bluuebunny/crossref_metadata_2025 huggingface dataset:
Prevalence of Hypoparathyroidism in Children and Young Adults with Thalassemia Major.
Authors: Yadav Satya Prakash , Sachdeva Anupam , Arya Subash C. , Khanna Virender K. , Arya Archana D.
Published: April 2020

Endocrinopathies are frequently reported in children with thalassemia major. Affected children at our center are transfused regularly to maintain hemoglobin of at least 8–9 gm/dl. Most subjects are chelated with Desferrioxamine or Deferiprone or a combination of the two drugs, although a majority of children have poor compliance and are poorly chelated. Hypoparathyroidism usually occurs in thalassemia major due to excess iron deposition in the parathyroid glands. All patients suffering from thalassemia major are screened routinely at our clinic for their bone metabolism. The fasting serum calcium, phosphorus, intact parathormone and alkaline phosphatase levels are checked annually to screen for hypoparathyroidism and Vitamin D deficiency. A diagnosis of hypoparathyroidism is made if the serum calcium is low, phosphorus is high, alkaline phosphatase level is normal and serum parathormone level (intact) is low normal. Mean S. ferritin levels of all the subjects were calculated by averaging the ferritin values over the last 5 years. We analyzed the screening results for hypoparathyroidism in 110 subjects with Thalassemia Major between the ages of 10–25 years. Of these 110 subjects 19 (17.3%) were found to be suffering from hypoparathyroidism and 91 patients had a normal screen. Mean S. ferritin level was 5997ng/ml in the affected subjects (ranging from 2900 to 10,904 ng/ml), compared with the mean S. ferritin level of 3793ng/ml in the unaffected subjects, which was clinically significant, showing a direct correlation between the poor chelation and development of hypoparathyroidism. Only 2 subjects from our affected group had an average ferritin of 2000–3000 ng/ml, although they also had other endocrine disorders such as short stature and hypothyroidism. Of the affected subjects, 9 were males and 10 were females (M: F = 1.1:1). The reported male: female ratio for hypoparathyroidism in thalassemics is 3:1. Age of onset of hypoparathyroidism ranged from 10.9 years to 23 years with mean age of onset being 16 years. The youngest subject (10.9 years) had an average ferritin of 2908 ng/ml. All the affected subjects above 15 years of age had hypogonadism, 7 of the 19 affected subjects (36.84%) had diabetes, 4 (21%) of them had an abnormal glucose tolerance test, and 5 (26.3%) had hypothyroidism. The incidence of hypoparathyroidism reported in our group of patients is higher compared to those reported from other continents (3.6–4.5%) probably due to poor chelation., both males and females are equally affected and the age of onset is earlier compared to reports from other countries.. This suggests that high ferritin levels due to poor chelation is a major factor in the development of hypoparathyroidism and most subjects who develop it, have multiple endocrine disorders.
Left Ventricular Function Declines with Increasing Myocardial Ferritin Iron in Thalassemia Major.
Authors: Sheth Sujit , Tang Haiying , Jensen Jens H. , Altmann Karen , Prakash Ashwin , Printz Beth F. , Brown Anthony L. , Hordof Allan J. , Tosti Christina L. , Azabagic Andjela , Swaminathan Srirama , Brown Truman R. , Olivieri Nancy F. , Brittenham Gary M.
Published: April 2020

Using a new magnetic resonance method that separately estimates the two principal forms of storage iron, ferritin and hemosiderin, in the heart, we examined the relationship between myocardial storage iron fractions and left ventricular function in thalassemia major. In patients with iron overload, the amount of iron in functional and transport pools changes only slightly. Virtually all of the excess is sequestered in storage forms of iron, as ferritin, a diffuse, soluble fraction, and as hemosiderin, an aggregate, insoluble fraction. The two storage forms of iron strongly affect signal intensity in both T2 and T2* weighted images but influence MRI signal decay through different means because of their differences in solubility and in intracellular distribution (Magn Reson Med2002; 47:1131–8). Separate estimates of the iron concentrations of the two forms of storage iron may be obtained by measuring two distinct relaxation parameters, the “ferritin iron index” (“reduced” transverse relaxation rate) RR2, and the “hemosiderin iron index”, A. We studied 14 patients with thalassemia major, all being treated with subcutaneous deferoxamine. Study participants were examined with a Philips 1.5 T Intera scanner using three Carr-Purcell-Meiboom-Gill (CPMG)-like multi-echo spin echo sequences with varied inter-echo times, using electrocardiographic triggering and respiratory navigator gating to estimate RR2 and A. The left ventricular shortening fraction was measured using standard echocardiographic methods. The Figure shows the relationship (R=0.91, p<0.0001) between the ferritin index, RR2, and the left ventricular shortening fraction.

Figure Figure

Overall, variation in the ferritin index explained more than 80% of the variation in ventricular function. For comparison, variation in the hemosiderin index, A, accounted for only about 33% of the variation in shortening fraction. Using an empirical calibration to estimate iron concentrations, variation in total (ferritin + hemosiderin) iron accounted for only about 40% of the variation in ventricular function. In patients with thalassemia major, the concentration of ferritin iron may provide a better indicator of the magnitude of the toxic iron pool than the total storage iron concentration. Magnetic resonance determinations of the partition of storage iron between ferritin and hemosiderin may be clinically valuable in evaluating tissue iron toxicity in patients with transfusional iron overload.
Vibrio parahaemolyticus Flagellin Stimulates the Maturation of Human Monocyte-Derived Dendritic Cells and Elicits Th1-Type Immune Response.
Authors: Kang Hyun-Kyu , Park Myong-Suk , Lee Shee-Eun , Rhee Joon-Haeng , Park Jung-Sun , Nguyen Thanh-Nhan , Zhu Xiao-Wei , Cho Duck , Nam Jong-Hee , Kim Young-Jin , Kim Hyeoung Joon , Lee Je-Jung
Published: April 2020

