bluuebunny/crossref_metadata_2025_split huggingface dataset:
dHPLC as a Rapid and Reliable Screening Method for A-Thalassemia Screening.
Authors: Bernasconi Luca , Herklotz Roberto , Hergersberg Martin , Huber Andreas 
Published: October 2019

Abstract
Background: a-thalassemia, the most prevalent of all thalassemias, is an inherited single gene disorder of the hemoglobin synthesis characterized by the absence or the reduced expression of a-globin genes resulting in an imbalanced ratio between a- and b-gobin chain synthesis. While some DNA alterations lead to microcytic anemia, the most others cause severe anemia and intrauterine death if co-inherited in a homozygous constellation. The most common genetic defects leading to the a-thalassemia phenotype are deletions of one or more of the two highly homologous a-globin genes. Less frequently, a-thalassemia derives from nondeletional mutations located in parts of the a-globin gene that are critical for its normal expression. So far the identification of the genetic background of a-thalassemic patients has included specific amplification of each a-globin gene followed by laborious and expensive mutation analysis methods such as denaturing gradient gel electrophoresis (DGGE), single-strand conformation polymorphism (SSCP) or direct sequencing of the entire a-genes.
Method: We attempted to evaluate the performance of denaturing high performance liquid chromatography (dHPLC) technique to identify nondeletional mutations located in the a1- or a2-globin gene. Due to the high sequence homology of a1- and a2-globin genes, separate amplification of the two genes followed by nested PCR were performed. This generated four overlapping amplicons (a-dHPLC1 to 4) covering the whole a1/a2-globin locus including the 3′- and 5′-untranslated regions. The formation of DNA hetero-/homoduplexes was ensured by denaturing and slow reannealing of crude PCR products. Through a DNA separation column under partial-denaturing conditions, DNA homoduplexes were retained longer than their corresponding heteroduplexes generating distinguishable chromatographic profiles and hence allowing for the differentiation between wildtype and mutant DNA. Samples which differed from the wildtype in their elution profile were sequenced in both directions.
Results: 50 patients showing a presumptive a-thalassemic hematological profile not due to deletional mutations (excluded by gap PCR technique) were tested. 10 carriers of a-thalassemia nondeletional alleles (previously identified by endonuclease restriction analysis) and 10 healthy patients were also included in the evaluation.
Conclusion: Our results show that dHPLC is a reliable, sensitive, and specific screening method to detect pointmutations and small deletions located in the a-globin genes. Furthermore, the low costs of analysis and the rapidity of the screening (about 6 min/sample) make the dHPLC technique an appropriate alternative to technically demanding and time consuming mutation screening methods such as DGGE or SSCP analysis.
Comparative Benefits of the Non-Human Primate in Long-Term Toxicity Studies with Iron Chelators: 12-Month Studies with Deferiprone.
Authors: Connelly John , Spino Michael , Tricta Fernando 
Published: October 2019

Abstract
Cynomolgus monkeys and rats have been the species of choice for evaluation of general toxicity during the non-clinical development of iron chelators. Each model has its advantages in safety assessment studies, although the relatively poor conservation of iron in rats and other differences in their iron handling compared with primates, including man, have raised questions about their appropriateness. It was reported that in a study in cynomolgus monkeys conducted during the early development of deferiprone, doses greater than 150 mg/kg caused deaths when given orally once daily for c. 20 days of a scheduled 3-month treatment period. We present results from a 12-month oral toxicity study of deferiprone in naive and iron-loaded cynomolgus monkeys, using twice daily dosing and compare them with findings in a similar study in rats. Cynomolgus monkeys (4–6/sex/group) and rats (20/sex/group), iron loaded by intraperitoneal injection of iron dextran, were given deferiprone as two equal daily oral doses totalling 0, 75, 150 and 200 (250 after 3 months in monkeys) mg/kg/day; naive monkeys were given 0 and 150 mg/kg/day for 12 months. Measurement of all standard parameters, including toxicokinetics, was included. No mortalities, no adverse clinical signs, and no effects on body weight gain, food intake, cardiovascular function or eye morphology, were observed in either iron-loaded or naive monkeys. Haematological and plasma chemistry parameters were largely unaffected by treatment with deferiprone; intermittent increases in serum activities of some hepatic enzymes were related to iron loading. Similarly, histopathological changes were limited to those associated with iron deposition in tissues. Plasma deferiprone concentrations achieved were significantly in excess of clinical exposures. Both naive and iron-loaded rats were less tolerant of long-term administration of deferiprone.
Both divided daily dosing and iron loading may have contributed to the markedly better survival of monkeys in the 12-month study compared with the earlier investigation, and the results of this work confirm the value of the model in the evaluation of the safety of iron chelators.
Clinical Application of Matrix Metalloproteinase-8 and Matrix Metalloproteinase-9 Expression in Patients with CML.
Authors: Lin Liang-In , Lee Cheng-Yeh , Lin Dong-Tsamn , Wen Hwei-Wen , Tien Hwei-Fang 
Published: October 2019

