bluuebunny/crossref_metadata_2025 huggingface dataset:
Constructing Seasonal Climograph Overlap Envelopes from Holocene Packrat Midden Contents, Dinosaur National Monument, Colorado
Authors: Sharpe Saxon E.
Published: September 2002

Five Neotoma spp. (packrat) middens are analyzed from Sand Canyon Alcove, Dinosaur National Monument, Colorado. Plant remains in middens dated at approximately 9870, 9050, 8460, 3000, and 0 14C yr B.P. are used to estimate Holocene seasonal temperature and precipitation values based on modern plant tolerances published by Thompson et al. (1999a, 1999b). Early Holocene vegetation at the alcove shows a transition from a cool/mesic to a warmer, more xeric community between 9050 and 8460 14C yr B.P. Picea pungens, Pinus flexilis, and Juniperus communis exhibit an average minimum elevational displacement of 215 m. Picea pungens and Pinus flexilis are no longer found in the monument.

Estimates based on modern plant parameters (Thompson et al., 1999a) suggest that average temperatures at 9870 14C yr B.P. may have been at least 1° to 3°C colder in January and no greater than 3° to 10°C colder in July than modern at this site. Precipitation during this time may have been at least 2 times modern in January and 2 to 3 times modern in July. Discrepancies in estimated temperature and precipitation tolerances between last occurrence and first occurrence taxa in the midden record suggest that midden assemblages may include persisting relict vegetation.
The pure anti‐oestrogen ICI 182,780 (Faslodex™) activates large conductance Ca2+‐activated K+ channels in smooth muscle
Authors: Dick Gregory M
Published: September 2002

Oestrogen and tamoxifen activate large conductance Ca2+‐activated K+ (BKCa) channels in smooth muscle through a non‐genomic mechanism that depends on the regulatory β1 subunit and an extracellular binding site. It is unknown whether a ‘pure’ anti‐oestrogen such as ICI 182,780 (Faslodex™), that has no known oestrogenic properties, would have any effect on BKCa channels. Using single channel patch clamp techniques on canine colonic myocytes, the hypothesis that ICI 182,780 would activate BKCa channels was tested. ICI 182,780 increased the open probability of BKCa channels in inside‐out patches with an EC50 of 1 μM. These data suggest that molecules with the ability to bind nuclear oestrogen receptors, regardless of oestrogenic or anti‐oestrogenic nature, activate BKCa channels through this nongenomic, membrane‐delimited mechanism. The identity and characteristics of this putative binding site remain unclear; however, it has pharmacological similarity to oestrogen receptors α and β, as ICI 182,780 interacts with it.

British Journal of Pharmacology (2002) 136, 961–964. doi:10.1038/sj.bjp.0704807
TNF‐α, inefficient by itself, potentiates IL‐1β‐induced PGHS‐2 expression in human pulmonary microvascular endothelial cells: requirement of NF‐κB and p38 MAPK pathways
Authors: Said Fatima Ait , Werts Catherine , Elalamy Ismaïl , Couetil Jean‐Paul , Jacquemin Claude , Hatmi Mohamed
Published: September 2002

Prostaglandin H synthase‐2 (PGHS‐2), is an inducible enzyme involved in various inflammatory responses. We established here that interleukin‐1β (IL‐1β) but not tumour necrosis factor‐α (TNF‐α) increased its expression in human pulmonary microvascular endothelial cells (HPMEC). However, associated with IL‐1β, TNF‐α greatly potentiated this enzyme induction.

Although unable to induce PGHS‐2 expression by itself, TNF‐α promoted a similar transcription nuclear factor‐κB (NF‐κB) activation to IL‐1β. This effect was more pronounced when cells were co‐exposed to both cytokines. HPMEC pre‐treatment with MG‐132, a proteasome inhibitor, prevented NF‐κB activation as well as more distal signalling response, indicating that NF‐κB activation is required but not sufficient for PGHS‐2 expression.

Both IL‐1β and TNF‐α failed to activate c‐Jun NH2‐terminal kinase (JNK). In addition, PD98059, a p42/44 mitogen‐activated protein kinase (MAPK) phosphorylation inhibitor, did not decrease PGHS‐2 expression. However, SB 203580, a p38 MAPK inhibitor, suppressed PGHS‐2 induction by IL‐1β alone or combined with TNF‐α, demonstrating that p38 MAPK but not p42/44 MAPK or JNK cascades are required for PGHS‐2 up‐regulation.

Finally, TNF‐α, unlike IL‐1β, was unable to promote p38 MAPK phosphorylation, indicating that the failure of TNF‐α to induce PGHS‐2 expression is linked, at least in part, to its inability to activate p38 MAPK signalling pathway. Altogether, these data enhanced our understanding of PGHS‐2 regulation in HPMEC and emphasize the heterogeneity of cellular responses to proinflammatory cytokines.

British Journal of Pharmacology (2002) 136, 1005–1014. doi:10.1038/sj.bjp.0704811
Evidence for a GABAB receptor component in the spinal action of Substance P (SP) on arterial blood pressure in the awake rat
Authors: Brouillette Jonathan , Couture Réjean
Published: September 2002

The activation of tachykinin NK1 receptors in the rat spinal cord produced a transient drop in arterial blood pressure followed by a more prolonged pressor effect which is mediated by the stimulation of the sympatho‐adrenal system. This study aims at characterizing the spinal mechanism of that initial hypotension occurring in awake unrestrained rats.

The initial hypotension (−18±2.0 mmHg at 1 min) and the tachycardia (110±10 b.p.m.) produced by the intrathecal (i.t.) injection of the stable NK1 receptor agonist [Sar9, Met(O2)11]‐SP (Sar9, 0.65 nmol) at T‐9 spinal cord level was inhibited by the prior injection of 65 nmol LY306740 or LY303870 (NK1 receptor antagonists). No inhibition was seen when a similar dose of antagonists was given intravenously.