Flagellin, the principal component of bacterial flagella, interacts with Toll-like receptor (TLR5) and induces the generation of a pro-inflammation response and activation of host dendritic cells (DCs) in vivo. In this study, we investigated the role of Vibrio parahaemolyticus (V. parahaemolyticus)-derived flagellin as a DC maturation-inducing molecule. V. parahemolyticus-derived flagellin (100–1,000 ng/ml) induced the maturation of human monocyte-derived dendritic cells in a concentration-dependent manner with maximal effect at 500 ng/ml of flagellin as determined by increased levels of surface markers, namely, CD1a, CD80, CD86, CD83, and HLA-DR, a response which could be compared with the phenotypic change in immature DCs (iDCs) treated with lipopolysaccharide (LPS) or cytokine cocktails (CC) with TNF-α, IL-1β, IL-6, and PGE2. Moreover, V. parahaemolyticus-derived flagellin also reduced phagocytic activity, and increased IL-12 production in a polymyxin B-insensitive manner and DC-mediated T cell proliferation, which is comparable with that of LPS- or CC-treated iDCs at several responder to stimulator ratios, suggesting the functional maturation of DCs by V. parahaemolyticus-derived flagellin. Maturation of DCs by V. parahaemolyticus-derived flagellin also elicited a significant increase in specific cytotoxic activity against target cells at several effector to target cells ratios as determined by 51Cr-release assay, and induced Th1-type immune response, such as increase in INF-γ producing cells, determined by ELISPOT assay and analysis of intracellular cytokine staining assay. Taken together, this study demonstrates the role of V. parahaemolyticus-derived flagellin in the functional maturation of DCs, and suggests that V. parahaemolyticus-derived flagellin as a useful molecule for the development of a DC-based immunotherapy against tumors.
In Vivo Tracking of Macrophage Activated Killer Cells to Sites of Metastatic Ovarian Carcinoma.
Authors: Ritchie David S. , Mileshkin Linda , Wall Dominic M. , Bartholyens Jacques , Thompson Mick , Hicks Rod , Prince Miles
Published: April 2020

Radio-labelling of blood cells is an established technique for evaluating in vivo migration to sites of pathology such as infection and haemorrhage. We describe the use of two methods of cell labelling for tracking the destination of autologous-derived macrophage activated killer (MAK) cells linked to the bi-specific anti-her2neu antibody MDX-H210 delivered either by intravenous (iv) or intraperitoneal (ip) injection in ten patients with peritoneal relapse of epithelial ovarian carcinoma. Our results demonstrate the feasibility of generating high numbers and purity of GMP quality MAK cells, which can be efficiently radiolabeled. MAK cell administration produced minimal infusional toxicity and demonstrated a reproducible pattern of in vivo distribution to the lungs, liver and spleen in addition to active in vivo tracking to sites of known tumor following either iv (8 of 16 infusions) or ip (4 of 6 infusions) using either 18F-FDG or 111In-labelled cells. The addition of MDX-H210 monoclonal antibody did not alter the distribution of cells to tumor sites, but did alter the in vivo distribution of cells administered by iv injection. This study demonstrates that cellular cancer immunotherapies may be successfully delivered to sites of active tumor and allows selection of those patients who may benefit from these approaches.
Effect of Enzyme Replacement Therapy on Bone Mineral Density in Type 1 Gaucher Disease.
Authors: Wenstrup Richard , Kacena Katherine , Kaplan Paige , Pastores Gregory , Prakash-Cheng Ainu , Zimran Ari , Hangartner Thomas
Published: April 2020

Objective: To determine the effect of enzyme replacement therapy (ERT; Cerezyme®, imiglucerase for injection) on bone mineral density (BMD) in patients with type 1 Gaucher disease (GD).

Methods: The GD population included all adults (males 18–70y, females 18–50y) with type 1 GD enrolled in the International Collaborative Gaucher Group (ICGG) Gaucher Registry for whom serial spine BMD measurements were submitted. BMD data with up to 12 years of follow-up were analyzed for 111 patients not on ERT, 218 patients on ERT only and 34 patients on ERT plus bisphosphonates. BMD was measured by dual energy X-ray absorptiometry (DXA) Z-scores (age and sex-adjusted) in the lumbar spine of each patient. All measurements were compared to a reference population (Hologic reference standard).

Results: DXA Z-scores for GD patients not on ERT were significantly lower than the reference population and remained approximately one standard deviation below the reference population over time (y intercept = −1.01, p<0.001; slope= +0.012, p=0.63). The DXA Z-scores for GD patients receiving ERT-only were also significantly lower than the reference population at baseline, but improved significantly over time, and the model suggests that on average patients do achieve the same values as the reference population after approximately 12 years of ERT (y-intercept = −1.21, p<0.001; slope= +0.092, p<0.001). The DXA Z-scores for GD patients receiving ERT plus bisphosphonates had an even more rapid increase, on average approaching the reference population after approximately 6 years of treatment (y-intercept = −1.23, p<0.001; slope = +0.202, p=.0003).

