bluuebunny/crossref_metadata_2025 huggingface dataset:
Donor CD8+ T cells facilitate induction of chimerism and tolerance without GVHD in autoimmune NOD mice conditioned with anti-CD3 mAb
Authors: Liang Yaming , Huang Tammy , Zhang Chunyan , Todorov Ivan , Atkinson Mark , Kandeel Fouad , Forman Stephen , Zeng Defu
Published: September 2004

Prevention of autoimmune diabetes and induction of islet transplantation tolerance in nonobese diabetic (NOD) mice can be reached by induction of mixed chimerism via bone marrow transplantation (BMT), but this procedure requires total body irradiation (TBI) conditioning of the recipients. The toxicity of radiation and potential for graft-versus-host disease (GVHD) prevents its clinical application. Donor CD8+ T cells play a critical role in facilitation of engraftment but also contribute to induction of GVHD in TBI-conditioned recipients. Here, we showed that high doses of donor CD8+ T cells in combination with bone marrow (BM) cells induced mixed chimerism without GVHD in NOD recipients conditioned with anti-CD3 monoclonal antibody (mAb). The prevention of GVHD in those recipients was associated with low-level production of inflammatory cytokines (ie, tumor necrosis factor α [TNF-α]), high-level production of anti-inflammatory cytokines (ie, interleukin 4 [IL-4] and IL-10), and confining of the donor CD8+ T-cell expansion to lymphohematopoietic tissues. The chimeric NOD recipients showed donor-specific tolerance and reversal of insulitis. These results demonstrate that donor CD8+ T-cell–mediated facilitation of engraftment can be separated from GVHD in nonirradiated recipients. This regimen may have potential application in the treatment of autoimmune disorders as well as induction of transplantation tolerance.
High-resolution tracking of cell division demonstrates differential effects of TH1 and TH2 cytokines on SCF-dependent human mast cell production in vitro:correlation with apoptosis and Kit expression
Authors: Kulka Marianna , Metcalfe Dean D.
Published: September 2004

T-helper 1 (TH1) (interferon-γ [IFN-γ]) and TH2 (interleukin-4 [IL-4] and IL-5) cytokines have been variably reported to alter human mast cell numbers in complex culture systems. The effects of these cytokines on the kinetics of cell division and cell death are unknown, and their effect on mast cell behavior is relevant to anticipate the consequences of in vivo strategies that alter cytokine levels. To determine the effect of these cytokines on stem cell factor (SCF)–dependent human mast cell production, we used highresolution tracking of cell division and correlated the results with cell apoptosis, expression of Kit, and mast cell degranulation. When IFN-γ, IL-5, or IL-4 was administered over 8 weeks, we found each cytokine decreased the mast number through a different mechanism. IFN-γ inhibited early progenitor cell division, IL-4 down-regulated early Kit expression, and IL-5 blocked later cell division. Further, IL-4 and IFN-γ had the greatest suppressive effect on degranulation and FcϵRI expression. When these cytokines were administered to mature mast cells, IFN-γ and IL-5 had no effect on degranulation and cell division, but IL-4 induced division and potentiated FcϵRI-mediated degranulation. Thus, exposure of human mast cells to IL-4, IL-5, and IFN-γ during growth and differentiation generally down-regulated mast cell number and function, whereas IL-4 increased mature mast cell division and degranulation.
Proinflammatory role of inducible nitric oxide synthase in acute hyperoxic lung injury
Authors: Hesse Anne-Karin , Dörger Martina , Kupatt Christian , Krombach Fritz
Published: September 2004

Background

Hyperoxic exposures are often found in clinical settings of respiratory insufficient patients, although oxygen therapy (>50% O2) can result in the development of acute hyperoxic lung injury within a few days. Upon hyperoxic exposure, the inducible nitric oxide synthase (iNOS) is activated by a variety of proinflammatory cytokines both in vitro and in vivo. In the present study, we used a murine hyperoxic model to evaluate the effects of iNOS deficiency on the inflammatory response.

Methods

Wild-type and iNOS-deficient mice were exposed to normoxia, 60% O2 or >95% O2 for 72 h.

Results

Exposure to >95% O2 resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. No significant effects of iNOS deficiency on cell differential in bronchoalveolar lavage fluid were observed. However, hyperoxia induced a significant increase in total cell count, protein concentration, LDH activity, lipid peroxidation, and TNF-α concentration in the bronchoalveolar lavage fluid compared to iNOS knockout mice. Moreover, binding activity of NF-κB and AP-1 appeared to be higher in wild-type than in iNOS-deficient mice.

Conclusion

Taken together, our results provide evidence to suggest that iNOS plays a proinflammatory role in acute hyperoxic lung injury.
Tissue detection of natural killer cells in colorectal adenocarcinoma
Authors: Papanikolaou Ioannis S , Lazaris Andreas Ch , Apostolopoulos Periklis , Kavantzas Nikos , Papas Maria G , Mavrogiannis Christos , Patsouris Efstratios S , Archimandritis Athanasios
Published: September 2004

Background

Natural killer (NK) cells represent a first line of defence against a developing cancer; however, their exact role in colorectal cancer remains undetermined. The aim of the present study was to evaluate the expression of CD16 and CD57 [immunohistochemical markers of natural NK cells] in colorectal adenocarcinoma.

