bluuebunny/crossref_metadata_2025 huggingface dataset:
Recombinant Interleukin-11 (rhIL-11) in Women with Refractory Menorrhagia and Von Willebrand Disease.
Authors: Jankowitz Rachel C. , Ragni Margaret V. , Chapman Holly L. , Merricks Elizabeth P. , Kloos Mark T. , Dillow Aaron M. , Franck Helen , Nichols Timothy C.
Published: April 2020

Von Willebrand disease (VWD) is the single most common congenital bleeding disorder, and among women, menorrhagia is the most common bleeding symptom. Although DDAVP is the treatment of choice for those with type 1 VWD, its use may be limited by tachyphylaxis, allergic reactions, or unresponsiveness. We previously showed that recombinant human IL-11 (rhIL-11, Neumega), a gp-130 signaling cytokine with hematopoietic and anti-inflammatory activity, increases VWF levels 2–3 fold when given subcutaneously daily in individuals with mild VWD, and is well tolerated. We, therefore, initiated a Phase II clinical trial to determine the efficacy and safety of rhIL-11 in women with VWD and refractory menorrhagia, despite estrogen, DDAVP, and/or other treatment. We report the results of the first four subjects, age 28–40, with VWF:RCo 0.22–0.37 U/ml at diagnosis, normal VWF multimers, and bleeding severity scores (BSS) 4–6. rhIL-11 was given in a daily subcutaneous dose of 25 mcg/kg for 4 days, followed by monthly home self-injection for up to 7 days during each of six consecutive menstrual cycles, beginning on the first day of each cycle. Menstrual bleeding severity was measured by

a validated bleeding pictorial assessment chart (BPAC) (Janssen, Obstet Gynecol, 1995) and cycle severity rating (CSR), as compared with two pre-treatment baseline menstrual cycles. BPAC scores were compared with standardized cutoff score for menorrhagia of 185.

Cycle severity ratings (CSR) were rated on a nominal scale, with 0=mild bleeding; 1=moderate bleeding, less than usual cycle; 2= moderately severe bleeding, not as severe as worst cycle; and 3=severe bleeding, as severe as worst cycle. The drug was well tolerated with less than grade 1 toxicity, including mild conjunctival erythema and mild fluid retention (fingers) in one each. Following treatment with rhIL-11, there was a significant reduction in BPAC and cycle severity rating during the seven days the drug was given, as compared with the first seven days in cycles prior to rhIL-11 (see Table). Cycle length also shortened, but this did not reach significance. These was an associated increase in VWF:RCo, VWF:Ag, and FVIII:C on day 4 of rhIL-11 dosing, as compared with pre-rhIL-11 levels day 1. These data suggest that rhIL-11 improves hemostasis and reduces bleeding severity when used during the first seven days of menses in women with VWD. Further studies are needed to confirm these findings.

Mensural Cycle Bleeding Severity in Women with VWD With and Without rhIL-11

No. Cycles Bleeding Pictorial Chart (BPAC) No. >185 Cycle Severity Rating (CSR) Cycle Length (Day) VWF:Rco U/ml VWF:Ag U/ml FVIII:C U/ml Significance, *p<0.05; †p<0.01; ‡p=0.014 CYCLES WITHOUT rhIL-11 7 6/7 (85.7%) 2.0±5 12±5 0.92±0.13 1.16 ± 0.06 0.60±0.09 CYCLES WITH rhIL-11 15 4/15 (23.5) ‡ 1.1±0.2† 7±1 1.71±0.17† 1.70±0.17† 1.02 ± 0.12*
Biomarkers of Cartilage and Bone Damage as a Measure of Joint Damage in Haemophilia.
Authors: Jansen Nathalie W.D. , Roosendaal Goris , Lundin Bjorn , Heijnen Lily , Theobald Matthias , Bijlsma Johannes W.J. , Lafeber Floris P.J.G.
Published: April 2020

Purpose Biomarkers of bone and cartilage turnover have frequently been evaluated for joint diseases such as rheumatoid arthritis (RA) and osteoarthritis (OA). Results have thus fare not been very conclusive. Some biomarkers such as urinary CTXII and serum COMP appear to correlate with severity of joint degeneration, whereas other are less distinctive. Hemophilic arthropathy (HA) is a very progressive joint degeneration as a result of frequent joint bleeds. From clinical practice it is concluded that the rate of degeneration exceeds that of OA and RA joints. This degeneration has characteristics of both inflammation mediated (as seen in RA) and degenerative (as seen in OA) joint disease. Furthermore, the joint damage is largely restricted to 3 major joints (ankle, knees, and elbows). Therefore, it might be that this rapidly progressive, localized joint degeneration can be used for the evaluation and validation of biomarkers of cartilage and bone turnover. In the present study we therefore investigated whether commercially available biomarkers of cartilage and bone in blood and/or urine are associated with severity of joint damage in patients with haemophilic arthropathy.

Methods Blood and urine were collected from 36 patients suffering from haemophilia. Urine samples were assessed for the amount of CTX-I and CTX-II. Serum samples were assessed for the amount of CTX-I, CTX-II, COMP, C1,2C, C2C, and CS846. Radiographs of ankles, knees and elbows were scored according to Pettersson, a radiographic joint score specific for haemophilic arthropathy based on cartilage and bone changes.

Results U-CTX-II (R=0.39; p=0.01), C1,2C (R=0.31; p=0.04) and CS846 (R=0.31; p=0.03) showed (marginal) correlations with the Pettersson score. Slightly better correlations were obtained when only narrowing of joint space width (JSW) as one of the items in the Pettersson score was used. The other biomarkers showed no correlation with the Pettersson score. Also the bone biomarkers did not correlate with specific bone changes. Interestingly, combined indexes of different markers, based on linear stepwise regression analysis, increased the correlation significantly up to R=0.65; p≤0.001) for the combination of U-CTX-II, COMP and CS846.

