Identification and characterization of the product encoded by ORF69 of Kaposi's sarcoma-associated herpesvirus.

We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.

[A simplified occupational health and safety management system designed for small enterprises. Initial validation results]. / Modello semplificato di sistema di gestione della sicurezza sul lavoro per piccole imprese. Primi risultati della sperimentazione.

BACKGROUND: Occupational Health and Safety Management Systems (OHSMS) are known to be effective in improving safety at work. Unfortunately they are often too resource-heavy for small businesses. OBJECTIVES: The aim of this project was to develop and test a simplified model of OHSMS suitable for small enterprises. METHODS: The model consists of 7 procedures and various operating forms and check lists, that guide the enterprise in managing safety at work. The model was tested in 15 volunteer enterprises. RESULTS: In most of the enterprises two audits showed increased awareness and participation of workers; better definition and formalisation of respon sibilities in 8 firms; election of Union Safety Representatives in over one quarter of the enterprises; improvement of safety equipment. The study also helped identify areas where the model could be improved by simplification of unnecessarily complex and redundant procedures.

EBV reduces autophagy, intracellular ROS and mitochondria to impair monocyte survival and differentiation.

EBV has been reported to impair monocyte in vitro differentiation into dendritic cells (DCs) and reduce cell survival. In this study, we added another layer of knowledge to this topic and showed that these effects correlated with macroautophagy/autophagy, ROS and mitochondrial biogenesis reduction. Of note, autophagy and ROS, although strongly interconnected, have been separately reported to be induced by CSF2/GM-CSF (colony stimulating factor 2) and required for CSF2-IL4-driven monocyte in vitro differentiation into DCs. We show that EBV infects monocytes and initiates a feedback loop in which, by inhibiting autophagy, reduces ROS and through ROS reduction negatively influences autophagy. Mechanistically, autophagy reduction correlated with the downregulation of RAB7 and ATG5 expression and STAT3 activation, leading to the accumulation of SQSTM1/p62. The latter activated the SQSTM1-KEAP1- NFE2L2 axis and upregulated the anti-oxidant response, reducing ROS and further inhibiting autophagy. ROS decrease correlated also with the reduction of mitochondria, the main source of intracellular ROS, achieved by the downregulation of NRF1 and TFAM, mitochondrial biogenesis transcription factors. Interestingly, mitochondria supply membranes and ATP required for autophagy execution, thus their reduction may further reduce autophagy in EBV-infected monocytes. In conclusion, this study shows for the first time that the interconnected reduction of autophagy, intracellular ROS and mitochondria mediated by EBV switches monocyte differentiation into apoptosis, giving new insights into the mechanisms through which this virus reduces immune surveillance. Abbreviations: ACTB: actin beta; ATG5: autophagy related 5; BAF: bafilomycin A1; BECN1: beclin 1; CAT: catalase; CSF2: colony stimulating factor 2; CT: control; CYCS (cytochrome C: somatic); DCs: dendritic cells; EBV: Epstein-Barr virus; GSR: glutathione-disulfide reductase; KEAP1: kelch like ECH associated protein 1; IL4: interleukin 4; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MET: metformin; NAC: N-acetylcysteine; NFE2L2/NRF2 nuclear factor: erythroid 2 like 2; NRF1 (nuclear respiratory factor 1); clPARP1: cleaved poly(ADP-ribose) polymerase; Rapa: Rapamycin; ROS: reactive oxygen species; SQSTM1/p62: sequestosome 1; TFAM: (transcription factor A: mitochondrial); TUBA1A: tubulin alpha 1a.

Cochlear microphonic potential recorded by transtympanic electrocochleography in normally-hearing and hearing-impaired ears.

