bluuebunny/crossref_metadata_2025 huggingface dataset:
Olfactory ensheathing glia transplantation: A therapy to promote repair in the mammalian central nervous system
Authors: Santos‐Benito Fernando F. , Ramón‐Cueto Almudena
Published: March 2003

A therapy to treat injuries to the central nervous system (CNS) is, to date, a major clinical challenge. The devastating functional consequences they cause in human patients have encouraged many scientists to search, in animal models, for a repair strategy that could, in the future, be applied to humans. However, although several experimental approaches have obtained some degree of success, very few have been translated into clinical trials. Traumatic and demyelinating lesions of the spinal cord have attracted several groups with the same aim: to find a way to promote axonal regeneration, remyelination, and functional recovery, by using a simple, safe, effective, and viable procedure. During the past decade, olfactory ensheathing glia (OEG) transplantation has emerged as a very promising experimental therapy to promote repair of spinal cords, after different types of injuries. Transplants of these cells promoted axonal regeneration and functional recovery after partial and complete spinal cord lesions. Moreover, olfactory ensheathing glia were able to form myelin sheaths around demyelinated axons. In this article, we review these recent advances and discuss to what extent olfactory ensheathing glia transplantation might have a future as a therapy for different spinal cord affections in humans. Anat Rec (Part B: New Anat) 271B:77–85, 2003. © 2003 Wiley‐Liss, Inc.
Asymptomatic avascular necrosis in patients with primary antiphospholipid syndrome in the absence of corticosteroid use: A prospective study by magnetic resonance imaging
Authors: Tektonidou Maria G. , Malagari Katerina , Vlachoyiannopoulos Panayiotis G. , Kelekis Dimitris A. , Moutsopoulos Haralampos M.
Published: March 2003

Objective

To evaluate the prevalence of avascular necrosis (AVN), using magnetic resonance imaging (MRI), in patients with primary antiphospholipid syndrome (APS) and in patients with systemic lupus erythematosus (SLE), with or without anticardiolipin antibodies (aCL), who are asymptomatic for AVN and have not taken corticosteroids.

Methods

Seventy‐nine subjects who were asymptomatic for AVN were evaluated by MRI of the femoral heads: 30 patients with primary APS who had never received corticosteroids, 19 SLE patients who had never received steroids (divided into 2 groups, aCL positive and aCL negative, in order to examine any association between AVN and aCL), and 30 healthy subjects who were age‐ and sex‐matched with patients with primary APS. Established MRI criteria were used for a diagnosis of AVN.

Results

Asymptomatic AVN was evident in 6 (20%) of 30 patients with primary APS: 3 of them (1 man, 2 women) had intermediate bilateral AVN, and 3 (all women) had early AVN (bilateral in 1 patient). Results of hip and pelvis radiography and dynamic scintigraphy were negative. Followup MRI 6 months later revealed no changes. At the time of the initial MRI examination, the mean (±SD) age of patients in whom AVN was identified was 31.2 ± 7.3 years, and that of patients without AVN was 42.4 ± 11.9 years (P = 0.036). Livedo reticularis occurred significantly more commonly in the group with AVN (P = 0.041). None of the healthy subjects and none of the patients with SLE demonstrated AVN on MRI.

Conclusion

AVN can be detected by MRI in 20% of patients with primary APS. Younger patients tend to develop AVN more frequently than do older patients, and the presence of livedo reticularis may identify individuals at risk for AVN. Clinicians should be aware of this possible clinical manifestation of primary APS, because early diagnosis can lead to early intervention.
Novel genetic markers in the 5′‐flanking region of ANKH are associated with ankylosing spondylitis
Authors: Tsui Florence W. L. , Tsui Hing Wo , Cheng Emily Y. , Stone Millicent , Payne Ursula , Reveille John D. , Shulman Marc J. , Paterson Andrew D. , Inman Robert D.
Published: March 2003

Objective

To use a candidate gene approach for the identification of genetic markers that are significantly linked to and associated with ankylosing spondylitis (AS).

Methods

We searched for novel polymorphisms in the ANKH gene (human homolog of the murine progressive ankylosis gene) and genotyped 2 polymorphic sites, one in the 5′‐noncoding region and the other in the promoter region of ANKH, using DNA from affected (n = 273) and unaffected (n = 112) individuals from 124 AS families. We used these ANKH and other nearby polymorphisms to perform linkage and family‐based association analyses.

Results

We identified 2 novel polymorphic sites: one in the 5′‐noncoding region of ANKH involving 1–2 copies of an 8‐bp repeat (denoted as ANKH‐OR), and the other in the promoter region involving different copy numbers of a triplet repeat (denoted as ANKH‐TR). ANKH‐OR and ANKH‐TR were in complete linkage disequilibrium. Five markers (D5S1953, ANKH‐TR, ANKH‐OR, D5S1954, and D5S1963) were used for both the linkage and association analyses. Multipoint linkage analysis of 124 AS families showed a modest level of significance (nonparametric linkage score 2.15; P = 0.015) at the ANKH region. The contribution of ANKH to AS susceptibility (λs) was 1.9. A family‐based association study on the same AS families revealed that both ANKH‐OR allele 1 and ANKH‐TR allele 7 were significantly associated with disease, assuming an additive model (for ANKH‐OR allele 1, P = 0.03; for ANKH‐TR allele 7, P = 0.04).

