bluuebunny/crossref_metadata_2025 huggingface dataset:
Nestedness in fragmented landscapes: birds of the box‐ironbark forests of south‐eastern Australia
Authors: Mac Nally Ralph , Horrocks Gregory , Bennett Andrew F.
Published: March 2003

Nestedness in biota as a function of species richness – biota of depauperate assemblages being non‐random subsets of richer biotas – has been widely documented in recent years (see Wright et al. 1998, Oecologia 113: 1–20). Ordering sites by richness maximizes nestedness indices; however, ordering by other criteria such as area or isolation may be more ecologically interpretable. We surveyed birds in true fragments (35 in all), and in “reference areas” in large extant forest blocks (30 locations), of the same range of areas (10, 20, 40, 80 ha). The avifauna was divided into “bush birds”– species dependent on forest and woodland, and “open country” species. We looked at nestedness in four data sets: “bush birds” in fragments and reference areas, and “all birds” in fragments and in reference areas. All data sets were significantly nested. Ordering by area in all cases was not significantly less nested than ordering by richness. Ordering by area in fragments was significantly greater than in reference areas, but the differences in standardized nestedness indices were small (<15%). We identified those birds that had distributions among fragments that conformed strongly with area, those that were more randomly distributed and some species that were more likely to occupy the smallest fragments. Among the latter was a hyperaggressive, invasive, colonial native species (noisy miner Manorina melanocephala). A suite of small, insectivorous birds were more likely to strongly conform with expected distributions in relation to area, which was consistent with observations of their vulnerability to the effects of the noisy miner in smaller fragments.
Acute parvovirus B19 infection mimicking juvenile myelomonocytic leukemia
Authors: Yetgin Sevgi , Çtin Mualla , Yenicesu İdil , Özaltın Fatih , Uçkan Duygu
Published: March 2003

Abstract: An 11‐month‐old patient with parvovirus infection mimicking juvenile myelomonocytic leukemia (JMML) is presented. The patient's history, presenting physical and laboratory features, was suggestive of JMML and consisted of fever, hepatosplenomegaly, lymphadenopathy, desquamation of the skin, anemia, leukocytosis with monocytosis and trilineage dysplastic findings of the peripheral blood and bone marrow. However, positive IgM titers for parvovirus B19 followed by seroconversion, negative cytogenetics and the benign follow‐up of the patient suggested acute parvovirus infection as an etiologic factor for development of dysplastic features in the patient, and thus is recommended for consideration in the differential diagnosis of MDS. Although parvovirus B19 infection mimicking MDS has previously been shown in two patients with spherocytosis and one with subclinical immune deficiency; to our knowledge, the present report is the first describing the association of acute parvovirus B19 infection with dysplastic features mimicking myelodysplasia (MDS) in a child without a demonstrable underlying hematolymphoid disorder.
Phenotype–genotype correlation in Sicilian patients with Hb H
Authors: Mirabile E. , Samperi P. , Di Cataldo A. , Poli A. , La Spina M. , Schilirò G.
Published: March 2003

Abstract: We studied 15 Sicilian subjects with Hb H disease correlating clinical examinations with hematological and molecular data. Seven different α‐thal mutations were identified: four deletion types (‐‐MED, ‐‐CAL, ‐α3.7, ‐α4.2) and three nondeletion types (αNcolα, αHphα, αCSα). All the patients had a zero‐gene chromosome (‐‐MED or ‐‐CAL), while the third α gene was deleted (‐α3.7, ‐α4.2) or inactive (αNcolα, αHphα, αCSα). In patients with the nondeletion genotype the analysis of hematological values revealed lower levels of RBC and Hb A2 and significantly higher levels of Hb H. The clinical variability was remarkable, ranging from totally asymptomatic conditions, casually diagnosed, to severe thalassemia intermedia with marked hemolytic crises, liver and spleen enlargement and the necessity for frequent transfusions. The genotype did not justify the gravity of the phenotype in every case, and the differences in clinical manifestations, also notable, are not easily explainable in subjects who apparently have the same genotype.
Serum oncostatin M in multiple myeloma: impact on disease severity and prognosis
Authors: Koskela K. , Pelliniemi T‐T. , Rajamäki A. , Pulkki K. , Remes K.
Published: March 2003

Abstract: Since high levels of serum IL‐6 predict a poor prognosis of patients with multiple myeloma (MM), we investigated if a related cytokine, oncostatin M (OSM), correlates with clinical or biochemical findings or has prognostic significance in patients with MM. Among 82 newly diagnosed MM patients, OSM was detected in the sera in 45 (55%). Serum OSM had a borderline statistical correlation with serum IL‐6 (r=0.198, p=0.074) and C‐reactive protein (r=0.199, p=0.074) concentrations. However, OSM did not have prognostic significance alone or in combination with other factors. The median survival of patients with detectable serum OSM concentration was 41 months (range 2–124 months) and of OSM negative patients 35 months (1–75 months). Serum OSM concentration was not associated with clinical factors or severity of bone disease at diagnosis. We conclude that serum OSM concentration is not a prognostic factor in MM patients.
Unexpectedly high incidence of hypoplastic/aplastic foci in bone marrow biopsies of hairy cell leukemia patients in remission following 2‐chlorodeoxyadenosine therapy
Authors: Gillis Shmuel , Amir Gail , Bennett Michael , Polliack Aaron
Published: March 2003

