Analysis by confocal microscopy of the behavior of heat shock protein 70 within the nucleus and of a nuclear matrix polypeptide during prolonged heat shock response in HeLa cells.

By means of confocal laser scanning microscopy and indirect fluorescence experiments we have examined the behavior of heat-shock protein 70 (HSP70) within the nucleus as well as of a nuclear matrix protein (M(r) = 125 kDa) during a prolonged heat-shock response (up to 24 h at 42 degrees C) in HeLa cells. In control cells HSP70 was mainly located in the cytoplasm. The protein translocated within the nucleus upon cell exposure to hyperthermia. The fluorescent pattern revealed by monoclonal antibody to HSP70 exhibited several changes during the 24-h-long incubation. The nuclear matrix protein showed changes in its location that were evident as early as 1 h after initiation of heat shock. After 7 h of treatment, the protein regained its original distribution. However, in the late stages of the hyperthermic treatment (17-24 h) the fluorescent pattern due to 125-kDa protein changed again and its original distribution was never observed again. These results show that HSP70 changes its localization within the nucleus conceivably because it is involved in solubilizing aggregated polypeptides present in different nuclear regions. Our data also strengthen the contention that proteins of the insoluble nucleoskeleton are involved in nuclear structure changes that occur during heat-shock response.

Changes in nuclear 3,5,3'-triiodothyronine receptor expression in rat dorsal root ganglia and sciatic nerve during development: comparison with regeneration.

The action of the thyroid hormones on responsive cells in the peripheral nervous system requires the presence of nuclear triiodothyronine receptors (NT3R). These nuclear receptors, including both the alpha and beta subtypes of NT3R, were visualized by immunocytochemistry with the specific 2B3 monoclonal antibody. In the dorsal root ganglia (DRG) of rat embryos, NT3R immunoreactivity was first discretely revealed in a few neurons at embryonic day 14 (E14), then strongly expressed by all neurons at E17 and during the first postnatal week; all DRG neurons continued to possess clear NT3R immunostaining, which faded slightly with age. The peripheral glial cells in the DRG displayed a short-lived NT3R immunoreaction, starting at E17 and disappearing from the satellite and Schwann cells by postnatal days 3 and 7 respectively. In the developing sciatic nerve, Schwann cells also exhibited transient NT3R immunoreactivity restricted to a short period ranging from E17 to postnatal day 10; the NT3R immunostaining of the Schwann cells vanished proximodistally along the sciatic nerve, so that the Schwann cells rapidly became free of detectable NT3R immunostaining. However, after the transection or crushing of an adult sciatic nerve, the NT3R immunoreactivity reappeared in the Schwann cells adjacent to the lesion by 2 days, then along the distal segment in which the axons were degenerating, and finally disappeared by 45 days, when the regenerating axons were allowed to re-occupy the distal segment.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinct profiles of cytotoxic granules in memory CD8 T cells correlate with function, differentiation stage, and antigen exposure.

Cytotoxic CD8 T cells exert their antiviral and antitumor activity primarily through the secretion of cytotoxic granules. Degranulation activity and cytotoxic granules (perforin plus granzymes) generally define CD8 T cells with cytotoxic function. In this study, we have investigated the expression of granzyme K (GrmK) in comparison to that of GrmA, GrmB, and perforin. The expression of the cytotoxic granules was assessed in virus-specific CD8 T cells specific to influenza virus, Epstein-Barr virus (EBV), cytomegalovirus (CMV), or human immunodeficiency virus type 1 (HIV-1). We observed a dichotomy between GrmK and perforin expression in virus-specific CD8 T cells. The profile in influenza virus-specific CD8 T cells was perforin(-) GrmB(-) GrmA(+/-) GrmK(+); in CMV-specific cells, it was perforin(+) GrmB(+) GrmA(+) GrmK(-/+); and in EBV- and HIV-1-specific cells, it was perforin(-/+) GrmB(+) GrmA(+) GrmK(+). On the basis of the delineation of memory and effector CD8 T cells with CD45RA and CD127, the GrmK(+) profile was associated with early-stage memory CD8 T-cell differentiation, the perforin(+) GrmB(+) GrmA(+) profile with advanced-stage differentiation, and the GrmB(+) GrmA(+) Grmk(+) profile with intermediate-stage differentiation. Furthermore, perforin and GrmB but not GrmA and GrmK correlated with cytotoxic activity. Finally, changes in antigen exposure in vitro and in vivo during primary HIV-1 infection and vaccination modulated cytotoxic granule profiles. These results advance our understanding of the relationship between distinct profiles of cytotoxic granules in memory CD8 T cells and function, differentiation stage, and antigen exposure.

Differential distribution of thyroid hormone receptor isoform in rat dorsal root ganglia and sciatic nerve in vivo and in vitro.

