bluuebunny/crossref_metadata_2025 huggingface dataset:
Fetal hemoglobin expression in the compound heterozygous state for−117 (G→A) Aγ HPFH and IVS‐1 nt 110 (G→A) β+ thalassemia: a case study
Authors: Dedoussis G. V. Z. , Sinopoulou K. , Gyparaki M. , Loutradis A.
Published: March 2003

Abstract: The splicing defect at IVS‐I‐110 is by far (43.15%) the most common β‐thalassaemia mutation in Greece. The −117 (G→A) Aγ hereditary persistence of fetal hemoglobin (Greek HPFH) is also the most frequent nondeletional HPFH in Greece. We report a case in which these two defects co‐segragates. She is a healthy female where the total Hb is 12.3 g/dl with 51% HbF and normal HbA2. Her Gγ/Aγ ratio is 35:65 differing from that of 10 simple heterozygotes for the Greek HPFH who have ratio of 8:92. Molecular analysis of the β‐globin genotype revealed the presence of the IVS‐I‐110 β+ mutation in trans to the −117 G→A Greek HPFH. Both mutations are linked to Ia. Her father has Greek HPFH in trans to the −158 C→T on the Gγ promoter, which is linked with haplotype IIIα. He has 13% HbF with a Gγ/Aγ ratio 32:68. Her sister is a compound heterozygote for the IVS‐I‐110 mutation in trans to the −158 C→T, with HbF levels of 3% and a Gγ/Aγ ratio 72:28.
Interferon alpha is an effective therapy for congenital dyserythropoietic anaemia type I
Authors: Shamseddine Ali , Taher Ali , Jaafar Hassan , Haidar Joud H. , Nasr Rihab , Arzoumanian Varant , Salem Ziad , Bazarbachi Ali
Published: March 2003

Abstract: The efficacy of interferon‐alpha (IFN) was reported in three patients with congenital dyserythropoietic anaemia (CDA) type I. We describe two additional cases treated with IFN, which normalized the haemoglobin level in both patients with a dramatic decrease in the size of the spleen in one. Haemoglobin remained stable more than 6 months after discontinuation of treatment. IFN induced more than 50% decrease in the number of BFU‐E in both patients' bone marrow cultures, suggesting an indirect effect of IFN on erythropoiesis in vivo. We conclude that a trial of IFN therapy should be considered in patients with CDA type I.
Incidence of iron‐deficiency anaemia in infants in a prospective study in Jordan
Authors: Kilbride Julia , Baker Terry G. , Parapia Liakat A. , Khoury Sami A.
Published: March 2003

Abstract: A high prevalence of iron‐deficiency anaemia has been reported in Jordanian infants.A prospective study of infants in downtown Amman examined the relationship between anaemia in pregnancy and iron deficiency in infancy. The iron status of infants born to 107 anaemic (Hb <11 g/dl) and 125 non‐anaemic mothers was reviewed at 3, 6, 9 and 12 months. Indicators to define iron‐deficiency anaemia were Hb <11 g/dl and either plasma ferritin <12 μg/l or zinc protoporphyrin (ZPP) >35 μg/dl whole blood. Haemoglobin electrophoresis excluded haemoglobinopathy. There was 72% iron‐deficiency anaemia throughout the year, significantly higher in infants born to anaemic mothers (81%; n=91) compared with controls (65%; n=112). At 12 months, 72% of the infants tested (n=195) were anaemic. While 57% were identified as iron‐deficient by research criteria of either ferritin or ZPP, only 37% were identified by ferritin alone, 40% by ZPP alone and 29% if both ferritin and ZPP were required to meet criteria. Most infant anaemia was identified as due to iron deficiency, supporting contextual setting as assisting diagnosis: infants in developing countries are recognised as vulnerable to iron deficiency. Using multiple criteria, more cases were identified when either ferritin or ZPP were abnormal than when one alone, or both parameters were required to meet research criteria.
Acquired Factor VIII autoantibody: four cases demonstrating the heterogenous nature of this condition and problems involved in diagnosis and treatment
Authors: Hall Rachel L. , Leahy Michael F.
Published: March 2003

Abstract: The development of an autoantibody to human Factor VIII is rare and presents many problems for diagnosis and treatment. We have seen several cases at our institution recently with widely heterogenous clinical and laboratory presentations. A wide range of treatment modalities were used in these cases with no gold standard of treatment or widely accepted guidelines existing. This has prompted us to examine all cases of this condition presenting at Fremantle Hospital over the last decade. We describe four cases which demonstrate the heterogeneity of this condition and its treatment and review the recent literature on the subject.
Comparison between deferoxamine and deferiprone (L1) in iron‐loaded thalassemia patients
Authors: Taher Ali , Sheikh‐Taha Marwan , Koussa Suzanne , Inati Adlette , Neeman Roger , Mourad Fadi
Published: March 2003

Abstract: Introduction: Iron‐chelating therapy with deferoxamine in patients with thalassemia major has dramatically improved the prognosis of this disease. However, the limitations of this treatment have stimulated the design of alternative orally active iron chelators. Objective: To compare the effectiveness and safety of, and compliance with, oral deferiprone (L1), and deferoxamine, in thalassemia major patients. Methods: All patients were followed up in one center in Lebanon. Sixteen patients were on L1 (75 mg/kg/d), and 40 patients on subcutaneous deferoxamine (20–50 mg/kg/d). Serum ferritin level, urinary iron excretion (UIE) and side effects were monitored over a two year period. Results: Patients on L1 had an initial serum ferritin concentration of 3663±566 µg/l (mean±SEM), that dropped to 2599±314 at 6 months (p<0.02; paired t‐test), and stabilised at that level over the 24 months follow up. Patients on deferoxamine had an initial mean serum ferritin concentration of 3480±417 (NS compared to the L1 group), which dropped gradually to 3143±417 (p<0.05) and 2819±292 (p<0.02) at 6 and 24 months, respectively. The most common adverse reactions associated with L1 were arthralgia and nausea, but they did not necessitate stopping the drug. Conclusion: L1 had comparable efficacy as deferoxamine with minimal side effects and better compliance. Provided long term side effects are not encountered, L1 seems to be a valuable alternative iron chelator for patients unable or unwilling to use deferoxamine effectively.
Treatment of plasma cell leukemia with vincristine, liposomal doxorubicin and dexamethasone
Authors: Christou Leonidas , Hatzimichael Eleftheria , Chaidos Aristidis , Tsiara Stavroula , Bourantas Konstantinos L.
Published: March 2003

