Outcome of surgery for chronic suppurative otitis media with resistant Pseudomonas aeruginosa.

OBJECTIVE: To assess the effectiveness of surgical treatment of active chronic suppurative otitis media (CSOM) with ciprofloxacin-resistant Pseudomonas aeruginosa (P. aeruginosa) that failed comprehensive local and systemic treatment. STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral center. PATIENTS: Eleven patients with ciprofloxacin-resistant P. aeruginosa CSOM that remained active despite comprehensive local and systemic treatment. All patients were operated by a single surgeon between February 2016 and July 2019 INTERVENTION(S): Tympanoplasty alone was performed in seven cases and accompanied by mastoidectomy in the other four cases. MAIN OUTCOME MEASURE(S): Resolution of infection and tympanic graft take on otoscopy. The secondary outcome measure is hearing. RESULTS: Tympanic graft take was successful and the infectious process was resolved in 8 out of the 11 cases, yielding a success rate of 73%. The average follow-up was 20 months. No surgical complications occurred. CONCLUSIONS: Tympanoplasty, with or without mastoidectomy, is safe and yields acceptable anatomical and functional success rates when intensive local and systemic treatment fails to stop the purulent discharge.

Increased incidence of Susac syndrome: a case series study.

BACKGROUND: Susac syndrome (SuS) is a rare condition characterized by a clinical triad of sensorineural hearing loss, branch artery occlusion and encephalopathy. This study reports an increased incidence of SuS in Israel. We describe the clinical characteristics of these patients, diagnostic procedures and the use and subsequent outcomes of newly published treatment guidelines. METHODS: This is a single center retrospective study. Patients who were diagnosed with SuS between July 2017 and August 2018 were enrolled in this study. RESULTS: Seven patients were diagnosed with SuS according to the diagnostic criteria in a time period of 13 months. The annual incidence was recently evaluated in Austria to be 0.024/100000, therefore, our case series represent at least a 5.4- fold increase in the annual incidence of SuS expected in Israel and a 7-fold increase in the annual incidence expected in our medical center. Mean time from the onset of the symptoms to diagnosis was three weeks and follow-up time was twenty four months. Recent exposure to cytomegalovirus was serologically evident in three patients and one patient had high titer of anti-streptolysin antibody. All patients underwent brain MRI, fluorescein angiography and audiometry. All patients were treated according to the newly recommended guidelines. All patients achieved clinical and radiological stability. CONCLUSIONS: We report of an increased incidence of SuS in Israel. Infectious serological findings may imply a post infectious mechanism. The use of the recommended diagnostic procedures reduced the time to diagnosis. Newly published treatment guidelines led to favorable clinical outcomes.

Integrin-based therapy of pancreatic adenocarcinoma: current status and future perspectives.

Pancreatic ductal adenocarcinoma (PDAC), although tenth in cancer incidence, holds the dubious distinction of being the fifth cause of cancer deaths in the Western countries and possibly the deadliest malignancy. Inoperable PDAC is characterized by late diagnosis, extensive metastases, extremely poor response to chemotherapy and, consequently, poor patients' prognosis-6.7% 5-year survival. PDAC reflects the failure of the medical profession to significantly prolong patients' lives and modest expectations for future cure. PDAC is characterized by extensive desmoplastic reaction, resulting in approximately 50% of tumor's volume consisting of non-tumor cells and extracellular matrix (ECM) stroma. These properties imply an important role for cell-ECM interaction, making cell-matrix adhesion molecules, such as integrins, of special interest as possible candidate targets for future anti-PDAC therapies. This review will attempt to overview the status of studies dealing with the involvement of integrins in the unique aggressive character of PDAC, the current status of experimental cancer therapies targeted at integrins, and the possible application of these preliminary clinical experiments to future PDAC therapy. I will also try to delineate the reasons for the failures of PDAC therapies and make some modest suggestions that might improve the health scientific community approaches to this extremely difficult problem.

