bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of G proteins involved in activation of nonselective cation channels by endothelinB receptor
Authors: Kawanabe Yoshifumi , Hashimoto Nobuo , Masaki Tomoh
Published: September 2002

We recently demonstrated that endothelin‐1 (ET‐1) activates two types of Ca2+‐permeable nonselective cation channels (NSCC‐1 and NSCC‐2) in Chinese hamster ovary cells expressing endothelinB receptors (CHO‐ETBR) that couple with Gq and Gi. The purpose of the present study was to identify the G proteins involved in the activation of these Ca2+ channels by ET‐1. For this purpose, we constructed CHO cells expressing an unpalmitoylated (Cys402Cys403 Cys405→Ser402Ser403Ser405) ETBR (CHO‐SerETBR) and ETBR truncated at the cytoplasmic tail downstream of Cys403 (CHO‐ETBRΔ403).

Based on the data obtained from actin stress fibre formation, CHO‐ETBR couple with G13. Therefore, CHO‐ETBR couple with Gq, Gi and G13. CHO‐SerETBR and CHO‐ETBRΔ403 couple with G13 and Gq, respectively.

ET‐1 activated NSCC‐1 in CHO‐ETBR preincubated with phospholipase C (PLC) inhibitor, U73122, and in CHO‐SerETBR. On the other hand, ET‐1 failed to activate Ca2+ channels in CHO‐ETBRΔ403. Microinjection of dominant negative mutants of G13 (G13G225A) abolished activation of NSCC‐1 and NSCC‐2 in CHO‐ETBR and that of NSCC‐1 in CHO‐SerETBR.

Y‐27632, a specific Rho‐associated kinase (ROCK) inhibitor, did not affect the ET‐1‐induced transient and sustained increase in [Ca2+]i in CHO‐ETBR.

These results indicate that (1) the cytoplasmic tail downstream of the palmitoylation sites of ETBR, but not the palmitoylation site itself, is essential for coupling with G13, (2) the activation mechanism of each Ca2+ channel by ET‐1 is different in CHO‐ETBR. NSCC‐1 activation depends on G13‐dependent cascade, and NSCC‐2 activation depends on both Gq/PLC‐ and G13‐dependent cascades. Moreover, ROCK‐dependent cascade is not involved in the activation of these channels.

British Journal of Pharmacology (2002) 136, 1015–1022. doi:10.1038/sj.bjp.0704805
Actions of orphanin FQ/nociceptin on rat ventral tegmental area neurons in vitro
Authors: Zheng Fang , Grandy David K , Johnson Steven W
Published: September 2002

Non‐dopamine (putative GABAergic) neurons in the ventral tegmental area are in a position to influence mesolimbic functions by their inhibitory terminals that impinge locally on dopamine neurons and via their GABAergic efferents that innervate mesolimbic structures. In the present study we investigated responses of non‐dopamine and dopamine neurons, recorded intracellularly in the rat midbrain slice, to orphanin FQ/nociceptin, the endogenous ligand for opioid receptor‐like orphan receptors.

When recording in either non‐dopamine or dopamine neurons, orphanin FQ/nociceptin reduced the frequency of spike firing and caused membrane hyperpolarization under current‐clamp, or produced outward current under voltage‐clamp. Such responses were concentration‐dependent and reversed at −108 mV and −102 mV in non‐dopamine and dopamine neurons, respectively.

Hyperpolarizations to orphanin FQ/nociceptin were not altered by tetrodotoxin or the opioid receptor antagonist naloxone, but were reduced by the opioid receptor‐like orphan receptor antagonist [Phe11φCH2‐NH)Gly2]NC(1–13)NH2 (1 μM).

In dopamine neurons, orphanin FQ/nociceptin reduced the frequency of bicuculline‐ and tetrodotoxin‐sensitive spontaneous inhibitory postsynaptic potentials, and reduced the amplitude of stimulus‐evoked inhibitory postsynaptic potentials.

Taken together, the above data provide evidence that both non‐dopamine and dopamine neurons are important substrates for orphanin FQ/nociceptin within the ventral tegmental area. Simultaneous inhibition of both non‐dopamine and dopamine pathways by orphanin FQ/nociceptin may account for its influences on various ventral tegmental area‐related functions.

British Journal of Pharmacology (2002) 136, 1065–1071. doi:10.1038/sj.bjp.0704806
Renal targeting of captopril selectively enhances the intrarenal over the systemic effects of ACE inhibition in rats
Authors: Haverdings R Folgert G , Haas Marijke , Navis Gerjan , van Loenen‐Weemaes Anne‐miek , Meijer Dirk K F , de Zeeuw Dick , Moolenaar Frits
Published: September 2002

In previous studies on the renal targeting of the ACE inhibitor captopril, we demonstrated that a 6 fold increased concentration of this drug could be obtained in the kidney after conjugation to the low‐molecular‐weight protein lysozyme. In this study, we investigated in unrestrained rats whether systemic administration of captopril–lysozyme also results in an enhanced effect on renal parameters, relative to the systemic effects.

Renal effects: intravenous infusion of captopril–lysozyme for 6 h resulted in a more pronounced increment of renal blood flow (31±2% vs 17±4% at 0.5 mg kg−1 6h−1, P<0.01) and an approximately 5 fold enhanced natriuresis (167±17% vs 36±7% at 1 mg kg−1 6 h−1, P<0.001) in comparison with equimolar amounts of captopril as a free drug. In correspondence with these findings, renal ACE inhibition was potentiated approximately 5 fold (−50±4% vs −22±3% at 1 mg kg−1 6 h−1, P<0.001).