Methods

Presence of NK cells was investigated in 82 colorectal adenocarcinomas. Immunohistochemical analysis was performed, using 2 monoclonal antibodies (anti-Fc Gamma Receptor II, CD16 and an equivalent to Leu-7, specific for CD-57). The number of immunopositive cells (%) was evaluated by image analysis. The cases were characterized according to: patient gender and age, tumor location, size, grade, bowel wall invasion, lymph node metastases and Dukes' stage.

Results

NK cells were detected in 79/82 cases at the primary tumor site, 27/33 metastatic lymph nodes and 3/4 hepatic metastases; they were detected in levels similar to those reported in the literature, but their presence was not correlated to the clinical or pathological characteristics of the series, except for a negative association with the patients' age (p = 0.031).

Conclusions

Our data do not support an association of NK cell tissue presence with clinical or pathological variables of colorectal adenocarcinoma, except for a negative association with the patients' age; this might possibly be attributed to decreased adhesion molecule expression in older ages.
Frequency of myasthenic crisis in relation to thymectomy in generalized myasthenia gravis: A 17-year experience
Authors: Soleimani Ali , Moayyeri Alireza , Akhondzadeh Shahin , Sadatsafavi Mohsen , Shalmani Hamidreza Tavakoli , Soltanzadeh Akbar
Published: September 2004

Background

Myasthenic crisis is the most serious life-threatening event in generalized myasthenia gravis (MG) patients. The objective of this study was to assess the long-term impact of thymectomy on rate and severity of these attacks in Iranian patients.

Methods

We reviewed the clinical records from 272 myasthenic patients diagnosed and treated in our neurology clinic during 1985 to 2002. Fifty-three patients were excluded because of unconfirmed diagnosis, ocular form of MG, contraindication to surgery, concomitant diseases and loss to follow-up. The Osserman classification was used to assess the initial severity of the disease. Frequency and severity of the attacks were compared between two groups with appropriate statistical tests according to the nature of variables. Multivariate logistic regression analysis was used to assess the predictors of myasthenic crisis in the group of patients without thymoma.

Results

110 patients were in thymectomy group and the other 109 patients were on medical therapy. These two groups had no significant differences with respect to age at onset, gender, Osserman score in baseline and follow up period. 62 patients (28.3% of all 219 patients) had reported 89 attacks of myasthenic crisis. 20 patients of 62 (32%) were in thymectomy group and 42 (68%) were in the other group. There was significant difference between the two groups in number of patients with crisis (P = 0.001; odds ratio = 2.8 with 95% CI of 1.5 to 5.2). In addition, these attacks were more severe in group of non-thymectomized patients as the duration of ICU admission was longer and they needed more ventilatory support during their attacks. Regression model showed thymectomy and lower age at onset as two predictors of decrement in myasthenic crisis rate in non-thymomatous MG patients.

Conclusions

It is suggested that frequency and severity of myasthenic attacks as important endpoints in evaluation of MG patients. Thymectomy seems to have a preventive role on rate and severity of these attacks.
B-Raf specific antibody responses in melanoma patients
Authors: Fensterle Joachim , Becker Jürgen C , Potapenko Tamara , Heimbach Veronika , Vetter Claudia S , Bröcker Eva B , Rapp Ulf R
Published: September 2004

Background

Mutations of the BRAF gene are the most common genetic alteration in melanoma. Moreover, BRAF mutations are already present in benign nevi. Being overexpressed and mutated, B-Raf is a potential target for the immune system and as this mutation seems to be an early event, a humoral immune response against this antigen might serve as a diagnostic tool for detection of high risk patients.

Methods

372 sera of 148 stage IV melanoma patients and 119 sera of non-melanoma patients were screened for B-Raf, B-Raf V599E and C-Raf specific antibodies by an ELISA assay. Sera were screened for specific total Ig and for IgG. Serum titers were compared with a two tailed Mann-Whitney U test. Sera with titers of 1:300 or higher were termed positive and groups were compared with a two tailed Fisher's exact test.

Results

B-Raf specific antibodies recognizing both B-Raf and B-Raf V599E were detected in 8.9% of the sera of melanoma patients and in 2,5% of the control group. Raf specific IgG was detected in some patients at very low levels. B-Raf specific antibody responses did not correlate with clinical parameters but in some cases, B-Raf antibodies emerged during disease progression.

Conclusion

These findings imply that B-Raf is immunogenic in melanoma patients and that it might serve as a potential target for immunotherapy. However, B-Raf specific antibodies emerge at rather late stages of melanoma progression and are present only with a low frequency indicating that spontaneous B-Raf specific antibodies are not an early marker for melanoma, but rather may serve as a therapeutic target.
Scoliosis treatment using a combination of manipulative and rehabilitative therapy: a retrospective case series
Authors: Morningstar Mark W , Woggon Dennis , Lawrence Gary
Published: September 2004

Background

The combination of spinal manipulation and various physiotherapeutic procedures used to correct the curvatures associated with scoliosis have been largely unsuccessful. Typically, the goals of these procedures are often to relax, strengthen, or stretch musculotendinous and/or ligamentous structures. In this study, we investigate the possible benefits of combining spinal manipulation, positional traction, and neuromuscular reeducation in the treatment of idiopathic scoliosis.