Abstract: Treatment with the purine analog 2‐chlorodeoxyadenosine (2‐CDA) achieves a complete response in close to 90% of patients with hairy cell leukemia, with approximately 75% remaining in prolonged remission. Recently, we unexpectedly noted foci of hypoplasia and aplasia in routine follow‐up bone marrow biopsies of several hairy cell leukemia patients in remission with normal blood counts. Because of this finding we examined all available biopsies to assess the incidence of this phenomenon. A total of 94 biopsies in 31 patients were reviewed. Of these, 23 were prior to 2‐CDA therapy and 71 (in 30 patients) were obtained 2–76 (mean 22) months following one or more courses of treatment. Nine patients had also received prior interferon and 7 (of whom 3 had also received interferon) had undergone splenectomy. Hypocellular foci were found in only 3 (13%) of the pre‐therapy biopsies. Forty‐seven of the 71 post‐therapy biopsies (in 23 patients) (66%) had a total of 176 hypocellular foci. Of these 47 biopsies, 39 were without evidence of disease. A simultaneous complete blood count was normal in 34 of the 47 hypoplastic biopsies (72%). This suggests that these hypoplastic areas may not be representative of the entire bone marrow and that normal hematopoiesis may take place at other sites. However, since the longest follow‐up is less than 7 yr, the potential long‐term significance of these findings, such as progressive bone marrow aplasia or dysplasia, may still be unrecognised.
Iron status markers in 224 indigenous Greenlanders: influence of age, residence and traditional foods
Authors: Milman Nils , Byg Keld‐Erik , Mulvad Gert , Pedersen Henning Sloth , Bjerregaard Peter
Published: March 2003

Abstract: Objective: To evaluate iron status in indigenous Greenlanders and its relationship to gender, age and intake of traditional Greenlandic foods. Methods: Serum ferritin, serum transferrin saturation and haemoglobin were evaluated in a population survey in 1993–1994 comprising 224 Greenlandic individuals (109 men) aged 19–82 yr. The participants were residents in the capital Nuuk (n=73) with a predominantly Western style of living, the town Ilulissat (n=60) with a mixture of Western and Greenlandic style of living, and the small town Uummannaq (n=91) with a predominantly Greenlandic style of living. Consumption of traditional foods was assessed by questionnaire. Results: Intake of traditional foods was more prevalent among elderly than among young individuals and more frequent in Uummannaq than in Ilulissat and Nuuk. Ferritin levels were higher in men than in women (p<0.0001). Median ferritin levels were lowest in Nuuk (men, 92 µg/L; women, 40 µg/L), higher in Ilulissat (men, 104 µg/L; women, 69 µg/L) and in Uummannaq (men, 118 µg/L; women, 46 µg/L) (p<0.001). The prevalence of iron load (ferritin >200 µg/L) was lowest in Nuuk (men: 13.8%, women: 2.3%) intermediate in Ilulissat (men, 11.1%; women, 9.1%) and highest in Uummannaq (men, 32.1%; women, 21.1%). The prevalence of iron depletion (ferritin <16 µg/L) was high in Nuuk (men, 0%; women, 20.5%), and lower in Ilulissat (men, 3.7%; women, 6.1%) and in Uummannaq (men, 0%; women, 10.5%). The prevalence of iron deficiency anaemia (ferritin <13 µg/L and Hb <5th percentile for iron‐replete men and women) was 0.92% in men and 0.87% in women. Correlations between age and ferritin were lowest in Nuuk (men, rs=0.26, p=0.2; women, rs=0.50, p=0.001) intermediary in Ilulissat (men, rs=0.37, p=0.06; women, rs=0.73, p<0.0001) and highest in Uummannaq (men, rs=0.59, p<0.0001; women, rs=0.74, p<0.0001). Intake of traditional foods was correlated with ferritin in men (rs=0.29, p=0.01) and women (rs=0.40, p<0.0001). Conclusion: The observed differences in estimated body iron stores in Greenlanders from the three residential areas can be explained by differences in the dietary intake of haem iron.
Glucosamine sulfate does not crossreact  with the antibodies of patients with  heparin‐induced thrombocytopenia
Authors: Weimann Gerrit , Lubenow Norbert , Selleng Kathleen , Eichler Petra , Albrecht Dorothea , Greinacher Andreas
Published: March 2003

Abstract: Objective: To assess the crossreactivity of glucosamine sulfate, used for treatment of degenerative joint disease with antibodies induced in heparin‐induced thrombocytopenia (HIT). Background: HIT is a severe adverse effect of heparin therapy induced by an immunological mechanism. The antibodies in HIT are induced by a complex of heparin and, in most cases, platelet factor 4. Hereby generation of the antigen is not strictly dependent on heparin. Heparin can be substituted by a variety of polysulfated carbohydrates. In vitro and in vivo crossreactivity of HIT antibodies has been demonstrated for a chemically polysulfated chondroitin‐like substance (Arteparon®, Luitpoldwerke, Munich, Germany), formerly used for chondroprotection. Another drug widely used in the treatment of degenerative joint disease is glucosamine sulfate. Glucosamine is a building block of glycosaminoglycans, of which heparin is the clinically most important. Many patients with degenerative joint disease use glucosamine sulfate. This group is also at the highest risk to develop HIT following joint replacement surgery. Methods: We examined the interactions of glucosamine sulfate (DONA 200‐S, Opfermann, Wiehl, Germany) with platelets and antibodies of patients with HIT in and without the presence of heparin. Sera of 5 HIT patients and platelets of 4 healthy donors were used. The binding of HIT antibodies to PF4/glucosamine sulfate complexes was assessed by an ELISA. Results: HIT antibodies did not activate platelets in the presence of glucosamine sulfate in a serotonin‐release assay. Preincubation with glucosamine sulfate did not inhibit platelet activation by HIT antibodies in the presence of heparin (0.2 IU/ml). Antibodies bonded to PF4/heparin but not to PF4/glucosamine sulfate complexes. Conclusions: In contrast to sulfated glycosaminoglycans, there is no evidence for an immunological crossreactivity of HIT antibodies between heparin and glucosamine sulfate.
Pretreatment leukaemia cell drug resistance is correlated to clinical outcome in acute myeloid leukaemia
Authors: Nørgaard Jan M. , Langkjer Sven Tyge , Palshof Torben , Pedersen Bent , Hokland Peter
Published: March 2003

