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.

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.

Auditory brainstem responses to clicks and tone bursts in C57 BL/6J mice.

In auditory research, hearing function of mouse mutants is assessed in vivo by evoked potential recording. Evaluation of the response parameters should be performed with reference to the evoked responses recorded from wild-type mice. This study reports normative data calculated on auditory brainstem responses (ABRs) obtained from 20 wild-type C57 BL/6J mice at a postnatal age between 21 and 45 days. Acoustic stimuli consisted tone bursts at 8, 14, 20, 26, 32 kHz, and clicks. Each stimulus was delivered in free field at stimulation intensity starting from a maximum of 100 dB peak equivalent SPL (dB peSPL) at decreasing steps of 10 dB with a repetition rate of 13/sec. Evoked responses were recorded by needle electrodes inserted subcutaneously. At high intensity stimulation, five response waveforms, each consisting of a positive peak and a subsequent negative valley, were identified within 7 msec, and were labelled with sequential capital Roman numerals from I to V. Peak IV was the most robust and stable at low intensities for both tone burst and click stimuli, and was therefore utilized to estimate hearing thresholds. Both latencies and amplitudes of ABR peaks showed good reproducibility with acceptable standard deviations. Mean wave IV thresholds measured across all animals ranged from a maximum of 23 dB peSPL for clicks to a minimum of 7 dB peSPL for 20 kHz-tone burst stimuli. Statistical analysis of the distribution of latencies and amplitudes of peaks from I to V performed for each stimulus type yielded a normative data set which was utilised to obtain the most consistent fitting-curve model. This could serve as a reference for further studies on murine models of hearing loss.

HSP70 inhibition by 2-phenylethynesulfonamide induces lysosomal cathepsin D release and immunogenic cell death in primary effusion lymphoma.

Heat-shock protein (HSP) 70 is aberrantly expressed in different malignancies and has a cancer-specific cell-protective effect. As such, it has emerged as a promising target for anticancer therapy. In this study, the effect of the HSP70-specific inhibitor (PES), also Pifitrin-µ, on primary effusion lymphoma (PEL) cell viability was analyzed. PES treatment induced a dose- and time-dependent cytotoxic effect in BC3 and BCBL1 PEL cells by inducing lysosome membrane permeabilization, relocation of cathepsin D in the cytosol, Bid cleavage, mitochondrial depolarization with release and nuclear translocation of apoptosis-activating factor. The PES-induced cell death in PEL cells was characterized by the appearance of Annexin-V/propidium iodide double-positive cells from the early times of treatment, indicating the occurrence of an additional type of cell death other than apoptosis, which, accordingly, was not efficiently prevented by the pan-caspase inhibitor Z-VAD-fmk. Conversely, PES-induced cell death was robustly reduced by pepstatin A, which inhibits Bid and caspase 8 processing. In addition, PES was responsible for a block of the autophagic process in PEL cells. Finally, we found that PES-induced cell death has immunogenic potential being able to induce dendritic cell activation.

Cochlear implant electrode array misplacement: a cautionary case report.

OBJECTIVE: To report a series of pitfalls and complications in a case of cochlear implantation. METHOD: Case report. RESULTS: An 11-year-old boy affected by auditory neuropathy underwent cochlear implantation. Intra-operative assessment was apparently consistent with correct insertion of the electrode array into the cochlea. However, subsequent high resolution computed tomography revealed that the entire electrode array was curled up within the vestibule. Revision surgery was complicated by cerebrospinal fluid leakage. A straight probe was repeatedly inserted into the internal auditory canal, before conversion to a canal wall down procedure and appropriate positioning of the electrode array. CONCLUSION: In this case, mild anteriorisation of the facial nerve created an awkward insertion angle for the electrode array via the retro-facial route, which may have triggered the described series of adverse events.

Sudden hearing loss in sarcoidosis: otoneurological study and neuroradiological correlates.

Sarcoidosis is an inflammatory multisystem disorder of unknown cause. Approximately 5-7% of patients manifest symptoms of central nervous system involvement, or neurosarcoidosis. Cranial neuropathy usually entails facial nerve palsy and optic neuritis. Sudden hearing loss has been reported in fewer than 20 cases. Herewith, two new cases of sudden hearing loss due to probable neurosarcoidosis are reported, each having a quite different clinical course. In one case, unilateral sudden hearing loss and facial palsy were the presenting symptoms of systemic sarcoidosis, while in the second, unilateral sudden deafness occurred despite ongoing immunosuppressive treatment for systemic sarcoidosis.

[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.

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.

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.

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.

Direct correlation between human herpesvirus-8 seroprevalence and classic Kaposi's sarcoma incidence in Northern Sardinia.