Conclusion

Our results indicate that ANKH‐OR and ANKH‐TR are novel genetic markers that are significantly associated with AS.
Down‐regulation of the nonspecific and antigen‐specific T cell cytokine response in ankylosing spondylitis during treatment with infliximab
Authors: Zou Jianxiang , Rudwaleit Martin , Brandt Jan , Thiel Andreas , Braun Jürgen , Sieper Joachim
Published: March 2003

Objective

Treatment of active ankylosing spondylitis (AS) with the monoclonal tumor necrosis factor α (TNFα) antibody infliximab is highly clinically effective. This study was undertaken to investigate the precise mechanism of action of anti‐TNFα treatment in AS.

Methods

Cytokine expression of CD4+ and CD8+ T cells was investigated before and 6 and 12 weeks after the start of treatment in 10 patients treated with infliximab, and before and after 6 weeks of treatment and 6 weeks after placebo was switched to infliximab in 10 patients treated initially with placebo. Peripheral blood mononuclear cells (PBMCs) were stimulated for 6 hours either nonspecifically with phorbol myristate acetate (PMA)/ionomycin or antigen specifically with a pool of 46 overlapping 18‐mer peptides derived from the G1 domain of aggrecan. Cells were stained for T cell surface markers CD4 and CD8 and for the intracellular cytokines interferon‐γ (IFNγ), TNFα, interleukin‐4 (IL‐4), and IL‐10. Positive cells were quantified by flow cytometry. For monocyte‐derived cytokines, PBMCs were stimulated with lipopolysaccharide (LPS) for 18 hours and TNFα and IL‐10 in the supernatant were measured by enzyme‐linked immunosorbent assay.

Results

Compared with baseline, infliximab treatment induced a significant decrease at 12 weeks in the number of CD4+ and CD8+ T cells that were positive for IFNγ and TNFα upon PMA/ionomycin stimulation (P = 0.005). A significant reduction had already begun to occur at 6 weeks. No change in the percent IFNγ or TNFα positivity among CD4+ and CD8+ subpopulations was observed after 6 weeks in patients treated with placebo. However, when these patients began infliximab treatment after 6 weeks of receiving placebo, there was a similar significant decrease in IFNγ and TNFα production by CD4+ and CD8+ T cells (P < 0.05). Furthermore, infliximab treatment induced a significant reduction in the number of IFNγ+ and TNFα+ CD8+ T cells (P = 0.005 at week 6 and week 12) after antigen‐specific in vitro stimulation with G1‐derived peptides. Between‐group analysis showed that the change in the expression of IFNγ and TNFα in both CD4+ and CD8+ T cells was significantly different between the infliximab and placebo groups (P = 0.001 for all variables). There was no change in the number of IL‐10+ or IL‐4+ T cells during treatment. No significant change in the production of TNFα and IL‐10 upon in vitro stimulation of PBMCs with LPS was detectable during infliximab treatment.

Conclusion

Infliximab down‐regulates both IFNγ and TNFα secreted by T cells but does not induce a change in cytokines produced by monocytes during 3 months of treatment. This is likely to be a relevant mechanism for the clinical efficacy of this therapy.
Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by toll‐like receptor signaling
Authors: Kyburz Diego , Rethage Janine , Seibl Reinhart , Lauener Roger , Gay Renate E. , Carson Dennis A. , Gay Steffen
Published: March 2003

Objective

To test the hypothesis that bacterial products acting as adjuvants, such as CpG oligodeoxynucleotides (ODNs) and peptidoglycans (PGs), are able to activate synoviocytes, and to determine the involvement of Toll‐like receptors (TLRs) in this activation process.

Methods

Cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) were stimulated with CpG ODNs or PGs. The expression of various integrins was determined by fluorescence‐activated cell sorting. TLR and matrix metalloproteinase (MMP) messenger RNA (mRNA) was measured by real‐time polymerase chain reaction. Additionally, levels of interleukin‐6 (IL‐6) and IL‐8 in the culture supernatants were assessed by enzyme‐linked immunosorbent assay. Blocking experiments were performed by adding anti–TLR‐2 and anti–TLR‐4 monoclonal antibodies to cultures stimulated with bacterial PGs.

Results

Incubation of synovial fibroblasts with CpG ODNs resulted in neither up‐regulation of the expression of integrins on the cell surface, up‐regulation of MMP mRNA expression, nor IL‐6 and IL‐8 production. However, incubation of RA synovial fibroblasts as well as OA synovial fibroblasts with staphylococcal PGs led to an up‐regulation of CD54 (ICAM‐1) surface expression and to increased expression of MMP‐1, MMP‐3, and MMP‐13 mRNA. Furthermore, production of the proinflammatory cytokines IL‐6 and IL‐8 was increased by treatment with PGs. We demonstrated that cultured synovial fibroblasts express low levels of TLR‐2 and TLR‐9 mRNA. TLR‐2 was up‐regulated after stimulation with PGs, whereas TLR‐9 mRNA remained at baseline levels after stimulation with CpG ODNs. Anti–TLR‐2 monoclonal antibodies significantly inhibited production of IL‐6 and IL‐8 induced by stimulation with PGs.

Conclusion

We demonstrate that bacterial PGs activate synovial fibroblasts, at least partially via TLR‐2, to express integrins, MMPs, and proinflammatory cytokines. Inhibition of TLR signaling pathways might therefore have a beneficial effect on both joint inflammation and joint destruction.
Microenvironment regulation of extracellular signal–regulated kinase activity in chondrocytes: Effects of culture configuration, interleukin‐1, and compressive stress
Authors: Li Kelvin W. , Wang Aaron S. , Sah Robert L.
Published: March 2003

Objective

To compare extracellular signal–regulated kinase (ERK) activity in response to interleukin‐1 (IL‐1) in chondrocytes under various culture configurations designed for the study of cartilage biology and repair, and also in response to dynamic load for chondrocytes in cartilage.