Video head impulse test for the assessment of vestibular function in patients with idiopathic sudden sensorineural hearing loss without vertigo.

OBJECTIVE: Idiopathic sudden sensorineural hearing loss may be accompanied by dizziness without true vertigo. This study used the video head impulse test to evaluate vestibular function in idiopathic sudden sensorineural hearing loss patients who described experiencing dizziness and not true vertigo. METHODS: A prospective study was conducted of 30 consecutive patients diagnosed with idiopathic sudden sensorineural hearing loss with dizziness without true vertigo. A comparison of the video head impulse test results of the patients who complained of dizziness (symptomatic group) with a group of patients with idiopathic sudden sensorineural hearing loss and no dizziness (asymptomatic) was performed. RESULTS: Nine patients (30 per cent) were symptomatic. Two of those patients had abnormal video head impulse test findings. Seven patients in the asymptomatic group (7 out of 21, 33 per cent) presented with abnormal video head impulse test results. No significant difference in vestibular function between the two groups was detected by the video head impulse test. CONCLUSION: The site of insult in patients with idiopathic sudden sensorineural hearing loss without true vertigo is usually limited to the cochlea or the cochlear nerve.

Non-melanoma skin cancer diagnosis: a comparison between dermoscopic and smartphone images by unified visual and sonification deep learning algorithms.

PURPOSE: Non-melanoma skin cancer (NMSC) is the most frequent keratinocyte-origin skin tumor. It is confirmed that dermoscopy of NMSC confers a diagnostic advantage as compared to visual face-to-face assessment. COVID-19 restrictions diagnostics by telemedicine photos, which are analogous to visual inspection, displaced part of in-person visits. This study evaluated by a dual convolutional neural network (CNN) performance metrics in dermoscopic (DI) versus smartphone-captured images (SI) and tested if artificial intelligence narrows the proclaimed gap in diagnostic accuracy. METHODS: A CNN that receives a raw image and predicts malignancy, overlaid by a second independent CNN which processes a sonification (image-to-sound mapping) of the original image, were combined into a unified malignancy classifier. All images were histopathology-verified in a comparison between NMSC and benign skin lesions excised as suspected NMSCs. Study criteria outcomes were sensitivity and specificity for the unified output. RESULTS: Images acquired by DI (n = 132 NMSC, n = 33 benign) were compared to SI (n = 170 NMSC, n = 28 benign). DI and SI analysis metrics resulted in an area under the curve (AUC) of the receiver operator characteristic curve of 0.911 and 0.821, respectively. Accuracy was increased by DI (0.88; CI 81.9-92.4) as compared to SI (0.75; CI 68.1-80.6, p < 0.005). Sensitivity of DI was higher than SI (95.3%, CI 90.4-98.3 vs 75.3%, CI 68.1-81.6, p < 0.001), but not specificity (p = NS). CONCLUSION: Telemedicine use of smartphone images might result in a substantial decrease in diagnostic performance as compared to dermoscopy, which needs to be considered by both healthcare providers and patients.

Calculated versus measured pure tone bone conduction 3 kHz thresholds in sudden sensorineural hearing loss.

OBJECTIVE: To compare the measured bone conduction threshold at 3 kHz with the calculated threshold in newly diagnosed sudden sensorineural hearing loss. METHODS: A retrospective chart review was conducted of pure tone audiograms in confirmed sudden sensorineural hearing loss cases. RESULTS: Of 157 patients with sudden sensorineural hearing loss, 144 had idiopathic hearing loss, 8 had vestibular schwannoma and 5 had Ménière's disease. The r value for the correlation between the two methods of 3 kHz assessment for all patients was 0.887 (p < 0.001). The mean difference between the measured and calculated 3 kHz thresholds was 0.76 ± 7.96 dB, 0.4 ± 8.08 dB and 1.5 ± 1.8 dB in the sudden sensorineural hearing loss, idiopathic and Ménière's disease groups, respectively. The mean difference between the measured and calculated 3 kHz thresholds was significantly greater in the vestibular schwannoma group (6.86 ± 4.38 dB) than in the idiopathic group (p = 0.013). CONCLUSION: The 3 kHz frequency may encompass important audiometric information. A discrepancy between the measured and calculated bone conduction 3 kHz thresholds raises suspicion of an underlying vestibular schwannoma as an aetiology for sudden sensorineural hearing loss, and these thresholds should therefore be measured independently and routinely.