Conclusions The present results show that even despite this rapidly progressive degeneration of 6 large joints, from the individual biomarkers determined only U-CTX-II, C1,2C and CS846 show correlation with the severity of arthropathy. Importantly, a relation improved when the markers were related to the process they are supposed to describe (cartilage degeneration markers with JSW narrowing). Most important, combination of markers, significantly improve the relation with the radiographically determined joint degeneration. In general however, it may be concluded that these markers alone seem not of sufficient value for evaluation of joint damage yet.
Unraveling the Danger Signal Necessary for the Immune Response to Therapeutic Factor VIII.
Authors: Skupsky Jonathan , Zhang Ai-Hong , Scott David W.
Published: April 2020

It is well established that mice which do not produce endogenous factor VIII (fVIII−/−) can manifest a robust immune response to exogenous fVIII treatments. They form B-cell and T-cell responses even when they encounter fVIII through traditionally tolerogenic routes (e.g., intravenous or intraperitoneal). In the fVIII−/− mouse, repeated administration of recombinant human fVIII has emerged as a useful model for studying the physiologic response in hemophilic patients iatrogenically immunized to therapeutic factor VIII treatments. While environmental factors likely offer some co-stimulatory signals, nonetheless, the ability to respond effectively in the absence of extrinsic adjuvant begs the questions of what is the “danger signal” required for immune responsiveness to fVIII? We have previously shown that when factor VIII is heat inactivated (56°, 30′), it completely losses function and much of its immunogenicity (Skupsky and Scott, Blood110: 2685 Abstract, 2007). Heated fVIII lacks several of its B-cell epitopes (we did not find a subsequent response to neo-epitopes), but retains its T-cell epitopes. We concluded that fVIII’s immunogenicity is inherently tied to its function. To explore this topic further, we have immunized hemophilic mice with rfVIII and compared the response to mice treated with both rfVIII and Hirudin. Hirudin is the reactive agent found in medicinal leech saliva and its anti-coagulant activity is based on its ability to inhibit thrombin. We found that T cell responses to rfVIII in mice protected with Hirudin are significantly reduced (p<0.05) and the anti-fVIII antibody concentration has decreased by 25%. As a control, we injected a third group of mice i.v. with an equivalent amount of another foreign protein, ovalbumin (OVA) in PBS. As expected, the mice did not respond to this historically tolerogenic treatment. Interestingly, when mice were injected simultaneously with rfVIII and OVA, they did form a humoral response to both the fVIII (200 μg/ml) and the OVA (30μg/ml). This suggests that fVIII may have adjuvant properties remaining to be discovered. Overall, these data suggest that the activation of thrombin provides co-stimulatory signals necessary for the immune response. Activated thrombin does this directly or indirectly through the activation of other blood components, including platelets.
Screening Tool for Bleeding Disorders in Women with Menorrhagia: Evaluation in a Prospective US Multi-Site Cohort.
Authors: Philipp Claire S. , Faiz Ambarina , Byams Vanessa , Miller Connie H. , Heit John A. , Kouides Peter A. , Kulkarni Roshni , Lukes Andrea , Stein Sidney F.
Published: April 2020

Menorrhagia is a common clinical problem among reproductive age women and annually 5% of reproductive age women seek medical attention, usually with gynecologists and other primary care physicians, for this symptom. Since underlying bleeding disorders are common in women presenting with menorrhagia, and referral for comprehensive hemostatic testing of substantial numbers of women with otherwise unexplained menorrhagia is problematic from the public health and cost perspective, a short, easy-to-administer screening tool comprised of 8 questions for identifying women with menorrhagia for hemostatic evaluation was previously developed (Am J Ob Gyn2008;198:e1–163e8). In the present study, the validity of the screening tool was evaluated in a multi-site, prospectively recruited cohort of women with menorrhagia. 232 women with menorrhagia age 18 and older with a pictorial blood assessment chart (PBAC) score > 100 were recruited from 5 US centers as potential subjects for a prospective cross-over study for evaluation of intranasal DDAVP versus tranexamic acid. All subjects underwent comprehensive laboratory testing for bleeding disorders, including VWF, platelet aggregation/ATP release, and factor assays. Study participants were administered a questionnaire which included the 8 screening tool questions in 4 categories, including history of duration and severity of menorrhagia, anemia treatment, excessive bleeding with hemostatic challenges, and family diagnosis of bleeding disorder. A screening tool was considered positive if there was a positive response for any of the questions in the four categories. Sensitivity of the screening tool with 95% confidence interval was calculated for bleeding disorders and also separately for low VWF (ristocetin cofactor < 50%), and platelet function defects.217 women with menorrhagia including 78% white and 16% black women with complete data were evaluated. In this population, a positive screening tool had a sensitivity of 89% (95% CI, 83–93) for bleeding disorders, 89% for platelet function defects (95% CI, 82–94), and 73% for low VWF (95% CI, 39–94). The sensitivity for bleeding disorders was 87% (95% CI, 79–92) among white women and 94% (95% CI, 79–99) among black women. Adding a PBAC score > 185 increased the sensitivity of the screening tool for bleeding disorders to 95% (95% CI, 90–98). Using a multi-site US population of adult women with menorrhagia, this study confirms the benefit of a short screening tool to assist primary care physicians in the selection of women with menorrhagia to refer for comprehensive hemostatic testing and evaluation. population of adult women with menorrhagia, this study confirms the benefit of a short screening tool to assist primary care physicians in the selection of women with menorrhagia to refer for comprehensive hemostatic testing and evaluation.
CD300f Triggering Modifies Myeloid Cell Function
Authors: Hart Derek , Modra Courtney , Clark Georgina
Published: April 2020

