bluuebunny/crossref_metadata_2025 huggingface dataset:
Substantially High Frequency of Chromosome 7q Interstitial Deletion in B-Cell Lymphoproliferative Disorders.
Authors: Lu Gary , Zhang John , Latorre Katherine , Hibbard Michele , Chen Sing-Tsung
Published: October 2019

It has been well known that cytogenetics study plays an important role in cancer patient care, in particular in the diagnosis, treatment and prognosis of hematological malignancies. The MCIM test system, a combination of studies of cell morphology (M), cytogenetics (C), immunophenotype (I) and molecular genetics (M), has become a useful test system in hematologic disease diagnosis. It provides physicians more comprehensive and accurate diagnostic laboratory information. Deletions in the long arm of chromosome 7 are among the most common abnormalities observed in hematologic disorders. These changes were usually viewed as markers for myeloid malignancies, in particular myelodysplastic syndrome and acute myeloid leukemia. Recently, interstitial 7q deletions, a subset of the 7q deletions, have been found to be associated with B-cell lymphoproliferative disorders (LPDs). The correlation of narrow 7q deletions with specific subsets of B-cell LPDs has been extensively studied. However, the frequency of interstitial 7q deletions associated with B-cell LPDs has not been well investigated. We recently studied 7q deletions collected from our clinical cases done by the MCIM system at US Labs over the past two years. Deletions of chromosome 7q were observed in 85 of 19,483 cases. Interstitial 7q deletions were detected in 46 of the 85 cases with 7q deletions. In combination with the findings of flow cytometry, interstitial 7q deletions were found to be associated with B-cell LPDs in 10 out of the 46 cases, accounting for 21.7% of all the observed interstitial 7q deletions and 11.8% of all the 7q deletions. The B-cell LPDs associated with 7q interstitial deletion are diverse, including hairy cell leukemia, atypical chronic lymphocytic leukemia, splenic marginal zone lymphoma, and large B-cell lymphoma. Interestingly, the deleted region in the 10 cases with B-cell LPDs in this study was solely confined to 7q22-32. This is so far, to our knowledge, the largest series reported in the literature. Our investigation showed that the frequency of 7q interstitial deletions associated with B-cell LPDs is substantially high. We conclude that (1) association of B-cell LPDs must be taken into account when an interstitial 7q deletion is observed, in particular when the deleted region is confined to 7q22-32; (2) together with the flow cytometry analysis in the MCIM system within a clinical laboratory diagnostic facility, cytogenetics study can provide clinicians more accurate diagnostic information, thus strengthening patient care by improving the management of hematological malignancies. (The authors are grateful to Deanna Collins for her cooperation to collect the data in this study.)
VTD (Velcade, Thalidomide, Dexamethasone) as Primary Therapy for Newly-Diagnosed Multiple Myeloma.
Authors: Wang Luhua M. , Weber Donna M. , Delasalle Kay B. , Alexanian Raymond
Published: October 2019

The combination of thalidomide and dexamethasone (TD) has induced disease remission in 69% of 132 newly diagnosed patients with multiple myeloma (MM) treated at our center, with preventable and/or manageable side effects, and represents our primary treatment of choice. By adding bortezomib (Velcade), the 3-drug combination (VTD) has induced remission in 55% of patients with myeloma resistant to standard therapies, twice the frequency observed with bortezomib alone (Zangari, Barlogie et al, 2003). From 6/03 to 6/04, we combined bortezomib with thalidomide (100–200 mg each evening) and dexamethasone (20 mg/m2 on days 1–4, 9–12, 17–20) with therapeutic anticoagulation for 25 previously untreated patients with MM. Bortezomib was given at a dose of 1.0 (n=2), 1.3 (n=11), 1.5 (n=7), 1.7 (n=4) or 1.9 (n=1) mg/m2 IV on days 1, 4, 8 and 11, every 4 weeks to patients for 2–3 cycles of VTD. Median age was 63 (39–81), median B2M 5.1 mg/L (1.7–19), Hgb <10.5 g/dl in 60%, corrected serum calcium >11.5 mg/dl in 17%, and high tumor mass in 32% of patients. Grade 3 or 4 toxicities included non-neutropenic infection (2), orthostatic hypotension (1), DVT (1), neutropenia (1), thrombocytopenia (1); grade 3 or 4 neuropathy was not seen. Median nadir of ANC was 2360/ul and of platelets 135,000/ul with no correlation between bortezomib dose and myelosuppression. An initial dose of bortezomib of 1.5 mg/m2 IV x 4 was safe unless age > 70 and/or drugs for hypertension were taken concurrently. Responses were confirmed (>75% reduction serum myeloma protein and/or >95% reduction Bence Jones protein) in 19 patients (76%) and when defined by less stringent Blade criteria in 21 patients (84%); median time to remission was 0.6 mo (range 0.3–1.8) by either criteria in comparison with 1.1 mo (range 0.3–8.1) after TD (p<.01). Disease resistance was recognized by one month in 5 of 6 patients rated as unresponsive. Autologous blood stem cells were collected easily with G-CSF alone in 12 patients who were intensified a median 3.6 months after initial therapy; all 4 patients with primary resistant disease who received such therapy achieved partial remission. The frequency of CR to VTD could not be assessed because of early intensification. All patients are alive after median follow-up of 6 months (range 2–14). VTD induced a slightly higher frequency of PR, and significantly more rapid onset of remission, than those observed in similar patients treated previously with TD. No more than 2 courses of therapy were necessary before intensification or maintenance, so that the potential side effects and cost of more therapy were avoided. Results appeared superior to those observed with any prior program for previously untreated patients with MM at our center.
Effects of B7.2−/− Mature Dendritic Cells on Tolerance Induction to Alloantigens in Fetal Mice Following In Utero Transplantation with Lineage Depleted Bone Marrow.
Authors: Bhattacharyya Swati , Cowan Morton J.
Published: October 2019