The prior i.t. injection of the GABAB receptor antagonist CGP52432 (100 nmol) reduced the hypotension evoked by Sar9 (0.65 nmol) and by the GABAB receptor agonist baclofen (100 nmol). The GABAA receptor antagonist bicuculline (25 nmol, i.t.) was without effect against Sar9, and the GABAA agonist muscimol (100 nmol, i.t.) had no cardiovascular effect.

The putative involvement of other mediators (dopamine, serotonine, glycine and glutamate) in Sar9‐induced hypotension was made unlikely on the basis of various pharmacological treatments. Thus data, suggest that the transient hypotension which occurs upon the activation of NK1 receptors in the spinal cord is due to the release of GABA which in turn activates GABAB receptors to inhibit sympathetic pre‐ganglionic fibres. This mechanism may have a physiological significance in the spinal reflex autonomic control of arterial blood pressure.

British Journal of Pharmacology (2002) 136, 1169–1177. doi:10.1038/sj.bjp.0704813
Comparison of vasodilators in human internal mammary artery: ghrelin is a potent physiological antagonist of endothelin‐1
Authors: Wiley Katherine E , Davenport Anthony P
Published: September 2002

The potential vasodilator function of the peptide ghrelin, recently identified as the endogenous ligand of the growth hormone secretagogue orphan receptor (GHS‐R), was investigated in human endothelium‐denuded internal mammary artery. The peptide endothelin‐1 (ET‐1) is a potent and long‐lasting vasoconstrictor. Comparisons were made with established and putative endogenous vasodilators to determine if any could reverse ET‐1‐induced vasoconstriction in this vessel.

Ghrelin (0.1–300 nM) potently dilated 10 nM ET‐1‐induced constrictions (pD2 8.39±0.29; EMAX 63±5.6%; n=9/14, responders/total).

ANP (pD2 7.75±0.14; EMAX 106±2.0; n=5/5) and CGRP (pD2 8.08±0.17; EMAX 76±15% n=5/6) both produced complete reversal of the constrictor response to ET‐1 (EMAX not significantly different from 100%, P>0.05 one‐sample t‐test).

The following caused partial reversal of the ET‐1 response: Adrenomedullin (n=9/9) and two peptides derived from proadrenomedullin, PAMP‐12 (n=6/7) and PAMP‐20 (n=9/9) (pD2 values 7.63±0.28, 7.97±0.23 and 8.51±0.29; EMAX 58±7.3, 54±10 and 51±7.8% respectively). Unexpectedly, amylin was only 2 fold less potent than CGRP, although there was less than 50% reversal of the ET‐1 constriction (pD2 7.86±0.30; EMAX 41±5.4%; n=7/9). CNP (n=6/6) also partially reversed constrictions to ET‐1 (EMAX 53±6.3; pD2 8.07±0.38).

BNP (n=4/5) and PGI2 (n=6/8) were weak vasodilators, since concentration‐response curves failed to reach a maximum within the range tested. PGE2 caused a small dilatation in some vessels (EMAX 17±2.1%; pD2 8.63±0.36; n=4/8).

We have demonstrated ghrelin to be an effective, endothelium‐independent vasodilator of the long‐lasting constrictor ET‐1 in human arteries producing responses similar to those of adrenomedullin (P>0.05, ANOVA).

British Journal of Pharmacology (2002) 136, 1146–1152. doi:10.1038/sj.bjp.0704815
The role of gap junctions in mediating endothelium‐dependent responses to bradykinin in myometrial small arteries isolated from pregnant women
Authors: Kenny Louise C , Baker Philip N , Kendall David A , Randall Michael D , Dunn William R
Published: September 2002

Endothelium‐dependent responses were assessed in myometrial small arteries isolated from pregnant women, using pressure myography. Responses to bradykinin were unaffected by the combined presence of the nitric oxide synthase (NOS) inhibitor, N‐nitro‐L‐arginine methyl ester (L‐NAME, 100 μM) and the cyclo‐oxygenase inhibitor, indomethacin (10 μM). The additional presence of clotrimazole (50 μM) attenuated, but did not abolish, vasodilator responses to bradykinin. Raising extracellular [K+] (by between 1 and 15 mM) did not evoke a vasodilator response, nor did the cannabinoids, anandamide and methanandamide. Responses to bradykinin were attenuated in the presence of the gap junction inhibitors 18‐α‐glycyrrhetinic acid (18‐α GA, 100 μM), carbenoxolone (100 μM) and palmitoleic acid (50 μM). SR141716A, the CB1 receptor antagonist attenuated responses to bradykinin, but only at high concentrations (10 μM). These results suggest that gap junctional communication is involved in the nitric oxide (NO)‐ and prostanoid‐independent vasodilator responses to bradykinin in myometrial small arteries in normal pregnancy.

British Journal of Pharmacology (2002) 136, 1085–1088. doi:10.1038/sj.bjp.0704817
Effects of histamine on the contractile and electrical activity in isolated lymphatic vessels of the guinea‐pig mesentery
Authors: Fox James L R , von Der Weid Pierre‐Yves
Published: September 2002

The effect of histamine on the rate of lymphatic vessel constrictions and lymphatic smooth muscle membrane potential was examined in the guinea‐pig mesentery.

Histamine (0.01–5 μM) increased the frequency and decreased the amplitude of constrictions in lymphatic vessels under intraluminal perfusion. This response was accompanied by a depolarization of the smooth muscle membrane potential, an increase in the activity of spontaneous transient depolarizations (STDs), the proposed pacemaker for constrictions in these vessels, and an increase in the occurrence of action potentials.

Responses to histamine were inhibited by the H1 receptor antagonist pyrilamine (0.2 μM), but unaffected by NO synthase inhibition with NG‐nitro L‐arginine (L‐NOARG, 100 μM) and lysis of the endothelium.

