bluuebunny/crossref_metadata_2025 huggingface dataset:
High efficiency gene transfer into cultured primary rat and human hepatic stellate cells using baculovirus vectors
Authors: Gao Runping , McCormick Christopher J. , Arthur Michael J. P. , Ruddell Richard , Oakley Fiona , Smart David E. , Murphy Frank R. , Harris Mark P. G. , Mann Derek A.
Published: December 2004

Abstract: Background/aims: Gene transfer into hepatic stellate cells (HSC) is inefficient when using plasmid‐based transfection methods; viral‐based systems are therefore being developed. A baculovirus system has recently been shown to be useful for expressing genes in mammalian cells. The aim of this study was to determine if baculovirus vectors can infect and express target genes in rat and human HSC and to assess potential cytotoxic and modulatory effects of infection. Methods: A recombinant baculovirus vector (AcCALacZ) carrying the LacZ gene was used to infect HSC. β‐Galactosidase assays and electron microscopy were used to determine efficiency of infection and gene expression. Counting of trypan blue negative cells was used to assess cytotoxic/cytostatic effects of infection. Measurement of protein content of cells and α‐smooth muscle actin expression were performed to assess the effects of baculovirus on cell function/phenotype. Results: Baculovirus infection of activated HSC was highly efficient (> 90%) and provided long‐term LacZ gene expression (15 days) in the absence of cytotoxic, cytostatic or modulatory effects. Infection of freshly isolated cells was also observed but at lower levels (20%). Conclusions: Baculovirus vectors can therefore be used to deliver target genes to cultured rat and human HSC with high efficiency and longevity in the absence of detrimental effects on cell function.
The detection of HBV antigens and HBx‐transcripts in an Indian fibrolamellar carcinoma patient: a case study
Authors: Dadke Disha , Jaganath P. , Krishnamurthy Shanta , Chiplunkar Shubhada
Published: December 2004

Abstract: Fibrolamellar carcinoma (FLC) of the liver is a rare variant of hepatocellular carcinoma (HCC). Here we report the case of a 12‐year‐old Indian male with typical FLC with no apparent hepatitis B virus (HBV) infection and a non‐cirrhotic liver. The patient, though seronegative for HBsAg, showed expression of HBcAg in both the liver and tumour tissue. RT‐PCR analysis revealed the presence of full‐length HBx‐transcripts in both liver/tumour tissue, along with truncated HBx‐transcripts only in the tumour tissue. The lymphocytes in both peripheral and liver/tumour compartments showed a proliferative response to either/or HBcAg and HBxAg, which could be further augmented on addition of rIL‐2. This is the first study to show not only the presence of HBcAg in the liver/tumour tissue but also prior exposure of the FLC patient's lymphocytes to HBV antigens. Also, the presence of the full‐length and truncated HBx‐transcripts in the tumour tissue, a proposed tumorigenic marker for hepatocarcinogenesis in chronic HBV patients, suggests an oncogenic role of HBV in this rare variant of HCC.
Sclerosed hemangioma and sclerosing cavernous hemangioma of the liver: a comparative clinicopathologic and immunohistochemical study with emphasis on the role of mast cells in their histogenesis
Authors: Makhlouf Hala R. , Ishak Kamal G.
Published: December 2004

Abstract: Background/Aims: Sclerosed hemangiomas of the liver are rare. To date, their histopathology, immunohistochemistry, and the role of mast cells (MC) in their histogenesis have not been systematically studied. Patients/Methods: Clinical, histopathologic and immunohistochemical features of 20 sclerosed hemangiomas were compared with those of 18 sclerosing cavernous hemangiomas. The number of MC was quantified and compared in all cases, using a tryptase immunostain. Results: Compared to patients with sclerosed hemangiomas, those with sclerosing hemangiomas were younger (mean age, 63 versus 71 years); had larger tumors (mean 6 ± 4.73 versus 3 ± 2.2 cm); presented with a mass more frequently, and epigastric pain less frequently. Sclerosing hemangiomas, but not sclerosed hemangiomas, were more frequent in males than in females. Sclerosing hemangiomas occurred much more frequently in the right lobe than sclerosed hemangiomas. Sclerosing hemangiomas had less fibrosis, hyalinization, and elastic fibers than sclerosed hemangiomas (p = 0.00004). Numerous thick‐walled blood vessels were a feature of sclerosed hemangiomas but not of sclerosing hemangiomas. Collagen IV, and laminin were more uniformly positive in sclerosing hemangiomas than in sclerosed hemangiomas. Increased immunoreactivity for smooth muscle actin was present in sclerosed hemangiomas more often than in sclerosing hemangiomas. FVIII‐R Ag, CD34, and CD31 were more diffusely positive in sclerosing hemangiomas than in sclerosed hemangiomas. In sclerosing hemangiomas, the mean number of tryptase‐positive MC per high power field (MC/HPF) varied from 8.25 ± 6.23 in vascular areas to 1.6 ± 4.01 in sclerotic areas. In comparison, the mean number of MC in sclerosed hemangiomas, was 4.3 ± 5.01 in vascular areas, and 0.86 ± 0.58 in sclerotic areas (p = 0.0095). The number of MC was significantly correlated with vascular proliferation and inversely related to the degree of fibrosis (p < 0.0001). Conclusions: This study demonstrates certain distinct clinical and histopathologic differences between sclerosing cavernous hemangiomas and sclerosed hemangiomas of the liver. We have established the presence of MC in those tumors, and suggest possible involvement of the MC in angiogenesis, and the regression process and development of fibrosis.
Long‐term asymptomatic biochemical cholestasis after fulminant or subfulminant liver failure is associated with extensive postnecrotic collapse with regeneration of the liver
Authors: Ngassa Michèle , Van Beers Bernard E. , Geubel André P.
Published: December 2004