Eustachian tube dysfunction in candidates for surgery for obstructive sleep apnoea syndrome.

BACKGROUND: Defining the risk factors for Eustachian tube dysfunction can facilitate its prevention. It is hypothesised that Eustachian tube dysfunction as measured by the Eustachian Tube Dysfunction Questionnaire-7 is associated with obstructive sleep apnoea syndrome. METHODS: The questionnaire was systematically translated into Hebrew and validated in the accepted manner. This questionnaire was applied to obstructive sleep apnoea syndrome patients before and after expansion sphincter pharyngoplasty, in pre-set time intervals. The results were compared to those of controls from the general population. RESULTS: Thirty-one patients (males:females = 19:12) were enrolled in the obstructive sleep apnoea syndrome group. Mean age was 43 years (range, 31-55 years) and mean body mass index was 28 kg/m2 (range, 27-30 kg/m2). Median apnoea-hypopnea index (pre-operatively) was 34 events per hour. The questionnaire scores in expansion sphincter pharyngoplasty candidates were significantly worse than in controls (p < 0.001). Expansion sphincter pharyngoplasty did not change Eustachian tube function in the long term, but was associated with additional self-limiting Eustachian tube dysfunction in the first two post-operative months. CONCLUSION: Eustachian tube dysfunction is significantly worse in patients with obstructive sleep apnoea syndrome compared to controls. Expansion sphincter pharyngoplasty is not associated with Eustachian tube function improvement.

Decreased TRH receptor mRNA activity precedes homologous downregulation: assay in oocytes.

Ligand-induced decrease in cell-surface receptor number (homologous downregulation) is often due to rapid receptor internalization. Thyrotropin-releasing hormone (TRH), however, causes a slow downregulation of TRH receptors (TRH-Rs), with a half-time of approximately 12 hours, in GH3 rat pituitary cells. The mechanism of TRH-R downregulation was studied by monitoring TRH-evoked depolarizing currents in Xenopus oocytes injected with GH3 cell RNA as a bioassay for TRH-R messenger RNA (mRNA) activity. In GH3 cells, TRH caused a rapid decrease in TRH-R mRNA activity to 15 percent of control within 3 hours. Because the half-life of TRH-R mRNA activity in control cells was approximately 3 hours, the rapid decrease in mRNA activity was not due to inhibition of mRNA synthesis alone and may represent a post-transcriptional effect.

Growth advantage and enhanced toxicity of Escherichia coli adherent to tissue culture cells due to restricted diffusion of products secreted by the cells.

This study was undertaken to examine whether Escherichia coli adherent to tissue cells gain advantages over nonadherent bacteria due to their proximity to the cells. We used tissue culture cells and isogenic derivatives of a proline auxotrophic strain of E. coli that were fimbriated (Fim+) or nonfimbriated (Fim-), and were heat-labile enterotoxin producing (Tox+) or toxin nonproducing (Tox-). We found that the Fim+ bacteria; which were capable of adhering to tissue culture cells, initiated growth much sooner than did nonadherent Fim- bacteria; the adherent bacteria used tissue cell-derived proline, which was available at high concentrations only in the zone of bacterial adherence. Likewise, cyclic AMP secreted by adherent (Fim+) bacteria was maintained at high concentration on the tissue cell surfaces. As few as 2 X 10(5) adherent Fim+ Tox+ bacteria exert toxic activity upon Y1 adrenal cells, whereas toxin secreted in the medium by 6 X 10(6) Fim- Tox+ bacteria was undetectable. The results suggest that the growth advantage and enhanced toxicity of adherent E. coli is due to restricted diffusion of products secreted by the tissue culture and bacterial cells, respectively.

