bluuebunny/crossref_metadata_2025 huggingface dataset:
Combined therapy in advanced stages (III and IV) of follicular lymphoma increases the possibility of cure: results of a large controlled clinical trial
Authors: Avilés Agustin , Delgado Serafin , Fernández Raúl , Talavera Alejandra , Neri Natividad , Huerta‐Guzmán Judith
Published: March 2003

Abstract: Objectives: We evaluate the long‐term results of a randomized clinical trial in patients with advanced stages (III and IV) of follicular lymphoma using chemotherapy or combined therapy (chemotherapy following by adjuvant radiotherapy in patients with nodal bulky disease). Material and methods: Between 1981 and 1995, patients with follicular lymphoma were treated with combined chemotherapy, mostly anthracycline‐based regimens; patients who achieved complete response were randomly assigned either to receive adjuvant radiotherapy to sites or to nodal bulky disease or not (control group). Results: Four hundred and sixty‐nine patients were randomized; in an intent‐to‐treat analysis all were evaluable for efficacy and toxicity. Actuarial curves at 20 yr showed that event‐free survival (EFS) and overall survival (OS) in the control group were 41% [95% confidence interval (CI) 36–56%) and 71% (95% CI 65–78%), respectively; these were statistically different from results for the patients who received adjuvant radiotherapy: 68% (95% CI 62–72%) and 89% (95% CI 79–96%), respectively (P < 0.01). Acute and late toxicity were minimal; only four patients (< 1%) developed myelodysplastic syndrome/acute leukemia. Cardiac toxicity was 2%, but one case was lethal. Thirty‐six patients (8%) died secondary to unrelated causes, in complete remission. Conclusions: The use of adjuvant radiotherapy in patients with poor‐prognosis follicular lymphoma increases EFS and OS with minimal toxicity. We feel that follicular lymphoma should be treated curatively because < 80% of patients will be in first complete response at < 20 yr. The use of adjuvant radiotherapy will be considered in the first line of treatment in this set of patients.
A novel case of haemoglobin H disease associated with clinical and morphological characteristics of congenital dyserythropoietic anaemia type I
Authors: Antonelou M. , Papassideri I. S. , Karababa F. , Gyparaki M. , Loutradi A. , Margaritis L. H.
Published: March 2003

Abstract: We report, for the first time, an unusual case of congenital anaemia with the clinical diagnosis of haemoglobin H disease complicated by morphological features at the light and electron microscopy level very similar to those of CDA‐I. The red cell indices and the globin chain biosynthetic ratio were not characteristic of the defective haemoglobin genotype. The haematological, clinical and morphological data strongly suggest the novel coexistence of the two defects in a patient. The disease is characterised by a unique dyserythropoietic phenotype of diagnostic importance, which possibly brings new data regarding the reciprocal interaction between the two diseases, especially concerning a specific abnormality in globin chain synthesis in CDA‐I, as previously suggested.
Activity of all‐trans‐retinoic acid in a case of central nervous system extramedullary relapse of acute promyelocytic leukemia
Authors: Patriarca Francesca , Filì Carla , Geromin Antonella , Sperotto Alessandra , Prosdocimo Simonetta , Fanin Renato
Published: March 2003

Abstract: We describe a patient with an acute promyelocytic leukemia (APL) previously treated with two courses of cytarabin, idarubicin and all‐trans retinoic acid (ATRA), who presented a medullary and meningeal relapse after 8 months of complete remission. A diagnosis of central nervous system (CNS) involvement was based on the appearance of APL blasts in the cerebrospinal fluid (CSF); magnetic resonance (MR) imaging was negative. The neurological symptoms were not evident at the time of recognition of the medullary recurrence, but appeared a few days later, when the patient had already received a reinduction treatment. When the CSF was first examined, showing atypical promyelocytes, there was no excess of blasts on bone‐marrow examination. The patient was treated with ATRA and intrathecal administrations of cytoxic drugs, achieving a complete long‐lasting CNS remission. The appearance of mature myeloid cells in the CSF during this treatment suggested a possible differentiating effect of ATRA towards extramedullary relapse.
Ki‐67 expression level, histological subtype, and the International Prognostic Index as outcome predictors in mantle cell lymphoma
Authors: Räty Riikka , Franssila Kaarle , Joensuu Heikki , Teerenhovi Lasse , Elonen Erkki
Published: March 2003

Abstract: Objectives
: Mantle cell lymphoma (MCL) is characterised by translocation t(11;14) (q13;q32) leading to rearrangement of bcl1/CCND1 and overexpression of the cell‐cycle regulatory protein cyclin D1. We assessed the significance of the cell proliferation rate as an outcome predictor with the five components of the International Prognostic Index (IPI) and the histological subtypes of MCL.
Patients and methods
: The hospital case records and histopathological material of 127 patients diagnosed with MCL in a single centre were reviewed. The cell proliferation rate was assessed by Ki‐67 immunostaining and mitosis counting. Cox's multivariate regression model was used in multivariate survival analyses. The median follow‐up time was 87 months.
Results
: The mantle zone/nodular subtype of the common variant (19%) was associated with median survival of 70 months, the diffuse subtype of the common variant (64%) of 35 months, and the blastoid subtype (17%) of 11 months (
P

 < 0.001). Patients with Ki‐67 expression in ≥ 26% (the upper tertile) of the lymphoma cells had median survival of only 13 months as compared with 45 months in the rest of the patients (
P

