Multimodal decoding of human liver regeneration.

The liver has a unique ability to regenerate1,2; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option3-5. Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2+ migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut-liver barrier may advance new areas of therapeutic discovery in regenerative medicine.

Resolving the fibrotic niche of human liver cirrhosis at single-cell level.

Liver cirrhosis is a major cause of death worldwide and is characterized by extensive fibrosis. There are currently no effective antifibrotic therapies available. To obtain a better understanding of the cellular and molecular mechanisms involved in disease pathogenesis and enable the discovery of therapeutic targets, here we profile the transcriptomes of more than 100,000 single human cells, yielding molecular definitions for non-parenchymal cell types that are found in healthy and cirrhotic human liver. We identify a scar-associated TREM2+CD9+ subpopulation of macrophages, which expands in liver fibrosis, differentiates from circulating monocytes and is pro-fibrogenic. We also define ACKR1+ and PLVAP+ endothelial cells that expand in cirrhosis, are topographically restricted to the fibrotic niche and enhance the transmigration of leucocytes. Multi-lineage modelling of ligand and receptor interactions between the scar-associated macrophages, endothelial cells and PDGFRα+ collagen-producing mesenchymal cells reveals intra-scar activity of several pro-fibrogenic pathways including TNFRSF12A, PDGFR and NOTCH signalling. Our work dissects unanticipated aspects of the cellular and molecular basis of human organ fibrosis at a single-cell level, and provides a conceptual framework for the discovery of rational therapeutic targets in liver cirrhosis.

αv integrins on mesenchymal cells regulate skeletal and cardiac muscle fibrosis.

Mesenchymal cells expressing platelet-derived growth factor receptor beta (PDGFRß) are known to be important in fibrosis of organs such as the liver and kidney. Here we show that PDGFRß+ cells contribute to skeletal muscle and cardiac fibrosis via a mechanism that depends on αv integrins. Mice in which αv integrin is depleted in PDGFRß+ cells are protected from cardiotoxin and laceration-induced skeletal muscle fibrosis and angiotensin II-induced cardiac fibrosis. In addition, a small-molecule inhibitor of αv integrins attenuates fibrosis, even when pre-established, in both skeletal and cardiac muscle, and improves skeletal muscle function. αv integrin blockade also reduces TGFß activation in primary human skeletal muscle and cardiac PDGFRß+ cells, suggesting that αv integrin inhibitors may be effective for the treatment and prevention of a broad range of muscle fibroses.

Growth and the Growth Hormone-Insulin Like Growth Factor 1 Axis in Children With Chronic Inflammation: Current Evidence, Gaps in Knowledge, and Future Directions.

Growth failure is frequently encountered in children with chronic inflammatory conditions like juvenile idiopathic arthritis, inflammatory bowel disease, and cystic fibrosis. Delayed puberty and attenuated pubertal growth spurt are often seen during adolescence. The underlying inflammatory state mediated by proinflammatory cytokines, prolonged use of glucocorticoid, and suboptimal nutrition contribute to growth failure and pubertal abnormalities. These factors can impair growth by their effects on the GH-IGF axis and also directly at the level of the growth plate via alterations in chondrogenesis and local growth factor signaling. Recent studies on the impact of cytokines and glucocorticoid on the growth plate further advanced our understanding of growth failure in chronic disease and provided a biological rationale of growth promotion. Targeting cytokines using biological therapy may lead to improvement of growth in some of these children, but approximately one-third continue to grow slowly. There is increasing evidence that the use of relatively high-dose recombinant human GH may lead to partial catch-up growth in chronic inflammatory conditions, although long-term follow-up data are currently limited. In this review, we comprehensively review the growth abnormalities in children with juvenile idiopathic arthritis, inflammatory bowel disease, and cystic fibrosis, systemic abnormalities of the GH-IGF axis, and growth plate perturbations. We also systematically reviewed all the current published studies of recombinant human GH in these conditions and discussed the role of recombinant human IGF-1.

A randomized trial of nortriptyline for severe chronic tinnitus. Effects on depression, disability, and tinnitus symptoms.