CD300f, a member of the CD300 family of immunoregulatory molecules, is capable of signalling through association with both SHP-1 phosphatase and the p85α subunit of phosphoinositide 3-kinase. On normal cells, CD300f is expressed on monocytes and dendritic cells in the blood and bone marrow. CD300f is expressed on myeloid derived cell lines and Acute Myeloid Leukaemias (AMLs) and is an acknowledged candidate for antibody targeting of AML (Modra CJ et al. Blood2006;108:225B-225B and Zhao X et al Blood2007;110:531a–532a). We have generated a monoclonal antibody, MMRI- 23, specific for the extracellular domain of CD300f. MMRI-23 immunoprecipitates a 57kd protein from the myeloid derived cell lines HEL, THP-1 and U937 and this protein binds to a polyclonal antibody to CD300f (LMIR3) in Western blot analysis. Binding of MMRI-23 to myeloid cells was blocked by the CD300f recombinant proteins or the polyclonal CD300f antibody. The MMRI-23 mAb was used as a surrogate ligand to study the functional consequences of crosslinking CD300f on normal monocytes and myeloid derived cell lines. Purified peripheral blood monocytes were cultured for 18 hours in the presence of immobilised MMRI-23 or control mAb. MMRI-23 binding was not altered by activation of peripheral blood monocytes but crosslinking monocytes with MMRI- 23 induced downregulation of CD14 and CD33. There was significant inhibition of IL-6 but not IL-1β, IL-8 or TNFα secretion. Crosslinking monocytes or the U937 cell line with MMRI-23 increased specific chemotaxis towards CXCL12 (SDF-1). This increased migration index following MMRI-23 crosslinking was reversed in the presence of wortmannin indicating that MMRI-23 induces signalling through phosphoinositide 3-kinase. The effect of crosslinking did not enhance CXCR4 upregulation but did induce localization of CD300f and CXCR4 to the lipid rafts in myeloid cell line, U937. Thus CD300f plays a significant role in the regulation of monocyte migration to CXCR4. As CD300f is upregulated on around 70% of AMLs, this regulation of homing has major implications for the treatment of AML.
Ficolin-1 Is Present in a Highly Mobilizable Subset of Human Neutrophil Granules and Associates with the Cell Surface after Stimulation with fMLP.
Authors: Roervig Sara , Honore Christian , Larsson Lars-Inge , Ohlsson Sophie , Pedersen Corinna cavan , Jacobsen Lars Christian , Cowland Jack B. , Garred Peter , Borregaard Niels
Published: April 2020

Ficolins are soluble pattern recognition molecules that bind carbohydrate structures on the surface of microorganisms. Three ficolins have been identified in man: ficolin-1, ficolin-2, and ficolin-3. Ficolin-1 is derived from the FCN1 gene on chromosome 9 and is synthesized in monocytes and type II alveolar epithelial cells. Ficolin-1 has been shown to be present in secretory granules of human neutrophils, but which subset of the neutrophils secretory granules harbors ficolin-1 is not known. In order to determine the exact subcellular localization of ficolin-1 in neutrophils, recombinant ficolin-1 was expressed in Chinese hamster ovary cells and used for generation of polyclonal antibodies. This allowed detection of ficolin-1 in subcellular fractions of human neutrophils by ELISA and western blotting, and by immunohistochemistry. Real time PCR examination of normal human bone marrow showed FCN1 gene expression largely in myelocytes, metamyelocytes, and band cells with a profile quite similar to that of gelatinase. In accordance with this, immunohistochemistry and subcellular fractionation demonstrated that ficolin-1 is primarily localized in gelatinase granules, but also in highly exocytosable gelatinase poor granules, not previously described.

Ficolin-1 is released from neutrophil granules by stimulation with fMLP or PMA, and a significant part becomes associated with the surface membrane of the cells and can be detected by flow cytometry. Our studies show that neutrophils are a major source of ficolin-1, which can be readily released to the surroundings by stimulation.

Figure. Left: Distribution profile of ficolin-1 in unstimulated (control) neutrophils and neutrophils stimulated with fMLP or PMA. Right: Distribution profile of ficolin-1 and granule marker proteins in subcellular fractions of unstimulated human neutrophils. Figure. Left: Distribution profile of ficolin-1 in unstimulated (control) neutrophils and neutrophils stimulated with fMLP or PMA. Right: Distribution profile of ficolin-1 and granule marker proteins in subcellular fractions of unstimulated human neutrophils.
The Role of Endothelial PECAM-1 Polymorphism (S536N) as Heterophilic Counter Receptor for Neutrophil Specific CD177 in Transendothelial Cell Migration.
Authors: Bayat Behnaz , Andrei-Selmer Cornelia , Werth Silke , Newman Peter , Santoso Sentot
Published: April 2020

The human neutrophil-specific adhesion antigen CD177 (NB1, HNA-2a or PRV-1) becomes upregulated on the cell surface in a number of inflammatory settings, and human alloantibodies specific for CD177 have been implicated in the development of transfusion related acute lung injury (TRALI). Recent studies have shown that CD177 functions as an important counter-receptor for the endothelial cell-cell junctional protein PECAM-1 (CD31) – a heterophilic adhesive interaction that appears to be mediated by Ig domain 6 of PECAM-1 and a still-to-be-identified site on CD177. Interestingly, a common single nucleotide polymorphism (SNP) exists within the PECAM-1 gene that produces an S536N amino acid substitution within Ig domain 6 that is proximal to the CD177 binding site. Whether this polymorphism influences PECAM-1/CD177 interactions and subsequent neutrophil transendothelial cell migration, however, is not known. To investigate the potential for the S536N PECAM-1 SNP to affect functionally important interactions between PECAM-1 and CD177, recombinant CD177 and PECAM-1 S536 or N536 allelic isoforms were produced in CHO mammalian cells, purified by affinity chromatography, and subjected to surface plasmon resonance analysis. We found that the S536 isoform of PECAM-1 bound with much higher affinity to CD177 than did its N536 counterpart (Ka for PECAM-1S536/CD177 = 5×10−7 versus a Ka for PECAM-1N536/CD177=1×10−7). To analyze the possible functional impact of the S536N polymorphism on neutrophil transendothelial migration, fMLP-stimulated neutrophils were allowed to transmigrate across human umbilical vein endothelial cell (HUVEC) monolayers that had been genotyped to be either PECAM-1 homozygous SS, heterozygous NS, or homozygous NN. We found that neutrophils migrated significantly faster across HUVECs expressing the PECAM-1S536, rather than the PECAM-1N536, allelic isoform. In addition, western blot analysis using a phospho-specific antibody revealed that cytoplasmic PECAM-1 ITIM tyrosines became phosphorylated to a greater extent in PECAM-1S536- versus PECAM-1N536-expressing HUVECs that had been incubated with either soluble recombinant CD177 or an anti-PECAM-1 mAb specific for Ig domain 6. Taken together, these data demonstrate that