Abstract: Fulminant or subfulminant liver failure usually leads to liver failure or to recovery. In rare instances, patients who recover exhibit prolonged asymptomatic biochemical cholestasis which coincides with the development into the parenchyma of large postnecrotic collapse with regeneration. This hitherto poorly recognized form of recovery may now be assessed by noninvasive techniques such as magnetic resonance imaging. We report the case of three patients who recovered from fulminant or subfulminant liver failure and in whom investigation of long‐term biochemical cholestasis led to that unusual diagnosis.
HLA and cytokine gene polymorphisms in biliary atresia
Authors: Donaldson Peter T. , Clare Michael , Constantini Patrizia K. , Hadzic Nedim , Mieli‐Vergani Giorginia , Howard Edward , Kelley Dympna
Published: December 2004

Background/aims:

Extrahepatic biliary atresia remains one of the major hepatic causes of death in early childhood. Though a number of hypotheses have been developed to account for this disease, its aetiopathogenesis is poorly understood. One possibility is that this is an immune mediated disease which occurs following either toxic or infectious insult in a genetically susceptible host. Earlier studies suggested weak HLA associations but these remain unconfirmed. More recently studies of viral and autoimmune liver disease have begun to investigate non‐MHC immunoregulatory gene polymorphisms.

Methods:

In the present study we used molecular genotyping to investigate selected HLA A, B, DRB1, DQA1, DQB1 and DPB1 alleles as well as polymorphisms in the interleukin‐1 gene family, interleukin‐10 promoter sequence and tumour necrosis factor alpha promoter genes in 101 children referred for surgical assessment with extra hepatic biliary atresia. Genotyping data were compared with those of 134 racially and geographically matched healthy adult health care workers.

Results and conclusions:

Overall there were no statistically significant differences in the distribution of any of the genes tested comparing patients and controls. These data suggest that biliary atresia is not an HLA‐associated disease and that polymorphisms in both the interleukin‐1 and interleukin‐10 genes are not risk factors for this disease.
Subsampling the mean of heavy‐tailed dependent observations
Authors: Kokoszka Piotr , Wolf Michael
Published: December 2004

Abstract. We establish the validity of subsampling confidence intervals for the mean of a dependent series with heavy‐tailed marginal distributions. Using point process theory, we focus on GARCH‐like time series models. We propose a data‐dependent method for the optimal block size selection and investigate its performance by means of a simulation study.
Monitoring woodland deer populations in the UK: an imprecise science
Authors: SMART JAMES C. R. , WARD ALASTAIR I. , WHITE PIRAN C. L.
Published: December 2004

1. The need to assess population size and change is central to any population monitoring programme. A range of monitoring techniques is available for deer, but few studies have addressed the performance of these techniques in terms of their accuracy and their power to detect population changes reliably. This study compares the performance of three commonly used techniques to monitor woodland deer populations in terms of their accuracy, precision and statistical power using field data and simulation modelling.

2. Faecal Standing Crop (FSC) was found to provide the most accurate estimates at all deer densities studied, but was outperformed by distance sampling using thermal imaging in terms of precision and statistical power. Faecal Accumulation Rate (FAR) performed worst in terms of precision and statistical power, and was inferior in terms of accuracy to FSC. The accuracy of FAR and distance sampling using thermal imaging was similar.

3. Annual surveying is not the best use of resources, since 10% per annum increases or decreases in a population could not be detected by any of the techniques within a 3‐year time period. Even with the best technique under the most favourable conditions of deer density simulated in this study, a population change could only be detected reliably once the population had increased by 33% or decreased by 27%. In the least favourable conditions, the respective figures were a 380% increase or extinction.

4. All the techniques studied are relatively poor at detecting population change, so their use in practical deer management needs to be treated with caution. The consequences of relying on similar techniques to detect population change for rare species could be severe.
Monitoring cetaceans in European waters
Authors: EVANS PETER G. H. , HAMMOND PHILIP S.
Published: December 2004

1. Monitoring spatial and temporal patterns in cetacean abundance involves a variety of approaches depending upon the target species and the resources available. As a first step, the collection of incidental sightings or strandings information aids the construction of a species list and a rough measure of status and seasonal variation in abundance. These often make use of networks of volunteer observers although the wide variation in abilities and experience means that special attention must be paid to training and to data quality control. More robust monitoring of numbers requires quantification of effort and some correction for factors that influence detectability, such as sea state.

2. The presence of cetaceans may be recorded visually, or indirectly by acoustics. Each has advantages and disadvantages, and their applicability may vary between species. The use of fixed stations tends to allow sustained monitoring at relatively low cost but coverage is limited to the immediate vicinity. For more extensive coverage, mobile platforms are necessary. Platforms of opportunity such as ferries, whale‐watching boats, etc. are often used to survey areas at low cost. These may allow repeat observations to be made over time, but with no control over where the vessel goes, it is typically not possible to sample wide areas, thus limiting abundance estimation.