 < 0.001). In a multivariate analysis high Ki‐67 expression, Ann Arbor stage III–IV, and age over 60 yr had independent influence on survival, whereas serum lactate dehydrogenase level, the number of extranodal disease sites, and performance status did not.
Conclusions : The IPI may not be an optimal tool for outcome prediction in MCL, and a better prognostic index may be obtained by including Ki‐67 expression and possibly the histological subtype in the index.
Hodgkin's lymphoma in first relapse following chemotherapy or combined modality therapy: analysis of outcome and prognostic factors after conventional salvage therapy
Authors: Vassilakopoulos Theodoros P. , Angelopoulou Maria K. , Siakantaris Marina P. , Kontopidou Flora N. , Dimopoulou Maria N. , Boutsis Dimitrios E. , Anargyrou Konstantinos , Kokoris Styliani I. , Giannakakis Antonia , Karkantaris Christos , Kyrtsonis Marie‐Christine , Tsaftaridis Panayiotis , Rombos John , Variamis Eleni , Korkolopoulou Pinelopi , Kittas Christos , Pangalis Gerassimos A.
Published: March 2003

Abstract:Objectives: To investigate the prognosis of patients with Hodgkin's lymphoma (HL) who relapse following a complete remission (CR) achieved by chemotherapy with or without radiotherapy (CT±RT), and to identify prognostic factors for freedom from second progression (FF2P).Methods: We analyzed the prognostic significance of the initial CT regimen (4 vs. 7–8 drugs), treatment‐free interval (TFI), and demographic, clinical, and laboratory factors at the time of relapse and diagnosis, in 113 patients with HL, who relapsed after a CR achieved by CT±RT.Results: Conventional salvage CT±RT was administered in 107 patients, while six received RT only. The 5‐yr FF2P was 24%, while the 10‐yr survival after relapse (O2S) was 39% and was not afffected by the initial CT regimen. Multivariate analysis revealed that extranodal disease at relapse (P<0.001), TFI<6 month (P<0.001), 5 involved sites at diagnosis (P=0.04) and anemia at relapse (P=0.03) were independent predictors of FF2P. 55% of patients had 0 or 1 of these adverse prognostic factors. The 5‐yr FF2P of patients with 0, 1 or 2 adverse factors was 58%, 34% and 5% (P<0.0001). The corresponding rates for 10‐yr O2S were 68%, 51% and 25%, respectively (P=0.002).Conclusions:Our data confirmed the significance of TFI and extranodal relapse and demonstrated a potential role for anemia at relapse and number of involved sites at diagnosis, for the prognosis of patients with HL relapsing after CT±RT. The combination of these prognostic factors defines a sizeable subgroup of patients with favorable outcome following conventional salvage therapy.
Idiopathic autoimmune thrombocytopenia and neutropenia in siblings
Authors: Patel Ashwin P.
Published: March 2003

Abstract: Familial incidences of autoimmune disorders involving red cells, white cells and platelets are rare. Two cases of autoimmune neutropenia and thrombocytopenia occurring in two adult brothers are reported here. One patient showed prompt recovery of white cells with steroids but needed IV IgG+ splenectomy for durable platelet recovery. His brother required splenectomy for durable recovery of white cells and platelets as he showed transient recovery with steroids and IV IgG.
Epstein–Barr virus‐associated lymphoproliferative disease occurring in a patient with sarcoidosis treated by methotrexate and methylprednisolone
Authors: Theate I. , Michaux L. , Dardenne S. , Guiot Y. , Brière J. , Emile F.J. , Fabiani B. , Detry R. , Gaulard P.
Published: March 2003

Abstract: We describe the case of a 51‐yr‐old man with systemic sarcoidosis, complicated by the occurrence of a lymphoproliferative disease following a 36‐month (duration) immunosuppressive treatment with methotrexate (MTX) and methylprednisolone. Four years after the onset of sarcoidosis, the patient presented a large necrotizing anal fistula. Pathological examination of this lesion showed a diffuse polymorphic infiltrate containing large Epstein–Barr virus (EBV)‐positive lymphoid cells associated with areas of necrosis, all features similar to classical B‐cell lymphoproliferative disorders occurring in immunosuppressed solid‐organ recipients. MTX has been recently implicated in the development of lymphoproliferative disease in connective tissue diseases. This case supports the hypothesis that immunosuppression therapy may contribute to an increased risk for the development of EBV‐associated lymphoproliferative disorders in patients suffering from sarcoidosis.
Standard‐dose anti‐CD20 antibody rituximab has efficacy in chronic lymphocytic leukaemia: results from a Nordic multicentre study
Authors: Itälä M. , Geisler CH. , Kimby E. , Juvonen E. , Tjonnfjord G. , Karlsson K. , Remes K.
Published: March 2003

