bluuebunny/crossref_metadata_2025 huggingface dataset:
Successful treatment of collagen‐induced arthritis in mice and rats by targeting extracellular high mobility group box chromosomal protein 1 activity
Authors: Kokkola R. , Li J. , Sundberg E. , Aveberger A.‐C. , Palmblad K. , Yang H. , Tracey K. J. , Andersson U. , Harris H. Erlandsson
Published: July 2003

Objective

Extracellular high mobility group box chromosomal protein 1 (HMGB‐1) is a recently identified, endogenous, potent tumor necrosis factor– and interleukin‐1 (IL‐1)–inducing protein detectable in inflamed synovia in both human and experimental disease. In the present study, we examined clinical effects in collagen‐induced arthritis (CIA) using therapeutic administration of neutralizing HMGB‐1 antibodies or truncated HMGB‐1–derived A‐box protein, a specific, competitive antagonist of HMGB‐1.

Methods

CIA was induced in DBA/1j mice or dark agouti rats, and animals were examined daily for signs of arthritis. Treatment with polyclonal anti–HMGB‐1 antibodies or the A‐box protein was initiated at the onset of disease and was administered intraperitoneally twice daily for 7 days. Animals were killed 8 days after initiation of therapy, and immunohistochemical analysis of synovial tissue specimens was performed.

Results

Systemic administration of anti–HMGB‐1 antibodies or A‐box protein significantly reduced the mean arthritis score, the disease‐induced weight loss, and the histologic severity of arthritis. Beneficial effects were observed in both mice and rats. Immunohistochemical analysis revealed pronounced synovial IL‐1β expression and articular cartilage destruction in vehicle‐treated mice. Both these features were significantly less manifested in animals treated with anti–HMGB‐1 antibodies or A‐box protein.

Conclusion

Counteracting extracellular HMGB‐1 with either neutralizing antibodies or a specific HMGB‐1 antagonist may offer a new method for the successful treatment of arthritis. Inflammation and tissue destruction were suppressed in CIA after HMGB‐1 blockade.
Bone edema scored on magnetic resonance imaging scans of the dominant carpus at presentation predicts radiographic joint damage of the hands and feet six years later in patients with rheumatoid arthritis
Authors: McQueen Fiona M. , Benton Nick , Perry David , Crabbe Jeff , Robinson Elizabeth , Yeoman Sue , McLean Lachy , Stewart Neal
Published: July 2003

Objective

Magnetic resonance imaging (MRI) is capable of revealing synovitis and tendinitis in early rheumatoid arthritis (RA), as well as bone edema and erosion. These features are visible before radiographic joint damage occurs. We sought to examine whether MRI of one body region (the wrist) can be used to predict whole‐body radiography scores reflecting joint damage at 6 years.

Methods

We conducted a 6‐year prospective study of a cohort of patients who fulfilled the criteria for RA at presentation, using clinical parameters, radiographs, and MRI scans of the dominant wrist. Of the 42 patients enrolled at baseline, full MRI, radiographic, and clinical data were available for 31 at 6‐year followup. MRI scans were scored by 2 radiologists, using a validated scoring system. Radiographs of the hands and feet were graded using the modified Sharp scoring method. MRI and radiography scores obtained at baseline and 6 years were compared, and baseline MRI scores were examined for their ability to predict radiographic outcome at 6 years.

Results

At 6 years, the total Sharp score correlated significantly with the total MRI score and the MRI erosion score (r = 0.81, P < 0.0001 and r = 0.79, P < 0.0001, respectively). The 6‐year Sharp score also correlated with the baseline total MRI and MRI erosion scores (r = 0.56, P < 0.0001 and r = 0.33, P = 0.03, respectively). MRI synovitis and bone edema scores remained constant for the group as a whole over 6 years, but bone erosion scores progressed (P = 0.0001), consistent with radiographic deterioration. Erosions on 6‐year MRI scans were frequently preceded by MRI bone edema at baseline (odds ratio 6.5, 95% confidence interval 2.78–18.1). Regression models indicated that the baseline MRI bone edema score was predictive of the 6‐year total Sharp score (P = 0.01), as was the C‐reactive protein (CRP) level (P = 0.0002). Neither shared epitope status nor swollen or tender joint counts predicted radiographic outcome in this cohort. A model incorporating baseline MRI scores for erosion, bone edema, synovitis, and tendinitis plus the CRP level and the erythrocyte sedimentation rate explained 59% of the variance in the 6‐year total Sharp score (R2 = 0.59, adjusted R2 = 0.44).

Conclusion

MRI scans performed at the first presentation of RA can be used to help predict future radiographic damage, allowing disease‐modifying therapy to be targeted to patients with aggressive disease.
Anti–tumor necrosis factor α therapy in fifteen patients with AA amyloidosis secondary to inflammatory arthritides: A followup report of tolerability and efficacy
Authors: Gottenberg Jacques‐Eric , Merle‐Vincent Florence , Bentaberry Fabrice , Allanore Yannick , Berenbaum Francis , Fautrel Bruno , Combe Bernard , Durbach Antoine , Sibilia Jean , Dougados Maxime , Mariette Xavier
Published: July 2003

Objective

Because anti–tumor necrosis factor α (anti‐TNF) has emerged as a highly effective treatment for numerous inflammatory arthritides, which are a common cause of AA amyloidosis, we retrospectively evaluated the safety and efficacy of anti‐TNF in a nationwide study.

Methods

The rheumatology departments of all French teaching hospitals were contacted by mail to obtain the files of patients with histologically proven secondary AA amyloidosis and renal involvement who were treated with anti‐TNF. Efficacy was assessed as a sustained decrease in 24‐hour proteinuria and a stable/improved glomerular filtration rate (GFR).