In utero hematopoietic stem cell transplantation (IUT) has the potential to cure a variety of marrow stem cell defects without using marrow ablative therapy. However IUT for diseases other than SCID has been unsuccessful. To better understand the barriers to successful IUT we wanted to define the role of the B7.1/B7.2 co-stimulatory molecules in inducing tolerance to allogeneic donor bone marrow cells in the fetal murine recipient. We studied the relative role of B7.1 and B7.2 expression on dendritic cells (DC) on engraftment and in generating donor specific tolerance in fetal mice. Mature DC (mDC) from B7.1−/− or B7.2−/− donors and wild type (wt) lineage depleted (lin−) C57Bl/6 (B6) bone marrow (BM) were injected into gestational day (GD) 14 Balb/c fetuses. Recipients of lin− wt BM and B7.1−/− mDC had a significantly lower survival (47.4%, p<0.01) associated with mild-moderate GvHD compared to the recipients of B7.2−/− mDC and lin− BM (82.3%) where none developed GvHD. Engraftment results in blood at 6 weeks post IUT showed, B7.1−/− recipients had multilineage engraftment (4.7±0.8% T cells and 5.7± 1.1% granulocytes) in their blood, but by 12 weeks, only donor CD3+ (predominantly CD8+) cells (2.1±1.3%) were present. The percent H2Kb+ (donor) T cells (predominantly CD4+) in the blood of recipients of lin− wt BM and B7.2−/− was 11.8±8.5% at 6 weeks p<0.001 and 6.5±2.5% at 12 weeks, p=0.006. The circulating donor CD4+ cells were Th2 (CD4+CD25−IL4+IL10+) and Treg (CD4+CD25+IL4−IL10−). Both fractions inhibited the T cell proliferative response in the MLR. Long term engraftment in thymic tissues was found in the tolerant recipients of lin− wt BM and B7.2−/− mDC (13.4±8.3% donor CD3+ T cells). We also found prolonged (rejection by day 36) acceptance of donor skin grafts in 7 of 12 recipients of B7.2−/− mDC and 2 of 5 recipients of B7.2−/− mDC and lin−BM. All third party C3H grafts were rejected by day 14 and 80% of the Balb/c (self) skin grafts were permanently accepted. We hypothesized that tolerized animals would behave similarly to recipients of megadoses of syngeneic BM with an increase in multilineage engraftment. We injected a total of 200x106 male wt B6 lin− BM cells over 5 days into adult IUT recipients of B7.1−/− or B7.2−/− mDC ± lin− wt BM and wt age-matched allogeneic and syngeneic (female) controls. Mice that had received B7.2−/− mDC + lin− BM in utero showed multi-lineage engraftment in the blood. In contrast, the in utero recipients of B7.1−/− mDC + lin− BM showed no significant engraftment (p<0.05). In conclusion, donor DC costimulatory molecules significantly affect survival, engraftment and GvHD; and these responses to B7.2−/− mDC and lin− BM appear to be mediated by both Th2 and Treg donor cells.
HDAC3, a Class I Histone Deacetylase, Interacts with the P38 Pathway and Suppresses MAPK11-Mediated ATF-2 Activation and TNF Gene Expression.
Authors: Mahlknecht Ulrich , Will Jutta , Varin Audrey , Herbein Georges
Published: October 2019

During inflammatory events, the induction of immediate-early genes, such as tumor necrosis factor α (TNF), is regulated by signaling cascades including the JAK/STAT, NF-kB, and the p38 mitogen-activated protein kinase (MAPK) pathways, which result in phosphorylation-dependent activation of transcription factors. We observed the direct interaction of HDAC3, a class I histone deacetylase, with MAPK11 (p38 beta isoform) by west-western-based screening analysis, pull-down assay and two-hybrid system analysis. Results further indicated that HDAC3 decreases the MAPK11 phosphorylation state and inhibits the activity of the MAPK11-dependent transcription factor ATF-2. LPS-mediated activation of ATF-2 was inhibited by HDAC3 in a time- and dose-dependent manner. Inhibition of HDAC3 expression by RNA interference resulted in increased ATF-2 activation in response to LPS stimulation. In agreement with decreased ATF-2 transcriptional activity by HDAC3, HDAC3 repressed TNF gene expression and TNF protein production observed in response to LPS stimulation. Therefore, our results indicate that HDAC3 interacts directly and selectively with MAPK11, represses ATF-2 transcriptional activity and acts as a regulator of TNF gene expression in LPS stimulated cells, especially in mononuclear phagocytes.
Activation of the P38 MAPK Pathway Mediates Resistance to Ara-C y Bcr/Abl Positive Cells.
Authors: Sanchez Victor J. , Aceves Clara I. , Gomez Juan C. , Manso Felix , Romero Juan R. , Melo Junia V. , Sanchez-Prieto Ricardo
Published: October 2019

The chimeric protein Bcr/Abl has been connected to several signalling pathways such as AKT, JNK or ERK1/2. Here, we present evidence showing that Bcr/Abl is able to modulate the p38 MAPK pathway. Transient transfection experiments indicated that over-expression of Bcr/Abl in 293T is able to activate p38 MAPK or induce p73 stabilization, suggesting that c-Abl and Bcr/Abl share some biological substrates. Interestingly, the control exerted by Bcr/Abl on the p38 MAPK pathway was not only mediated by the tyrosine kinase activity of Bcr/Abl. In fact, Bcr alone is able to induce P38 MAPK activation specifically through MKK3. Supporting these observations Chronic Myeloid Leukaemia-derived K562 cells or BaF 3 cells stably transfected with Bcr/Abl showed higher levels of phosphorylated p38 MAPK compared to Bcr/Abl-negative cells. Interestingly, Bcr/Abl-negative cells activated p38 MAPK in response to Ara-C, while Bcr/Abl-positive cells were unable to activate p38 MAPK. Furthermore, inhibition of p38 MAPK activation by SKF860002 induces a resistant phenotype in Bcr/Abl negative cells. Our results demonstrate that the interference of Bcr/Abl in the p38 MAPK pathway is a key mechanism for explaining resistance to Ara-C in Bcr/abl positive cells.
Biochemical and Cellular Characterization of the Novel Proteasome Inhibitor PR-171.
Authors: Demo Susan D. , Buchholz Tonia J. , Laidig Guy J. , Parlati Francesco , Shenk Kevin D. , Smyth Mark S. , Sullivan Carol M. , Sun Congcong M. , Vallone Marcy K. , Woo Tina , Bennett Mark K.
Published: October 2019