Methods

Isolated bovine articular chondrocytes were maintained in serum‐supplemented medium under 4 culture configurations: high‐density monolayer, attached to a cut surface of cartilage, within tissue‐engineered constructs, or within intact cartilage explants. Samples were subjected to a change of medium with or without IL‐1. Cartilage explants were also subjected to dynamic compression.

Results

In chondrocyte monolayers, both basal and IL‐1–stimulated ERK activities were similarly elevated at 0.5 hours after medium change, diminishing by 74% after 16 hours. In contrast, chondrocytes in other culture configurations exhibited lower basal levels of ERK activity and a moderate activation of ERK in response to IL‐1 that was sustained over the 16‐hour treatment time. The dynamic component of loading of cartilage explants led to a 5‐fold activation of ERK, compared with free‐swelling controls, that was indistinguishable from the effects of IL‐1.

Conclusion

ERK signaling in response to IL‐1 in chondrocyte monolayers exhibited a pattern that was distinct from that in other culture systems, suggesting that the extracellular matrix plays an important regulatory role in modulating the response to extracellular stimuli. Since IL‐1 and dynamic loading have distinct effects on chondrocyte biosynthesis, signaling pathways other than ERK participate in the chondrocyte responses to these stimuli.
Placental immunomodulator ferritin, a novel immunoregulator, suppresses experimental arthritis
Authors: Weinberger Abraham , Halpern Marisa , Zahalka Muayad A. , Quintana Francisco , Traub Leonid , Moroz Chaya
Published: March 2003

Objective

To determine the effect of treatment with C48, the recombinant cytokine‐like domain of the novel human placental immunomodulator ferritin (PLIF) immunoregulator, on zymosan‐induced arthritis (ZIA) in mice and on adjuvant‐induced arthritis (AIA) in rats.

Methods

The in vitro effect of PLIF/C48 was tested in mixed lymphocyte cultures (MLCs) of allogeneic mouse splenocytes. Arthritis was induced by intraarticular injection of zymosan into naive mice and by subcutaneous injection of Mycobacterium tuberculosis into rats. C48 was injected intraperitoneally daily from day 3 to day 9 or from day 7 to day 13 after induction of synovitis by zymosan, and every other day from day 2 to day 14 after induction of AIA. Swelling of the joints and histologic features of the synovium were assessed. Th1 and Th2 cytokines were quantified by enzyme‐linked immunosorbent assay.

Results

Both PLIF and C48 significantly inhibited the in vitro immunoreactivity of mouse splenocytes in MLCs. Treatment of ZIA mice and AIA rats with C48 effectively reduced joint swelling. C48 treatment reduced synovial lining thickening, numbers of mononuclear cells and histiocytes, as well as cartilage destruction and bone erosions. In vitro, activated splenocytes from C48‐treated ZIA and AIA animals produced significantly higher levels of interleukin‐10 (IL‐10). In animals with ZIA, this was accompanied by lower levels of tumor necrosis factor and IL‐2.

Conclusion

Human PLIF and C48 were shown to exert cross‐species immunosuppressive activity in vitro. The in vivo suppression of articular inflammation in the experimental models of ZIA and AIA was the result of treatment with the antiinflammatory human C48. These results suggest that treatment with C48 may offer an effective immunotherapeutic means of controlling inflammatory polyarthritis.
Attenuation of autoimmune disease in fas‐deficient mice by treatment with a cytotoxic benzodiazepine
Authors: Bednarski Jeffrey J. , Warner Roscoe E. , Rao Tharaknath , Leonetti Francesco , Yung Raymond , Richardson Bruce C. , Johnson Kent J. , Ellman Jonathan A. , Opipari Anthony W. , Glick Gary D.
Published: March 2003

Objective

Elimination of autoreactive cells relies on Fas‐dependent activation‐induced cell death mechanisms, an important component of peripheral tolerance. Defects in Fas or its cognate ligand lead to inefficient activation‐induced cell death and are specific causes of lymphoproliferative and autoimmune diseases. The present study was undertaken to investigate a novel 1,4‐benzodiazepine (Bz‐423) that induces apoptosis and limits autoimmune disease in NZB/NZW mice, to determine its activity against lupus‐like disease associated with defective Fas expression. We investigated the Fas‐dependence of its cytotoxic actions, its therapeutic potential in mice deficient in Fas, and its therapeutic mechanism of action.

Methods

Primary lymphocytes isolated from Fas‐deficient MRL/MpJ‐Faslpr (MRL‐lpr) mice were tested for sensitivity to Bz‐423. Bz‐423 was administered to MRL‐lpr mice for short (1‐week) or long (14‐week) periods, and its effects on cell survival were determined along with measures of nephritis, arthritis, antibody titers, and Th subpopulations. BALB/c mice were similarly treated to determine if Bz‐423 alters normal immune functions in vivo.

Results

Administration of Bz‐423 to MRL‐lpr mice significantly reduced autoimmune disease including glomerulonephritis and arthritis. Treatment was associated with decreases in CD4+ T cells and an alteration in the Th1/Th2 balance. At the therapeutic dosage, Bz‐423 did not interfere with normal T and B cell responses in BALB/c mice, suggesting that this agent is not globally immunosuppressive.