Abstract: Objectives: This prospective multicentre study was conducted to assess the efficacy of the monoclonal anti‐CD20 antibody rituximab in patients with chronic lymphocytic leukaemia (CLL). Secondary objectives were defined as the tolerability and feasibility of rituximab in patients with CLL. Methods: Twenty‐four heavily pretreated patients with CLL were treated with a standard dose of 375 mg m−2 of rituximab given once weekly for four doses. Results: The overall response rate was 35% and all the responses were partial as defined by the revised NCI criteria. In 17 (85%) of 20 patients with initially measurable peripheral lymph nodes the size of lymph nodes decreased by at least 50%, while an improvement of the bone marrow infiltration was observed only in two (11%) of 18 evaluable patients. The median duration of the overall response was 12.5 wk. Rituximab was relatively well tolerated. Although side‐effects were common (75%) they were usually mild or moderate. There was only one grade 3 adverse event and no grade 4 events. Conclusions: Standard‐dose rituximab has activity in heavily pretreated patients with CLL, although the response is mainly limited to the lymph nodes and of short duration. Since rituximab has in vitro synergism with chemotherapeutic agents and is well tolerated by CLL patients, it is reasonable to investigate rituximab in combination with other treatments.
Bisphosphonate therapy in multiple myeloma: past, present, future
Authors: Jantunen Esa
Published: March 2003

Abstract: Bone disease characterised by osteolytic lesions, pathological fractures and hypercalcaemia is an important clinical feature in multiple myeloma. Pain, decreased performance status, and the need for palliative radiotherapy and surgical interventions are common sequelae. Bisphosphonates act primarily on osteoclasts to inhibit excessive bone resorption, and have therefore been investigated in myeloma patients to ameliorate the clinical consequences of the bone disease. Bisphosphonates are currently the therapy of choice in myeloma patients with hypercalcaemia. In long‐term management, both oral clodronate and intravenous pamidronate are effective in reducing skeletal‐related events. Zoledronic acid seems to be as effective as pamidronate. Whether bisphosphonates have antimyeloma activity is currently unknown. Cost–benefit analyses have shown reasonable efficacy with acceptable costs. Bisphosphonate therapy is now accepted as an important part of care in myeloma patients, although much still has to be learned in order to optimise this therapy in multiple myeloma.
β‐Globin gene cluster haplotypes and HbF levels are not the only modulators of sickle cell disease in Lebanon
Authors: Inati A. , Taher A. , Bou Alawi W W. , Koussa S. , Kaspar H. , Shbaklo H. , Zalloua P. A.
Published: March 2003

Abstract: Sickle cell disease (SCD) is an inherited autosomal recessive disorder of the β‐globin chain. Despite the fact that all subjects with SCD have the same single base pair mutation, the severity of the clinical and hematological manifestations is extremely variable. This study examined for the first time in Lebanon the correlation between the clinical manifestation of SCD and the β‐globin gene haplotypes. The haplotypes of 50 patients diagnosed with SCD were determined using polymerase chain reaction amplification of fragments containing nine polymorphic restriction sites around and within the ε–Gγ–Aγ–ψβ–δ–β‐globin gene complex. Most reported haplotypes were found in our population with the Benin haplotype as the most prevalent one. When the patients were divided according to their HbF levels into three groups (Group A: HbF < 5%, Group B: HbF between 5 and 15%, and Group C: HbF > 15%), surprisingly, the highest levels of HbF were associated with the most severe clinical cases. Our findings suggest that fetal hemoglobin levels are important but not the only parameters that affect the severity of the disease. In addition, the high levels of HbF in patients with CAR haplotypes did not seem to ameliorate the severity of symptoms, suggesting that genetic factors other than haplotypes are the major determinants of increased HbF levels in Lebanon.
Effects of UVA radiation on an established immune response in humans and sunscreen efficacy
Authors: Moyal Dominique D. , Fourtanier Anny M.
Published: March 2003

Abstract: It is well established that ultraviolet radiation has immunomodulatory effects which may be involved in skin cancer. Recent studies have shown that UVA radiation (320–400 nm) as well as UVB (290–320 nm) is immunosuppressive. This means that sunscreens which mainly absorb UVB (protection against erythema) may be less effective in preventing UVR‐induced immunosuppression than broad‐spectrum products. We have studied the effects of UVA exposure on the human delayed‐type hypersensitivity response (DTH) and compared the efficacy of sunscreens having different levels of UVA protection under both solar‐simulated radiation (SSR) chronic exposures or acute exposure and outdoor real‐life solar exposure conditions. DTH was assessed using recall antigens. Our studies clearly demonstrate the role of UVA in the induction of photoimmunosuppression together with the need for sunscreen products providing efficient photoprotection throughout the entire UV spectrum. These data suggest that sun protection factor may not be sufficient to predict the ability of sunscreens for protection from UV‐induced immune suppression. Determining the level of UVA protection is particularly necessary, because UVA seems to have a relatively low contribution to erythema but is highly involved in immunosuppression.
Comet assay combined with p53 detection as a sensitive approach for DNA photoprotection assessment in vitro
Authors: Marrot Laurent , Belaïdi Jean‐Philippe , Meunier Jean‐Roch
Published: March 2003

Abstract: A simple in vitro approach where sun formulations are spread on a quartz slide and placed over human skin cells in culture is proposed as a convenient test for photoprotection assessment at the DNA level. Using the comet assay, DNA strand breaks and oxidative DNA damage were detected. Then, accumulation of p53 protein was studied as a marker for UV‐induced genotoxic stress. Such a method was used to compare formulations with different photostability. Spectroradiometry showed that a photo‐unstable formulation lost its effectiveness in UVA screening when pre‐irradiated by simulated sunlight. As a consequence, such a formulation was not as protective as a photostable one at the genomic level.
A three‐dimensional skin culture model for mouse keratinocytes: application to transgenic mouse keratinocytes
Authors: Carroll J. M. , Molès J.‐P.
Published: March 2003

