Genome Sequences and Structures of Two Biologically Distinct Strains of <Emphasis Type="Italic">Grapevine leafroll</Emphasis>-<Emphasis Type="Italic">associated virus</Emphasis> 2 and Sequence Analysis

Grapevine leafroll-associated virus 2 (GLRaV-2), a member of the genus Closterovirus within Closteroviridae, is implicated in several important diseases of grapevines including “leafroll”, “graft-incompatibility”, and “quick decline” worldwide. Several GLRaV-2 isolates have been detected from different grapevine genotypes. However, the genomes of these isolates were not sequenced or only partially sequenced. Consequently, the relationship of these viral isolates at the molecular level has not been determined. Here, we group the various GLRaV-2 isolates into four strains based on their coat protein gene sequences. We show that isolates “PN” (originated from Vitis vinifera cv. “Pinot noir”), “Sem” (from V. vinifera cv. “Semillon”) and “94/970” (from V. vinifera cv. “Muscat of Alexandria”) belong to the same strain, “93/955” (from hybrid “LN-33”) and “H4” (from V. rupestris “St. George”) each represents a distinct strain, while Grapevine rootstock stem lesion-associated virus.     

Mechanical properties of arteries cryopreserved at −80 °C and −150 °C

A new protocol for cryopreservation of arteries frozen at −80 °C was compared to the reference protocol for cryopreservation at −150 °C and to freshly harvested arteries. The aim of the study is to evaluate both protocols as global procedures to freeze and thaw arteries commonly used in tissue banks. Changes in mechanical properties of rabbit common carotid arteries were studied. Vascular segments were tested in vitro under dynamics loading conditions. Pressure and diameter were recorded simultaneously by a high fidelity transducer and an echotracking device, respectively. The pressure–diameter relationship was fitted by the arctangent Langewouters’ model and the arterial thickness was derived from histological measurements. Histological sections showed that the fresh and −80 °C groups were less damaged by hemodynamic load and histological preparation than the −150 °C group (p < 0.05). No differences between fresh and cryopreserved arteries regarding the structural (diameter, intimal-media thickness) and mechanical parameters (distensibility, circumferential stress, elastic modulus) were found. The isobaric circumferential stress was reduced in frozen arteries. These results demonstrate that the cryopreservation at −80 °C preserves the histological structure and mechanical properties better than the cryopreservation at −150 °C, suggesting that the new cryopreservation protocol at −80 °C is a method of choice for treating vessel replacement in vascular surgery.     

Splenocyte response to T cell-derived cytokines in thymectomized Xenopus

A miniaturized, “hanging-drop” bioassay reveals that splenocytes from earlythymectomized (Tx) Xenopus can respond (by enhanced thymidine incorporation) to thymicdependent “cytokines” generated in PHA- or alloantigen-stimulated cultures. Preliminary evidence, using fluorescence activated cell sorting, indicates that surface IgM⁻ splenocytes, rather than sIgM⁺ cells, from Tx toads are sensitive to the crude, splenocyte-derived, active supernatants. Although these responsive cells display residual, but low, reactivity to PHA, their thymus independence is suggested by flow cytometric observations using the anti-T cell monoclonal antibody XT-1. The development of “T-like” cells in Tx Xenopus is discussed.     

Anti-nuclear antibody reactivity in lupus may be partly hard-wired into the primary B-cell repertoire

When monoclonal ANAs and non-ANAs generated from a genetically simplified mouse model of lupus, B6.Sle1, were recently compared, the ANAs exhibited three sequence motifs in their immunoglobulin heavy chains, including increased cationicity in CDR3 (“motif A”), reduced anionicity in CDR2 (“motif B”) and increased aspartate at H50 (“motif C”). The present study was designed to elucidate the extent to which these ANA-associated sequence motifs might be hard-wired into the primary B-cell repertoire in lupus. The immunoglobulin heavy chain sequence of total splenic B-cells, follicular B-cells and marginal zone B-cells from B6.Sle1 congenic mice and C57BL/6 controls were amplified by single-cell PCR and compared. Analysis of the primary immunoglobulin heavy chain repertoire indicated that the first two sequence motifs “A” and “B” were already encoded in the naïve repertoire of B6.Sle1^z mice, whereas the third motif “C” was introduced in part by somatic mutation. Site-directed mutagenesis confirmed that non-anionic CDR2 and cationic CDR3 residues in the immunoglobulin heavy chain facilitated nuclear antigen binding in concert, whereas aspartate at H50 strongly vetoed DNA-binding, while preserving nucleosome reactivity. Hence, anti-nuclear antibodies appear to arise as a consequence of two distinct processes—genetically programmed selection of specific CDR charge motifs into the primary immunoglobulin repertoire, with secondary contribution from somatic mutation. Polymorphisms in the lupus susceptibility gene Ly108 that impair central B-cell tolerance may be mechanistically responsible for these early repertoire differences in lupus.     