3. Line transect surveys using dedicated platforms allow representative coverage of areas from which abundance estimates can be made (either using indices or absolute measures derived from density estimation). Assumptions relating to detectability and responsiveness need to be addressed and various methods (such as two‐platform surveys) have been developed to accommodate these.

4. For some cetacean species, mark‐recapture methods can be applied using photo‐identification of recognizable individuals. Again, a number of assumptions are made, particularly relating to recognizability, representativeness of sampling and capture probabilities. Capturing, on film, as many animals in the population as possible helps to reduce the problem of heterogeneity of capture probabilities. Mark‐recapture methods require at least two sampling occasions. If multiple sampling is employed, either open or closed population models can be used.

5. Measuring population change represents a particular challenge for mobile animals such as cetaceans. Changes in ranging patterns may have a large impact on abundance estimates unless very large areas are adequately covered. Power analysis is a useful method to indicate the ability of the data to detect a trend of a given magnitude. Increasingly, spatial modelling using GLMs and GAMs is being used to provide a better understanding of the biotic and hydrographic factors influencing cetacean distribution.
Methods of monitoring red foxesVulpes vulpesand badgersMeles meles: are field signs the answer?
Authors: SADLIER LINDA M. J. , WEBBON CHARLOTTE C. , BAKER PHILIP J. , HARRIS STEPHEN
Published: December 2004

1. A national monitoring scheme for recording the abundance of foxes and badgers in Britain would have to utilize a technique or techniques that could detect a wide range of animal densities in structurally different habitats. Furthermore, the likely reliance on volunteers for data collection means that these techniques must be easily applied by people with different levles of field expertise.

2. Direct methods that rely on counts of the animals themselves (e.g. capture‐mark‐recapture, radio‐tracking, spotlight counts) are generally unsuitable because of cost, manpower and licensing requirements, are not readily applied to all habitats and cannot easily be used by volunteers. However, density estimates derived from capture‐mark‐recapture and radio‐tracking methods are likely to represent the benchmark against which other estimates of abundance are measured.

3. The number of foxes killed per unit area is currently collated by non‐governmental organisations for some patterns of land use, e.g. game estates. No such data are available for badgers, as this species is legally protected in Britain. However, the applicability of hunting statistics for monitoring fox abundance is limited by differences in culling effort, the non‐independence of different culling practices applied in the same region, possible future changes in the legal status of different culling methods and changes in the ratio of land where foxes are and are not culled.

4. Indirect methods that rely on counts of the signs of the animals (e.g. droppings, breeding refugia) are less expensive than direct methods, can be applied to the range of habitats found in Britain and can easily be used by volunteers. To date, indirect methods have been utilised to derive estimates of relative animal density or the density of social groups. However, the major factor currently limiting the use of indirect methods is that their relationship with absolute animal density has not been validated. The preliminary results of two projects quantifying the use of field signs as a measure of absolute fox and badger abundance suggest that indirect methods could be applicable for monitoring changes in fox and badger numbers at a national scale.
Angiogenesis in intracranial meningiomas: immunohistochemical and molecular study
Authors: Pistolesi S. , Boldrini L. , Gisfredi S. , Ieso K. De , Camacci T. , Caniglia M. , Lupi G. , Leocata P. , Basolo F. , Pingitore R. , Parenti G. , Fontanini G.
Published: December 2004

Much of the morbidity of intracranial meningiomas is related to the degree of tumour vascularity and the extent of peritumoural vasogenic oedema. Several studies have shown that vascular endothelial growth factor (VEGF) is up‐regulated in meningiomas, although its relationship with tumour vasculature is still unclear. In order to better understand the angiogenic assessment of intracranial meningiomas, we analysed its vascular pattern, both as number and as morphologic configuration of microvessels. Moreover, we investigated the mRNA‐VEGF expression, relating this expression to vascular pattern. A total of 40 intracranial meningiomas, classified as benign (31 cases), atypical (7 cases), and anaplastic (2 cases) were analysed. RT‐PCR analyses of mRNA‐VEGF and competitive‐PCR were performed. VEGF expression and microvessel density (MVD) were also immunohistochemically investigated. Grade II–III meningiomas showed numerous small microvessels (mean: 34), while the majority of Grade I showed few larger vessels (mean: 13.09) (P = 0.000003). A microvessel pattern overlapping into atypical subtype was found in eignt of the 31 (25.8%) Grade I meningiomas. A significant association was found between grading and vascular pattern (P = 0.0002), as well as between the MVD and the immunohistochemical expression of VEGF (P = 0.0005). The expression of mRNA agreed with the immunohistochemical expression of the protein (P < 0.0001). A total of 39 cases expressed the 121 VEGF isoform and, among these, 28 cases also expressed the 165 isoform. Only 9 cases expressed both isoforms 165 and 189. Grade II and III meningiomas showed a preponderant expression of soluble isoforms (121 and 165). These results prompt us to speculate that the microvessel pattern could underlie a higher metabolic demand, probably due to a rapid growth with a consequent worse clinical behaviour of the tumour. In this sense, the vascular pattern may be used as a prognostic factor, in order to mostly focus attention on those Grade I meningiomas which have a higher likelihood of either recurrence or development of perilesional oedema. The pattern of vasculature itself seems to be dependent on the types of VEGF isoforms: the Grade II–III meningiomas (that presented numerous microvessels) expressed the soluble isoforms 121 and 165, while the isoform 189 was more frequently detected in Grade I meningiomas.
Infection of stationary human brain aggregates with HIV‐1 SF162 and IIIB results in transient neuronal damage and neurotoxicity
Authors: Trillo‐Pazos G. , Kandanearatchi A. , Eyeson J. , King D. , Vyakarnam A. , Everall I. P.
Published: December 2004