Abstract: In 85 adult patients diagnosed with acute myeloid leukaemia (AML) and treated at the same institution during a 5‐yr period, the clinical significance of in vitro cellular drug resistance to the anthracyclines aclarubicin (Acla) and daunorubicin (Dau) as well as the nucleoside analogue cytarabine (Ara‐C) was investigated using a 4‐d MTT (3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide) assay. In 59 patients of whom 40 were treated by the combination of Acla and Ara‐C we found that leukaemia cell drug resistance towards Acla was higher (by a factor 2.80) in patients who failed to enter complete remission (CR) after the first cycle of induction chemotherapy as compared to patients who entered complete remission. The relationship was significant in univariate as well as multivariate analysis (p=0.02 and 0.03, respectively). By contrast, no in vitro single drug resistance values were consistently correlated to other parameters of clinical outcome (overall CR rate, overall survival (OS), or continuous complete remission (CCR)), whereas the combined Acla and Ara‐C drug resistance profile (Acla/Ara‐C DRP) was of prognostic significance to overall survival of all 85 patients (p=0.004) as well as to the CCR of 39 complete responders (p=0.04). These findings remained statistically significant in multivariate analyses correcting for other variables influencing clinical outcome including patient age, leukocyte count, karyotype, FAB‐subtype, and presence/absence of secondary AML. We conclude that the in vitro drug resistance of leukaemia cells at time of disease presentation appears to be independent of prognostic significance to short‐ and long‐term clinical outcome in AML.
Kinetics of BCR‐ABL fusion transcript levels in chronic myeloid leukemia patients treated with STI571 measured by quantitative real‐time polymerase chain reaction
Authors: Stentoft Jesper , Pallisgaard Niels , Kjeldsen Eigil , Holm Mette Skov , Nielsen Johan Lanng , Hokland Peter
Published: March 2003

Abstract: The activated tyrosine kinase, which arises as a result of the balanced t(9,22) translocation in chronic myeloid leukemia (CML), is thought to be essential for the development of the leukemic phenotype. Recently, designer drugs have been introduced which specifically inhibit such specific kinases. Among these, STI571 (GlivecTM) has entered clinical trials and shown promising activities in chronic phase (CP), accelerated phase (AP) and blast crisis (BC) as evidenced by significant hematological and cytogenetic responses in CML patients. To evaluate the effect of STI571 at the molecular level we have employed quantitative real‐time PCR (RQ‐PCR) to measure the amount of BCR‐ABL fusion transcript in a series of 19 patients treated with STI571, either in CP(11) or in (AP)(8) of the disease for 3–9 months (median 6 months). Employing this method, which is able to detect at least one BCR‐ABL+ cell in 500 000, in serial blood and bone marrow specimens we found decreases in transcript levels in 10/11 CP patients, but only in 1/8 of the AP patients. When present such decreases were gradual and became evident only after 3 months of STI571 treatment, and their kinetics in blood closely mirrored those seen in parallel marrow samples. Moreover, decreases were between 10‐ and 100‐fold in 11/13 patients, with only two patients reaching residual disease levels below 10−2 (a 900‐fold decrease). Thus, no patient reached PCR negativity. We conclude that the RQ‐PCR method is a highly suitable tool for following the effect of STI571 in CML and that further validation of the method, performed in a prospective manner, will contribute significantly to the elucidation of the proper role of STI571 in CML.
Analysis of the stem cell sparing properties of cyclophosphamide
Authors: Gardner Renee V. , McKinnon E. , Astle C. M.
Published: March 2003

Abstract: Objectives: Cyclophosphamide was examined for its ability to spare the most primitive hematopoietic stem cell (PHSC). Methods: C57BL6/J mice (Groups A and B) were sacrificed 24 h and 4–6 wk, respectively, after a single or second injection of low‐dose cyclophos‐phamide (90 mg/kg) on days 1, 3, 7, or 15. A competitive repopulation assay was then performed, using B6‐HbbdGpi‐1a competitor cells, to determine the repopulating ability of exposed PHSC. Results and Conclusions: PHSC function was preserved after a single injection of cyclophosphamide and after a second injection on days 7 and 15 in both groups. In Group A, PHSC repopulating ability declined after a second injection on days 1 and 3 (p<0.05 only for day 1), as did repopulating units [RU]; PHSC numbers did not. In Group B, an insignificant decrease in repopulating ability and RU numbers was observed after a second injection on days 1 and 3, suggesting different etiologies for losses in the 2 groups, or correction of drug‐induced defects within 1 month of cyclophosphamide administration. Total RU increased in single, day 1, 7 and 15 treatment groups. A significant number of marrow cells entered the S phase after cyclophosphamide dosing on day 3, and it is possible that a relationship exists between cell cycling and replicative damage. DNA damage was also increased 1 and 3 d after cyclophosphamide administration, although the significance of differences from controls was not definitive. Conclusion: Low‐dose cyclophosphamide can spare stem cells, depending upon the timing of subsequent doses.
Important region in the β‐spectrin C‐terminus for spectrin tetramer formation
Authors: Luo Bing‐Hao , Mehboob Shahila , Hurtuk Michael G. , Pipalia N. H. , Fung L. W.‐M.
Published: March 2003