“Low-risk” and “High-risk” HPV-infection and K-ras Gene Point Mutations in Human Cervical Cancer: A Study of 31 Cases

To analyze the coexistence of human papilloma virus (HPV) infection and K-ras gene activation in cervical neoplasia, we investigated 31 (seven pre-invasive and 24 invasive) cervical carcinomas for “low-risk” (types 6 and 11) and “high-risk” (types 16 and 18) HPVs and K-ras point mutations using PCR-based technology. “Low-risk” HPVs were not detected in the group investigated; however, 20 of 31 (64%) cases were HPV 16 positive, while HPV 18 was found in only three (9.7%) samples (HPV 6/11 v. HPV 16/18, p < 0.0001; HPV 16 v. HPV 18, p < 0.0001; Fisher's exact test). There was a K-ras codon 12 point mutation in two of 31 (6.4%) neoplasms, with none of the cases showing a K-ras codon 13 point mutation. Two moderately differentiated squamous carcinomas showed K-ras exon 2 gene alterations. Interestingly, none of the pre-invasive cervical carcinomas displayed K-ras gene point mutations. The mean patient age did not differ significantly in the number of HPV-positive and -negative cases. A coexistence of “high-risk” human papillomavirus DNA with K-ras gene alterations was observed in three of 31 (9.7%) neoplasms (one IIA and two IB moderately differentiated cervical carcinomas). Our results suggest that “high-risk” HPVs coexist with K-ras gene alterations in a subset of moderately differentiated carcinomas of the cervix uteri.     

Comparison of antigenic and allergenic composition of two partially puriflied extracts from Dermatophagoïdes farinae and Dermatophagoïdes pteronyssinus mite cultures

Dermatophagoïdes farinae (Df 80d) and Dermatophagoïdes pteronyssinus (Dp 80d) extracts were analyzed,for their antigenic and allergenic composition by means of crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE). By CIE, II antigens could be numbered in Df 80d (Df I to Df II) and seven antigens in Dp 80d (Dp I to Dp 7). This technique allowed us also to define antigens with common as well as specific parts for the two mite species. Among the antigens of D. farinae and D. pteronyssinus, only the antigen corresponding to DO and Dp 5 seems to bear common epitopes to the two mite species, whereas Df 6 and Dp 4 appear to, bear, respectively, specific epitopes 4f each species. Moreover, Df lI appears to bear specific epitopes of D. farinae, although it shows a partial identity with Dp 7. By CRIE, on 20 mite-sensitive patients' sera, we identified, for each mite extract, the allergens responsive to human specific IgE. The allergograms show that the majority of mite-sensitive patients react with Df 11 and Df 6 and with Dp 7 and Dp 4. Tines, these antigens can be considered as major allergens. The minor allergens were also identified. None of these antigens was recognized by the control sera. Moreover, we observed that, for one antigen (antigen 5) there exist antigenic determinants common to the two species of mites toward the rabbit serum and specific allergenic determinants to the human IgE response. A significant correlation was found between the specific IgE binding in CRIE and in RAST (Spearman coefficient: “rs” = 0.61 p < 0.0l ,for Df; “rs” = 0.78 p < 0.01 for Dp).     

Significance of a “hot patella” in total knee replacement without primary patellar resurfacing

A “hot patella” is a bone scan finding of increased tracer uptake in the patella, greater than the ipsilateral distal femur or the proximal tibia. Increased patellar uptake on the bone scans is a relatively frequent finding; this is often not commented upon. The aim of our study was to define the incidence of a “hot patella” on bone scans following total knee replacements with ongoing symptoms unrelated to sepsis. We wanted to investigate the correlation between the adverse retropatellar symptomatology following total knee replacement with bone scan findings.We undertook a retrospective study of 55 symptomatic total knee replacement patients, who had bone scans. The incidence of a “hot patella” in patients without primary patellar resurfacing was 51% (28/55). Ninety-five percent patients with anterior knee pain and 21% patients with diffuse knee pain had a “hot patella”. Statistical analysis showed a positive association between “hot patella” and anterior knee pain (p value of < 0.001). The patients with “hot patella” who underwent secondary patellar resurfacing had symptomatic relief of symptoms.Our study has shown that the finding of a “hot patella” on a bone scan in patients with anterior knee pain following total knee replacement suggests a problem related to the patellofemoral joint. This study would appear to indicate that a “hot patella” in a patient with clinically defined anterior knee pain is likely to benefit from secondary patellar resurfacing.     