Systemic effects: conjugated captopril did not affect blood pressure in dosages up to 5 mg kg−1 6 h−1. This effect coincided with a less pronounced inhibition of the pressor response to intravenously administered angiotensin I (−12±3% vs −66±5% at 1 mg kg−1 6 h−1, P<0.001), and a markedly attenuated plasma ACE inhibition (−19±2% vs −37±3% at 1 mg kg−1 6 h−1, P<0.001) compared to an equivalent dose of free captopril.

An experiment of continued intravenous administration of captopril–lysozyme for 7 days in nephrotic syndrome demonstrated that the conjugate is also active in renal disease: the antiproteinuric response was substantially augmented (−67±5% vs −15±7% at 4 mg kg−1 24 h−1, P<0.001) compared to the free drug, in the absence of blood pressure reduction.

These data demonstrate that intravenous administration of a captopril–lysozyme conjugate leads to more selective renal ACE inhibition and enhanced renal effects as well as less systemic effects compared to captopril itself.

British Journal of Pharmacology (2002) 136, 1107–1116. doi:10.1038/sj.bjp.0704814
Protein kinase mediated upregulation of endothelin A, endothelin B and 5‐hydroxytryptamine 1B/1D receptors during organ culture in rat basilar artery
Authors: Hansen‐Schwartz Jacob , Svensson Carl‐Lennart , Xu Cang‐Bao , Edvinsson Lars
Published: September 2002

Organ culture has been shown to upregulate both endothelin (ET) and 5‐hydroxytryptamine 1B/1D (5‐HT1B/1D) receptors in rat cerebral arteries. The purpose of the present study was to investigate the involvement of protein kinases, especially protein kinases C (PKC) and A (PKA) in this process.

The effect of inhibiting protein kinases during organ culture with staurosporine (unspecific protein kinase inhitor), RO 31‐7549 (specific inhibitor of classical PKC's) and H 89 (specific inhibitor of PKA) was examined using in vitro pharmacological examination of cultured vessel segments with ET‐1 (unspecific ETA and ETB agonist), S6c (specific ETB agonist) and 5‐CT (5‐HT1 agonist). Levels of mRNA coding for the ETA, ETB, 5‐HT1B and 5‐HT1D receptors were analysed using real‐time RT–PCR.

Classical PKC's are critically involved in the appearance of the ETB receptor; co‐culture with RO 31‐7549 abolished the contractile response (6.9±1.8%) and reduced the ETB receptor mRNA by 44±4% as compared to the cultured control. Correlation between decreased ETB receptor mRNA and abolished contractile function indicates upstream involvement of PKC.

Inhibition of PKA generally had an enhancing effect on the induced changes giving rise to a 7–25% increase in Emax in response to ET‐1, S6c and 5‐CT as compared to the cultured control.

Staurosporine inhibited the culture induced upregulation of the response of both the ETA and the 5‐HT1B/1D receptors, but had no significant effect on the mRNA levels of these receptors. This lack of correlation indicates an additional downstream involvement of protein kinases.

British Journal of Pharmacology (2002) 137, 118–126. doi:10.1038/sj.bjp.0704838
A comparison of the actions of BIBN4096BS and CGRP8–37 on CGRP and adrenomedullin receptors expressed on SK‐N‐MC, L6, Col 29 and Rat 2 cells
Authors: Hay Debbie L , Howitt Stephen G , Conner Alex C , Doods Henri , Schindler Marcus , Poyner David R
Published: September 2002

The ability of the CGRP antagonist BIBN4096BS to antagonize CGRP and adrenomedullin has been investigated on cell lines endogenously expressing receptors of known composition.

On human SK‐N‐MC cells (expressing human calcitonin receptor‐like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1)), BIBN4096BS had a pA2 of 9.95 although the slope of the Schild plot (1.37±0.16) was significantly greater than 1.

On rat L6 cells (expressing rat CRLR and RAMP1), BIBN4096BS had a pA2 of 9.25 and a Schild slope of 0.89±0.05, significantly less than 1.

On human Colony (Col) 29 cells, CGRP8–37 had a significantly lower pA2 than on SK‐N‐MC cells (7.34±0.19 (n=7) compared to 8.35±0.18, (n=6)). BIBN4096BS had a pA2 of 9.98 and a Schild plot slope of 0.86±0.19 that was not significantly different from 1. At concentrations in excess of 3 nM, it was less potent on Col 29 cells than on SK‐N‐MC cells.

On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 μM. CGRP8–37 had a pA2 of 6.72 against adrenomedullin.

BIBN4096BS shows selectivity for the human CRLR/RAMP1 combination compared to the rat counterpart. It can discriminate between the CRLR/RAMP1 receptor expressed on SK‐N‐MC cells and the CGRP‐responsive receptor expressed by the Col 29 cells used in this study. Its slow kinetics may explain its apparent ‘non‐competive’ behaviour. At concentrations of up to 10 μM, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP8–37.