Abstract: The study of mouse epidermal biology has been hampered by the lack of a good in vitro model for the culture of mouse keratinocytes which allowed the reconstruction of a fully differentiated epidermis. We adapted the Pruniéras' model, also called the Dead de‐Epidermized Dermis model (DED), to mouse keratinocytes and showed that a neo‐epidermis can be reconstructed exhibiting a complete differentiation program. We also used this model to culture transgenic mouse keratinocytes. We observed that transgene expression occurred in the correct location and that the neo‐epidermis mimed previous in vivo observations obtained with integrin skin‐targeted transgenic mice. Therefore, this model will be a powerful tool to further investigate normal mouse and transgenic keratinocyte biology.
Substance P induction of murine keratinocyte PAM 212 interleukin 1 production is mediated by the neurokinin 2 receptor (NK‐2R)
Authors: Song I.‐S. , Bunnett N. W. , Olerud J. E. , Harten B. , Steinhoff M. , Brown J. R. , Sung K. J. , Armstrong C. A. , Ansel J. C.
Published: March 2003

Abstract: The neurological system plays an important role in modulating some inflammatory skin diseases. Neuro‐cutaneous interactions may be mediated by the release of neuropeptides such as substance P (SP) which activate immunocompetent cells in the skin by binding to high affinity neurokinin receptors (NKR). Since epidermal keratinocytes produce a variety of cytokines and are intimately associated with cutaneous sensory fibers, we tested the ability of these cells to participate in the cutaneous neuroimmune system by the secretion of potent cytokines such as interleukin 1 (IL‐1) in response to released SP. RT‐PCR studies demonstrated that cultured PAM 212 murine keratinocytes expressed mRNA for NK‐2R but not NK‐1R. Correspondingly, the addition of SP to these cells resulted in a rapid increase in intracellular Ca2+ levels that could be specifically blocked by an NK‐2R antagonist. NK‐2R was also shown in normal mouse epidermis by immunohistochemistry. SP augmented the expression of PAM 212 keratinocyte IL‐1α mRNA in a dose and time dependent manner and this induction was inhibited by an NK‐2R antagonist. Secretion of bioactive IL‐1α by the PAM 212 keratinocytes was likewise stimulated by SP in a dose dependent manner. These data support the hypothesis that SP released from cutaneous sensory nerves contributes to neuroimmune inflammatory responses in the skin by modulating the expression and release of cytokines from epidermal keratinocytes.
Calcium release activated calcium entry in a human skin derived cell line (HaCaT)
Authors: Csernoch L. , Hunyadi J. , Kovács L.
Published: March 2003

Abstract: Using isolated cells from a spontaneously immortalized human keratinocyte cell line (HaCaT) loaded with Fura‐2 the regulation of intracellular Ca2+ concentration ([Ca2+]i) was studied. The continuous presence of ATP induced a biphasic response in high external Ca2+. The first component reflected the release of calcium from intracellular stores since it was present after the removal of external calcium with ethylene‐glycol‐bis‐N,N,N′,N′‐tetraacetic acid (EGTA). The second phase of [Ca2+]i increase was not detectable in the absence of external calcium and raising the extracellular [Ca2+] increased the rate of rise in [Ca2+]i suggesting that it was influenced by the external environment. Furthermore, after adenosine triphosphate (ATP) had emptied the intracellular store in a calcium‐free milieu, the elevation of external Ca2+ induced a secondary increase in [Ca2+]i that did not require the presence of ATP. Depleting the intracellular calcium store with a Ca‐ATP‐ase inhibitor (cyclopiasonic acid, 10 μM) also induced calcium entry. The depletion induced calcium entry was inhibited by econazole (100 μM). These findings indicate the presence of a calcium release activated calcium influx pathway in HaCaT keratinocytes.
IL‐15 and IL‐16 overexpression in cutaneous T‐cell lymphomas: stage‐dependent increase in mycosis fungoides progression
Authors: Asadullah K. , Haeußler‐Quade A. , Gellrich S. , Hanneken S. , Hansen‐Hagge T. E. , Döcke W.‐D. , Volk H.‐D. , Sterry W.
Published: March 2003

Abstract: Cytokines are of major importance for the pathogenesis of cutaneous T‐cell lymphomas (CTCL). Recent data suggested that IL‐15 and IL‐16 are survival/growth factors for the malignant T cells in these entities. To investigate the expression of IL‐15 and IL‐16 in mycosis fungoides (MF) and CD30+ pleomorphic T‐cell lymphoma in vivo, we established a competitive RT‐PCR technique. Analyzing skin biopsies from CTCL patients at different stages in comparison to psoriatic and healthy skin, we found IL‐15 and IL‐16 mRNA overexpression in both CTCL entities. Remarkably, there was some evidence for a stage‐dependent increase during MF progression. We found only slight overexpression in early stage MF, when only few tumor cells are detectable within the infiltrates, whereas marked overexpression was found in more advanced lesions, which are characterized by a higher density of malignant cells. These results suggested that CTCL cells themselves might produce the cytokines. To further elucidate this hypothesis, two CTCL cell lines were analyzed but gave conflicting results. Therefore, the cellular origin of the IL‐15 and IL‐16 overexpression in CTCL remains unclear. Considering the significant overexpression of IL‐15 and IL‐16 and their biological capacities it is likely that these cytokines contribute to the tumor development. So, they might be involved in growth and skin homing of CTCL cells.
Preimplantation genetic diagnosis in two families at risk for recurrence of Herlitz junctional epidermolysis bullosa
Authors: Cserhalmi‐Friedman P. B. , Tang Y. , Adler A. , Krey L. , Grifo J. A. , Christiano A. M.
Published: March 2003