Conclusion

Bz‐423 is a novel immunomodulatory agent that is active against disease even in the context of defective Fas signaling. It is a leading compound for further investigation into the development of selective therapies for lupus.
Why do we need algorithmic historiography?
Authors: Garfield Eugene , Pudovkin A. I. , Istomin V. S.
Published: March 2003

This article discusses the rationale for creating historiographs of scholarly topics using a new program called HistCite™, which produces a variety of analyses to aid the historian identify key events (papers), people (authors), and journals in a field. By creating a genealogic profile of the evolution, the program aids the scholar in evaluating the paradigm involved.
Indicator‐assisted evaluation and funding of research: Visualizing the influence of grants on the number and citation counts of research papers
Authors: Boyack Kevin W. , Börner Katy
Published: March 2003

This article reports research on analyzing and visualizing the impact of governmental funding on the amount and citation counts of research publications. For the first time, grant and publication data appear interlinked in one map. We start with an overview of related work and a discussion of available techniques. A concrete example—grant and publication data from Behavioral and Social Science Research, one of four extramural research programs at the National Institute on Aging (NIA)—is analyzed and visualized using the VxInsight® visualization tool. The analysis also illustrates current existing problems related to the quality and existence of data, data analysis, and processing. The article concludes with a list of recommendations on how to improve the quality of grant‐publication maps and a discussion of research challenges for indicator‐assisted evaluation and funding of research.
Human cytochromes mediating gepirone biotransformation at low substrate concentrations
Authors: Greenblatt David J. , von Moltke Lisa L. , Giancarlo Gina M. , Garteiz Daniel A.
Published: March 2003

Biotransformation of gepirone to 1‐(2‐pyrimidinyl)‐piperazine (1‐PP) and 3'‐OH‐gepirone, as well as two other hydroxylated metabolites, was studied in vitro using a human liver microsomal preparation and heterologously expressed human CYP3A4 and CYP2D6. The focus was on a low range of gepirone concentrations (1000 nM and below). Liver microsomes formed 1‐PP and 3'‐OH‐gepirone with similar reaction velocities. Two other hydroxylated metabolites (2‐OH‐ and 5‐OH‐gepirone) were also formed, but pure reference standards were not available for purposes of quantitative analysis. The CYP3A inhibitor ketoconazole completely eliminated 1‐PP formation, reduced 3'‐OH‐gepirone formation to less than 20% of control, and reduced 2‐OH‐gepirone formation to 7% of control. All metabolites were formed by expressed CYP3A4; however, CYP2D6 formed 3'‐OH‐ and 5‐OH‐gepirone, but not 1‐PP or 2‐OH‐gepirone. Based on estimated relative abundances of the two isoforms in human liver, CYP3A4 was predicted to account for more than 95% of net clearance of gepirone in vivo at low concentrations approaching the therapeutic range. CYP2D6 would account for less than 5% of net clearance. The findings are consistent with previous in vitro studies of gepirone using higher substrate concentrations. Copyright © 2003 John Wiley & Sons, Ltd.
Are there teratogenic risks associated with antidotes used in the acute management of poisoned pregnant women?
Authors: Bailey Benoit
Published: March 2003

OBJECTIVE

We reviewed evidence suggesting teratogenic risk associated with the use of antidotes in the acute management of poisoned pregnant women.

METHODS

Medline, Toxline, and DART/ETIC searches; references of retrieved articles, pertinent databases and textbooks were also searched.

RESULTS

There are case reports or case series of women who received antidotes for poisoning during (*) or after (+) the period of organogenesis who showed no fetal adverse effects. Some antidotes, however, have no teratogenic risk: atropine (cohort/surveillance studies)+, calcium (oral supplement: cohort study)+ and pyridoxine (Bendectin studies). Also, ethanol+, methylene blue (intra‐amniotic injection but not oral) and penicillamine* can be considered teratogens but their risks in the treatment of poisonings are unknown. There is no epidemiologic study evaluating the risk of the following antidotes during pregnancy: N‐acetylcysteine*+, BAL (dimercaprol)+, black widow spider antivenin+, calcium EDTA+, crotalidae antivenin, crotalidae polyvalent immune FAB, cyanide antidote kit (amyl and sodium nitrate, sodium thiosulfate), deferoxamine*+, digoxin immune FAB+, DMSA+, flumazenil+, fomepizole, methylene blue (IV), naloxone, physostigmine, pralidoxime+, protamine+; and parenteral pharmacologic doses of calcium+, folinic acid*, glucagon+, hydroxycobalamin, phytonadione (vitamin K), and pyridoxine.

CONCLUSIONS

Despite the limited evidence supporting the risk of antidote use during pregnancy, antidotes should be used when there is a clear maternal indication to decrease the morbidity or mortality associated with poisoning. The only exception may be penicillamine, which is a teratogen. Better antidotes exist for most poisonings that penicillamine could potentially treat. At this time, there is no known fetal indication for all antidotes. Reporting the use of an antidote during pregnancy should be encouraged, especially if used during the critical period of organogenesis. Birth Defects Research (Part A) 67:133–140, 2003. © 2003 Wiley‐Liss, Inc.
Birth prevalence and pattern of osteochondrodysplasias in an inbred high risk population
Authors: Al‐Gazali Lihadh I. , Bakir Mahmood , Hamid Zuhair , Varady Elizabeth , Varghes Mani , Haas Danialla , Bener Abdulbari , Padmanabhan Rengasamy , Abdulrrazzzaq Yeusef M. , Dawodu Aden Kula
Published: March 2003

BACKGROUND

Define the pattern and birth prevalence of the different types of osteochondrodysplasias in newborn infants in the United Arab Emirates (UAE) population, which is highly inbred and where termination of pregnancy is not accepted.