Using autoradiographic techniques carried out under precise conditions we previously demonstrated that both sensory neurons and peripheral glial cells in dorsal root ganglia (DRG) or sciatic nerve, possess specific [125I]-labeled T3 binding sites. Thyroid hormone receptors (TR) include several isoforms (TR alpha(1), TR alpha(2), TR beta(1), TR beta(2...)) The present study demonstrates that while sensory neurons and peripheral glial cells both possess functional TR, they express a differential expression of TR isoforms. Using a panel of antisera to specific for the TR alpha-common (alpha(1) and alpha(2)), TR alpha-1 or TR beta-1 isoforms, we detected TRs isoform localization at the cellular level during DRG and sciatic nerve development and regeneration. Immunohistochemical analysis revealed that during embryonic life, sensory neurons express TR alpha-common and TR beta-1 rather than TR alpha-1. The number of TR alpha-common and TR beta-1 positive neurons as well as the intensity of labeling increased during the first two postnatal weeks and remained more or less stable in adult life. TR alpha-1 immunoreactivity, which was undetectable in embryonic sensory neurons, became discreetly visible in neurons after birth. In developing DRG and sciatic nerves, Schwann cells exhibited TR alpha-common and TR alpha-1 rather than TR beta-1 immunolabeling. The appearance of TR alpha-common and alpha-1 isoform immunoreactivity in the sciatic nerve was restricted to a short period ranging from E17 up to two postnatal weeks. By comparing TR alpha-common and TR alpha-1 immunostaining we can deduce that Schwann cells primarily express TR alpha-1. Afterwards, in adult rat sciatic nerve TR alpha isoforms was no more detected. However transection of sciatic nerve caused a reexpression of TR alpha isoforms in degenerating nerve. The prevalence of TR alpha in Schwann cells in vivo was correlated with in vitro results. The differential expression of TR alpha and beta by sensory neurons and Schwann cells indicates that the feedback regulation of circulating thyroid hormone could occur by binding to either the alpha or beta TR isoforms. Moreover, the presence of multiple receptor isoforms in developing sensory neurons suggests that thyroid hormone uses multiple signaling pathways to regulate DRG and sciatic nerve development.

Staff eye lens and extremity exposure in interventional cardiology: Results of the ORAMED project

Within the ORAMED project a coordinated measurement program for occupationally exposed medical staff was performed in different hospitals in Europe. The main objectives of ORAMED were to obtain a set of standardized data on doses for staff in interventional cardiology and radiology and to optimize staff protection. Doses were measured with thermoluminescent dosemeters on the ring finger and wrist of both hands, on legs and at the level of the eyes of the main operator performing interventional procedures. In this paper an overview of the doses per procedure measured during 646 interventional cardiology procedures is given for cardiac angiographies and angioplasties (CA/PTCA), radiofrequency ablations (RFA) and pacemaker and defibrillator implantations (PM/ICD). 31% of the monitored procedures were associated with no collective protective equipment, whereas 44% involved a ceiling screen and a table curtain. Although associated with the smallest air kerma - area product (KAP), PM/ICD procedures led to the highest doses. As expected, KAP and doses values exhibited a very large variability. The left side of the operator, most frequently the closest to the X-ray scattering region, was more exposed than his right side. An analysis of the effect of parameters influencing the doses, namely collective protective equipment, X-ray tube configuration and catheter access route, was performed on the doses normalized to KAP. Ceiling screen and table curtain were observed to reduce normalized doses by atmost a factor 4, much smaller than theoretical attenuation factors typical for such protections, i.e. from 10 to 100. This observation was understood as their inappropriate use by the operators and their non-optimized design. Configurations with tube above the patient led to higher normalized doses to the operator than tube below, but the effect of using a biplane X-ray suite was more complex to analyze. For CA/PTCA procedures, the upper part of the operator's body received higher normalized doses for radial than for femoral catheter access, by atmost a factor 5. This could be seen for cases with no collective protection. The eyes were observed to receive the maximum fraction of the annual dose limit almost as frequently as legs and hands, and clearly the most frequently, if the former 150 mSv and new 20 mSv recommended limits for the lens of the eye are considered, respectively.