Evidence for a tinnitus subgroup responsive to somatosensory based treatment modalities.

Studies have established that the somatosensory system of the upper cervical region and head can be intimately involved in tinnitus. Tinnitus can arise directly from a disorder of the head and upper neck through activation of the somatosensory system. "Somatic testing" (a series of strong muscle contractions of the head and neck) can (1) modulate the tinnitus percept of approximately 80% of people with ongoing tinnitus, and (2) elicit a sound percept in approximately 50% of people with no tinnitus. These somatic phenomena are equally prevalent among people with or without functioning cochlea. Likely neural pathways underlying both the induction and modulation of tinnitus have been revealed in animal studies. Because somatic influences are fundamental to the operation of the auditory system, in general, and to tinnitus, in particular, somatic testing should be incorporated into all evaluations of tinnitus (1) to improve understanding of the role of the somatosensory system in any individual and (2) to identify subgroups of tinnitus patients who may respond to a particular treatment modality (as has already been shown for the tinnitus associated with temporomandibular disorder). Our clinical experience and review of reports of treatment modalities directed toward the somatosensory system supports the hypothesis that these modalities can benefit individuals with symmetric hearing thresholds but asymmetric widely fluctuating tinnitus. Treatment modalities involving the somatosensory system should be re-assessed by targeting this tinnitus subgroup.

Genetic synthetic lethality screen at the single gene level in cultured human cells.

Recently, we demonstrated the feasibility of a chemical synthetic lethality screen in cultured human cells. We now demonstrate the principles for a genetic synthetic lethality screen. The technology employs both an immortalized human cell line deficient in the gene of interest, which is complemented by an episomal survival plasmid expressing the wild-type cDNA for the gene of interest, and the use of a novel GFP-based double-label fluorescence system. Dominant negative genetic suppressor elements (GSEs) are selected from an episomal library expressing short truncated sense and antisense cDNAs for a gene likely to be synthetic lethal with the gene of interest. Expression of these GSEs prevents spontaneous loss of the GFP-marked episomal survival plasmid, thus allowing FACS enrichment for cells retaining the survival plasmid (and the GSEs). The dominant negative nature of the GSEs was validated by the decreased resident enzymatic activity present in cells harboring the GSEs. Also, cells mutated in the gene of interest exhibit reduced survival upon GSE expression. The identification of synthetic lethal genes described here can shed light on functional genetic interactions between genes involved in normal cell metabolism and in disease.

Mechanism of membrane electrical response to thyrotropin-releasing hormone in Xenopus oocytes injected with GH3 pituitary cell messenger ribonucleic acid.

TRH evoked a complex electrical membrane response in Xenopus laevis oocytes injected with either total cytosolic or poly(A)(+)-enriched RNA from GH3 pituitary cells but not in uninjected oocytes. A typical response consisted of a transient, rapid depolarizing current followed by a prolonged depolarizing current with superimposed current fluctuations. The reversal potentials of the rapid and the slow components of the response were -23.0 and -22.6 mV, respectively, and were markedly affected by CI- concentration indicating that the TRH response was mainly an increase in Cl- conductance. The response to TRH was dose dependent and was inhibited by the TRH antagonist, chlordiazepoxide. TRH caused rapid hydrolysis of labeled phosphatidylinositol 4,5-bisphosphate and a marked, prolonged increase in 45Ca2+ efflux from injected oocytes. The depolarizing response to TRH was not diminished in oocytes incubated in a Ca2(+)-free medium, but was inhibited by microinjection of EGTA. These data suggest that TRH evokes an electrophysiological response in oocytes injected with RNA from GH3 cells via activation of the same biochemical pathway that mediates its actions in GH3 cells. This pathway involves hydrolysis of phosphatidylinositol 4,5-bisphosphate, forming inositol trisphosphate that causes mobilization of cellular Ca2+. We suggest that oocytes injected with GH3 cell RNA, because of their large size and easy access to their intracellular milieu, will be a useful intact cell model in which to define the molecular details of signal transduction by TRH.

