In vivo therapeutic efficacy of intra-renal CD40 silencing in a model of humoral acute rejection.

The humoral branch of the immune response has an important role in acute and chronic allograft dysfunction. The CD40/CD40L costimulatory pathway is crucial in B- and T- alloresponse. Our group has developed a new small interfering RNA (siRNA) molecule against CD40 that effectively inhibits its expression. The aim of the present study was to prevent rejection in an acute vascular rejection model of kidney transplant by intra-graft gene silencing with anti-CD40 siRNA (siCD40), associated or not with sub-therapeutic rapamycin. Four groups were designed: unspecific siRNA as control; sub-therapeutic rapamycin; siCD40; and combination therapy. Long-surviving rats were found only in both siCD40-treated groups. The CD40 mRNA was overexpressed in control grafts but treatment with siCD40 decreased its expression. Recipient spleen CD40+ B-lymphocytes were reduced in both siCD40-treated groups. Moreover, CD40 silencing reduced donor-specific antibodies, graft complement deposition and immune-inflammatory mediators. The characteristic histological features of humoral rejection were not found in siCD40-treated grafts, which showed a more cellular histological pattern. Therefore, the intra-renal effective blockade of the CD40/CD40L signal reduces the graft inflammation as well as the incidence of humoral vascular acute rejection, finally changing the type of rejection from humoral to cellular.

N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel.

Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure-function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology.

Chronic exposure to an environmental noise permanently disturbs sleep in rats: inter-individual vulnerability.

Chronic exposure to an environmental noise (EN) induces sleep disturbances. However, discrepancies exist in the literature since many contradictory conclusions have been reported. These disagreements are largely due to inappropriate evaluation of sleep and also to uncontrolled and confounding factors such as sex, age and also inter-individual vulnerability. Based on a recently validated animal model, aims of the present study were (i) to determine the effects of a chronic exposure to EN on sleep and (ii) to evaluate the inter-individual vulnerability of sleep to EN. For this purpose, rats were exposed during 9 days to EN. Results show that a chronic exposure to EN restricts continually amounts of slow wave sleep (SWS) and paradoxical sleep (PS) and fragments these two sleep stages with no habituation effect. Results also evidence the existence of subpopulations of rats that are either resistant or vulnerable to these deleterious effects of EN on sleep and especially on SWS amounts, bouts number and bout duration. Furthermore, importance of SWS debt and daily decrease of SWS bout duration are correlated to each others and both correlate to the amplitude of the locomotor reactivity to novelty, a behavioral measure of reactivity to stress. This last result suggests that this psychobiological profile of subjects, known to induce profound differences in neural and endocrine systems, could be responsible for their SWS vulnerability under a chronic EN exposure.

Deleterious effects of an environmental noise on sleep and contribution of its physical components in a rat model.

Sleep disturbances induced by environmental noise (EN) exposure are now well admitted. However, many contradictory conclusions and discrepancies have been reported, resulting from uncontrolled human factors or the use of artificial noises (pure tone). Thus, the development of an animal model appears to be a useful strategy for determining whether EN is deleterious to sleep. The aims of this study were: (i) to confirm the effects of noise on sleep in a rat model; and (ii) to determine the most deleterious physical component of noise regarding sleep structure. For this purpose, rats were exposed during 24 h either to EN or to artificial broad-band noises [either continuous broad-band noise (CBBN) or intermittent broad-band noise (IBBN)]. All the noises decrease both slow wave sleep (SWS) and paradoxical sleep (PS) amounts during the first hours of exposure. However, CBBN acts indirectly on PS through a reduction of SWS bout duration, whereas IBBN and EN disturb directly and more strongly both SWS and PS. Finally, EN fragments SWS and decreases PS amount during the dark period, whereas IBBN only fragments PS. These results demonstrate the validity and suitability of a rodent model for studying the effects of noise on sleep and definitively show that sleep is disturbed by EN exposure. Two physical factors seem to be implicated: the intermittency and the frequency spectrum of the noise events, which both induce long-lasting sleep disturbances. An additive effect of frequency spectrum to intermittency tends to abolish all possible adaptations to EN exposure. Since sleep is involved in cognitive processes, such disturbances could lead to cognitive deficits.

Changes in electrovestibular brainstem responses after aminoglycoside intoxication in guinea pigs.