Abstract: Primary plasma cell (PCL) leukemia is a rare lymphoproliferative disorder characterized by a malignant proliferation of plasma cells in the bone marrow and peripheral blood. Survival with standard therapy using melphalan is very poor. Doxorubicin encapsulated with liposomes has less cardiotoxicity, is at least as efficient and has fewer side effects than conventional doxorubicin. Two female patients (69 and 54 yr old) with primary PCL are described in this study. They both received a modified form of VAD (vincristine, doxorubicin and dexamethasone), a regimen which includes liposomal doxorubicin (40 mg/m2for 1 d), vincristine (2 mg for 1 d) and dexamethasone 40 mgper oson days 1–4, 9–12 and 17–20.

A disease evaluation of the first patient after six courses of the modified \VAD regimen showed no plasma cells in the peripheral blood, a decrease in the serum M protein level and a plasma cell infiltration in the bone marrow of less than 5%. The patient died from a cardiac episode 24 months post‐diagnosis, while she was in complete hematological remission. The second patient also exhibited good tolerance to liposomal doxorubicin with no side effects, achieved complete haematological remission and remains in good condition 7 months after the last VAD administration. These results suggest that this modified form of VAD regimen also seems to work in PCL and is well tolerated with no side effects.
Clinical and laboratory manifestations of congenital dyserythropoietic anemia type I in young adults
Authors: Shalev H. , Kapleushnik Y. , Haeskelzon L. , Degani O. , Kransnov T. , Sphilberg O. , Moser A. , Yaniv I. , Tamary H.
Published: March 2003

Abstract: Objectives: Congenital dyserythropoietic anemia (CDA) type I is a rare autosomal recessive macrocytic anemia whose natural history is not well documented. The aim of the present study was to evaluate the clinical picture of the disease in young adults. Methods: The study sample consisted of 17 patients of mean age 11.9±5.4 yr (range 18–33 yr) and one older patient (age 44 yr), all Israeli Bedouins. The degree of anemia was evaluated as well as the extent of development of gallstones and iron overload. In each subject we determined the hemochromatosis gene mutations and the uridine dyphosphate‐glucoronosyltransferase (UGT‐1A) gene polymorphism associated with Gilbert's syndrome. Results: The patients were found to have moderate anemia, with the women displaying lower mean hemoglobin levels than the men (8.2±0.9 g dL−1 vs. 10±1.3 g dL−1; P=0.0059). The majority of patients (59%) had received at least one blood transfusion, with the women having a significantly higher transfusion requirement. Although delayed puberty was noted, final height and weight were within normal limits, and eight patients had progeny. Biliary stones were found in three of 16 patients, two of whom were homozygous for UGT‐1A gene polymorphism. None of the patients carried the common hemochromatosis gene mutation, although serum ferritin levels were moderately elevated (788±332 ng mL−1). Conclusions: CDA type I in young adults is characterized by moderate macrocytic anemia, more severe in women, and a tendency to cholelithiasis and secondary progressive iron overload. We suggest that iron overload in this patient population should be monitored and chelation therapy initiated when indicated to prevent organ damage.
Aplastic anemia as a late complication of thymoma in remission
Authors: Ritchie David S. , Underhill Craig , Grigg Andrew P.
Published: March 2003

Abstract:  Thymoma has many auto‐immune associations, including aplastic anemia and myasthenia gravis. The pathophysiologic relationship between thymoma and its auto‐immune sequelae are yet to be fully elucidated, but are thought to be as a result of the production of auto‐reactive T cell clones by the thymic epithelium. We report a case in which aplastic anemia did not develop until sometime after remission from thymoma had been induced. This observation suggests that auto‐reactive T cells may be produced by the thymoma but not induce clinical auto‐immune disease until a later time, even after eradication of thymoma has been achieved.
Macrophage‐dependent regulation of neutrophil mobilization and chemotaxis during development of sterile peritonitis in the rat
Authors: Knudsen Eirunn , Iversen Per Ole , Van Rooijen Nico , Benestad Haakon Breien
Published: March 2003