Recent clinical studies have identified the proteasome as an important therapeutic target for hematologic malignances. The proteasome inhibitor, bortezomib, has been approved for the treatment of relapsed or refractory multiple myeloma and ongoing clinical trials suggests a potential benefit for the treatment of Non-Hodgkin’s lymphoma. PR-171 is a novel epoxomicin derivative that is a potent and irreversible inhibitor of the human proteasome. It inhibits the chymotrypsin-like activity of purified human 20S proteasome with a kinact/Ki of 34,000 M−1s−1 and is >300-fold selective over the other proteasome catalytic activities. In addition, PR-171 has been found to exhibit minimal activity in a broad diversity panel of biochemical assays that includes 67 receptor/ligand and 37 enzyme assays. PR-171 retains its potency for inhibition of the proteasome chymotrypsin-like activity in mammalian cells displaying IC50 values <10 nM in multiple tumor cell lines. Although PR-171 is a covalent irreversible inhibitor, proteasome activity in cells recovers with a t1/2 of approximately 24 hr after removal of the compound. This recovery is likely due to induction of de novo synthesis since other proteasome inhibitors have been shown to promote transcription of multiple proteasome subunit genes. The cellular consequences of proteasome inhibition by PR-171 include accumulation of polyubiquitinated proteins, cell cycle arrest, and induction of apoptosis. Potent cytotoxic activity of PR-171 is observed across a broad panel of human tumor cell lines (IC50 range: 2–40 nM). Pulsatile exposure studies designed to mimic the anticipated drug exposure in vivo have demonstrated that cytotoxicity is dependent upon the magnitude and duration of proteasome inhibition. These studies have also shown that hematological tumor lines are most sensitive to brief PR-171 exposure, with solid tumor lines exhibiting intermediate sensitivity and non-transformed cells being the least sensitive to such treatment. Finally, PR-171 has been found to retain its cytotoxic potential on cells made resistant to bortezomib in vitro. These studies motivate the clinical investigation of PR-171 in hematological malignances.
The Role of Stromal Cells in Multiple Myeloma: Actors or Spectators?.
Authors: Corso A. , Ferretti E. , Gallì A. , Tenore A. M. , Pascutto C. , Zappasodi P. , Mangiacavalli S. , Varettoni M. , Rusconi C. , Lazzarino M.
Published: October 2019

Multiple myeloma (MM) is a B cell neoplasia characterized by an accumulation of clonal plasma cells (PCs) in the bone marrow (BM). The growth and survival of MM plasma cells is regulated by their network with the microenvironment, mainly with the stromal cells. However, although bone marrow stromal cells have been shown to take part in the pathogenesis of the disease, it is still unknown whether these cells play an active or passive role. Namely, whether normal stromal cells simply supply the demand of MM plasma cells, or, during the course of the disease, they acquire abnormal characteristics becoming pathological. To address this question, we designed an in vitro co-culture model in which PCs isolated by immuno-magnetic separation from MGUS and MM patients are crossed with BM stromal cells (BMSCs) derived from MGUS and MM patients. As a result, four type of co-cultures were obtained: MM-BMSCs/MM-PCs, MM-BMSCs/MGUS-PCs, MGUS-BMSCs/MM-PCs, MGUS-BMSCs/MGUS-PCs. After two days of co-culture in a serum free medium, we evaluated the survival of MM-PCs or MGUS-PCs for each combination. We also quantified by ELISA assays in the supernatants of the same cultures, the level of several growth factors (IL-6, IL-8, VEGF, MIP-1a, MIP-1b, RANTES, MCP-1, TGF-b, SDF-1) to evaluate the possible influence of these cytokines on plasma cells. Multivariate general linear models were applied to compare survival in the different combinations of BMSCs and PCs, also accounting for the various growth factors. MM-BMSCs showed to support the survival of both MM-PCs and MGUS-PCs significantly better than MGUS-BMSCs (p=0.0007). However, in the combination MGUS-PCs/MGUS-BMSCs plasma cells survived statistically better than in that MM-PCs/MGUS-BMSCs (p=0.00003). As regards the cytokines, IL-6, IL-8, VEGF, MIP-1a, MIP-1b, and RANTES did not show to be significantly associated with plasma cell survival in all settings. TGF-B and SDF-1 levels were significantly associated with better survival of both MM-PCs and MGUS-PCs when cultured with MM-BMSCs compared to MGUS-BMSCs (p=0.0001 and p=0.038, respectively), while MCP-1 was significantly associated with reduced survival of MM-PCs and MGUS-PCs in the same setting (p=0.006). In conclusion, these data favours the concept that the behaviour of stromal cells may change during the transition from the condition of MGUS to the overt state of myeloma, evolving from a simple role of a spectator to that of an actor. It also appears that overt MM plasma cells have the highest need for cytokine supply and therefore are more dependent on BMSCs activity.
Disparities in Criteria for Initiating Chelation Therapy for Iron Overload in Patients with Myelodysplastic Syndromes (MDS).
Authors: Bennett John M. , List Alan F. ,   for the Myelodysplastic Syndromes Foundation Inc.
Published: October 2019