Abstract: The Herlitz type of junctional epidermolysis bullosa (H‐JEB) is a severe inherited bullous disease which leads to the early demise of the affected newborn. Mutations in the genes encoding the 3 polypeptides of the anchoring filament protein laminin 5 underlie this condition. We studied 2 families with affected children who previously died from H‐JEB. Mutation screening using heteroduplex analysis and direct sequencing of the PCR products revealed a previously described hotspot mutation in LAMB3 (R635X), and a novel delayed termination codon in LAMB3 in the first proband. In the second proband, we found a novel initiation codon mutation in LAMB3, and a novel 2 bp deletion in LAMB3. For preimplantation genetic diagnosis (PGD) in these families, we developed nested multiplex PCR assays, amplifying the mutations and informative intragenic polymorphisms in the probands. Single embryonic cells were biopsied from 8‐cell embryos using standard techniques, and subjected to the multiplex PCR assay followed by restriction enzyme digestion. Embryos found not to carry either mutation were transferred to the mothers, and a pregnancy was established in the second family as evidenced by the elevated level of HCG, although the pregnancy did not persist. This study illustrates the feasibility of PGD for an inherited skin disorder for the first time.
What is the most promising strategy for the treatment of metastasizing melanoma?
Authors: Cascinelli N. , Heerlyn M. , Schneeberger A. , Kuwert C. , Slominski A. , Armstrong C. , Belli F. , Lukiewcz S. , Maurer D. , Ansel J. , Stingl G. , Saida T.
Published: March 2003

Abstract: The treatment of patients with metastasizing melanoma, still one of the most deadly diseases in modern medicine, ranks among the greatest challenges that a clinician has to face. Metastatic melanoma also is one of the most profound sources of clinical frustration, since it provides far more ultimately defeating experiences than clinical victories. At the same time, the fascinating biology of melanoma has invited the study of this neuroectodermal tumor as a model system for dissecting many of the key problems of modern oncology, ranging from molecular oncogenesis via the controls of tumor proliferation, apoptosis, invasion, metastasis, and angiogenesis to tumor immunosurveillance and tumor drug resistance. Together with the dire need to develop more effective treatment modalities for improving both life expectancy and quality of life of affected patients, this has made metastatic melanoma a favorite model for the exploration of innovative strategies for tumor management. Encouragingly, many of these have already generated very promising results in animal models. However, this impressive level of research progress in conquering melanoma in the animal room contrasts rather pitifully with the actual progress made on the ward. This CONTROVERSIES feature, therefore, critically and soberly reviews the state of the art of treating metastatic melanoma today (distinguishing between nodal and distant metastases), and sharply defines unresolved or comparatively neglected key problems. In addition, this feature highlights several novel, provocative, hitherto underappreciated, yet potentially promising treatment approaches that deserve systematic exploration. Hopefully, this will offer further inspiration for the design and pursuit of innovative anti‐melanoma strategies off‐the‐beaten‐track.
Serum‐free cultured keratinocytes fail to organize fibronectin matrix and possess different distribution of beta‐1 integrins
Authors: Altankov G. , Hecht J. , Dimoudis N.
Published: March 2003

Abstract: The development of serum free medium formulation for culturing keratinocytes was a breakthrough in achieving a high number of epidermal cells for experimental and therapeutic studies, in particular to support the wound healing process. It is not clear, however, if switching the cells to highly proliferative phenotype may reflect change in other cellular functions important for the wound repair as their adhesive interactions with the extracellular matrix components. Remodelling of the extracellular matrix, particularly of fibronectin plays an essential role for guiding the cells during wound healing. The molecular mechanisms for organization of this provisional fibronectin matrix, however, are still not clear. We found that keratinocytes in serum containing medium, although in fewer numbers than fibroblasts, were able to remove adsorbed fluorescent labelled fibronectin from the substratum and reorganize it in a fibrilar pattern along the cell periphery. After 3 days the secreted fibronectin had also been organized as matrix‐like fibers and as clusters deposited on the substratum after migrating cells. In contrast, serum free cultured keratinocytes fail to organize pre‐adsorbed fluorescent labelled fibronectin, as well as the secreted fibronectin, although they grow very well under these conditions. Switching the cells to serum containing medium initiates the removal of fluorescent labelled fibronectin from the substratum, however without reorganization in fibrillar pattern. Most likely, these keratinocytes remove fluorescent labelled fibronectin by the expression of proteolytic activity, rather than with the mechanical function of β1 integrins. The latter were diffusely dispersed in serum containing conditions and tend to organize in focal adhesions in serum free cultured cells. We assumed their transient expression and different affinity state might be important for the keratinocyte migration and matrix assembly mechanism.
Isolating a pure population of epidermal stem cells for use in tissue engineering
Authors: Dunnwald M. , Tomanek‐Chalkley A. , Alexandrunas D. , Fishbaugh J. , Bickenbach J. R.
Published: March 2003

Abstract: Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label‐retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and nonproliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.
An improved and rapid method to construct skin equivalents from human hair follicles and fibroblasts
Authors: Hoeller D. , Huppertz B. , Roos T. C. , Poblete Gutiérrez P. , Merk H. F. , Frank J. , Jugert F. K.
Published: March 2003