Abstract:Pro‐inflammatory cytokines attract leukocytes to inflamed tissues and activate them. Few attempts have been made to identify the sources of cytokinesin vivo. We examined the importance of peritoneal macrophages in the mobilization and homing of neutrophils to a sterile peritonitis in the rat, with emphasis on their cytokine production. Macrophages, present in virtually all tissues, are known to be easily activated and to serve as an important source of cytokines. Flow cytometric analysis of cells stained intracellularly with tagged antibodies against various cytokines revealed that the peritoneal macrophages were stimulated to produce the following cytokines: interleukin (IL)‐1β, macrophage inflammatory protein‐2 (MIP‐2), and keratinocyte‐derived cytokine (KC). High numbers of neutrophils, activated on arrival into the peritoneal cavity, also produced IL‐1β, whereas lower numbers contained interleukin‐6, tumor necrosis factor‐α, MIP‐2, KC, and MIP‐1α. This marked activation of peritoneal neutrophils was also reflected by increased surface expression of CD11b. On the other hand, peritoneal macrophages expressed high basal levels of CD11b, which were reduced 24 h after the onset of inflammation. In rats selectively depleted of macrophages by i.p. injection of liposome‐containing clodronate, the massive influx of neutrophils to the peritoneal cavity was markedly reduced, as was the rapid mobilization of mature bone marrow neutrophils. Local macrophages are important both for the accumulation of neutrophils in the inflamed peritoneal cavity and for the early mobilization of neutrophils from the bone marrow. Macrophage‐derived IL‐1β, MIP‐2, and KC are possible mediators of neutrophil homing to inflamed tissues.
Fulminant tumour lysis syndrome in acute myelogenous leukaemia with inv(16)(p13;q22)
Authors: Seftel M.D. , Bruyere H. , Copland M. , Hogge D.E. , Horsman D.E. , Nantel S.H. , Shepherd J.D. , Lavoie J.C. , Le A. , Sutherland H.J. , Toze C.L. , Nevill T.J.
Published: March 2003

Abstract: Tumour lysis syndrome (TLS) is caused by rapid breakdown of malignant cells resulting in electrolyte disturbances and acute renal failure. TLS has rarely been described in patients with acute myelogenous leukaemia (AML). Between November 1997 and July 2001, 114 consecutive adult AML patients aged <60 yr received induction chemotherapy consisting of cytosine arabinoside 1.5 g m−2 q 12 h × 12 doses and daunorubicin 45 mg m−2 d−1 × 3 doses. During induction chemotherapy (CT), seven patients (6.1%, 95% CI 2.5–12.2) developed fulminant TLS, resulting in acute renal failure; five of these seven patients had inversion of chromosome 16 [inv(16)(p13;q22)], and one patient had a biological equivalent [t(16,16)(p13;q22)]. Four of the TLS patients underwent leukapheresis for a presenting white blood cell (WBC) count > 100 × 109 L−1 prior to commencing chemotherapy, and six patients subsequently required haemodialysis for a median of 2 (range 1–8) wk. One TLS patient died of intracerebral hemorrhage on day 10 and another patient of multiorgan failure on day 17. Of the other five patients, all entered a complete remission (CR) and recovered normal renal function. Four patients remain in continuous CR [median follow‐up 20 (range 12–25) months]. One patient relapsed at 12 months and again developed TLS on re‐induction. In univariate analysis, TLS patients were more likely to have an elevated presentation and pre‐chemotherapy WBC counts, elevated serum creatinine, and uric acid levels at presentation, as well as an inv(16). In multivariate analysis, only serum creatinine and inv(16) remained statistically significant (P < 0.001 for each). Patients with an inv(16) are a unique AML subgroup at high risk for fulminant TLS.
Initial exposed phosphatidylserine levels correlate with cellular response to cytotoxic drugs
Authors: Drucker Liat , Ciobotaro Pnina , Kimchi Oded , Tohami Tali , Yarkoni Shai , Radnay Judith , Shapira Hava , Lishner Michael
Published: March 2003

Abstract: Phosphatidylserine's (PS) membranal distribution is associated with an expanding variety of biological processes. We studied the relevance of preliminarily exposed membranal PS levels to cellular effects of cytotoxic agents. PBL of normal controls (n = 18) and patients with doxorubicin‐treated breast carcinoma (n = 27) or 5′‐fluorouracil‐treated colorectal cancer (n = 32) were assayed before and after drug infusion. Membranal expression levels of PS, adhesion molecules (CD18, CD11a–c, CD63) and Fas‐R of leukocyte subtypes were assessed by flow cytometer. Statistical analysis was implemented. Our results demonstrate external expression of PS on all leukocyte subpopulations despite non‐apoptotic light scatter characteristics. Several distinct features were observed of which the more prominent were: leukocyte subtypes each display characteristic PS levels; cancer patients’ PBL display higher preliminary PS levels than normal controls in all cell groups; and existence of negative correlations between initial membranal PS levels and drug‐induced changes in its expression. Our findings underscore the complex involvement of PS in PBL apoptosis and possibly drug resistance.
Surface antigen expression and correlation with variable heavy‐chain gene mutation status in chronic lymphocytic leukemia
Authors: Vilpo Juhani , Tobin Gerard , Hulkkonen Janne , Hurme Mikko , Thunberg Ulf , Sundström Christer , Vilpo Leena , Rosenquist Richard
Published: March 2003

Abstract: Recent studies have demonstrated that B‐cell chronic lymphocytic leukemia (CLL) consists of two clinical entities with either somatically hypermutated (M‐CLL) or unmutated (UM‐CLL) immunoglobulin variable heavy‐chain (VH) regions. In view of the fact that the cellular biology of these two subsets of disease is currently unexplored, we performed an extensive analysis of the surface antigen expression and correlated this with the VH gene mutation status in a cohort of 32 CLL patients. Using polymerase chain reaction amplification and nucleotide sequencing, the VH genes were shown to be mutated in 10 cases (31%) and unmutated in 22 (69%). The expression of 27 surface membrane antigens in peripheral blood leukemic cells was analyzed by flow cytometry, measuring both the percentage of positive cells as well as the geometric mean fluorescence intensity (GMF). Most of the surface membrane antigens (CD5, CD11c, CD19, CD20, CD21, CD22, CD23, CD25, CD40, CD45, VD79b, CD80, CD95, CD122, CD124, CD126, CD130, CD154, IgM, and IgD) showed a similar expression pattern in both UM‐CLL and M‐CLL patients. The similarity of M‐CLL and UM‐CLL, as demonstrated here for the first time with many protein markers, indicates a considerably homogenous phenotype in both subsets. Furthermore, CD27 was strongly expressed in all cases, which may suggest a memory cell phenotype for both M‐CLL and UM‐CLL. More positive cells in the UM‐CLL group were observed regarding CD38, but CD38 was not a good predictor of VH gene mutation status. Seventy percent of the M‐CLL cases, but only 36% of UM‐CLL cases, were Ig‐λ+. The most striking differential expression, however, was observed in the two slicing variants of the common leukocyte antigen CD45, namely CD45RO and CD45RA. CD45RO expression was significantly associated with M‐CLL, whereas the GMF intensity of CD45RA tended to be associated with UM‐CLL. The role of these CD45 splicing variants in the pathogenesis of CLL deserves further investigation.
Role of variant Creutzfeldt‐Jakob disease for safety of treatment with blood components: screening of lymphatic tissue is a potential tool for risk assessment
Authors: Sachse C. , Groschup M. H. , Warzok R. , Greinacher A.
Published: March 2003