heterophilic interactions between neutrophil CD177 and endothelial cell PECAM-1 play an important role in neutrophil transendothelial migration, and the naturally-occurring S536N polymorphism within Ig domain 6 likely affects ligand binding-induced downstream PECAM-1 signaling.

Allele-specific, PECAM-1-mediated effects on neutrophil transendothelial migration and inflammatory events such as TRALI may represent an important area for future investigation.
Supplemental Use of Inferior Vena Cava Filters: A Review of An Institutional Experience.
Authors: Singh Parminder , Lerner Robert G. , Chugh Tarun , Lai Hoang , Aronov Wilbert S
Published: April 2020

Introduction: Increasing use of inferior vena cava (IVC) filters in recent years as a preventative measure against pulmonary embolism (PE) has raised concern for usage outside of accepted guidelines. Based on the American College of Chest Physicians 2004 guidelines for the initial treatment of deep vein thrombosis (DVT) and PE, and the Eastern Association for the Surgery of Trauma 2002 guidelines for prophylaxis of PE, placement of an IVC filter is indicated in patients who either have, or are at high risk for thromboembolism, but have a contraindication for anticoagulation, a complication of anticoagulant treatment, or recurrent thromboembolism despite adequate anticoagulation. The purpose of our study is to identify patients who meet the guidelines for IVC filter placement and to compare clinical outcomes with those who did not meet the guidelines.

Methods: Charts of 558 patients who received IVC filter placement were reviewed from Jan 1, 2004 to Dec 31, 2007. Patients were divided into two groups called within-guidelines or supplemental. The within-guidelines group included patients that met the criteria described above. The supplemental indication group included patients who did not have a contraindication or failure of anticoagulation. Patient characteristics and clinical outcomes between the two groups were compared and analyzed.

Results: The within-guidelines group had 362 patients and the supplemental group had 196 patients. While there were more males in the within-guidelines group, age, race, length of stay, and in-hospital mortality were comparable between the two groups. Clinical follow-up in patients with a supplemental indication showed 1 (0.5%) case of post-filter PE, 2 (1%) cases of IVC thrombosis, 7 (3.6%) cases of DVT. Patients who were in the within-guidelines indication group had 4 (1.1%) cases of post-filter PE, 13 (3.6%) cases of IVC thrombosis, and 34 (9.4%) cases of DVT. All patients who developed post-filter PE had a prior DVT at the time of filter placement, and the risk of developing post-filter IVC thrombosis and PE is higher in patients with prior thromboembolic disease. Conversely, patients who did not have a VTE event before filter placement were at a significantly lower risk of developing IVC thrombosis and PE.

Conclusion: Anticoagulation should be initiated at the earliest possible time in patients treated with an IVC filter to prevent subsequent venous thromboembolic disease. Our data does not support the use of IVC filter in patients who can tolerate anticoagulation and have no prior venous thromboembolic event due to the low risk of developing pulmonary embolism
Comprehensive Geriatric Assessment (CGA) in Elderly Patients with Hematological Neoplasia: CGA and Not Age Predicts Mortality - Results from in-GHO®-Registry
Authors: Wedding Ulrich , Honecker Friedemann , Rettig Klaus , Huschens Susanne , Bokemeyer Carsten
Published: April 2020

Background: The internet based registry of IN-GHOα prospectively collects data of elderly cancer patients (pts) 370 yrs. We report data from 450 pts with hematological neoplasia (HE) out of 1580 cancer patients registered currently. To gain more insight into feasibility of treatment in elderly patients, results of a comprehensive geriatric assessment (CGA), physicians’ and pts’ judgement of fitness, and outcome measures were studied.

Methods: Besides demographic data, activities of daily living (ADL), instrumental activities of daily living (IADL), Karnofsky-Performance-Statuts (KPS), co-morbidity (Charlson score), medication (number of drugs not related the HE treatment), mobility (Timed-Up&Go-Test), cognition (MMSE), and depression (SCID screening) results were collected. Age, results of CGA, physicians’ rating (fit vs. compromised vs. frail) and pts’ self rated fitness (lickert scale for 1= very fit to 6 very unfit) for treatment were analysed for their association with death. Follow up assessment was scheduled 8–12 weeks and 5–7 months after inclusion.