In the majority of MDS cases, isolated anemia is the only clinical evidence of impaired hematopoiesis at the time of MDS diagnosis. It is estimated that >40% of MDS patients require regular red blood cell transfusions during some stage of their disease. Findings from The MDS Foundation’s 2004–2005 Practices & Treatment Survey indicate that a substantial proportion of MDS patients in all International Prognostic Scoring System (IPSS) risk groups are transfusion-dependent: Low, 30%; Intermediate-1, 50%; Intermediate-2, 70%; High, 90% (median values). Seventy (38 US and 22 international) of the MDS Foundation’s102 Centers of Excellence responded to the survey as of July 1, 2005. Survey responses revealed that an average of 30% of transfusion-dependent patients receive parenteral iron chelation therapy (median, 20%; range: 3–100%) and that the criteria for initiating chelation therapy are not uniform. The number of transfusions was reported as a determining criterion by 46% of respondents, with a mean number of 35 transfusions (median, 30; range: 4–100). 15% of respondents indicated at the number of transfusions was the sole criterion used. Serum ferritin levels were reported as a determining criterion by 57% of respondents, with the following cutoff values:

Ferritin concentrations for initiating chelation therapy *% respondents using this cutoff as criterion >1000 ng/mL 41%* >1500 ng/mL 11% >2000 ng/mL 37% Other (>3000, unspecified) 9%

32% of respondents indicated that serum ferritin was the sole criterion used. (Irrespective of chelation therapy, 92% indicated that they monitored ferritin levels in transfusion-dependent patients.) Other criteria used to determine start of chelation therapy included age/life expectancy, MDS subtype, clinical signs of hemochromatosis, quantitative CT liver iron estimation, liver function, transferrin saturation >50%, BMT, anticipated chronic transfusion need, and logistical issues and insurance coverage. A combination of criteria was reported to be used by 38% of respondents. The majority of respondents (90%) reported that they would increase the number of patients on chelation therapy if an oral agent were available. Although the decision for initiating chelation therapy in transfusion-dependent anemic MDS patients is individualized because of the heterogeneous patient population, this data analysis suggests a need for the standardization of select criteria (e.g., number of transfusions, serum ferritin).
Heparin Synergistically Enhances Interleukin-11 Signaling through Upregulation of the Map Kinase Pathway.
Authors: Rajgopal Raghav , Butcher Martin , Walton Kimberly J. , Shaughnessy Stephen G.
Published: October 2019

Using an animal model of heparin-induced osteoporosis we previously demonstrated that heparin causes bone loss, in part, by increasing osteoclast number and activity. In an effort to account for these findings, we examined the effect of heparin on osteoclast formation in vitro. We found that while heparin alone had no effect, it was able to synergistically enhance Interleukin-11 (IL-11) - induced STAT3 (Signal Transducer and Activator of Transcription) activation and thus, increase both gp130 expression and osteoclast formation. In the present study, we examine the effect of various serine kinase inhibitors on heparin’s ability to synergistically enhance both STAT3 activation and gp130 expression. As assessed by electrophoretic mobility shift assays (EMSAs), treatment of murine calvaria cells with heparin was found to increase IL-11-induced STAT3-DNA complex formation by 2 fold. Inhibition of the c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein (MAP) kinase, or the phosphatidylinositol 3-kinase (PI3-K) pathway had no effect on heparin’s ability to promote STAT3-DNA complex formation. In contrast, the mitogen-activated protein kinase kinase (MEK) inhibitor, PD098059, completely abolished the ability of heparin to act synergistically with IL-11. Similar results were obtained when northern blot analysis was used to assess heparin’s ability to enhance IL-11-induced gp130 expression. In an attempt to resolve the mechanism by which this was occurring, we examined the ability of heparin to influence STAT3 Ser727 phosphorylation in the presence or absence of IL-11. Heparin alone was found to have no effect on Ser727 phosphorylation, nor did heparin alter the phosphorylation status of Ser727 in the presence of IL-11. Heparin was, however, found to increase ERK1/2 (extracellular signal regulated kinase) activation in both a time and dose-dependent manner. When taken together, these findings suggest that heparin enhances IL-11-induced STAT3 activation and thus, gp130 expression, by a mechanism that is independent of STAT3 Ser727 phosphorylation, but which involves upregulation of the MAP Kinase pathway.
Marker Expression in Peripheral T-Cell Lymphoma Unspecified: Proposal of a Clinical-Pathologic Prognostic Score.
Authors: Piccaluga Pier Paolo , Went Philip , Agostinelli Claudio , Gallamini Andrea , Ascani Stefano , Sabattini Elena , Bacci Francesco , Falini Brunangelo , Motta Teresio , Paulli Marco , Artusi Tullio , Zinzani Pier Luigi , Baccarani Michele , Pileri Stefano A. ,   on Behalf of the Intergruppo Italiano Linfomi
Published: October 2019

Peripheral T-cell lymphoma unspecified (PTCL/U) represents the commonest form of T-cell tumor in Western Countries according to the WHO Classification. So far, no concrete attempts have been made in order to apply a wide panel of markers to a large series of PTCLs and to assess the impact of phenotype on prognosis and survival. We then studied the protein expression and outcome of 148 PTCL/U cases, along with 45 tumors of the AILD type, utilizing highly standardized high-throughput technology.

Tissue micro-arrays corresponding to the above mentioned cases were constructed and analyzed with a panel of 18 commercially available markers. In 93 patients with PTCLs/U clinical data were available and were matched with the protein expression profile. Interestingly, most of these patients had been included in a previous study that proposed a prognostic index for PTCL/U (PIT) (Gallamini et al. Blood2004, 103:2474–9).

