YEAR 2 YEARS 3 YEARS *n refers to number of patients Bone crisis n = 219(100%) 38(17%) 10(5%) 1(0%) 6(3%) Bone pain n = 244(100%) 119(49%) 74(30%) 71(29%) 73(30%)

These results provide strong clinical evidence for ERT’s effectiveness in the reduction of bone crises and bone pain in type 1 GD.
Incidence, Natural History and Management of Cytomegalovirus (CMV) Dnaemia and Disease in Patients with Haematological Malignancies in the Non-Allogeneic Transplantation Setting.
Authors: Seymour John Francis , Ng Ashley , Worth Leon , Slavin Monica , Prince H. Miles , Thursky Karin
Published: October 2019

There is little information regarding CMV reactivation in the non-allogeneic transplantation setting. We report the incidence of CMV-DNAemia and CMV-associated disease in a cohort of patients treated for haematological malignancies at the Peter MacCallum Cancer Center from 6/99-6/04. Among the 36 patients identified, potential predictive factors were analysed, including underlying malignancy, previous therapy and therapy immediately preceding CMV-DNAemia/disease. Patients received a median of 2 prior therapies (range 1–9), age ranged from 35–77 years, with 19 males. Diseases were: acute leukaemia(6), CLL(6), NHL(21), HD(1), myeloma(2). Therapies immediately preceding CMV-DNAemia/disease were: “conventional chemotherapy” (39%, including HyperCVAD-14%), fludarabine-based (19%), autologous BMT (22%), and antibody therapy (20%, including denileukin diftitox, 6%, alemtuzumab, 14%). Risk of CMV-DNAemia/disease was calculated from the total number of patients treated over the period of analysis: alemtuzumab, (6/12; 50%), denileukin (2/33; 6%), autologous BMT (9/223; 4%), fludarabine-based (8/177; 4.5%), HyperCVAD (5/77; 6.5%). 94% were symptomatic on investigation (fever 92%, CXR changes 25%, abnormal LFT’s 3%, GI symptoms 8%, rash 6%). 7 had CMV-disease (3 pneumonitis, 2 oesophagitis, 2 enteritis/colitis). 11 had isolated fever. 14/19 treated with ganciclovir had possible/proven CMV-related illness; in 12, fever, CMV-DNAemia or symptoms resolved. 2 died from underlying disease or treatment complications. 3 asymptomatic patients with CMV-DNAemia on >1 occasion were observed until DNA negativity. 17/19 with CMV-DNAemia on only 1 occasion did not develop CMV-related illness. We recommend that patients receiving alemtuzumab, should have CMV surveillance to facilitate early therapy, though the role of ongoing surveillance/prophylaxis is unknown. Patients with persistent/rising CMV-DNA titres or evidence of CMV-disease, should be treated.
Natural Killer Cell Changes To Moderate Exercise on Arterial Compliance and Autonomic Modulation in Patients with Human Immunodeficiency Virus-1 (HIV-1).
Authors: Spierer D.K. , McPherson Eugene , Kleinfeld J.I.M. , Hazel E.
Published: October 2019