METHODS

All infants with a birth weight of 500 gm and above in the three hospitals in Al Ain Medical District of the UAE were studied prospectively over a period of 5 years. For each live birth or stillbirth with suspected skeletal dysplasia, a detailed clinical and radiological examination was carried out. Pregnancy history and information regarding parental age, ethnic origin, family history, and level of consanguinity were obtained and a pedigree was constructed.

RESULTS

Among the 38,048 births during the study period, 36 (9.46/10,000 births) had some type of skeletal dysplasia. Eighteen cases were attributed to autosomal recessive genes (4.7/10,000 births), 10 were due to apparent new dominant mutations (2.62/10,000), five were autosomal dominant type (1.3/10,000) and one was X‐linked dominant type (0.26/10,000). In three cases, inheritance was unknown. The most common recessive type of skeletal dysplasia in our series was fibrochondrogenesis (1.05/10,000), followed by chondrodysplasia punctata (0.78/10,000). The birth prevalence rate of skeletal dysplasia doubled in the last 2 years of the 5‐year observation period (6.74/10,000 in 1996 vs. 12.86/10,000 in 1999, and 13.45/10,000 in 2000). This increase involved cases caused by new dominant mutations, and occurred mainly in the first half of 1999.

CONCLUSION

This prospective study has identified a high birth prevalence of skeletal dysplasia, and risk factors are postulated. These findings represent an accurate birth prevalence figure and a useful baseline for this group of birth defects in the UAE. Birth Defects Research (Part A) 67:125–132, 2003. © 2003 Wiley‐Liss, Inc.
Bendectin and birth defects II: Ecological analyses
Authors: Kutcher Jeffrey S. , Engle Arnold , Firth Jacqueline , Lamm Steven H.
Published: March 2003

BACKGROUND

Bendectin was the primary pharmaceutical treatment of nausea and vomiting of pregnancy (NVP) in the United States until the early 1980s. Its manufacture was then discontinued after public allegations that it was causing birth defects. Subsequently, meta‐analyses of the many epidemiological cohort and case/control studies used to examine that hypothesis have demonstrated the absence of a detectable teratogenic effect. This study presents an ecological analysis of the same hypothesis that examines specific malformations.

METHODS

Annual birth defect prevalence data for the 1970s to the 1990s have been obtained for specific birth defects from the Center for Disease Control's nationwide Birth Defect Monitoring Program. These data for the US have been compared graphically to the annual US Bendectin sales for the treatment of NVP. Data have also been obtained for annual US rates for hospitalization for NVP. The three data sets have been temporally compared in graphic analysis.

RESULTS

The temporal trends in prevalence rates for specific birth defects examined from 1970 through 1992 did not show changes that reflected the cessation of Bendectin use over the 1980–84 period. Further, the NVP hospitalization rate doubled when Bendectin use ceased.

CONCLUSIONS

The population results of the ecological analyses complement the person‐specific results of the epidemiological analyses in finding no evidence of a teratogenic effect from the use of Bendectin. Birth Defects Research (Part A) 67:88–97, 2003. © 2003 Wiley‐Liss, Inc.
Estimation of Treatment Difference and Standard Deviation with Blinded Data in Clinical Trials
Authors: Chen Michael , Kianifard Farid
Published: March 2003

In an ongoing clinical trial and while the randomized treatment codes remain blinded, it is often desirable to estimate the treatment difference and standard deviation for a normally‐distributed response variable. This is particularly useful for estimating the sample size for future trials or for adjusting the sample size for the ongoing trial. We describe the limitations of an available EM algorithm‐based procedure to reestimate the standard deviation without unblinding the codes for the two treatments. We introduce a new procedure and propose a clinical trial design for estimating both the treatment difference and standard deviation without unblinding. The performance of the proposed procedure is evaluated in a simulation study.
Estimation and Confidence Intervals after Adjusting the Maximum Information
Authors: Lawrence John , Hung H.M. James
Published: March 2003

In a comparative clinical trial, if the maximum information is adjusted on the basis of unblinded data, the usual test statistic should be avoided due to possible type I error inflation. An adaptive test can be used as an alternative. The usual point estimate of the treatment effect and the usual confidence interval should also be avoided. In this article, we construct a point estimate and a confidence interval that are motivated by an adaptive test statistic. The estimator is consistent for the treatment effect and the confidence interval asymptotically has correct coverage probability.
Cumulative Damage Survival Models for the Randomized Complete Block Design
Authors: Gregory Gavin G.
Published: March 2003

A continuous time discrete state cumulative damage process {X(t), t ≥ 0} is considered, based on a non‐homogeneous Poisson hit‐count process and discrete distribution of damage per hit, which can be negative binomial, Neyman type A, Polya‐Aeppli or Lagrangian Poisson. Intensity functions considered for the Poisson process comprise a flexible three‐parameter family. The survival function is S(t) = P(X(t) ≤ L) where L is fixed. Individual variation is accounted for within the construction for the initial damage distribution {P(X(0) = x) | x = 0, 1, …,}. This distribution has an essential cut‐off before x = L and the distribution of L – X(0) may be considered a tolerance distribution. A multivariate extension appropriate for the randomized complete block design is developed by constructing dependence in the initial damage distributions. Our multivariate model is applied (via maximum likelihood) to litter‐matched tumorigenesis data for rats. The litter effect accounts for 5.9 percent of the variance of the individual effect. Cumulative damage hazard functions are compared to nonparametric hazard functions and to hazard functions obtained from the PVF‐Weibull frailty model. The cumulative damage model has greater dimensionality for interpretation compared to other models, owing principally to the intensity function part of the model.
The Effect of Design Imbalance on the Power of the F‐test of a Variance Component in the One‐Way Random Model
Authors: Lee Juneyoung
Published: March 2003

The ANOVA‐based F‐test used for testing the significance of the random effect variance component is a valid test for an unbalanced one‐way random model. However, it does not have an uniform optimum property. For example, this test is not uniformly most powerful invariant (UMPI). In fact, there is no UMPI test in the unbalanced case (see Khuri, Mathew, and Sinha, 1998).