OBJECTIVE: To determine whether the antidepressant, nortriptyline, is effective for treatment of depression, tinnitus-related disability, and tinnitus symptoms in patients with severe chronic tinnitus. DESIGN: A 12-week, double-blind, randomized controlled trial. SETTING: A university otolaryngology clinic. PATIENTS: Ninety-two subjects with severe chronic tinnitus: 38 with current major depression and 54 with depressive symptoms and significant tinnitus-related disability. INTERVENTION: Nortriptyline (maintained at 50 to 150 mg/mL for 6 weeks) or placebo. MAIN OUTCOME MEASURES: Hamilton Depression Rating Scale, Tinnitus Disability Measures, and Audiometric Measures. RESULTS: Nortriptyline was superior to placebo by multivariate analysis of covariance for depression (10.6 vs 14.3 final Hamilton Depression score), for tinnitus-related disability (1.8 vs 2.4 final MPI Tinnitus Interference), and tinnitus loudness (13.6 vs 20.0 dB final loudness match [in worst ear at tinnitus frequency]). When major depression and depressive symptoms groups were considered separately, nortriptyline was superior to placebo on these same measures but differences did not achieve statistical significance. CONCLUSIONS: The antidepressant nortriptyline decreases depression, functional disability, and tinnitus loudness associated with severe chronic tinnitus. What appears to be irreversible disability of otologic origin may, in part, be reversible disability of psychiatric origin.

Psychiatric and otologic diagnoses in patients complaining of dizziness.

BACKGROUND: Dizziness is a common and disabling symptom in primary care practice, especially among the elderly. Though there are many organic causes of dizziness, the results of medical workups are negative in the majority of patients. METHODS: A total of 75 patients with dizziness who were referred to a community otolaryngology practice received a structured psychiatric diagnostic interview (National Institute of Mental Health Diagnostic Interview Schedule) and questionnaires that assessed psychological distress as well as a complete otologic evaluation, including electronystagmogram. Patients with evidence of a peripheral vestibular disorder were compared with those without such evidence. RESULTS: While psychiatric diagnoses were present in both those with and without evidence of a peripheral vestibular disorder, those without such evidence had a greater mean number of lifetime psychiatric diagnoses as defined by the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition, and specifically, a greater lifetime prevalence of major depression and panic disorder. This group also more frequently met criteria for somatization disorder, had more current and lifetime unexplained medical symptoms, and had more severe current depressive, anxiety, and somatic symptoms. CONCLUSIONS: Psychiatric diagnoses are common among patients with dizziness referred for otologic evaluation who do not show evidence of a peripheral vestibular disorder. Specific psychiatric disorders should be part of the differential diagnosis of patients who present with dizziness.

Evoked electromyography of the eleventh nerve and trapezius muscle.

Values and criteria for abnormality of the eleventh nerve evoked electromyography (EEMG) have been determined. The nerve conduction velocity and duration appear to be relatively consistent from subject to subject. Amplitude of response, while varying significantly from subject to subject, remains relatively constant when the right shoulder is compared to the left shoulder and when a retest is performed.

Depressive symptoms and measures of disability: a prospective study.

OBJECTIVE: The purpose of this study was to test the hypothesis that decreases in severity of depressive symptoms in patients with chronic tinnitus would correlate with reductions in measures of functional disability. METHOD: This study describes the correlations between several measures of functional disability and Hamilton depression scores in two groups of patients with major depression and depressive symptoms (D-NOS) who were patients enrolled in a 12-week placebo-controlled, double-blind trial of nortriptyline. These patients had chronic severe tinnitus which was associated in most patients with high frequency hearing loss. The effect of whether affective symptoms improved and the patient's initial depression status (major depression versus depressive symptoms) are examined in order to increase understanding about the correlations between depressive symptoms and functional disability. RESULTS: Most measures of functional disability decreased synchronously with Hamilton Depression Scale scores in both patients with major depression and those with subclinical depressive symptoms. Patients whose depression improved had a significantly greater change in each disability measure than patients whose depression did not improve. This significant decrease in functional disability was seen in 11 of 13 scales in the improved D-NOS group, versus only 3 of the 13 scales in the unimproved D-NOS group; 9 of 13 scales of functional disability significantly decreased in the improved major depression group, versus 3 of 13 scales in the unimproved major depression group. CONCLUSIONS: The results suggest that there was a significant correlation between improvement in both major depression and depression-NOS symptoms, and decreases in measures of functional disability in an aging population with a chronic medical illness.

Coping and marital support as correlates of tinnitus disability.