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

Abstract
Enzyme replacement therapy (ERT) with exogenous glucosylceramide β-glucosidase (alglucerase, [Ceredase®] or imiglucerase, [Cerezyme®]) has been shown to improve anemia, thrombocytopenia, hepatosplenomegaly, bone symptoms and quality of life in patients with Gaucher disease. However, the impact of ERT on mortality has not been assessed since the inception of ERT in 1991. Data from the epoch prior to the availability of ERT were obtained from the University of Pittsburgh Gaucher Disease Registry. Data from the time period following the advent of ERT were obtained from the International Collaborative Gaucher Group (ICGG) Gaucher Registry and the Ceredase/Cerezyme pharmacovigilance database (Genzyme Corporation). Age of death was obtained for patients with reported Type 1 (non-neuronopathic) Gaucher disease. Only patients with a known cause of death were included in this analysis.
Pre-ERT Era (R.E.L.) Post-ERT Era Number of patients 31 137 Treated with ERT No Yes Mean age at death (years) 53.2 55.6 Range of age at death (years) 3–85 0.2–89 Cause of death due to: n % n % • Gaucher disease 4 12.9% 1 0.7% • Leukemia 3 9.7% 7 5.1% • Lymphoma 1 3.2% 3 2.2% • Myeloma 3 9.7% 1 0.7% • Solid tumor 12 38.7% 17 12.4% • Hemorrhage 1 3.2% 14 10.2% • Thromboembolism 0 0.0% 4 2.9% • Other cardiovascular disease 2 6.5% 26 19.0% • Infectious disease 3 9.7% 6 10.9% • Other causes 2 6.5% 49 35.8%
These descriptive data collected prior to the advent of ERT and following approval of ERT in 1991 raise intriguing questions about the changing pattern of mortality in Gaucher disease. Any direct comparison of these populations must be qualified by the possibility of detection bias or a cohort effect. Therefore, pending further information, it is difficult to attribute significance to the difference in mean age at death between the two populations. However, there have been shifts in the pattern of the causes of death. Most notably, deaths due to the primary manifestations of Gaucher disease and to hematologic cancers and solid tumors appear substantially less common in the post-ERT era whereas the proportion of deaths due to cardiovascular disease and other causes appears to be increasing. More accurate estimates of the patterns of mortality in Gaucher disease remain to be determined, particularly for the pre-ERT era. Additional studies of the changing patterns of mortality in Gaucher disease are ongoing.
Down-Regulation of Toll-Like Receptor 4 Expression in Transforming Growth Factor β1 Treated Murine Dendritic Cells: Implications of Maturation Resistant to Lipopolysaccharide.
Authors: Lin Maofang , Mou Haibo , Cen Hong 
Published: October 2019

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

Abstract
On the basis of promising results obtained by imatinib mesilate in a patient with cerebral LCH with high expression of PDGFRb in the affected tissue (Case Report in press in the New Engl J Med), we evaluated PDGFRb expression by immunohistochemistry on banked LCH specimens.
Methods: A 4 m m section obtained from formalin-fixed, paraffin-embedded specimens was incubated in a microwave oven for 15 min in 10 mmol/L buffered citrate pH 6.0, followed by immunohistochemical procedures to detect PDGFRb (using a rabbit polyclonal antibody, Santa Cruz Biotechnology Inc., CA) and KIT (CD117, Dako, Carpinteria, CA), diluted 1:50 and 1:300, respectively. A conventional avidin-biotin complex procedure was then applied, according to manufacturer’s protocol. Briefly, the sections were washed and incubated with the primary antibody overnight at 4°C, then incubated with the secondary antibody. Positive staining was revealed by the DAB chromogen, followed by counterstaining with Mayer’s hematoxilin. The slide was cover-slipped with a xilene-based mounting medium. The staining was scored as Neg, +1 (low expression), +2 (intermediate expression), or +3 (strong expression).
Results. Up to now, we have analysed specimens from 9 patients (see Table). A strong PDGFRb expression with a predominant cytoplasmic localization was detected in 5 cases; two specimens had intermediate expression and two low expression. KIT expression was never observed. The study is ongoing, to evaluate a larger number of cases; however, in the limited series presented, 70% of cases had a significant PDGFRb expression. This finding may stimulate further clinical investigation on the use of tirosin kinase inhibitors in LCH.
PDGFRß and KIT expression in LCH specimens
Case Gender/Age Disease site KIT PDGFRß C: cytoplasmic; N: nuclear 1 M/10 ocular soft tissue Neg +1C 2 F/9 skin Neg +3C/N 3 M/76 skin Neg +2C 4 M/13 bone Neg +3C 5 M/19 bone Neg +3C 6 M/1 bone Neg +1C 7 M/45 oral mucosa Neg +3C 8 F/23 brain Neg +2C 9 M/45 oral mucosa Neg +3C
Enzyme Replacement Therapy in Gaucher Disease: Beneficial Effects on Bone Crisis and Bone Pain.
Authors: Grabowski Gregory , Charrow Joel , Weinreb Neal J. , Dulisse Brian 
Published: October 2019