Abstract: Background: Transmissible spongiform encephalopathies (TSEs) are neuropathological diseases caused by prions. Prions are infectious particles (PrPSc) which can induce bovine spongiform encephalopathy and most likely also the related infectious disease, variant of Creutzfeldt‐Jakob disease (vCJD). The exposure of humans to orally ingested BSE agent in contaminated meat products presumably led to the emergence of vCJD. In vCJD, prions can be detected immunohistochemically not only in neuronal tissue but also in lymphoreticular tissue. vCJD is of significance in transfusion medicine because of the hypothetical transmission of prions by blood products. Methods: An immunohistochemistry method was used to allow screening for vCJD in human lymphoreticular tissue. Results: PrPSc can be detected in the cerebrum and cerebellum of patients with sporadic Creutzfeldt‐Jakob disease (sCJD) and in the lymph nodes, tonsils, and spleen of vCJD patients. This method has the major advantage of working in fixed specimens which are routinely saved in departments of pathology and therefore allows screening of large numbers of archived human lymphoreticular tissues in different regional areas and from different time points. Scrapie‐positive lymphoreticular sheep tissue reacts similarly to human tissue of vCJD affected patients and is available in sufficient amounts to be used as positive control in screening programs. Conclusion: A method is provided which is a feasible tool for an epidemiological screening program to assess the prevalence of the assumed infectious agent of vCJD, PrPSc, in various populations.
Inhibition of UV‐induced p53 mutations and skin cancers by sunscreens: implication for skin cancer prevention
Authors: Ananthaswamy Honnavara N. , Ullrich Stephen E. , Kripke Margaret L.
Published: March 2003

Abstract: The relationship between exposure to UVB radiation and development of skin cancer has been well established. Several studies have shown that UVB induces unique mutations (C to T and CC to TT transitions) in the p53 tumor suppressor gene that are not commonly induced by other carcinogens. Our studies have demonstrated that UV‐induced mouse skin cancers contain p53 mutations at a high frequency and that these mutations can be detected in UV‐irradiated mouse skin well before the appearance of skin tumors. This observation suggested that it might be possible to use p53 mutations as a biological endpoint for testing the efficacy of sunscreens in photoprotection studies. Indeed, application of SPF 15 sunscreens to mouse skin before each UVB irradiation resulted in 88–92% reduction in the number of p53 mutations. Because p53 mutations represent an early essential step in photocarcinogenesis, these results imply that inhibition of this event may protect against skin cancer development. This hypothesis is confirmed by our finding that sunscreens used in p53 mutation inhibition experiments also protected mice against UVB‐induced skin cancer.
Cloning of the mouse desmoglein 3 gene (Dsg3): interspecies conservation within the cadherin superfamily
Authors: Ishikawa H. , Li K. , Tamai K. , Sawamura D. , Uitto J.
Published: March 2003

Abstract: Desmoglein 3 is a cadherin‐like calcium‐dependent cell adhesion molecule expressed primarily in suprabasal keratinocytes of the epidermis. In this study, we have cloned the full‐length cDNA and characterized the entire gene structure for the mouse desmoglein 3 gene (Dsg3). Isolation of overlapping cDNA clones, together with 5′ and 3′ rapid amplification of cDNA ends (RACE), allowed delineation of the entire coding sequence. The transcriptional initiation site was confirmed by primer extension and reverse transcription polymerase chain reaction analysis. The entire cDNA consisted of 6407 bp with an open reading frame of 2979 bp, and the deduced polypeptide contained 993 amino acids. Comparison of mouse and human desmoglein 3 amino acid sequences demonstrated 85.6% homology. Computer analysis suggested the presence of a transmembrane segment, 5 potential calcium binding sites, and a RAL motif which corresponds to the HAV motif, the potential site for homophilic interaction of typical cadherins. The mouse desmoglein 3 gene consisted of 15 exons in chromosome 18. Comparison of the intron–exon organization of Dsg3 with various cadherins from different species revealed remarkable conservation. This relatively high level of conservation both at the protein and genomic level suggests that desmoglein 3 plays an important role in keratinocyte cell–cell adhesion.
Note
Flow‐cytometric characterization of normal versus psoriatic epidermis using improved cell separation methodology
Authors: Seegers B. A. M. P. A. , Glade C. P. , Van Hooijdonk C. A. E. M. , Van Erp P. E. J. , De Jongh G. J. , Van De Kerkhof P. C. M.
Published: March 2003