Results: 450 patients, mean age 77.7 years (SD 5.7; range 69–97), 43.3%male, were included. Main diagnoses were aggressive lymphoma n = 135 (30%), chronic lymphocytic leukemia n = 66 (14.7), multiple myeloma n = 54, acute myeloid leukemia n = 27 (6.0%), and others. Physicians rating of patientś fitness for treatment: fit 54.4%, compromised 39.1%, frail 5.5%. Patients rating of patientś fitness for treatment: 1 = 19.6%, 2 = 31.8%, 3 = 25.1%, 4 = 13.6%, 5 = 3.8%, 6 = 1.1%. During follow up (median 178 days), 98 pts (21.8%) patients died: mean age (77.5 vs. 78.5; p=0.219), mean BMI (25.6 vs. 25.3; p=0.228), mean Charlson-score (2.0 vs. 2.3; p=0.28) and mean number of drugs (4.0 vs. 4.2; p=0.14) were not associated with death. However significantly associated with death during follow up were mean ADL score (94.0 vs. 87.4; p=0.047), mean IADL score (7.1 vs. 6.3; p<0.001), mean KPS score (83 vs. 74; p=0.001), mean MMSE score (26 vs. 24; p=0.03), categorical Timed-Up&Go-Test (< 10 sec 18% vs. > 20 sec 38%; p<0.001), categorical SCID screening (no depression 18% vs. depression 31%; p<0.001), physicianś rating of fitness (fit 18.8%, compromised 21.6%, frail 52.0%; p=0.001) and patientś rating of fitness (lickert scale 2.3 vs. 3.4; p<0.001).

Conclusion: Patients characteristics as assessed by CGA and not age are associated with death in elderly patients with hematological malignancies. Results of CGA should be validated in treatment algorithm for this population.
Adipocytes Secrete Factors That Cause Leukemia Drug Resistance.
Authors: Behan James W , Yun Jason P , Proektor Marina P , Ehsanipour Ehsan A , Butturini Anna , Heisterkamp Nora , Mittelman Steven D
Published: April 2020

We have previously shown that obesity is an independent predictor of leukemia relapse in children. We have also shown that obese mice transplanted with syngeneic leukemia cells have poorer survival after chemotherapy, even when they are dosed proportional to body weight. Since interactions between leukemia cells and cells of the bone marrow niche are considered important for chemotherapy resistance and relapse, and adipocytes can comprise ~50% of the bone marrow niche, we developed in vivo and in vitro models to investigate the role of adipocytes in the leukemia microenvironment.

Obese C57Bl/6J mice were transplanted with GFP+ murine preB cell ALL (“8093”) cells and then treated with vincristine (0.5 mg/kg/week × 3 weeks). At the time of relapse, we found that GFP+ leukemia cells persisted in the fat pads of the mice. We then developed an in vitro co-culture system in which human or murine leukemia cells were cultured together with adipocytes (differentiated 3T3-L1s). Undifferentiated 3T3-L1 cells, which are fibroblastic in nature, were used as a control. In this model, adipocytes severely diminished the anti-leukemic effect of all chemotherapeutics tested against murine 8093 cells, including vincristine, dexamethasone, nilotinib, daunorubicin, and L-asparaginase. Adipocytes also protected murine T-cell ALL and human SD-1, RCH-ACV, and BV173 cells from vincristine and daunorubicin. Adipocyte protection of leukemia cells occurred independent of cell contact.

Further experiments demonstrated that media conditioned by adipocytes was able to protect 8093 cells from a 3-day exposure to 25 nM dexamethasone (viable cells were at 40±12% of their plated value in regular media, 66±17% in fibroblast-conditioned media, and 109±24% in adipocyte-conditioned media, p<0.05). Surprisingly, adipocyte-conditioned media did not protect leukemia cells from daunorubicin. However, media conditioned by the presence of both adipocytes and leukemia cells simultaneously conferred a high degree of resistance to the leukemia cells (n=3, p<0.05 compared to all other media types).

In summary, adipose tissue is a reservoir for relapsed leukemia cells in vivo. Adipocytes engender protection from multiple chemotherapies in murine and human leukemia cell lines. Adipocytes secrete factor(s) that confer dexamethasone and daunorubicin resistance to leukemia cells, though for the latter drug it appears that a two-way communication between leukemia and adipocytes may be necessary for this protection.

Figure Figure
TLR4-Agonist Stimulated Human Multipotent Mesenchymal Stromal Cells Enhance Both Hematopoietic Progenitors and Myeloid Differentiation
Authors: Ziegler Patrick , Boettcher Steffen , Garavaglia Guido , Manz Markus G.
Published: April 2020

Human mesenchymal stromal cells (MSCs) can replicate as undifferentiated cells in vitro and are capable of differentiating into multiple mesenchymal tissues. In the bone marrow, MSCs are thought to contribute to nurturing stroma involved in maintaining and modulating hematopoiesis. Toll-like receptors (TLRs) function as receptors for different conserved pathogen associated products as well as certain host derived molecules. TLRs are expressed in several hematopoietic and non-hematopoietic cells, and their activation plays a key role in innate and adaptive immune responses to infectious agents, as well as in the development of pathologic conditions like autoimmune disease. In inflammatory conditions like sepsis, cytokines like G-CSF are elevated in serum, CD34+ cells increase in circulation, and bone marrow myelopoiesis is enhanced. We hypothesized that MSCs express TLRs and are capable to respond to TLR agonists by changing their cytokine expression pattern in order to more efficiently support hematopoiesis according to respective needs. In vitro cultured bone marrow MSCs were analyzed for mRNA expression of TLRs. TLR 1, 3, 4, 5, 6, and 9 expression, but not TLR 2, 7, 8, and 10 expression was detectable. Compared to human conventional and plasmacytoid blood dendritic cells, MSCs expressed TLR-3 and TLR-4 at levels up to 2 log higher than did conventional DCs, while TLR 9 expression was low. Upon in vitro stimulation with TLR-4 agonists, mRNA transcripts of flt3-ligand and thrombopoietin increased, and MSCs secreted high amounts of G-CSF, GM-CSF, and M-CSF, whereas stimulation with TLR-9 agonists did not lead to changes in cytokine levels measured in supernatants. TLR-4 stimulated MSC conditioned media enhanced myeloid colony formation from human CD34+ cells about 2.5× fold compared to non-stimulated MSC conditioned media. Furthermore, in co-culture assays of MSCs with CD34+ cells, TLR-4 stimulated MSCs were capable to retain 8 fold more CD34+ cells in divisions 0–3 and maintained overall 2 fold higher numbers of CD34+ cells. In secondary colony formation assays, CD34+ cells from TLR-4 stimulated MSC co-cultures produced about 2.5 fold more myeloid colonies, with some being CFU-GEMM. Moreover, hematopoietic cells taken from TLR-4 stimulated MSC co-cultures were able to engraft conditioned newborn Rag 2−/− gc−/− mice and differentiated into B, T, and myeloid cells, while CD34+ cells from co-cultures of non-stimulated MSCs did not engraft.