Exposition au VIH, à l'hépatite B et C au cabinet médical et à l'hôpital. Prévention et prophylaxie postexpositionnelle [Exposure to HIV, hepatitis B and C in office practice and hospital. Prevention and post-exposure prophylaxis]

Suite à un accident exposant à du sang (piqûre; coupure), provenant d'un patient infecté, le risque d'infection par VIH est d'environ 0,3% et par le virus de l'hépatite C (VHC) d'environ 0,5%. Chez les personnes vaccinées avec une réponse immunitaire adéquate (titre d'anticorps HBs >100 mUI/ml), aucune infection professionnelle par hépatite B n'a été reconnue en Suisse. La plupart des infections par VIH et VHB peuvent être prévenues par un traitement d'urgence et une prophylaxie postexpositionnelle (PEP). Il n'y a actuellement aucune prophylaxie postexpositionnelle pour le VHC. En cas de transmission de VHC, un traitement rapide par peginterféron et ribavirine est à envisager. Chaque hôpital et cabinet médical doivent mettre sur pied un système pour assurer une prise en charge optimale et en urgence des blessures par piqûres ou coupures. Lors de blessures accidentelles avec du sang de patients séropositifs pour le VIH et dans des situations complexes, il est recommandé de consulter un médecin du personnel ou un infectiologue expérimenté. The risk of infection after an occupational needle stick injury with blood from an infected source patient is approximately 0.3% for HIV and 0.5% for hepatitis C virus (HCV). In Switzerland no cases of occupational HBV infection have been recorded in fully vaccinated persons with a documented adequate vaccine response (HBsantibody titer >100 mIU/mL). Most occupational HIV und HBV infections can be prevented by appropriate emergency measures and post-exposure prophylaxis (PEP). No HCV-PEP is currently available. Early therapy with peginterferon and ribavirin should be considered in cases of occupational HCV seroconversion. Every hospital and office practice should establish a system for 24 h/24 h emergency management of occupational needle stick injuries. In the setting of an HIV-seropositive source patient and in complex situations, early consultation with a specialist in occupational medicine or infectious diseases should be considered.

The clinical profile of idiopathic and cat scratch neuroretinitis: who is at risk for recurrence?

Background: Most cases of neuroretinitis (NR) are idiopathic or due to cat scratch disease and occur as a single episode but a subgroup of patients experience recurrent attacks with cumulative visual loss. We reviewed our cases of NR to better characterize the clinical features of these subgroups in an effort to predict the risk of recurrence. Methods: Retrospective study of NR patients from a single institution. Sixty-seven patients were divided into three groups: 22 cases due to cat scratch disease (CSD-NR), 24 with idiopathic neuroretinitis (I-NR) and 21 (23 eyes) with recurrent neuroretinitis (R-NR). Results: Preceding systemic symptoms, predominantly central visual field (VF) loss and the combination of poor acuity with small relative afferent pupillary defect at presentation were common features of CSD-NR. There were no cases of recurrent CSD-NR. In the first attack of R-NR, the magnitude of VF loss at presentation was greater compared to the other two groups. While 39% of R-NR had a pattern of VF loss other than a central or cecocentral scotoma, only 13.6% of CSD-NR and 17% of I-NR showed this pattern. Visual recovery was least substantial for the R-NR group (average gain of 3.7 lines of Snellen acuity vs. 5 and 6.4 lines for CSD-NR and I-NR, respectively, and an average gain in VF score of 5.1 in the R-NR group compared to 8.2 and 11.5 for the other two groups). Conclusion: The main predictive factors for recurrence are absence of systemic symptoms, significant VF loss at presentation, particularly loss outside the central 30°, and less substantial visual recovery.

Effects of a 5-h hilly running on ankle plantar and dorsal flexor force and fatigability

This study aimed to examine the effects of a 5-h hilly run on ankle plantar (PF) and dorsal flexor (DF) force and fatigability. It was hypothesised that DF fatigue/fatigability would be greater than PF fatigue/fatigability. Eight male trail long distance runners (42.5 ± 5.9 years) were tested for ankle PF and DF maximal voluntary isokinetic contraction strength and fatigue resistance tests (percent decrement score), maximal voluntary and electrically evoked isometric contraction strength before and after the run. Maximal EMG root mean square (RMS(max)) and mean power frequency (MPF) values of the tibialis anterior (TA), gastrocnemius lateralis (GL) and soleus (SOL) EMG activity were calculated. The peak torque of the potentiated high- and low-frequency doublets and the ratio of paired stimulation peak torques at 10 Hz over 100 Hz (Db10:100) were analysed for PF. Maximal voluntary isometric contraction strength of PF decreased from pre- to post-run (-17.0 ± 6.2%; P < 0.05), but no significant decrease was evident for DF (-7.9 ± 6.2%). Maximal voluntary isokinetic contraction strength and fatigue resistance remained unchanged for both PF and DF. RMS(max) SOL during maximal voluntary isometric contraction and RMS(max) TA during maximal voluntary isokinetic contraction were decreased (P < 0.05) after the run. For MPF, a significant decrease for TA (P < 0.05) was found and the ratio Db10:100 decreased for PF (-6.5 ± 6.0%; P < 0.05). In conclusion, significant isometric strength loss was only detected for PF after a 5-h hilly run and was partly due to low-frequency fatigue. This study contradicted the hypothesis that neuromuscular alterations due to prolonged hilly running are predominant for DF.