An aberrant phenotype with frequent loss of CD5 and/or CD7 was typical for all PTCLs, irrespectively of the subtype (unspecified or AILD-type). CD20 and CD15 were rarely aberrantly expressed, at times simultaneously with CD30. EBER positivity and CD15 expression emerged as adverse prognostic factors, while CD56 and CD57 were unremarkable. Among PTCLs/U, the proliferation-associated protein Ki-67 was found to be prognostically relevant and was then integrated in a new prognostic score, including age (>60 years), high serum lactate dehydrogenase, poor performance status, and Ki-67 3 80%. Such score was associated with the overall survival (p<0.0001) and was more a powerful predictor than PIT that - however - maintained its relevance.

Our retrospective analysis shows a wide range of protein expression in PTCLs and candidates a new prognostic index. The latter represents one of the first examples of mixed score (including patient- and tumor-specific factors) applied to malignant lymphomas and may be the basis for future prospective therapeutic trials.
Autologous Non-Myeloablative Hematopoietic Stem Cell Transplantation for Diffuse Scleroderma.
Authors: Oyama Yu , Statkute Laisvyde , Barr Walter , Traynor Ann , Burt Richard
Published: October 2019

Background &Aims Scleroderma is an immune mediated vasculopathy of unknown etiology. There is no known therapy that could alter the natural course of the disease. Patients with diffuse cutaneous disease or visceral involvement has an overall 10-year survival of 35 to 68%. Autologous hematopoietic stem cell transplantation (HSCT) with myeloablative regimen has been performed, but with substantial mortality and morbidity especially pulmonary and renal toxicity. We have performed autologous HSCT utilizing non-myeloablative, but lymphoablative conditioning.

Methods

We conducted a phase 1 HSCT study in 9 patients with diffuse scleroderma with poor clinical features. Candidates were less than 65 years old with Rodman score of more than 14 or diffusion capacity less than 80%, interstitial lung disease, elevated ESR, renal involvement, or abnormal electrocardiogram. Patients with pulmonary hypertension (systolic pressure > 45 mm Hg) were excluded. Peripheral blood stem cells were mobilized with cyclophosphamide and granulocyte colony-stimulating factor. The graft was not manipulated. The conditioning regimen consisted of 200mg/kg cyclophosphamide (CY), and 7.5mg/kg rabbit antithymocyte globulin (ATG).

Results

The procedure was well tolerated with anticipated cytopenias, neutropenic fever, and fluid overload that were easy to manage and without mortality. The median days for neutrophil and platelet engraftment were 8 (range 7–9) and 8 (range 0–10), respectively. The median infused CD34+ and CD3+ cell counts were 8.31 x 106/kg (range 2.35–14.7) and 2.03 x 108/kg (range 0.41–6.83), respectively. There was a marked improvement of skin score in all subjects, whereas cardiac (ejection fraction, pulmonary arterial pressure), pulmonary function (DLCO) and renal function (creatinine) remained stable. One patient with advanced disease with poor performance status died 2 years after the transplant with progressive disease. After median follow-up of 20 months (range 5–32), the overall survival is 89% (eight out of nine) and progression-free survival with continuing improvement is 67% (six out of nine. 2 patients developed recurrence of skin tightness without compromising organ function. Both of them were placed on mycophenolate mofetil with gradual improvement of skin tightness.

Conclusion

Autologous HSCT with CY/ATG is safe and is effective. A randomized study (ASSIST: American Scleroderma Stem Cell vs. Immune Suppression Trial), comparing HSCT to intravenous pulse cyclophosphamide is now enrolling patients.

Figure Figure
Intratumoral CD4+CD25+ Regulatory T-Cell-Mediated Suppression of Infiltrating CD4+ T-Cells in B-Cell Non-Hodgkin Lymphoma.
Authors: Yang Zhi-Zhang , Novak Anne J. , Stenson Mary J. , Witzig Thomas E. , Ansell Stephen M.
Published: October 2019

Background: Non-Hodgkin lymphomas (NHL) are increasing in incidence and are now the fifth most common tumor diagnosed each year in the United States. Most NHLs are of B-cell origin but the tumor tissue is variably infiltrated with T-cells. Our group has shown in diffuse B-cell large cell lymphoma, that a high number of intratumoral CD4+ T-cells predicts a better overall survival. It has been shown that recently-characterized CD4+CD25+ regulatory T-cells (Treg cells) played an important role in the mediation of anti-tumor immunity. However, there is no data on the role of intratumoral Treg cells in suppression of autologous infiltrating CD4+ T-cells in B-cell NHL.

Goal: To investigate the effect of intratumoral Treg cells on the proliferation of tumor-infiltrating CD4+CD25- T-cells, to determine the underlying mechanism of the T-cell suppression, and evaluate the role that malignant B-cells may play in the recruitment of Treg cells to the site of B-cell NHL.

Results: We identified a subset of CD4+CD25+ T-cells over-represented in biopsy specimens of B-cell NHL (these cells comprise 17% of cells in lymphoma biopsies, compared 7% of peripheral blood mononuclear cells, 12% of cells in inflammatory tonsil and 6% of cells in tumor free lymph nodes; p-value =0.001). These CD4+CD25+ T-cells are memory-like T-cells (CD45RO+ and CD45RA−) and express high levels of CTLA-4 and Foxp3 when compared to autologous tumor-infiltrating CD4+CD25- T-cells. Importantly, these CD4+CD25+ T-cells displayed the ability to suppress the proliferation and cytokine (IFN-g and IL-4) production of tumor-infiltrating CD4+CD25- T-cells in response to PHA stimulation. Treatment with anti-B7-H1 antibody or PD-1 fusion protein enhanced the proliferation of infiltrating CD4+CD25- T-cells when co-cultured with intratumoral CD4+CD25+ T-cells. Our results suggest that interaction between B7-H1 and PD-1 accounts for about 30% of intratumoral Treg cell-mediated inhibition of autologous infiltrating CD4+CD25- T-cells in tumor sites of B-cell NHL. Lastly, we found that CCL22 secreted by lymphoma B-cells is involved in the chemotaxis and migration of intratumoral CD4+CD25+ T-cells which express chemokine receptor CCR4, but not CCR8.