British Journal of Pharmacology (2002) 137, 80–86. doi:10.1038/sj.bjp.0704844
A Leu7Pro mutation in the signal peptide of platelet glycoprotein (GP)IX in a case of Bernard–Soulier syndrome abolishes surface expression of the GPIb–V–IX complex
Authors: Lanza François , De La Salle Corinne , Baas Marie‐Jeanne , Schwartz Agnès , Boval Bernadette , Cazenave Jean‐Pierre , Caen Jacques P.
Published: September 2002

Summary. This paper describes the molecular defect of the second case of Bernard–Soulier syndrome, initially reported in 1957. Analysis of the patient's platelets by flow cytometry and Western blotting failed to detect surface expression of any of the four subunits of the glycoprotein (GP)Ib–V–IX complex and revealed small amounts of intracellular GPIbα, GPIbβ and GPV but no GPIX. DNA sequencing revealed a novel missense mutation in the GPIX gene which replaced Leu (CTG) by Pro (CCG) at position 7 of the signal peptide. This mutation is, to date, the only known example of a leader sequence defect in Bernard–Soulier syndrome. The change occurred in a prototypic alpha‐helical hydrophobic core region, typically enriched in leucine and devoid of proline residues. Co‐transfection of GPIXPro7 with normal GPIbα and GPIbβ into Chinese hamster ovary cells reproduced the platelet phenotype, resulting in no detectable GPIX, low intracellular levels of GPIbα and GPIbβ, and an absence of surface expression. This mutation presumably leads to an abnormal conformation and, hence, incorrect insertion of GPIX into the endoplasmic reticulum and/or to defective signal peptide cleavage, both of which are required for correct transport to the cell membrane. This provides further evidence for a critical role of GPIX in controlling biosynthesis of the GPIb–IX complex.
Appearance of del(11q) in two patients with acute promyelocytic leukaemia treated with all‐trans retinoic acid and combination chemotherapy
Authors: Campbell Lynda J. , Rayeroux Kathleen C. , Arkell Kathryn , Catalano John V. , Cole‐Sinclair Merrole F.
Published: September 2002

Summary. Two acute promyelocytic leukaemia patients, treated with all‐trans retinoic acid and combination chemotherapy, acquired a deletion of 11q within 12 months of diagnosis. One patient died in relapse, with both t(15;17) and del(11q) cell lines co‐existing. Patient 2 remains in remission with del(11q) in 70% metaphases, despite normal marrow morphology. No deletion of the MLL gene was identified in the latter patient. The early appearance of a del(11q) is unusual, particularly without morphological evidence of myelodysplasia. We hypothesize that the del(11q) was therapy‐induced but the absence of other genetic lesions has resulted in no accompanying morphological changes.
The safety and effectiveness of deferiprone in a large‐scale, 3‐year study in Italian patients
Authors: Ceci Adriana , Baiardi Paola , Felisi Mariagrazia , Cappellini Maria Domenica , Carnelli Vittorio , De Sanctis Vincenzo , Galanello Renzo , Maggio Aurelio , Masera Giuseppe , Piga Antonio , Schettini Francesco , Stefàno Ippazio , Tricta Fernando
Published: September 2002

Summary. In 1997, the Italian Ministry of Health created a special programme for the controlled distribution of deferiprone to collect data and to evaluate its safety and effectiveness in long‐term use. Five hundred and thirty‐two thalassaemia patients from 86 treatment centres were enrolled in this programme. One hundred and eighty‐seven patients (32%) experienced a total of 269 events that led to a temporary interruption or, in some cases, to a discontinuation of treatment. The incidence of agranulocytosis and milder neutropenias were 0·4/100 and 2·1/100 patient‐years respectively. Neutropenia occurred predominantly in younger and non‐splenectomized patients. Transient alanine transaminase increase, gastrointestinal discomfort and arthralgia were the other most commonly reported events. Ferritin levels showed a significant decrease in time after 3 years of therapy. This is the largest number of deferiprone‐treated patients to have been reported to date. These data show that the drug was effective in reducing serum ferritin levels and the incidence of adverse events was not greater than the frequency reported in clinical trials.
Hyperlipidaemia and venous thromboembolism in patients lacking thrombophilic risk factors
Authors: Vayá Amparo , Mira Yolanda , Ferrando Fernando , Contreras MaTeresa , Estelles Amparo , España Francisco , Corella Dolores , Aznar Justo
Published: September 2002

Summary. To ascertain the potential contribution of serum lipids to the development of deep vein thrombosis (DVT), a case–control study was conducted in 143 DVT patients lacking thrombophilic risk factors and in 194 age‐ and sex‐matched controls. DVT patients showed significantly higher body mass indices (BMI), and triglyceride levels than did controls (P < 0·001 and P = 0·045 respectively). Using multivariate analysis, BMI was the only variable which remained statistically different, thus the risk of DVT was associated with obesity (odds ratio = 2·49). These results were confirmed when additional control for fibrinogen and plasminogen activator inhibitor type 1 (PAI‐1) was carried out in a subgroup of cases and controls. When idiopathic (n = 39) and secondary (n = 104) patients with DVT were compared, the former showed a higher mean age, a higher proportion of men, and higher cholesterol levels. Age, sex and total cholesterol were statistically different by multivariate analysis. After age was dichotomized as ≥ 50 years and cholesterol ≥ 5·69 mmol/l, all three variables constituted independent risk factors for idiopathic DVT, with odds ratios of 2·73 for ages ≥ 50 years; 3·72 for men and 2·67 for cholesterolaemia ≥ 5·69 mmol/l. Obesity thus constitutes an independent risk factor for DVT, possibly in part mediated through triglyceride, fibrinogen and PAI‐1 effects on haemostasis. In addition, cholesterolaemia levels of ≥ 5·69 mmol/l constitute an independent risk factor for idiopathic DVT.
Prolonged inhibition of von Willebrand factor‐cleaving protease after splenectomy in a 22‐year‐old patient with acute and plasma refractory thrombotic thrombocytopenic purpura
Authors: Langer Florian , Bergmann Frauke , Budde Ulrich , Hegewisch‐Becker Susanna , Hossfeld Dieter Kurt
Published: September 2002