Abstract: Many hereditary hemolytic anemias are due to spectrin mutations at the C‐terminal region of β‐spectrin (the βC region) that destabilize spectrin tetramer formation. However, little is known about the βC region of spectrin. We have prepared four recombinant β‐peptides of different lengths from human erythrocyte spectrin, all starting at position 1898 of the C‐terminal region, but terminating at position 2070, 2071, 2072 or 2073. Native polyacrylamide gel electrophoresis showed that the two peptides terminating at positions 2070 and 2071 did not associate with an N‐terminal region α‐peptide (Spα1–156) in the micromolar range. However, the peptides that terminated at positions 2072 and 2073 associated with the α‐peptide. Circular dichroism results showed that the unassociated helices in both α‐ and β‐peptides became associated, presumably to form a helical bundle, for those β‐peptides that formed an αβ complex, but not for those β‐peptides that did not form an αβ complex. In addition, upon association, an increase in the α‐helical content was observed. These results showed that the β‐peptides ending prior to residue 2072 (Thr) would not associate with α‐peptide, and that no helical bundling of the partial domains was observed. Thus, we suggest that the C‐terminal segment of β‐spectrin, starting from residue 2073 (Thr), is not critical to spectrin tetramer formation. However, the C‐terminal region ending with residue 2072 is important for its association with α‐spectrin in forming tetramers.
Pulmonary embolism associated with tranexamic acid in severe acquired haemophilia
Authors: Taparia Minakshi , Cordingley Frank T. , Leahy Michael F.
Published: March 2003

Abstract: Tranexamic acid (an antifibrinolytic agent) is of proven benefit in the treatment of bleeding in patients with congenital and acquired coagulation disorders. We report the case of a patient with an acquired Factor VIII inhibitor, who was on a prophylactic dose of tranexamic acid because of recurrent bleeding episodes and developed a pulmonary embolism. Although studies using tranexamic acid have not shown a definite increased risk for the development of venous thrombosis, this is the likely cause of the pulmonary embolism in this patient.
Remission of transformed myelodysplastic syndrome with fibrosis after danazol therapy
Authors: Damaj G. , Lefrère F. , Canioni D. , Rubio M.T. , Radford‐Weiss I. , Valensi F. , Varet B. , Hermine O.
Published: March 2003

Abstract: Danazol has been used with success in some hematological diseases, but there is no report of this treatment in acute leukemia. We report here a case of remission of myelodysplastic syndrome with myelofibrosis in transformation after danazol therapy in a 72‐yr‐old man. The role of danazol in remission induction is briefly discussed.
The prognostic significance of CA 125 in patients with non‐Hodgkin's lymphoma
Authors: Zacharos Ioannis D. , Efstathiou Stamatis P. , Petreli Elisa , Georgiou George , Tsioulos Dimitrios I. , Mastorantonakis Stylianos E. , Christakopoulou Ioulia , Roussou Paraskevi P.
Published: March 2003

Abstract: Objective: To assess the association of serum CA 125 in patients with non‐Hodgkin's lymphoma (NHL) with prognostic parameters of the disease, response to treatment, and survival. Patients and methods: Sixty‐eight patients [38 males, median age 56 (range 17–82) yr] with NHL were evaluated. CA 125 was measured by an enzyme immunoradiometric assay at diagnosis and at the end of first‐line treatment. Results: Median overall CA 125 was 49 (1–963) U mL−1, whereas 49 patients had initially abnormal (>35 U mL) CA 125 levels. High CA 125 was found to correlate with failure of treatment (P = 0.001) and relapse (P = 0.01), and to be independently associated with bulky disease, effusions, LDH, and the International Prognostic Index (IPI) score (P<0.01 for each of these four variables). An initially abnormal CA 125 value was associated with poorer 5‐yr survival [median survival of patients with CA 125>35 U mL−1 33 (18–72) months compared to 58 (20–77) months for those with CA 125 = 35 U mL−1, P = 0.012]. Moreover, CA 125>35 U mL−1 (among stage III/IV and LDH>460 mU mL−1) emerged as an independent predictor of death within 5 yr from diagnosis (Relative Risk (RR) 3.1, 95% CI 1.5–12.8, P = 0.02). Conclusion: Measurement of serum CA 125 is useful for staging, monitoring, and estimating prognosis in patients with NHL.
Cytogenetic, FISH, and molecular studies in a case of B‐cell chronic lymphocytic leukemia with karyotypic evolution
Authors: Chena Christian , Cerretini Roxana , Noriega María Fernanda , Narbaitz Marina , Scolnik Mariano , Palacios María Fernanda , Neme Daniela , Bruno Salvador , Slavutsky Irma
Published: March 2003

Abstract:  We report the clinical, cytogenetic, fluorescence in situ hybridization (FISH) and molecular findings in a 54‐yr‐old male patient diagnosed with B‐cell chronic lymphocytic leukemia (B‐CLL), who showed progression to a diffuse large B‐cell lymphoma (Richter's syndrome). Genetic studies were performed at diagnosis and during the Richter's transformation (RT). A clonal karyotype with two dicentric chromosomes, psu dic(12,21)(q24;q10) and dic(17,18)(p11.2;p11.2), was found. Both rearrangements were confirmed by FISH. Molecular cytogenetics analysis using p53 probe showed monoallelic loss of this tumor suppressor gene in 43.8% and 77.3% of cells for the first and the second studies, respectively). In both studies, deletions of D13S319 (18% and 12% of cells) and D13S25 loci (13% and 12% of cells) at 13q14 were found. Polymerase chain reaction analysis showed the MBR/JH rearrangement of the bcl‐2 gene. FISH studies using LSI bcl‐2/IgH probe allowed quantifying the clonal cell population with this rearrangement (4% and 6.6% of cells at diagnosis and RT, respectively). To our knowledge, this is the first case with a psu dic(12,21) described in B‐CLL. The low percentage of cells with the 13q14 deletion and bcl‐2/IgH rearrangement suggests that they were secondary events that resulted from clonal evolution. Our patient had a short survival (9 months) and a clear lack of response to several therapeutic agents, confirming the association of p53 gene deletion and karyotypic evolution with disease progression.
Anti‐HPA‐1a‐mediated platelet phagocytosis by monocytes in vitro and its inhibition by Fc gamma receptor (FcγR) reactive reagents
Authors: Wiener E , Abeyakoon O , Benchetrit G , Lyall M , Keler T , Rodeck C. H.
Published: March 2003

Abstract: The study was undertaken to delineate mechanisms of platelet destruction by phagocytosis during fetal/neonatal alloimmune thrombocytopenia (FAIT/NAIT) because of maternal antibodies against human platelet antigen 1a (HPA‐1a). By employing a platelet phagocytosis assay based on the ORPEGEN flow cytometric bacterial phagocytosis test, we measured monocyte ingestion of platelets mediated by anti‐HPA‐1a antibodies. Moreover, we tested, as potential therapeutic agents, FcγR reactive reagents, for their inhibition of this process.