The cochlear microphonic is a receptor potential believed to be generated primarily by outer hair cells. Its detection in surface recordings has been considered a distinctive sign of outer hair cell integrity in patients with auditory neuropathy. This report focuses on the results of an analysis performed on cochlear microphonic recorded by transtympanic electrocochleography in response to clicks in 502 subjects with normal hearing threshold or various degrees of hearing impairment, and in 20 patients with auditory neuropathy. Cochlear microphonics recorded in normally-hearing and hearing-impaired ears showed amplitudes decreasing by the elevation of compound action potential Cochlear microphonic responses were clearly detected in ears with profound hearing loss. After separating recordings according to the presence or absence of central nervous system pathology (CNS+ and CNS-, respectively), cochlear microphonic amplitude was significantly higher in CNS+ than in CNS- subjects with normally-hearing ears and at 70 dB nHL compound action potential threshold. Cochlear microphonic responses were detected in all auditory neuropathy patients, with similar amplitudes and thresholds to those calculated for normally-hearing CNS- subjects. Cochlear microphonic duration was significantly higher in auditory neuropathy and normally-hearing CNS+ patients compared to CNS- subjects. Our results show that: 1. cochlear microphonic detection is not a distinctive feature of auditory neuropathy; 2. CNS+ subjects showed enhancement in cochlear microphonic amplitude and duration, possibly due to efferent system dysfunction; 3. long-lasting, high frequency cochlear microphonics with amplitudes comparable to those obtained from CNS- ears were found in auditory neuropathy patients. This could result from a variable combination of afferent compartment lesion, efferent system dysfacilitation and loss of outer hair cells.

KSHV reduces autophagy in THP-1 cells and in differentiating monocytes by decreasing CAST/calpastatin and ATG5 expression.

We have previously shown that Kaposi sarcoma-associated herpesvirus (KSHV) impairs monocyte differentiation into dendritic cells (DCs). Macroautophagy/autophagy has been reported to be essential in such a differentiating process. Here we extended these studies and found that the impairment of DC formation by KSHV occurs through autophagy inhibition. KSHV indeed reduces CAST (calpastatin) and consequently decreases ATG5 expression in both THP-1 monocytoid cells and primary monocytes. We unveiled a new mechanism put in place by KSHV to escape from immune control. The discovery of viral immune suppressive strategies that contribute to the onset and progression of viral-associated malignancies is of fundamental importance for finding new therapeutic approaches against them.

SV40 T-antigen induces breast cancer formation with a high efficiency in lactating and virgin WAP-SV-T transgenic animals but with a low efficiency in ovariectomized animals.

The whey acid protein (WAP) is a major mouse milk protein and its gene expression is induced by various lactotrophic hormones (eg, estrogen, progesterone). Transgenic animals harboring the early SV40 coding region (T/t-antigen) under the transcriptional control of the WAP promoter develop breast cancer after the first lactation period. The tumor cells synthesize the SV40 T-antigen with a high efficiency indicating that WAP-SV-T expression escapes down-regulation after the lactation period. However about 5-10% of the tumors became T-antigen negative during tumor progression and WAP-SV-T expression was only demonstrable by PCR analysis. Both T-antigen positive and negative tumor cells expressed the estrogen and progesterone receptor at a comparable rate, indicating that hormone receptor levels do not determine expression of the WAP-SV-T transgene. Furthermore, WAP and WAP-SV-T gene expression are not restricted to the pregnancy-lactation period. Virgin animals also express both genes with a low efficiency and about 70% of these animals also developed T-antigen positive breast tumors. The tumor rate however was strongly reduced in ovariectomized animals, indicating that the ovary hormones play a critical role in breast cancer formation.

The SV40 T-antigen induces premature apoptotic mammary gland involution during late pregnancy in transgenic mice.

Transgenic animals of the line 8 contain the WAP-SV-T transgene. Females of this line synthesise the SV40 T-antigen in mammary gland epithelial cells during pregnancy and the lactation period. All females are 'milk-less' and the offspring have to be nursed by foster mothers. The reason for this phenomenon is a premature apoptosis during late pregnancy. Nontheless a significant number of mammary epithelial cells escape apoptosis and all transgenic females devlop breast cancer after the first lactation period.