These findings thus suggest a regulatory mechanism how, e.g. in gram-negative infection, inflammatory signals are translated via stromal cells into sustained early hematopoiesis and myeloid differentiation to efficiently meet the requests of an ongoing innate immune response.
Histone Deacetylases Are Essential for Chromatin Condensation and Enucleation in Mammalian Erythroblasts
Authors: Ji Peng , Sánchez-Rivera Francisco J , Lodish Harvey F.
Published: April 2020

In the last steps of differentiation mammalian erythroid cells undergo chromatin condensation and enucleation; the latter process does not occur in other vertebrates. Enucleation was critical for the evolution of mammals, as it permits an enhanced hemoglobin concentration – and thus oxygen- carrying capacity – in mammalian red blood cells. We previously reported that Rac GTPases and their downstream forming target mDia2 are required for mouse fetal erythroblast enucleation. We also found that the nucleus undergoes a gradual ~10- fold decrease in volume during erythropoiesis. Since histone deacetylases (HDACs) play important roles in chromatin condensation, we hypothesized that HDACs are involved in mammalian erythroblast enucleation. To test this hypothesis, we purified E13.5 mouse fetal TER119 negative erythroblasts and cultured them in fibronectin-coated plates. Cells were then treated with Trichostatin A (TSA), a pan-HDAC inhibitor, at different times during erythropoiesis. TSA completely blocked enucleation and kinetic studies showed this inhibitory effect occurred earlier than the step catalyzed by the Rac-mDia2 pathway. This indicates that chromatin condensation is required for the final extrusion of the nucleus. We further investigated the activity ofHDAC6 specifically, since HDAC6 is known to interact with mDia2. We found that mDia2is acetylated in vitro and in vivo and that HDAC6 interacts with and deacetylates mDia2. Treatment of purified TER119 negative mouse fetal erythroblasts with a specific HDAC6inhibitor partially blocked enucleation. We conclude that histone deacetylase activities are essential for mammalian erythroblasts to undergo enucleation and that HDACs act both on chromatin condensation and mDia2 deacetylation to promote enucleation.
Dominant Role for SDF-1 in the Vasculo-Angiogenesis Phenotype in Children with Sickle Cell Disease.
Authors: Gee Beatrice E , Manlove-Simmons Jerry M , Huang Yao , Wilson Nana , Stiles Jonathan , Ofori-Acquah Solomon Fiifi
Published: April 2020

BACKGROUND: Vasculogenesis is critically important to restoring and maintaining vascular homeostasis in patients who have sickle cell disease. Emerging evidence indicates vasculo-angiogenesis is a complex process involving recruitment of endothelial progenitor cells (EPCs) from the bone marrow and homing of EPCs to sites of vascular injury. Both recruitment and homing of EPCs are intimately regulated by cytokines and growth factors that are released in response to vascular insult. Hitherto, the phenotype of growth factors and cytokines intimately involved in EPC recruitment and homing in patients who have sickle cell disease remains poorly understood.

METHODS: A total of 50 children aged 6–18 years were studied, 30 with Hemoglobin SS, including 11 on chronic red blood cell transfusions, and 20 with normal hemoglobin. We measured plasma concentrations of angiogenesis growth factors/cytokines using a multiplex assay, which permitted analysis of nine factors concurrently, and determined the number and phenotype of EPC colonies derived from peripheral blood samples.

RESULTS: Plasma concentrations of stromal derived growth factor (SDF-1, 1.7-fold), angiopoietin-2 (Ang-2, 2-fold) and erythropoietin (7-fold) were significantly elevated (p<0.001) in children with sickle cell disease compared to control subjects. On the contrary, plasma leptin was significantly lower (2-fold, p<0.001) in the sickle cell disease group, while no significant differences were found for several other angiogenic growth factors including VEGF, HGF, IL-8, PGF and PDGF between children with sickle cell disease and the control group. Peripheral blood-derived EPC colonies were significantly higher (almost 3-fold) in patients who have sickle cell disease compared to the control group. Interestingly, EPC colony number was lower in sickle cell patients on chronic transfusion therapy compared to those who were not on chronic transfusion. There were significant correlations between EPC colony number and plasma concentrations of SDF-1 (r=0.65, p<0.001) and Ang-2 (r=0.36, p<0.05), but no correlation between EPC colony number and plasma erythropoietin and leptin.

CONCLUSION: These data demonstrate for the first time enhanced vasculogenic potential in patients with sickle cell disease at steady-state. SDF-1 enhances the homing of EPCs and is highly expressed in ischemic tissues. The association of elevated SDF-1 and Ang-2 levels with EPC colony number is consistent with the hypothesis that sickle cell disease causes ongoing ischemic vascular injury. SDF-1 may be potentially used as a marker for vascular injury and a therapeutic target that can be developed to augment vascular repair in sickle cell disease.
Differential Effects of TRAFs in the Activation of NF-KappB in Hodgkin’s Lymphoma Cells.
Authors: Guo Feng , Chen Zi-Xing , Sun Aining , Zhou Peng , Wang Wenjuan
Published: April 2020