Four of six anti‐HPA‐1a sera tested mediated phagocytosis of HPA‐1a‐positive platelets in a concentration‐dependent manner. Monocyte ingestion of platelets was almost completely inhibited by cytochalasin D. No anti‐HPA‐1a‐mediated phagocytosis was observed with anti‐HPA‐1a‐negative platelets. The humanised anti‐FcγRI monoclonal antibody H22 at concentrations 1–100 μg/ml, completely inhibited anti‐HPA‐1a‐mediated phagocytosis as did similar concentrations of ivIg. By contrast, a mouse monoclonal anti‐FcγRII (IV.3, Fab) at 10 μg/ml caused little or no suppression of platelet phagocytosis mediated by two anti‐HPA‐1 sera. Furthermore, the addition of anti‐FcγRII (10 μg/ml) to sub‐optimal concentrations of H22 did not significantly increase the inhibitory effect of the latter compound. Monomeric IgG (0.1–10 μg/ml) failed to suppress anti‐HPA‐1 mediated platelet ingestion by the phagocytes, as did anti‐FcγRIII.

To our knowledge this is a rare example of an assay that measures platelet phagocytosis in vitro. The results suggest that FcγRI plays a major role in anti‐HPA‐1a‐mediated platelet phagocytosis by monocytes while FcγRIIa, is of little or minor importance only. Moreover, the findings indicate the use of H22 as an alternative to interavenous Ig (ivIg) in the management of FAIT/NAIT.
Quantitative real‐time reverse‐transcription polymerase chain reaction for diagnosis of BCR‐ABL positive leukemias and molecular monitoring following allogeneic stem cell transplantation
Authors: Neumann Frank , Herold Caroline , Hildebrandt Barbara , Kobbe Guido , Aivado Manuel , Rong Astrid , Free Maren , Rössig Renate , Fenk Roland , Schneider Peter , Gattermann Norbert , Royer‐Pokora Brigitte , Haas Rainer , Kronenwett Ralf
Published: March 2003

Abstract: Real‐time reverse‐transcription polymerase chain reaction (RT‐PCR) (qPCR) of the BCR‐ABL mRNA is a suitable technique to measure the amount of circulating leukemic cells in chronic myelogenous leukemia (CML). In this study, we evaluated a BCR‐ABL‐specific qPCR method using the LightCycler technology in 95 patients with Philadelphia chromosome positive acute leukemia (n = 7) or CML in different stages (n = 88). Primers and hybridization probes were chosen to detect the most prevalent variants of BCR‐ABL (b2a2, b3a2, b2a3, b3a3, e19a2, e1a2) with a sensitivity of 10−5 for b2a2 and b3a2. With median BCR‐ABL/G6PDH ratios of 10.7% in the untreated chronic phase, 43.2% in the newly diagnosed accelerated phase, and 131.4% in newly diagnosed blast crisis the BCR‐ABL mRNA levels varied significantly between different stages of CML whereas no difference was found between blast crisis and untreated acute leukemias (136.9%). There was a strong relationship between qPCR results and cytogenetics in patients treated with imatinib, interferon‐α, or following allografting. Thirteen patients with CML were sequentially examined by qPCR following myeloablative or non‐myeloablative allogeneic peripheral blood stem cell transplantation. Five patients received donor lymphocytes and became BCR‐ABL negative as confirmed by nested RT‐PCR. The gradual disappearance of BCR‐ABL positive cells could be monitored by qPCR following non‐myeloablative transplantation. Comparison of BCR‐ABL levels with the degree of donor chimerism showed that 91% of samples with complete donor chimerism were BCR‐ABL negative. In 22% of BCR‐ABL negative samples chimerism between 71% and 98% was observed, indicating the persistence of normal recipient's hematopoietic cells. In conclusion, the qPCR protocol used in this study is a reliable and fast method for monitoring molecular response in CML.
A novel missense mutation (C622G) in the zinc‐finger domain of the human hairless gene associated with congenital atrichia with papular lesions
Authors: Aita V. M. , Ahmad W. , Panteleyev A. A. , Kozlowska U. , Kozlowska A. , Gilliam T. C. , Jablonska S. , Christiano A. M.
Published: March 2003

Abstract: Congenital atrichia with papular lesions is a rare, recessively inherited form of hair loss characterized by a complete absence of all body hair shortly after birth. Mutations in the human ortholog of the mouse hairless (hr) gene have been implicated in the pathogenesis of this disorder. In this study, we screened, by direct sequence analysis, the hairless gene in a family of Polish descent and identified a novel missense mutation (C622G). The mutation alters the third of four invariant cysteins in the zinc‐finger domain, which has high homology to the C‐X‐X‐C‐(X)17‐C‐X‐X‐C structure of the zinc‐fingers of the GATA family of transcription factors. The human hairless gene encodes a putative transcription factor with restricted expression in the brain and skin, which is involved in the regulation of apoptosis during catagen remodeling in the hair cycle.
Electrophysiological characterization of human keratinocytes using the patch‐clamp technique
Authors: Wohlrab D. , Wohlrab J. , Markwardt F.
Published: March 2003