Global problem of drug-induced hearing loss.

Clinically used drugs and chemical agents may potentially cause adverse effects to the human auditory and vestibular systems. Many of them, such as aminoglycosides and cisplatin, can play a critical role in the treatment of serious or life-threatening diseases; others, like loop diuretics or salycilates, offer such important therapeutical effects compared to the ototoxic side effects that the ototoxicity risk can be considered to be of minor importance. The problem of ototoxic side effects is more acute in developing countries, where highly effective and low-cost drugs are more easily prescribed without adequate monitoring. Medical awareness of doses, forms of administration, populations at risk, and possible synergism is necessary in order to develop appropriate care in the prescription of drugs with ototoxic side effects. Relatively recent issues such as risk-benefit analysis, patient-informed consent, and quality-of-life considerations, particularly when life expectancy can be low, are also to be considered. At present, a uniform method of monitoring for all potentially ototoxic therapeutics does not seem reasonable or practical. It is recommended, however, that individual auditory function be noted for a particular drug being employed. Protocols and exams should be easy, quick, sensitive, reliable, and as objective as possible. Benefits of audiological monitoring include the opportunity to change the patient's treatment course, improvement of patient and family awareness of the impact of hearing impairment, and timely prescription of amplification devices. Finally, particular attention should be paid to high-risk populations such as neonatal intensive care unit patients.

Auditory steady-state responses to click trains from the rat temporal cortex.

In order to investigate the mechanisms underlying the generation of steady-state responses (SSRs), auditory evoked potentials elicited by click trains presented at several stimulation rates (30, 40, 50, 60 Hz) were recorded in 7 awake rats by means of epidural electrodes placed over the temporal cortex. Mean amplitude-rate function calculated on the recorded responses appeared almost flat and showed the maximum value at 50 Hz, while mean phases showed a linear increase when increasing the stimulation rate. In each rat, predictions of the recorded responses at 30, 40, 50 and 60 Hz were synthesized by superimposing middle-latency auditory evoked potentials (MAEPs) at suitable time intervals at each rate. Mean amplitudes calculated on the predicted curves decreased linearly when increasing the stimulation rate and appeared higher in comparison to those obtained from the recorded SSRs. Predicted phases showed a linear increase when increasing the stimulation rate and were leading with respect to corresponding phase values calculated for recorded SSRs. Our findings indicate that the MAEP superimposition mechanism does not adequately predict the generation of temporal recorded SSRs in rats. This was explained by admitting that phenomena related to the recovery cycle and, to a lesser extent, to rate-dependent facilitating effects come into play.

Possible modulation of auditory middle latency responses by nitric oxide in the inferior colliculus of anaesthetized rats.

Nitric oxide (NO) is a short-lived radical species endowed with intercellular signalling functions in the mammalian brain. In the present study we have investigated the effects of focal injection into one inferior colliculus of N omega-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, on the acoustic middle latency responses (MLRs) evoked by click stimuli and recorded from the auditory cortex in anaesthetized rats. Microinfusion of L-NAME (1.0 mM) did not alter the latency of MLRs nor did it affect the evoked brain stem responses (ABRs). By contrast, L-NAME reduced P1a-N1 amplitude of MLRs by 51.7 +/- 6.6% (mean +/- SEM; n = 5) and almost complete recovery to background amplitude was obtained 15-25 min after treatment. The less active isomer, D-NAME (1.0 mM; n = 5), failed to produce consistent effects on the evoked MLRs. A higher concentration of L-NAME (5.0 mM; n = 5) yielded a 69.0 +/- 13.3% inhibition whereas maximum inhibition produced by 0.5 mM (n = 3) L-NAME was approximately equal to 10% of control value. The inhibitory effect typically evoked by 1.0 mM L-NAME was prevented by treating rats with L-arginine (5.0 mM; n = 5), the endogenous precursor of NO synthesis. Reduction of MLR amplitude was also obtained in rats receiving intracollicular injection of dizocilpine (MK801; 1.0 microM) and LY274614 (1.0 mM), two selective N-methyl-D-aspartate (NMDA) receptor antagonists. In conclusion, the present data support a role for intracollicular NO in the processing and transmission of the acoustic input to the auditory cortex in the rat.