Results

Among the 15 patients studied, the 24‐hour proteinuria was 4.5 ± 3.6 gm (mean ± SD), creatininemia was 178.4 ± 74.9 μmoles/liter, and GFR was 46 ± 23 ml/minute before starting anti‐TNF. Ten patients received infliximab, 4 received etanercept, and 1 received both types of treatment. The mean followup was 10.4 months. No severe adverse events were recorded; one episode of herpes zoster in the first branch of the trigeminal nerve occurred after one infusion of infliximab. Amyloidosis progressed in 7 patients and was stabilized in 5 patients. Three patients (receiving infliximab alone, infliximab plus methotrexate [MTX], or etanercept plus MTX) experienced rapid, dramatic, and sustained decreases in proteinuria (≥80%), with the GFR increasing 15–78%.

Conclusion

Anti‐TNF was well‐tolerated and safe in the 15 patients with AA amyloidosis and renal involvement. The pathogenic role of TNF in AA amyloidosis, the sustained proteinuria decrease in 3 patients, and the stabilization of renal parameters in 5 other patients make anti‐TNF a promising candidate to treat AA amyloidosis secondary to inflammatory arthritides.
Monocyte chemoattractant protein 3 as a mediator of fibrosis: Overexpression in systemic sclerosis and the type 1 tight‐skin mouse
Authors: Ong Voon H. , Evans Lowri A. , Shiwen Xu , Fisher Ivan B. , Rajkumar Vineeth , Abraham David J. , Black Carol M. , Denton Christopher P.
Published: July 2003

Objective

To determine the gene‐expression profile in dermal fibroblasts from type 1 tight‐skin (Tsk1) mice, and to examine the expression and potential fibrotic activity of monocyte chemoattractant protein 3 (MCP‐3) in Tsk1 mouse and human systemic sclerosis (SSc) skin.

Methods

Complementary DNA microarrays (Atlas 1.2) were used to compare Tsk1 fibroblasts with non‐Tsk1 littermate cells at 10 days, 6 weeks, and 12 weeks of age. Expression of MCP‐3 protein was assessed by Western blotting of fibroblast culture supernatants, and localized in the mouse and human skin biopsy samples by immunohistochemistry. Activation of collagen reporter genes by MCP‐3 was explored in transgenic mouse fibroblasts and by transient transfection assays.

Results

MCP‐3 was highly overexpressed by neonatal Tsk1 fibroblasts and by fibroblasts cultured from the lesional skin of patients with early‐stage diffuse cutaneous SSc. Immunolocalization confirmed increased expression of MCP‐3 in the dermis of 4 of 5 Tsk1 skin samples and 14 of 28 lesional SSc skin samples, compared with that in matched healthy mice (n = 5) and human controls (n = 11). Proα2(I) collagen promoter–reporter gene constructs were activated by MCP‐3 in transgenic mice and by transient transfection assays. This response was maximal between 16 and 24 hours of culture and mediated via sequences within the proximal promoter. The effects of MCP‐3 could be diminished by a neutralizing antibody to transforming growth factor β.

Conclusion

We demonstrate, for the first time, overexpression of MCP‐3 in early‐stage SSc and in Tsk1 skin, and suggest a novel role for this protein as a fibrotic mediator activating extracellular matrix gene expression in addition to promoting leukocyte trafficking. This chemokine may be an important early member of the cytokine cascade driving the pathogenesis of SSc.
Regulation of p53 by macrophage migration inhibitory factor in inflammatory arthritis
Authors: Leech Michelle , Lacey Derek , Xue Jin Rong , Santos Leilani , Hutchinson Paul , Wolvetang Ernst , David John R. , Bucala Richard , Morand Eric F.
Published: July 2003

Objective

To study the capacity of macrophage migration inhibitory factor (MIF) to regulate proliferation, apoptosis, and p53 in an animal model of rheumatoid arthritis (RA) and in fibroblast‐like synoviocytes (FLS) from humans with RA.

Methods

Antigen‐induced arthritis (AIA) was induced in MIF–/– mice and littermate controls. FLS were obtained from patients with RA. Western blotting and immunohistochemistry were used to measure p53 in cells and tissues. Apoptosis was detected in cells by flow cytometry using TUNEL and annexin V/propidium iodide labeling. Apoptosis in tissue was detected using TUNEL. Proliferation was assessed in cultured cells and tissue by 3H‐thymidine incorporation and Ki‐67 immunostaining, respectively.

Results

MIF inhibited p53 expression in human RA FLS. Levels of p53 were correspondingly increased in MIF–/– mouse tissues and cells. Spontaneous and sodium nitroprusside–induced apoptosis were significantly increased in MIF–/– cells. In vitro exposure of FLS to MIF reduced apoptosis and significantly induced FLS proliferation. Synoviocyte proliferation in MIF–/– mice was correspondingly reduced. A decrease in the severity of AIA in MIF–/– mice was associated with an increase in p53 and apoptosis in synovium. Evidence of in situ proliferation was scant in this model, and no difference in in situ proliferation was detectable in MIF–/– mice compared with wild‐type mice.

Conclusion

These results indicate a role for MIF in the regulation of p53 expression and p53‐mediated events in the inflamed synovium and support the hypothesis that MIF is of critical importance in the pathogenesis of RA.
Functional characterization of adherent synovial fluid cells in rheumatoid arthritis: Destructive potential in vitro and in vivo
Authors: Neidhart Michel , Seemayer Christian A. , Hummel Klaus M. , Michel Beat A. , Gay Renate E. , Gay Steffen
Published: July 2003

Objective

To characterize the morphologic and immunologic features of adherent synovial fluid cells derived from patients with rheumatoid arthritis (RA), and to explore their potential function in vitro and in vivo by focusing on cartilage destruction.