The power of the F‐test depends not only on the design used, but also on the true values of the variance components. As Khuri (1996) noted, we can gain a better insight into the effect of data imbalance on the power of the F‐test using a method for modelling the power in terms of the design parameters and the variance components. In this study, generalized linear modelling (GLM) techniques are used for this purpose. It is shown that GLM, in combination with a method of generating designs with a specified degree of imbalance, is an effective way of studying the behavior of the power of the F‐test in a one‐way random model.
Determination of laccase activity in mixed solvents: Comparison between two chromogens in a spectrophotometric assay
Authors: Cantarella Gaetano , d'Acunzo Francesca , Galli Carlo
Published: March 2003

Spectrophotometric determination of laccase activity with ABTS acting as chromogen yields exceedingly low values whenever conducted in a water–organic mixed solvent. Nevertheless, there is firm evidence that laccase is able to oxidize substrates such as phenols and amines quantitatively in these mixed solvents. We show that the apparently small rate of ABTS oxidation by laccase in a mixed solvent, such as buffered water–dioxane 1:1, is not amenable to the denaturation of laccase but rather to the decreased stability of ABTS·+. We propose HAA as a more reliable chromogen for the determination of laccase activity in mixed solvents. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 395–398, 2003.
Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris
Authors: Trinh L. B. , Phue J. N. , Shiloach Joseph
Published: March 2003

Pichia pastoris, a methylotrophic yeast, is an efficient producer of recombinant proteins in which the heterologous gene is under the control of the methanol‐induced AOX1 promoter. Hence, the accepted production procedure has two phases: In the first phase, the yeast utilizes glycerol and biomass is accumulated; in the second phase, the yeast utilizes methanol which is used both as an inducer for the expression of the recombinant protein and as a carbon source. Since the yeast is sensitive to methanol concentration, the methanol is supplied gradually to the growing culture. Three methanol addition strategies were evaluated for the purpose of optimizing recombinant endostatin production. Two strategies were based on the yeast metabolism; one responding to the methanol consumption using a methanol sensor, and the other responding to the oxygen consumption. In these two strategies, the methanol supply is unlimited. The third strategy was based on a predetermined exponential feeding rate, controling the growth rate at 0.02 h−1, in this strategy the methanol supply is limited. Throughout the induction phase glycerol, in addition to methanol, was continuously added at a rate of 1 g L h−1. Total endostatin production was similar in all three strategies, (400 mg was obtained from 3 L initial volume), but the amount of methanol added and the biomass produced were lower in the predetermined rate method. This caused the specific production of endostatin per biomass and per methanol to be 2 times higher in the predetermined rate than in the other two methods, making the growth control strategy not only more efficient but also more convenient for downstream processing. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 438–444, 2003.
High‐density seeding of myocyte cells for cardiac tissue engineering
Authors: Radisic Milica , Euloth Michelle , Yang Liming , Langer Robert , Freed Lisa E. , Vunjak‐Novakovic Gordana
Published: March 2003

Tissue engineering of 1‐ to 5‐mm‐thick, functional constructs based on cells that cannot tolerate hypoxia for prolonged time periods (e.g., cardiac myocytes) critically depends on our ability to seed the cells at a high and spatially uniform initial density and to maintain their viability and function. We hypothesized that rapid gel–cell inoculation in conjunction with direct medium perfusion through the seeded scaffold would increase the rate, yield, viability, and uniformity of cell seeding. Two cell types were studied: neonatal rat cardiomyocytes for feasibility studies of seeding and cultivation with direct medium perfusion, and C2C12 cells (a murine myoblast cell line) for detailed seeding studies. Cells were seeded at densities corresponding to those normally present in the adult rat heart ([0.5–1] × 108 cells/cm3), into collagen sponges (13 mm × 3 mm discs), using Matrigel® as a vehicle for rapid cell delivery. Scaffolds inoculated with cell–gel suspension were seeded either in perfused cartridges with alternating medium flow or in orbitally mixed Petri dishes. The effects of seeding time (1.5 or 4.5 h), initial cell number (6 or 12 million cells per scaffold), and seeding set‐up (medium perfusion at 0.5 and 1.5 mL/min; orbitally mixed dishes) were investigated using a randomized three‐factor factorial experimental design with two or three levels and three replicates. The seeding cell yield was consistently high (over 80%), and it appeared to be determined by the rapid gel inoculation. The decrease in cell viability was markedly lower for perfused cartridges than for orbitally mixed dishes (e.g., 8.8 ± 0.8% and 56.3 ± 4%, respectively, for 12 million cells at 4.5 h post‐seeding). Spatially uniform cell distributions were observed in perfused constructs, whereas cells were mainly located within a thin (100–200 μm) surface layer in dish seeded constructs. Over 7 days of cultivation, medium perfusion maintained the viability and differentiated function of cardiac myocytes, and the constructs contracted synchronously in response to electrical stimulation. Direct perfusion can thus enable seeding of hypoxia‐sensitive cells at physiologically high and spatially uniform initial densities and maintain cell viability and function. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 403–414, 2003.
Accelerated process development for protease production in continuous multi‐stage cultures
Authors: Raninger A. , Steiner W.
Published: March 2003