Sympathetic control of skeletal muscle function: possible co-operation between noradrenaline and neuropeptide Y in rabbit jaw muscles.

Stimulation of the cervical sympathetic nerve at 10/s increases by 12.9 +/- 0.7% peak tension of maximal twitches in the directly stimulated jaw muscles and markedly depresses (41.6 +/- 1.3%) the tonic vibration reflex (TVR) elicited in the same muscles by vibration of the mandible. Both effects are not significantly influenced by administration of beta-adrenoceptor antagonists. When both alpha- and beta-adrenergic receptors are blocked, sympathetic stimulation induces a very small increase in twitch tension (3.8 +/- 0.7%), while no detectable change in the TVR is observed. Close arterial injection of alpha 1-adrenoceptor agonist phenylephrine mimics the effects induced by sympathetic stimulation on twitch tension and TVR, dose-dependently. The noradrenaline co-transmitter neuropeptide Y also produces a long-lasting, dose-dependent increase in the twitch tension which is unaffected by blockade of adrenergic receptors as well as of the neuromuscular junctions. Contribution of neuropeptide Y to the sympathetically-induced reduction of the stretch reflex is not clearly demonstrated. These data suggest that co-operation between noradrenaline and neuropeptide Y may be effective in determining sympathetic modulation of skeletal muscle function.

Inhibition of nitric oxide synthase prevents magnesium-free-induced epileptiform activity in guinea-pig piriform cortex neurones in vitro.

The effects of N-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase inhibitor, were examined on Mg2+-free-induced epileptiform activity, in guinea-pig piriform cortex slices in vitro. L-NAME (0.1-1 mM) had no effect on neuronal membrane properties or electrically-evoked postsynaptic potentials (PSPs). In contrast, during superfusion of the slices with Mg2+-free solution neurones exhibited spontaneous and stimulus-evoked epileptiform potentials that were suppressed in the presence of L-NAME (100 microM) or the selective NMDA receptor antagonist DL-APV (100 microM). The inhibitory effects induced by L-NAME were reversibly reduced by L-arginine (1 mM), but not D-arginine (1 mM), the latter drug not being a substrate for NO formation. It was concluded that L-NAME can suppress epileptiform activity induced by Mg2+-free exposure primarily through a decrease in presynaptic transmitter release, although additional actions on the NMDA-receptor complex were also considered.

Generation of human auditory steady-state responses (SSRs). II: Addition of responses to individual stimuli.

In order to investigate the generation of the 40 Hz steady-state response (SSR), auditory potentials evoked by clicks were recorded in 16 healthy subjects in two stimulating conditions. Firstly, repetition rates of 7.9 and 40 Hz were used to obtain individual middle latency responses (MLRs) and 40 Hz-SSRs, respectively. In the second condition, eight click trains were presented at a 40 Hz repetition rate and an inter-train interval of 126 ms. We extracted from the whole train response: (1) the response-segment taking place after the last click of the train (last click response, LCR), (2) a modified LCR (mLCR) obtained by clearing the LCR from the amplitude enhancement due to the overlapping of the responses to the clicks preceding the last within the stimulus train. In comparison to MLRs, the most relevant feature of the evoked activity following the last click of the train (LCRs, mLCRs) was the appearance in the 50-110 ms latency range of one (in 11 subjects) or two (in 2 subjects) additional positive-negative deflections having the same periodicity as that of MLR waves. The grand average (GA) of the 40 Hz-SSRs was compared with three predictions synthesized by superimposing: (1) the GA of MLRs, (2) the GA of LCRs, (3) the GA of mLCRs. Both the MLR and mLCR predictions reproduced the recorded signal in amplitude while the LCR prediction amplitude resulted almost twice that of the 40 Hz-SSR. With regard to the phase, the MLR, LCR and mLCR closely predicted the recorded signal. Our findings confirm the effectiveness of the linear addition mechanism in the generation of the 40 Hz-SSR. However the responses to individual stimuli within the 40 Hz-SSR differ from MLRs because of additional periodic activity. These results suggest that phenomena related to the resonant frequency of the activated system may play a role in the mechanisms which interact to generate the 40 Hz-SSR.