Discussion and Conclusion: Osteopenia and osteoporosis are common manifestations of type 1 GD. ERT with Cerezyme® significantly improves BMD in patients with GD. Response to treatment with ERT for BMD may be slower than other aspects of GD. On average, GD patients achieve age- and sex-adjusted normal BMD after approximately 12 years of ERT. The addition of bisphosphonates to ERT may produce an additive effect, with patients achieving a normal BMD more rapidly. A normal (age- and sex-adjusted) BMD should be a therapeutic goal for patients with type 1 GD.
Prognostic Factors for Neutropenic Patients Admitted to Intensive Care.
Authors: Silva Madalena , Checa Luisa , Costa Fatima , Moreno Rui , Silva Eduardo G. , Sousa Aida B.
Published: April 2020

The admission of neutropenic patients (pts) to intensive care units (ICU) is controversial, especially when mechanical ventilation is required. Knowledge of the relative prognostic impact of factors related to the underlying disease and to the severity of acute organ failures might help avoiding futile admissions. We retrospectively assessed predictors of 30-day mortality in neutropenic (<1000/ul) pts referred from a single Hematology unit to the 2 ICUs of the institution over a 10-year period. Of 66 consecutive pts, median age 48 (15–73), 82% had acute leukemia (AL) and 21% were in complete remission (CR). On ICU admission 62% of the pts had a neutrophil count ≤500/ul; microbiologically documented infection was found in 42%. The main reason for ICU referral was severe sepsis or septic shock in 62% of the cases and respiratory failure in 38%. Seventy per cent of the pts were already on vasopressor agents. At ICU entry the median Simplified Acute Physiology Score (SAPS) II was 63 and 26% of pts had ≥ 2 acute organ system failures (OSF). Coma was present in 23%. Mechanical ventilation was eventually needed in 89% and dialysis in 9% of the pts. Mortality at 30 days was 73%. By univariate logistic regression analysis mortality was not significantly related to age, to status of underlying disease (CR vs no CR/not yet known) to duration of neutropenia nor to depth of neutropenia at entry (≤ 500 vs >500/ul). Pts who died were more likely to have non-M3 AL subtype vs M3 (p=0.037), to have ≥ 2 acute OSF vs < 2 (p=0.012) and a higher SAPS II score (p< 0.001). In multivariate analysis only the latter 2 variables remained significant. In conclusion, our data show that 27% of neutropenic pts admitted to ICUs are alive at 30 days; that selection for admission should not be based on the characteristics of the underlying malignancy; and that the 30-day mortality is highly predictable by initial acute illness severity scores.
Autoimmunity Caused by Cell Type-Specific Deficiency in Apoptosis.
Authors: Chen Min , Wang Yui-Hsi , Wang Yihong , Sandoval Hector , Liu Yong-Jun , Wang Jin
Published: April 2020

Apoptosis in the immune system is essential for maintaining self-tolerance. Defective apoptosis causes systemic autoimmune diseases in humans and mice. However, inhibition of apoptosis in lymphocytes alone does not induce significant autoimmune responses, indicating that impaired apoptosis in other cell types plays a critical role in the breakdown of self-tolerance. To study whether apoptosis in dendritic cells (DCs) regulates self-tolerance, we generated transgenic mice expressing a potent caspase inhibitor from baculovirus, p35, in dendritic cells (DC-p35). DC-p35 transgenic mice displayed defective apoptosis in DCs, leading to DC accumulation, chronic lymphocyte activation and systemic autoimmune manifestations resembling the phenotypes in Fas-deficient lpr mice. Our results suggest that apoptosis in DCs is critical for limiting lymphocyte activation and preventing autoimmunity.
Quantification of CD52 Antigen Expression by Normal and Leukemic Lymphoid Cells: Indications for Anti-CD52 Antibody Therapy?.
Authors: Guarini Anna , De Propris Maria Stefania , Intoppa Stefania , Milani Maria Laura , Lisci Alessandro , Martinelli Marilisa , Vitale Antonella , Mauro Francesca Romana , Foà Robert
Published: April 2020

CD52 is a glycosyl-phosphatidylinositol-anchored protein expressed on the surface of lymphoid cells. Polyclonal and monoclonal anti-CD52 antibodies have been utilized to purge T-lymphocyte populations from the peripheral blood and bone marrow in allogeneic transplantation procedures to prevent graft-versus-host disease. Nowadays, the anti-CD52 monoclonal antibody represents an established tool for the treatment of acute and chronic lymphoproliferative disorders. In this study, we have quantified the levels of expression of the CD52 antigen on the surface of normal B- and T-cell populations, as well as on different acute and chronic leukemic cells of B- and T-cell origin. The quantification of the CD52 antigen was performed by flow cytometry utilizing the QuantiBRITE system and the QuantiCALC software (all instruments, reagents and software came from Becton Dickinson, Immunocytometry System, San Jose, CA). The leukemic cell populations analyzed were obtained from primary B-cell chronic lymphocytic leukemia (CLL) and B- and T-lineage acute lymphoid leukemia (ALL) patients. All analyses were conducted on freshly drawn samples from untreated patients.

In a population of 20 normal adult donors, the CD20+ and CD19+ peripheral B lymphocytes showed 9,581 (median value, range 6,234 – 26,078) and 9,495 (7,259 – 26,196) molecules/cell of the CD52 antigen, respectively. T-cell populations had significantly (p = 0.0001) lower levels of expression of the CD52 antigen: 5,803 molecules/cell on CD3+/CD7+ cells (range 2,917 – 11,056 ). Interestingly, the CD4+ population expressed a significantly higher (p<0.0001) number of CD52 molecules/cell than CD8+ lymphocytes (7,531 median value, range 4,340–12,092 vs 3,178, 2,204–7,591). In 98 CLL samples, the CD19+ leukemic cells showed a median number of CD52 molecules comparable to normal values: 10,205 (range 3,154–36,801). On the contrary, in ALL samples the levels of expression of the CD52 antigen by the leukemic cells was significantly (p = 0.01) lower: in the 8 T-ALL cases analyzed the median value was 1,939 molecules/cell (range 1,110–34,509) and in the 27 B-lineage ALL 1,678 molecules/cell (range 164–7,319). These data can explain the efficacy of the anti-CD52 antibody in the treatment of CLL patients and underline that a close monitoring of the CD4+ population should be performed, particularly in patients who have also received fludarabine. In ALL patients, the clinical results obtained following the administration of the anti-CD52 antibody need to be correlated with the levels of expression of the CD52 antigen on the leukemic cells.
Evidence That Functional Neural Tissue-Committed Stem Cells (NTCSC) Reside in the Human Bone Marrow and Are Mobilized into Peripheral Blood in a Patients after Stroke.
Authors: Kucia Magda , Paczkowska Edyta , Majka Marcin , Machalinski Bogdan , Ratajczak Mariusz Z.
Published: April 2020