The cellular basis of HIV associated dementia has been correlated with microglial activation and neuronal dysfunction in symptomatic HIV‐1 disease. As a cellular model of HIV‐1 infection of brain tissue in vitro, we established a stationary human brain aggregate (SHBA) system to compare infection of HIV‐1 SF162 (R5 virus) to that of IIIB (X4 virus). Aggregates were analysed by immunohistochemistry, morphometry, flow cytometry and p24 ELISA. SHBAs had a 1 mm3 size with a mixed cellular composition of 36% neurones, 27% astrocytes, 2% macrophages/microglia and 14% oligodendrocytes. Infection of SHBA's with the R5 HIV‐1 SF162 virus led to the expression of HIV‐1 p24 antigen in 6% of cells. Infection with this R5 using virus culminated in transient neuronal damage and a decrease in mitotically active progenitor cells within aggregates. Infection with X4 using HIV‐1 IIIB was associated with astrocytosis and neurotoxicity. We propose that: (1) the pattern of cellular damage elicited by HIV‐1 infection of brain tissue in vitro depends on virus subtype as determined by its preferential use of R5 or X4 chemokine receptors for entry into cells; (2) SHBAs are a reliable and readily established model of the cellular complexity of human brain tissue in vitro.
Selective changes in nicotinic acetylcholine receptor subtypes related to tobacco smoking: an immunohistochemical study
Authors: Teaktong T. , Graham A. J. , Johnson M. , Court J. A. , Perry E. K.
Published: December 2004

Increases in neuronal nicotinic receptors (nAChRs) in response to nicotine exposure have been reported in cell cultures, rodent brains, and in the brains of human smokers. The present study examines alterations in α4 and α7 nAChR subunit cellular expression in human hippocampus and entorhinal cortex from normal elderly individuals with known smoking history. There were significant increases in the intensity of α4 immunoreactive neuropil, but not the number of cell bodies, in many regions of hippocampus and entorhinal cortex in smokers compared to age‐matched non‐smokers and ex‐smokers. There was also an increase in α7 immunoreactive perikarya in the granular cell layer of dentate gyrus in smokers but not other regions examined. There was, in contrast, a significant reduction in α7 immunoreactive astrocytes in smokers and ex‐smokers compared to non‐smokers. These findings suggest exposure to tobacco smoke acutely up‐regulates α4 receptors in axon terminals and dendrites but not perikarya, whereas tobacco smoking induced down‐regulation of α7 expression on astrocytes is a long‐term effect. As the α4 subunit decreases with ageing and degenerative diseases such as Alzheimer's disease, whereas α7 increases in astrocytes in Alzheimer's disease, the findings further indicate the therapeutic relevance of nicotinic agonists such as nicotine.
Expression of the β‐adhesin part of HRgpA in Sprague Dawley rats induces a specific antibody response
Authors: Vågnes K. S. , Vågnes Ø. B. , Vasstrand E. N. , Bakken V.
Published: December 2004

The β‐adhesin part of the Porphyromonas gingivalis W50 (ATCC 53978) protease HRgpA was cloned in an eukaryotic expression vector and expressed in COS‐7 cells. The monoclonal antibody MAb (61BG1.3), specific for the hemagglutinating domain of β‐adhesin, recognized the expressed β‐adhesin in the transfected cells both by immunoblot and immunofluorescence. Sprague Dawley rats were immunized intramuscularly with β‐adhesin encoding expression plasmid and expression plasmid without β‐adhesin insert. Skeletal muscle tissue at the site of immunization in the β‐adhesin immunized animals was shown to express this protein. The immunization induced a β‐adhesin‐specific antibody response. Sera from the immunized animals were tested for hemagglutination inhibiting activity. Due to high natural inhibiting activity in all rat sera tested, no increased hemagglutination inhibition was detected in sera from the β‐adhesin immunized animals.
Human cytomegalovirus and Epstein‐Barr virus type 1 in periodontal abscesses
Authors: Saygun I , Yapar M , Özdemir A , Kubar A , Slots J
Published: December 2004

Objectives:  Recent studies have linked herpesviruses to severe types of periodontal disease, but no information exists on their relationship to periodontal abscesses. The present study determined the presence of human cytomegalovirus (HCMV) and Epstein‐Barr virus type 1 (EBV‐1) in periodontal abscesses and the effect of treatment on the subgingival occurrence of these viruses.

Material and methods:  Eighteen adults with periodontal abscesses participated in the study. Subgingival samples were collected from each patient with sterile curettes from an abscess‐affected site and a healthy control site. HCMV and EBV‐1 were identifed by polymerase chain reaction at the time of the abscess and at 4 months after surgical and systemic doxycycline therapy.

Results:  HCMV was detected in 66.7% of periodontal abscess sites and in 5.6% of healthy sites (P = 0.002). EBV‐1 occurred in 72.2% of abscess sites but not in any healthy site (P < 0.001). HCMV and EBV‐1 co‐infection was identified in 55.6% of the abscess sites. Posttreatment, HCMV and EBV‐1 were not found in any study site.