Abstract: To produce sufficient amounts of high quality skin equivalents (SE), either allogenic for dermatopharmacological and dermatotoxicological studies or autologous for transplantation purposes, we established a rapid, easy and cost effective three‐dimensional SE model on the basis of human dermal fibroblasts, collagen and freshly plucked hair follicles. Acidic liquid collagen was polymerized with sodium hydroxide in the presence of fibroblasts to form a dermal equivalent (DE) resembling normal human dermis. At 24 h later, freshly plucked hair follicles were implanted into the surface of these DEs after cutting their bulbs off. Another 48 h later, the surface of the SEs was lifted to the air–liquid interface. Fourteen days after implantation, outgrowing keratinocytes from the outer root sheath of the hair follicles completely covered the surface of the SE and built a fully developed, multi‐layered and cornified epidermis. Histology and immunofluorescence studies with specific antibodies directed against components of keratinocytes, fibroblasts, cell‐adhesion molecules, different extracellular matrix and basement membrane proteins revealed the similarity of our three‐dimensional SEs to the in vivo situation in normal human skin. Using autologous cell sources and cell culture media enriched with serum from the respective cell donor, it will be possible to use these SEs for autologous transplantation, thereby reducing the risk of transplant rejection.
Regulation of epidermal growth factor receptor expression in human melanocytes
Authors: Gordon‐Thomson C. , Mason R. S. , Moore G. P. M.
Published: March 2003

Abstract: The epidermal growth factor receptor (EGFR) and its ligand, transforming growth factor alpha (TGFα), are reportedly involved in autocrine growth of melanoma cells. The signal pathway has also been implicated in early events of transformation, suggesting a function for EGFR in normal cells. This study reports the presence of EGFR in cultured melanocytes and examines some cellular responses to TGFα. Western analysis revealed 170 kDa bands in extracts of cultured neonatal human melanocytes, corresponding to the receptor Mr. Protein expression was more pronounced in cells during active growth. EGFR were less evident in cultures populated predominantly by melanized cells, indicating that receptor expression became reduced in differentiating cells. Immunocytochemistry confirmed these observations and also showed that EGFR reactivity was predominantly localized in the cell body but absent from dendrites. Addition of TGFα to early cultures induced a rapid increase in phosphotyrosine signal of the 170 kDa protein. Longer treatment (24–48 h) increased the intensity of the EGFR signal, suggesting that receptors had been upregulated. However, inclusion of TGFα in cultures did not result in an increase in cell numbers when compared to controls. The observations provide evidence of the existence of a receptor‐mediated pathway in melanocytes which has transforming potential in vivo.
Expression of dipeptidyl‐peptidase IV (CD26) on CD8+ T cells is significantly decreased in patients with psoriasis vulgaris and atopic dermatitis
Authors: Bock O. , Kreiselmeyer I. , Mrowietz U.
Published: March 2003

Abstract: T cells play a major role in inflammatory skin disorders such as psoriasis vulgaris and atopic dermatitis. They are both active on the level of cell‐to‐cell interaction and by the secretion of pro‐inflammatory mediators. CD26 is a lymphocyte membrane‐associated dipeptidyl peptidase IV (DPP IV), which is able to inactivate chemokines such as RANTES or eotaxin by cleaving dipeptides from the NH2−terminus of proteins. We investigated the expression of CD26 on CD4+ and CD8+ peripheral blood T cells in patients with psoriasis and atopic dermatitis. In addition PASI and SCORAD as a measure of disease severity were determined in each patient at the time of blood drawing. Thirty patients with psoriasis, 15 with atopic dermatitis and 17 age‐ and sex‐matched healthy persons were investigated by two‐colour flow cytometry using epitope‐specific monoclonal antibodies. Our results revealed, that there is a significant decrease (P<0.05) of CD26 expression on CD8+ T cells in both psoriasis (7.7%±3.3, mean and SD, n=30) and atopic dermatitis patients (7.9%±3.7, mean and SD, n=15) compared to the control population (11.58%±5.0, mean and SD, n=17). However, there was no correlation to disease severity as determined by PASI and SCORAD, respectively. Since CD26 can be regarded as an anti‐inflammatory principle the decreased expression in psoriasis and atopic dermatitis patients may lead to a dysbalance in favour of pro‐inflammatory mediators in both clinical conditions.
Effect of protein kinase C on transmembrane calcium fluxes in HaCaT keratinocytes
Authors: Gönczi Mónika , Papp Helga , Bíró Tamás , Kovács László , Csernoch László
Published: March 2003

Abstract: Capacitive calcium influx is associated with the release of calcium from internal stores and participates in intracellular calcium homeostasis. In keratinocytes, its activation is linked to the stimulation of the phospho‐inositide (PI) pathway and seems to be altered in psoriasis. An overnight treatment of isolated HaCaT keratinocytes with phorbol 12‐myristate 13‐acetate (PMA) selectively downregulated the classical, calcium‐dependent protein kinase C (PKC) isoenzyme PKCα in preconfluent cells. This was parallelled by an increased capacitative calcium influx with no effects on the PI pathway. These observations were strengthened in measurements using cyclopiazonic acid which revealed a 47% increase in PMA pretreated as compared with control cells in the calcium influx rate through store‐operated calcium channels (SOC‐s) following the emptying of the intracellular calcium stores. In confluent as compared with preconfluent cultures PKCε was markedly increased, while other isoenzymes were not affected. In parallel, the kinetics of capacitative calcium influx were altered, showing clear inactivation. PMA pretreatment in these cells had little effect on PKCα but downregulated both PKCβ and PKCε, and did not increase the influx through SOC‐s. These observations support the differential regulation of SOC‐s by PKC and suggest the involvement of several PKC isoenzymes in human keratinocytes.
Similar UV responses are seen in a skin organ culture as in human skin in vivo
Authors: Bäckvall Helena , Wassberg Cecilia , Berne Berit , Ponten Fredrik
Published: March 2003