The concept that bone marrow (BM)-derived cells participate in neural regeneration remains highly controversial and the identity of the specific cell type(s) involved remains unknown. We recently reported that murine BM contains a highly mobile population of CXCR4+ cells that express mRNA for various markers of early tissue-committed stem cells (TCSC), including neural TCSC (Leukemia2004:18;29–40). Here we show that these cells could be isolated from murine BM as a population of Sca-1+lin− CD45− cells that not only express neural lineage markers (β-III-tubulin, Nestin, NeuN and GFAP), but more importantly form neurospheres in vitro and cells from these neurospheres after replating into secondary cultures differentiate into neuronal (β-III tubulin+, nestin+) and macroglia (O4+, MBP+, GFAP+) lineages. Our data demonstrate that neural TCSC i) are very rare (0.01% of BMMNC) (ii) present in BM harvested from young (1–2 month) while being barely detectable in older (1-year) mice; iii) are mobilized from BM into peripheral blood (PB) during pharmacological mobilization and iv) are chemoattracted for potential brain regeneration in an SDF-1-CXCR4, dependent manner. In humans the corresponding population of cells is present among CXCR4+CD34+AC133+CD45− BMMNC. Our studies performed on 14 patients with stroke (age 48–75 years) demonstrated in the peripheral blood an increase in the number of CXCR4+CD34+AC133+ cells expressing neural TCSC. The maximal elevation of these cells was observed 24–72 hours after stroke and remained elevated up to one week. The degree of mobilization correlated with younger age, smaller size of the stroke, was best in the patients suffering from a less extensive stroke and worse in patients with dyslipidemias. Thus, we conclude that bone marrow is a potential source of CD45- neural TCSC for brain repair and since purified CD45+ HSC neither express neuronal markers nor differentiate in vitro into neurospheres, we provide for the first time evidence that neural TCSC residing in bone marrow but not trans-dedifferentiated HSC account for neural differentiation of BM-derived cells. Furthermore, our observation that the number of BM-derived neural TCSC decreases with age provides a novel insight into aging and may explain why the brain regeneration process becomes less effective in older individuals. These observations provide rationale for further studies aimed at optimizing therapeutic brain regeneration by BM-derived neural TCSC.
CD4+CD28+ T Lymphoctye Subsets Decreased in Healthy Donors after G-CSF Mobilization.
Authors: Weng Jianyu , Du Xin , Zhang Jianjun , Wu Suijin , Lu Zesheng , Zhou Maohua , Lin Wei , Huang Zilun
Published: April 2020

Objective Allogeneic transplantation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) -mobilized peripheral blood stem cells (PBSCs) is now being increasingly performed, but safety considerations for hematologically normal PBSC donors have not been fully addressed. The effection of G-CSF on donors’ lymphocytes have not been defined clearly. Our study was to detect and analyze the phenotypical and functional properties of lymphocytes from allo-PBSC donors treated with recombinant human G-CSF by flow cytometry. Methods Thirty-four HLA-identical sibling donors (13male, 21female; median age, 35 years) were treated by subcutaneous injection with rhG-CSF at a dose of 5 μg/kg twice daily for 4–6 consecutive days to mobilize HSCs to the peripheral blood. Leukapheresis was performed using a continuous flow blood cell separator (COBE Spectra, Lakewood, CO) on 1 to 2 consecutive days beginning on day 4 of rhG-CSF administration. Donor blood samples, which were obtained before the first administration of G-CSF (pre-G), on day 4 of G-CSF administration (post-G), and one week after the last rhG-CSF were analyzed by 3-color flow cytometry. Monoclonal antibodies included: CD3, CD4, CD8, CD20, CD16, CD56, CD25, CD69, CD45RA, CD45RO, CD28, CD95. Results Absolute counts of lymphocytes, B cells, T cells, CD4+ and CD8+cells post-G were significantly elevated two more times than pre-G (P<0.05), but these changes recovered when the last G-CSF administrated one week later. The percentage of CD3+, CD4+, CD8+ and the ratio of CD4 and CD8 T cells had no significantly difference between pre-G and post-G. The percentage of CD4+CD25+, CD4+CD45RO+ and CD4+CD28+ T cells were significantly decreased post-G (45.26±9.68% to 37.34±11.12%; 65.53±13.74% to 52.32±13.47%; 94.75±4.02% to 91.74±8.72%, P=0.00329, 0.0003 and 0.0947, respectively). We found that CD4+CD28+ T cells(91.77 ± 6.15, P=0.00218) were still lower than pre-G when stopped administrate G-CSF one week later, while others changes have recovered to the Pre-G level. Conclusion: The study shows that CD4+CD28+ T lymphoctye subsets were still lower than pre-G, although most of other change recover one week later after mobilization
Analysis of PRAME Protein Expression in Normal Lymphoid Tissues and during B Ontogenesis.
Authors: de Figueiredo-Pontes Lorena L. , do Nascimento Fabio M. , de Abreu e Lima Rodrigo S. , Proto-Siqueira Rodrigo , Garcia Aglair B. , Matos Daniel M. , Gout Ivan T. , Filonenko Valery V. , Zago Marco A. , Falcao Roberto P. , Rego Eduardo M.
Published: April 2020