Methods

Synovial fluid adherent cells obtained from patients with RA and from control subjects were characterized by immunohistochemistry, flow cytometry, and electron microscopy. In vitro, these cells were cultured in the presence of cartilage particles. Cartilage destruction was monitored by the release of sulfated glycosaminoglycans (sGAG) into the medium, and the level of matrix metalloproteinase 1 (MMP‐1) in the cell culture supernatant was measured by enzyme‐linked immunosorbent assay. To inhibit cartilage destruction in vitro, the MMP inhibitor marimastat was tested in this system. In vivo, in the SCID mouse coimplantation model, RA synovial fluid adherent cells and RA synovial fibroblasts (as positive controls) were coimplanted with human cartilage under the kidney capsule and maintained there for 60 days.

Results

In vitro, the synovial fluid adherent cells consisted of 2 subpopulations, large round‐shaped macrophage‐like cells (CD68+) and spindle‐shaped fibroblast‐like cells (Thy‐1+). When passaged, the latter cells proliferated and organized themselves into 3‐dimensional formations. This allowed them to reach collagen particles fixed with agarose. Fibroblasts derived from synovial tissues could not be used in this assay because they grew only in monolayers and not on agarose. The majority (>90%) of passaged RA synovial fluid adherent cells expressed the Thy‐1+,CD45−,CD68−,CD86− phenotype. Electron microscopy did not reveal important morphologic differences between the 2 types of fibroblasts, those from synovial tissue or those from synovial fluid. However, synovial fluid adherent cells expressed lower levels of adhesion molecules, including CD54 and galectin 3, as well as the complement‐regulatory molecule CD55. The in vitro release of sGAG associated with cell activity was 2.5‐fold higher from RA synovial fluid adherent cells in comparison with that from negative control cells. The release of sGAG correlated with the concentration of MMP‐1 and was inhibited by the broad‐range MMP inhibitor marimastat in a dose‐dependent manner. RA synovial fluid adherent cells coimplanted with cartilage in SCID mice showed the same invasive behavior as that displayed by tissue‐derived RA synovial fibroblasts.

Conclusion

Similar to tissue‐derived RA synovial fibroblasts, RA synovial fluid adherent cells, which contain “floating” anchorage‐independent fibroblast‐like cells, mediate cartilage destruction independent of the hyperplastic synovial tissue.
Polymorphism at position −308 of the tumor necrosis factor α gene influences outcome of infliximab therapy in rheumatoid arthritis
Authors: Mugnier Benedicte , Balandraud Nathalie , Darque Albert , Roudier Chantal , Roudier Jean , Reviron Denis
Published: July 2003

Objective

To test whether the G‐to‐A polymorphism at position −308 in the promoter of the tumor necrosis factor α (TNFα) gene influences response to infliximab therapy in patients with rheumatoid arthritis (RA).

Methods

We genotyped 59 RA patients by polymerase chain reaction and subdivided them into two groups: those with the A/A or A/G genotype and those with the G/G genotype. We compared the groups' clinical responses to infliximab treatment after 22 weeks, using the Disease Activity Score in 28 joints (DAS28).

Results

We found that 42% of patients in the A/A and A/G group and 81% of patients in the G/G group had improvement of at least 1.2 in the DAS28 score (P = 0.0086). The average improvement in the DAS28 score was 1.24 in the A/A and A/G patients and 2.29 in the G/G patients (P = 0.029).

Conclusion

These data suggest that patients with a TNFα −308G/G genotype are better infliximab responders than are patients with A/A or A/G genotypes. TNFα −308 genotyping may be a useful tool for predicting response to infliximab treatment.
Five‐year followup of rheumatoid arthritis patients after early treatment with disease‐modifying antirheumatic drugs versus treatment according to the pyramid approach in the first year
Authors: Verstappen Suzan M. M. , Jacobs Johannes W. G. , Bijlsma Johannes W. J. , Heurkens Anton H. M. , van Booma‐Frankfort Christina , Borg Evert Jan ter , Hofman Dick M. , van der Veen Maaike J.
Published: July 2003

Objective

To evaluate whether the clinical advantages observed after 1 year in a randomized controlled clinical trial, in which 2 treatment strategies were compared (the early disease‐modifying antirheumatic drug [DMARD] approach versus the pyramid approach), persist after 5 years.

Methods

In this study, 238 patients with recently diagnosed rheumatoid arthritis (RA) were randomized to either the pyramid group (n = 56) or the early DMARD group (n = 182). Patients assigned to the pyramid group received nonsteroidal antiinflammatory drugs for at least 1 year after inclusion (the mean ± SD lag time until first prescription of a DMARD was 14 ± 9 months). Patients in the early DMARD group were treated with a DMARD immediately after inclusion.

Results

After 5 years, data were available for 44 patients in the pyramid group (79%) and 145 patients in the early DMARD group (80%). No prolongation of the clinical advantages in favor of the early DMARD group, as observed after the first year, was demonstrated. Nevertheless, a significantly shorter delay time until complete response and a higher number of patients with overall clinically relevant improvement at several assessment points were observed in the early DMARD group compared with the pyramid group.

Conclusion

The clinical results in favor of the early DMARD group, as observed after the first year, were not as evident after 5 years. This indicates that a more aggressive treatment approach in early RA is required, and that treatment should be continued for a prolonged period of time, in order to maintain the advantages obtained in the first year.
Expression of activation‐induced, T cell–derived, and chemokine‐related cytokine/lymphotactin and its functional role in rheumatoid arthritis
Authors: Blaschke Sabine , Middel Peter , Dorner Brigitte G. , Blaschke Volker , Hummel Klaus M. , Kroczek Richard A. , Reich Kristian , Benoehr Peter , Koziolek Michael , Müller Gerhard A.
Published: July 2003

Objective

To evaluate the possible role of activation‐induced, T cell–derived, and chemokine‐related cytokine (ATAC)/lymphotactin (Lptn) in the pathogenesis of rheumatoid arthritis (RA).

Methods

ATAC/Lptn levels in serum and synovial fluid samples were measured by sandwich enzyme‐linked immunosorbent assay. Expression of messenger RNA for ATAC/Lptn in synovial tissues was analyzed by reverse transcription–polymerase chain reaction (PCR) and by in situ hybridization, and was quantitated by real‐time PCR. The phenotype of peripheral blood mononuclear cells (PBMCs) expressing ATAC/Lptn was analyzed by intracellular cytokine staining and flow cytometry.