Abstract: Recently a new approach for epidermal cell characterization was developed: three‐parameter flow cytrometrical analysis of pure and complete epidermal cell suspensions prepared from punch biopsies followed by dermoepidermal separation by thermolysin. The aim of the present communication is the comparison between psoriatic lesional skin and normal skin using this new approach with respect to the percentage of suprabasal keratinocytes (keratin 10+ cells), mesenchymal cells, including the infiltrate cells (vimentin+ cells) and the percentage of basal cells in SG2 M phase, in order to validate this methodology in studies on psoriatic skin. Punch biopsies were taken from 7 healthy volunteers and in 7 psoriatic patients 4 biopsies were taken in each of them from comparable lesions. The present study reconfirmed that the percentage of basal keratinocytes in psoriasis was increased and the percentage of keratin 10+ cells was substantially decreased as compared to normal skin. The new methodology revealed data with a narrow range. In psoriatic lesional skin the intra individual variation was less compared to the inter individual variation.
Bovine gingival lysate: a novel substrate for rapid diagnosis of autoimmune vesiculo‐bullous diseases
Authors: Engineer L. , Johnson R. E. , Bhol K. C. , Ahmed A. R.
Published: March 2003

Abstract: Immunoblot assays have been developed to characterize the autoantigens and to detect autoantibodies in muco‐cutaneous autoimmune vesiculo‐bullous diseases using different substrates. However the results have been inconsistent, because availability and standardization of different substrates has been a major problem. The aim of this study was to develop an immunoblot assay using bovine gingival lysate as substrate because it is easily and readily available as well as inexpensive. Sera from patients with different vesiculo‐bullous diseases were studied. These included 25 patients with pemphigus vulgaris (PV), 8 with paraneoplastic pemphigus (PNP), 12 with pemphigus foliaceus (PF), 25 with bullous pemphigoid (BP), and 22 with cicatricial pemphigoid (CP). Serum samples from 40 normal human volunteers were also studied. The autoantibody titers were determined based on the binding pattern of each disease and compared to those obtained by routine indirect immunofluorescence (IIF). Our observations suggest that the titers from immunoblot assays were significantly higher than titers obtained by IIF (P<0.0001). When the autoantibody titers were compared using bovine gingival lysate and human epidermal lysate as substrate, statistically significant differences were not observed. The use of bovine gingival lysate as a substrate will facilitate the rapid and early serological diagnosis of patients with vesiculo‐bullous diseases. It may also be of benefit to laboratory investigators studying these autoantibodies.
Genomic organization and amplification of the human plakoglobin gene (JUP)
Authors: Whittock N. V. , Eady R. A. J. , McGrath J. A.
Published: March 2003

Abstract: Plakoglobin is a globular protein common to the intracellular plaques of adhesive junctions, predominantly desmosomes and adherens junctions. Recently, a number of pathogenic mutations have been described in other components of desmosomes, specifically in plakophilin 1, desmoplakin and desmoglein 1. The phenotype of affected patients mainly involves thickening of palm and sole skin (keratoderma). Although no human mutations in plakoglobin have been described thus far, this protein represents an excellent candidate for other human genetic disorders, possibly involving skin and heart, sites of high plakoglobin expression. To facilitate future mutation detection analyses in such conditions, we have characterized the intron–exon organization of the human plakoglobin gene, which comprises 13 distinct exons spanning approximately 17 kb on 17q21. We have also developed a PCR‐based mutation detection strategy using primers placed on flanking introns followed by direct sequencing of the PCR products.
The effect of ultraviolet B irradiation on nitric oxide synthase expression in murine keratinocytes
Authors: Sasaki M. , Yamaoka J. , Miyachi Y.
Published: March 2003

Abstract: Nitric oxide (NO), which has several physiological functions in skin, is generated by NO synthase (NOS). NOS has at least three isoforms; endothelial NOS (eNOS), brain NOS (bNOS), and inducible NOS (iNOS). Ultraviolet B (UVB) irradiation has been reported to stimulate NO production in skin via induction or activation of NOS, however, the exact mechanism of NOS induction by UVB irradiation remains obscure. In this study, we investigated the direct effect of UVB on the expression of NOS isoforms in murine keratinocytes, and found a significant increase in NO production within 48 h. mRNA and protein expressions of bNOS were both enhanced by UVB irradiation in murine keratinocytes, whereas iNOS mRNA expression was suppressed at 4 and 12 h after UVB irradiation. These results suggest that the enhancement of NO production observed after UVB irradiation in murine keratinocytes may be explained in part by the upregulation of bNOS expression, but not iNOS expression.
Changes in the ultrastructure of cytoskeleton and nuclear matrix during HaCaT keratinocyte differentiation
Authors: Gniadecki R. , Olszewska H. , Gajkowska B.
Published: March 2003

Abstract:The cellular scaffold that comprises nuclear matrix and cytoskeleton provides mechanical support for the cell and plays a crucial role in motility, cellular signaling, regulation of gene transcription and DNA replication. In this study we examined the structure of cytoskeleton and nuclear matrix in the keratinocyte cell line HaCaT using a recently developed technique, embedment‐free electron microscopy. With this method the three‐dimensional structure of cellular scaffold is visualized in the cells extracted from soluble proteins and the chromatin. In actively proliferating cells the cytoskeleton appeared to consist of a continuous meshwork of 10–15 nm filaments with a smaller amount of thin (5 nm) and ultrathin (1–2 nm) filaments. In contrast to what could be expected from earlier immunofluorescence and electron microscopy studies, the cytoskeleton in HaCaT keratinocytes did not consist of superposed autonomous networks of different filaments but was a highly integrated, continuous structure filling whole cytoplasmic territory. Moreover, cytoskeletons of adjacent cells were in a direct physical contact. Nuclear matrix consisted of globular ribonucleoprotein aggregates attached to the meshwork of 20–40 nm filaments. Nuclear envelope was firmly fastened to the cytoskeleton. In keratinocytes induced to differentiation by calcium switch both the cytoskeleton and nuclear matrix were drastically rearranged and comprised a monomorphic, dense and regular meshwork of 10–15 nm filaments. Our data underscore the fact that in HaCaT keratinocyte monolayerin vitro, and probably also in the epidermisin vivo, the nuclear matrices and the cytoskeletons of adjacent cells seemed to form a continuous, highly ordered structure which is rapidly rearranged during cell differentiation. This feature may be crucial for the understanding of how the signal initiated by, e.g. mechanical forces generated through the cell–cell and cell–matrix interaction is transmitted to the nucleus producing ultimately changes in the pattern of gene expression.
Metalloelastase (MMP‐12) and 92‐kDa gelatinase (MMP‐9) as well as their inhibitors, TIMP‐1 and ‐3, are expressed in psoriatic lesions
Authors: Suomela S. , Kariniemi A.‐L. , Snellman E. , Saarialho‐Kere U.
Published: March 2003