Methods

A total of 22 patient files were selected to participate in the protocol. Of these, 19 met the study criterion required for analysis of treatment benefits. Anteroposterior radiographs were taken of each subject prior to treatment intervention and 4–6 weeks following the intervention. A Cobb angle was drawn and analyzed on each radiograph, so pre and post comparisons could be made.

Results

After 4–6 weeks of treatment, the treatment group averaged a 17° reduction in their Cobb angle measurements. None of the patients' Cobb angles increased. A total of 3 subjects were dismissed from the study for noncompliance relating to home care instructions, leaving 19 subjects to be evaluated post-intervention.

Conclusions

The combined use of spinal manipulation and postural therapy appeared to significantly reduce the severity of the Cobb angle in all 19 subjects. These results warrant further testing of this protocol.
Radiation enteropathy and leucocyte-endothelial cell reactions in a refined small bowel model
Authors: Johnson Louis Banka , Riaz Amjid Ali , Adawi Diya , Wittgren Lena , Bäck Sven , Thornberg Charlotte , Osman Nadia , Gadaleanu Virgil , Thorlacius Henrik , Jeppsson Bengt
Published: September 2004

Background

Leucocyte recruitment and inflammation are key features of high dose radiation-induced tissue injury. The inflammatory response in the gut may be more pronounced following radiotherapy due to its high bacterial load in comparison to the response in other organs. We designed a model to enable us to study the effects of radiation on leucocyte-endothelium interactions and on intestinal microflora in the murine ileum. This model enables us to study specifically the local effects of radiation therapy.

Method

A midline laparotomy was performed in male C57/Bl6 mice and a five-centimetre segment of ileum is irradiated using the chamber. Leucocyte responses (rolling and adhesion) were then analysed in ileal venules 2 – 48 hours after high dose irradiation, made possible by an inverted approach using intravital fluorescence microscopy. Furthermore, intestinal microflora, myeloperoxidase (MPO) and cell histology were analysed.

Results

The highest and most reproducible increase in leucocyte rolling was exhibited 2 hours after high dose irradiation whereas leucocyte adhesion was greatest after 16 hours. Radiation reduced the intestinal microflora count compared to sham animals with a significant decrease in the aerobic count after 2 hours of radiation. Further, the total aerobic counts, Enterobacteriaceae and Lactobacillus decreased significantly after 16 hours. In the radiation groups, the bacterial count showed a progressive increase from 2 to 24 hours after radiation.

Conclusion

This study presents a refinement of a previous method of examining mechanisms of radiation enteropathy, and a new approach at investigating radiation induced leucocyte responses in the ileal microcirculation. Radiation induced maximum leucocyte rolling at 2 hours and adhesion peaked at 16 hours. It also reduces the microflora count, which then starts to increase steadily afterwards. This model may be instrumental in developing strategies against pathological recruitment of leucocytes and changes in intestinal microflora in the small bowel after radiotherapy.
A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods
Authors: Yarrow Justin C , Perlman Zachary E , Westwood Nicholas J , Mitchison Timothy J
Published: September 2004

Background

Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. Investigation of cell migration and its underlying biology is of interest to basic scientists and those in search of therapeutics. Current migration assays for screening small molecules, siRNAs, or other perturbations are difficult to perform in parallel at the scale required to screen large libraries.

Results

We have adapted the commonly used scratch wound healing assay of tissue-culture cell monolayers to a 384 well plate format. By mechanically scratching the cell substrate with a pin array, we are able to create characteristically sized wounds in all wells of a 384 well plate. Imaging of the healing wounds with an automated fluorescence microscope allows us to distinguish perturbations that affect cell migration, morphology, and division. Readout requires ~1 hr per plate but is high in information content i.e. high content. We compare readouts using different imaging technologies, automated microscopy, scanners and a fluorescence macroscope, and evaluate the trade-off between information content and data acquisition rate.

Conclusions

The adaptation of a wound healing assay to a 384 well format facilitates the study of aspects of cell migration, tissue reorganization, cell division, and other processes that underlie wound healing. This assay allows greater than 10,000 perturbations to be screened per day with a quantitative, high-content readout, and can also be used to characterize small numbers of perturbations in detail.
Screening for Parkinson's disease with response time batteries: A pilot study
Authors: Johnson Andrew M , Vernon Philip A , Almeida Quincy J , Grantier Linda L , Singarayer Rene , Jog Mandar S
Published: September 2004

Background

Although significant response time deficits (both reaction time and movement time) have been identified in numerous studies of patients with Parkinson's disease (PD), few attempts have been made to evaluate the use of these measures in screening for PD.

Methods

Receiver operator characteristic curves were used to identify cutoff scores for a unit-weighted composite of two choice response tasks in a sample of 40 patients and 40 healthy participants. These scores were then cross-validated in an independent sample of 20 patients and 20 healthy participants.

Results

The unit-weighted movement time composite demonstrated high sensitivity (90%) and specificity (90%) in the identification of PD. Movement time was also significantly correlated (r = 0.59, p < 0.025) with the motor score of the Unified Parkinson's Disease Rating Scale (UPDRS).