OBJECTIVE: To verify the usefulness of short-latency vestibular responses evoked by a combination of round window electrical stimulation and sinusoidal rotation (electrovestibular brainstem responses; EVBRs) as a new monitoring tool of the vestibular function in animal experiments. METHODS: EVBRs were obtained before, during, and after treatment with aminoglycosides, along with compound action potential (CAP) audiograms. The changes in EVBRs were compared with morphological changes observed by scanning electron microscopy. RESULTS: EVBR amplitudes did not change in the group of guinea pigs treated with amikacin, but markedly decreased in the streptomycin and gentamicin- treated groups. CAP audiograms indicated a significant threshold elevation in the amikacin group, a moderate elevation in the gentamicin group, and no change in the streptomycin group. Under scanning electron microscopy, the loss of the sensory hair cells observed in the cristae ampullares was slight to moderate in the amikacin group, moderate to severe in the streptomycin group, and severe in the gentamicin group. CONCLUSION: EVBRs reflect overall pathological changes undergone by vestibular hair cells, and support the vestibular specificity of EVBRs.

Effects of contralateral acoustical stimulation on three measures of cochlear function in the guinea pig.

The magnitudes of suppression of the click-evoked compound action potential of the auditory nerve (CAP), transient click-evoked otoacoustic emissions (TEOAEs) and ensemble background activity of the auditory nerve (EBA), elicited by contralateral acoustical stimulation, were compared in awake or lightly sedated guinea pigs. The contralateral ear was stimulated either by continuous broad-band noise or by low-pass or high-pass noise (intensity 41-62 dB SPL) with cut-off frequencies of 2, 8 and 12 kHz. The maximal suppression of TEOAEs was achieved by contralateral noise containing mainly low frequencies, whereas for suppression of the CAP it was necessary for middle frequencies to be present in the contralateral noise (less than 8 kHz). In contrast to this, EBA was suppressed mainly by high-frequency noise (higher than 8 kHz) whereas low- and middle-frequency noise was ineffective in suppressing EBA. Evaluation of the root mean square voltage of the EBA (filtered in frequency range 0.75-1.25 kHz) enabled the evaluation of fast and slow components of olivocochlear activation. Both fast and slow effects were proportionally suppressed by individual types of contralateral stimulation. The mechanisms of TEOAEs and CAP generation has been confirmed in many earlier studies, but the origin of EBA has yet to be fully elucidated. The obtained data support the hypothesis that a large part of EBA is formed by spontaneous activity of high-frequency-tuned auditory nerve fibres. Suppression of the EBA magnitude during contralateral stimulation may be caused either by a reduced spontaneous firing rate or by a decrease in possible synchronised neuronal firing.

Discharge rate of the auditory nerve during noise revealed by electrocochlear stimulation.

The purpose of this study was to evaluate the average discharge rate of all fibres in the whole auditory nerve (R(wn)) when a broad-band noise with steady-state effects is applied to the ear. We assessed the R(wn) parameter by detecting the state of refractoriness of the nerve during noise stimulation using an electric stimulus (ES) as a probe. The technique, applied in awake pre-implanted guinea pigs (Charlet de Sauvage et al., 1994), made it possible to obtain electro-acoustic responses (EARs), from which an estimate of the R(wn) parameter could be deduced. Negative current pulses of 100 micros duration, each followed by an identical pulse of positive polarity for charge balance, were applied between round window and indifferent vertex electrodes at intervals of 160 ms. The 120 ms wide-band noise masker started 92 ms before every other negative ES. The signal on the stimulating electrodes was averaged over a 5.12 ms window in synchrony with the negative pulse. EARs were obtained by alternately subtracting recordings during noise from those during silence. The R(wn) parameter was determined by comparing experimental and computed EAR patterns. For this purpose, a model of unit response incorporating changes in amplitude and conduction velocity during the relative refractory period was designed. The recovery function of the firing probability in response to ES was evaluated. Fibres were classified in different categories according to their background discharge rates. The probability of response of single fibres to ES in each category was calculated on the basis of their interval histograms during silence and noise. Individual spikes were combined accordingly to obtain the computed EAR waveform. R(wn) was determined by adjusting the EAR amplitude of the model in relation to that of the experimental EAR. R(wn) generally increases in a linear fashion with respect to noise intensity expressed in dB, thus following the increase in loudness perception estimated by Weber's law. At the highest noise levels, R(wn) tends to saturate. The estimated saturation rate was found to be about 380 spikes/s.

Role of the efferent medial olivocochlear system in contralateral masking and binaural interactions: an electrophysiological study in guinea pigs.