In about 50% of the vessels, a decrease in constriction frequency, STD activity and a smooth muscle hyperpolarization were observed in response to dimaprit (10 μM), suggesting the presence of H2 receptors. These vessels had also a significantly lower basal contractile rate. Lymphatic vessel pumping was not affected by R‐α‐methylhistamine (10–50 μM), ruling out a role for H3 receptor stimulation in the histamine response.

The present results suggest a direct action of histamine on the lymphatic smooth muscle via stimulation of H1 (and in some vessels H2) receptors. H1 receptors enhance and H2 receptors slow down lymphatic pumping, the dominant effect being an increased contractile activity. Correlation of these effects with histamine‐induced changes in membrane potential and STD activity suggests the involvement of these electrical changes in the initiation of the contractile response.

British Journal of Pharmacology (2002) 136, 1210–1218. doi:10.1038/sj.bjp.0704820
Participation of 5‐HT1B receptors in the inhibitory actions of serotonin on masculine sexual behaviour of mice: pharmacological analysis in 5‐HT1B receptor knockout mice
Authors: Rodríguez‐Manzo Gabriela , López‐Rubalcava Carolina , Hen Rene , Fernández‐Guasti Alonso
Published: September 2002

The role of the 5‐Hydroxytryptamine1B (5‐HT1B) receptor subtype in masculine sexual behaviour in mice was analysed in both 5‐HT1B receptor knockout (KO1B) and wild‐type (WT) animals.

Comparison of male copulatory behaviour of WT and KO1B strains revealed that KO1B mice become interested earlier in sexual behaviour, but require more stimulation to achieve ejaculation than its corresponding WT strain.

The pharmacological manipulation of male sexual activity in the WT strain showed that the serotonin precursor 5‐Hydroxytryptophan (5‐HTP), the 5‐HT1B agonist (1‐(m‐trifluoromethylphenyl) piperazine (TFMPP) and the 5‐Hydroxytryptamine1A (5‐HT1A) receptor agonist 8‐hydroxy‐2‐di‐n‐propylamino‐tetralin (8‐OH‐DPAT) all inhibited male copulatory behaviour in mice.

In KO1B mice, TFMPP lacked an effect, 5‐HTP exerted a mild inhibitory effect while 8‐OH‐DPAT provoked only a tendency towards a reduction in the percentage of animals that achieved ejaculation. In general, KO1B mice were less sensitive to the inhibitory actions of 5‐HTP and 8‐OH‐DPAT than the WT strain.

Based on these results, we can suggest that serotonin plays a general inhibitory role in the sexual behaviour of male mice and that both 5‐HT1B and 5‐HT1A receptor subtypes participate in the inhibitory actions of this neurotransmitter.

The absence of the 5‐HT1B receptor subtype affected both components of mouse masculine sexual behaviour, motivation and execution, further confirming the involvement of this receptor subtype in the control of this behaviour. In addition, the diminished sensitivity to serotonergic stimulation exhibited by KO1B mice suggests the occurrence of compensatory changes as a consequence of the absence of the 5‐HT1B receptor subtype.

British Journal of Pharmacology (2002) 136, 1127–1134. doi:10.1038/sj.bjp.0704827
Potentiation of slow component of delayed rectifier K+ currentby cGMP via two distinct mechanisms: inhibition of phosphodiesterase 3 and activation of protein kinase G
Authors: Shimizu Kentaro , Shintani Yutaka , Ding Wei‐Guang , Matsuura Hiroshi , Bamba Tadao
Published: September 2002

Regulation of the slowly activating component of delayed rectifier K+ current (IKs) by intracellular guanosine 3′5′ cyclic monophosphate (cGMP) was investigated in guinea‐pig sino‐atrial (SA) node cells using the whole‐cell patch‐clamp method.

When a cell was dialyzed with pipette solution containing 100 μM cGMP, IKs started to gradually increase and reached a maximum increase of a factor of 2.37±0.39 (n=4) about 10–15 min after rupture of patch membrane. Atrial natriuretic peptide (ANP, 100 nM) also potentiated IKs, consistent with intracellular cGMP‐induced enhancement of IKs.

Bath application of a selective blocker of the cGMP‐inhibited phosphodiesterase (PDE3) milrinone (100 μM) enhanced IKs by a factor of 1.50±0.09 (n=4) but failed to further enhance IKs after a maximum stimulation by intracellular cGMP (100 μM), suggesting that blockade of PDE3 activity is involved in the enhancement of IKs. A potent but nonspecific PDE inhibitor 3‐isobutyl‐1‐methylxanthine (IBMX, 100 μM) further increased IKs stimulated by 100 μM milrinone, indicating that PDE subtypes other than PDE3 are also involved in the regulation of basal IKs in guinea‐pig SA node cells.

Bath application of 100 μM 8‐bromoguanosine 3′5′ cyclic monophosphate (8‐Br‐cGMP) increased IKs by a factor of 1.48±0.11 (n=5) and this stimulatory effect was totally abolished by cGMP‐dependent protein kinase (PKG) inhibitor KT‐5823 (500 nM), suggesting that the activation of PKG also mediates cGMP‐induced potentiation of IKs.

These results strongly suggest that intracellular cGMP potentiates IKs not only by blocking PDE3 but also by activating PKG in guinea‐pig SA node cells.