Conclusions

Measures of chronometric speed, assessed without the use of biomechanically complex movements, have a potential role in screening for PD. Furthermore, the significant correlation between movement time and UPDRS motor score suggests that movement time may be useful in the quantification of PD severity.
Synchronously diagnosed pre-sacral neurofibroma and cutaneous spitzoid melanoma: a fortuitous association?
Authors: Fadare Oluwole , Hileeto Denise
Published: September 2004

Background

At a U.S prevalence of 1 in 3000, Neurofibromatosis type-1 (NF-1) is a relatively common disorder. Amongst a variety of others, occurrence of 2 or more neurofibromas in the same patient represents one of the major diagnostic criteria for this disorder. Rarely, ocular, cutaneous or anorectal malignant melanomas may be identified in patients with NF-1, This rare association has caused controversy as to whether patients with NF-1 have an inherently higher risk for melanomas or whether the associations can be explained by chance alone.

Case presentation

The purpose of this report is to highlight the unusual confluence of rare clinicopathologic features in a patient without NF-1. The patient was diagnosed with an 8.5 cm pre-sacral neurofibroma and was shortly thereafter diagnosed with a cutaneous malignant melanoma showing spitzoid features. Pre-sacral neurofibromas are rare in patients without NF-1; likewise, malignant spitzoid melanoma, a controversial histopathological entity, is distinctly uncommon.

Conclusions

The synchronous diagnoses of these neural crest derived tumor entities in a patient without neurofibromatosis lends credence to the view that when these two lesions occur in patients with NF-1, the association is coincidental.
Ambiguous allele combinations in HLA Class I and Class II sequence-based typing: when precise nucleotide sequencing leads to imprecise allele identification
Authors: Adams Sharon D , Barracchini Kathleen C , Chen Deborah , Robbins FuMeei , Wang Lu , Larsen Paula , Luhm Robert , Stroncek David F
Published: September 2004

Sequence-based typing (SBT) is one of the most comprehensive methods utilized for HLA typing. However, one of the inherent problems with this typing method is the interpretation of ambiguous allele combinations which occur when two or more different allele combinations produce identical sequences. The purpose of this study is to investigate the probability of this occurrence. We performed HLA-A,-B SBT for Exons 2 and 3 on 676 donors. Samples were analyzed with a capillary sequencer. The racial distribution of the donors was as follows: 615-Caucasian, 13-Asian, 23-African American, 17-Hispanic and 8-Unknown. 672 donors were analyzed for HLA-A locus ambiguities and 666 donors were analyzed for HLA-B locus ambiguities. At the HLA-A locus a total of 548 total ambiguous allele combinations were identified (548/1344 = 41%). Most (278/548 = 51%) of these ambiguities were due to the fact that Exon 4 analysis was not performed. At the HLA-B locus 322 total ambiguous allele combinations were found (322/1332 = 24%). The HLA-B*07/08/15/27/35/44 antigens, common in Caucasians, produced a large portion of the ambiguities (279/322 = 87%). A large portion of HLA-A and B ambiguous allele combinations can be addressed by utilizing a group-specific primary amplification approach to produce an unambiguous homozygous sequence. Therefore, although the prevalence of ambiguous allele combinations is high, if the resolution of these ambiguities is clinically warranted, methods exist to compensate for this problem.
Empirical Attenuation Equations for Vertical Ground Motion in Turkey
Authors: Kalkan Erol , Gülkan Polat
Published: September 2004

In the aftermath of two destructive urban earthquakes in 1999 in Turkey, empirical models of strong motion attenuation relationships that have been previously developed for North American and European earthquakes have been utilized in a number of national seismic hazard studies. However, comparison of empirical evidence and estimates present significant differences. For that reason, a data set created from a suite of 100 vertical strong ground motion records from 47 national earthquakes that occurred between 1976 and 2002 has been used to develop attenuation relationships for strong ground motion in Turkey. A consistent set of empirical attenuation relationships was derived for predicting vertical peak and pseudo-absolute vertical acceleration spectral ordinates in terms of magnitude, source-to-site distance, and local geological conditions. The study manifests the strong dependence of vertical to horizontal (V/H) acceleration ratio on spectral periods and relatively weaker dependence on site geology, magnitude, and distance. The V/H ratio is found to be particularly significant at the higher frequency end of the spectrum, reaching values as high as 0.9 at short distances on soil sites. The largest long-period spectral ratios are observed to occur on rock sites where they can reach values in excess of 0.5. These results raise misgivings concerning the practice of assigning the V/H ratio a standard value of two-thirds. Hence, nonconservatism of this value at short periods and its conservatism at long periods underline the need for its revision, at least for practice in Turkey.
On the Use of Spectra to Establish Damage Control in Regular Frames during Global Predesign
Authors: Teran-Gilmore Amador
Published: September 2004

