Clinician Interruptions and Patient-Rated Clinician Empathy in Specialty Visits.

INTRODUCTION: Clinicians tend to interrupt patients when they are describing their problem, which may contribute to feeling unheard or misunderstood. Using transcripts of audio and video recordings from musculoskeletal (MSK) specialty visits, we asked what factors are associated with (1) Perceived clinician empathy, including the time a patient spends describing the problem and time to the first interruption, (2) duration of patient symptom description, and (3) duration between the end of greeting and first nonactive listening interruption. METHODS: We analyzed transcripts of 194 adult patients seeking MSK specialty care with a median age (Interquartile range [IQR]) of 47 (33 to 59) years. Participants completed postvisit measures of perceived clinician empathy, symptoms of depression, accommodation of pain, and health anxiety. A nonactive listening interruption was defined as the clinician unilaterally redirecting the topic of conversation. Factors associated with patient-rated clinician empathy, patient problem description duration, and time until the first nonactive listening interruption were sought in bivariate and multivariable analyses. RESULTS: The patient's narrative was interrupted at least one time in 144 visits (74%). The duration of each visit was a median of 12 minutes (IQR 9 to 16 minutes). The median time patients spent describing their symptoms was 139 seconds before the first interruption (IQR 84 to 225 seconds). The median duration between the end of the initial greeting and the first interruption was 60 seconds (IQR 30 to 103 seconds). Clinician interruption was associated with shorter duration of symptom description. Greater perceived clinician empathy was associated with greater accommodation of pain (regression coefficient [95% confidence interval] = 0.015 [0.0005-0.30]; P = 0.04). DISCUSSION: Clinician interruption was associated with shorter symptom presentation, but not with diminished perception of clinician empathy. Although active listening and avoidance of interruption are important communication tactics, other aspects of the patient-clinician relationship may have more effect on patient experience.

Correlation of the I-HaND Scale with Other Musculoskeletal Patient-Reported Outcome Measurement Scores.

Background Experiments can determine if nerve-specific patient-reported outcome measures (PROMs) can outperform regional or condition-specific PROMs. We compared a nerve-specific PROM of the upper extremity, the Impact of Hand Nerve Disorders (I-HaND) scale, to other validated measures quantifying activity intolerance and sought to assess interquestionnaire correlations and factors independently associated with activity intolerance and pain intensity. Methods One hundred and thirty patients with any upper extremity nerve-related condition completed measures of demographics, psychological limitations, quality of life, activity intolerance, and pain intensity. To quantify activity intolerance, we used the I-HaND, Patient-Reported Outcomes Measurement Information System Physical Function Upper Extremity, and Disabilities of the Arm, Shoulder and Hand short form. Results Strong interquestionnaire correlations were found between the activity intolerance measures ( r between 0.70 and 0.91). Multivariable analysis revealed that greater activity intolerance and greater pain intensity correlated most with greater symptoms of depression on all scales, with symptoms of depression accounting for 53 to 84% of the variability in the PROMs. Conclusion There is no clear advantage of the nerve-specific I-HaND over shorter, regional PROMs, perhaps because they are all so closely tied to mental health. Unless an advantage relating to responsiveness to treatment is demonstrated, we support using a brief arm-specific PROM for all upper extremity conditions. Level of Evidence Level II; Prognostic.

When Musculoskeletal Clinicians Respond to Empathetic Opportunities, do Patients Perceive Greater Empathy?