Conclusion: Our results suggest that tumor microenvironmental CD4+CD25+ regulatory T-cells are important regulators of tumor immunity and that these cells are recruited to the area of lymphoma involvement by the malignant B-cells.
Liver Hepcidin mRNA Is down Regulated at High Liver Iron Burdens and up Regulated in HCV Infection in Thalassaemia.
Authors: Farrukh Shah T. , Monica Mascarenhas , Roozina Rafique , Kaila Srai S. , John Porter B.
Published: October 2019

Rationale. Hepcidin is an important negative regulator of iron absorption from the gut and iron release from macrophages. Synthesis is increased by inflammation and decreased in iron deficiency. Iron overload conditions might be predicted to up-regulate hepcidin synthesis, thereby decreasing dietary iron absorption. However in adult onset herediditary hemochromatosis, there is paradoxical down regulation of liver hepcidin mRNA (Bridle et al.,Lancet 2003). Urinary hepcidin is decreased thalassaemia major (Papanikolaou et al., Blood 2005) but expression of mRNA has not been described. However, hepcidin mRNA may be increased in iron overloaded in patients with chronic HCV infection (Aoki, et al., J Clin Gasteroenterol 2005).

Methodology. mRNA was extracted on 20 liver biopsies from thalassaemia patients and RT PCR performed from cDNA as described by Leung et al., Blood 2005. 14 biopsies were obtained from thalassaemia major (TM) patients on regular transfusion programmes and the remaining 6 from thalassaemia intermedia (TI) patients, 2 of whom were not currently receiving transfusion, 3 were on exchange transfusion regimes and one was on 6–8 weekly top up transfusions. All samples were calculated as the hepcidin/actin ratio and were measured simultaneously with liver biopsies obtained from healthy control subjects at the time of surgery. Hepcidin mRNA was then expressed relative to control values from normal liver tissue in healthy control subjects.

Results. Mean hepcidin mRNA was significantly lower in patients with liver iron concentrations >10mg/g/dw compared to those with lower burdens (85 vs 131%, p=0.05). Hepcidin mRNA did not vary significantly with anaemia, with ferritin > 3000ng/ml or <1500ng/ml (p=0.99). Hepcidin mRNA was not significantly different between the 2 thalassemia groups; TI (123 ± 47%) compared to (110 ± 73%)TM. 3 patients with high transaminase levels (>80 iu/ml) all had low hepcidin mRNA levels (77 ± 16 %) and increased levels of fibrosis compared to those with lower transaminase levels (106 ± 55%) and only mild fibrosis. 4 patients who were HCV antibody positive but with only modestly elevated transaminase levels (<160iu/ml) and mild/moderate fibrosis had raised hepcidin mRNA (163 + 70 %) p=0.02. There was no difference in the HCV positive and HCV negative groups for ferritin (2530 ng/ml: 2483 ng/ml), ALT (61 iu/l :53 iu/l) or liver iron (7.5 mg/g/dw versus 8.2 mg/g/dw).

Conclusions. This suggests a critical level of iron burden (LIC) above which hepcidin expression becomes dysregulated, failing to increase with further elevations in the iron burden and paradoxically decreases. This could lead to inappropriately high iron absorption in heavily iron-loaded patients. Of other interacting factors affecting hepcidin synthesis, this study suggests that active HCV may increase hepcidin synthesis.
Klf12 Is Required for Vessel Organization in Zebrafish Embryos.
Authors: Perkins Andrew C. , Gunaratnam Dinushka , Gardiner Melissa R. , Robinson Kathleen C.
Published: October 2019

The process of forming a lumenised vessel from an angioblast cord is a crucial part of both vasculogenesis and angiogenesis. The Krüppel like factors (klfs) are a family of zinc finger transcription factors which play important roles in many aspects of differentiation. Klf2 knockout mice die in utero from haemorrhaging due to arterial wall defects. Due to the similarity of vascular and haematopoietic systems to those of mammals, the zebrafish was chosen as a model in which to study the function of klf12, the only zebrafish representative of the repressor subfamily of mammalian klfs which includes klf3, klf8 and klf12. Klf12 is first detected by WISH at 12 somites in the lateral plate mesoderm (LPM) and continues in these cells as they from the ICM at 18–22hpf, the site of vasculogenesis and haematopoiesis. From 24hpf klf12 is expressed in short stripes extending from the ICM dorsally towards the notochord. This expression pattern is similar but not identical to that of fli1 and flk1, two vascular markers. Expression subsequently decreases and is absent by 30hpf.

Targeted knockdown of klf12 by translantion-inhibiting and splicing morpholinos (MOs) produces a vascular defect. At 24hpf morphants display correct expression of primitive blood, kidney and vascular markers such as gata1, βE3globin, biklf, pax2.1 and fli1. However, circulation is absent in 65% of embryos at 48 hpf and reduced in most, as seen by both brightfield microscopy and fluorescent microbead microangiography. Embryos also display a slower heartbeat, pericardial oedema and oedema over the yolk/duct of Cuvier, all likely to be secondary effects due to the circulatory defect. Injection of klf12 MOs into fli1-eGFP transgenic embryos reveals correct differentiation of endothelial cells, but disorganised angiogenesis.