A reliable seismic design requires the formulation of explicit design requirements for structural and nonstructural damage control. Recently, several methods for structural damage control have been established. While the majority of them take into consideration the maximum deformation demand, a few of them account for the effect of cumulative plastic deformation demands. From the comparison of the spectral values of displacement and plastic energy evaluated at the fundamental period of vibration and their corresponding demands in regular frames, it can be concluded that reliable estimates of these demands can be obtained for regular frames through the use of response spectra. Furthermore, within the context of performance-based design that accounts for low cycle fatigue, response spectra can be used for the conception of regular frames and, through the use of the static method of analysis, for their global predesign. Any reduction in lateral strength (base shear) that may be obtained from the use of dynamic methods of analysis should be carefully assessed.
Surface Rupture of the 2002 Denali Fault, Alaska, Earthquake and Comparison with Other Strike-Slip Ruptures
Authors: Haeussler Peter J. , Schwartz David P. , Dawson Timothy E. , Stenner Heidi D. , Lienkaemper James J. , Cinti Francesca , Montone Paola , Sherrod Brian , Craw Patricia
Published: September 2004

On 3 November 2002, an M7.9 earthquake produced 340 km of surface rupture on the Denali and two related faults in Alaska. The rupture proceeded from west to east and began with a 40-km-long break on a previously unknown thrust fault. Estimates of surface slip on this thrust are 3–6 m. Next came the principal surface break along ∼218 km of the Denali fault. Right-lateral offsets averaged around 5 m and increased eastward to a maximum of nearly 9 m. The fault also ruptured beneath the trans-Alaska oil pipeline, which withstood almost 6 m of lateral offset. Finally, slip turned southeastward onto the Totschunda fault. Right-lateral offsets are up to 3 m, and the surface rupture is about 76 km long. This three-part rupture ranks among the longest strike-slip events of the past two centuries. The earthquake is typical when compared to other large earthquakes on major intracontinental strike-slip faults.
A Teleseismic Study of the 2002 Denali Fault, Alaska, Earthquake and Implications for Rapid Strong-Motion Estimation
Authors: Ji Chen , Helmberger Don V. , Wald David J.
Published: September 2004

Slip histories for the 2002 M7.9 Denali fault, Alaska, earthquake are derived rapidly from global teleseismic waveform data. In phases, three models improve matching waveform data and recovery of rupture details. In the first model (Phase I), analogous to an automated solution, a simple fault plane is fixed based on the preliminary Harvard Centroid Moment Tensor mechanism and the epicenter provided by the Preliminary Determination of Epicenters. This model is then updated (Phase II) by implementing a more realistic fault geometry inferred from Digital Elevation Model topography and further (Phase III) by using the calibrated P-wave and SH-wave arrival times derived from modeling of the nearby 2002 M6.7 Nenana Mountain earthquake. These models are used to predict the peak ground velocity and the shaking intensity field in the fault vicinity. The procedure to estimate local strong motion could be automated and used for global real-time earthquake shaking and damage assessment.
Trans-Alaska Pipeline System Performance in the 2002 Denali Fault, Alaska, Earthquake
Authors: Honegger Douglas G. , Nyman Douglas J. , Johnson Elden R. , Cluff Lloyd S. , Sorensen Steve P.
Published: September 2004

The Trans-Alaska Pipeline System is one of the most significant engineering achievements of the 20th century and the first major pipeline system for which considerable attention was focused on the identification and quantification of potential seismic hazards and the implementation of design and operational features to address those hazards. One of these special design features included the concept for an above-ground supporting system for the pipeline crossing of the Denali fault. The 2002 M7.9 Denali fault earthquake represents the first successful test of a structure specifically designed for fault displacement. The earthquake also demonstrated the benefits of the multi-tiered earthquake preparedness and response strategy in place at the time of the earthquake.
Transgenic Expression of Enhanced Green Fluorescent Protein Enables Direct Visualization for Physiological Studies of Vasopressin Neurons and Isolated Nerve Terminals of the Rat
Authors: Ueta Yoichi , Fujihara Hiroaki , Serino Ryota , Dayanithi Govindan , Ozawa Hitoshi , Matsuda Ken-ichi , Kawata Mitsuhiro , Yamada Junko , Ueno Shinya , Fukuda Atsuo , Murphy David
Published: September 2004

We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.
Characterization of the Potent Luteinizing Hormone-Releasing Activity of KiSS-1 Peptide, the Natural Ligand of GPR54
Authors: Navarro V. M. , Castellano J. M. , Fernández-Fernández R. , Tovar S. , Roa J. , Mayen A. , Nogueiras R. , Vazquez M. J. , Barreiro M. L. , Magni P. , Aguilar E. , Dieguez C. , Pinilla L. , Tena-Sempere M.
Published: September 2004