Summary. We report a 22‐year‐old woman with acute, plasma refractory thrombotic thrombocytopenic purpura (TTP) in whom splenectomy led to consistent stabilization of platelet counts, but who showed complete inhibition of vonWillebrand factor‐cleaving protease (VWF‐cp) after 6 months of follow up. Persistent protease deficiency and resolved clinical and haematological TTP symptoms resulted in the transient appearance of unusually large VWF multimers in the patient plasma. As low but significant protease activity (10%) was first detectable as late as 9 months after splenectomy, we conclude tentatively that, at least in a subgroup of patients with acquired TTP, the beneficial effect of splenectomy is not exclusively due to the removal of splenic B lymphocytes producing an inhibitor of VWF‐cp.
Interleukin 6 expression by Hodgkin/Reed–Sternberg cells is associated with the presence of ‘B’ symptoms and failure to achieve complete remission in patients with advanced Hodgkin's disease
Authors: Reynolds Gary M. , Billingham Lucinda J. , Gray Laura J. , Flavell Joanne R. , Najafipour Sohrab , Crocker John , Nelson Paul , Young Lawrence S. , Murray Paul G.
Published: September 2002

Summary. Interleukin 6 (IL‐6) is a potent immunomodulatory cytokine that has pathogenic and prognostic significance in a number of disorders. Previous studies in Hodgkin's disease (HD) have demonstrated the association between elevated serum levels of IL‐6 and unfavourable prognosis, including advanced stage and the presence of ‘B’ symptoms and with reduced survival. Although IL‐6 expression has been demonstrated in both the malignant Hodgkin/Reed–Sternberg (HRS) cells and in the various non‐malignant cells present in HD biopsies, a relationship between expression of IL‐6 by the tumour and outcome measures has not been established. The study group comprised of 97 patients with advanced HD who were recruited to two related clinical trials. IL‐6 expression was determined on paraffin‐wax sections of biopsy material by means of an immunohistochemical assay. Of the 97 patients, 27 (28%) showed staining for IL‐6 in HRS cells. IL‐6 expression by HRS cells was significantly correlated with a decreased likelihood of achieving a complete response to chemotherapy (P = 0·02) and with an increased prevalence of ‘B’ symptoms (P = 0·04). IL‐6 expression by HRS cells was not associated with Epstein–Barr virus status (P = 0·57). In summary, the results suggest that IL‐6 expression by HRS cells may contribute to the presence of ‘B’ symptoms and to a decreased likelihood to achieve a complete remission in HD patients.
Low‐dose doxorubicin‐induced necrosis in Jurkat cells and its acceleration and conversion to apoptosis by antioxidants
Authors: Sugimoto Koichi , Tamayose Kenji , Sasaki Makoto , Hayashi Keiko , Oshimi Kazuo
Published: September 2002

Summary. We treated rapidly growing Jurkat cells with 40 nmol/l of doxorubicin for 72 h. After 36 h, the G2‐arrested cells became larger and some of them started endoreplication. Nuclear staining with Hoechst 33342 combined with propidium iodide (PI) exclusion revealed that about 90% of the cells were necrotic at 72 h, although apoptotic cells accounted for only 8%. Incubation with 40 nmol/l of aclarubicin or cytosine β‐d‐arabinofuranoside for 60 h induced necrosis both in Jurkat and ml‐1 cells. Pre‐necrotic Jurkat cells incubated with 40 nmol/l of doxorubicin had much higher intracellular reactive oxygen species (ROS) levels than pre‐apoptotic ones. Addition of Tempol or Desferal accelerated doxorubicin‐induced necrosis and partially converted it into apoptosis. Both antioxidants reduced surviving colony numbers of prenecrotic Jurkat cells. n‐acetyl‐l‐cysteine had little effect on the apoptotic conversion but profoundly accelerated necrosis. Because an apoptosis‐resistant Jurkat subclone was also refractory to doxorubicin‐induced necrosis, apoptosis and necrosis might share some common pathways. Low‐dose doxorubicin increased micronuclei‐positive cell percentages and also suppressed high‐dose doxorubicin‐induced apoptosis in Jurkat and ml‐1 cells. Some of the prenecrotic cells, therefore, might survive and obtain genomic instability. Antioxidants may be useful to suppress, at least to some extent, this vicious consequence.
Factor V Leiden in central venous catheter‐associated thrombosis
Authors: Fijnheer Rob , Birgit Paijmans  , Verdonck Leo F. , Nieuwenhuis H. Karel , Roest Mark , Dekker Adriaan W.
Published: September 2002

Summary. Subclavian vein thrombosis is a well‐recognized complication following central venous catheter insertion and is associated with significant morbidity. The factor V Leiden mutation is an important risk factor for deep venous thrombosis and pulmonary embolism. Whether this mutation also predisposes patients fitted with a central venous catheter to subclavian vein thrombosis is not known. The occurrence of central venous catheter‐associated thrombosis was investigated in 277 consecutive patients receiving an allogeneic bone marrow transplantation. All patients received a tunnelled double or triple catheter positioned in the subclavian vein. Catheter‐associated thrombosis was diagnosed on the basis of clinical signs of thrombosis, i.e. swelling and/or redness of the limb or venous engorgement and was confirmed with a colour‐flow Doppler ultrasound. Thirteen patients were heterozygous for the factor V Leiden mutation. Seven of these patients had a subclavian vein thrombosis (54%), while this occurred in only 9% of the factor V Leiden‐negative patients, corresponding with a relative risk of 7·7 (95% CI 3·3–17·9). Factor V Leiden is attributable for 17·3% of all thrombosis in patients with central venous catheters. The majority of patients with the factor V Leiden mutation with a central venous catheter will develop thrombosis. Patients with a factor V Leiden mutation should receive adequate thrombosis prophylaxis upon catheter introduction and the catheter should be removed immediately after the treatment. Based on this very high risk, we advise testing for factor V Leiden in all bone marrow transplantation patients receiving a central venous catheter.
The modified International Prognostic Index can predict the outcome of localized primary intestinal lymphoma of both extranodal marginal zone B‐cell and diffuse large B‐cell histologies
Authors: Cortelazzo Sergio , Rossi Andrea , Oldani Elena , Motta Teresio , Giardini Roberto , Zinzani Pier Luigi , Zucca Emanuele , Gomez Henry , Ferreri Andrés J.M. , Pinotti Graziella , Chini Claudio , Devizzi Liliana , Gianni Alessandro M. , Cavalli Franco , Barbui Tiziano ,   the International Extranodal Lymphoma Study Group (IELSG)
Published: September 2002