Conclusions:  HCMV and EBV‐1 genomes are commonly found in periodontal abscesses. These data favor a model in which a herpesvirus infection of the periodontium impairs the host defense and serves as a platform for the entrance of bacterial pathogens into gingival tissue with subsequent risk of abscess development.
l‐arginine‐dependent nitric oxide production of a murine macrophage‐like RAW 264.7 cell line stimulated with Porphyromonas gingivalis lipopolysaccharide
Authors: Sosroseno W. , Herminajeng E. , Bird P. S. , Seymour G. J.
Published: December 2004

The aim of this study was to determine nitric oxide (NO) production of a murine macrophage cell line (RAW 264.7 cells) when stimulated with Porphyromonas gingivalis lipopolysaccharides (Pg‐LPS). RAW264.7 cells were incubated with i) various concentrations of Pg‐LPS or Salmonella typhosa LPS (St‐LPS), ii) Pg‐LPS with or without l‐arginine and/or NG‐monomethyl‐l‐arginine (NMMA), an arginine analog or iii) Pg‐LPS and interferon‐γ (IFN‐γ) with or without anti‐IFN‐γ antibodies or interleukin‐10 (IL‐10). Tissue culture supernatants were assayed for NO levels after 24 h in culture. NO was not observed in tissue culture supernatants of RAW 264.7 cells following stimulation with Pg‐LPS, but was observed after stimulation with St‐LPS. Exogenous l‐arginine restored the ability of Pg‐LPS to induce NO production; however, the increase in NO levels of cells stimulated with Pg‐LPS with exogenous l‐arginine was abolished by NMMA. IFN‐γ induced independent NO production by Pg‐LPS‐stimulated macrophages and this stimulatory effect of IFN‐γ could be completely suppressed by anti‐IFN‐γ antibodies and IL‐10. These results suggest that Pg‐LPS is able to stimulate NO production in the RAW264.7 macrophage cell model in an l‐arginine‐dependent mechanism which is itself independent of the action of IFN‐γ.
Inhibitory effect of green tea catechins on cysteine proteinases in Porphyromonas gingivalis
Authors: Okamoto M. , Sugimoto A. , Leung K. ‐P. , Nakayama K. , Kamaguchi A. , Maeda N.
Published: December 2004

The purpose of this study was to examine the effects of catechins and their derivatives on the activities of Arg‐gingipain (Rgp) and Lys‐gingipain (Kgp) in Porphyromonas gingivalis. Catechin derivatives, which included (−)‐epigallocatechin gallate, (−)‐epicatechin gallate, (−)‐gallocatechin gallate, and (−)‐catechin gallate, significantly inhibited the Rgp activity. The 50% inhibitory concentrations (IC50s) of these catechin derivatives for Rgp ranged from 3 to 5 μm. While (−)‐epigallocatechin and (−)‐gallocatechin moderately inhibited Rgp activity (IC50s, 20 μm), (−) ‐epicatechin, (+)‐catechin, and gallic acid were not effective, with IC50s greater than 300 μm. Further, some of the catechin derivatives tested also inhibited the Kgp activity, though to a lesser extent than inhibition of the Rgp activity. These findings suggest that green tea catechins may have the potential to reduce periodontal breakdown resulting from the potent proteinase activity of P. gingivalis.
Differential cytokine induction by two types of Porphyromonas gingivalis
Authors: Sugano N. , Ikeda K. , Oshikawa M. , Sawamoto Y. , Tanaka H. , Ito K.
Published: December 2004

The presence of Porphyromonas gingivalis with type II fimA is strongly associated with adult periodontitis. However, the importance of specific fimA types in the immune response is unknown. Two types of P. gingivalis (type I and type II) and Actinomyces naeslundii were assessed for their degree of cytokine induction in the macrophage‐like human cell line U937. Real‐time reverse transcriptase polymerase chain reaction was used to determine mRNA expression of 12 cytokines. Significant levels of interleukin (IL)‐8 induction and a similar cytokine expression pattern were observed at 6 h postinfection for all three bacterial strains. However, type II P. gingivalis infection showed statistically higher levels of IL‐1β, IL‐8, IL‐12 and tumor necrosis factor‐α mRNA induction than those of control at 24 h postinfection, whereas type I P. gingivalis and A. naeslundii showed no significant induction of these cytokines. These data suggest that compared with A. naeslundii and type I P. gingivalis, type II P. gingivalis prolongs the cytokine response. Although other factors may also be involved, the sustained cytokine response induced by type II P. gingivalis may play an important role in enhanced periodontal tissue inflammation and destruction.
The effect of oxygen on the growth and physiology of Porphyromonas gingivalis
Authors: Diaz P. I. , Rogers A. H.
Published: December 2004

Oxygen constitutes a constant challenge for the survival of strict anaerobes in the oral environment. The aim of this study was to investigate the effect of oxygen on the physiology and growth of Porphyromonas gingivalis in a continuous culture system when grown under conditions of hemin limitation and excess. Results showed that, when grown in the presence of hemin at 0.5 mg/l, P. gingivalis could tolerate low levels of oxygen, being able to reach steady‐state when 6% oxygen was present in the incoming gas mixture. When the hemin concentration was increased to 5 mg/l, the culture tolerated 10% oxygen. Anaerobically‐grown cells were coccoid in shape, whereas those grown in the presence of oxygen were bacillary. Acetate was the predominant end‐product in cultures grown in the presence of oxygen or in cultures hemin‐limited. Despite some changes in the activity of Arg‐ and Lys‐gingipain, most of the proteolytic activity was retained in the presence of oxygen. Activity of each of the three anti‐oxidant enzymes tested (NADH oxidase, NADH peroxidase and SOD) was detected under all conditions and usually increased under oxygenated environments. Higher activities were also seen in the hemin‐limited cultures. These results show some of the changes that occur in the physiology of P. gingivalis as a result of oxidative stress and confirm that hemin has a protective effect on the growth of the microorganism in the presence of oxygen.
Roles of Streptococcus mutans dextranase anchored to the cell wall by sortase
Authors: Igarashi T. , Asaga E. , Goto N.
Published: December 2004