Contralateral broadband noise (BBN) elevates ipsilateral auditory thresholds (central masking) and reduces the amplitude of ipsilateral brainstem auditory evoked potentials (BAEPs). Binaural interactions are complex psychophysical phenomena, but binaural interaction components are easily extracted from BAEPs to monaural versus binaural click stimulation. However, contralateral, or binaural, acoustical stimulation is known to activate simultaneously the crossed and uncrossed medial olivocochlear (MOC) efferent systems and decrease activity in both cochleas. Particularly, contralateral BBN stimulation suppresses in part ipsilateral peripheral activity. What is the role of such contralaterally induced peripheral suppression in the overall changes in central BAEPs observed during contralateral masking or binaural stimulation? Compound action potentials (CAPs) of the auditory nerve and BAEPs were recorded simultaneously in awake guinea pigs from electrodes chronically implanted on the round window of the cochlea and the surface of the brain. Peripheral and central measures of contralateral masking and binaural interactions were obtained from responses to monaural or binaural clicks, with or without contralateral BBN, recorded before, during, and after the reversible blockade of the MOC function following a single intramuscular injection of gentamicin. Contralateral BBN effectively reduced the amplitudes of CAP and of all BAEP peaks. CAP to ipsilateral click did not, however, change significantly from monaural to binaural click stimulation; still, normal binaural interaction components developed in the BAEPs. When the medial efferent function was blocked by gentamicin, the normal contralateral BBN suppression of CAP and of the earliest BAEP peak was lost; however, the later BAEP peaks were suppressed by contralateral BBN as before gentamicin, and the central binaural interaction components were unchanged. In these experimental conditions, the MOC efferent system seems to play little role in centrally recorded contralateral masking and binaural interactions.

Retroviral transfer of human MDR1 gene to hematopoietic cells: effects of drug selection and of transcript splicing on expression of encoded P-glycoprotein.

Protection of hematopoietic cells of patients undergoing anticancer chemotherapy by MDR1 gene transfer is currently being studied in clinical trials. From animal studies, it has been suggested that aberrant splicing due to cryptic donor and acceptor sites in the MDR1 cDNA could be a major reason for failure to obtain high-level expression of P-glycoprotein in bone marrow. We investigated effects of drug selection on protein expression levels and on splicing of MDR1 transcripts in murine bone marrow cells (BMCs) in vitro. To this end, retroviruses were generated through an identical plasmid, pHaMDR1/A, introduced into different packaging cells. GP + E86- but not PA317-derived producer cells were found to express truncated in addition to full-length message. In BMCs transduced with GP + E86-derived viruses, both messages were increased after treatment with colchicine or daunomycin. Similar results were obtained with NIH 3T3 fibroblasts. However, transduced and drug-selected BMCs displayed the spliced transcript even if the respective PA317-derived producer cells contained no truncated RNA as detected in transduced NIH 3T3 fibroblasts. Short-term drug selection in BMCs transduced with either ecotropic or amphotropic retroviruses resulted in a striking increase in P-glycoprotein expression. Thus, aberrant splicing failed to abrogate P-glycoprotein expression in BMCs. We also studied a vector in which MDR1 was coexpressed with glucocerebrosidase, using an internal ribosomal entry site. Although chemoprotection was less efficient than with pHaMDR1/A, augmentation of protein expression was observed at low selecting drug concentrations. Our study shows that drug selection can partially compensate for inefficient transduction of hematopoietic cells, and may help to develop strategies by which unstable expression of transduced genes can be overcome.

Xenogeneic ossicular implants: an experimental study of heterotopic, demineralized, lyophilized, porcine implants in the guinea-pig.

This study was done to compare the outcome of porcine ossicular implants in the middle ear and the subcutaneous dorsal region of the guinea-pig to those of allo-implants implanted in parallel in the dorsal region. The implants were heteropic, xenogeneic, demineralized (HCl), lyophilized and sterilized. The evaluation was histological (light microscopy and scanning electron microscopy) and immunological (immunofluorescence staining). Fifty-four guinea-pigs were implanted in the middle ear and 14 of them were also implanted subcutaneously in the dorsal region with xeno-implants and allo-implants. The middle ear implants were found to be constantly reossified and coated with normal mucosa with only a minimal immune reaction. In contrast, the dorsal xeno-implants were found to be the target of mononucleic infiltration, fibrous encapsulation and an influx of immunoglobulins resulting in segregation. The corresponding allo-implants were found to be partially reoccupied and reossified. These findings highlight the value of HCl demineralization in the induction of non-species-specific Bone Morphogenetic Protein and the failure of attempts at immuno-despecification. It appears that the fate of the implant depends less on its antigenic load than on the site of implantation. In this regard the middle ear is apparently very advantageous. The very good short-term tolerance and recovery observed in the middle ear xeno-implant suggest that these implants offer sufficiently good results to warrant clinical testing.