Summary. We have previously reported on the efficacy of a modified International Prognostic Index (MIPI) in predicting the outcome of patients with primary gastric lymphoma. This prompted the retrospective analysis of a large series of patients with primary intestinal lymphoma (PIL) of both diffuse large B‐cell (DLCL) and low‐grade (extranodal marginal zone B‐cell lymphoma, MZL) histology. Clinical records of 122 patients with localized primary intestinal lymphoma of MZL (n=35) and DLCL (n=87) histology, confirmed by an ad hoc expert panel of pathologists, were reviewed. Forty‐nine patients were treated with single therapy, while 72 received combined‐modality treatment, which included surgery followed by a short‐term chemotherapy. MIPI was included in a multivariate prognostic analysis for overall survival (OS) and event‐free survival (EFS). Sixty‐five patients (75%) with DLCL and 22 with MZL(65%) achieved complete remission. After a median follow‐up of 42 months (range 6–163 months), 5‐year estimates of OS and EFS were 68% and 50% for DLCL and 65% and 26% for MZL. OS varied according to MIPI, from, respectively, 86% and 87% for DLCL and MZL patients with 0–1 risk factor to 50% and 32% for patients with > 1 risk factor (P=0·01 and P=0·02). Similar results were obtained for EFS. Cox regression analysis showed an unfavourable MIPI to be the only independent predictor of shorter EFS. This retrospective study shows that stage‐MIPI can be a reliable prognostic indicator for PIL of both low‐grade MZL and diffuse large B‐cell histology, enabling the early identification of patients at higher risk of failure.
Analysis of T‐cell repertoire and mixed chimaerism in a patient with aplastic anaemia after allogeneic bone marrow transplantation
Authors: Tsutsumi Yutaka , Tanaka Junji , Sugita Junichi , Kato Naoko , Zhang Li , Yonezumi Masakatsu , Chiba Kouji , Toyosima Nobuyasu , Kondo Takeshi , Ohta Shuichi , Mori Akio , Hasino Satoshi , Asaka Masahiro , Imamura Masahiro
Published: September 2002

Summary. We analysed 26 T‐cell receptor (TCR) β chain subfamilies (VB) of a patient with aplastic anaemia (AA) who underwent allogeneic bone marrow transplantation (allo‐BMT). The patient developed pancytopenia at d 80. The patient's T cells were skewed in 10 of 26 TCR‐VB on d 83. These TCR‐VB, especially VB15, which were almost entirely CD8‐positive cells, were skewed throughout her clinical course. Chimaerism analysis of the CD8‐positive cells indicated that they were of recipient origin. Therefore, some immune responses induced by the recipient CD8‐positive T cells had an important role in pancytopenia in AA patients after allo‐BMT.
Granulocyte colony‐stimulating factor mobilized whole blood containing over 0·3 × 106/kg CD34+ cells is a sufficient graft in autologous transplantation for relapsed non‐Hodgkin's lymphoma
Authors: Jonkhoff Andries R. , De Kreuk Arne M. , Franschman Gaby , Van Der Lelie Johannes , Schuurhuis Gerrit J. , Dräger Angelika M. , Zweegman Sonja , Huijgens Peter C. , Ossenkoppele Gert J.
Published: September 2002

Summary. The feasibility of unprocessed, granulocyte colony‐stimulating factor (G‐CSF)‐mobilized whole blood (WB) as an alternative stem cell source for autologous stem cell transplantation was studied. Forty‐seven relapsed non‐Hodgkin's lymphoma (NHL) patients entered the study. After two or three ifosfamide, methotrexate and etoposide (IMVP) courses, 1 l of G‐CSF‐mobilized WB was collected and stored refrigerated for 72 h. Meanwhile, BAM conditioning was given: BCNU (carmustine) 300 mg/m2, high‐dose cytarabine 6000 mg/m2 and melphalan 140 mg/m2. Toxicity, haematological recovery and survival were assessed and compared with peripheral blood stem cell transplantation (PBSCT) and bone marrow transplantation (BMT) reference groups. High‐dose G‐CSF (2 × 12 µg/kg/d) gave the best mobilization results. Haematological recovery was related to the WB CD34+ content. A CD34+ threshold of ≥ 0·3 106/kg, obtained in 90% of patients using high‐dose G‐CSF, correlated with adequate recovery: absolute neutrophil count (ANC) > 0·5 × 109/l: median 12 d (range 9–19). Platelet recovery > 20 and > 50 × 109/l was 19 (11–59) and 30 d (14 not reached) respectively. Overall survival of patients < 60 years was 57% at 4 years and event‐free survival was 32%. Survival was comparable with PBSCT and BMT after BEAM (BCNU, etoposide, cytarabine, melphalan). Remarkably, haematological recovery after BAM + WB was rapid and comparable (ANC) or slightly prolonged (platelets) in comparison with BEAM + PBSCT, despite a 10–20 times lower CD34+ cell dose in the WB graft. In conclusion, transplantation of WB containing ≥ 0·3 × 106/kg CD34+ cells after BAM conditioning is a safe procedure, and offers a fully equivalent and less costly alternative for PBSC.
A method to fix lipids for staining fat embolism in paraffin sections
Authors: Tracy R E , Walia P
Published: September 2002

A method to fix lipids for staining fat embolism in paraffin sections

Aims: To develop a method to preserve lipids in formalin‐fixed tissues for staining in paraffin sections, and to illustrate its use in lung and brain of a fat embolism case, and in examples of fatty liver and atheroma.