A fermentation process was developed and optimized for the production of a specific protease from Bacillus licheniformis PWD‐1. Media formulations were constructed and crucial environmental parameters were optimized to enhance growth and product formation. Process dynamics of substrate consumption, biomass‐, product‐, as well as by‐product formation were determined under controlled conditions in a bioreactor. Using kinetic data from batch‐ and continuous‐culture experiments, a fed‐batch process was developed producing proteolytic activities 10 times those found during regular batch culture. In one stage continuous stirred tank culture protease formation was completely decoupled from sporulation. Shift experiments in one‐stage continuous cultures led to the development of a two‐stage continuous stirred tank fermentation process using optimized conditions for growth in the first stage and protease formation in the second stage. Accordingly, the basis for a continuous production of the enzyme on a pilot scale was accomplished. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 517–524, 2003.
Expression and characterization of a humanized cocaine‐binding antibody
Authors: Redwan El‐Rashdy M. , Larsen Nicholas A. , Zhou Bin , Wirsching Peter , Janda Kim D. , Wilson Ian A.
Published: March 2003

The murine immunoglobulin G (IgG) cocaine‐binding monoclonal antibody (mAb), GNC92H2, is notable for its exquisite specificity for cocaine, as opposed to chemically‐related cocaine metabolites, and for its moderately high affinity (Kd ≈ 200 nM) for cocaine. Recently, we described the crystal structure of a mouse/human chimeric Fab construct at 2.3 Å resolution. Herein, we report the successful framework humanization of a single‐chain Fv (scFv) GNC92H2 construct without loss of affinity for cocaine. In brief, we compared the mAb GNC92H2 sequence to human antibody sequences, and used structure‐based design to incorporate mutations (total = 49) that would humanize the framework region without affecting the overall shape of the binding pocket or the key cocaine‐contact residues. The codons of the rationally designed sequence were optimized for E. coli expression, and the gene was synthesized by a de novo PCR reaction using 14 overlapping primers. Expression of the scFv construct was significantly improved in E. coli by fusion to thioredoxin. Intriguingly, this construct apparently refolds to form soluble active antibody in the reducing environment of the cytoplasm. Competitive ELISA and equilibrium dialysis demonstrated comparable binding activity between the humanized scFv and the whole IgG. The successful humanization of mAb GNC92H2 should enhance its potential therapeutic value by reducing its overall. immunogenicity. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 612–618, 2003.
Measurement of trehalose loading of mammalian cells porated with a metal‐actuated switchable pore
Authors: Acker Jason P. , Lu Xiao‐ming , Young Vernon , Cheley Stephen , Bayley Hagan , Fowler Alex , Toner Mehmet
Published: March 2003

Efforts to improve the tolerance of mammalian cells to desiccation have focused on the role that sugars have in protecting cells from lethal injury. Among the key determinants of desiccation tolerance is the intracellular trehalose concentration, and thus quantifying the amount and rate of trehalose accumulation has now become very critical to the success of these desiccation approaches. We introduced trehalose into 3T3 fibroblasts, human keratinocytes, and rat hepatocytes using a genetically engineered mutant of the pore‐forming α‐hemolysin from Staphylococcus aureus. Manipulating the extracellular Zn2+ concentration selectively opens and closes this pore (∼2 nm) and enables controlled loading of cells with sugars. We quantified intracellular trehalose using gas chromatography–mass spectroscopy (GC‐MS) to examine the trimethylsilyl derivative of intracellular trehalose. Using the GC‐MS method, we demonstrate that the switchable characteristics of H5 α‐hemolysin permit controlled loading of the high concentrations of trehalose (up to 0.5 M) necessary for engineering desiccation tolerance in mammalian cells. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 525–532, 2003.
Oil/water and pre‐emulsified oil/water (PIT) dispersions in a stirred vessel: Implications for fermentations
Authors: Erler S. T. , Nienow A. W. , Pacek A. W.
Published: March 2003

This study examines dispersions of rapeseed oil (RSO) in water by mechanical agitation under conditions mimicking those found in certain antibiotic fermentations; for example, in the presence of air, antifoam, and finely divided CaCO3 particles. A problem with residual oil has been reported for such fermentations, and it has been suggested that the use of pre‐emulsified oil can reduce this problem. Hence, the dispersion of a pre‐emulsified oil produced by the “phase inversion temperature (PIT) method” has been evaluated. In both cases, the volume fraction of oil was 2%. For the RSO systems, a relatively high agitation speed was required to disperse the oil, especially in the presence of the particles and, when the agitation was stopped, separation occurred rapidly. The Sauter mean drop diameters depended on the system, being at an average energy dissipation rate of approximately 0.9 W kg−1, 180 μm for RSO/water, 130 μm for RSO/water(antifoam)/air, 580 μm for RSO/water/CaCO3, and 850 μm for RSO/water(antifoam)/air/CaCO3. For the same four systems, the PIT emulsion, once dispersed, was very stable and the drop size was essentially independent of the operating conditions, with a Sauter mean diameter of ∼0.3 μm. The implications of these findings for fermentations in which oil is used as a carbon source are assessed. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 543–551, 2003.
Photocrosslinked hyaluronic acid hydrogels: Natural, biodegradable tissue engineering scaffolds
Authors: Baier Leach Jennie , Bivens Kathryn A. , Patrick Jr. Charles W. , Schmidt Christine E.
Published: March 2003