Results

Levels of ATAC/Lptn were similar in sera and synovial fluids from RA patients (n = 20) and osteoarthritis controls (n = 15). In phorbol myristate acetate/ionomycin–stimulated PBMCs, ATAC/Lptn expression was detected in CD8+ T cells and in a significantly increased proportion of CD4+,CD28− T cells from RA patients as compared with healthy controls. In synovial tissues, ATAC/Lptn was predominantly localized in CD3+ T cells in the sublining layer. Lymphocytes, synovial macrophages, and, unexpectedly, fibroblast‐like synoviocytes (FLS) were identified as major target cells for ATAC/Lptn in RA synovium, as determined by analysis of the ATAC/Lptn receptor XCR1. In vitro, ATAC/Lptn stimulation of FLS resulted in a marked down‐regulation of matrix metalloproteinase 2 production.

Conclusion

These data indicate that in RA synovium, ATAC/Lptn is mainly produced by T cells. Considering its function as a lymphocyte‐specific chemoattractant, ATAC/Lptn might be a key modulator for T cell trafficking in the pathogenesis of RA. In addition, functional studies suggest that ATAC/Lptn may exert additional immunomodulatory effects in RA.
Mechanical effects of the intraarticular administration of high molecular weight hyaluronic acid plus phospholipid on synovial joint lubrication and prevention of articular cartilage degeneration in experimental osteoarthritis
Authors: Kawano Tsutomu , Miura Hiromasa , Mawatari Taro , Moro‐Oka Takaaki , Nakanishi Yoshitaka , Higaki Hidehiko , Iwamoto Yukihide
Published: July 2003

Objective

To examine in vivo the effects of a mixture of high molecular weight hyaluronic acid (HA) plus phospholipids on joint lubrication and articular cartilage degeneration.

Methods

Experimental osteoarthritis (OA) of the right knee was induced by anterior cruciate and medial collateral ligament transection in 40 rabbits. The animals were subjected to 8 consecutive weekly intraarticular administrations of high molecular weight HA (the HA200 group), conventional molecular weight HA (the HA80 group), or high molecular weight HA plus L‐δ dipalmitoyl phosphatidylcholine liposomes (the PHA group) and were killed 1 week after the final injection. The remaining transected right knees (the OA group) and randomly selected nontransected contralateral left knees (the control group) were collected simultaneously. Each group (n = 10) was divided into 2 equal subgroups, one of which was evaluated histologically while the other was subjected to a lubricating ability test using a pendulum friction tester.

Results

The injected knees had a tendency to demonstrate less damage to the articular cartilage compared with the OA group, and the histologic findings in all groups except for the PHA group differed significantly from the control group. There was a significant difference in the mean ± SD friction coefficient between the control group (0.0100 ± 0.00300) and the OA (0.0206 ± 0.00649), HA200 (0.0190 ± 0.00427), and HA80 (0.0177 ± 0.00712) groups (P < 0.05 for each comparison), but not between the control group and the PHA group (0.0150 ± 0.00330) (P = 0.15).

Conclusion

To our knowledge, this is the first in vivo study to examine whether intraarticular injections of phospholipids influence joint lubrication by acting as a boundary lubricant, thus protecting articular cartilage from degenerative changes.
Analysis of systemic sclerosis in twins reveals low concordance for disease and high concordance for the presence of antinuclear antibodies
Authors: Feghali‐Bostwick Carol , Medsger Thomas A. , Wright Timothy M.
Published: July 2003

Objective

To examine concordance for systemic sclerosis (SSc) in monozygotic (MZ) and dizygotic (DZ) twins.

Methods

MZ and DZ twins were recruited nationwide. Zygosity was confirmed by DNA fingerprint analysis. The presence of antinuclear antibodies (ANAs) was determined using indirect immunofluorescence with HEp‐2 cells as substrate. Identification of SSc‐associated serum autoantibodies was performed by immunoprecipitation and double immunodiffusion. Major histocompatibility complex class II alleles were identified by polymerase chain reaction–restriction fragment length polymorphism analysis.

Results

Concordance for SSc was found to be similar in MZ and DZ twins. Overall concordance for SSc was low in the twins (4.7%). Concordance for the presence of ANAs was significantly higher in MZ twins compared with DZ twins. SSc‐associated serum autoantibodies occurred exclusively in patients with SSc. The distribution of SSc‐associated serum autoantibodies was similar to that observed in our large database of SSc patients. Increased HLA allele sharing was detected in DZ twins, irrespective of disease concordance.

Conclusion

These results indicate that inherited genetic factors are not sufficient to explain the development of SSc. Rather, these data indicate that inheritance may play a role in the development of serum autoantibodies in the “healthy” twin sibling of an SSc patient.
The spontaneous remission of juvenile idiopathic arthritis is characterized by CD30+ T cells directed to human heat‐shock protein 60 capable of producing the regulatory cytokine interleukin‐10
Authors: de Kleer I. M. , Kamphuis S. M. , Rijkers G. T. , Scholtens L. , Gordon G. , de Jager W. , Häfner R. , van de Zee R. , van Eden W. , Kuis W. , Prakken B. J.
Published: July 2003

Objective

To test the hypothesis that T cell reactivity to self heat‐shock protein 60 (Hsp60) in patients with remitting juvenile idiopathic arthritis (JIA) is part of an antiinflammatory, regulatory mechanism.

Methods

Using peripheral blood–derived mononuclear cells (PBMCs) and synovial fluid–derived mononuclear cells (SFMCs) obtained from patients with JIA, we analyzed the expression of CD30 and the induction of regulatory cytokines in response to human and mycobacterial Hsp60.