Abstract: The living, not‐cornified part of the epidermis consists mainly of keratinocytes. The control mechanisms of proliferation and differentiation are only partly understood. Similarly, the influence of ionic channels of the cell membrane on the proliferation of keratinocytes remains unclear. Preliminary investigations point to a relation between the opening of ionic channels and keratinocyte proliferation. Therefore, voltage clamp experiments were performed to gain further knowledge of the electrophysiological characteristics of human keratinocyte cellular membranes. In‐vitro cultured keratinocytes of the cell line HaCaT were characterized by means of the voltage‐clamp technique. As measured in the whole‐cell configuration, changing the extracellular K+ or Cl−‐concentration shifted the membrane potential of HaCaT cells. Application of the patch‐clamp‐technique in the cell‐attached and inside–out configuration revealed an ionic channel with a conductance of multiples of 200 pS. The reversal potential of the single channel current was shifted by substituting of intracellular Cl− by aspartate−. Channel openings disappeared after addition of 0.1 mM of the anion channel blocker 4′,4′ diisothiocyanato‐stilbene‐2‐2′ disulfonic acid (DIDS). It is concluded that this channel contributes to the Cl−‐conductance of the cellular membrane and is a determinant of the membrane potential of human keratinocytes. This channel may represent a target for pharmacological manipulation of the membrane potential and possibly the growth of human keratinocytes in dermatological proliferation disorders.
Identification of desmoglein 1 as autoantigen in a patient with intraepidermal neutrophilic IgA dermatosis type of IgA pemphigus
Authors: Kárpáti Sarolta , Amagai Masayuki , Liu Wen Li , Dmochowski Marian , Hashimoto Takashi , Horváth Attila
Published: March 2003

Abstract: In a 51‐year‐old female patient with intraepidermal neutrophilic IgA dermatosis (IEN) type of IgA pemphigus, circulating IgA, but not IgG, autoantibodies were detected to bind to the cell surface of the whole epidermis, being much stronger in the upper epidermis. In the patient's skin a heavy intraepidermal IgA staining was observed throughout the whole epidermis, accompanied by a weak IgG and a more prominent C3 staining. IgA from the patient's serum showed no reactivity either with epidermal proteins by immunoblot analysis, or with COS 7 cells transiently transfected with mammalian cell expression constructs containing full length human Dsc1, Dsc2 and Dsc3. Our patient's IgA specifically reacted with conformational epitopes of human desmoglein (Dsg) 1 but not Dsg 3, when studied in a previously established, here for IgA antibody detection modified enzyme‐linked immunoabsorbent assay (ELISA) of baculovirus expression system. The immunoreactivity against keratinocyte cell surface was completely removed from the serum of the patient by pre‐incubation with recombinant Dsg1 baculoprotein. This finding indicates that the sera possess only IgA antibodies against the extracellular domain of Dsg1 baculoprotein, but no antibodies against components of keratinocyte cell surface other than Dsg1. This is the first case of IgA pemphigus where Dsg1 has been identified as the autoantigen.
Lectin and proteoglycan histochemistry of Merkel cell carcinomas
Authors: Sames K. , Schumacher U. , Halata Z. , Van Damme E. J. M. , Peumans W. J. , Asmus B. , Moll R. , Moll I.
Published: March 2003

Abstract: Changes in carbohydrate residue expression and in proteoglycan distribution occur during different stages of tumor development and progression. However, few data concerning carbohydrate residue analysis as performed by lectin histochemistry and proteoglycan distribution of Merkel cell carcinoma, a rare malignant tumor of the skin, have been reported. Hence, lectin‐ and proteoglycan immunohistochemistry was performed on paraffin wax material of 9 cases of Merkel cell carcinomas characterized by cytokeratin and neurofilament immunohistochemistry. The lectin binding pattern of tumor cells varied between lectins with different sugar binding specificities, while within a given nominal sugar specificity intensities were remarkably similar between tumors from different patients. The most intensive reaction was observed using Con A (mannose/glucose‐specific) followed by LCA with the same specificity and the N‐Acetyl glucosamine‐specific lectins (WGA, UDA, CMA), while no fucose binding sites were detected (UEA‐I). In addition, N‐Acetyl galactosamine residues were only occasionally detected. The lectin binding pattern of Merkel cell carcinoma cells indicated that predominantly N‐linked glycans and not O‐linked glycans, typical for mucins of most epithelia, were present. Hence these tumor cells were relatively undifferentiated and resembled stem cells more closely than differentiated epithelia. The tumor stroma was especially evaluated in this study and showed a lectin reaction, which was intermediate between the tumor cells and extra‐tumoral stroma. For example, the reactions of N‐Acetyl galactosamine‐specific lectins were intensive in the extra‐tumoral stroma but nearly negative in tumor cells, while the lectin reaction of the intra‐tumoral stroma was similar to the cellular reaction. These results indicated an influence of tumor cells on the stromal constituents. Antibodies against chondroitin type glycosaminoglycans reacted with the tumor stroma and the pericellular substance around the tumor cells most intensely in – and around the major tumor septae which, in general, were well vascularized. The most intensive immunoreactivity was detected using the chondroitin‐6‐sulfate antibody. The cellular and membrane‐associated reaction for heparan sulfate was less intensive in comparison to epidermal cells. In conclusion the pattern of lectin‐binding sites, the high chondroitin(sulfate) specific reactivity and the relatively low intensity of heparan sulfate immunohistochemistry indicate a low degree of differentiation and high malignity of the tumors, which is consistent with the clinical behavior of Merkel cell carcinomas.
Human skin mast cells rapidly release preformed and newly generated TNF‐α and IL‐8 following stimulation with anti‐IgE and other secretagogues
Authors: Gibbs B. F. , Wierecky J. , Welker P. , Henz B. M. , Wolff H. H. , Grabbe J.
Published: March 2003