British Journal of Pharmacology (2002) 137, 127–137. doi:10.1038/sj.bjp.0704843
Platinum(iv)-mediated coupling of dione monoximes and nitriles: a novel reactivity pattern of the classic oxime-based chelating ligands
Authors: Makarycheva-Mikhailova Anastassiya V. , Haukka Matti , Bokach Nadezhda A. , Garnovskii Dmitrii A. , Galanski Mathea S. , Keppler Bernhard K. , Pombeiro Armando J. L. , Kukushkin Vadim Yu.
Published: September 2002

The reaction between dione monoximes and platinum(iv) nitrile complexes leads, instead of the conventional substitution, to metal-mediated coupling, giving iminoacylated species which, on being liberated, undergo disintegration to the nitrile and the oxime.
Clinical relevance of vascular endothelial growth factor receptors 1 and 2 in acute myeloid leukaemia and myelodysplastic syndrome
Authors: Verstovsek Srdan , Estey Elihu , Manshouri Taghi , Giles Francis J. , Cortes Jorge , Beran Miloslav , Rogers Anna , Keating Michael , Kantarjian Hagop , Albitar Maher
Published: September 2002

Summary.  We have previously reported that high levels of cellular vascular endothelial growth factor (VEGF) protein correlated with short survival of patients with acute myeloid leukaemia (AML). As VEGF exerts its effects via two receptors, VEGF receptor 1 (VEGFR‐1) and VEGFR‐2, we evaluated the significance of VEGFR‐1 and VEGFR‐2 protein levels in AML and myelodysplastic syndrome (MDS), and their relationship to VEGF protein levels. Western blot analysis and radioimmunoassay confirmed and quantified specific protein levels in bone marrow samples from 41 MDS and 66 AML previously untreated patients. VEGFR‐1 levels were significantly higher in AML than in MDS (P = 0·0004), but no significant difference was found in the VEGFR‐2 levels (P = 0·5). No significant correlation between VEGFRs levels and duration of survival was found. VEGF protein levelsweresignificantly higher in MDS than in AML (P < 0·0001). A Cox proportional‐hazard regression model showed increasing VEGF levels to significantly correlate with shorter survival of patients with MDS (P = 0·008), a finding similar to our previous report of the inverse relationship between VEGF levels and survival of AML patients. We found a significant correlation between VEGF and VEGFR‐2 levels in both AML and MDS (P < 0·0000001 andP < 0·0002 respectively) but not between VEGF and VEGFR‐1 levels. These data suggest that VEGF expression, rather than the expression of its receptors, is the determining factor in the biological behaviour of AML and MDS, and that VEGFRs are differentially expressed in AML and MDS.
Reciprocal alterations of Th1/Th2 function in γδ T‐cell subsets of human immunodeficiency virus‐1‐infected patients
Authors: Dobmeyer Thomas S. , Dobmeyer Rita , Wesch Daniela , Helm Eilke B. , Hoelzer Dieter , Kabelitz Dieter
Published: September 2002

Summary.  While T cells that express Vγ9 as a variable T‐cell receptor chain dominate among peripheral blood γδ T cells in healthy adults, Vδ1 cells are the major subpopulation of γδ T cells in human immunodeficiency virus (HIV)‐infected patients. We used intracellular cytokine staining and flow cytometry to analyse whether an imbalance of T helper 1 (Th1)/T helper 2 (Th2) cytokine patterns, as observed in αβ T cells, also occurs in γδ T cells. When compared with healthy HIV‐negative subjects, HIV+ patients had a decreased number of interferon‐γ (IFN‐γ)+γδ T cells, which showed a linear relation to the CD4+ cell count but not to the plasma viral load. Similar results were obtained when Vγ9 cells were analysed. In contrast, in the Vδ1 subpopulation, the number of IFN‐γ+ cells was increased in HIV+ donors when compared with healthy subjects. Even though less impressive, the number of interleukin 4 (IL‐4)‐ and IL‐10‐producing cells was uniformly inversely correlated with the number of tumour necrosis factor‐α+ and IFN‐γ+ cells. The increased IFN‐γ‐producing capacity of Vδ1 cells might represent a compensatory mechanism for the progressive loss of Vγ9 γδ T cells during the course of HIV infection.
Manumycin inhibits farnesyltransferase and induces apoptosis of drug‐resistant interleukin 6‐producing myeloma cells
Authors: Frassanito Maria Antonia , Cusmai Antonio , Piccoli Claudia , Dammacco Franco
Published: September 2002

Summary.  Interleukin 6 (IL‐6) is an important survival and growth factor for myeloma cells and exerts its effects by activating several transduction pathways, including the Ras cascade. As farnesylation of the activated Ras oncogene product by protein farnesyltransferase (FTase) is a critical step for Ras functional activity, FTase has emerged as a potential target for the development of new anti‐cancer agents. Based on our previous demonstration that IL‐6‐producing myeloma cells are refractory to drug‐induced apoptosis, we have analysed the effect of manumycin, a natural FTase inhibitor, on IL‐6‐producing myeloma cells resistant to Fas‐, dexamethasone‐ and doxorubicin‐induced apoptosis. Treatment of myeloma cells with manumycin prevented cell proliferation and induced apoptosis. Western blotting experiments demonstrated that this effect was related to inhibition of the post‐translational Ras processing.Further analysis showed that manumycin‐induced apoptosis involved caspase‐3. Activation of caspase‐3, in fact, was observed in 6 h‐treated myeloma cells expressing Apo 2·7 antigen, the marker of early apoptosis, whereas their treatment with cell‐permeable DEVD‐fmk, that irreversibly inhibits caspase‐3 activity, prevented their apoptosis. Over‐expression of caspase‐3 was also demonstrated by reverse transcription–polymerase chain reaction. Finally, over‐expression of Bcl‐2 and its homologue Bcl‐xL was observed in manumycin‐treated cells as well as in control myeloma cells, implying that the Bcl‐2 family is not involved. FTase inhibitors may thus be proposed as a potential pharmacological weapon, as they block the Ras pathway and induce the apoptosis of drug‐resistant IL‐6‐producing myeloma cells.
Variable product purity and functional capacity after CD34 selection: a direct comparison of the CliniMACS® (v2·1) and Isolex® 300i (v2·5) clinical scale devices
Authors: Watts Michael J. , Somervaille Tim C. P. , Ings Stuart J. , Ahmed Forhad , Khwaja Asim , Yong Kwee , Linch David C.
Published: September 2002