Receptor number determines latency and amplitude of the thyrotropin-releasing hormone response in Xenopus oocytes injected with pituitary RNA.

TRH evokes depolarizing membrane electrical responses in Xenopus laevis oocytes injected with RNA from pituitary cells. We have shown previously that the amplitude of this response is directly proportional to the dose of TRH and the amount of RNA injected. Herein we show that the number of TRH receptors expressed on oocytes after injection of rat pituitary (GH3) cell RNA or mouse thyrotropic (TtT) tumor RNA determines the latency as well as the amplitude of the response. In oocytes injected with a maximally effective amount of GH3 cell RNA, the latency of the response decreased from a maximal duration of 103 +/- 16 to 10 +/- 1 sec when the TRH concentration was increased from 5 to 3000 nM. When oocytes injected with different amounts of GH3 cell RNA were stimulated with 3000 nM TRH, the latency decreased from 31 +/- 4 to 11 +/- 0.5 sec when the amount of RNA injected was increased from 30 to 400 ng. Specific binding of [3H]methylhistidine-TRH increased when increasing amounts of TtT poly(A)+ RNA was injected, and binding correlated with increased response amplitude. To show that these effects were caused by mRNA for the TRH receptor and did not depend on other mRNAs, TtT poly(A)+ RNA was fractionated on a sucrose gradient. Using RNA from each fraction, there was an inverse correlation between response amplitude and latency. For size-fractionated RNA, as for unfractionated RNA, there was a direct correlation between specific [3H]methylhistidine-TRH binding and response amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)

A constitutively active mutant thyrotropin-releasing hormone receptor is chronically down-regulated in pituitary cells: evidence using chlordiazepoxide as a negative antagonist.

A carboxyl-terminus truncated mutant of the guanine nucleotide-binding (G) protein-coupled TRH receptor (TRH-R) was previously shown to exhibit constitutive, i.e. TRH-independent, activity (C335Stop TRH-R). Chlordiazepoxide (CDE), a known competitive inhibitor of TRH binding to wild-type (WT) TRH-Rs, is shown to compete for binding to C335Stop TRH-Rs also. More importantly, CDE is shown to be a negative antagonist of C335Stop TRH-Rs. CDE rapidly caused the basal rate of inositol phosphate second messenger (IP) formation to decrease in AtT-20 pituitary cells stably expressing C335Stop TRH-Rs (AtT-C335Stop cells), but not in cells expressing WT TRH-Rs (AtT-WT cells). Similar observations were made in HeLa cells transiently expressing C335Stop or WT TRH-Rs. CDE inhibition of IP formation was shown to be specific for TRH-Rs using GH4C1 cells expressing both TRH-Rs and receptors for bombesin. In these cells, CDE inhibited TRH-stimulated IP formation, but had no effect on bombesin-stimulated IP formation. The effects of chronic administration of CDE were studied. Preincubation of AtT-C335Stop cells, but not AtT-WT cells, with CDE for several hours caused an increase in cell surface receptor number (up-regulation) that led to increased TRH stimulation of inositol phosphate formation and elevation of intracellular free Ca2+. Preincubation with CDE did not affect methyl-TRH binding affinity or TRH potency in cells expressing AtT-C335Stop or in AtT-WT cells. We conclude that CDE is a negative antagonist of C335Stop TRH-Rs and that constitutively active C335Stop TRH-Rs are down-regulated in AtT-20 pituitary cells in the absence of agonist.

Calcium control of glycogen synthase activities in mouse diaphragms, rat adipocytes and rat hepatocytes.