Methods and results: A saturated solution of linoleic acid in 70% ethylene glycol was prepared and tissues were exposed to this for 3 days at 56°C. These tissues were treated with 2% chromic acid at 4°C for 24 h followed by 24 h in 5% sodium bicarbonate, with appropriate rinsing between solutions. Paraffin sections of these tissues were stained with a lipid‐soluble dye such as Oil Red O. Examples of fat embolism, fatty liver, and atheroma were shown photographically as illustrations of expected results.

Conclusions: The demonstration of fat embolism with good quality tissue detail is made practical by the method, which is convenient and inexpensive. The method appears to be generally applicable to tissue lipids of various sorts, as exemplified by adipose tissue, fatty liver, and atheroma.
Granulomatous ulcerative colitis: a re‐appraisal of the mucosal granuloma in the distinction of Crohn's disease from ulcerative colitis
Authors: Mahadeva U , Martin J P , Patel N K , Price A B
Published: September 2002

Granulomatous ulcerative colitis: a re‐appraisal of the mucosal granuloma in the distinction of Crohn's disease from ulcerative colitis

Aims: To determine whether the presence and location of giant cells or granulomas in relation to crypts distinguishes between ulcerative colitis and Crohn's disease.

Methods and results: Twenty‐nine large bowel mucosal biopsy specimens showing giant cells and/or granulomas in a background more typical of ulcerative colitis than Crohn's disease were collected between 1986 and 1996. Each was subject to detailed independent analysis by three histopathologists. Follow‐up of the cases was by examination of all previous and subsequent gastrointestinal surgical or biopsy material and by scrutiny of the clinical notes by a gastroenterologist. On the basis of the accumulated histological data 10 of these 29 cases were accorded the diagnosis of ulcerative colitis. In nine of these 10 cases the clinical diagnosis, where known, was in keeping with this and all nine contained only crypt‐associated giant cells and/or granulomas. The tenth case contained a solitary free‐standing granuloma and clinically the patient had perianal disease, suggesting that the true diagnosis was Crohn's disease.

Conclusions: Isolated giant cells and well‐defined epithelioid granulomas distant from crypts do not, as a rule, occur in ulcerative colitis, and hence their presence in a colonoscopic biopsy showing features of chronic inflammatory bowel disease is a strong pointer towards the diagnosis of Crohn's disease. Crypt‐associated giant cells and granulomas can occur in ulcerative colitis and in themselves are unreliable features for the discrimination between Crohn's disease and ulcerative colitis.
Inhibition of cell proliferation by chronic wound fluid
Authors: Bucalo Brian , Eaglstein William H. , Falanga Vincent
Published: September 2002

It has been proposed that occlusive wound dressings may enhance chronic wound repair by the stimulatory action of the fluid accumulating beneath the dressings. In this report, we investigated the in vitro proliferative effects of chronic wound fluid obtained from under a polyurethane membrane applied for 24 hours to venous ulcers in the ambulatory setting. By measuring cell counts and DNA synthesis, we found that chronic wound fluid inhibited the proliferation of human dermal fibroblasts (p = 0.008) and failed to stimulate the proliferation of microvascular endothelial cells (p = 0.03) and keratinocytes (p = 0.03). The inhibitory activity of chronic wound fluid on fibroblast proliferation was blocked after the fluid was heated to 100° C, but not 56° C, and was mainly restricted to a fraction of chronic wound fluid enriched in components less than 30 kd in molecular weight (p = 0.028). At concentrations ranging from 1% to 4% and in the presence of serum, chronic wound fluid decreased the viability of fibroblasts, as shown by a decreased ability of the cells to exclude trypan blue (p = 0.02), and the viability of endothelial cells, as shown by an increased release of tritiated adenine (p = 0.03). We conclude that the wound fluid obtained from beneath occlusive dressings applied to chronic wounds inhibits cell proliferation.
Influence of human growth hormone on granulation tissue formation, collagen deposition, and the aminoterminal propeptide of collagen type III in wound chambers in rats
Authors: Rasmussen Lars H. , Garbarsch Charly , Schuppan Detlef , Moe Dennis , Hørslev‐Pedersen Kim , Gottrup Finn , Steenfos Henrik
Published: September 2002

The influence of growth hormone on granulation tissue formation was investigated in wire mesh cylinders implanted subcutaneously in rats. Two groups of 10 rats (study 1) and 1 group of 12 rats (study 2) were used for the investigation. Growth hormone, 0.02 and 0.2 IU (study 1), 0.05 and 0.2 IU (study 2), or vehicle only, was injected into the cylinders every third day for 16 days. In study 2, wound fluid was aspirated before injection of growth hormone and saved for later analysis of the aminoterminal propeptide of collagen type III. In both studies, growth hormone significantly increased the formation of granulation tissue and of total collagen content dose‐dependently, whereas the relative amount of collagen was unaffected by growth hormone treatment. Wound fluid aminopropeptide increased significantly after implantation of the cylinders until day 7, before declining slightly, with no difference between the groups. We conclude that growth hormone stimulated granulation tissue formation and collagen deposition dose‐dependently in the wound cylinders when injected every third day. The results suggest that growth hormone treatment does not cause excessive collagen deposition in newly formed granulation tissue.
Role of hypoxia in growth factor responses: differential effects of basic fibroblast growth factor and platelet‐derived growth factor in an ischemic wound model
Authors: Mustoe Thomas A. , Tae Ahn Sang , Tarpley John E. , Pierce Glenn F.
Published: September 2002