Summary. The two clinical scale devices currently available for CD34+ cell selection from peripheral blood stem cells (PBSC) apheresis products, the CliniMACS and the Isolex 300i, were compared directly by pooling and splitting two PBSC harvests collected on sequential days from 10 patients and processing half of each pooled harvest on each device. The CliniMACS product had significantly higher median CD34+ purity (90%vs 78%; P = 0·004) and lower median T‐cell content (0·06%vs 0·44%; P = 0·003) compared with the Isolex 300i product. The median CD34+ yields were similar (64% and 60% respectively). However, when the functional capacities of the products were compared, the median recovery of colony‐forming units was significantly greater from the Isolex 300i product (48%vs 38%; P = 0·035), as was expansion of cells in either erythroid or granulocytic lineage‐specific liquid culture (2·1‐fold more erythroid and 1·5‐fold more granulocytic lineage progenitors on d 9 (P = 0·03 and 0·03 respectively). This was due to a higher proportion of apoptotic cells in the CliniMACS product (28%vs 18%; P = 0·007, annexin V binding). Hence, although the CliniMACS device yielded a higher purity product with fewer T cells, the Isolex 300i product contained fewer apoptotic cells and consequently had greater functional capacity in culture.
Correlation of blood lymphocyte CTLA‐4 (CD152) induction in Hodgkin's disease with proliferative activity, interleukin 2 and interferon‐gamma production
Authors: Kosmaczewska Agata , Frydecka Irena , Boćko Dorota , Ciszak Lidia , Teodorowska Renata
Published: September 2002

Summary.  Expression of the downregulatory CTLA‐4 molecule was determined on unstimulated and anti‐CD3 + recombinant interleukin 2 (rIL‐2)‐stimulated peripheral blood T cells in Hodgkin's disease (HD) and correlated with the T‐cells' proliferative activity, IL‐2 and interferon (IFN)‐gamma production. There was a negligible percentage of CTLA‐4+/CD3+ cells before culture. The mean percentage of CTLA‐4+/CD3+ lymphocytes increased gradually, peaked after 72 h of stimulation and returned to basal values after 96 h of stimulation. The mean proportion of CTLA‐4+/CD3+ cells from untreated patients was significantly higher after 24, 48 and 72 h of stimulation compared with controls. The mean percentage of CTLA‐4+/CD3+ cells from patients in clinical remission (CR) was lower than that of untreated patients, but remained significantly higher compared with controls. Lymphocytes from untreated HD patients showed impaired proliferative activity, IL‐2 and IFN‐gamma production compared with controls. The proliferative activity of the lymphocytes, IL‐2 and IFN‐gamma production remained significantly lower in CR compared with controls. The proportion of CTLA‐4+/CD3+ cells negatively correlated with proliferative activity, IL‐2 and IFN‐gamma production in HD patients and controls. However, some untreated patients as well as patients in CR with normal mean fluorescence intensity values of CTLA‐4 showed unimpaired T‐cell function tests. Our study provides the first evidence of an increased expression of downregulatory CTLA‐4 molecule on stimulated T‐cells in HD, which could be one of the mechanisms of immune deficiency in this disease.
Severe aplastic anaemia and Grave's disease in a paediatric patient
Authors: Kumar Manjusha , Goldman Jeffrey
Published: September 2002

Summary.  Severe aplastic anaemia (SAA) is considered to be an autoimmune disorder affecting the haematopoietic cells and most often is idiopathic. An association between SAA and other autoimmune diseases is rare and has been described in adults for eosinophilic fasciitis, thymomas, systemic lupus erythematosus and thyroid disorders. We describe the first paediatric patient with chronic relapsing SAA and Grave's disease. We discuss the difficulty in diagnosis of Grave's disease, the possibility of its manifestation due to withdrawal of immunosuppressants, and issues to consider in the treatment of this disease in the setting of bone marrow failure.
Determinants of survival after human leucocyte antigen‐matched unrelated donor bone marrow transplantation in adults
Authors: Barker Juliet N. , Davies Stella M. , DeFor Todd E. , Burns Linda J. , McGlave Philip B. , Miller Jeffrey S. , Weisdorf Daniel J.
Published: September 2002

Summary. Unrelated donor (URD) bone marrow transplantation (BMT) in adults can be associated with high non‐relapse mortality (NRM). Therefore, factors determining survival in 136 human leucocyte antigen (HLA)‐A, B, DRB1‐matched adult BMT recipients were reviewed. Fifty‐four per cent of patients had chronic myelogenous leukaemia (CML) and 36% had acute leukaemia or myelodysplasia. Graft‐versus‐host disease (GvHD) prophylaxis was either cyclosporin A (CSA)/methotrexate (64%) or T‐cell depletion and CSA/corticosteroids (34%). The probability of donor engraftment by d 45 was 97% (95% CI: 94–100). Incidence of grades III–IV acute GvHD was 18% (95% CI: 12–24) at 100 d, and chronic GvHD was 42% (95% CI: 32–52) at 2 years. At 2 years, 14% (95% CI: 8–20) had relapsed. Multiple regression analysis showed that adverse risk factors for survival were non‐CML diagnosis, age > 35 years, diagnosis to transplant time of > 18 months [chronic‐phase CML (CML‐CP) only]; and grades III–IV acute GvHD. Patients ≤ 35 years with early CML‐CP had a 2 year survival of 77% (95% CI: 54–100), which compared with a survival in advanced CML patients ≤35 years of 67% (95% CI: 37–97) and 37% (95% CI: 20–54) in non‐CML patients. Two year survival for patients > 35 years with early CML‐CP was 55% (95% CI: 33–77), 40% (95% CI: 19–61) in advanced CML and 14% (95% CI:1–27) in non‐CML. Future efforts should focus on improving the outcome for older BMT recipients, especially those with diagnoses other than CML.
Expression of multidrug resistance‐associated protein 2 (MRP2) in normal human tissues and carcinomas using tissue microarrays
Authors: Sandusky G E , Mintze K S , Pratt S E , Dantzig A H
Published: September 2002

Aims: Using tissue microarrays, this study analysed the expression of the multidrug resistance protein, MRP2, by immunohistochemistry with two different MRP2 antibodies. This is the first study to address the expression of MRP2 in various common human neoplasms.