BACKGROUND: Patient use of verbal and nonverbal communication to signal what is most important to them can be considered empathetic opportunities. Orthopaedic surgeons may have mixed feelings toward empathetic opportunities, on one hand wanting the patient to know that they care, and on the other hand fearing offense, prolonged visit duration, or discussions for which they feel ill prepared. Evidence that action about empathetic opportunities does not harm the patient's experience or appreciably prolong the visit could increase the use of these communication tactics with potential for improved experience and outcomes of care. QUESTIONS/PURPOSES: Using transcripts from musculoskeletal specialty care visits in prior studies, we asked: (1) Are there factors, including clinician attentiveness to empathetic opportunities, associated with patient perception of clinician empathy? (2) Are there factors associated with the number of patient-initiated empathetic opportunities? (3) Are there factors associated with clinician acknowledgment of empathetic opportunities? (4) Are there factors associated with the frequency with which clinicians elicited empathetic opportunities? METHODS: This study was a retrospective, secondary analysis of transcripts from prior studies of audio and video recordings of patient visits with musculoskeletal specialists. Three trained observers identified empathetic opportunities in 80% (209 of 261) of transcripts of adult patient musculoskeletal specialty care visits, with any uncertainties or disagreements resolved by discussion and a final decision by the senior author. Patient statements considered consistent with empathetic opportunities included relation of emotion, expression of worries or concerns, description of loss of valued activities or loss of important roles or identities, relation of a troubling psychologic or social event, and elaboration on daily life. Clinician-initiated empathetic opportunities were considered clinician inquiries about these factors. Clinician acknowledgment of empathetic opportunities included encouragement, affirmation or reassurance, or supportive statements. Participants completed post-visit surveys of perceived clinician empathy, symptoms of depression, and health anxiety. Factors associated with perceived clinician empathy, number of empathetic opportunities, clinician responses to these opportunities, and the frequency with which clinicians elicited empathetic opportunities were sought in bivariate and multivariable analyses. RESULTS: After controlling for potentially confounding variables such as working status and pain self-efficacy scores in the multivariable analysis, no factors were associated with patient perception of clinician empathy, including attentiveness to empathetic opportunities. Patient-initiated empathetic opportunities were modestly associated with longer visit duration (correlation coefficient 0.037 [95% confidence interval 0.023 to 0.050]; p < 0.001). Clinician acknowledgment of empathetic opportunities was modestly associated with longer visit duration (correlation coefficient 0.06 [95% CI 0.03 to 0.09]; p < 0.001). Clinician-initiated empathetic opportunities were modestly associated with younger patient age (correlation coefficient -0.025 [95% CI -0.037 to -0.014]; p < 0.001) and strongly associated with one specific interviewing clinician as well as other clinicians (correlation coefficient -1.3 [95% CI -2.2 to -0.42]; p = 0.004 and -0.53 [95% CI -0.95 to -0.12]; p = 0.01). CONCLUSION: Musculoskeletal specialists can respond to empathic opportunities without harming efficiency, throughput, or patient experience. CLINICAL RELEVANCE: Given the evidence that patients prioritize feeling heard and understood, and evidence that a trusting patient-clinician relationship is protective and healthful, the results of this study can motivate specialists to train and practice effective communication tactics.

Does Addressing Mental Health During a Musculoskeletal Specialty Care Visit Affect Patient-rated Clinician Empathy?

BACKGROUND: Unhelpful thoughts and feelings of worry or despair about symptoms account for a notable amount of the variation in musculoskeletal symptom intensity. Specialists may be best positioned to diagnose these treatable aspects of musculoskeletal illness. Musculoskeletal specialists might be concerned that addressing mental health could offend the patient, and avoidance might delay mental health diagnosis and treatment. Evidence that conversations about mental health are not associated with diminished patient experience might increase specialist confidence in the timely diagnosis and initial motivation to treat unhelpful thoughts and feelings of worry or despair. QUESTIONS/PURPOSES: Using transcripts of videotaped and audiotaped specialty care visits in which at least one instance of patient language indicating an unhelpful thought about symptoms or feelings of worry or despair surfaced, we asked: (1) Is clinician discussion of mental health associated with lower patient-rated clinician empathy, accounting for other factors? (2) Are clinician discussions of mental health associated with patient demographics, patient mental health measures, or specific clinicians? METHODS: Using a database of transcripts of 212 patients that were audio or video recorded for prior studies, we identified 144 transcripts in which language reflecting either an unhelpful thought or feelings of distress (worry or despair) about symptoms was detected. These were labeled mental health opportunities. Patients were invited on days when the researcher making video or audio records was available, and people were invited based on the researcher's availability, the patient's cognitive ability, and whether the patient spoke English. Exclusions were not tracked in those original studies, but few patients declined. There were 80 women and 64 men, with a mean age of 45 ± 15 years. Participants completed measures of health anxiety, catastrophic thinking, symptoms of depression, and perceived clinician empathy. Factors associated with perceived clinician empathy and clinician discussion of mental health were sought in bivariate and multivariable analyses. RESULTS: Greater patient-rated clinician empathy was not associated with clinician initiation of a mental health discussion (regression coefficient 0.98 [95% confidence interval 0.89 to 1.1]; p = 0.65). A clinician-initiated mental health discussion was not associated with any factors. CONCLUSION: The observation that a clinician-initiated mental health discussion was not associated with diminished patient ratings of clinician empathy and was independent from other factors indicates that generally, discussion of mental health does not harm patient-clinician relationship. Musculoskeletal clinicians could be the first to notice disproportionate symptoms or misconceptions and distress about symptoms, and based on the evidence from this study, they can be confident about initiating a discussion about these mental health priorities to avoid delays in diagnosis and treatment. Future studies can address the impact of training clinicians to notice unhelpful thoughts and signs of distress and discuss them with compassion in a specialty care visit; other studies might evaluate the impact of timely diagnosis of opportunities for improvement in mental health on comfort, capability, and optimal stewardship of resources.