Although there is increasing awareness that depression can add significantly to the disability associated with chronic medical illness, it is not clear whether all of the impact of psychosocial factors upon medical disability are mediated by or moderated by depression. It has not been determined whether treating depression alone is an adequate strategy for addressing psychosocial magnification of medical disability. We analyzed data collected at initiation of a treatment trial from 92 subjects with chronic severe tinnitus to assess the role of coping, and 49 subject-spouse pairs to assess the role of marital interaction in tinnitus-related role dysfunction. Three multiple regression models were developed. After accounting for gender, tinnitus loudness, and depressive severity among the 92 subjects, greater role dysfunction was associated appraisal of tinnitus as salient, and less role dysfunction with coping through avoidance or seeking social support. Marital interaction was assessed from patient and spouse perspectives. In the patient-rated set, less marital cohesion was associated with greater tinnitus-related role dysfunction. In the spouse-rated set, more punishing responses to subject illness behavior were associated with greater tinnitus-related role dysfunction. In each case the disabling effect was greater in the face of high levels of subject depression. This study provides evidence for the oft-stated analogy between chronic tinnitus and chronic pain, and provides justification for a similar multimodal treatment strategy. Reducing depression is an important means to reduce medical disability but should be supplemented by clinical attention to appraisal of the illness, modes of coping with the illness, and spousal response to the illness.

Disabling tinnitus. Association with affective disorder.

Forty consecutive patients with disabling tinnitus were interviewed using a structured psychiatric interview and were asked to complete the Hopkins Symptom Checklist (SCL-90), the Chronic Illness Problem Inventory, and the Revised Ways of Coping Checklist. They were compared to a control group of 14 patients attending the same otolaryngologic clinic with a complaint of hearing loss. The tinnitus patients had a significantly greater lifetime prevalence of major depression (78% vs 21%) than controls and a significantly higher prevalence of current major depression (60% vs 7%). The currently depressed tinnitus patients had significantly higher scores on all subscales of the SCL-90 compared to the nondepressed tinnitus group and to the controls. The number of psychological problems as measured by the Chronic Illness Problem Inventory was significantly greater in the tinnitus group than in controls. This difference in psychosocial disability was due to the high psychologic and social impairment in the depressed tinnitus group, as there were no significant differences in psychosocial problems between the nondepressed tinnitus group and the controls. These results demonstrate that tinnitus disability is strongly associated with major depression and suggest that treatment of the concurrent affective illness may reduce disability due to tinnitus.

Chronic tinnitus: association with psychiatric diagnoses.

Twenty-one consecutive patients with severe tinnitus were interviewed using a structured psychiatric interview (the National Institute of Mental Health Diagnostic Interview Schedule) and were asked to complete the Hopkins Symptom Checklist (SCL-90) and the Chronic Illness Problem Inventory. They were compared to a control group of 14 patients attending an otolaryngological clinic with a complaint of hearing loss. The tinnitus patients had a significantly greater lifetime prevalence of major depression (62% vs 21%) than controls and a significantly higher prevalence of current major depression (48% vs 7%). The currently depressed tinnitus patients had significantly higher scores on all subscales of the SCL-90, except the phobia and paranoid subscales, compared to the non-depressed tinnitus group and on all scales compared to the controls. The number of psychosocial problems and thus the resulting disability experienced was significantly greater in the tinnitus group compared to controls and in the currently depressed tinnitus patients when compared to non-depressed tinnitus patients. In view of our results treatment should aim at not only alleviation of tinnitus, but also the frequently co-existing major depression.

Symptoms as a clue to otologic and psychiatric diagnosis in patients with dizziness.

Dizziness is a common symptom that often remains unexplained despite extensive medical evaluation. Psychiatric disorders are usually considered only after all medical causes of dizziness have been ruled out. Sixty-five patients referred to an otolaryngology practice received a structured psychiatric interview, an otologic evaluation, and a dizziness questionnaire modified to assess psychiatric symptoms. They were divided into four diagnostic groups: psychiatric diagnosis only, otologic diagnosis only, both diagnoses, or neither diagnosis. Eleven questionnaire items were significantly associated with diagnostic groupings. Stepwise discriminant function analysis utilizing age, gender, rapid/irregular heartbeat, extremity weakness, nausea/vomiting, and difficulty with speech resulted in correct group classification for 70% of subjects. The presence of dizziness symptoms like vertigo or lightheadedness was not significantly different between groups. This study suggests that assessment of psychiatric and autonomic symptoms should accompany, not follow, otologic evaluation of dizziness. These symptoms may be more important diagnostically than dizziness quality.