Abstract: Several groups have previously reported that rodent or human leukemic mast cells produce inflammatory cytokines such as TNF‐α and IL‐8 as well as the pro‐allergic cytokines IL‐4, IL‐5 and IL‐13. Comparatively little is known, however, regarding the ability of normal human skin mast cells to secrete these factors following either IgE‐dependent or IgE‐independent modes of activation. We therefore investigated whether normal human skin mast cells produce these cytokines following stimulation by a variety of secretagogues. Enriched isolated skin mast cells released both TNF‐α and IL‐8 following activation with either anti‐IgE, SCF, substance P, compound 48/80 or A23187. This release was dose‐ and time‐dependent, with maximal levels being reached within 4 h of stimulation involving, in part, the secretion of preformed stores of both cytokines. In accordance with this, using lysates of highly purified (>90%) skin mast cells, we could demonstrate that both TNF‐α and IL‐8 mRNA and protein were present in both unstimulated as well as stimulated mast cells. In stark contrast to these results, no significant levels of either IL‐4, IL‐5 or IL‐13 were detected, regardless of the secretagogue used or the period of stimulation. These results show that human skin mast cells are capable of rapidly secreting pro‐inflammatory cytokines like TNF‐α and IL‐8 following IgE‐dependent activation and stimulation by the neuropeptide substance P, SCF and the basic polypeptide analogue compound 48/80. In contrast to other types of human mast cells however, human skin mast cells were incapable of secreting IL‐4, IL‐5 or IL‐13 in these settings.
Different pathways leading to cutaneous leukocytoclastic vasculitis in mice
Authors: Sunderkötter C. , Seeliger S. , Schönlau F. , Roth J. , Hallmann R. , Luger T. A. , Sorg C. , Kolde G.
Published: March 2003

Abstract: To investigate the pathomechanisms of leukocytoclastic vasculitis (LcV) we compared mouse models of LcV with non‐vasculitic irritant contact dermatitis (ICD). Criteria for LcV as met by the immune complex‐mediated Arthus reaction (Art‐r) were also fulfilled by the localized Shwartzman reaction (Shw‐r) and by cutaneous Loxoscelism (Lox) (injection of venom from Loxosceles reclusa containing sphingomyelinase D). After depletion of PMN (by γ‐irradiation) vessel damage could not be elicited in these models, distinguishing them from models of direct endothelial insult (necrotizing ICD). Depletion of complement could only delay, but not inhibit the Art‐r, and did not change ICD, Lox or the Shw‐r. The Shw‐r exclusively revealed a sustained local expression of vascular adhesion molecules for 24 h in the preparatory phase (LPS s.c.), not observed in the Art‐r, in Lox or ICD. Subsequent challenge with LPS i.p. was associated with upregulation of Mac‐1 and ICAM‐1 on PMN, but not of VLA‐4 or LFA‐1 (FACS analysis). Cytokines which were able to replace LPS in priming for LcV in the Shw‐r (TNF‐α and IL‐1β) also induced sustained expression of adhesion molecules, whereas IL‐12 and IFN‐γ did neither. Neutralizing IL‐12 or IFN‐γ also inhibited neither LcV nor sustained expression of adhesion molecules, whereas anti‐TNF‐α inhibited both. Anti‐TNF‐α had no marked inhibitory effects in the Art‐r, in Lox or ICD. Combined (but not separate) neutralization of both E‐selectin and VCAM‐1 by antibodies suppressed LcV independent from reducing influx of PMN, proving that their sustained expression is decisive for the Shw‐r and interferes with normal diapedesis. Since Loxosceles venom is known to dysregulate diapedesis and degranulation of PMN in vitro, since adherent immune complexes activate PMN at the vessel wall, and since adhesion molecules are dysregulated in the Shw‐r, we suggest that LcV develops when activation of PMN coincides with vascular alterations which interfere with normal diapedesis.
Effect of skin barrier competence on SLS and water‐induced IL‐1α expression
Authors: Gibbs Susan , Vietsch Helene , Meier Ursi , Ponec Maria
Published: March 2003

Abstract: For screening of a potential irritant it is essential that an early marker for irritation should be chosen which could be detected before the physiological signs of irritation occur. Interleukin 1 alpha (IL‐1α) is widely accepted as such a marker in both in vivo and in vitro test systems. In this study, we have determined the mRNA levels of IL‐1α in the epidermis after topical application of sodium dodecyl sulphate (SLS) in both a commercially available epidermal kit (EpiDerm) and in excised skin. Furthermore, we have determined the effect of water, the vehicle for SLS, on IL‐1α mRNA levels. Topical application of water to excised skin increases IL‐1α mRNA levels sixfold in the epidermis whereas topical application of water to EpiDerm cultures did not alter IL‐1α mRNA levels. This is explained by the finding that EpiDerm cultures have a sub‐optimal barrier function when compared with excised skin – topical application of SLS was clearly toxic at much lower concentrations in EpiDerm cultures (0.2% SLS) than in excised skin (5% SLS). Also caffeine penetration was 10‐fold higher through EpiDerm cultures than through the excised skin. Therefore, incubation of control EpiDerm cultures at 100% humidity effectively mimics topical exposure to water. An additional increase in IL‐1α mRNA levels observed between topical application of water and SLS is similar (about threefold) in both experimental systems. In conclusion, in vitro reconstructed epidermis models, such as EpiDerm, can be used as a predictive model for irritancy screening. However, great care should be taken when interpreting the results due to the fact that EpiDerm cultures do not have a competent barrier function and therefore lower irritant concentrations are required than in in vivo or ex vivo studies in order to induce cytotoxic effects. Furthermore, the irritant effects of the vehicle should not be neglected. Our results show clearly that the topical application of water to excised skin results in increased levels of IL‐1α mRNA in the epidermis. This is a cytokine that is widely used as an early marker for skin irritation.
Expression of the C‐C chemokine MIP‐3α/CCL20 in human epidermis with impaired permeability barrier function
Authors: Schmuth M. , Neyer S. , Rainer C. , Grassegger A. , Fritsch P. , Romani N. , Heufler C.
Published: March 2003