Are the Pathologic Features of Enthesopathy, Tendinopathy, and Labral and Articular Disc Disease Related to Mucoid Degeneration? A Systematic Review.

BACKGROUND: Tendinopathy, enthesopathy, labral degeneration, and pathologic conditions of the articular disc (knee meniscus and ulnocarpal) are sometimes described in terms of inflammation or damage, while the histopathologic findings are often consistent with mucoid degeneration. A systematic review of the histopathology of these structures at diverse locations might reconceptualize these diseases as expected aspects of human aging. The potential benefits of this evolution might include healthier patient and clinician mindsets as well as a reduced likelihood of overdiagnosis and overtreatment resulting from greater awareness of base rates of pathology. QUESTION/PURPOSE: In this systematic review of studies of surgical specimens, we asked: Are there are any differences in the histopathologic findings of structural soft tissue conditions (mucoid degeneration, inflammation, and vascularity) by anatomic site (foot, elbow, or knee) or structure (tendon body, muscle or tendon origin or insertion [enthesis], labrum, or articular disc)? METHODS: Studies between 1980 and 2021 investigating the histopathologic findings of specimens from surgery for trigger digit, de Quervain tendinopathy, plantar fasciitis, lateral and medial elbow enthesopathy, rotator cuff tendinopathy, posterior tibial tendinopathy, patellar tendinopathy, Achilles tendinopathy, or disease of the hip labrum, ulnocarpal articular disc, or knee meniscus were searched for in the PubMed, EMBASE, and CINAHL databases. Inclusion criteria were the prespecified anatomic location or structure being analyzed histologically and any findings described with respect to inflammation, vascularity, or mucoid degeneration. Studies were excluded if they were nonhuman studies or review articles. Search terms included "anatomy," "pathology," and "histopathology." These terms were coupled with anatomic structures or disorders and included "trigger finger," "de Quervain," "fasciitis, plantar," "tennis elbow," "rotator cuff tendinopathy," "elbow tendinopathy," "patellar tendonitis," "posterior tibial tendon," and "triangular fibrocartilage." This resulted in 3196 studies. After applying the inclusion criteria, 559 articles were then assessed for eligibility according to our exclusion criteria, with 52 eventually included. We recorded whether the study identified the following histopathologic findings: inflammatory cells or molecular markers, greater than expected vascularity (categorized as quantitative count, with or without controls; molecular markers; or qualitative judgments), and features of mucoid degeneration (disorganized collagen, increased extracellular matrix, or chondroid metaplasia). In the absence of methods for systematically evaluating the pathophysiology of structural (collagenous) soft tissue structures and rating histopathologic study quality, all studies that interpreted histopathology results were included. The original authors' judgment regarding the presence or absence of inflammation, greater than expected vascularity, and elements of mucoid degeneration was recorded along with the type of data used to reach that conclusion. RESULTS: Regarding differences in the histopathology of surgical specimens of structural soft tissue conditions by anatomic site, there were no differences in inflammation or mucoid degeneration, and the knee meniscus was less often described as having greater than normal vascularity. There were no differences by anatomic structure. Overall, 20% (10 of 51) of the studies that investigated for inflammation reported it (nine inflammatory cells and one inflammatory marker). Eighty-three percent (43 of 52) interpreted increased vascularity: 40% (17 of 43) using quantitative methods (14 with controls and three without) and 60% (26 of 43) using imprecise criteria. Additionally, 100% (all 52 studies) identified at least one element of mucoid degeneration: 69% (36 of 52) reported an increased extracellular matrix, 71% (37 of 52) reported disorganized collagen, and 33% (17 of 52) reported chondroid metaplasia. CONCLUSION: Our systematic review of the histopathology of diseases of soft tissue structures (enthesopathy, tendinopathy, and labral and articular disc) identified consistent mucoid degeneration, minimal inflammation, and imprecise assessment of relative vascularity; these findings were consistent across anatomic sites and structures, supporting a reconceptualization of these diseases as related to aging (senescence or degeneration) rather than injury or activity. CLINICAL RELEVANCE: This reconceptualization supports accommodative mindsets known to be associated with greater comfort and capability. In addition, awareness of the notable base rates of structural soft tissue changes as people age might reduce overdiagnosis and overtreatment of incidental, benign, or inconsequential signal changes and pathophysiology.