PRAME (Preferentially Expressed Antigen in Melanoma) gene was originally isolated in melanoma. A significant increase in the number of PRAME transcripts has been demonstrated in hematologic malignancies such as acute myeloid and lymphoid leukemias, multiple myeloma and chronic lymphoproliferative diseases. Furthermore, our group generated an anti-PRAME monoclonal antibody (MoAb) and by quantitative flow cytometry has demonstrated that PRAME protein was aberrantly expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma. However, the expression of this antigen in normal lymphoid tissues and during B cells ontogeneis has not been characterized. To address this question, PRAME protein expression was studied by flow cytometry in peripheral blood (PB, n=15) and bone marrow (BM, n=6) from healthy donors, lymphonodes (n=4) and spleen (n=4) from patients submitted to lymphonode excision or splenectomy for non malignant diseases. First, we determined in which hematopoietic lineage PRAME was expressed by concomitantly staining PB, BM, lymphonode and spleen mononuclear cells (MCs) with anti-PRAME and a panel of MoAbs specific to B(CD19)/ T(CD3)/ NK(CD16/56), monocytic(CD14) and granulocytic(CD33) markers. PRAME was detected exclusively in CD19+ cells. The median percenatge of PRAME positive cells was 5,31% (2,55–12,34%), 13,01% (8,47–38,15%), 12,79% (3,15–23,06%) and 17,5% (12,67–27,43%) in PB, BM, lymphonode and spleen MCs, respectively. Amongst CD19+ cells, we have observed that PRAME was expressed by 42,39% (16,16–75,72%), 16% (13–69,5%), 15,16% (5,49–41,20%) and 48,82%(12,67–58,89%) in PB, BM, lymphonode and spleen, respectively. To establish in which stage of B ontogenesis PRAME was expressed on, cell suspensions stained with anti-CD19 were submitted to positive magnetic separation and labeled with anti-PRAME, CD5, CD27, CD38, CD34, CD10 and IgD MoAbs. PRAME+/CD19+ cells were CD5−, CD27+, CD38+, CD34−, CD10− and IgD+, thus suggesting that PRAME is expressed by the memory B cell compartment of the normal lymphoid tissues. This study defines PRAME as a B cell antigen that may accompany the neoplastic clone proliferation of mature B cell neoplasms. Although PRAME is mainly an embryonic antigen, expressed by carcinomas of immature phenotype, it is expressed by mature B cells in normal and pathological lymphoid tissues. Our findings suggest that maturational events occurring at the germinal center of lymphoid follicles affects PRAME expression.
Circulating Cytokeratin 5+ Progenitor Epithelial Cells Also Express Other Progenitor Cell Markers and Are Necessary for Normal Airway Repair.
Authors: Gomperts Brigitte N. , Burdick Marie D. , Belperio John A. , Strieter Robert M.
Published: April 2020

Background: Human lung transplants have demonstrated recipient epithelial cells in donor organs, supporting the notion of a circulating epithelial progenitor cell. Resident progenitor epithelial cells in the proximal airway are located in the submucosal gland ducts and basal epithelium and express cytokeratin (CK) 5/14. We have identified circulating progenitor CK5/14 positive epithelial cells in the bone marrow and buffy coat of mice. These cells appear to traffic via the CXCR4/CXCL12 biologic axis.

Purpose of study: To further determine additional markers of these circulating progenitor epithelial cells and to disrupt the CXCR4/CXCL12 axis to prevent recruitment of these circulating progenitor cells after airway injury.

Methods used: Flow cytometric analysis of cytokeratin 5 (CK5), CXCR4, CD45, CD34, Sca-1, c-kit expressing cells in the buffy-coat and bone marrow of naive mice and CK5-GFP transgenic mice. Subcutaneous heterotopic implantation of dissected female wild type tracheas into male GFP recipient mice. Immunohistochemical and dual immunofluorescence analysis of GFP and CK5 as well as p63 in tracheal transplants.

Summary of results: FACS analysis of bone marrow and buffy coat from CK5-GFP transgenic mice and wild type mice revealed that these circulating CK5+ progenitor epithelial cells also express Sca-1,CD34 and c-kit. In the heterotopic tracheal transplant model, wild type tracheal transplantation in CK5-GFP recipient mice demonstrated that the recipient circulating CK5-GFP cells contributed to repair of the airway epithelium. Passive immunization of animals bearing tracheal transplants with specific neutralizing anti-CXCL12 F(Ab)2 antibodies resulted in the unexpected phenotype of the airway epithelium demonstrating squamous metaplasia at day 21 post-transplant.. In contrast, animals bearing tracheal transplants passively treated with control antibodies demonstrated normal pseudostratified epithelium at day 21 post-transplantation. Immunofluorescence for p63 in the tracheal transplants demonstrated that all of the cells of the squamous metaplasia were p63 positive in tracheal transplants from animals that had CXCL12 depleted. Analysis of these tracheal transplants with immunohistochemistry and immunofluorescence for GFP and CK5 demonstrated that the squamous metaplasia was solely derived from resident progenitor epithelial cells.

Conclusions: There is a population of CK5+ cells in the bone marrow and circulation of naive mice that also express Sca-1, CD34 and c-kit, which is compatible with a stem cell. These circulating CK5 positive cells contribute to regeneration of the airway epithelium and their recruitment to the progenitor cell niche in the airway appears to be critical for the development of normal pseudostratified columnar epithelium.
Tissue Factor-Exposing Microparticles at the Site of Hemostasis Activation.
Authors: Gartner Verena , Lubscyk Barbara , Kollars Marietta , Kyrle Paul A. , Weltermann Ansgar
Published: April 2020

Introduction: Tissue factor exposing microparticles (TF+ MPs) are capable to activate hemostasis in vitro. The aim of this study was to investigate the impact of TF+ MPs during physiologic activation of hemostasis in vivo in human.

Methods: In a double-blind, cross-over study healthy male volunteers (n=13) were randomized to 7 days treatment with 100 mg aspirin and placebo, respectively. At the end of each treatment period TF+ MPs were determined in venous blood and in blood emerging from a skin incision reflecting hemostasis in an activated state (shed blood). TF+ MPs were analyzed by flow cytometry after staining with annexin V, a TF-antibody and cell-specific antibodies.