Abstract: In skin biology, matrix metalloproteinases (MMPs) have been implicated in inflammatory matrix remodeling, neovascularization, wound healing and malignant transformation. Psoriasis is histologically characterized by keratinocyte hyperproliferation, infiltration of inflammatory cells, neoangiogenesis and production of cytokines, such as TNF‐α, IL‐1β, TGF‐α, and IFN‐γ, also capable of regulating MMP transcription. To investigate the role of stromelysins‐1 and ‐2, matrilysin, metalloelastase, collagenases‐1 and ‐3 and 92‐kDa gelatinase as well as their inhibitors, TIMPs‐1 and ‐3, in psoriasis, we performed in situ hybridization using 35S‐labeled cRNA probes on 29 psoriatic lesions and 9 samples of normal looking skin from psoriatic patients. Metalloelastase mRNA was detected in 21/27 samples in macrophages that had migrated into the epidermis or in the inflammatory infiltrates of the superficial dermis. A quantity of 92‐kDa gelatinase was found in macrophages and neutrophils (25/27). Stromelysin‐1 mRNA was detected in basal keratinocytes in 4/21 lesions. Intracellular laminin‐5 immunosignal in basal keratinocytes of the same samples, suggested that stromelysin‐1 might participate in remodeling of the basement membrane zone. No signal for stromelysin‐2 or collagenase‐3 was found and only sweat glands were positive for matrilysin. TIMP‐1 was more abundantly expressed than TIMP‐3 in the inflammatory infiltrates and endothelial cells of dermal papillae (22/29). TIMP‐3 was expressed perivascularly in 9/16 samples. Our results suggest that overexpression of the investigated MMPs by keratinocytes is not associated with psoriasis. However, macrophages express MMPs in psoriatic skin. Also TIMPs, particularly TIMP‐1, were abundantly expressed, suggesting that mere MMP overexpression is unlikely to contribute to psoriatic tissue changes.
Doxycycline‐inducible retroviral expression of green fluorescent protein in immortalized human keratinocytes
Authors: Gill Pritmohinder S. , Krueger Gerald G. , Kohan Donald E.
Published: March 2003

Abstract: Keratinocytes have a great potential to deliver systemically therapeutic genes, and a regulatable switch technology for transgene expression in this cell type would greatly enhance their clinical value for cutaneous gene therapy. We describe a method wherein immortalized human keratinocytes (IMKc) are transduced with high efficiency with retroviral vectors of the RetroTet‐Art system, which confers stable doxycycline (Dox)‐regulated green fluorescent protein (GFP) expression. In this RetroTet‐Art system the TCN transactivators and TCN transrepressors are coexpressed in cells. After one round of transduction, approximately 50% of IMKc expressed GFP; after puromycin selection over 90% of cells expressed GFP. With this retroviral vector system no baseline expression of GFP was observed in the genetically modified IMKcs. Dox treatment of these transduced cells induced GFP expression in a dose‐ and time‐dependent manner. Peak GFP expression occurred after 72 h of Dox treatment and dropped to baseline when Dox was removed. These multiply transduced cells formed differentiated epidermis in vitro and the Dox treatment did not induce evidence of toxicity in the architecture of the epidermis. Our observations demonstrate an efficient method for achieving stable Dox‐regulatable transgene expression in human keratinocytes.
mRNA expression of tumor‐associated antigens in melanoma tissues and cell lines
Authors: Eichmüller Stefan , Usener Dirk , Jochim Anita , Schadendorf Dirk
Published: March 2003

Abstract: Tumor‐associated antigens (TAA) are increasingly used as specific targets for immune therapy of malignant melanoma. The aim of the present study was to provide a basis for selecting the most suitable TAA by analyzing the mRNA expression of a large panel of TAA by RT‐PCR and Northern blotting. We have chosen primers differentiating four groups of TAA (MAGE‐A, MAGE‐B, and two groups of GAGE‐genes) and 13 individual TAA (MAGE‐A2 and ‐A3, RAGE‐1, ‐2, ‐3, and ‐4, LAGE‐1a and ‐1b, NY‐ESO‐1, GAGE‐1, SSX‐2, SCP‐1, and cTAGE‐1) based on most recent sequence data. In addition, the RAGE‐gene family has been separated into its four members by a novel designed nested PCR, which was confirmed by Northern analysis. Furthermore, the chromosomal organization and relationship between the RAGE‐family and MOK was analyzed. RAGE‐4 mRNA could be shown for the first time to be present in testis tissue. The most frequently expressed TAA were the MAGE‐A and the GAGE‐3,‐4,‐5,‐6,‐8 group, whereas among individual TAA MAGE‐A2, ‐A3, RAGE‐1, ‐3, and LAGE‐1a/b were found within most specimens and are thus promising candidates for immune therapy. In comparison, melanoma metastatic specimens and cell lines show similar profiles of TAA expression, but individual TAA differ notably between both types of samples indicating that results from cell lines are not always applicable to tumor specimen.
Effect of parathyroid hormone‐related protein on fibroblast proliferation and collagen metabolism in human skin
Authors: Maioli Emanuela , Fortino Vittoria , Torricelli Claudia , Arezzini Beatrice , Gardi Concetta
Published: March 2003