Both recombinant basic fibroblast growth factor and platelet‐derived growth factor‐BB homodimer are potent inducers of new tissue generation in models of normal dermal repair. However, their therapeutic targets include chronic wounds, which are frequently characterized by local tissue hypoxia. To explore the potential of recombinant basic fibroblast growth factor and platelet‐derived growth factor‐BB homodimer to stimulate more clinically relevant repair, we created an ischemic dermal wound on the rabbit ear by ligating two of the arteries which feed the ear. Both recombinant basic fibroblast growth factor and platelet‐derived growth factor‐BB homodimer stimulated marked neovascularization of the wound (p < 0.0001), but only recombinant platelet‐derived growth factor‐BB homodimer accelerated and augmented granulation tissue formation (p = 0.01) and reepithelialization. This study is the first demonstration of a direct angiogenic effect of recombinant platelet‐derived growth factor‐BB homodimer in vivo. India ink perfusion coupled with endothelial cell‐specific histochemistry showed that nearly all the neovessels in all wounds were functional, indicating rapid capillary morphogenesis. In the nonischemic (normal) rabbit ear, recombinant basic fibroblast growth factor and platelet‐derived growth factor‐BB homodimer accelerated healing comparably, as expected. Higher doses of recombinant basic fibroblast growth factor also failed to elicit stimulatory effects in ischemic wounds. These results indicate that differential responsiveness to growth factors is related to local tissue hypoxia, angiogenesis alone is an insufficient stimulus for repair. These data also suggest new therapeutic approaches for the treatment of chronic wounds.
Exchange and the mechanical properties of the skin: Oncotic and hydrostatic forces control by blood supply and lymphatic drainage
Authors: Ryan Terence J.
Published: September 2002

Unwounded healthy skin can survive several hours without oxygen. The rich blood supply of the skin is important for thermoregulation, mostly only above the waist. Why then, does the skin of even the big toe have a rich blood supply? That exchange requirements are more for fluid in support of turgor and that this process is essential for the mechanical properties of the skin is suggested. It is argued that the supposed richness of blood supply is not sufficient for wound healing, and thus a new organ, granulation tissue, has to be grown. However, granulation tissue is mechanically inappropriate for the protective mechanical function of the skin and must be completely removed before healing is complete.
Quantitative analysis of wound healing
Authors: Bardsley William G. , Sattar Abdul , Armstrong James R. , Shah Mamta , Brosnan Patti , Ferguson Mark W. J.
Published: September 2002

In this article, we initially review several problems associated with the design and interpretation of certain types of experiments currently used to study wound healing, drawing attention to the fact that applications for standard statistical techniques in the analysis of the experimental results are often of limited value. We then argue that, because of the special nature of wound healing data, curve fitting of empirical model equations can often provide a convenient way to summarize treatment effects with large data sets. The various ways in which this technique could be used to facilitate the interpretation of experimental wound healing results are then explored. To illustrate this approach, we then took several wound healing experiments and introduced possible models that could be used, paying particular attention to simple equations with the smallest possible number of parameters. For each equation, the way that the parameters of the model could be interpreted with regard to the biologic effects represented is given. Examples are given to show the application of each model discussed theoretically in the interpretation of some typical experimental data sets.
A population of cells resident within embryonic and newborn rat skeletal muscle is capable of differentiating into multiple mesodermal phenotypes
Authors: Lucas Paul A. , Calcutt Andrew F. , Southerland Sheila S. , Alan Wilson J. , Harvey Richard L. , Warejcka Debra , Young Henry E.
Published: September 2002

We have previously shown a population of putative mesenchymal stem cells in the connective tissue surrounding embryonic avian skeletal muscle. These cells differentiate into at least five recognizable phenotypes in culture: fibroblasts, chondrocytes, myotubes, osteoblasts, and adipocytes. We have now isolated a similar population of cells from fetal and newborn rat skeletal muscle. Cells from rat leg muscle were dissected, minced, and then enzymatically digested with a collagenase‐dispase solution. The dissociated cells were plated and allowed to differentiate into two recognizable populations: myotubes and stellate mononucleated cells. The cells were then trypsinized, filtered through a 20 µm filter to remove the myotubes, frozen at −80° C, then thawed and replated. In culture the cells maintained their stellate structure. However, under treatment with dexamethasone, a nonspecific differentiating agent, seven morphologic conditions emerged: cells with refractile vesicles that stained with Sudan black B (adipocytes), multinucleated cells that spontaneously contracted in culture and stained with an antibody to myosin (myotubes), round cells whose extracellular matrix stained with Alcian blue, pH 1.0 (chondrocytes), polygonal cells whose extracellular matrix stained with Von Kossa's stain (osteoblasts), cells with filaments that stained with an antibody to smooth muscle a‐actin (smooth muscle cells), cells that incorporated acetylated low density lipoprotein (endothelial cells), and spindle‐shaped cells that grew in a swirl pattern (fibroblasts). The initial population is tentatively classified as putative mesenchymal stem cells. The presence of these cells point to the existence of stem cells in the postembryonic mammal that could provide a basis for tissue regeneration as opposed to scar tissue formation during wound healing.
Covering by a flap induces apoptosis of granulation tissue myofibroblasts and vascular cells
Authors: Garbin Stefania , Pittet Brigitte , Montandon Denys , Gabbiani Giulio , Desmoulière Alexis
Published: September 2002