Generation of human auditory steady-state responses (SSRs). I: Stimulus rate effects.

Auditory evoked responses were recorded in 16 normally hearing subjects in order to investigate the mechanisms underlying the generation of the 40 Hz steady-state response (SSR). In the first part of our study, auditory potentials were evoked by 0.1 ms clicks presented at 105 dB p.e. SPL with repetition rates of 7.9 (to obtain middle latency response, MLR), 20, 30, 40, 50, 60 Hz. In each subject predictions of the responses recorded at stimulus repetition rates of 30, 40, 50, 60 Hz were synthesized by superimposing MLRs at suitable time intervals. The calculated mean amplitude/rate and phase/rate functions behaved similarly for the recorded and predicted curves, showing the highest amplitude at 40 Hz and a linear increase of phase values when increasing the stimulus rate. Nevertheless the synthetic curves closely predicted amplitude and phase values of the recorded responses only at 40 Hz. At frequencies below 40 Hz, the mean amplitude of the predicted curve was lower than that of the recorded one while at frequencies above 40 Hz the mean amplitude was higher. Predicted phase values were found lagging at 30 Hz, and leading at 50 Hz and 60 Hz in comparison to phase values calculated on the recorded responses. Our findings suggest that a model based on the linear addition of transient MLRs is not able to adequately predict steady-state responses at stimulus rates other than at 40 Hz. Other mechanisms related to the recovery cycle of the activated system come into play in the steady-state response generation causing a decrease in amplitude and an increase in phase lag when increasing the stimulus repetition rate.

Method for the deconvolution of auditory steady-state responses.

The potential evoked by a 'train' of N equally spaced auditory clicks, with an inter-click period shorter than the duration of the response to an isolated click, is said to be a steady-state response (SSR). Extracting the individual responses evoked by the clicks of the train during steady state can be key to understanding of the neurophysiological mechanisms underlying SSR generation. In the literature, this task has been dealt with only under the (unwarranted) assumption that the response of the system does not vary during the presentation of the clicks, i.e. no neurophysiological adaptation is present. In this work, a new, non-parametric algorithm is proposed that, relaxing the time-invariance hypothesis, allows the extraction from the SSR of the N waveforms individually evoked by the N clicks of the train. The performance of the approach is evaluated on simulated SSRs and on real data recorded from the temporal cortex of awake rats. Results show that the method is able to detect and assess possible adaptation of the neurophysiological system in the generation of SSRs.

Compound action potential and cochlear microphonic extracted from electrocochleographic responses to condensation or rarefaction clicks.

In electrocochleography (ECochG) compound action potential (CAP) and summation potential (SP) are usually separated from the cochlear microphonic (CM) by the CM cancellation technique consisting in averaging the responses evoked by rarefaction and condensation clicks. With the aim of analysing the ECochG responses evoked by monophasic clicks, we developed a numerical method based on the theory of optimal filtering, which makes no assumptions about the unknown potentials. The application of the filtering technique to the ECochG recordings obtained from 6 normally hearing children and 10 children with cochlear hearing loss allowed us to perform CAP extraction in cases where CM was not cancelled by the conventional method. Differences in SP amplitude and polarity were found between rarefaction and condensation click-evoked responses in cochlear hearing losses.