Abstract: The parathyroid hormone‐related protein (PTHrp), structurally similar to the parathyroid hormone (PTH) in its NH2‐terminal part, was first identified as a tumour‐derived peptide responsible for a paraneoplastic syndrome known as humoral hypercalcemia of malignancy. The PTHrp gene is expressed not only in cancer but also in normal tissues during adult and/or fetal life, where it plays predominantly paracrine and/or autocrine roles. In the skin PTHrp produced by keratinocytes acts on fibroblasts by complex cooperative circuits involving cytokines and growth factors. In this report, we studied the direct effects of synthetic PTHrp 1–40 on proliferation and collagen synthesis and matrix metalloproteinase‐2 (MMP‐2) activity in cultures of fibroblasts isolated from normal human skin. Fibroblasts exposure to varying doses of PTHrp for 48 h, significantly and dose‐dependently inhibited proliferation evaluated by [3H]‐thymidine incorporation into DNA. A dose‐dependent stimulation of cAMP released into the medium was concomitantly observed. In contrast, PTHrp had no effect on collagen synthesis evaluated either by [3H]‐proline incorporation or by radioimmunoassay (RIA) of the carboxyterminal fragment of type I procollagen (PICP). MMP‐2 activity, evaluated by quantitative zymographic analysis, was significantly increased by PTHrp treatment at doses of 160 and 320 nM. These findings indicate that PTHrp may play a role in normal dermal physiology by controlling both fibroblast proliferation and extracellular matrix degradation.
17α‐estradiol induces aromatase activity in intact human anagen hair follicles ex vivo
Authors: Hoffmann R. , Niiyama S. , Huth A. , Kissling S. , Happle R.
Published: March 2003

Abstract: For topical treatment of androgenetic alopecia (AGA) in women, solutions containing either estradiol benzoate, estradiol valerate, 17β‐ or 17α‐estradiol are commercially available in Europe and some studies show an increased anagen and decreased telogen rate after treatment as compared with placebo. At present it is not precisely known how estrogens mediate their beneficial effect on AGA‐affected hair follicles. We have shown recently that 17α‐estradiol is able to diminish the amount of dihydrotestosterone (DHT) formed by human hair follicles after incubation with testosterone, while increasing the concentration of weaker steroids such as estrogens. Because aromatase is involved in the conversion of testosterone to estrogens and because there is some clinical evidence that aromatase activity may be involved in the pathogenesis of AGA, we addressed the question whether aromatase is expressed in human hair follicles and whether 17α‐estradiol is able to modify the aromatase activity. Herewith we were able to demonstrate that intact, microdissected hair follicles from female donors express considerably more aromatase activity than hair follicles from male donors. Using immunohistochemistry, we detected the aromatase mainly in the epithelial parts of the hair follicle and not in the dermal papilla. Furthermore, we show that in comparison to the controls, we noticed in 17α‐estradiol‐incubated (1 nM) female hair follicles a concentration‐ and time‐dependent increase of aromatase activity (at 24 h: 1 nM = +18%, 100 nM = +25%, 1 µM = +57%; 24 h: 1 nM = +18%, 48 h: 1 nM = +25%). In conclusion, our ex vivo experiments suggest that under the influence of 17α‐estradiol an increased conversion of testosterone to 17β‐estradiol and androstendione to estrone takes place, which might explain the beneficial effects of estrogen treatment of AGA.
Keratinocyte gene therapy: cytokine gene expression in local keratinocytes and in circulation by introducing cytokine genes into skin
Authors: Meng Xianmin , Sawamura Daisuke , Ina Shinsuke , Tamai Katsuto , Hanada Katsumi , Hashimoto Isao
Published: March 2003

Abstract: Using the plasmid DNA injection method, we introduced cytokine genes into skin to determine whether systemic expression of cytokine genes is possible. Eight human cytokine [interleukin‐4 (IL‐4), IL‐6, IL‐10, transforming growth factor β1 (TGF‐β1), monocyte chemotactic and activating factor (MCAF), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), tumor necrosis factor α (TNF‐α) and interferon γ (IFN‐γ)] gene expression vectors were constructed and injected into rat skin. Transgenic cytokines in local keratinocytes and in the sera were assayed with ELISA. Our results showed that transgenic cytokines were markedly increased in keratinocytes at the injection site. The serum concentrations of IL‐4, 6, 10 and ΤGF‐β1 reached levels high enough to have systemic biologic effects. However, other cytokines used in this study could not be detected in the sera. Moreover, the serum transgenic IL‐10 level after subcutaneous injection was significantly higher than after intramuscular injection. We suggest that keratinocytes can be used as a bioreactor to achieve systemic expression of cytokine genes by DNA injection, but the transgenic protein level in circulation depends on different kinds of cytokine. This level also depends on different target cells used for gene transfer.
A colorimetric bead‐binding assay for detection of intermolecular interactions
Authors: Irvine A. D. , Sun P. , Kos L. , Wang X. Q. , Paller A. S.
Published: March 2003