Results: Compared to venous blood, the median (interquartile range) number of TF+ MPs was significantly higher in shed blood: 16.2 (11.3 – 20.1) k/ml vs. 8.2 (5.9 – 14.8) k/ml (p = 0.004). TF+ MPs from platelets were higher in shed blood than in venous blood [4.7 (3.7 – 8.6) k/ml vs. 3.3 (2.0 – 5.0) k/ml, p = 0.03], whereas TF+ MPs from endothelial cells were not [1.7 (1.2 – 5.8) k/ml vs. 2.7 (1.7 – 4.5) k/ml, p = 0.9]. Aspirin did not significantly influence the level of TF-MPs both in venous blood and in shed blood.

Conclusion: High levels of TF+ MPs can be detected at the site of physiologic activation of hemostasis in vivo in human.
An Anti-Phosphatidylserine (PS) Antibody, Tarvacin™ That Targets Tumor Blood Vessels Does Not Affect Platelet Function In Vitro.
Authors: Dyszkiewicz-Korpanty Anna M. , Sarode Ravindra , Thorpe Philip E. , Frenkel Eugene P.
Published: April 2020

Tarvacin™ is a chimeric anti-PS antibody that is currently in Phase I clinical trials in cancer patients. It acts by targeting PS that becomes exposed on vascular endothelium in tumors in response to oxidative stress in the tumor microenvironment. Tarvacin™ recognizes a complex of PS and the PS-binding protein, β2 glycoprotein I. Host leukocytes are induced to bind to the complex in tumor vessels and destroy tumor vessels by antibody-dependent cellular cytotoxity. However, antibodies directed against PS-associated proteins are also known to elicit anti-phospholipid syndromes (APS). Anti-PS antibodies possibly cause APS by displacing anticoagulant proteins from PS on activated cell or by enhancing the binding of prothrombin; another explanation might be a direct activation of endothelial cells and platelets. The aim of the study was to determine whether Tarvacin ™ induces or interferes with platelet activation caused by ADP, collagen type I or calcimycin in vitro. Blood was drawn from 3 healthy volunteers, aged 31–54, who have not taken any antiplatelet medication for 14 days prior to the study. Dual channel whole blood aggregometer (Chronolog, Havertown, PA, USA) was employed for platelet aggregation studies in whole blood (WB/impedance method) and platelet rich plasma (PRP/optical method). Platelet count in PRP was adjusted to 200 K/μL. Platelet agonists (PS exposure triggers) used in the experiments were as follows: collagen (0.5, 1, 2 μg/mL), ADP (1.25, 2.5, 5, 10 μM), Calcimycin (10, 20, 30 μM) and Calcium ions (1, 2 mmol/L). Tarvacin™ was provided by Peregrine Pharmaceuticals Inc, Tustin, CA. The Anti-CD 20 antibody, Rituxan ™ and physiologic saline were used as controls. Specimens (WB diluted with saline in 1:1 ratio or PRP) with the addition of Tarvacin™ (100 μg/mL) or Rituxan ™ (100 μg/mL) or saline were first incubated on a gentle mixer for 10 minutes; incubation was then continued at 37 ° in the aggregometer well for another 5 minutes. Agonist-induced platelet aggregation was subsequently examined. Platelet aggregation studies in both WB and PRP showed that Tarvacin™ neither induced platelet activation, nor inhibited platelet activation in response to ADP, collagen or calcimycin in vitro. In conclusion, Tarvacin™ does not affect platelet function in the present in vitro assays. Possibly, the epitope on the PS -β2 glycoprotein I complex does not orientate the antibody in a manner that interferes with platelet activation. Alternatively, activated endothelial cells or other factors may be critical to support platelet activation.
2-Oxoglutarate Down-Regulates Expression of Vascular Endothelial Growth Factor and Erythropoietin through Decreasing Hypoxia Inducible Factor-1 α and Inhibits Angiogenesis.
Authors: Imagawa Shigehiko , Matsumoto Ken , Obara Naoshi , Suzuki Norio , Nagasawa Toshiro , Yamamoto Masayuki
Published: April 2020

In oxygenated cells, hypoxia inducible factor-1 (HIF-1)α subunits are rapidly degraded by a mechanism that involves ubiquitination by the von Hippel-Lindau tumor suppressor (pVHL) E3 ligase complex using 2-oxoglutarate as a substrate. This process is suppressed by hypoxia and iron chelation, allowing transcriptional activation. The interaction between human pVHL and a specific domain of the HIF-1α subunit is regulated through hydroxylation of proline residues 402 and 564 by HIF-1α proryl-hydroxylase (PHD). N-oxalyl glycine acts as a competitive inhibitor of HIF-PHDs and this inhibition is in competition with 2-oxoglutarate. We examined the effect of 2-oxoglutarate on the production of vascular endothelial growth factor (VEGF) and erythropoietin (Epo). The expression of VEGF and Epo protein were dose-dependently downregulated in Hep3B cells by the addition of 2-oxoglutarate. The enhancer activity of the HIF-1 binding site of Epo and the promoter activity of VEGF-luciferase were also dose-dependently downregulated by the addition of 2-oxoglutarate. Gel mobility shift assays revealed that the addition of 2-oxoglutarate dose-dependently inhibited HIF-1 binding activity, but did not affect GATA binding activity. Western blot analysis revealed that 2-oxoglutarate dose-dependently inhibited HIF-1α protein in Hep3B, Hela and SW480 cells in hypoxic conditions. However MG132 (the proteasome inhibitor) rescued the inhibition of HIF-1α protein expression by 2-oxoglutarate. Furthermore, under hypoxic conditions, 2-oxoglutarate dose-dependently inhibited tube formation in in vitro angiogenesis assays. These results indicate that 2-oxoglutarate treatment may be useful for the inhibition of tumor angiogenesis through decreasing HIF-1α protein, HIF-1 binding activity, the promoter activity of VEGF and enhancer activity of Epo, and the production of VEGF and Epo. More studies to determine if 2-oxoglutarate inhibits tumor angiogenesis in vivo mouse assay are in progress.
Production of Tissue Factor Pathway Inhibitor in Endothelial Cell Cultures Is Reduced by Estrogens, Selective Estrogen Receptor Modifiers, and a Selective Estrogen Receptor Downregulator.
Authors: Dahm Anders E.A. , Iversen Nina , Birkenes Baard , Sandset Per Morten
Published: April 2020