Canine airway responses to acetylcholine, prostaglandin F2α, histamine, and serotonin after chronic antigen exposure

Dose-response curves were obtained for aerosols of acetylcholine (ACh), prostaglandin F _2α (PGF_2α), histamine (H), and 5-hydroxytryptamine (5-HT) on pulmonary resistance (Rp) and dynamic lung compliance (Cdyn) in Ascaris-hypersensitive dogs. Previously, these animals had been subjected to chronic biweekly “allergic asthmatic” episodes by aerosol administration of Ascaris antigen. When examined either one week before or after antigen provocation the airways were not hyperreactive to ACh, H, or 5-HT but did demonstrate a modest hyperreactivity to PGF _2α. When aerosol dose-response curves were obtained for these agonists immediately following an “allergic asthmatic” episode, the airways were hyporeactive to PGF_2α H, and 5-HT, but not to ACh. Studies with atropine indicated that the hyporeactivity was the result of decreased airway responsiveness to both direct and indirect effect of PGF_2α and H. It is concluded that, in dogs, chronic antigen challenge is not accompanied by a general increase in airway reactivity to pharmacologic agents.     

Les supports épidémiologiques de l'hémovigilance

“Hémovigilance” is a continuous and standardized system for data collection and analysis, and diffusion of results to clinical and public health decisionmakers. This system has different objectives than clinical and epidemiological research on labile blood products. A published study of characteristics of donnors that are infected by the human immunodeficiency virus illustrates the need for information on temporal patterns of events relevant to transfusion safety. Two cohort studies on post-transfusion hepatitis and a randomized study on cytomegalovirus infections illustrate the importance of surveillance for detecting events that are causally linked to transfusion. The interest of “hémovigilance” is also discussed in the context of epidemics and new hazards, when surveillance is the only possibility of designing ad-hoc epidemiologic studies, yielding results within delays compatible with decisions that are timely and effective for improving transfusion safety.     

Light Hygiene: Time to make preventive use of insights – old and new – into the nexus of the drug light, melatonin, clocks, chronodisruption and public health

Light is, clearly, a key to life on Earth and light, equally clearly, determines biological rhythmicity in organisms. Light does the latter by setting internal or endogenous clocks which allow a multitude of species, including man, to adjust their lives to changing external or environmental conditions. Critical changes over time occur from day to night and throughout the year. In this paper, we sum up how visible light provides electromagnetic information about environmental “time” via the ocular interface of newly discovered photoreceptive cells to a master clock in our brain, viz the suprachiasmatic nuclei [SCN], and how the SCN translate this input, with melatonin as a key biologic intermediary, into endogenous or biological time. We summarize experimental and epidemiological evidence suggesting how chronodisruption, a relevant disturbance of the temporal organization or order of physiology, endocrinology, metabolism and behaviour, is probably detrimental for human beings. On the basis of our synthesis, and in line with suggestions by other researchers voiced decades ago, light must, functionally, be considered as a drug equivalent. In this vein, the very timing, quality (wavelength), quantity (dose) and side effects, including chronodisruption, of light exposures can be critically important for health and disease in man. As a promising means to foster public health, we advocate an appropriate balance of exposures to the key Zeitgeber light in terms of “light hygiene”, implying strong and appropriate rather than weak and confusing temporal information. This focus on “light hygiene”, and thus on the key Zeitgeber light, does not mean to ignore that there are multiple entrainment pathways for our circadian clocks. Indeed, when dealing with light, chronodisruption and a multitude of adverse health effects, we ultimately need to consider Zeitgeber cues, and their possible interplay, beyond light alone. Confusions of the temporal programmes in humans can also stem from physical and social activities, stress and facets of food intake. And yet, since light possesses a rather unique and exclusive Zeitgeber role and in view of its ubiquitous nature, a specific, preventative focus on “light hygiene”, as a contribution to a general “Zeitgeber hygiene”, is warranted.     