Abstract
Bone crises and bone pain are major causes of morbidity in type 1 Gaucher disease (GD), patients. Enzyme replacement therapy (ERT) for GD has proven to be of clinical benefit in resolving hematologic abnormalities and organomegaly. However, the clinical effects of ERT on bone crisis and bone pain have not been fully evaluated. This paper reports the results of the first retrospective data analysis undertaken to examine the association between ERT in this population and the reduction of bone crisis and bone pain.
Data were analyzed from the International Collaborative Gaucher Group Registry, established in 1991 as a longitudinal database to track the clinical outcomes of patients with GD, irrespective of treatment status. Inclusion criteria were GD type 1, treatment by ERT, and data available on the occurrence of bone crises and/or bone pain for 1 year prior to and for each of the 3 years following the initiation of ERT. A bone crisis was defined as pain with acute onset requiring immobilization, narcotics for pain, and periosteal elevation, leucocytosis, fever or debilitation. For bone crises and bone pain, 219 and 244 patients, respectively, met the criteria.
TYPE 1 GAUCHER DISEASE PATIENTS WITH BONE CRISES AND BONE PAIN 1 YEAR PRE- AND 1, 2 AND 3 YEARS POST-ERT
The numbers of patients with reports of bone crises and bone pain in each of the time periods are presented in the table. Bone crisis frequency, at 17% prior to ERT, declined to 5% or less in each of the 3 years following the initiation of ERT (t-test, p <0.0001). Similarly, bone pain, reported in 49% of patients prior to ERT, declined to approximately 30% in each of the 3 years following the initiation of ERT (t-test, p <0.0001)
Yr from ERT initial infusion −1 YEAR 1 YEAR 2 YEARS 3 YEARS *n refers to number of patients Bone crisis n = 219(100%) 38(17%) 10(5%) 1(0%) 6(3%) Bone pain n = 244(100%) 119(49%) 74(30%) 71(29%) 73(30%)
These results provide strong clinical evidence for ERT’s effectiveness in the reduction of bone crises and bone pain in type 1 GD.
Incidence, Natural History and Management of Cytomegalovirus (CMV) Dnaemia and Disease in Patients with Haematological Malignancies in the Non-Allogeneic Transplantation Setting.
Authors: Seymour John Francis , Ng Ashley , Worth Leon , Slavin Monica , Prince H. Miles , Thursky Karin 
Published: October 2019

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

Abstract
Human immunodeficiency virus-1(HIV-1) is associated with cardiovascular abnormalities and autonomic dysfunction. Exercise training is recommended to enhance functional capacity, as it is known that fitness is icommensurate with a improved autonomic profile and can reduce cardiovascular malfunctions. Although it is well established that physical activity in HIV-1 pts results in increased aerobic capacity, the effect of moderate exercise training on vascular mechanisms and autonomic modulations in HIV-1 disease is still unknown. The purpose of this investigation is to examine the effects of regular physical activity on arterial compliance, autonomic modulation and alterations in NK cell number and function in HIV-1 disease. Subjects were matched for age, body mass index, waist/hip ratio, and fitness. Arterial compliance (AC), heart rate variability (HRV), and baroreflex sensitivity (BRS) were collected and analyzed via traditional methods using power spectral analysis during seated rest. NK cell number was measured via flow cytometry. Four groups of men (N=12), mean age 38.8 ± 5.3) were studied. Trained subjects completed three weekly sessions of supervised aerobic exercised at 60–75% VO 2max for 10 weeks. Controls performed activities of daily living (ADL) with no formal exercise training. Subjects were categorized as follows: HIV negative untrained (HNU), HIV negative trained (HNT), HIV positive untrained (HPU), and HIV positive trained (HPT). HIV positive subjects were seropositive for HIV disease within 24 months of this investigation. Preliminary data demonstrate a strong trend toward augmented arterial compliance and a higher autonomic profile with increased NK cell number and activity [ HPU mean NK cell level, 160.42/cu mm(10.7%); and HPT was 251.8/cu mm (15.4%)] in response to moderate regular exercise. However, cessation of training exercises resulted in return to near baseline NK cell number, percentage (191.8/cu mm (11.2%).
HNU HNT HPU HPT AC (mmHgxsec) 7.0±3.1 9.4±5.4 4.0±1.5 7.8±1.1 HRV (msec²) 6.1± 2.1 7.6±1.1 3.9±1.5 7.4±0.3 BRS (msec/mmHg) 7.9±6.6 13.9±7.5 8.1±2.9 19.2±8.7
CONCLUSIONS: Enhanced fitness and its effect on vascular compliance and autonomic modulation with moderate regular exercise induces alterations in NK cell number and activity may help improve quality of life, morbidity and mortality, survival and response to therapy in pts with HIV-1 disease.