Distribution and anatomical localization of the glucose transporter 2 (GLUT2) in the adult rat brain--an immunohistochemical study.

The aim of this work was to study the distribution and cellular localization of GLUT2 in the rat brain by light and electron microscopic immunohistochemistry, whereas our ultrastructural observations will be reported in a second paper. Confirming previous results, we show that GLUT2-immunoreactive profiles are present throughout the brain, especially in the limbic areas and related nuclei, whereas they appear most concentrated in the ventral and medial regions close to the midline. Using cresyl violet counterstaining and double immunohistochemical staining for glial or neuronal markers (GFAp, CAII and NeuN), we show that two limited populations of oligodendrocytes and astrocytes cell bodies and processes are immunoreactive for GLUT2, whereas a cross-reaction with GLUT1 cannot be ruled out. In addition, we report that the nerve cell bodies clearly immunostained for GLUT2 were scarce (although numerous in the dentate gyrus granular layer in particular), whereas the periphery of numerous nerve cells appeared labeled for this transporter. The latter were clustered in the dorsal endopiriform nucleus and neighboring temporal and perirhinal cortex, in the dorsal amygdaloid region, and in the paraventricular and reuniens thalamic nuclei, whereas they were only a few in the hypothalamus. Moreover, a group of GLUT2-immunoreactive nerve cell bodies was localized in the dorsal medulla oblongata while some large multipolar nerve cell bodies peripherally labeled for GLUT2 were scattered in the caudal ventral reticular formation. This anatomical localization of GLUT2 appears characteristic and different from that reported for the neuronal transporter GLUT3 and GLUT4. Indeed, the possibility that GLUT2 may be localized in the sub-plasmalemnal region of neurones and/or in afferent nerve fibres remains to be confirmed by ultrastructural observations. Because of the neuronal localization of GLUT2, and of its distribution relatively similar to glucokinase, it may be hypothesized that this transporter is, at least partially, involved in cerebral glucose sensing.

Effects of a 5-h hilly running on ankle plantar and dorsal flexor force and fatigability.

This study aimed to examine the effects of a 5-h hilly run on ankle plantar (PF) and dorsal flexor (DF) force and fatigability. It was hypothesised that DF fatigue/fatigability would be greater than PF fatigue/fatigability. Eight male trail long distance runners (42.5 ± 5.9 years) were tested for ankle PF and DF maximal voluntary isokinetic contraction strength and fatigue resistance tests (percent decrement score), maximal voluntary and electrically evoked isometric contraction strength before and after the run. Maximal EMG root mean square (RMS(max)) and mean power frequency (MPF) values of the tibialis anterior (TA), gastrocnemius lateralis (GL) and soleus (SOL) EMG activity were calculated. The peak torque of the potentiated high- and low-frequency doublets and the ratio of paired stimulation peak torques at 10 Hz over 100 Hz (Db10:100) were analysed for PF. Maximal voluntary isometric contraction strength of PF decreased from pre- to post-run (-17.0 ± 6.2%; P < 0.05), but no significant decrease was evident for DF (-7.9 ± 6.2%). Maximal voluntary isokinetic contraction strength and fatigue resistance remained unchanged for both PF and DF. RMS(max) SOL during maximal voluntary isometric contraction and RMS(max) TA during maximal voluntary isokinetic contraction were decreased (P < 0.05) after the run. For MPF, a significant decrease for TA (P < 0.05) was found and the ratio Db10:100 decreased for PF (-6.5 ± 6.0%; P < 0.05). In conclusion, significant isometric strength loss was only detected for PF after a 5-h hilly run and was partly due to low-frequency fatigue. This study contradicted the hypothesis that neuromuscular alterations due to prolonged hilly running are predominant for DF.

Towards a consensus view on understanding nanomaterials hazards and managing exposure

The aim of this article is to present an overview of salient issues of exposure, characterisation and hazard assessment of nanomaterials as they emerged from the consensus-building of experts undertaken within the four year European Commission coordination project NanoImpactNet. The approach adopted is to consolidate and condense the findings and problem-identification in such a way as to identify knowledge-gaps and generate a set of interim recommendations of use to industry, regulators, research bodies and funders. The categories of recommendation arising from the consensual view address: significant gaps in vital factual knowledge of exposure, characterisation and hazards; the development, dissemination and standardisation of appropriate laboratory protocols; address a wide range of technical issues in establishing an adequate risk assessment platform; the more efficient and coordinated gathering of basic data; greater inter-organisational cooperation; regulatory harmonization; the wider use of the life-cycle approaches; and the wider involvement of all stakeholders in the discussion and solution-finding efforts for nanosafety.