In summary, klf12 morphants display correct specification of angioblasts and differentiation of endothelial cells but a defect in tubulogenesis of endothelial cords.
Knockdown of ABCme Impairs Heme Biosynthesis as Revealed by Integrating of RNAi and the LiveCell® Array.
Authors: Haigh Sarah E. , Elorza Alvaro A. , Biran Israel , Zurgil Naomi , Deutsch Motti , Mikkola Hanna , Shirihai Orian S.
Published: October 2019

Heme synthesis begins and ends in the mitochondrion, whilst intermediate steps are performed in the cytosol. Consequently, heme precursors must be transported across the mitochondrial membranes, however, the transporters of these intermediates have not been identified. Conditions such as sideroblastic anemia, porphyria and MDS are all characterized by abnormal heme synthesis and accumulation of heme intermediates and iron. Thus, impaired function of mitochondrial transporters is thought to play a key role in these conditions.

We have previously reported that over-expression of murine ABCme increases hemoglobin production; however the identity of the heme intermediate transported by ABCme is as yet unclear (Shirihai et al. 2000).

We have used lentivirus delivered shRNA, harboring a GFP marker, to knockdown ABCme mRNA in human K562 erythroid cells. Interpreting RNAi experiments in heterogeneous cell populations, such as differentiating cells, can be problematic. Specific challenges which must be addressed are: 1) transgene and RNAi expression is hard to achieve in erythroid cells; 2) cell to cell expression efficiency is variable; 3) differentiation is a stochastic process; 4) erythroid cells are non-adherent rendering imaging of living cells difficult. Taken together, these factors lead to pronounced cellular heterogeneity, which may mask biological effects. Furthermore, the correlation of RNAi expression and heme synthesis requires the use of benzidine staining, which results in protein oxidation and elimination of all fluorescent signals. To address these challenges we developed a new assay that takes advantage of a novel array which corrals living cells into individual wells. The LiveCell® Array is a densely packed array of micron-sized wells, each accommodating an individual cell, embedded within a microscope slide. Sequential measurements of multiple cellular activities can be taken and the images analyzed using Metamorph image analysis software.

Using this system we identified a population of cells which expressed high levels of RNAi by screening for GFP. The stage of differentiation of each cell was determined using antibodies against extra-cellular makers (i.e. Glycophorin A) conjugated to fluorescent moieties thus identifying maturing subpopulations of cells. To measure heme content, benzidine was subsequently applied and light absorbance was quantized at the resolution of the individual cell. Heme content was then cross correlated with the differentiation and expression fluorescent markers. By integrating RNAi with the LiveCell® Array we have found that knockdown of ABCme results in reduced heme production in differentiating erythroid cells. Further analysis of heme intermediates using this model is being pursued in order to identify the substrate of ABCme.
The Co-Recessive Inheritance Model: A Paradigm for Fanconi Anemia and Other Bone Marrow Failure Syndromes.
Authors: Lambert W. Clark , Brown Monique M. , Centurion Santiago A.
Published: October 2019

One of us (WCL) has previously proposed a mathematical model, Co-Recessive Inheritance, for inherited diseases associated with DNA repair deficiencies (Lambert WC, Lambert MW: Mutat. Res., 1985;145:227–234; Lambert WC: Keynote Address, 21st Anniversary Celebration, MRC Cell Mutation Unit, University of Sussex, UK. Mutat. Res., 1992;273:179–102). The model is also applicable to diseases associated with defective cell cycle modulation following specific types of DNA damage, such as Fanconi Anemia, with or without additional defects in DNA repair. The model proposes that in some complementation groups of these diseases defective alleles at more than one locus are required for the disease phenotype to be expressed. It follows from the model (A readily understandable derivation will be presented.) that the carrier frequencies of the genes involved are very much higher than would be predicted based on classical population genetics. This may impact on recent observations of higher than expected co-inheritance of defective alleles of Fanconi Anemia and Bloom Syndrome genes along with BRCA genes in certain populations (e.g., Koren-Michowitz, M, et al.: Am. J. Hematol., 2005;78:203–206), and provides an explanation for the lower than expected incidence of cancer in these individuals. It also provides an explanation for finding biallelic defects in the same DNA repair genes in more than one complementation group of Fanconi Anemia (Howlett NG, et al.: Science, 2002;297:606–609). The Co-Recessive Model predicts that other findings of this nature are to be expected, and provides some guidelines that may be helpful in the process of gene discovery in Fanconi Anemia. Among the more important of these are 1) that the search for defective genes in each complementation group should not cease when one such gene is found, even if one or more patients in the group is homozygous or compound heterozygous for defective alleles of that gene, and 2) that carrier frequencies for some Fanconi Anemia genes may be much higher than would otherwise be anticipated, with a significant proportion of the normal population being carriers. If the latter hypothesis is correct, it follows that the relevance of these rare diseases and their associated genes to disease, including bone marrow failure, in the general population is dramatically greater than has been generally believed.
Serum Cystatin C Is a Better Marker for Renal Function Than Serum Creatinine in Children with Sickle Cell Disease.
Authors: Alvarez Ofelia A. , Wright Dale , Lopez Gabriela , Zilleruelo Gaston
Published: October 2019

High serum creatinine is a late manifestation for patients (pts) with sickle cell disease (SCD) who develop severe renal dysfunction, because creatinine is secreted by the renal tubules. Serum cystatin C is a cysteine proteinase inhibitor, that is produced by all nucleated cells in the body, does not undergo tubular secretion, and reflects glomerular filtration rate (GFR)accurately. Normal levels for serum cystatin C are 0.5–1.3 mg/L for children 1–17 years, and 0.5–1 mg/L for adults. The purpose of this study was to compare serum cystatin C and serum creatinine as markers of GFR in children with SCD with different levels of albuminuria. Twenty pts (mean age 16.4, range 9–21 years) had serum creatinine, serum cystatin, and 24-hour urine for creatinine clearance. Pts with normoalbuminuria (N=11) had mean serum creatinine of 0.55±0.14 mg/L, creatinine clearance of 168±36 ml/min/1.73m2, normal serum cystatin C 0.78±0.16 mg/L, and normal estimated GFR derived from cystatin of 106±27 ml/min/1.73m2. In contrast, pts with proteinuria (N=4) had higher or abnormal serum cystatin C (mean 1.25 ±0.34, range 0.9–1.7 mg/L) and reduced estimated GFR (mean 59±21, range 35–85) consistent with poor kidney function; nevertheless, the serum creatinine (0.7±0.2) and creatinine clearance remained normal (125±27). Pts with only microalbuminuria (N=5) maintained normal levels of cystatin C and estimated GFR by cystatin. We conclude that serum cystatin C discriminated better for kidney dysfunction than serum creatinine and creatinine clearance with significantly different values between patients with normoalbuminuria and macroalbuminuria (p=0.038). More studies are warranted in order to investigate further the value of serum cystatin C in the monitoring of patients with SCD and albuminuria.
Reduced Cholesterol Levels in Adults with Sickle Cell Disease.
Authors: Nagajothi Nagaprasad , Sanmugarajah Jasotha
Published: October 2019