It has recently been shown that during the healing of an open wound, apoptosis mediates the decrease in cellularity during the transition between granulation tissue and scar. Because reduced contraction and a decrease in the number of fibroblastic cells have been described in wounds covered with a successful skin graft, we hypothesized that apoptosis could be responsible for these phenomena. Using in situ labeling of fragmented DNA, immunohistochemistry for α‐smooth muscle actin, and electron microscopy, we have studied in rats the evolution of 10‐day‐old wound tissue covered with a total skin flap (containing epidermis, dermis, and the cutaneous muscle). In 10‐day‐old wound tissue, few apoptotic vascular cells and rare apoptotic myofibroblasts were present; the number of apoptotic cells increased slightly 72 hours later. In wounds covered with total skin flaps, the number of apoptotic vascular and myofibroblastic cells increased drastically 6 hours after flap application with a maximum at 24 and 48 hours, respectively. A decrease of apoptotic cell number was noted at 72 hours; at this time, the size of the granulation tissue was greatly reduced and showed extracellular matrix remodeling. Total flaps were more efficient in the induction of granulation tissue cell apoptosis compared with dermo‐epidermal flaps. Moreover, the control application of full‐thickness skin autografts, which were not viable 7 days later, did not induce apoptosis 24 hours after implantation. Our results indicate that covering granulation tissue with a skin flap results in a massive apoptotic process, possibly by means of a (some) locally released substance(s).
Modulation of α‐smooth muscle actin expression in fibroblasts by transforming growth factor‐β isoforms: an in vivo and in vitro study
Authors: Serini Guido , Gabbiani Giulio
Published: September 2002

Myofibroblasts are granulation tissue fibroblasts bearing ultrastructural and biochemical features of smooth muscle cells, such as cytoplasmic microfilaments and α‐smooth muscle actin expression. They appear transiently during wound healing and more permanently during several pathologic situations such as fibrotic diseases. Transforming growth factor‐β1 has been suggested to be an important promoter of the myofibroblastic phenotype. Here we show that (1) transforming growth factor‐β2, like transforming growth factor‐β1, induces myofibroblast formation in vivo and in vitro; (2) transforming growth factor‐β3 acts as a negative regulator of the myofibroblastic phenotype in vivo but not in vitro; and (3) in vitro, the three different transforming growth factor‐β isoforms are equally able to induce α‐smooth muscle actin messenger RNA and protein expression in growing and quiescent cultured human and rat subcutaneous tissue fibroblasts. These data confirm that in vitro the behavior of the three different transforming growth factor‐β isoforms is similar, whereas in vivo transforming growth factor‐β isoforms possibly play different but complementary roles in myofibroblast modulation during wound repair.
Centrally and locally mediated thermoregulatory responses alter subcutaneous oxygen tension
Authors: Sheffield Clark W. , Sessler Daniel I. , Hopf Harriet W. , Schroeder Marc , Moayeri Azita , Hunt Thomas K. , West Judith M.
Published: September 2002

Mild perianesthetic hypothermia decreases resistance to infections. Decreased resistance likely results in part from direct immune inhibition. However, decreased tissue oxygen partial pressure also decreases resistance to infection by impairing oxidative killing by neutrophils and collagen deposition. Thermoregulatory vasoconstriction decreases skin blood flow and may also decrease subcutaneous tissue oxygen tension. Accordingly, we determined the influence of centrally and locally mediated thermoregulatory vasomotion on subcutaneous oxygen tension. We also compared subcutaneous oxygen tension to other potential markers of tissue perfusion: laser Doppler flowmetry and transcutaneous oxygen tension. Arterial oxygen tension was maintained near 325 mm Hg in five volunteers. Control subcutaneous oxygen tension values were recorded after 1 hour of euthermia (no sweating or vasoconstriction). Volunteers were then cooled with a circulating‐water mattress positioned under the trunk and legs. After 1.5 hours of cooling sufficient to produce shivering, the right upper arm was covered for 1 hour with a small circulating water blanket set to 40° C while systemic cooling continued. The volunteers were then systematically warmed to produce sweating, and the right arm was locally cooled. There was no correlation among laser Doppler flowmetry, transcutaneous oxygen tension, and subcutaneous oxygen tension. Systemic cooling significantly decreased subcutaneous oxygen tension, but subcutaneous oxygen tension in the right arm returned to control values during local heating. Systemic warming significantly increased subcutaneous oxygen tension, and 1 hour of local cooling failed to fully reverse the increase. These data indicate that thermoregulatory vasoconstriction significantly decreases tissue oxygen availability. Decreased subcutaneous oxygen tension may be one mechanism by which mild perianesthetic hypothermia facilitates development of surgical wound infections.
Interactions of cytokines, growth factors, and proteases in acute and chronic wounds
Authors: Mast Bruce A. , Schultz Gregory S.
Published: September 2002

A healing wound represents a complex series of interactions between cells, soluble mediators, and extracellular matrix. Within this multifaceted environment, there are multiple regulatory points which control the ordered series of events that lead to normal tissue repair. An alteration in this physiologic network can lead to the development of a chronic wound. This article presents an update on the numerous mediators that exist within the wound environment in both acute normal healing and chronic nonhealing wounds. We also present a hypothesis which may provide a conceptual pathophysiologic mechanism with which to understand all chronic wounds.