The following article provides evidence that cellular calcium controls the activity of glycogen synthase in all three major glycogen storage tissues; muscle, fat, and liver. Depletion of cellular calcium resulted in a moderate increase of glycogen synthase %I activities in intact mouse diaphragms, in isolated rat adipocytes, and in rat hepatocytes. The increase in %I activity of glycogen synthase was more pronounced when the uridine di-phosphoglucose concentration in the glycogen synthase assay was lowered from 4.4 mM to 0.2 mM. Calcium depletion resulted in an approximately two-fold decrease in the Ka values for glucose-6-phosphate in all three tissues. The activities of glycogen synthase also correlated well with the content of cell-associated calcium in rat hepatocytes. The glucose-6-phosphate independent activities of glycogen synthase in extracts of calcium-replete and calcium-depleted tissue approached the same value following the exposure to crude phosphoprotein phosphatase. The activities of glycogen phosphorylase decreased in calcium-depleted tissues and cells. Insulin stimulated the activity of glycogen synthase in muscle and fat in the absence of added sugar and in the absence of extracellular calcium. It is concluded that glycogen synthase is under the control of calcium in the three main glycogen storage tissues. The actions of calcium are probably mediated through the actions of calcium-sensitive protein kinase(s).

Agonist-evoked calcium efflux from a functionally discrete compartment in Xenopus oocytes.

Agonist-induced calcium (Ca) mobilization is accompanied by Ca efflux, presumably reflecting the rise in Ca concentration at the cytosolic surface of the cell membrane. We studied the relationship between Ca efflux and intracellular Ca mobilization in Xenopus oocytes. Elevation of cytosolic Ca by a direct injection of 1 nmol 45CaCl2 resulted in a typical Ca-activated chloride current, but not in 45Ca efflux. This demonstrated that a Ca rise at the cytoplasmic surface of the membrane is not sufficient to produce an increased efflux. Co-injection of inositol 1,4,5-trisphosphate (InsP3), to prevent rapid Ca sequestration, also failed to cause Ca efflux. Smaller amounts of labelled Ca (0.05 nmol) equilibrated with Ca stores in a time-dependent pattern with an optimum at 2 h after injection. In contrast, Ca taken up from the medium was immediately available for agonist- or InsP3-induced efflux. Emptying the agonist-sensitive stores with thapsigargin (TG) did not affect chloride currents induced by Ca injection, indicating that these currents were due to direct elevation of Ca at the plasma membrane, rather than Ca-induced Ca release from InsP3-sensitive stores. Agonist-induced depletion of Ca stores enhanced uptake from the extracellular medium and the subsequent release of the label by an agonist. Similar protocol when the label was injected into the oocytes, failed to affect agonist induced efflux. We suggest that, under physiological conditions, agonist-dependent Ca extrusion or uptake in oocytes is executed exclusively via a functionally restricted compartment, which is closely associated with both agonist-sensitive Ca stores and the plasma membrane.

Calcium entry in Xenopus oocytes: effects of inositol trisphosphate, thapsigargin and DMSO.

Agonist- and inositol 1,4,5-trisphosphate (InsP3)-evoked responses in Xenopus oocytes utilize calcium mobilized from cellular stores as well as from the medium. We studied the effect of the status of Ca stores on InsP3-induced Ca entry. Thapsigargin (TG) caused a net increase of 45Ca2+ efflux from oocytes in a time and dose dependent manner (31 and 54% of total label, at 30 and 60 min, respectively). Incubation with TG (60 min) resulted in a complete loss of the response to InsP3 implying that InsP3-sensitive Ca stores were depleted. Challenge with 1.8 mM Ca2+ resulted in a large depolarizing chloride current (1231 +/- 101 nA) which was not further potentiated by InsP3. This suggested that extensive depletion of cellular Ca stores is sufficient to induce maximal entry of extracellular Ca (Cao). Following the injection of InsP3, a much more limited loss of cellular Ca was sufficient to produce large Ca entry. Dimethyl sulfoxide (DMSO) alone, the vehicle used to dissolve TG, did not cause increase in either efflux of 45Ca2+, nor in the Cao-evoked Cl- current. It did, however, markedly potentiate this current following the injection of InsP3. DMSO moderately inhibited InsP3-induced 45Ca2+ efflux from oocytes. Hence, apparent potentiation of Ca entry can be observed without additional depletion of cellular Ca. We conclude that Ca entry may be induced via either stimulation with InsP3 and limited Ca depletion or depletion of a specific and, possibly small, cellular Ca store alone. The mechanism of DMSO potentiation is unknown, but may be important in view of the universal use of this solvent as vehicle.