Results

In oligoarticular JIA patients, in vitro activation of PBMCs and SFMCs with Hsp60 induced a high expression of CD30 on CD4+, activated (HLA–DR–positive), memory (CD45RO+) T cells. The expression of CD30 induced by human Hsp60 was much higher than that induced by mycobacterial Hsp60. In oligoarticular JIA patients with active disease, the expression of CD30 in response to human Hsp60 was paralleled by a high interleukin‐10 (IL‐10):interferon‐γ (IFNγ) ratio. In addition, restimulated human Hsp60–specific T cell lines from oligoarticular JIA patients showed a high production of IL‐10 and a low production of IFNγ. In contrast, PBMCs and SFMCs from polyarticular JIA patients responded to human Hsp60 with virtually no expression of CD30 and a low IL‐10:IFNγ ratio.

Conclusion

The results show that T cells responding to human Hsp60 in oligoarticular JIA patients express CD30, and during active phases of the disease, these T cells have a cytokine profile with a high IL‐10:IFNγ ratio. These findings suggest that in oligoarticular JIA patients, human Hsp60–specific CD4+ cells have a regulatory function and contribute to disease remission.
Will this paper ever be cited?
Authors: Burrell Quentin L.
Published: July 2003

A recently proposed stochastic model to describe the citation process in the presence of obsolescence is used to answer the question: If a paper has not been cited by time t after its publication, what is the probability that it will ever be cited?
Clinical and physiological findings, and possible aetiological factors of rectal hyposensitivity
Authors: Gladman M A , Scott S M , Williams N S , Lunniss P J
Published: July 2003

Background

Rectal hyposensitivity (RH) relates to insensitivity of the rectum on anorectal physiological investigation and appears common in functional bowel disorders. The clinical significance of this physiological abnormality is unclear.

Method

RH was defined as one or more sensory threshold volumes raised beyond the normal range (mean plus two standard deviations) on rectal balloon distension. Clinical information and results of other anorectal physiological investigations were evaluated in 261 patients with RH.

Results

Patients with RH most commonly presented with constipation (48 per cent), constipation and incontinence in combination (27 per cent), or faecal incontinence (20 per cent). Thirty-eight per cent of patients had a history of previous pelvic surgery, 22 per cent a history of anal surgery and 13 per cent a history of spinal trauma. In patients with RH presenting with symptoms of constipation or incontinence, impaired rectal sensation was the only abnormality on physiological investigation in 48 per cent and 31 per cent respectively.

Conclusion

Patients with RH display marked heterogeneity in terms of presenting symptoms. The exact causes of RH are unknown, but there is evidence to suggest that pelvic nerve injury and spinal trauma are possible aetiological factors. RH appears important in the aetiology of both constipation and faecal incontinence, and may be useful as a predictor of surgical outcome.
Platinum(II)‐Based Coordination Compounds as Nucleic Acid Labeling Reagents: Synthesis, Reactivity, and Applications in Hybridization Assays
Authors: Heetebrij R. J. , Talman E. G. , Velzen M. A. v. , Gijlswijk R. P. M. v. , Snoeijers S. S. , Schalk M. , Wiegant J. , Rijke F. v.d. , Kerkhoven R. M. , Raap A. K. , Tanke H. J. , Reedijk J. , Houthoff H.‐J.
Published: July 2003

The synthesis, characterization, and molecular interactions of platinum(II) coordination compounds, which contain a distal nonradioactive reporter molecule, with mono‐ and polynucleotides are described. A [Pt(II)(en)(NH2(CH2)6NH‐tBoc)Cl](NO3) (en=ethylenediamine) entity has been coupled, after removal of the tBoc group, to a number of hapten and fluorophore molecules through succinimide derivatives. The influence of the various tethered reporter groups within these complexes on the reactivity towards guanosine 5′‐monophosphate (5′‐GMP), as a model for polynucleotide sequences, was investigated to shed light on the use of these reagents in hybridization assays. Reactivity turned out to be strongly dictated by the chemical nature of the distal reporter molecule present. At pH 7.0 the sequence of reactivity is cationic ≈ aromatic (stacking) > neutral > anionic; there is approximately an order of magnitude difference between the fastest reacting complex (k=10.2×10−2 M−1 s−1) and the slowest reacting complex (k=0.93×10−2 M−1 s−1) under these conditions. Platination of an oligodeoxynucleotide (30‐mer), dsDNA, or an RNA transcript, shows that a Pt/nucleotide ratio between 1:10 and 1:20 (established by using flameless atomic absorption spectroscopy) results in probes with excellent hybridization characteristics. In terms of applicability and detection limits these platinated nucleic acid probes perform equally well compared to conventionally generated nucleic acid probes, that is, through enzymatic incorporation of covalently labeled nucleotide triphosphates. Applications of these reagents to in situ hybridization assays and gene expression profiling on microarrays illustrate the potential of these monofunctional binding platinum triamine compounds.
Modified DNA Bearing 5(Methoxycarbonylmethyl)‐2′‐deoxyuridine: Preparation by PCR with Thermophilic DNA Polymerase and Postsynthetic Derivatization
Authors: Mehedi Masud Mohammad , Ozaki‐Nakamura Akiko , Kuwahara Masayasu , Ozaki Hiroaki , Sawai Hiroaki
Published: July 2003

A thymidine analogue bearing a methyl ester at the C5 position was accepted as a substrate by the thermophilic family B DNA polymerases, KOD Dash, Pwo, and Vent(exo−), to form the corresponding PCR product, but not by the thermophilic family A DNA polymerases, Taq, Tth, and T7 thermosequenase. Modified DNA containing this analogue was prepared by PCR on a large scale with KOD Dash DNA polymerase and 5(methoxycarbonylmethyl)‐2′‐deoxyuridine 5′‐triphosphate as a substrate. The methyl ester of the modified DNA was further allowed to react with tris(2‐aminoethyl)amine or histamine by an ester–amide exchange reaction to form the corresponding derivatized DNA bearing a tris(2‐aminoethyl)amine or histamine moiety. Hydrolysis of the methyl ester of the modified DNA gave a functionalized DNA bearing an anionic carboxyl group. The derivatized DNA could act as a template for the PCR with KOD Dash DNA polymerase and the natural 2′‐deoxythymidine 5′‐triphosphate or the modified thymidine analogue as a substrate. The postsynthetic derivatization of the modified DNA may expand the variety of structurally modified DNA produced by PCR.
A Homology Model of Penicillin Acylase from Alcaligenes faecalis and In Silico Evaluation of its Selectivity
Authors: Braiuca Paolo , Ebert Cynthia , Fischer Lutz , Gardossi Lucia , Linda Paolo
Published: July 2003