Methods and results: Immunohistochemistry was performed on zinc formalin‐fixed tissue to evaluate normal tissues and carcinomas using two antibodies against MRP2 (EAG5, a polyclonal antibody, and M2‐lll‐6, a monoclonal antibody). Immunostaining was localized in neoplastic cells mainly on the cell membrane with M2‐lll‐6 and cell membrane and cytoplasm with EAG5. In normal tissues MRP2 was expressed in liver, gastrointestinal tract, and kidney tubular epithelial cells with both antibodies. MRP2 was seen in nine of 22 renal cell carcinomas, eight of 13 gastric carcinomas, 25 of 49 breast carcinomas, 14 of 32 lung carcinomas, 39 of 50 colon carcinomas, and 16 of 17 ovarian carcinomas. There was < 10% variability between the two antibodies. MRP2 expression was highest in moderate to poorly differentiated tumours from colon, lung, gastric, and ovarian carcinomas and in grade 2 and 3 breast and renal carcinomas.

Conclusion: The expression of MRP2 in many solid human tumours indicates that inherent drug resistance may play an important role as a biomarker for predictive chemotherapy treatment.
Breast hamartomas: clinicopathological and immunohistochemical studies of 24 cases
Authors: Herbert M , Sandbank J , Liokumovich P , Yanai O , Pappo I , Karni T , Segal M
Published: September 2002

Aims:  The aim of our study was to investigate the clinical, pathological and immunohistochemical features of breast hamartomas, in order to obtain a detailed profile of this somewhat uncommon lesion.

Methods and results:  This study presents a clinical, pathological and immunohistochemical analysis of 24 breast hamartomas. Four cases of fibrocystic breast changes were used as a control group. The immunohistochemical panel induced oestrogen (ER) and progesterone (PR) receptors, c‐erbB‐2 protein, p53, Ki67, CK‐MNF116, and vimentin. The patients ranged in age from 30 to 78 years (mean 48 years). All patients presented with a palpable unilateral mass. Three patients showed two hamartomas in the same breast. Tumours ranged from 20 to 50 mm in diameter. Typical macroscopic and microscopic features were noted. Immunohistochemical studies showed ER and PR receptor positivity in epithelial cells as well as in the stromal cells in all 24 cases. No c‐erbB‐2 protein over‐expression was noted. p53 expression was not observed. Ki67 showed 2–3% positivity in epithelial cells and not in stromal cells in most cases.

Conclusions:  There was no association with side, ethnic origin or dietary habits. The immunohistochemistry of hamartomas is generally similar to normal breast or fibrocystic breast tissue. Ki67 together with receptor positivity may reflect some proliferative activity and explain observed faster growth of hamartoma during pregnancy and lactation.
Gestational age‐specific birthweight of twins in vital records
Authors: Cooperstock Michael
Published: September 2002

Analysis of singleton preterm birth from vital statistics data is hampered by inaccurate gestational age dating. This is most notably evidenced by the large fraction of implausibly high birthweights for gestational age given in birth records. Using natality statistics for New England, 1977–88, birthweight distributions were plotted for representative preterm gestations. Confounding by improbable birthweights was observed to be considerably less among preterm twin births than among preterm singleton births.
Long‐term follow‐up of participants in the Collaborative Perinatal Project: tracking the next generation
Authors: Klebanoff Mark A. , Zemel Babette S. , Buka Stephen , Zierler Sally
Published: September 2002

This report describes the methods used in conducting a long‐term follow‐up study and the factors associated with the successful location of subjects. In 1987–91, we selected, from among the women who were born in 1959–66 as members of the Philadelphia and Providence cohorts of the Collaborative Perinatal Project (CPP), 391 who were preterm at birth, 413 who were small‐for‐gestational age (SGA) and 978 who were term and appropriately grown; 1314 were from Philadelphia and 468 from Providence. Tracing was accomplished using original CPP records, telephone directories, voter registration and driver's licence records, birth certificate files and credit bureaux. A total of 61% of the women were located, 55% at Philadelphia and 77% at Providence. After adjustment, the odds ratio for ease of location, compared with women who were term and appropriately grown, was 0.85 [95% CI 0.66–1.11] for women who were preterm and 1.02 [0.78–1.34] for women who were SGA. In Philadelphia, African–American women were more likely than white women to be located, but in Providence, African–American women were less likely than white women to be located. Women whose own mothers were older at the time of their birth were more likely to be located, as were those who had more siblings followed in the CPP. Compared with women who did not complete their original CPP examinations at ages 4 and 7 years, those whose original CPP follow‐up was more complete were more likely to be located. These findings demonstrate that long‐term follow up in a US urban setting can be accomplished, and that successful location was not associated with birth status.
A Peptide‐Binding Assay for the Disease‐Associated HLA‐DQ8 Molecule
Authors: Straumfors  , Johansen  , Vartdal  , Sollid  , Thorsby  , Buus
Published: September 2002

The study of peptide binding to HLA class II molecules has mostly concentrated on DR molecules. Since many autoimmune diseases show a primary association to particular DQ molecules rather than DR molecules, it is also important to study the peptide‐binding properties of DQ molecules. Here we report a biochemical peptide‐binding assay for the type I diabetes‐associated DQ8, i.e. DQ (α1*0301, β1*0302), molecule. Affinity‐purified DQ8 molecules were tested in peptide‐binding assays using a radiolabelled influenza haemagglutinin (Ha) peptide encompassing positions 255–271(Y) as an indicator peptide. The Ha 255–271(Y) peptide bound to DQ8 in a pH‐dependent fashion showing optimal binding around pH 5. The association kinetics were relatively slow and the resulting complexes were heat labile. The specificity of peptide binding to DQ8 was investigated in competitive inhibition experiments with a panel of 43 peptides of different lengths and sequences. The DQ8 molecules showed a different pattern of peptide binding compared to a previously studied DQ2 molecule. Peptides derived from thyroid peroxidase, HLA‐DQ(α1*0301), HLA‐DQ(α1*0302), retinol receptor and p21ras were among the high‐affinity binders, whereas peptides derived from myelin basic protein were among the low‐affinity binders. The sequence of the high‐affinity peptides conformed with a previously published peptide‐binding motif of DQ8.
Airway remodelling in asthma
Authors: Beasley R. , Page C. , Lichtenstein L.
Published: September 2002