Ideally, rationally designed tissue engineering scaffolds promote natural wound healing and regeneration. Therefore, we sought to synthesize a biomimetic hydrogel specifically designed to promote tissue repair and chose hyaluronic acid (HA; also called hyaluronan) as our initial material. Hyaluronic acid is a naturally occurring polymer associated with various cellular processes involved in wound healing, such as angiogenesis. Hyaluronic acid also presents unique advantages: it is easy to produce and modify, hydrophilic and nonadhesive, and naturally biodegradable. We prepared a range of glycidyl methacrylate‐HA (GMHA) conjugates, which were subsequently photopolymerized to form crosslinked GMHA hydrogels. A range of hydrogel degradation rates was achieved as well as a corresponding, modest range of material properties (e.g., swelling, mesh size). Increased amounts of conjugated methacrylate groups corresponded with increased crosslink densities and decreased degradation rates and yet had an insignificant effect on human aortic endothelial cell cytocompatibility and proliferation. Rat subcutaneous implants of the GMHA hydrogels showed good biocompatibility, little inflammatory response, and similar levels of vascularization at the implant edge compared with those of fibrin positive controls. Therefore, these novel GMHA hydrogels are suitable for modification with adhesive peptide sequences (e.g., RGD) and use in a variety of wound‐healing applications. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 578–589, 2003.
Controling the rate of protein release from polyelectrolyte complexes
Authors: Kamiya Noriho , Klibanov Alexander M.
Published: March 2003

Extended protein release from readily prepared, water‐insoluble complexes with oppositely charged polyions is explored. Using hen egg‐white lysozyme as a model, its sustained release from such complexes with a number of polyanions under physiological conditions has been demonstrated and rationalized. The rate of release varies orders of magnitude and is controlled by the nature of the polyanion (decreasing upon increase in its linear charge density, length, and hydrophobicity) and the complex particle size (the larger the particles, the slower the release). © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 82: 590–594, 2003.
Early indicators of child abuse and neglect: a multi‐professional Delphi study
Authors: Powell Catherine
Published: March 2003

Through the application of the Delphi technique, this study draws on the expertise of British child protection academics and practitioners from a wide range of disciplines in seeking to develop a consensus opinion on possible early indicators of child abuse and neglect. The search for early indicators is described in the context of a secondary preventative approach to the problem of child maltreatment. A very tentative conclusion arising from the study is that the early indicators of child abuse and neglect that achieved consensus of agreement may help in diagnosing child abuse and neglect at an earlier stage, although they are not necessarily diagnostic. Alternative explanations, differential diagnoses and information‐gathering are paramount, as is a willingness and ability to act on concerns. Although great caution is urged, it is suggested that the findings from the study are credible and of interest to those who are working towards more timely recognition and referral of abused and neglected children. Copyright © 2003 John Wiley & Sons, Ltd.
Difficulties with age estimation of internet images of south‐east Asian girls
Authors: Stathopulu Eleni , Antony Hulse J. , Canning David
Published: March 2003

Community paediatricians are increasingly asked to provide expert opinion on internet child pornography related to ‘sex tourism’, mostly in south‐east Asian countries. The girls involved are often prepubertal, and the main legal question is to establish whether they are under 13 years of age. This paper provides a review of the literature related to this question. Although there are limited data of age of menarche from the countries at risk of ‘sex tourism’, one recent study from the Philippines showed that the mean age of menarche was similar to the mean age of British girls born between 1982 and 1986. There are no data in the above countries on the early pubertal changes in breast development, but studies from India and China suggested that these might be more prolonged than for British girls. In particular, the interval between B2 stage (early pubertal breast development) and menarche was nearly 3 years in these developing countries, compared with 2 years for the British girls. Maternal malnutrition and girls small for gestational age at birth resulted in an earlier menarche, while chronic malnutrition, severe enough to cause stunting in preschool years, caused a delay in menarche. Taking into account information from the existing studies, we estimated that 97.5% of south‐east Asian girls will show pubertal changes of B2 stage by the age of 13.7 years. Copyright © 2003 John Wiley & Sons, Ltd.
Cover Picture: NMR Structure of the Single QALGGH Zinc Finger Domain from the Arabidopsis thaliana SUPERMAN Protein (ChemBioChem 2‐3/2003)
Authors: Isernia Carla , Bucci Enrico , Leone Marilisa , Zaccaro Laura , Di Lello Paola , Digilio Giuseppe , Esposito Sabrina , Saviano Michele , Di Blasio Benedetto , Pedone Carlo , Pedone Paolo V. , Fattorusso Roberto
Published: March 2003

The cover picture shows the NMR structure of the SUPERMAN zinc finger domain, which is the first high‐resolution structure of a classical zinc finger domain from a plant protein. The structure consists of a very well‐defined ββα motif, typical of all the other Cys2–His2 zinc fingers so far structurally characterized. As a consequence, the QALGGH sequence, which is highly conserved in plant protein classical zinc finger domains, is located at the N terminus of the α helix. Interestingly, this domain region, in animal protein zinc fingers, is constituted of hypervariable residues deputed to the recognition of the DNA bases. Therefore, a peculiar DNA recognition code for the QALGGH zinc finger domain is proposed in the article by Fattorusso and co‐workers on p. 171 ff.