Abstract: External assault to the skin is followed by an epidermal response including synthesis of DNA, lipids, cytokines and migration of antigen presenting cells. MIP‐3α (CCL20, LARC, Exodus‐1, Scya20) is a recently described C‐C chemokine, predominantly expressed in extralymphoid tissue, which is known to direct migration of dendritic cell precursors and memory lymphocytes to sites of antigen invasion. We assessed the expression of MIP‐3α in human skin using semi‐quantitative polymerase chain reaction. In vivo, MIP‐3α mRNA was constitutively expressed at low levels in untreated human epidermis. After acute disruption of the epidermal permeabiltiy barrier MIP‐3α mRNA was upregulated in the epidermal fraction, whereas dermal MIP‐3α mRNA levels remained unchanged. In vitro, MIP‐3α was increased in cultured keratinocytes treated with IL‐1α and TNF‐α and was present in immature and mature dendritic cells, THP‐1 monocytic cells and activated T cells. Finally, skin biopsies from patients with psoriasis, contact dermatitis and mycosis fungoides showed abundant expression. In biopsies from atopic dermatitis and graft vs. host disease a weak signal was present, whereas no expression was found in scleroderma and toxic epidermal necrolysis. We conclude that regulation of MIP‐3α mRNA is part of the epidermal response to external assault. Its upregulation may represent a danger signal for increased immunosurveillance in barrier disrupted skin and inflammatory skin conditions with impaired barrier function to counteract potential antigen invasion.
Stimulation of Kaposi's sarcoma cell growth by urine from women in early pregnancy, the current source for clinical‐grade human chorionic gonadotropin preparations
Authors: Simonart T. , Hermans P. , Delogne‐Desnoeck J. , Van Vooren J. P. , Meuris S.
Published: March 2003

Abstract: Clinical‐grade preparations of human chorionic gonadotropin (hCG) have been shown to be toxic to Kaposi's sarcoma (KS) cells. However, the results of clinical studies using commercial hCG preparations KS remain highly contradictory. More particularly, some hCG preparations could have a paradoxical growth effect on KS. Such discrepant results may be explained by the fact that the anti‐KS activity is not associated with hCG itself but with one or more factors that are co‐purified with the hormone. We found here that crude urine from first trimester pregnant women, the current source for commercial hCG, had a growth stimulatory effect on KS cells. By contrast, urine from last trimester pregnant women, from non‐pregnant young women, from menopausal women and from men exhibited neither a growth stimulatory nor a growth inhibitory effect on KS cells. The amplitude of this pregnancy urine‐associated pro‐KS activity/hCG unit was higher than that achieved with clinical‐grade hCG preparations. Partial co‐purification of pregnancy‐associated factors during the extraction procedure of commercial hCG from urine may explain the pro‐KS activity achieved with some hCG preparations. We, therefore, suggest a cautious use of hCG purified from pregnancy urine for the treatment of KS.
Efficacy of naltrexone on acetylcholine‐induced alloknesis in atopic eczema
Authors: Heyer G. , Groene D. , Martus P.
Published: March 2003

Abstract: Atopic eczema (AE) is a chronically pruritic inflammatory skin disease. Although the mediators and exact mechanisms eliciting and sustaining pruritus are not completely known, AE patients in clinical trials have been shown to benefit under treatment with morphine antagonists. Naltrexone (NAL) is a relatively pure morphine antagonist that blocks the effects of opioids twice as much as naloxone. NAL exhibits minimal pharmacological activity and displaces endorphines at mu‐ and kappa‐receptors without its own intrinsic activity. NAL's excellent oral bioavailability and linear increases in the area under plasma concentration‐time curve make it ideal for use in experimental studies. We designed our present experiments similar to former experiments evaluating both peripheral cutaneous sensations and central itch procession in order to gain more information about the possible distribution of opioid receptors and their involvement in the pathophysiology of pruritus. Eleven AE patients participated in our double‐blind study. Either 25 mg of NAL (Nemexin®) or a placebo (PLA) was given to the participants 60 min prior to the acetylcholine (ACH) injection [intracutaneous (i.c.) injection of 0.02 ml of 0.55 M]. A PLA stimulus with buffered saline served as control on the opposite forearm. We used laser Doppler flowmetry to measure the vasomotoric changes after ACH injection and recorded the duration and intensity of itch with a visual analogue scale (VAS). Following the evaluation of wheal and flare sensation, we obtained the area of itchy skin around the injection site (alloknesis) by gently stroking the surrounding skin with a brush in the centripetal direction towards the injection site. The results were planimetrically evaluated. Oral NAL reduced the perifocal itch significantly (P < 0.009). In four of our observations the area of alloknesis completely disappeared. Itch duration was reduced by 20 s and the intensity of itch was diminished, yet not significantly. NAL had no significant effects on cholinergic vasoreactions measured by the laser Doppler (P > 0.50) and especially failed to decrease the initial flux response, which is a typical sign of an altered vascular reaction (P > 0.25). The decrease of wheal (P = 0.008) and flare (P = 0.01) extension indicates an appropriate dosage of our treatment for this experiment. The most significant effects of NAL were observed in parameters of itch processing such as alloknesis (P = 0.009) and flare extension (P = 0.01). Therefore we favour the concept that NAL might have a stronger impact on central nervous mechanisms than on peripheral nociceptive structures.