Airway remodelling is a relatively new concept in asthma research. It can be defined as the ‘structural changes in the airways that may affect their functional properties’. The structural changes usually comprise smooth muscle hyperplasia, hypertrophy of mucus glands, subepithelial fibrosis, angiogenesis and changes to the extracellular matrix. One of the most important consequences of these changes is narrowing of the airway lumen. The aetiology of remodelling has not been elucidated fully, but it is clear that there is an important association between remodelling and inflammation. However, the degree to which one process causes the other, and the nature of the causality, is not known. Numerous cells and mediators including epithelial cells, cytokines and growth factors all appear to play a part in the remodelling process, but the precise roles of specific cells and mediators are yet to be defined clearly. Because of the problems in assessing the extent of remodelling in living patients, research into the clinical consequences of this process has been difficult. However, it is clear that the extent of remodelling correlates with the severity of asthma, and patients with extensive remodelling may also be resistant to steroid treatment. It is not clear whether remodelling is generally reversible, although there is evidence that it is in some patients, and this reversal is associated with improvement in symptoms. However, remodelling represents an important target for the development of future drugs to treat asthma, and pharmacological treatment that could reverse the process of remodelling has the potential to play a key role in asthma management.
Wound healing in the transforming growth factor‐β1—deficient mouse
Authors: Brown Rebeccah L. , Ormsby Ilona , Doetschman Thomas C. , Greenhalgh David G.
Published: September 2002

To investigate the role of transforming growth factor‐β1 in tissue repair, we performed wound healing studies in the transforming growth factor‐β1—deficient mouse with targeted disruption of the transforming growth factor‐β1 gene. Transforming growth factor‐β1—deficient mice exhibit no obvious developmental defects and are phenotypically normal until approximately 3 weeks of age when a severe wasting syndrome develops, accompanied by an overwhelming inflammatory response resulting in multisystem organ failure and death. Full‐thickness 0.5 × 0.5 cm skin wounds were created on the backs of 10‐day‐old mice (wild type or heterozygous controls versus homozygous transforming growth factor‐β1—deficient mutants) and covered with a nonabsorbent dressing (OpSite). Serial wound measurements were made, and percentage of wound closure over time was determined. On day 10, wounds and liver were harvested for histologic and molecular analysis. Histologic scores were assigned (1 [no healing] to 12 [complete healing]) on the basis of granulation tissue formation, vascularity, collagen deposition, and epithelialization. Reverse transcription—polymerase chain reaction was performed to detect messenger RNA transcripts for transforming growth factor‐β1, transforming growth factor‐β2, platelet‐derived growth factor A‐chain and B‐chain, interleukin‐1β and ‐6, and tumor necrosis factor‐α in unwounded skin, day 10 wounds, and liver. No significant differences in wound closure were observed until day 10. Weight gain, however, was significantly decreased in the mutant animals as early as day 6. Histologic scores were significantly lower in the transforming growth factor‐β1—deficient mutants (5.4 ± 0.6 versus 11.1 ± 0.3, p < 0.01, Wilcoxon rank‐sum test) and showed decreased granulation tissue formation, vascularity, collagen deposition, and epithelialization and a marked inflammatory infiltrate. As expected, transforming growth factor‐β1 was expressed in controls but not mutants. Transforming growth factor‐β2, platelet‐derived growth factor A‐chain and B‐chain, and tumor necrosis factor‐α were constitutively expressed in unwounded skin, day 10 wounds, and liver of both controls and mutants. Interleukin‐1β and ‐6, however, were induced after wounding. Early wound healing in the transforming growth factor‐β1—deficient mouse proceeds relatively normally because of upregulation or functional redundancy of other growth factors or possibly because of maternal rescue by means of transforming growth factor‐β1 transmitted in milk. Loss of transforming growth factor‐β1 regulation ultimately results in a marked inflammatory response, as evidenced by the histologic appearance of the wound and increased expression of the inflammatory cytokines (tumor necrosis factor‐α, interleukin‐1β and 6). The severe wasting syndrome (marked by weight loss) undoubtedly has an adverse effect on wound healing.
Interferons gamma and alpha‐2b differentially regulate the expression of collagenase and tissue inhibitor of metalloproteinase‐1 messenger RNA in human hypertrophic and normal dermal fibroblasts
Authors: Ghahary Aziz , Shen You J. , Nedelec Bernadette , Scott Paul G. , Tredget Edward E.
Published: September 2002