Auditory responses to the envelopes of pseudorandom noise stimuli in humans.

Averaged scalp potentials evoked by continuous pseudorandom noise can be cross-correlated with the evoking stimulus, yielding a cross-correlation function (CCF) which reflects neural phase-locking and is quite sensitive for low-frequency stimulus components [M.J. Wilson and R.A. Dobie (1987) Electroencephalogr. Clin. Neurophysiol. 66, 529-538]. However, for higher frequency signals, replicable CCFs can only be obtained at moderate to high intensities. Since auditory neurons also respond to envelopes of complex sounds, even for high-frequency carriers, we compared scalp responses evoked by band-limited complex sounds to the envelopes of these sounds; the resultant envelope cross-correlation functions (ECCFs) contained replicable response components primarily below 1,000 Hz, regardless of the evoking stimulus spectrum. ECCF thresholds for three octave-band stimuli (830-1,562, 1,611-3,125, and 3,174-6,201 Hz) were more sensitive than CCF thresholds (P = 0.006), averaging 35 dB spectrum level for 10 normal subjects. When stimuli with only odd harmonics were used, replicable odd-component scalp responses were seen only in the spectral range of the stimuli, while even-component responses (presumably to stimulus envelope) were seen only in low-passed scalp responses.

Some aspects of temporal coding for single-channel electrical stimulation of the cochlea.

Estimates of the useful frequency range for single-channel electrical stimulation of the cochlea range from 400 to 4000 Hz. Psychophysical studies in single-channel implant patients are relevant not only to the practical problem of designing stimulation strategies, but also to questions of temporal processing of pitch in the normal auditory nervous system. Patients with single-channel extracochlear devices participated in several experiments involving stimuli differing in fine temporal structure. Stochastic pulse trains, in which the probability of pulse delivery (p) for a given cycle was less than 1.0, were readily discriminated from ordinary pulse trains. Frequency discrimination using stochastic pulse trains differing only in fine temporal structure (but identical average pulse rates) was as good as with ordinary pulse trains or sinusoids for P greater than or equal to 0.5, but deteriorated rapidly for P less than 0.5. Discrimination of triangular and trapezoidal waveforms from square waves was surprisingly good: rise-times (for 0 to maximum current) as low as 0.08 ms could be discriminated. Conversely, detection of jitter in pulse trains was almost an order of magnitude worse. The results show that frequency discrimination for single-channel electrical stimulation of the cochlea is based on discrimination of inter-pulse periods, and that pulse rates which would be unnatural for acoustically-evoked VIIIth nerve activity - up to 750 Hz - are more useful for coding mid-range frequencies than low-rate stochastic stimulations of normal VIIIth nerve firing patterns. The waveform discriminations reported would be obscured by low-pass filtering even at 2000 Hz, and probably depend on changes in relative synchrony among an array of VIIIth nerve units with different thresholds. In general, these results support the use of analog coding schemes with relatively large bandwidth.

Phase weighting: a method to improve objective detection of steady-state evoked potentials.

Objective response detection statistics such as magnitude-squared coherence (MSC) reflect the degree to which subaverage phases are clustered or dispersed, but not their agreement with an expected, or target, phase. Using signal-plus-noise simulations and human 40-Hz auditory evoked potentials, we tested MSC performance with and without phase weighting, in which MSC values were multiplied by weights related to the phase error between measured phase and target phase. Phase weighting improved MSC performance for both simulated and 40-Hz auditory evoked potential data. However, the improvement was greater for the simulations, probably because target phase was precisely known.

Amplitude-modulation following response (AMFR): effects of modulation rate, carrier frequency, age, and state.