Independent external calcium entry and cellular calcium mobilization in Xenopus oocytes.

We studied cellular calcium (Ca) mobilization and Ca entry from the medium following injection of various inositol phosphates (IPs) or activation of thyrotropin-releasing hormone receptors (TRH-Rs) in oocytes injected with TRH-R cRNA. We determined the order of potency of various IPs for evoking the rapid depolarizing current in Ca-free medium, which reflects the mobilization of cellular Ca. The most potent compound was inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), followed by inositol 1,2,4,5-tetrakisphosphate (Ins(1,2,4,5)-P4), which displayed 91% of the activity of Ins(1,4,5)P3, while inositol 1,3,4,6-tetrakisphosphate (Ins(1,3,4,6)P4) had only 29% effect. All other IPs used in the present study exhibited responses that were 40% or less than those elicited by Ins(1,4,5)P3. Cellular Ca mobilization was confirmed by 45Ca2+ efflux for Ins(1,4,5)P3, Ins(1,2,4,5)P4 and Ins(1,3,4,6)P4, or by Fura-2 ratio imaging studies for the latter. In parallel, we assayed the ability of these compounds to promote Ca entry into the cell, as reflected by Ca-evoked depolarizing current or Fura-2 imaging. These assays revealed a different order of potency, where Ins(1,4,5)P3 > inositol 4,5-bisphosphate (Ins(4,5)P2) > Ins(1,3,4,6)P4 = Ins(1,2,4,5)P4. All other inositol phosphates were largely ineffective. Heparin inhibited the response to TRH by 67% while Ca entry was inhibited only by 22%. The latency of the response to TRH was significantly shorter in the presence of extracellular Ca, suggesting Ca entry preceded the response, i.e. major depletion of Ca stores. These results strongly suggest that the activation of Ca entry is largely independent of cellular Ca mobilization and may be mediated by a receptor for an unidentified phosphorylated compound, different from that for Ins(1,4,5)P3 on the endoplasmic reticulum.

A hyperosmotic stimulus elevates intracellular calcium and inhibits proliferation of a human keratinocyte cell line.

Occlusion has previously been used to treat psoriatic plaques and was shown to improve the condition. We investigated the consequences of applying a mechanical stress, in vitro, on the HaCaT keratinocyte cell line. A mechanical load applied to cells can be mimicked by a hyperosmotic stimulus. Exposure of HaCaT keratinocytes to different hyperosmotic solutions (final osmolarity in the range 350-600 mOsm, produced by sucrose addition) resulted in an inhibition of cell proliferation after 96 h of treatment. As keratinocyte maturation is regulated by calcium levels, we measured hyperosmotic-stimulus-induced changes of intracellular calcium ([Ca2+]i) by single-cell image analysis employing FURA-2/AM. The hyperosmotic stimulus produced a rapid transient 2.6-fold elevation of [Ca2+]i followed by a gradual decay to the basal level. The transients originated from extracellular as well as from intracellular calcium pools and did not respond to voltage-sensitive calcium channel blockers. The hyperosmotic stimulus was shown to increase the cellular expression of involucrin, a differentiation marker, following 72 h of incubation, as measured by flow cytometry. Treatment of cells with the [Ca2+]i chelator BAPTA/AM almost completely blocked the [Ca2+]i elevation, but did not alter cellular growth or the induction of differentiation observed after hyperosmotic stimulus. It is suggested that treatment of keratinocytes with hyperosmotic stimulus can induce short-time effects (calcium transients) as well as long-term cellular maturation.