A three‐dimensional model of the relatively unknown penicillin acylase from Alcaligenes faecalis (PA‐AF) was built up by means of homology modeling based on three different crystal structures of penicillin acylase from various sources. An in silico selectivity study was performed to compare this homology model to the structure of the Escherichia coli enzyme (PA‐EC) in order to find any selectivity differences between the two enzymes. The program GRID was applied in combination with the principal component analysis technique to identify the regions of the active sites where the PAs potentially engage different interactions with ligands. These differences were further analyzed and confirmed by molecular docking simulations. The PA‐AF homology model provided the structural basis for the explanation of the different enantioselectivities of the enzymes previously demonstrated experimentally and reported in the literature. Different substrate selectivities were also predicted for PA‐AF compared to PA‐EC. Since no crystallographic data are available for PA‐AF to date, the three‐dimensional homology model represents a useful and efficient tool for fully exploiting this attractive and efficient biocatalyst, particularly in enantioselective acylations of amines.
Real‐time Detection of Nucleotide Incorporation During Complementary DNA Strand Synthesis
Authors: Krieg Alexander , Laib Stephan , Ruckstuhl Thomas , Seeger Stefan
Published: July 2003

Real‐time observation of DNA strand synthesis by using a supercritical angle fluorescence detection apparatus for surface‐selective fluorescence detection is described. DNA template molecules were immobilized on a glass surface and the synthesis of the complementary strand was observed after addition of enzyme, dTTP, dATP, dGTP, and fluorescently labeled dCTP (d, deoxy; TP, triphosphate; T, A, G, and C, nucleobases). The fluorescence increase during the Klenow‐fragment‐catalyzed polymerization depends on the number of labeled dCTP nucleotides incorporated. The efficiency of this reaction is of the same order of magnitude as that of a bimolecular hybridization reaction.
Complex Formation of Divalent Metal Ions with Uridine 5′‐O‐Thiomonophosphate or Methyl Thiophosphate: Comparison of Complex Stabilities with Those of the Parent Phosphate Ligands
Authors: Da Costa Carla P. , Okruszek Andrzej , Sigel Helmut
Published: July 2003

The stability constants of the 1:1 complexes formed in aqueous solution between Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Zn2+, or Cd2+ (M2+) and methyl thiophosphate (MeOPS2−) or uridine 5'‐O‐thiomonophosphate (UMPS2−) (PS2−=MeOPS2− or UMPS2−) have been determined (potentiometric pH titrations; 25 °C; I = 0.1 M, NaNO3). Comparison of these results for M(PS) complexes with those known for the parent M(PO) phosphate species, where PO2−=CH3OPO${_{3}^{2-}}$ or UMP2− (uridine 5′‐monophosphate), shows that the alkaline earth metal ions, as well as Mn2+, Co2+, and Ni2+ have a higher affinity for phosphate groups than for their thio analogues. However, based on the linear log K${_{{\rm M}({\rm R} \char45 {\rm PO}_{3})}^{{\rm M}}}$ versus pK${_{{\rm H}({\rm R} \char45 {\rm PO}_{3})}^{{\rm H}}}$ relationships (R‐PO${_{3}^{2-}}$=simple phosphate monoester or phosphonate ligands with a non‐interacting residue R) it becomes clear that the indicated observation is only the result of the lower basicity of the thiophosphate residue. In contrast, the thio complexes of Zn2+ and Cd2+ are more stable than their parent phosphate ones, and this despite the lower basicity of the PS2− ligands. This stability increase is identical for M(MeOPS) and M(UMPS) species and amounts to about 0.6 and 2.4 log units for Zn(PS) and Cd(PS), respectively. Since no other binding site is available in MeOPS2−, this enhanced stability has to be attributed to the S atom. Indeed, from the mentioned stability differences it follows that Cd2+ in Cd(PS) is coordinated by more than 99% to the thiophosphate S atom; the same value holds for Pb(PS), which was studied earlier. The formation degree of the Sbonded isomer amounts to 76±6 % for Zn(PS) and is close to zero for the corresponding Mg2+, Ca2+, and Mn2+ species. It is further shown that Zn(MeOPS)(aq)2+ releases a proton from a coordinated water molecule with pKa≈6.9; i.e., this deprotonation occurs at a lower pH value than that for the same reaction in Zn(aq)2+. Since Mg2+, Ca2+, Mn2+, and Cd2+ have a relatively low tendency for hydroxo complex formation, it was possible, for these M2+, to also quantify the stability of the binuclear complexes, M2(UMPS−H)+, where one M2+ is thiophosphate‐coordinated and the other is coordinated at (N3)− of the uracil residue. The impact of the results presented herein regarding M2+/nucleic acid interactions, including those of ribozymes (rescue experiments), is briefly discussed.
Acceptor Specificity and Inhibition of the Bacterial Cell‐Wall Glycosyltransferase MurG
Authors: Liu Haitian , Ritter Thomas K. , Sadamoto Reiko , Sears Pamela S. , Wu Min , Wong Chi‐Huey
Published: July 2003