Reduced levels of cholesterol was first reported in African children with sickle cell disease (SCD).More recently similar findings were reported in African American adults with SCD in the United States. Epidemiologic studies have identified a relation between low serum levels of total cholesterol (<130 mg/dL) and increased mortality from all causes. We wanted to study the cholesterol levels in adults with SCD especially as their average life expectancy is continuing to increase with advances in medical care.We conducted a retrospective study comparing the serum lipid profile with 15 adults with SCD seen at our hospital with 15 age matched controls who did not have SCD or hyperlipidemia. The mean age in the SCD group was 48±13 years and the mean age in the control group was 46±5years (P=0.73). The mean total cholesterol level in adults with SCD was 135±26 mg/dL and was significantly lower than the mean total cholesterol level in age matched controls which was 169±30 mg/dL (P=0.001). LDL levels were significantly lower in those with SCD compared to controls (67±16 mg/dL versus 90±29mg/dL, respectively, P=0.01). Adults with SCD also had lower HDL levels (45±15 mg/dL) compared with their controls (60±11 mg/dL, P=0.004). There was no difference in the mean triglyceride levels between the two groups. While there was no difference in the mean height between the two groups the SCD group had lower Body Mass Index (BMI) than the control group (23± 4mg/dL versus 27±4mg/dL, respectively, P=0.02). Our results suggest that adults with sickle cell disease have lower serum total cholesterol, LDL, HDL levels as well as lower BMI compared to their age matched controls. Larger studies to validate our observations and to study its clinical implications are warranted.

Lipid profile and Body Mass Index in Sickle Cell Disease and Controls Sickle Cell* Control* Mean(SD) Mean(SD) Pvalue *n=15 Age(years) 48±13 46±5 0.73 Total Cholsterol (mg/dL) 135±26 169±30 0.001 LDL(mg/dL) 67±16 90±29 0.01 HDL(mg/dL) 45±15 60±11 0.004 Triglycerides(mg/dL) 113±51 94±47 0.3 Height(cm) 167±7 167±6 0.7 Weight(lbs) 141±27 178±35 0.007 Body Mass Index 23±4 27±4 0.02
Platelet Binding to Leukocytes as a Determinant of Leukocyte Activation In Vivo.
Authors: Chirinos Julio A. , Jy Wenche , Tiede Maike , Castrellon Aurelio , Soriano Andres O. , Del Carpio Freddy , Arteaga Roque , Jimenez Joaquin J. , Horstman Lawrence , Bidot Carlos , Ahn Yeon S.
Published: October 2019

Background: Platelet binding to leukocytes has been shown to induce leukocyte activation in vitro. It is unknown to what extent platelet binding to leukocytes regulates leukocyte activation in vivo. We examined the correlation between platelet binding to leukocytes (platelet-leukocytes conjugates) and leukocyte activation in various clinical conditions.

Methods: We studied a total of 321 venous blood samples from 291 subjects with acute venous thromboembolism (n=25), atrial fibrillation (n=48), metabolic syndrome (n=37), congestive heart failure (n=44), antiphospholipid syndrome (n=70), early sepsis (n=35), late sepsis (n=30) and normal controls. Using flow cytometry, we measured leukocyte expression of activation marker CD11b. Platelet-leukocyte conjugate measurement was based on co-expression of CD41 and CD45.

Results: Levels of platelet-leukocyte conjugates strongly correlated with CD11b expression. This finding was present in congestive heart failure (r=0.47; p=0.001), atrial fibrillation (r=0.32; p=0.02), metabolic syndrome (r=0.51; p=0.001), venous thromboembolism (r=0.74; p<0.0001), early sepsis (r=0.70; p<0.0001), late sepsis (r=0.66, p=0.0002), and antiphospholipid antibody syndrome (r=0.53; p<0.0001). In normal controls, the relationship between platelet-leukocyte conjugates and CD11b expression was best described by a 2nd-degree polynomial model, which included the quadratic term platelet-leukocyte conjugates squared, suggesting a threshold effect. The quadratic term added significant prediction of CD11b expression, demonstrating a highly significant exponential increase in CD11b expression after a threshold level of platelet-leukocyte conjugates is reached (model R2=0.57; r2 increase from quadratic term=29%; p=0.0001), as shown in Figure 1. This threshold was not seen in any disease state examined (all p for quadratic terms > 0.05). For example, the relation between platelet-leukocyte conjugates and CD11b expression in venous thromboembolism is shown in Figure 2, and is best described by a straight line. The addition of quadratic term did not add any prediction of CD11b expression, as shown.

Conclusions: Our findings strongly suggest that platelet binding to leukocytes is a key determinant of leukocyte activation in vivo. The degree of platelet binding to leukocytes strongly correlates with leukocyte activation after a threshold is reached in normal adults. This threshold is lost in pro-thrombotic and pro-inflammatory disease states, in which a linear increase in leukocyte activation is seen with increasing platelet binding to leukocytes.

Figure 1. Figure 1.

Figure 2. Figure 2.