Reforming the coroner and death certification service

The intention of the Home Office is to introduce a new system that combines an independent check on all deaths and a professional oversight of death patterns, with, for the majority of cases, the minimum of bureaucracy. No public consultation is intended, so that reforms are not delayed. However as the proposals are developed in the coming months, the details, practicalities and costs will be discussed “with relevant professionals (not defined) and those with experience of the existing arrangements”. The imperfections of the present system are outlined. It is hoped the medical profession will have an input, since statements such as “ ‘hospital post mortems’, which are for medical research and public health protection purposes” need amendment. There should be a change in perception of the audit value of this procedure to BOTH the family and the treating doctor. Unfortunately it is proposed the new system in total should cost no more than at present. “Professionals” (not defined) will be involved in the financial detail.All deaths, after verification and certification of the medical cause of death (if known) would then be referred to the ‘medical examiner’ based in the coroner's office. He/she would be a qualified doctor employed by the new coroner service and independent of the Health Service. The medical examiner could provide supplementary advice on medical matters required by the coroner. “Retention of tissue should only take place where absolutely necessary and the coroner and his or her other staff should take account of the needs of families and friends carefully throughout the process.” “Coroners could take advice from their medical examiner to ascertain and prescribe the minimum level of invasiveness to establish the cause of death.” This issue is far from resolved, as signified by a recent call from the DoH giving a grant to study the value of MRI versus a full post mortem. No thought is given to systemic diseases, which may present in one organ system or another disease process, other than that causing death. Medical examiners will have to keep abreast of current developments in medicine BUT will be outside the NHS, which could cause problems. Medical examiners will be appointed (with an input from Regional Directors of Public Health) and managed from within the coroner service. They would work closely with the registrar of births and deaths.Deaths from unnatural causes or when the medical cause of death is unknown will result in judicial inquests. The medical examiner will have an input into causes of death and relevant investigations.Details of the proposed structure of the system are given, as well as the investigative and other roles of Coroner's officers.The establishment of medico-legal centres, as “examples of good practice” is advocated. The drawbacks of this system are stressed in this paper. There is at present an on-going review of forensic pathology services and it is hoped thought will be given to the increasing trend for sub-specialisation in medicine.     

Unifying classical and molecular taxonomic classification: analysis of the Podoviridae using BLASTP-based tools

We defined phage genera by measuring genome relationships by the numbers of shared homologous/orthologous proteins. We used BLAST-based tools (CoreExtractor.vbs and CoreGenes) to analyze 55 fully sequenced bacteriophage genomes from the NCBI and EBI databases. This approach was first applied to the T7-related phages. Using a cut-off score of 40% homologous proteins, we identified three genera within the T7-related phages, redefined the φ29-related phages, and introduced five novel genera. The T7- and φ29-related phages were given subfamily status and named “Autographivirinae” and “Picovirinae”, respectively. Our results confirm and refine the ICTV phage classification, enable elimination of errors in public databases, and provide a straightforward tool for the molecular classification of new phage genomes.     

Allergic bronchopulmonary aspergillosis: Reactivity of IgE and IgG antibodies with antigenic components of Aspergillus fumigatus (IgE/IgG antigen complexes)

Sera of patients with ABPA were tested by XRIE tests incorporating their own serum (self-XRIE) to detect the presence of IgG/IgE antigen complexes to a “reference” Aspergillus fumigatus preparation. Of the 32 sera studied, 29 (90%) had visible precipitin (IgG) peaks, and 27 of these 29 as well as the three apparently precipitin-negative sera, i.e., 30 (94%), showed binding of specific IgE by autoradiography. The two precipitin-positive sera that did not show IgE binding were also skin test negative and RAST negative to this A. fumigatus antigen. Specific IgG as determined in ELISA correlated well with the grading of the XIE precipitin peaks (p < 0.05). There was also a highly significant correlation between specific IgE by RAST and grading the radioactive uptake seen in the autoradiograph (p < 0.001) indicating, for each serum, the presence of IgG antibodies to most of the components to which there was specific IgE. In the self-XRIE tests there was considerable variation of reactivity from serum to serum, in numbers of antigen/antibody peaks observed, in relative peak heights, and in the intensity of the respective staining. By comparing each test to a “reference” pattern developed with the use of an ABPA serum pool, the antigenic components of A. fumigatus were found to be of two main types: (1) antigens that appeared to be poorly precipitating (possibly low-molecular-weight components) but showed strong IgE binding (these were apparently major allergenic components and with one exception proved to be the faster migrating components) and (2) antigens that produced the strongest precipitin reactions with only weak binding of specific IgE and therefore minor allergenic components.     