Human immunodeficiency virus-1(HIV-1) is associated with cardiovascular abnormalities and autonomic dysfunction. Exercise training is recommended to enhance functional capacity, as it is known that fitness is icommensurate with a improved autonomic profile and can reduce cardiovascular malfunctions. Although it is well established that physical activity in HIV-1 pts results in increased aerobic capacity, the effect of moderate exercise training on vascular mechanisms and autonomic modulations in HIV-1 disease is still unknown. The purpose of this investigation is to examine the effects of regular physical activity on arterial compliance, autonomic modulation and alterations in NK cell number and function in HIV-1 disease. Subjects were matched for age, body mass index, waist/hip ratio, and fitness. Arterial compliance (AC), heart rate variability (HRV), and baroreflex sensitivity (BRS) were collected and analyzed via traditional methods using power spectral analysis during seated rest. NK cell number was measured via flow cytometry. Four groups of men (N=12), mean age 38.8 ± 5.3) were studied. Trained subjects completed three weekly sessions of supervised aerobic exercised at 60–75% VO 2max for 10 weeks. Controls performed activities of daily living (ADL) with no formal exercise training. Subjects were categorized as follows: HIV negative untrained (HNU), HIV negative trained (HNT), HIV positive untrained (HPU), and HIV positive trained (HPT). HIV positive subjects were seropositive for HIV disease within 24 months of this investigation. Preliminary data demonstrate a strong trend toward augmented arterial compliance and a higher autonomic profile with increased NK cell number and activity [ HPU mean NK cell level, 160.42/cu mm(10.7%); and HPT was 251.8/cu mm (15.4%)] in response to moderate regular exercise. However, cessation of training exercises resulted in return to near baseline NK cell number, percentage (191.8/cu mm (11.2%).

HNU HNT HPU HPT AC (mmHgxsec) 7.0±3.1 9.4±5.4 4.0±1.5 7.8±1.1 HRV (msec²) 6.1± 2.1 7.6±1.1 3.9±1.5 7.4±0.3 BRS (msec/mmHg) 7.9±6.6 13.9±7.5 8.1±2.9 19.2±8.7

CONCLUSIONS: Enhanced fitness and its effect on vascular compliance and autonomic modulation with moderate regular exercise induces alterations in NK cell number and activity may help improve quality of life, morbidity and mortality, survival and response to therapy in pts with HIV-1 disease.
Oligoclonal Expansion of TCR V beta Subfamily T Cells in Patients with B-ALL.
Authors: Li Yangqiu , Huang Meijuan , Zhou Yubing , Chen Shaohua , Yang Lijian
Published: October 2019

Clonal expansion of TCR V beta subfamily T cells were identified in peripheral blood and tumor infiltrated tissues lymphocytes from some solid tumors and leukemias, which were thought to be relative to tumor associated antigen and be benefited for design of the strategy of specific immunotherapy. The aim of this study is to investigate the situation of TCR V beta gene repertoire and clonal expansion in peripheral blood T cells from patients with B-ALL. The complementarity determining region 3 (CDR3) of TCR V beta 24 subfamily genes in peripheral blood mononuclear cell from 13 cases with untreated B-ALL were amplified using RT-PCR. The PCR products were further labelled by fluorescein and then analyzed by genescan technique for detection of the CDR3 size, to determine the clonality of T cells. The results showed that only 2–18 V beta subfamilies were identified in 13 B-ALL cases respectively. The frequency expression of V beta subfamilies was V beat 13 (84.6%) and V beta 21 (76.9%). No positive products were detected in V beta 4, 6, 8, 18, 20 and V beta 24 subfamilies. Clonal expansive T cells in one or more V beta subfamilies were found in all 13 cases. The display frequency of clonal expansive T cells in V beta 21 (46.1%) and V beta 23 (30.8%) subfamilies were higher than others. In conclusions, the results indicated that the dominant utilization of TCR V beta repertoire could be found in peripheral blood T cells from patients with B-ALL, meanwhile clonal expansive T cells were existed. It may be a feature of the host immune response for leukemia associated antigen. The clonal expansive tendency of T cells in V beta 21 and V beta 23 subfamilies was rather obvious, which maybe correlated with certain malignant B-cell clone. Isolation and amplification of the clonal expanded V beta T cells in vitro may be necessary for identification of the specific cytotoxicity for leukemia cells.
Polyclonal IgG4 Hypergammaglobulinemia Associated with Lymphadenopathy and Renal Disease: A Novel Syndrome.
Authors: Boulanger Emmanuelle , Fuentes Vincent , Meignin Veronique , Mougenot Beatrice , Labaume Sylvaine , Cogne Michel , Aucouturier Pierre , Oksenhendler Eric , Clauvel Jean-Pierre , Ronco Pierre , Lassoued Kaiss
Published: October 2019