Abstract: Ultraviolet radiation (UVR) plays an important role in the development of non‐melanoma skin cancer. Most tumors develop in chronically sun‐exposed skin, most often in cosmetically sensitive locations, where in vivo experiments may be difficult to perform. In this study, we describe a skin organ culture model with preserved normal morphology and intact response to UVR. Skin explants from chronically sun‐exposed and non‐sun‐exposed skin were irradiated with artificial UVA+UVB with and without topical sunscreen. UV‐induced DNA damage, epidermal p53 response and repair kinetics were analyzed using immunohistochemistry. Four hours after UV‐irradiation epidermal keratinocytes showed a strong immunoreactivity for thymine‐dimers. Gradual repair during an incubation time resulted in few residual thymine‐dimers after 48 h. Repair appeared to be more efficient in chronically sun‐exposed skin compared with non‐sun‐exposed skin. There was also an accumulation of p53 protein in epidermal keratinocytes, peaking at 4–24 h after irradiation. Large interindividual differences with respect to formation and repair of thymine‐dimers as well as induction and duration of the p53 response were observed. Skin explants treated with topical sunscreen prior to UV‐irradiation showed a clear reduction of thymine‐dimers and p53 expression. The epidermal UV‐responses and repair kinetics in organ‐cultured skin were similar to what was found in vivo. Our data suggest that organ‐cultured skin provides a valuable tool for studies of UV‐induced epidermal responses in chronically sun‐exposed skin.
Transcriptional regulation of tumor necrosis factor‐α in keratinocytes mediated by interleukin‐1β and tumor necrosis factor‐α
Authors: Lisby S. , Hauser C.
Published: March 2003

Abstract: Irritant contact dermatitis (ICD) is an inflammatory skin reaction in which cytokines are thought to play a crucial role. In particular, tumor necrosis factor‐α (TNF‐α) has been implicated in the mechanism of this reaction. We report that interleukin‐1β (IL‐1β) that has been reported up‐regulated in many inflammatory skin conditions is capable of increasing TNF‐α mRNA and protein expression in murine keratinocytes. Furthermore, we show that TNF‐α is capable of up‐regulating itself in keratinocytes most likely in an autocrine manner. The signalling mechanisms involved in both IL‐1β‐ and TNF‐α‐mediated regulation of TNF‐α are critically dependent upon protein kinase C (PKC), as demonstrated by blocking studies using protein kinase inhibitors. Furthermore, the increase in TNF‐α mRNA expression seen after stimulation with rTNF‐α and rIL‐1β involved increased transcription of TNF‐α mRNA. This was demonstrated in a chloramphenicol acetyltransferase (CAT) assay using a CAT‐construct containing the full‐length TNF‐α promoter. These observations support the notion of keratinocytes functioning as an amplifier of pro‐inflammatory cytokine generation in the epidermis during ICD and other inflammatory skin conditions.
Genetic structure of New Zealand eels Anguilla dieffenbachii and A. australis with allozyme markers
Authors: Smith P. J. , Benson P. G. , Stanger C. , Chisnall B. L. , Jellyman D. J.
Published: March 2003

Abstract – Glass eels and adults of Anguilla australis and A. dieffenbachii were collected from rivers in the North and South Islands of New Zealand and tested for nine and ten allozyme loci, respectively. There was no heterogeneity among glass eel samples, but there was heterogeneity among adult samples within species. The results indicate that there may be single panmictic populations subject to sweepstake events or different selective pressures in the adult environments.
Using allometry with fish size to estimate production to biomass (P/B) ratios of salmonid populations
Authors: Randall R. G.
Published: March 2003

Abstract – The ratio of annual production rate to average biomass (P/B) is sometimes used as a shortcut method of estimating production if biomass is known.P/Bratios vary among salmonid populations in different areas depending on the species, fish size and growing conditions. The hypothesis that annualP/Bratios for salmonid populations can be predicted from the allometric equation:P/B = a W−0.35was investigated in this study. The allometric coefficient (−0.35) was obtained from the literature, andWwas the average weight (g) of fish in the population. Plots ofP/Bversus fish weight for three species of salmonids,Salvelinus fontinalis,Salmo truttaandSalmo salar, showed a declining trend, with a slope consistent with the hypothesis. The coefficientaof the allometric equation was species and habitat dependent. The efficacy of using the allometric formula to estimate theP/Bratio of specific salmonid populations is examined.
Expression of cytokeratins in human enamel organ
Authors: Domingues Manoela G. , Jaege Márcia M. M. , Araújo Vera C. , Araújo Ney S.
Published: March 2003