Loss-of-function mutations of the gene encoding GPR54, the putative receptor for the KiSS-1-derived peptide metastin, have been recently associated with hypogonadotropic hypogonadism, in both rodents and humans. Yet the actual role of the KiSS-1/GPR54 system in the neuroendocrine control of gonadotropin secretion remains largely unexplored. To initiate such analysis, the effects of KiSS-1 peptide on LH secretion were monitored using in vivo and in vitro settings under different experimental conditions. Central intracerebroventricular administration of KiSS-1 peptide potently elicited LH secretion in vivo over a range of doses from 10 pmol to 1 nmol. The effect of centrally injected KiSS-1 appeared to be mediated via the hypothalamic LHRH. However, no effect of central administration of KiSS-1 was detected on relative LHRH mRNA levels. Likewise, systemic (ip and iv) injection of KiSS-1 markedly stimulated LH secretion. This effect was similar in terms of maximum response to that of central administration of KiSS-1 and might be partially attributed to its ability to stimulate LH secretion directly at the pituitary. Finally, the LH-releasing activity of KiSS-1 was persistently observed after blockade of endogenous excitatory amino acid and nitric oxide pathways, i.e. relevant neurotransmitters in the neuroendocrine control of LH secretion. In summary, our results provide solid evidence for a potent stimulatory effect of KiSS-1 on LH release, acting at central levels (likely the hypothalamus) and eventually at the pituitary, and further document a novel role of the KiSS-1/GPR54 system as a relevant downstream element in the neuroendocrine network governing LH secretion.
Extracellular Superoxide Dismutase Functions as a Major Repressor of Hypoxia-Induced Erythropoietin Gene Expression
Authors: Zelko Igor N. , Folz Rodney J.
Published: September 2004

Hypoxia and biological responses to hypoxia are commonly encountered in both normal and pathologic cellular processes. Here we report that extracellular superoxide dismutase (EC-SOD) plays a major role in regulating the magnitude of hypoxia-induced erythropoietin (Epo) gene expression, thus implicating superoxide as an intermediary signal transduction molecule critical to this process. We found that mice which have the EC-SOD gene inactivated show a marked more than 100-fold elevation in hypoxia-induced Epo gene expression, compared with wild-type controls, which was both dose and time dependent. These mice also showed a significant increase in serum Epo levels after 1 d hypoxia. Interestingly, despite elevated Epo levels, reciprocal changes in hematocrit and reticulocyte counts were not found, suggesting that this newly synthesized Epo lacks functional hematopoietic effects. When EC-SOD was overexpressed in Hep3B cells, we found a significant reduction in Epo gene induction by both CoCl2 (50 μm) and hypoxia (1% O2). Similar findings were noted with another hypoxia-inducible gene, carbonic anhydrase IX. We conclude that EC-SOD functions as a major repressor of hypoxia-induced Epo gene expression, which implicates superoxide as a signaling intermediate whose downstream effects, at least in part, may be mediated by HIF-1α.
Enhanced Recovery from Thrombocytopenia and Neutropenia in Mice Constitutively Expressing a Placental Hematopoietic Cytokine
Authors: Zhou Beiyan , Kong Xiangrong , Linzer Daniel I. H.
Published: September 2004

Expression of the placental hormone, prolactin-like protein E (PLP-E), a potent cytokine that acts on multiple myeloid lineages, is normally restricted to pregnancy and certain hematopoietic disease states. We hypothesized that the restricted pattern of PLP-E expression is necessary to avoid hyperstimulation of myelopoiesis. To test this idea, we have produced PLP-E transgenic mice and analyzed their steady-state blood cell levels. We find that blood cell levels remain in the normal range, and thus the constitutive expression of a cytokine of pregnancy fails to overcome the tight control of hematopoietic set points for blood cell levels. In contrast, an effect of constitutive PLP-E expression is detected during the recovery from low blood platelet levels (acute thrombocytopenia) and from low granulocyte levels (acute neutropenia) but not from anemia. Mice producing high circulating concentrations of PLP-E recover more rapidly from both thrombocytopenia and neutropenia, as seen both by an earlier increase of progenitor numbers in the bone marrow and the earlier return to normal circulating blood cell levels.
Analysis of Ligand-Dependent Recruitment of Coactivator Peptides to Estrogen Receptor Using Fluorescence Polarization
Authors: Ozers Mary Szatkowski , Ervin Kerry M. , Steffen Corrine L. , Fronczak Jennifer A. , Lebakken Connie S. , Carnahan Kimberly A. , Lowery Robert G. , Burke Thomas J.
Published: September 2004

Ligand-dependent recruitment of coactivators to estrogen receptor (ER) plays an important role in transcriptional activation of target genes. Agonist-bound ER has been shown to adopt a favorable conformation for interaction with the LXXLL motifs of the coactivator proteins. To further examine the affinity and ligand dependence of the ER-coactivator interaction, several fluorescently tagged short peptides bearing an LXXLL motif (LXXLL peptide) from either natural coactivator sequences or random phage display sequences were used with purified ERα or ERβ in an in vitro high-throughput fluorescence polarization assay. In the presence of saturating amounts of ligand, several LXXLL peptides bound to ERα and ERβ with affinity ranging from 20–500 nm. The random phage display LXXLL peptides exhibited a higher affinity for ER than the natural single-LXXLL coactivator sequences tested. These studies indicated that ER agonists, such as 17β-estradiol or estrone, promoted the interaction of ER with the coactivator peptides, whereas antagonists such as 4-hydroxytamoxifen or ICI-182,780 did not. Different LXXLL peptides demonstrated different affinities for ER depending on which ligand was bound to the receptor, suggesting that the peptides were recognizing different receptor conformations. Using the information obtained from direct measurement of the affinity of the ER-LXXLL peptide interaction, the dose dependency (EC50) of various ligands to either promote or disrupt this interaction was also determined. Interaction of ER with the LXXLL peptide was observed with ligands such as 17β-estradiol, estriol, estrone, and genistein but not with ICI-182,780, 4-hydroxytamoxifen, clomiphene, or tamoxifen, resulting in distinct EC50 values for each ligand and correlating well with the ligand biological function as an agonist or antagonist. Ligand-dependent recruitment of the LXXLL peptide to ERβ was observed in the presence of the ERβ-selective agonist diarylpropionitrile, but not the ERα-selective ligand propyl pyrazole triol. This assay could be used to classify unknown ligands as agonists, antagonists, or partial modulators, based on either the receptor-coactivator peptide affinities or the dose dependency of this interaction in comparison with known compounds.
Dual antennular chemosensory pathways can mediate orientation by Caribbean spiny lobsters in naturalistic flow conditions
Authors: Horner Amy J. , Weissburg Marc J. , Derby Charles D.
Published: September 2004