Marked polyclonal IgG4 hypergammaglobulinemia has exceptionally been reported. Here we report on two patients who presented an unique syndrome including a predominant and massive increase in serum levels of the IgG4 subclass. Clincal, biological and pathological data were described. Production of IgG4 by patients’ peripheral blood lymphocytes was studied, as well as the capacity of patients’ sera and T lymphocytes to induce IgG4 production by normal B cells. The possible involvement of IL4 and IL13 in the increased IgG4 production was also investigated. The two patients had anemia, lymphadenopathy, renal manifestations and a marked polyclonal IgG4 hypergammaglobulinemia leading to hyperviscosity syndrome in one case. The IgG4-producing cell percentage was significantly increased in both cases. By contrast with normal sera, both patients’ sera significantly increased the IgG4-producing cell percentagae when incubated with CD40-stimulated normal B lymphocytes. Similar effects were obtained with the culture supernatants of patients’ activated T cells. IL4 and IL13 serum concentrations were normal in the two patients. Anti-IL4 and/or anti-IL13 antibodies were unable to antagonize the IgG4 synthesis. The increased IgG4 production was found to be mediated by soluble, heat-resistant factor(s) that did not require the Stat6 signaling pathway. We described here a novel syndrome characterized by a significantly increased IgG4 production, mediated by soluble, heat-stable factor(s) most probably secreted by activated T cells, and distinct from IL4 and IL13.
Deficient IL-10 Secretion by Dendritic Cells (DCs) and Activated Lymphocytes in Aplastic Anemia (AA) Patients.
Authors: Archuadze Shorena , Mikhailova Elena , Sadovnikova Elena , Parovichnikova Elena , Savchenko Valeri
Published: October 2019

Background: Immunoregulatory cytokines (IL-10 and IL-12) secreted by the activated DCs and lymphocytes (LCs) mediate Th1 and Th2 immune response, respectively. Characteristic increase in Th1/Th2 ratio in AA patients might be responsible for autoreactive T-cell mediated suppression of hematopoiesis. However, functional characteristics of DCs initiating the Th1/Th2 disbalance in AA patients remain unelucidated. Aims: To study the functional characteristics of DCs and activated LCs in AA patients. Methods: We have carried out the flow cytometry analysis of the expression of costimulatory molecules by DCs generated from peripheral blood mononuclear cells (PBMC) of 13 AA patients before and after the immunosuppressive therapy (IST). DCs from 4 healthy donors (HD) were studied as a control group. Supernatants (SNs) of LPS activated DC cultures and LC cultures with or without PHA stimulation were studied for IL-10 production by ELISA. CD4+CD25+ regulatory T cell population of AA patients and HDs has being evaluated by means of magnetic beads for the negative selection of CD4+ T cells and immunofluorescence staining with anti-CD25 monoclonal antibodies. Results: DCs of 53,4% (7/13) of AA patients exhibited lower than HDs’ DCs expression of CD83 - marker of mature DCs. Level of IL-10 production by DCs from AA patients was significantly lower compared with HDs; opposite to HDs’ LCs, unstimulated LCs from 88,8% (16/18) of untreated AA patients failed to produce IL-10, however, LPS stimulation resulted in IL-10 secretion though to significantly lower levels compared with HDs; IST enhanced IL-10 production by DCs as well as stimulated and unstimulated LCs from AA patients, though to lower amounts than those of HDs. Few experiments in AA patients after long-term successful IST showed that DCs of AA patients with complete remission tend to produce even more IL-10 than those of HDs. Conclusion: Altered expression of CD83 by the DC from AA patients might influence the activation of cytotoxic T-cells. IL-10 downregulation might inhibit the generation of CD4+CD25+ regulatory T cell population and consequently, contribute to the expansion of cytotoxic T-cells and to the development of AA.