Low plasma tissue factor pathway inhibitor (TFPI) is a weak risk factor for venous and arterial thrombotic disease. Female hormones strongly influence the level of TFPI in plasma. Oral contraceptives (OC) and hormone replacement therapy (HRT) reduce plasma levels of TFPI by 30–50%, and premenopausal women have lower TFPI than postmenopausal women and men. Moreover, the selective estrogen receptor modifiers (SERM) Tamoxifen and Raloxifen also reduce TFPI in plasma. The effect of these drugs on TFPI may in part explain their prothrombotic effects. The aim of the current study was to study the effects of estrogens and SERMs on the production of TFPI from endothelial cell cultures. Human coronary arterial endothelial cells from Cambrex Bio Science were seeded into 24 well plates and preincubated with 0 (control) or 10 nM of 17β-estradiol (E2), 17α-etinyl-estradiol (EE2), tamoxifen or raloxifene for 48 hours. The cell-medium was then changed and the cells were stimulated with the same concentrations of the same drug for 24 hours before harvesting. TFPI total antigen was measured using a commercial ELISA-kit, and cellular total protein was measured by an improved Lowry assay. The concentration of TFPI total antigen was normalized against the concentration of total protein from the cells. We found that 10 nM of E2, EE2, Tamoxifen and Raloxifene reduced TFPI in the cell medium with 39% (E2), 30% (EE2), 19% (Tamoxifen), and 35% (Raloxifene) compared to untreated cells. Fulvestrant is a selective estrogen receptor downregulater (SERD) known to have no agonist effects, and is also used as a drug against breast cancer. The effect of fulvestrant on TFPI-production was tested in order to later use it as a blocker of the estrogen receptor (ER), to decide whether the effects observed from the other compounds were ER-dependent. Surprisingly, 10 nM fulvestrant reduced TFPI in the cell medium by 12%, and 1000 nM fulvestrant reduced TFPI by 30%. We conclude that the effects of estrogens and SERMs, and interestingly also the SERD fulvestrant, reduce TFPI production in endothelial cells. Based on these results we propose that women using fulvestrant as a drug against breast cancer will have reduced levels of TFPI, and perhaps also increased risk for thrombotic dieseases.
Preparation and Charaterization of MoAb SZ118 and SZ118-scFv Against Platelet Glycoprotein VI.
Authors: Yu Ziqiang , Dong Ningzheng , Bai Xia , Shen Fei , Dai Lan , Ruan Changgeng
Published: April 2020

GlycoproteinVI (GPVI ), one of the platelet collagen receptors, plays an important role in platelet activation and thrombosis. GPVI participates in platelet adhesion to exposed collagen under high shear rate condition, and leads to platelet activation and thrombus formation. GPVI is widely recognized as a requisite factor for the formation of platelet aggregates on the collagen surface under blood flow. There were growing evidences showed that impairment of GPVI function could result in a long-term antithrombotic protection and less bleeding side-effect, which potentialized that GPVI might be an interesting target for safe anti-adhesion and anti-thrombotic therapy. Recently monoclonal antibodies directed against GPVI were developed, which impaired the interaction of platelets and collagen. A human soluble recombinant GPVI (rGPVI), consisting of extracellular domain of GPVI, was expressed in E. coli. After immunizing Balb/C mice with the purified rGPVI, a monoclonal antibody, SZ118, was developed. Meanwhile the gene encoding for the light-and heavy-chain variable regions of SZ118 had been cloned by RT-PCR from murine hybridoma cells. After ligated into the vector pET20b(+), expression plasmid pET20b(+)-SZ118-scFv was contructed, and SZ118-scFv fragment was expressed in E.coli. The parallel plate perfusion chamber and platelet aggregometer were used to study the effects of SZ118 and SZ118-scFv on the platelet adhesion and aggregation to collagen. The citrated whole blood with or without SZ118 was perfused over the surface of type I fibrillar collagen (Chrono Log, at 80μg/ml) at high shear rate (1000 s−1) for 5 min. The platelet aggregates were visualized by the phase-contrast microscope, the coverage rates of the platelet aggregates were measured. Monoclonal antibody SZ118 and SZ118-scFv could bind to human platelets exclusively by flow cytometry. SZ118 inhibited collagen-induced platelet aggregation in PRP in dose-dependent manner, nearly full inhibition being attained at the concentration of 110 ug•mL−1. Similarly SZ118-scFv inhibited collagen-induced platelet aggregation with maximum inhitition rate 84.3%±5.6%. And also SZ118 and SZ118-scFv had part effect on convulxin-induced platelet aggregation, but having little effect on ADP. After treating the citrated whole blood with SZ118 or SZ118-scFv at final concentration 50μg/ml at 37° for 10 min, the platelet adhesion on the surface of collagen was markedly inhibited with the coverage rates of 1.03%±0.22%, respectively (22.7%±5.7% for control), which indicated that the platelets adhesion to collagen was inhibited by 95.5%by SZ118 and 68.3% by SZ118-scFv. It was concluded that the MoAb SZ118 inhibited platelet aggregation to collagen and platelet adhesion to immobilized fibrillar collagen under high shear rate condition.
Therapy of Chronic ITP - The Patients’ Perspective in Germany.
Authors: Matzdorff Axel C. , Arnold Gabriele , Pralle Hans , Preiss Joachim
Published: April 2020

Introduction: Adult ITP is a chronic disease. Many patients do not achieve remission despite immunosuppressive therapy. After 1st line therapy with steroids and immunoglobulins splenectomy is usually offered as 2nd line therapy, even to thrombocytopenic patients without bleeding symptoms. However, several non-surgical treatment options have been available for many years and recently anti-CD20 antibodies (rituximab) and anti-D (Cooper, Blood 2002) have been added to this list. This study was intended to asses which treatments are usually offered to patients in Germany.