Benthic crustaceans rely on chemical stimuli to mediate a diversity of behaviors ranging from food localization and predator avoidance to den selection, conspecific interactions and grooming. To accomplish these tasks,Caribbean spiny lobsters (Panulirus argus) rely on a complex chemosensory system that is organized into two parallel chemosensory pathways originating in diverse populations of antennular sensilla and projecting to distinct neuropils within the brain. Chemosensory neurons associated with aesthetasc sensilla project to the glomerular olfactory lobes (the aesthetasc pathway), whereas those associated with non-aesthetasc sensilla project to the stratified lateral antennular neuropils and the unstructured median antennular neuropil (the non-aesthetasc pathway). Although the pathways differ anatomically, unique roles for each in odor-mediated behaviors have not been established. This study investigates the importance of each pathway for orientation by determining whether aesthetasc or non-aesthetasc sensilla are necessary and sufficient for a lobster to locate the source of a 2 m-distant food odor stimulus in a 5000-liter seawater flume under controlled flow conditions. To assess the importance of each pathway for this task, we selectively ablated specific populations of sensilla on the antennular flagella and compared the searching behavior of ablated animals to that of intact controls. Our results show that either the aesthetasc or the non-aesthetasc pathway alone is sufficient to mediate the behavior and that neither pathway alone is necessary. Under the current experimental conditions,there appears to be a high degree of functional overlap between the pathways for food localization behavior.
Development of pendulum mechanism and kinematic coordination from the first unsupported steps in toddlers
Authors: Ivanenko Yuri P. , Dominici Nadia , Cappellini Germana , Dan Bernard , Cheron Guy , Lacquaniti Francesco
Published: September 2004

The inverted pendulum model in which the centre of mass of the body vaults over the stance leg in an arc represents a basic mechanism of bipedal walking. Is the pendulum mechanism innate, or is it learnt through walking experience?We studied eight toddlers (about 1 year old) at their first unsupported steps,18 older children (1.3–13 years old), and ten adults. Two infants were also tested repeatedly over a period of 4 months before the onset of independent walking. Pendulum mechanism was quantified from the kinematics of the greater trochanter, correlation between kinetic and gravitational potential energy of the centre of body mass obtained from the force plate recordings, and percentage of recovery of mechanical energy. In toddlers,these parameters deviated significantly (P<10–5)from those of older children and adults, indicating that the pendulum mechanism is not implemented at the onset of unsupported locomotion. Normalising the speed with the Froude number showed that the percentage of recovery of mechanical energy in children older than 2 years was roughly similar to that of the adults (less than 5% difference), in agreement with previous results. By contrast, the percentage of recovery in toddlers was much lower (by about 50%). Pendulum-like behaviour and fixed coupling of the angular motion of the lower limb segments rapidly co-evolved toward mature values within a few months of independent walking experience. Independent walking experience acts as a functional trigger of the developmental changes,as shown by the observation that gait parameters remained unchanged until the age of the first unsupported steps, and then rapidly matured after that age. The findings suggest that the pendulum mechanism is not an inevitable mechanical consequence of a system of linked segments, but requires active neural control and an appropriate pattern of inter-segmental coordination.
Sedation Assessment in Critically Ill Adults: 2001–2004 Update
Authors: Watson Brian D , Kane-Gill Sandra L
Published: September 2004

OBJECTIVE

To review recently published literature on the validity and reliability of sedation assessment tools in critically ill adults and evaluate the potential advantages and disadvantages of each.

DATA SOURCES

A computerized search of MEDLINE and PubMed (2001–May 2004) was conducted.

STUDY SELECTION AND DATA EXTRACTION

Sedation assessment tools used in adult intensive care units (ICUs) were identified.

DATA SYNTHESIS

Six subjective and 3 objective assessment tools were identified. Four subjective assessment tools have reliability and 4 have validity data published that were not previously available. There are reliability data to further support the use of the previously published Motor Activity Assessment Scale. Additional reliability data exist for the Ramsay Scale and Glasgow Coma Scale. Conflicting evidence is available with the use of the Bispectral Index monitor in the ICU. Recently, the Patient State Index and Auditory Evoked Potentials were introduced for objective monitoring in critically ill patients.