CD30, a member of the TNF receptor (TNFR) superfamily, is a lymphocyte-specific receptor that is originally recognized as a surface molecule overexpressed on Hodgkin’s lymphoma cells. Engagement of CD30 with its ligand CD30L activates the nuclear factor kB (NF-kB), which are mediated by interactions with TNFR-associated factor (TRAFs), and supports proliferation of Hodgkin’s cells. However, the role of individual TRAFs in the CD30 signaling pathway in Hodgkin’s cells has not been fully addressed yet. In this study, we found that except TRAF3, all other TRAFs were expressed consistently in B cell-derived Hodgkin’s cell lines (L428 and KM-H2) either in mRNA or protein level. In L428 but not KM-H2 cells, the classical (p50-RelA) and alternative (p52-RelB) NF-kB activity were constitutively activated measured by Western Blotting and EMSA. To better understand the CD30 signaling properties in Hodgkin’s lymphoma cells, we silenced the expression of TRAFs individually by means of RNAi. Successful downregulation of TRAF1 protein expression led to the apoptosis of L428 cells, which was companied by the reduction of both classical and alternative NF-kB activity. Furthermore, the expression of targeting genes of NF-kB, such as A20, c-Flip, ICAM-1, and Cyclin D1 was also decreased. Using siRNA targeting TRAF2 expression, the classical NF-kB activity was reduced while the alternative NF-kB activity moderately induced especially upon CD30L treatment, which was companied by the induction of p100 processing and RelB nuclear localization. The survival rate was decreased when TRAF2 was knockdown. TRAF5 knockdown manifested similar results as that of TRAF2. These observations were only took place in L428 cells but not KM-H2 cells. In addition, the phosphorylation of the extracellular signal-regulated kinases (ERK) 1 and 2, and subsequent activation of JunB in Hodgkin’s cells were staying unchanged in individual TRAFs knockdown experiment. Taken together, the study here further investigates the features of CD30-TRAFs-NF-kB signaling pathway in Hodgkin’s lymphoma cells. At least TRAF1, 2, and 5 are involved in CD30 signaling pathway and protecting Hodgkin’s lymphoma cells from undergoing apoptosis. The interaction between TRAFs is necessary to facilitate the signaling and proper activation of classical NF-kB, which is responsible for the antiapoptotic effect of Hodgkin’s cells. Among TRAF1, 2, and 5, the redundant function is unlikely. Furthermore, the sequential activation of ERK and JunB is unlikely dependent on TRAFs-NF-kB pathway in Hodgkin’s lymphoma cells. Thus, these data reveal that the classical NF-kB activity downstream of CD30 signaling is TRAFs-dependent in Hodgkin’s lymphoma cells, which is regulated by the relative level of individual TRAF in a cell-specific manner.
VEBEP Regimen and Highly Active Antiretroviral Therapy (HAART) in Patients (pts) with HD and HIV Infection (HD-HIV): Final Results of the Italian Cooperative Group on AIDS and Tumors (GICAT) Study.
Authors: Spina Michele , Antinori Andrea , Neri Valentina , Rossi Giuseppe , Re Alessandro , Allione Bernardino , Chimienti Emanuela , Talamini Renato , Tirelli Umberto
Published: April 2020

Background: The outcome of pts with HD-HIV is still poor, because the duration of complete remission (CR) is short. To improve the prognosis of HD-HIV, a feasibility study with the VEBEP regimen and HAART was started in previously untreated HD-HIV pts.

Methods: Chemotherapy (CT) included epirubicin 30 mg/m2/day (days 1–3), cyclophosphamide 1000 mg/m2 (day 1), vinorelbine 25 mg/m2 (day 1), bleomycin 10 mg/m2 (day 3) and prednisone 100 mg/m2/day (days 1–3).

Results: Since September 2001, 71 pts have been enrolled. The median age was 41 yrs. The median CD4+ cell count was 248/mm3 and 51% of pts had a detectable HIV viral load. Stage III–IV was present in 50/71 (70%) pts. Histologic subtypes were: MC 70%, NS 20%, LD 4%, LP 2%, unknown 4%. Four toxic deaths were observed (septic shock, PCP, hepatic failure and pneumonia during neutropenia). An absolute neutrophil count <500 was noted in 60% of pts. Grade 3–4 anemia was observed in 38% of pts and severe thrombocytopenia in 22% of pts. Twenty-two per cent of pts had febrile neutropenia with 19 documented infections in 16 pts (4 varicella, 4 bacterial pneumonia, 3 bacterial sepsis, 2 PCP, 1 cerebral toxoplasmosis, 1 esophageal candidiasis, 1 HBV reactivation, 1 HCV reactivation, 1 prostatitis, 1 salmonellosis). CR was obtained in 47/71 pts (66%) and PR in 9/71 pts (13%). With a median follow up of 22 months, only 4 pts have relapsed. OS and TTF at 24 months are 69% and 59%, respectively.

Conclusions: Our preliminary data demonstrate that VEBEP regimen in combination with HAART is feasible and active in pts with HD-HIV. This study was supported by ISS grants.
Increase in the Incidence of Secondary Acute Myeloid Leukemia (2-AML): A Single Institution Experience Over 20 Years.
Authors: Jabbour Elias , Kantarjian Hagop M , Pierce Sherry , Garcia-Manero Guillermo , Cortes Jorge , Faderl Stefan , Ravandi Farhad
Published: April 2020