We have recently shown that both interferon gamma (γ) and interferon alpha‐2b (α‐2b) markedly depress the expression of messenger RNA for type I procollagen and fibronectin in postburn hypertrophic scar and normal dermal fibroblasts. In this article we examine the effects of these cytokines on the expression of mRNA for collagenase and its natural inhibitor, tissue inhibitor of metalloproteinase‐1. Twelve different fibroblast cell strains, six from postburn hypertrophic scar and six from the normal dermis of the same patients, were established in cell culture. The results of a dose response experiment showed increases in collagenase mRNA up to 4000 U/ml of interferon‐α‐2b, but maximal increases in tissue inhibitor of metalloproteinase mRNA expression and maximal decrease in mRNA for type I procollagen at 2000 U/ml. For subsequent experiments cells were treated with either interferon‐α‐2b (2000 U/ml) or ‐γ (1000 U/ml) for 96 hours. Quantitative analysis showed increases in tissue inhibitor of metalloproteinase‐1 and collagenase mRNA (81% and 54%, respectively) in interferon‐α‐2b‐treated hypertrophic scar fibroblasts. Under the same experimental conditions, interferon‐α‐2b had similar effects on normal dermal fibroblasts; however, interferon‐γ had a differential effect on the expression of mRNA for collagenase and tissue inhibitor of metalloproteinase‐1. Cells treated with interferon‐γ showed increases in tissue inhibitor of metalloproteinase‐1 mRNA (78% in hypertrophic scar and 56% in normal dermal fibroblasts) but decreases (59% and 42%, respectively) in collagenase mRNA. These effects appear to be selective because rehybridization of blots with a complementary DNA for tissue inhibitor of metalloproteinase‐2 mRNA showed no marked alteration in the abundance of this transcript. Significantly greater collagenase activity was found in conditioned medium from interferon‐α‐2b‐treated hypertrophic scar cells compared with that from interferon‐γ‐treated cells. These findings suggest that interferon α‐2b would have some advantages over interferon‐γ for the treatment of dermal fibroproliferative disorders, such as postburn hypertrophic scar.
Hypertrophic scar fibroblasts accelerate collagen gel contraction
Authors: Garner Warren L. , Rittenberg Toni , Ehrlich H. Paul , Karmiol Soverin , Rodriguez Jorge L. , Smith David J. , Phan Sem H.
Published: September 2002

Excessive contraction of hypertrophic scar and subsequent contracture formation are a formidable problem after thermal injury. A comparison between fibroblasts from hypertrophic scar and normal skin was made with the use of fibroblast‐populated collagen lattices as a measure of cellular generated contractile forces. Hypertrophic scar and normal skin fibroblasts were mixed with soluble tendon collagen and Dulbecco's modified Eagle medium supplemented with 10% serum, and contraction was measured by serial area measurements. Parallel experiments in the presence of transforming growth factor‐β or anti‐transforming growth factor‐β antibody examined the role of this cytokine on lattice contraction. Transforming growth factor‐β activity was measured in an additional set of 10 biopsy specimens. Hypertrophic scar fibroblasts contract lattices at a significantly faster rate than do normal skin fibroblasts. Exogenous transforming growth factor‐β increased lattice contraction by normal skin fibroblasts but had little effect on hypertrophic scar cell‐populated lattices. The addition of anti‐transforming growth factor‐β antibody decreased lattice contraction by both cell types. Transforming growth factor‐β activity was significantly increased in the hypertrophic scar biopsy specimens. Excessive scar contraction and post‐burn scar contracture result from increased contraction forces generated by hypertrophic scar cells. This increased contractility appears to be mediated by increased endogenous presence of transforming growth factor‐β.
Differential response to transforming growth factors‐β1 and β2: connective tissue deposition in animal models
Authors: Ogawa Yasushi , Gerhardt Carolyn O. , Ksander George A.
Published: September 2002

The ability of transforming growth factor (TGF)‐β1 and TGF‐β2 to promote connective tissue deposition were compared in different animal models. A single subcutaneous injection of TGF‐β2 in collagen/heparin gel carrier promoted markedly more extensive development of connective tissue than TGF‐β1 at the site of injection in both neonatal and adult mice. Both TGF‐β1 and TGF‐β2 promoted deposition of dense and well‐vascularized connective tissue matrix infiltrated with macrophages and fibroblasts. However, the results of immunohistochemical analyses suggested that TGF‐β2 promoted an accumulation of more macrophages in the connective tissue than TGF‐β1. Similar differences in the extent of connective tissue development were observed in neonatal mice when these factors were administered as a solution, without the collagen/heparin gel carrier. TGF‐β2 was also more potent than TGF‐β1 in domestic pigs. However, in guinea pigs, TGF‐β1 promoted more extensive connective tissue development than TGF‐β2. These results suggest that the differential connective tissue response to TGF‐β1 and TGF‐β2 is species dependent. However, the differences in the physical and chemical properties of these factors may account in part for the differential response as well.
Fetal wound healing: an overview
Authors: Olutoye Oluyinka O. , Cohen I. Kelman
Published: September 2002

The ability of fetal tissues to heal without scarring has prompted extensive research into the biochemical and molecular differences between fetal and postnatal wound healing. A thorough understanding of the basic mechanisms of fetal wound repair may to lead to approaches to correct or prevent the clinical problems encountered in abnormal adult wound healing and fetal surgery. This article contrasts the normal healing response in adults with fetal repair in animal models, highlighting investigations of extracellular matrix expression, cytokine profiles, and cellular dynamics.
Mesenchymal‐epithelial interactions regulate gene expression of type VII collagen and kalinin in keratinocytes and dermal‐epidermal junction formation in a skin equivalent model
Authors: Sahuc Florent , Nakazawa Keiko , Berthod François , Collombel Christian , Damour Odile
Published: September 2002

Anchoring fibrils constituted primarily of type VII collagen and anchoring filaments composed of kalinin are essential structural elements of the dermal‐epidermal junction and critical for its stability. The role of fibroblasts in the production of these structural elements and the formation of the dermal‐epidermal junction was studied by using a living skin equivalent model. This model had been modified such that keratinocytes and fibroblasts were allowed direct contact. After 2 weeks, immunohistochemical studies showed the linear deposition of type VII collagen and kalinin, as well as type IV collagen and α6 integrin at the dermal‐epidermal junction. By electron microscopy, anchoring fibrils, a continuous lamina densa, and numerous hemidesmosomes were noted. Reverse transcriptase‐polymerase chain reaction analysis showed an increased expression of both type VII collagen and kalinin genes in keratinocytes when they were in direct contact with fibroblasts. These results suggest that fibroblasts synthesize an extracellular matrix which favors keratinocyte adhesion and the formation of a dermal‐epidermal junction by increasing the production and the further arrangement of dermal‐epidermal junction components.