In order to clarify the role that sortase (SrtA) plays in anchoring dextranase (Dex) to the cell wall of Streptococcus mutans, both Dex− and SrtA− mutants were constructed by insertional inactivation of the respective genes. Western blot analysis with a Dex antiserum showed that in the srtA mutant the Dex was not bound to the cell wall but was secreted into the culture supernatant. In contrast, in the wild type, Dex remained cell‐wall‐associated. Biological properties of the srtA mutant were examined in dextran fermentation, colony morphology and adherence to a smooth surface. The srtA mutant, as well as the wild type, retained the ability to ferment dextran. However, the colony morphology of the srtA mutant on Todd Hewitt agar containing sucrose was much larger than that of the wild type and showed a ring‐like structure. In addition, the srtA mutant was more adhesive to a smooth surface than the wild type when sucrose was present. However, the adhesion of the srtA mutant remarkably decreased by addition of exogenous dextranase. These studies suggest that the SrtA mediates Dex‐anchoring to the cell wall in S. mutans, and cell wall‐anchored Dex plays a role in controlling both the adhesive properties of extracellular glucan and the ability to utilize extracellular glucan as a nutrient source. In contrast, extracellular Dex is only responsible for degrading extracellular glucan as a nutrient source.
Identification of γδT lymphocytes in human periapical lesions
Authors: McCutcheon J. A. , Yee H. , Hayashi R. , Licari B. , Lombardo D. , Rosenberg P. A. , Phelan J.
Published: December 2004

Endodontic (root canal) therapy is required when the pulp of a tooth becomes necrotic due to a bacterial infection or trauma. A proportion of patients who receive endodontic therapy subsequently have periapical (around the tooth root) lesions detected by radiolucency. Currently, there are no means to identify susceptible patients. Although tissue from periapical lesions has been described as inflammatory, inflammatory cell types and their functions have been poorly characterized. For example, T lymphocytes were identified using pan specific anti‐CD3 mAb, which recognizes both αβ and γδT cells. Using the current model of γδT cells as immunoregulatory cells; γδT cells can mediate protective or destructive milieus. We postulated that patients who have a periapical lesion, as identified by radiographic bone loss, mount a γδT cell response. We collected specimens removed by surgery from both periapical lesions and other oral tissues, generated total RNA and performed reverse‐transcriptase polymerase chain reaction to identify rearranged δ genes. Results were confirmed with semi‐nested polymerase chain reaction. In addition, we demonstrate that these lesions contain a population of CD3+ cells that are αβT cell receptor negative, implying that these cells are γδT cells. Here we show that 36/37 of periapical lesions and only 2/11 of other lesions contain γδT cells (P < 0.0001). Vδ2+ T cells were the most common subtype identified (30/36) in these samples. This is the first report in the literature of the presence of γδT cells in human periapical lesions.
The prevalence of atopic disorders in children with chronic otitis media with effusion
Authors: Alles Roshini , Parikh Abhi , Hawk Lorna , Darby Yvonne , Romero Jose Navas , Scadding Glenis
Published: December 2004

We studied 209 children, referred to a multi‐disciplinary ‘Glue ear/Allergy’ clinic at our hospital with a history of chronic or recurrent otitis media with effusion (OME), in order to determine the prevalence of atopic disease in this population. Referrals were made either from within the hospital by Ear, Nose and Throat (ENT) Surgeons and Audiological Physicians (internal), or by General Practitioners (external). Assessment of atopic status was based on medical history, physical examination, nasal smears and skin‐prick testing (SPT) in all children; and on blood eosinophil counts and total immunoglobulin E (IgE) levels in a randomly selected subset. The main outcome measures were number of children with rhinitis, asthma, eczema, positive SPT, raised IgE level (> 100 IU/l), and nasal and blood eosinophilia. We found allergic rhinitis in 89%, asthma in 36%, and eczema in 24%. SPTs were positive to one or more of eight common inhalant aeroallergens in 57% of children. Blood tests in the selected subset revealed eosinophilia in 40% and a raised serum IgE in 28%. The worldwide prevalence of allergic rhinitis in children has been estimated to be 20%. The 89% prevalence found in this study is very high and there are a number of reasons which suggest that there may be a causal relationship. Whole‐population studies will be required to confirm these findings, which could have important therapeutic implications for OME.
How mitochondrial DNA diversity can help to understand the dynamics of wild‐cultivated complexes. The case ofMedicago sativain Spain
Authors: Muller M. H. , Prosperi J. M. , Santoni S. , Ronfort J.
Published: December 2004