The human herpesvirus-8 (HHV-8) has been associated with the development of Kaposi's sarcoma. A high incidence of classic Kaposi's sarcoma has been described in Sardinia, an island West of Italy's mainland. Different seroepidemiological analyses have reported that prevalence of HHV-8 infection varies worldwide: a high HHV-8 seroprevalence has been shown in Italy. The present survey was carried out to evaluate the correlation between HHV-8 infection and classic Kaposi's sarcoma incidence in northern Sardinia. Blood samples were collected from 226 healthy donors born and resident in five different areas of North Sardinia. Seroprevalence to HHV-8 was determined searching antibodies to viral lytic proteins by immunofluorescence in sera diluted at 1:10. Classic Kaposi's sarcoma incidence data spanning a period of 23 years were examined in the areas studied. The present screening revealed that seroprevalence was 35%, within a range of 15.3-46.3% in the five areas, although it should be considered that the seroprevalence to HHV-8 can be established more accurately by the combined use of different assays. Age emerged as an important risk factor. Indeed, subjects aged > 50 years showed a higher seroprevalence to HHV-8 as compared with younger individuals. A strong direct correlation between HHV-8 prevalence and classic Kaposi's sarcoma incidence has been also observed. The wide diffusion of HHV-8 in Sardinia appears to represent an important factor in the high incidence of classic Kaposi's sarcoma reported in the island. However, additional co-factors, such as age, sex, genetic traits, or viral strain pathogenicity, are likely to play a role in the development of the disease.

Utility of postintubation chest radiographs in the intensive care unit.

OBJECTIVE: To determine the clinical usefulness of immediate (stat) chest radiographs after endotracheal intubation when performed by experienced critical care personnel. PATIENTS AND METHODS: This was a prospective study. Endotracheal intubations in an 11-bed intensive care unit and a nine-bed intermediate intensive care unit were included. After intubations were performed by an experienced critical care operator, that individual recorded demographic and procedural data, and predicted radiographic findings on a data collection sheet. Experience at intubation was stratified into four levels of lifetime experience: fewer than 10 procedures, 10-20 procedures, 20-50 procedures, and more than 50 procedures. Radiographic findings evaluated included endotracheal tube position and procedure-related complications. The postintubation chest radiograph was then reviewed and the actual findings were also recorded. RESULTS: A total of 101 evaluable intubations were recorded, two of which were predicted to show tube malposition. Actual radiographic findings revealed 10 malpositions, three of which were too high and seven were too low (one at the level of the carina). A single witnessed aspiration that occurred during intubation was not radiographically apparent until 24 h later. Only the tube positioned at the carina was felt to be of acute clinical significance or to place the patient at any acute risk. CONCLUSIONS: The incidence of endotracheal tube malposition after intubation was underestimated. However, when performed by experienced critical care personnel, acutely significant malpositions were rare (one out of 101 intubations). We conclude that, in the absence of specific pulmonary complications, endotracheal intubations performed by experienced operators may be followed by routine, rather than 'stat' chest radiographs.

The BFRF1 gene of Epstein-Barr virus encodes a novel protein.

Computer analysis of the Epstein-Barr virus (EBV) genome indicates there are approximately 100 open reading frames (ORFs). Thus far about 30 EBV genes divided into the categories latent and lytic have been identified. The BamHI F region of EBV is abundantly transcribed during lytic replication. This region is highly conserved among herpesviruses, thus suggesting that some common function could be retained in the ORFs encompassed within this viral fragment. To identify putative novel proteins and possible new markers for viral replication, we focused our attention on the first rightward ORF in the BamHI F region (BFRF1). Histidine and glutathione S-transferase-tagged BFRF1 fusion proteins were synthesized to produce a mouse monoclonal antibody (MAb). Analysis of human sera revealed a high seroprevalence of antibodies to BFRF1 in patients affected by nasopharyngeal carcinoma or Burkitt's lymphoma, whereas no humoral response to BFRF1 could be detected among healthy donors. An anti-BFRF1 MAb recognizes a doublet migrating at 37 to 38 kDa in cells extracts from EBV-infected cell lines following lytic cycle activation and in an EBV-negative cell line (DG75) transfected with a plasmid expressing the BFRF1 gene. Northern blot analysis allowed the detection of a major transcript of 3.7 kb highly expressed in EBV-positive lytic cycle-induced cell lines. Treatment with inhibitors of viral DNA polymerase, such as phosphonoacetic acid and acyclovir, reduced but did not abolish the transcription of BFRF1, thus indicating that BFRF1 can be classified as an early gene. Cell fractionation experiments, as well as immunolocalization by immunofluorescence microscopy, immunohistochemistry, and immunoelectron microscopy, showed that BFRF1 is localized on the plasma membrane and nuclear compartments of the cells and is a structural component of the viral particle. Identification of BFRF1 provides a new marker with which to monitor EBV infection and might help us better understand the biology of the virus.

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.

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.

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.

Generation of auditory steady-state responses: linearity assessment.

Auditory steady-state responses (SSRs) are believed to result from the superimposition of the middle latency responses (MLRs) evoked by individual stimuli. Our recent studies challenge this hypothesis in several regards. Surface-electrical recordings performed in 16 normal subjects showed that the prediction curves obtained by MLR linear addition failed to predict SSRs at rates other than 40 Hz. Amplitude and phase differences between actual and predicted responses point to the intervention of phenomena related to the recovery cycle of the neural networks underlying the SSR generation. A click train paradigm at a 40 Hz rate was utilized and an approximation to the response evoked by the last stimulus was obtained by an analytical handling. The most relevant feature of this response in comparison to the MLR was the appearance of an additional activity which could be related to the fast oscillations of auditory cortical neurons. Our findings suggest that the responses evoked by individual stimuli during steady-state stimulation change by increasing the repetition rate, thus contradicting the most widely accepted hypothesis of the MLR linear addition in the SSR generation.

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.