Abstract: We have developed a new technique that rapidly and reproducibly allows direct visualization of molecular interactions, including receptor‐ligand binding. The technique can be easily applied to examine binding between proteins and glycoproteins, or proteins and glycolipids, including gangliosides. In this novel bead‐binding assay, the suspected ‘ligand’ molecule is bound to 0.2‐ or 1.0‐µm colored FluoSphere beads. These coated beads are then mixed directly with 150‐µm Sepharose or 50‐ to 75‐µm agarose beads coated with the second ‘target’ molecule. Binding between molecules is easily detected by immunofluoresence microscopy as colored rosettes or aggregations formed by clustering of the smaller fluorescent beads around the larger non‐fluorescent bead. The validity of this technique for glycolipid binding to protein was verified through demonstration of the known interaction between the β subunit of cholera toxin with ganglioside GM1. The bead‐binding technique facilitated the novel observations of interaction between ganglioside GT1b with the α5 subunit of α5β1 integrin and the interaction of GM3 with the epidermal growth factor receptor. A modification of this technique, in which the coated beads are bound to protein fixed on plates, allows a quantifiable colorimetric assay of interaction. This versatile and rapid technique will have widespread applications for in vitro systems and may also be useful for in vivo analysis of binding to cell surface receptor molecules.
Seasonal changes in abundance and age structure of burbot Lota lota (L.) and stone loach Barbatula barbatula (L.) in the littoral zone of a large pre‐alpine lake
Authors: Hofmann N. , Fischer P.
Published: March 2003

Abstract
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Seasonal changes in the abundance and age structure of the stone loach and burbot populations in the littoral of Lake Constance, Germany were examined. The abundance of both species was lowest during the summer months and highest during autumn. The stone loach population in the littoral comprised age classes 1 to 3 plus, in autumn, age class 0. The burbot population showed distinct seasonal changes in both length and age composition. During spring and summer, only age class 1 was present in the littoral whereas, in autumn, only age class 0 was caught. This remarkably strict habitat segregation of two age classes by season is discussed with respect to a possible mechanism for avoiding conspecific predator risk in a cannibalistic species.
Piscivory in larval perch (Perca fluviatilis): mechanisms structuring larval roach (Rutilus rutilus) cohorts
Authors: Brabrand Å.
Published: March 2003

Abstract – Perch (Perca fluviatilis) can act as a piscivore from larval stage VI (body size 10.3 mm) on newly hatched larval roach (Rutilus rutilus), bream (Abramis brama) and smaller siblings of its own cohort. Consumption rates at this stage were approx. 0.5 prey/perch*h at 21°C. Larval perch predation was strongly gap‐limited, and the maximum size of roach consumed by perch (perch length interval 10.3–62.0 mm) under experimental conditions followed the linear regression, Pprey‐max.=0.478*LPred.+1.829 (r2=0.99, P<0.001, n=12). Under experimental conditions, predatory 0+ perch substantially affected the size distributions of 0+ roach prey cohorts, since smaller prey individuals were predated more frequently than larger ones. In both unimodal and bimodal size distributions of prey roach, the distributions changed according to the maximum prey size consumed by the added predatory perch. Unimodal prey distributions were positively skewed when piscivorous perch were added compared with controls without predators. According to the size distributions of lake‐living 0+ roach and 0+ perch and the relative size difference between prey and predator, the vulnerability of 0+ roach cohort to 0+ perch predation changed from June to September. Prey vulnerability was extremely sensitive to the relative size difference between predator and prey. Therefore differences in hatching time and growth rates between the two species will strongly influence the potential for predator‐prey interactions.Note
Diet dynamics of the juvenile piscivorous fish community in Spirit Lake, Iowa, USA, 1997–1998
Authors: Pelham M. E. , Pierce C. L. , Larscheid J. G.
Published: March 2003

Abstract – We assessed temporal dynamics and variation among species and age‐classes in the diets of age 0 and age 1 piscivorous fish species in Spirit Lake, Iowa, USA during 1997 and 1998. Species included walleye Stizostedion vitreum, yellow perch Perca flavescens, smallmouth bass Micropterus dolomieui, largemouth bass Micropterus salmoides, black crappie Pomoxis nigromaculatus and white bass Morone chrysops. Thirty taxa were identified in diets, including 12 species of fish. We found dramatic differences in diets among species, among age‐classes within species and over time. Walleye, largemouth bass, smallmouth bass and white bass were piscivorous at age 0. Black crappie began piscivory at age 1. Yellow perch also began piscivory at age 1, but fish were a very small fraction of age‐1 diets. The primary temporal pattern, seen in several species and age‐classes, was an increase in piscivory from spring to fall. This pattern was due to the lack of small, age‐0 prey fish in spring. Although some patterns were evident, the taxonomic composition of the diets of all species was highly variable over time, making generalizations difficult. A surprising result was the absence of yellow perch in the diet of age‐0 walleye, despite their abundance in Spirit Lake and prominence in diets of age‐1 walleye and other age 1‐piscivores. Age‐0 yellow perch were consistently too large to be eaten by age‐0 piscivores, which preyed primarily on invertebrates and smaller fish such as johnny darters Etheostoma nigrum and age 0 bluegill Lepomis macrochirus. This finding suggests that predator‐prey interactions and resulting population dynamics may be quite different in Spirit Lake than in other systems dominated by walleye and yellow perch.
Effects of environmental factors on size‐related growth efficiency of perch, Perca fluviatilis
Authors: Holmgren K. , Appelberg M.
Published: March 2003

Abstract – Two aspects of size‐dependent growth were addressed using perch, Perca fluviatilis L, from 22 Swedish lakes. Firstly, maximum annual growth decreased linearly with length after the previous year. Secondly, median or realized growth showed a non‐linear pattern, with minimum growth efficiency at intermediate length, probably related to the shift from invertebrate to fish diet. Between‐lake variation in size‐related growth efficiency was better explained by fish community characteristics than by abiotic factors. The growth of most size classes was negatively related to fish biomass, especially to biomass of 100 to 199‐mm perch. Growth efficiency of perch >200 mm was positively related to the proportion of large perch in the community. These size‐ and density‐dependent growth responses suggest that effects of competitive and predatory interactions will often mask the controlling or limiting effects of abiotic factors, such that realized growth of individuals and populations are poorly described by deterministic asymptotic growth models.