In order to clarify the relationships (genetic exchange and shared ancestry) between natural and cultivated populations of alfalfa (Medicago sativaL.) in Spain, we investigated the patterns of mitochondrial DNA variation (characterized through restriction fragment length polymorphism) for 248 individuals in seven natural and six cultivated populations of this species. Mitochondrial variation was evidenced in both natural and cultivated populations ofM. sativa. Among the seven mitotypes idengified in the species, two were specific of the natural populations, a result attesting the fact that the Spanish wild form ofM. sativais an original genetic pool compared to the cultivated one. Other mitotypes were observed in both natural and cultivated populations, suggesting the occurrence of gene flow through seeds from cultivated towards natural populations. Comparisons with previously gathered nuclear and phenotypic data give insights into the different evolutionary forces acting on the different kinds of Spanish natural populations examined so far.
A hybrid zone between hydrothermal vent mussels (Bivalvia: Mytilidae) from the Mid‐Atlantic Ridge
Authors: O’Mullan G. D. , Maas P. A. Y. , Lutz R. A. , Vrijenhoek R. C.
Published: December 2004

This study provides the first example of a hybrid zone between animal taxa distributed along the mid‐ocean ridge system. We examined the distribution and genetic structure of deep‐sea hydrothermal vent mussels (Bivalvia: Mytilidae) along a 2888‐km portion of the Mid‐Atlantic Ridge between 37°50′ N and 14°45′ N latitude. Mitochondrial DNA (mtDNA), allozymes and multivariate–morphometric evidence discriminated between individuals of a northern species,Bathymodiolus azoricus,and a southern species,B. puteoserpentis,that were separated by an intermediate ridge segment almost devoid of mussels. A small sample of mussels from Broken Spur, a vent locality along this intermediate zone, revealed a mixed population with gene frequencies and morphology that were broadly intermediate to those of the northern and southern species. Multilocus clines in mtDNA and allozyme frequencies were centred over the intermediate zone. We consider intrinsic and extrinsic processes that might limit genetic exchange across this hybrid zone.
MHC class II beta sequence diversity in the deer mouse (Peromyscus maniculatus): implications for models of balancing selection
Authors: Richman A. D. , Herrera L. G. , Nash D.
Published: December 2004

We studied population polymorphism at a major histocompatibility complex (MHC) class II beta gene in the deer mouse (Peromyscusmaniculatus). We found that: (i) a single population ofP. maniculatushas significantly higher levels of DNA and protein sequence diversity than worldwide samples from homologous genes in other taxa, including humans and mice; and (ii) the genealogy of allelic sequences inP. maniculatusdeviates significantly from theoretical expectation under a model of symmetric balancing selection, in that alleles are relatively more divergent than expected. We suggest that the observation of high levels of pairwise allelic sequence divergence and deviation of the genealogy from theoretical expectation inP. maniculatustogether provide support for a divergent allele advantage model for the maintenance of MHC polymorphism.
Source population of dispersing rock‐wallabies (Petrogale lateralis) idengified by assignment tests on multilocus genotypic data
Authors: Eldridge M. D. B. , Kinnear J. E. , Onus M. L.
Published: December 2004

The ability to confidently idengify or exclude a population as the source of an individual has numerous powerful applications in molecular ecology. Several alternative assignment methods have recently been developed and are yet to be fully evaluated with empirical data. In this study we tested the efficacy of different assignment methods by using a translocated rock‐wallaby (Petrogale lateralis) population, of known provenance. Specimens from the translocated population (n = 43), its known source population (n = 30) and four other nearby populations (n = 19–32) were genotyped for 11 polymorphic microsatellite loci. The results idengified Bayesian clustering, frequency and Bayesian methods as the most consistent and accurate, correctly assigning 93–100% of individuals up to a significance threshold of P = 0.01. Performance was variable among the distance‐based methods, with the Cavalli‐Sforza and Edwards chord distance performing best, whereas Goldstein et al.’s (δµ)2 consistently performed poorly. Using Bayesian clustering, frequency and Bayesian methods we then attempted to determine the source of rock‐wallabies which have recently recolonized an outcrop (Gardners) 8 km from the nearest rock‐wallaby population. Results indicate that the population at Gardners originated via a recent dispersal event from the eastern end of Mt. Caroline. This is only the second published record of dispersal by rock‐wallabies between habitat patches and is the longest movement recorded to date. Molecular techniques and methods of analysis are now available to allow detailed studies of dispersal in rock‐wallabies and should also be possible for many other taxa.
Prey specialization may influence patterns of gene flow in wolves of the Canadian Northwest
Authors: Carmichael L. E. , Nagy J. A. , Larter N. C. , Strobeck C.
Published: December 2004

This study characterizes population genetic structure among grey wolves (Canis lupus) in northwestern Canada, and discusses potential physical and biological determinants of this structure. Four hundred and ninety‐one grey wolves, from nine regions in the Yukon, Northwest Territories and British Columbia, were genotyped using nine microsatellite loci. Results indicate that wolf gene flow is reduced significantly across the Mackenzie River, most likely due to the north–south migration patterns of the barren‐ground caribou herds that flank it. Furthermore, although Banks and Victoria Island wolves are genetically similar, they are distinct from mainland wolf populations across the Amundsen Gulf. However, low‐level island–mainland wolf migration may occur in conjunction with the movements of the Dolphin‐Union caribou herd. Whereas previous authors have examined isolation‐by‐distance in wolves, this study is the first to demonstrate correlations between genetic structure of wolf populations and the presence of topographical barriers between them. Perhaps most interesting is the possibility that these barriers reflect prey specialization by wolves in different regions.
Social and genetic characteristics of geographically isolated populations in the antFormica cinerea
Authors: Goropashnaya A. V. , Seppä P. , Pamilo P.
Published: December 2004