Botulinum A toxin effects on rat jaw muscle spindles.

Botulinum A toxin (Botox) is used for the treatment of many muscular dystonias. However, the relief of the sustained and abnormal postures induced by Botox administration is not fully explained. In this work the possibility was considered that Botox can produce a block not only at the alpha motor endings, but also at the gamma motor endings, consequently reducing the spindle inflow to the alpha motoneurons, which have a great role in maintaining the tonic myotatic reflex. Jaw muscle spindle discharge was recorded before and after Botox injection in the deep masseter muscle. The drug consistently reduced the spindle afferent discharge. Such an effect is suggested to be direct on gamma endings as: i) muscle tension was not modified by Botox during the recording time; ii) saline administration never changed the spindle discharge. The Botox effect on muscle spindles suggests that the relief from dystonias could be due not only to a partial motor paralysis, but also to a decrease of the reflex muscular tone.

Gravity load related asymmetries in the sagittal vestibulo-collic reflex.

EMG recordings of the neck muscles (biventer cervicis, complexus, splenius, longus capitis) of decerebrate cats were obtained during pitch and roll stimulations (sinusoidal stimulation: 30 degrees p-p amplitude, 0.2 Hz frequency). Most of the EMG responses to pitch showed activation peaks leading the position stimuli by 56 degrees and inhibition peaks leading by 11 degrees. Conversely, in response to roll the activation peak led by 16 degrees and the inhibition peak by 10 degrees. The activation peaks of the pitch responses were, thus, more asymmetric and more leading than those of the roll responses. Consequently, the harmonic distortion coefficient was significantly higher in pitch than in roll. Moreover, when the vertical semicircular canals were activated in absence of otolithic modulation, the pitch and roll responses maintained the same difference in timing observed in the presence of otolithic coactivation. It appeared that the simultaneous stimulation of both anterior semicircular canals (pitch) induces a greater lead than that of combined anterior and posterior canals (roll). Thus the timing of neck muscle responses to vestibular stimulation depends on the pair of activated vertical semicircular canals.

Extraocular muscle proprioception and eye position.

In the lamb, acute unilateral section of the ophthalmic branch induced in the ipsilateral eye occasional oscillations of the resting position and misalignment of the horizontal vestibulo-ocular reflex (HVOR) with respect to the stimulus. Additional electrolytic lesion of the cells innervating the proprioceptors of the medial rectus muscle, or of the lateral rectus muscle in the contralateral semilunar ganglion, provoked a 4 degrees-7 degrees consensual eye deviation towards and away from the lesioned side, respectively. The optokinetic beating field was similarly deviated. Under these experimental conditions, HVOR showed enhanced gain and marked misalignment in both eyes. Therefore, the selective suppression of muscular proprioceptive input deviated both eyes towards the direction opposite to the muscle whose gangliar proprioceptive representation has been destroyed.

Postural control in benign paroxysmal positional vertigo before and after recovery.

Thirty-two patients affected by idiopathic benign paroxysmal positional vertigo (BPPV) of the posterior semicircular canal were studied before, 3 days and I month after a resolutive Semont manoeuvre by means of dynamic posturography. The overall postural control in BPPV patients was shown to be impaired, as demonstrated by the pathological equilibrium scores. Data obtained before treatment showed a specific pattern of vestibular involvement and a pathological composite score. After the liberatory manoeuvre the Sensory Organization Test indicated a significant improvement in the pathological composite and vestibular scores. However, significant differences from controls were still detected 3 days and 1 month after clinical recovery from BPPV. The results clearly show that, in BPPV patients, there is an impairment of the vestibular system, which seems unable to maintain a normal postural balance. This deficit can be particularly detected when dynamic posturography evaluates the vestibular cues. After the liberatory manoeuvre a consistent improvement in the overall postural control has been observed but the residual differences from controls seem to suggest that damage to the otoconial maculae influences postural control, even when there is significant improvement in the clinical signs.