Changes in auditory brainstem responses in alpha-linolenic acid deficiency as a function of age in rats.

Auditory brainstem responses (ABRs) to click stimuli have been compared in young (21-day-old), adult (6-month-old), and old (18-month-old) rats fed a normal (Arachid-Colza) or an alpha-linolenic acid deficient (Arachid only) diet. Wave I amplitude and latency did not show any significant change with either age or diet. However, wave III showed a progressive decrease in amplitude and latency from young to adult and from adult to old rats having a normal diet. With alpha-linolenic acid deficiency, wave III amplitude and latency values decreased faster than in the normal diet control groups. Although final values in the old groups with the two diets were similar, with alpha-linolenic acid deficiency values for wave III decreased to this final level in the adult group. These data indicate that the central auditory nervous system ages faster, or earlier, with a fatty acid deficiency.

Attenuation of aminoglycoside-induced cochlear damage with the metabolic antioxidant alpha-lipoic acid.

Free radical generation is increasingly implicated in a variety of pathological processes, including drug toxicity. Recently, a number of studies have demonstrated the ability of gentamicin to facilitate the generation of radical species both in vivo and in vitro, which suggests that this process plays an important role in aminoglycoside-induced ototoxicity. Free radical scavengers are compounds capable of inactivating free radicals, thereby attenuating their tissue damaging capacity. In this study we have determined the ability of the powerful free radical scavenger alpha-lipoic acid (100 mg/kg/day) to attenuate the cochlear damage induced by a highly ototoxic regimen of the aminoglycoside amikacin (450 mg/kg/day, i.m.). Experiments were carried out on pigmented guinea pigs initially weighing 200-250 g. Changes in cochlear function were characterized as shifts in compound action potential (CAP) thresholds, estimated every 5 days, by use of chronic indwelling electrodes implanted at the round window, vertex, and contralateral mastoid. Results showed that animals receiving alpha-lipoic acid in combination with amikacin demonstrated a significantly less severe elevation in CAP thresholds compared with animals receiving amikacin alone (P < 0.001; t-test). These results provide further evidence of the recently reported intrinsic role of free radical generation in aminoglycoside ototoxicity, and highlight a potential clinical therapeutic use of alpha-lipoic acid in the management of patients undergoing aminoglycoside treatment.

Acute and chronic effects of aminoglycosides on cochlear hair cells.

The first detectable effect on the auditory system after a single high-dose injection of an aminoglycosidic antibiotic (AA) like gentamicin (GM) is the reversible blockade of medial efferent function, probably via blockade of calcium channels at the base of the outer hair cells (OHC). The kinetics of this effect are compatible with that of the molecule in perilymph. In the course of chronic treatment with lower doses, however, ototoxicity develops only after several days of treatment. Still GM can be observed inside the OHCs as soon as 24 hours after the first injection, and will be still present in some OHCs as long as 11 months after a chronic, nonototoxic 6-day treatment. In vitro, the short-term viability of isolated OHCs is not affected by exposure to AAs, but their transduction channels and their response to acetylcholine are reversibly blocked. However, developing organs of Corti in culture are highly and rapidly affected by exposure to AAs. Yet during direct intracochlear perilymphatic perfusion of GM, 2-mM solutions are not ototoxic, and with perfusion with a 20-mM solution ototoxicity develops only after several days of perfusion. From these various observations one can describe some aspects of the mechanisms of ototoxicity of AAs, from their access to perilymph and endolymph, to penetration in the hair cells, likely via endocytosis at their apical pole, and intracellular cytotoxic events.

[Multi-frequency tympanometry in experimentally-induced cochlear lesions in chinchillas and guinea pigs]. / Données de l'impédancemétrie multifréquentielle dans les pathologies cochléaires. Approche expérimentale chez le chinchilla et le cobaye.