The antFormica cinereain northern Europe has geographically isolated populations that were examined using five microsatellite loci. The populations differ widely regarding the social organization of colonies. Based on genetic relatedness (r) among worker nest mates, the populations were classified as M type with monogynous (single queen) colonies (r > 0.59), as P type with polygynous colonial networks (r < 0.1), or as intermediate with weakly polygynous colonies (0.16 < r < 0.47). The social types showed weak geographical clustering, but the overall distribution indicated that the shift between the social types has occurred several times. The geographically isolated populations had slightly reduced levels of genetic diversity compared to populations from areas where the species is abundant and continuously distributed. Many of the isolated populations consisted of monogynous or weakly polygynous colonies, making their effective population sizes small, and some of them also showed weak bottleneck effects. The overall level of microsatellite diversity within populations was relatively high and differentiation among populations low, indicating recent connections. Isolation of populations may thus be a new phenomenon resulting from reduction of suitable habitats. At the local level, we obtained limited support from a group of nearby subpopulations in southern Finland to the hypothesis that the P type is connected to restricted dispersal. Other P type populations did not, however, show similar elevated levels of differentiation.
Relationships between fish assemblages, macrophytes and environmental gradients in the Amazon River floodplain
Authors: Petry P. , Bayley P. B. , Markle D. F.
Published: December 2004

During the flood season of 1992–1993, 139 species of fishes were collected from a floodplain lake system in the central Amazon Basin. Fish species distribution was examined relative to abiotic variables in seven vegetation strata on Marchantaria Island, Solimões River. Both environmental variables and species distributions were influenced by a river channel to floodplain‐interior gradient. Species diversity was significantly higher in vegetated areas than in unvegetated areas, with deeper water Paspalum repens stands harbouring the highest diversity. As a result, species richness and catches were positively related to habitat complexity, while catch was also negatively related to dissolved oxygen (DO) and water depth. Low DO and shallow waters appeared to act as a refuge from predation. Fish assemblages were related to water chemistry, but species richness was not. Canonical correspondence analysis provided evidence that floodplain fish assemblages formed by the 76 most common species were influenced by physical variables, macrophyte coverage and habitat complexity, which jointly accounted for 67% of the variance of fish species assemblages. Omnivores showed no pattern relative to the river channel to floodplain‐interior gradient while detritivores were more likely to be found at interior floodplain sites and piscivores closer to the river. Piscivores could be further separated into three groups, one with seven species associated with free‐floating macrophytes in deep water, a second with five species found in shallow waters with rooted grasses and a third with six open water orientated species. The results suggest that fish assemblages in the Amazon floodplain are not random associations of species.
Hypoxia tolerance of the mummichog: the role of access to the water surface
Authors: Stierhoff K. L. , Targett T. E. , Grecay P. A.
Published: December 2004

Low dissolved oxygen (DO) had a significant effect on specific growth rate (GS), length increment (IL) and haematocrit (Hct) of the mummichog Fundulus heteroclitus. Regardless of access to the water surface, F. heteroclitus maintained high growth rates (GS and IL) at DO concentrations as low as 3 mg O2 l−1. With access to the water surface, both GS and IL of F. heteroclitus decreased by c. 60% at 1·0 mg O2 l−1 compared to all higher DO treatments. When denied access to the water surface, a further decrease in GS (c. 90%) and IL (c. 75%) was observed at 1 mg O2 l−1. There was no effect of diel‐cycling DO (1–11 mg O2 l−1) with or without surface access on GS, IL or Hct of F. heteroclitus. Similar trends between GS and faecal production across DO treatments suggest that decreased feeding contributed significantly to the observed decrease in growth rate. Haematocrit was significantly elevated at 1 mg O2 l−1 for fish with and without access to the water surface. Increased Hct, however, was not sufficient to maintain high GS or IL at severely low DO. When permitted to respire in the surface layer, however, F. heteroclitus was capable of maintaining moderate growth rates at DO concentrations of 1 mg O2 l−1 (c. 15% saturation). Although aquatic surface respiration (ASR) was not quantified in this study, F. heteroclitus routinely swam in contact with the water surface and performed ASR at DO concentrations ≤3 mg O2 l−1. No hypoxia‐related mortality was observed in any DO or surface access treatment for as long as 9 days. This study demonstrates that surface access, and thus potential for ASR, plays an important role in providing F. heteroclitus substantial independence of growth rate over a wide range of low DO conditions commonly encountered in shallow estuarine environments.
Comparison of growth, feeding and cortisol level in Platichthys bicoloratus juveniles between estuarine and nearshore nursery grounds
Authors: Yamashita Y. , Tominaga O. , Takami H. , Yamada H.
Published: December 2004

Growth rates estimated using the RNA : DNA ratio of field‐collected stone flounder Platichthys bicoloratus juveniles were significantly higher in fish from brackish estuarine sites than from exposed nearshore sites. Gut fullness of juveniles indicated that prey availability was higher in estuaries than in nearshore areas. The whole‐body cortisol concentration of juveniles, however, was approximately four times higher in fish from the estuarine than the nearshore sites. This elevated cortisol level was possibly attributable to the much higher fluctuations in temperature and salinity over a diel cycle than in nearshore habitats where both environmental factors were comparatively stable. High growth rates, feeding conditions and densities of stone flounder juveniles in estuarine nursery grounds indicated that the elevated cortisol level was within a physiologically tolerable range and did not impair fish performance.