A continuous fluorescence coupled enzyme assay was developed to study the acceptor specificity of the glycosyltransferase MurG toward different lipid I analogues with various substituents replacing the undecaprenyl moiety. It was found that most lipid I analogues are accepted as substrates and, amongst these, the saturated C14 analogue exhibits the best activity. This substrate was used to evaluate the inhibition activity of such antibiotics as moenomycin, vancomycin, and two chlorobiphenyl vancomycin derivatives. A vancomycin derivative with a chlorobiphenyl moiety on the aglycon section was identified as a potent inhibitor of MurG.
Structures and Stabilities of Small DNA Dumbbells with Watson–Crick and Hoogsteen Base Pairs
Authors: Escaja Nuria , Gómez‐Pinto Irene , Rico Manuel , Pedroso Enrique , González Carlos
Published: July 2003

The structures and stabilities of cyclic DNA octamers of different sequences have been studied by NMR and CD spectroscopy and by restrained molecular dynamics. At low oligonucleotide concentrations, some of these molecules form stable monomeric structures consisting of a short stem of two base pairs connected by two mini‐loops of two residues. To our knowledge, these dumbbell‐like structures are the smallest observed to date. The relative stabilities of these cyclic dumbbells have been established by studying their melting transitions. Dumbbells made up purely of GC stems are more stable than those consisting purely of AT base pairs. The order of the base pairs closing the loops also has an important effect on the stabilities of these structures. The NMR data indicate that there are significant differences between the solution structures of dumbbells with G–C base pairs in the stem compared to those with A–T base pairs. In the case of dumbbells with G–C base pairs, the residues in the stem form a short segment of a BDNA helix stabilized by two Watson–Crick base pairs. In contrast, in the case of d〈pCATTCATT〉, the stem is formed by two A–T base pairs with the glycosidic angles of the adenine bases in a syn conformation, most probably forming Hoogsteen base pairs. Although the conformations of the loop residues are not very well defined, the thymine residues at the first position of the loop are observed to fold back into the minor groove of the stem.
Improvement of Hepatocyte‐Specific Gene Expression by a Targeted Colchicine Prodrug
Authors: van Rossenberg Sabine M. W. , Sliedregt‐Bol Karen M. , Koning Gerben , van den Elst Hans , van Berkel Theo J. C. , van Boom Jacques H. , van der Marel Gijs A. , Biessen Erik A. L.
Published: July 2003

Colchicine, an established tubulin inhibitor, interferes with the trafficking of endocytotic vesicles and thereby promotes the escape of lysosome‐entrapped compounds. To improve its potency and cell specificity, a targeted prodrug of colchicine was synthesized by conjugation to a high‐affinity ligand (di‐Nα,Nε‐(5‐(2‐acetamido‐2‐deoxy‐β‐D‐galactopyranosyloxy)pentanomido)lysine, K(GalNAc)2) for the asialoglycoprotein receptor on parenchymal liver cells. The resulting colchicine–K(GalNAc)2 conjugate bound to this receptor with an affinity of 4.5 nM. Confocal microscopy studies confirmed rapid uptake and receptor dependency of a prodrug conjugated with fluorescein isothiocyanate. Colchicine–K(GalNAc)2 substantially increased the transfection efficiency of polyplexed DNA in parenchymal liver cells in a concentration‐ and receptor‐dependent fashion. Colchicine–K(GalNAc)2 was found to enhance the transfection efficiency by 50‐fold at 1 nM, whereas the parental colchicine was ineffective. In conclusion, this nontoxic colchicine–K(GalNAc)2 conjugate can be a useful tool to improve the transfection efficiency of hepatic nonviral gene transfer vehicles.
Chemical Restriction: Strand Cleavage by Ammonia Treatment at 8‐Oxoguanine Yields Biologically Active DNA
Authors: Meyer Christoph , Meyer Dominik , Bickle Thomas A. , Giese Bernd
Published: July 2003

Cleavage of DNA single and double strands at an 8‐oxoguanine‐containing nucleotide occurs in 90 % yield if the modified oligonucleotide is treated with NH3 and O2 at 60 °C. The mechanism of this oxidative cleavage reaction was studied, and the reaction was applied to the generation of single‐stranded overhangs on PCR‐amplified DNA that can be ligated. As an example, the lac Z′ gene was amplified by PCR with 8‐oxoguanine modified primers, restricted by ammonia treatment, ligated into a plasmid vector, transformed in Escherischia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure with restriction enzymes, and it allows the design of any 3′‐overhang independent of the sequence of the cloned DNA.
Synthetic Potential of Molluscan Sulfatases for the Library Synthesis of Regioselectively O‐Sulfonated D‐Galacto‐Sugars
Authors: Uzawa Hirotaka , Nishida Yoshihiro , Sasaki Kenji , Minoura Norihiko , Kobayashi Kazukiyo
Published: July 2003

The substrate specificities of three molluscan sulfatases (E.C. 3.1.6.1; snail, abalone, and limpet origins) were investigated with assorted p‐nitrophenyl (pNP) di‐O‐sulfonated β‐D‐galactopyranosides and β‐lactosides [3,6‐SO3 Gal (1), 3′,6′‐SO3 Lac (2), 4, 6SO3 Gal (3), 2,6‐SO3 Gal (4), 3,4‐SO3 Gal (5), and 3,6‐SO3 GalNAc (6); Ac, acetyl; Gal, galactose; Lac, lactose] together with mono‐O‐sulfonated β‐D‐galactopyranoside [pNP 3SO3‐Gal (7)] and tri‐O‐sulfonated α‐D‐galactopyranoside [2,3,6‐SO3‐α‐Gal (11)]. Some notable differences between the substrate specificity of the three sulfatases were disclosed; snail sulfatase hydrolyzed the 3O‐ and 2O‐sulfo groups of 1 and 4, respectively, to afford 6SO3 Gal (9) in high yields, while the abalone enzyme did not act on 4. Only the limpet enzyme could cleave the 3O‐sulfo groups of 7 to give pNP β‐galactoside. In contrast, every enzyme could utilize 11 as a good substrate to afford a mixture of 6SO3‐α‐Gal (13) and 2,6‐SO3 α‐Gal (12). None of the enzymes could cleave the O‐sulfo groups of 5 and 6, which indicates that a primary 6O‐sulfo group tends to promote the enzymatic hydrolysis of O‐sulfo groups at the secondary positions.
Cover Picture: Chemical Restriction: Strand Cleavage by Ammonia Treatment at 8‐Oxoguanine Yields Biologically Active DNA (ChemBioChem 7/2003)
Authors: Meyer Christoph , Meyer Dominik , Bickle Thomas A. , Giese Bernd
Published: July 2003