Cytokeratin (CK) is a filament which plays a central role in epithelial tissue and, like the polypeptides of intermediate filaments in general, shows a high degree of tissue specificity. The CK expression patterns of odontogenic epithelia are still poorly described. We studied the distribution of individual CK polypeptides in the human enamel organ at bell stage and in remnants of the dental lamina. Our immunohistochemical study showed that epithelial cells stained for CKs 7, 13, 14 and 19 with slight changes in their pattern during the differentiation phase of odontogenesis. There was negative staining for all other CK polypeptides tested (CKs 8, 10, 16, 17 and 18). Most of the CKs in the enamel organ epithelia did not show differences related to the stage‐specific state of differentiation, except for CKs 14 and 19 at the inner enamel epithelium. A strong label for CK 14 was present at the inner dental epithelium at early bell stage, and this was substituted by CK 19 at the late bell stage when the ameloblasts were fully differentiated.
Fluoride effect on the activity of enamel matrix proteinases in vitro
Authors: Gerlach Raquel Fernanda , De Souza Ana Paula , Cury Jaime Aparecido , Line Sérgio Roberto Peres
Published: March 2003

Dental fluorosis is common in individuals exposed to different sources of fluoride during tooth development. The mechanism causing this enamel defect is still unknown. Enamel matrix proteinases play a central role in the maturation of dental enamel, and inhibition of these enzymes by fluoride has been one explanation for dental fluorosis. We have investigated the effect of fluoride on the activity of enamel matrix proteinases using a colorimetric assay, casein zymography, and an enamel protein degradation assay. Fluoride (625 μM to 10 mM) inhibited neither the enzymatic activity of the crude matrix extract nor the activity of individual enamel enzymes separated by SDS‐PAGE. The proposition that fluoride could directly inhibit enzymes was not confirmed in this study.
Total IgA and Porphyromonas gingivalis‐reactive IgA in the saliva of patients with generalised early‐onset periodontitis
Authors: Hägewald Stefan , Bernimoulin Jean‐Pierre , Köttgen Eckart , Kage Andreas
Published: March 2003

Generalised early‐onset periodontitis (GEOP) is characterized by acute inflammatory bursts, resulting in rapid destruction of the periodontal apparatus in young adults. An impaired host defense seems to play an improtant role as etilogical factor of periodontitis, especially in the development of GEOP. As the Gram‐negative Porphyromonas gingivalis has been indentified as one of the causative anaerobic bacteria, the humoral immune response to this micro‐organism is of particular interest in patients with GEOP. To evaluate the local immune status, we measured total and P. gingivalis‐ reactive salivary IgA in GEOP patients and in age‐ and gender‐matched periodontally normal controls. We found a significantly lower concentration and secretion rate of total salivary IgA in the GEOP group. Although no differences were detected in the concentration or secretion of P. gingivalis‐reactive IgA between groups, the specific fraction of P. gingivalis‐reactive IgA of the total IgA was significantly higher in the GEOP group. These findings indicate an inhibition of total secretory IgA in GEOP, while the P. gingivalis‐reactive humoral immune system in saliva is, however, activated. P. gingivalis seems to selectively activate IgA lymphocyte clones and induces a switch in the fraction of specific IgA.
One‐step sandwich enzyme immunoassay using monoclonal antibodies for detection of human enamelysin (MMP‐20)
Authors: Wang Ting , Aoki Takanori , Iwata Kazushi , Takata Takashi , Uchida Takashi , Knäuper Vera , Llano Elena , Okada Yasunori , Bartlett John D.
Published: March 2003

A one‐step sandwich enzyme immunoassay (EIA) system for human matrix metalloproteinase 20 (MMP‐20, enamelysin) was established by use of a solid‐phase monoclonal antibody and a separate peroxidase‐labeled monoclonal antibody. The EIA system was shown to be sensitive and quantitative for the detection of MMP‐20. As little as 1.0 ng/ml (50 pg/assay) of MMP‐20 protein could be reliably detected. The EIA system was linear over a range of 2.5–160 ng/ml (125–8,000 pg/assay), and the EIA system was versatile in that it was capable of detecting with equal sensitivity proMMP‐20, active MMP‐20, and MMP‐20 with COOH‐terminal deletions. The EIA system was validated by the successful detection of MMP‐20 in the culture medium of Chinese hamster ovary cells (CHO‐K1) that were transfected with an MMP‐20 expression vector. No MMP‐20 was detected in normal human serum, normal saliva, or in selected tumors. However, when recombinant human MMP‐20 was added to human saliva, the EIA system did detect quantifiable amounts of the MMP‐20, indicating that the system will work within the framework of complex in biological fluids.
Substance P expression in human tooth pulp in relation to caries and pain experience
Authors: Rodd Helen D. , Boissonade Fiona M.
Published: March 2003

The neuropeptide substance P (SP) is found within a subpopulation of nociceptive afferent nerve fibres and has been shown to be upregulated in a variety of sites following peripheral inflammation. The aim of this study was to investigate the expression of SP within human teeth, both in health and disease, and to seek a correlation between reported pain history and SP expression within pulpal nerves. Coronal pulps were removed from 62 permanent mandibular molars with a known pain history. Teeth were categorised as intact, moderately or grossly carious. Using indirect immunofluorescence, sections were double‐labelled for the general neuronal marker protein gene product 9.5 (PGP 9.5) and for SP. Image analysis was then used to quantify the percentage area of PGP 9.5‐labelled tissue which was also labelled for SP. Throughout the pulp, the expression of SP was found to be significantly increased with the progression of caries. Furthermore, SP expression was significantly greater in grossly carious painful specimens than in grossly carious asymptomatic specimens. These data would suggest that the expression of SP within pulpal nerves undergoes dynamic changes following caries, which may have an important clinical significance in terms of inflammation and pain experience.