Secondary acute myeloid leukemia (2-AML) is a distinctive clinical syndrome occurring after primary malignancy treated or not with chemotherapy (CT), radiotherapy (RT), surgery, or a multimodality strategy. We analyzed the data on 2898 consecutive patients (pts) with AML (WHO criteria) referred to our institution between 1985 and 2005, including 1642 males and 1256 females. The median age was 58 years (range, 13–89 years) for pts with de novo AML (1-AML) (n=2198) and 66 years (range, 18–89 years) for those with 2-AML (n=700). 171 pts (18%) were treated for 2-AML between 1985 and 1994 versus 529 patients (27%) treated between 1995 and 2005 (p<0.001). The predominant primary malignancies included lymphoma (18%), breast (16%), and prostate (13%). Pts had been administered various cytotoxic chemotherapy agents (185 pts, 26%) and RT (102 pts, 15%); 182 (26%) had undergone both modalities, and 231 pts (33%) had undergone surgery alone. At diagnosis, 181 (26%) pts with 2-AML had diploid cytogenetics versus 845 (38%) pts with 1-AML (p<0.001). 252 (32%) pts with 2-AML had clonal abnormalities involving chromosomes 5 and 7 versus 383 (17%) pts with 1-AML (p<0.001), and 49 (7%) pts with 2-AML had 11q abnormalities versus 73 (3%) with 1-AML (p<0.001). Pts undergoing CT/RT had a worse cytogenetic risk profile compared to those undergoing surgery alone with more clonal abnormalities involving chromosomes 5 and 7 (36% versus 24%; p=0.002) and chromosome 11 (10% versus 2%; p<0.001), and less diploid karyotype (19% versus 39%; p<0.001). Median survival time after diagnosis of 2-AML was 6 months with 2- and 5-year survival rates of 18% and 11%, respectively. The median survival for pts treated with surgery alone for the primary cancer was 8 months versus 5 months for those receiving CT/RT (p=0.007); their 2- and 5-year survival rates were 22% and 14% and 16% and 9%, respectively. The median survival for pts treated for their primary cancer between 1985 and 1994 was 4 months versus 6 months for those treated after 1994 (p=0.089); their 2-year and 5-year survival rates were 16% and 9% and 19% and 12%, respectively. In conclusion, the incidence of 2-AML is increasing; patients receiving CT/RT had worse cytogenetic profiles and clinical outcomes than those receiving surgical treatment alone. There remains to be seen whether increased use of targeted therapies as prime modality of therapy in primary cancers will be associated with a decreased risk of 2-AML.
Identification of a Human Natural Regulatory T Cell Micro-RNA Signature and Demonstration of the Major Role Played by miR31 in the Control of Foxp3 Expression
Authors: Rouas Redouane , Kazan Hussein Fayyad , Zein Nabil El , Lewalle Philippe , Rothé Françoise , Akl Haidar , Burny Arsène , Romero Pedro , Martiat Philippe , Badran Bassam
Published: April 2020

Regulatory T cells are the main mediators of dominant tolerance. Their mechanisms of action and applications are subjects of considerable debate at the moment. However, a micro-RNA human Treg signature has not been described yet. We investigated human natural regulatory T-cells derived from cord blood and identified, using TLDA and qPCR, a signature composed of five micro-RNAs (21, 31, 125a, 181c and 374). Among those five, two were considerably under-expressed (miR31 and miR125a). We identified a functional binding site for miR31 in the 3′ UTR of the Foxp3 mRNA. We show that Foxp3 gene expression can be directly regulated by miR31. However, our experiments demonstrate that two distinct mechanisms must cooperate to regulate play the global level of expression of FoxP3, one mediated via miR31, and the second one at the promoter site of the gene. This part of the work is still in progress. In conclusion, not only have we found and validated a miR signature for human natural Treg, but we also unveiled some of the mechanisms by which this signature was related to the control of the Foxp3 protein levels in these cells.
The Src-Like Adaptor Proteins, SLAP and SLAP2, Regulate Flt3-Dependent Dendritic Cell Development.
Authors: Liontos Larissa , McGlade C. Jane
Published: April 2020

The Src-like Adaptor Proteins, SLAP and SLAP2, are hematopoietic adaptor proteins that have been previously shown to act as negative regulators of T- and B-cell signaling. SLAP and SLAP2 work in conjunction with the E3 ubiquitin ligase, c-Cbl, to down regulate the T-cell receptor and other components of the T- and B-cell receptor signaling pathways including ZAP-70 and Syk. SLAP and SLAP2 are expressed in many hematopoietic cell types, including progenitor cells that give rise to cells of both myeloid and lymphoid lineages. Recent evidence indicates a role for SLAP and SLAP2 in regulating hematopoietic receptor tyrosine kinase (RTK) signaling. We have shown that SLAP and SLAP2 interact with CSF-1R/Fms and demonstrated that SLAP2 negatively regulates CSF-1 dependent differentiation of FD-Fms cells. Our recent work demonstrates that both SLAP and SLAP2 interact with the Flt3 receptor in an SH2 domain dependent manner and the interaction was mapped to pY589 and pY591 within the juxtamembrane region of the receptor. To examine the role of SLAP and SLAP2 in Flt3 regulation and signaling in vivo, we utilized Flt3 ligand (Flt3L) to generate dendritic cells from mice lacking both SLAP and SLAP2. Slap1−/− Slap2−/− mice have impaired development of Flt3L-dependent CD11c+ bone marrow-derived dendritic cells (BMDC). In contrast to wild-type mice, Slap1−/− Slap2−/− mice produce 47% ± 9.7% less CD11c+ BMDC after 10 days in culture with Flt3L. Whether the reduction in BMDC numbers is indicative of a defect in the proliferation of Slap1−/−Slap2−/− progenitor cells in response to Flt3L is currently being explored. Although the absolute number of Flt3L-generated BMDC from Slap1−/−Slap2−/− mice is reduced, there are no major differences in the subtype, myeloid (70–90% CD11b+) and lymphoid (4–8% B220+), of DC produced between wild-type and double knock-out mice. To determine whether the reduction in dendritic cell numbers is specific to those generated with Flt3L, we generated BMDC using GM-CSF and IL-4. Both wild-type and double knock-out mice produce similar numbers of CD11c+ BMDC when this combination of cytokines is used. The maturation and activation of Flt3L-generated BMDC from Slap1−/−Slap2−/− mice is being investigated. These data indicate a novel role for SLAP and SLAP2 in the differentiation of Flt3L-dependent dendritic cells.