The cover picture shows a chemical restriction method in which DNA double strands are cleaved at the position of the artificial base 8‐oxoguanine (Goxo). As as result of the mild cleavage conditions, the DNA fragments remain biologically active and can be used in gene cloning experiments. As an example, the lac Z′ gene was amplified by PCR with 8‐oxoguanine‐modified primers, restricted by treatment with ammonia, ligated into a plasmid vector, transformed in Escherichia coli cells, and screened for blue colonies. This method guarantees efficiencies comparable to the standard cloning procedure, and allows the design of any 3′ overhang, independent of the sequence of the cloned DNA. Further information can be found in the article by Giese et. al. on pp. 610–614
Automatic detection and tracking of moving image target with CNN‐UM via target probability fusion of multiple features
Authors: Kim Hyongsuk , Roska Támas , Chua Leon O. , Werblin Frank
Published: July 2003

A high speed target detection and tracking algorithm for a CNN‐UM chip is presented in this paper. The target confidence value is computed based on the fusion of target existence probabilities of features using products of weighted sums. The target decision is done with such a confidence value and target initiation is done through the temporal accumulation of the confidence. The probability of the target existence for each feature is created in the region of influence depending on the reliability and the strength of the feature. By virtue of the analogic parallel processing structure of the CNN‐UM (Roska T, Chua LO. The CNN universal machine: an analogic array computer. IEEE Trans. Circuits Systems II 1993;CAS‐40: 163–173), real time tracking can be achieved with presently available technologies with the speed of several kilo‐frames per second. Due to the utilization of multiple features of target, robust target detection is possible via the proposed algorithm. On‐chip experiments of the proposed target‐tracking algorithm have been done and properties of the proposed approach are disclosed through the various experiments. Copyright © 2003 John Wiley & Sons, Ltd.
Intercept measure of non‐linear stages
Authors: Larsen Torben
Published: July 2003

Intercept points are often used to describe the non‐linear performance of transceiver stages. This letter introduces an intercept measure. The intercept measure is a performance parameter including both intercept point and gain parameters. Particularly for low gain stages the intercept measure is useful and is better to use than the traditional output intercept point. The intercept measure is very useful as a single non‐linear performance parameter. Copyright © 2003 John Wiley & Sons, Ltd.
Base coupled differential amplifier: a new topology for RF integrated LNA
Authors: Pascoli S. Di , Fanucci L. , Neri B. , Zito D.
Published: July 2003

A new topology of bipolar low noise amplifier (LNA) for RF applications, named base coupled differential (BCD), is presented. The proposed approach is compared by simulation against most classical topologies. The BCD configuration has the key advantage to join an integrated matching on a single‐ended input with a differential output. This is done by using down‐bond wiring, so that no integrated inductors are needed. The main advantages of this new topology are a drastic area reduction and an increased linearity range (or a reduced biasing current with the same linearity) together with a noise figure (NF) and voltage supply reduction. Particularly, the BCD LNA presented in this paper has been designed for 2.44GHz frequency operation. It is characterized by a NF of 1.93dB, a voltage gain (Av) of 19.5dB, an input impedance of 50Ωa third Input‐referred Intercept Point (IIP3) of ‐7.25dBm and a dissipated power (PD) equal to 19mW. Copyright © 2003 John Wiley & Sons, Ltd.
A general approach to continuous‐time Gm‐C filters
Authors: Koziel S. , Szczepanski S. , Schaumann R.
Published: July 2003

A general topology for transconductance‐capacitor (Gm‐C) filters is presented that permits any continuous‐time analogGm‐Cfilter to be analysed via a matrix‐description. Based on these matrices, explicit formulas are derived for the transfer function and the sensitivities valid for anyGm‐Cfilter topology. The approach leads to a useful relationship between the passive capacitor network of the filter and the degree of its transfer function. The matrix‐based approach is formulated especially for efficient use in computer‐aided analysis and design of Gm‐C filters, but ‘hand designs’ ofGm‐CIFLF and LF filters are given as illustrative examples. A new, more general definition ofGm‐Cstate‐space filters is proposed. Connections between the state matrices for voltage‐ and current‐mode state‐spaceGm‐Cfilters are formulated and two canonical transformations are defined that convert state‐space filters into direct state‐space ones, i.e. those having only grounded capacitors. Copyright © 2003 John Wiley & Sons, Ltd.
Output universality in maximum efficiency linear power amplifiers
Authors: Rhodes J. D.
Published: July 2003

A general linear passive termination is considered for a typical field effect power transistor such that the first m odd harmonics, excluding the fundamental frequency, are open circuited with the remaining harmonics short‐circuited. Under this termination, with the appropriate resistive termination at the fundamental frequency, it is shown that an optimum maximum efficiency of

is universally achieved independent of non‐linearities in the transistor.

Furthermore, where higher ordered harmonics are terminated in finite reactive impedances, which is the case with any realizable network, it is shown that the same maximum efficiency is obtained with the correct complex termination at the fundamental frequency.

A prototype network is then defined including the output capacitance of the transistor and synthesized in a lowpass form which, when terminated in a shunt resonant circuit and load resistor, will provide the correct impedances at the fundamental and all of the harmonics. Remarkably, this optimum network has a simple formula for the element values in the general (2m+1) th degree network and a rigorous proof is presented in the appendix. Copyright © 2003 John Wiley & Sons, Ltd.