Methods: In June 2004 and February 2005 we sent out a questionnaire that was returned by 80 patients. 7 questionnaires were excluded because of patient age (<12 y) or minor thrombocytopenia (platelets >100.000/μl).

Results: Age (median 50 years) and gender distribution (38% m, 62% f) are as expected for chronic ITP. Median duration of disease is 5 years (range 1–43 years). 27% of the patients have experienced gastrointestinal/urogenital bleeding, 12% had CNS or eye bleeds. 92% of the patients had been treated with steroids and 24% were splenectomized. Only 31% had been informed by their physicians of rituximab and 12% of anti-D. 35% of the patients had exclusively been informed about steroids, immunoglobulins and splenectomy. 45% of patients had the impression that cost of treatment affected their therapy options.

Conclusions: ITP is a chronic condition and patient histories extend over many years. Standard therapies comprise steroids, i.v. immunoglobulins, and splenectomy. However, splenectomy has a considerable long-term failure rate (33%, Kojouri Blood 2004). Many patients are thrombocytopenic but have little or no bleeding symptoms. Particularly those patients are reluctant to undergo surgery as 2nd line therapy and prefer trying a non-surgical approach first. Anti-D and rituximab are not covered by public health insurance. It is possible that insurance reimbursement policies restrict treatments offered to ITP patients in Germany.
Treatment of Thrombotic Thrombocytopenic Purpura Using Solvent Detergent Treated Plasma.
Authors: Rock Gail , Anderson David , Benny Barrett , Clark William , Leblond Pierre , Sutton David , Sternbach Marion , Wells George ,   Members of the Canadian Apheresis Group,   Canadian Association of Apheresis Nurses
Published: April 2020

Background:

Patients with Thrombotic Thrombocytopenic Purpura require immediate therapy with large volumes of plasma. They have been reported to respond well to plasma exchange (PE) with FFP (78%)1 and CSP (95%)2. We initiated a randomized prospective trial with CSP and solvent detergent treated plasma (SDP) intended to show non inferiority.

Methods:

Patients were randomized to receive either of the exchange fluids at 1.5 PV x 3 days then 1 PV x 4–6 days. Laboratory values were followed with ADAMTS-13 levels measured according to Furlan3. The primary end point was survival at 6 months.

Results:

A total of 62 of the required 280 patients entered the study then the supply of SDP was discontinued. 3/35 patients receiving CSP and 1/27 on SDP died before 1 month. These values are not significant but the study is underpowered so statistical analysis is not possible. FVIII and vWF levels were elevated in all patients at entry. 34/50 patients tested also had antibodies to CD36. 9 patients had no ADAMTS-3 at entry, 19 had between 5–50%, 9 patients had 50–100% and 15/62 patients tested had normal levels. Inhibitors were present at between 5–50% levels in 22/50 patients tested whereas 9 patients had complete inhibition of ADAMTS-13 and 18 had normal enzyme activity.

Conclusions:

Patients with TTP appear to respond well to SDP thereby offering a relatively safer therapeutic option for PE. An insufficient number of patients were entered to achieve statistical significance. Both the ADAMTS-13 and the inhibitor levels show great variation and are not prognostic.
Earlier Administration of Rituximab Allows Higher Rate of Long-Lasting Response in Adult Patients with Autoimmune Thrombocytopenia.
Authors: Zaja Francesco F. , Vianelli Nicola N. , Battista Marta M. , Sperotto Alessandra A. , PAtriarca Francesca F. , Tomadini Valentina V. , Fili Carla C. , Tani M. , Baccarani Michele M. , Fanin Renato R.
Published: April 2020

Background

Previous reports highlighted the potential short and mid-term therapeutic activity and safety of rituximab in adult patients with autoimmune thrombocytopenias.

Objectives

Primary objectives of this study were to evaluate the long-term efficacy and toxicity profile. Patients and methods From October 1999 to April 2005, 37 adults patients, median age 54 years, with active and symptomatic autoimmune thrombocytopenias (30 idiopathic thrombocytopenic purpura, 1 idiopathic thrombocytopenia and neutropenia, 4 thrombocytopenia and concomitant undifferentiated connective tissue disease, 2 thrombocytopenia and concomitant B-cell lymphoprolipherative disorders) that had relapsed or were refractory to at least a full course of steroid therapy received weekly infusions of rituximab 375 mg/m2 for 4 weeks. The median interval from diagnosis to rituximab was 34 months (1–264 months) and the platelets median count was 11 x 109/L. The following parameters of efficacy and toxicity were considered: rate of complete and partial response, time to initial and maximum response, relapse rate, relapse free survival, treatment free survival, short and long-term toxicity. The possible prognostic influence of some clinical and laboratory parameters on the patients outcome were also analyzed.

Results

Complete and partial response (platelets count ≥ 100 x 109/L and ≥ 50 x 109/L) were 20/37 (54%) and 7/37 (19%), respectively. In most of the patients the time to initial and maximum response was prompt, already before the second administration of rituximab. The median period of observation from treatment was 22 months (1–48 months). Nine out 27 responding patients relapsed; 18/37 patients (49%) remained relapse free and 20/37 (54%) did not necessitated further therapy. A shorter interval period from the time of diagnosis and rituximab administration (≤ vs > 36 months) was associated with a lower relapse free survival (p= 0.03). During the period of rituximab administration, 2 patients experienced short term toxicity with one case of serum sickness syndrome; no infectious or other significant long term toxic complications were documented..

Conclusion

Rituximab administration may allow to achieve long-lasting remission in nearly 50% of patients suffering from autoimmune thrombocytopenia with good toxic profile. The possibility to achieved long lasting response appeared related with an earlier timing of administration.