CONCLUSIONS

Increasing data on sedation assessment were published over the last few years, probably in response to supporting evidence that goal-driven sedation therapy improves patient outcomes. Reliability and/or validity testing exists for many of these scales. Several useful tools are available to guide sedation therapy in critically ill patients.
Population Pharmacokinetics II: Estimation Methods
Authors: Ette Ene I , Williams Paul J
Published: September 2004

OBJECTIVE

To present, compare, and contrast the various approaches to estimating population pharmacokinetic (PPK) models with respect to the mathematical foundation, statistical aspects, software programs for implementation, and underlying assumptions.

DATA SOURCES

Information on PPK was retrieved from a MEDLINE search (1977–August 2004) of literature and a bibliographic review of review articles and books. This information is used in conjunction with experience to explain the various methodologic approaches to PPK.

STUDY SELECTION AND DATA EXTRACTION

All articles indentified from data sources were evaluated and relevant information was included in this review.

DATA SYNTHESIS

Over 80 articles dealing with PPK estimation methods and/or their implementation were identified and reviewed. Sixty-four of these were chosen for their direct relevance to the subject of this article. Different estimation methods ranging from the naïve averaging and naïve pooled approaches through the standard two-stage approach to the nonlinear mixed-effects modeling approaches for estimating PPK are reviewed with their advantages and limitations.

CONCLUSIONS

PPK estimation methods that rely on the characterizing of mixed (fixed and random) effects are known to produce PPK parameter estimates that are less biased than those obtained using the naïve and standard two-stage approaches. The NONMEM software is the most widely used software for the characterization of PPK.
Mutation Analysis in Spanish Patients with Hereditary Hemorrhagic Telangiectasia: Deficient Endoglin Up-regulation in Activated Monocytes
Authors: Sanz-Rodriguez Francisco , Fernandez-L. Africa , Zarrabeitia Roberto , Perez-Molino Alfonso , Ramírez Jose R , Coto Eliecer , Bernabeu Carmelo , Botella Luisa M
Published: September 2004

Background: Mutations in the endoglin (ENG) or ALK1 genes are responsible for hereditary hemorrhagic telangiectasia types 1 and 2 (HHT1 and HHT2), respectively, a dominant vascular dysplasia caused by haploinsufficiency. No formal mutation studies of patients with HHT have been conducted in Spain.

Methods: ENG and ALK1 mutation analyses were carried out in 13 Spanish HHT patients diagnosed according to the Curaçao criteria. Because endoglin is up-regulated at the cell surface during the monocyte-macrophage transition, endoglin concentrations in activated monocytes were determined by immunofluorescence flow cytometry in a systematic analysis. As controls, 40 non-HHT volunteers were studied for up-regulation of endoglin in activated monocytes.

Results: The mutation responsible for HHT was identified in eight patients belonging to two unrelated families. One of the families has a nonsense mutation in exon 4 (c.511C>T; R171X) of the ENG gene, and accordingly the disorder was identified as HHT1. The other family has a missense mutation affecting exon 8 (c.1120C>T; R374W) of the ALK1 gene, and hence is a HHT2 family. Interestingly, endoglin up-regulation was deficient in activated monocytes of both HHT1 and HHT2 patients compared with controls. By contrast, endoglin up-regulation was age-independent in control donors across a broad range of ages. The extent of endoglin up-regulation in activated monocytes was most diminished in those patients with the most severe symptoms.

Conclusions: Endoglin up-regulation in activated monocytes is impaired in HHT1 and HHT2 patients and is age-dependent in both HHT types. Endoglin expression may predict the clinical severity of HHT.
Evaluation of a Candidate Reference Measurement Procedure for Serum Free Testosterone Based on Ultrafiltration and Isotope Dilution–Gas Chromatography–Mass Spectrometry
Authors: Van Uytfanghe Katleen , Stöckl Dietmar , Kaufman Jean M , Fiers Tom , Ross H Alec , De Leenheer André P , Thienpont Linda M
Published: September 2004

Background: To assess the analytical validity of free testosterone (FTe) measurements, a reference measurement procedure (RMP) is required. For steroids, isotope dilution–mass spectrometry is accepted as state-of-the-art technology. Because FTe is defined as the hormone fraction in serum water in equilibrium with the protein-bound fraction, the RMP should include a physical separation step. The use of equilibrium dialysis (ED) or ultrafiltration (UF) is advocated. Our objective was to develop such a candidate RMP.

Methods: We selected UF combined with isotope dilution–gas chromatography–mass spectrometry (ID-GC/MS) for direct measurement of Te in the ultrafiltrate. After optimization of the UF process, the complete procedure was validated by use of split-sample comparisons with indirect ED (iED) and symmetric dialysis (SyD).

Results: The candidate RMP gave maximum within-day, between-day, and total CVs of 3.0%, 3.1%, and 4.3%. The Deming regression equations for the respective method comparisons were: UF-ID-GC/MS = 0.98(iED) − 53 pmol/L (r = 0.94; Sy|x= 42 pmol/L) and UF-ID-GC/MS = 0.92(SyD) + 21 pmol/L (r = 0.97; Sy|x= 31 pmol/L).

Conclusions: We achieved the objective of a state-of-the-art candidate RMP, which agreed well with iED and SyD. However, we also demonstrated that a degree of discordance remains, which may require a decision from an authoritative organization on the recommended procedure to measure free hormone concentrations.