Towards a unified theory for immunogenetic systems. IV. A complex-simple probe into the Ag system

The Ag system is a human β-lipoprotein polymorphism of medium complexity discussed in the present article mainly as a model for an analysis of the feedback processes between facts, artifacts and hypotheses. Two Caucasian population materials comprising a total of 530 unrelated individuals are explored at the six-reagent level. Through the new (complex-simple) conceptual framework (language and theory) these 6 × 530 or 3,180 experimental observables or information bits (plus(+) and minus(−) signs) are probed in a most sensitive manner. This probe also permits a perfectly transparent explanation for previously inexplicable-unnoticed pseudo-mysteries with regard to remarkable “clustering” and other relations of observed frequencies for (simple-complex) Ag “genes” and “antigens” within a specified population. The main ambition of this article is to introduce a new model and methodology permitting a more transparent discussion of the complex interrelations between “facts” or “visual sense data” and the (distorting) ability of reason to understand only that which it creates according to its own design. It is concluded that the serologic field (SEF) does indeed have considerable conceptual advantages over other scientific fields with regard to such fundamental epistemological studies concerning the growth of scientific knowledge. The present data illustrate the mutual feedbacks or dualisms between observation and hypotheses where one component has no meaningful existence without the other and where “facts” are not very trustworthy unless produced within a “guiding paradigm”.     

Segmentation de la fosse postérieure en IRM fœtale : application biométrique

Posterior fossa segmentation in fetal MR imaging: biometrical application.In order to complete ultrasound scanning and histological tests, magnetic resonance imaging is now considered for the study of prenatal development and fetal pathologies. Due to the recent implementation the quantitative data are still lacking, particularly concerning the central nervous system. Our study is dealing with fetal cerebral MR imaging, and, more precisely, with the biometrical analysis of the different structures of the posterior fossa. Its malformations and pathologies are often due to a trouble of “container-content” and could require a medical termination of pregnancy. So, the need of quantitative information about the relative growth of the different parts of the posterior fossa during the pregnancy is real. We have therefore developped a segmentation method based on region growing with pixel agregation, which has been tested on 67 sagital cross-section MR images. The good segmentation has permitted us to determine the regions of interest (posterior fossa, vermis, brain stem) and to calculate four relative surface measures between them. After technical and clinical validations, the analysis of our results shows that the relative development of these structures is constant and independent of the pregnancy term. It may thus be possible to find standard index for characterizing their relative growth.     

Patients with negative cervical biopsies after papanicolaou test interpretations of "atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion": comparative longitudinal follow-up

Approximately half of women with a Papanicolaou (pap) test interpretation of “atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion” (ASC-H) will have less than a high-grade dysplasia diagnosed on their follow-up cervical biopsy sample. Herein, we compared the follow-up findings in a group of women with a negative cervical biopsy after a pap test interpretation of ASC-H (study group) with those of a randomly selected control group of women whose pap tests were interpreted as “negative for intraepithelial lesion or malignancy” during the same period. The diagnoses in every follow-up sample in the control and study groups were tabulated. Patients with previous ASC-H or high-grade squamous intraepithelial lesion interpretations, and/or high-grade cervical intraepithelial neoplasia (CIN) diagnoses, were excluded. Both groups were then statistically compared regarding the diagnostic frequencies of each of the Bethesda 2001 categories and CIN grades. Of the 122 patients with ASC-H interpretations and documented histologic follow-up, the first follow-up biopsy was negative for dysplasia in 20 (16.4%). Seventy-six follow-up samples (70 pap tests, 6 biopsies) were obtained from these 20 patients. In the control group of 262 women with pap tests interpreted as “negative for intraepithelial lesion or malignancy,” 641 follow-up samples (629 pap tests, 12 biopsies) were obtained. Patients in the study group were significantly more likely than their control group counterparts to have a follow-up (1) cytologic and/or histologic abnormality (23/76 vs 76/641, respectively; P = .00006), (2) “atypical squamous cells of undetermined significance” (ASC-US) interpretation (13/76 vs 43/641; P = .005), and (3) CIN grade 1 diagnosis (4/76 vs 10/641; P = .05). Approaching statistical significance was the comparatively increased frequency of CIN grade 2 to 3 diagnoses in the study group (2/76 vs 2/641; P = .058). If the analysis is restricted to 1 follow-up sample (the most severe) per patient, patients in the study group were still more likely than those in the control group to have a follow-up cytologic and/or histologic abnormality (12/20 vs 40/262; P = .0002), ASC-US interpretation (6/20 vs 22/262; P = .008), and follow-up CIN grade 2 to 3 diagnosis (2/20 vs 2/262; P = .03). It is concluded that patients whose cervical biopsies are devoid of dysplasia after an ASC-H interpretation still require close surveillance and follow-up because their risk of being diagnosed with follow-up cervical abnormalities is significantly above baseline.