Scalp responses to continuous amplitude-modulated (AM) tones were recorded from adults and 1-month-old infants. The amplitude-modulation following (or envelope) response (AMFR) was quantified using magnitude-squared coherence. This measurement indicates the strength of the frequency-following response relative to background neural noise. The optimal modulation rate for generating the AMFR was determined by studying the effects of stimulus modulation rate on the response. Stimulus AM rate was varied between 10 and 80 Hz for continuous tonal stimuli of 500 Hz, and between 20 and 80 Hz for continuous tonal stimuli of 2000 Hz. Optimal modulation rate was defined as the AM rate that provided the highest coherence estimate. Adult AMFR coherence increased between 10 and 40 Hz (20-40 Hz for 2000 Hz), and decreased between 40 and 80 Hz in both carrier frequency conditions. Infant AMFR coherence, in contrast, monotonically increased between 10 and 80 H (20-80 Hz for 2000 Hz). Thus, within the frequency range examined, 40 Hz is optimal for generating the AMFR in adults, whereas 80 Hz is optimal in infants. Adults were tested while awake and infants were tested during periods of sleep. Given the observed age difference in effective modulation rate, we examined modulation rate effects in a group of adults in both awake and sedated states. As in sleeping infants, 80 Hz was optimal for generating AMFRs in the sedated adults.

Coherence analysis of envelope-following responses (EFRs) and frequency-following responses (FFRs) in infants and adults.

The frequency-following response (FFR) and the envelope-following response (EFR) were recorded in 1-month-old infants and in adults to examine the development of temporal coding. The stimuli were amplitude-modulated (AM) tones. A modulation frequency of 80 Hz was used in infants; modulation frequencies of 40 and 80 Hz were used in adults. The effects of intensity, carrier frequency, and modulation frequency on these responses were studied. Responses were analyzed using magnitude-squared coherence. The effect of intensity on the growth of FFR- and EFR-coherence were similar in infants and adults. In addition, the growth functions were not affected by the carrier frequency or the modulation frequency of the stimulus. FFR thresholds did not differ across age groups. 'Best frequency' (i.e., infant 80 Hz and adult 40 Hz) EFR thresholds were the same for infants and adults at 500 and 1000 Hz, but infant EFR thresholds were poorer than adult thresholds at 2000 Hz. Thus, although FFRs and EFRs are primarily adult-like at 1 month of age, there are some age differences in the EFR that deserve further study.

Endolymphatic hydrops in the rabbit: auditory brainstem responses and cochlear morphology.

A rabbit model of endolymphatic hydrops was studied using detailed functional and cytohistologic methods. Immediately following surgical destruction of the endolymphatic sac and the distal portion of the duct, measures of the evoked auditory brainstem response (ABR) revealed mild to profound losses specific to low- and high-frequency test stimuli while responses to mid-frequency signals remained unchanged for the majority of animals. Rabbits exhibited varying degrees of vestibular upset involving both overt behavior and reduced responses to caloric stimulation. Histologic processing of the plastic embedded cochleae demonstrated distended Reissner's membranes along with extensive damage to apical and basal turn sensory cells and myelinated afferent nerve fibers while the middle portion of the cochlear duct remained relatively unaltered. An atypical pattern of hair cell lesions involving a greater loss for inner than for outer hair cells was identified at the interface between damaged apical sensorineural elements and the normal appearing organ of Corti of the middle turns.

Industrial audiometry and the otologist.

The Hearing Conservation Amendment published by the Occupational Safety and Health Administration (OSHA) in 1983 requires hearing conservation programs to be provided for over 5 million workers whose daily noise exposures exceed 85 dBA time-weighted average. The permissible exposure level is 90 dBA, above which the use of hearing protectors is mandatory. Current policies rely heavily on periodic audiometry to detect early threshold shifts due to inadequate use of hearing protection or to individual variation in susceptibility to noise induced hearing loss (NIHL). Studies including over 1,000 noise exposed workers have shown that audiometry in the industrial setting is less reliable than clinical audiometry. Pure tone averaging markedly reduces test-retest variability and should be incorporated into rules for decision-making based on industrial audiometry. However, at least half of observed shifts are spurious. In addition, aging is an important confounding variable; in our data, about half of the population mean threshold shift seen was attributable to aging. Thus, only a minority of threshold shifts seen in industrial audiometry are due to NIHL. The large number of "false positive" shifts will inevitably eventually have the effect of a de facto lowering of the permissible exposure level to 85 dBA. Otologic referral and major administrative action should be reserved for large or repeated threshold shifts. OSHA does not specify rules for otologic referral, but the criteria recommended by the American Academy of Otolaryngology-Head and Neck Surgery are reasonable and should be supported. The necessity for otologic evaluation in the differential diagnosis of NIHL from other entities (particularly baseline audiometric abnormalities) is not universally appreciated; otolaryngologists need to educate their medical and nonmedical colleagues about this.