OBJECTIVES: To establish that susceptance-conductance tympanograms at a probe-tone frequency of 2 kHz reflects the status of the annular ligament (AL) and through it of the cochlea. METHODS: Experimental study in 5 chinchillas and 22 guinea pigs. Six validating experiments were used: blockages of the stapes and of the round window membrane (RWM), fistula of the RWM, fluid removal from the cochlea, injection of saline in the scala tympani (ST) and acoustic trauma (AT). Quantitative data (mean values of Y226, FR, Y2000, G2000 and B2000) and shape of the curves were analyzed before and immediately after lesions were done. RESULTS: Guinea pig was the most convenient provided bulla was vented and the same tip was used along the experiments. Only the shape of the curves are discriminant: 1/a supplementary sharp peak, centered around negative pressures, is observed in Y/G tympanograms in every case of RWM fistulas and in some case of AT. 2/injection of saline into ST induces immediate and reproducible Y2000, G2000, et B2000 curves modifications. 3/RWM and stapes blockages provoke foreseeable stiffening and sharpening of the tympanograms at 2 kHz. 4/on the contrary, fluid removal from the cochlea induces multiple peaks curves. CONCLUSIONS: Experimentally-induced modifications at the AL either direct (stapes blockage) or indirect by AT or decrease/increase of pressure load at the cochlear interface at the footplate result in noticeable, constant, reproducible changes of curves registered at 2 kHz. The stapes behaves both as the plotter of the curves and the interpreter of the inner ear pressure.

Short-term effectiveness of medial efferents does not predict susceptibility to temporary threshold shift in the guinea pig.

The 2f1-f2 distorsion product (DPOAE) was measured in conjunction with contralateral noise to evaluate the short-term effectiveness of the olivocochlear efferents in guinea pigs (GPs). An attenuation effect was observed predominantly between 1 and 6 kHz when primary tones were set at 65 dB SPL (contralateral noise of 55 dB SPL). Subsequently, GPs were exposed to a 2 kHz tone of 87 dB SPL for 40 min, using DPOAEs as an estimate of cochlear sensitivity. The response of the cochlea appeared variable. In order to investigate whether effectiveness of efferents plays a role in temporary threshold shift (TTS), the responses of the cochlea to overstimulation were classified into three groups: i) clear cochlear change with complete recovery or actual TTS (group A1); ii) clear cochlear change with incomplete recovery (group A2); iii) mild or no change in cochlear function (group B). No relationship was found between the attenuation effect measured before noise overexposure and the susceptibility to TTS. Animals with a significant attenuation effect could fit into any of the three groups. In addition, the recovery from loud sound exposure was not paralleled with the changes occurring over time in the attenuation effect. Therefore, the conclusion that short-term effectiveness of medial efferents does not predict susceptibility to TTS in the GP is suggested.

Taurine entry into perilymph of the guinea pig.

Taurine is a beta-aminosulfonic acid and is a ubiquitous amino acid whose role in the cochlea is not well established. In this study, its entry from blood into perilymph was investigated in the guinea pig as animal model. The penetration rate of [3H]taurine (molecular weight 125) into the perilymph of the scala vestibuli was measured 1 and 2 h after the intravenous infusion of [3H]taurine in nephrectomized animals. Results showed a rate of penetration in perilymph related to plasma at 36 +/- 4.7% (n = 5) after 1 h and 43 +/- 5.6% (n = 5) after 2 h. Compared to the penetration rate of urea (molecular weight 60) and mannitol (molecular weight 186) reported previously in rats, a passive entry of taurine into perilymph through the blood-perilymph barrier is suggested.

Construction and characterization of bicistronic retroviral vectors encoding the multidrug transporter and beta-galactosidase or green fluorescent protein.

Multidrug resistance (MDR) can be conferred by overexpression of the adenosine triphosphate-driven multidrug transporter P-glycoprotein (Pgp) known as MDR1. Thus, two potential applications of the MDR1 gene that may be useful in gene therapy are the protection of bone marrow cells from the cytotoxic effects of chemotherapy regimens in cancer patients and its possible use as an in vivo selectable gene when linked to a therapeutic gene. In this study, we have designed two retroviral bicistronic expression vectors by linking the MDR1 gene to the reporters known as beta-galactosidase and the red-shifted green fluorescent protein (GFP). We report the creation of stable producer cell lines that synthesize virus particles carrying the MDR-internal ribosomal entry site (IRES)-lacZ and the MDR-IRES-GFP transgenes. These transcriptional fusions allow coordinate expression of Pgp and the reporter gene product to easily mark the MDR phenotype. Using the MDR-IRES-lacZ retrovirus, we demonstrate that periodic pulses of cytotoxic drug selection with a Pgp substrate enable sustained, long-term expression of the reporter beta-galactosidase in otherwise unstable transductants. We have also incorporated the improved features of GFP as a reporter gene into our MDR-IRES-GFP retrovirus. This vector allows rapid and specific identification of MDR1 gene transfer and expression in living cells either by fluorescence microscopy or by fluorescence-activated cell sorter analysis. These two MDR/reporter gene systems should be useful for in vivo studies, the evaluation of the potential of the MDR1 gene in gene therapy applications, and as a monitor of the selective efficacy of its MDR phenotype.