Disruption of autophagy by the histone deacetylase inhibitor MGCD0103 and its therapeutic implication in B-cell chronic lymphocytic leukemia.

Evading apoptosis is a hallmark of B-cell chronic lymphocytic leukemia (CLL) cells and an obstacle to current chemotherapeutic approaches. Inhibiting histone deacetylase (HDAC) has emerged as a promising strategy to induce cell death in malignant cells. We have previously reported that the HDAC inhibitor MGCD0103 induces CLL cell death by activating the intrinsic pathway of apoptosis. Here, we show that MGCD0103 decreases the autophagic flux in primary CLL cells. Activation of the PI3K/AKT/mTOR pathway, together with the activation of caspases, and to a minor extent CAPN1, resulting in cleavage of autophagy components, were involved in MGCD0103-mediated inhibition of autophagy. In addition, MGCD0103 directly modulated the expression of critical autophagy genes at the transcriptional level that may contribute to autophagy impairment. Besides, we demonstrate that autophagy is a pro-survival mechanism in CLL whose disruption potentiates cell death induced by anticancer molecules including HDAC and cyclin-dependent kinase inhibitors. In particular, our data highlight the therapeutic potential of MGCD0103 as not only an inducer of apoptosis but also an autophagy suppressor in both combination regimens with molecules like flavopiridol, known to induce protective autophagy in CLL cells, or as an alternative to circumvent undesired immunomodulatory effects seen in the clinic with conventional autophagy inhibitors.

Medication error reporting in nursing homes: identifying targets for patient safety improvement.

BACKGROUND: Legislation enacted in the US State of North Carolina in 2003 requires all licenced nursing homes to report all medication errors. In 2007, nursing homes were encouraged to voluntarily convert from aggregate reporting to a new online system where they reported each individual error. METHODS: A new optional web-based reporting tool was made available to all 393 North Carolina nursing homes to submit error reports for each distinct medication error as they occurred during the year. RESULTS: A total of 5823 medication error reports were submitted by 203 sites (52%) using the new system during the reporting year, a median of 18 error reports per site. Of the 5823 error reports, 612 (10.5%) were categorised as serious. Serious errors were more likely to be caused by drugs given to the wrong patient (RR 4.39, CI 3.7 to 5.2), lab-work error (RR 2.40, CI 1.4 to 4.0), wrong product given (RR 2.22, CI 1.8 to 2.8) and medication overdoses (RR 1.49, 1.2 to 1.8). Serious errors were more likely to occur on second shift (RR 1.32, 1.1 to 1.5). Common medications that are involved in the most serious errors include warfarin (RR 2.58, CI 2.09 to 3.18) and insulin (RR 2.35, CI 1.86 to 2.97), and oxycodone combinations (RR 1.48, CI 1.07 to 2.06). CONCLUSIONS: Data collected from a nursing home medication error system can provide helpful information on serious errors that can be used to focus patient safety efforts to reduce harm. This improved information will be useful in nursing homes for continuous quality improvement efforts.

Phosphorus and ammonium concentrations in surface runoff from grasslands fertilized with broiler litter.

Application of broiler (Gallus gallus domesticus) litter to grasslands can increase ammonium (NH4-N) and dissolved reactive phosphorus (DRP) concentrations in surface runoff, but it is not known for how long after a broiler litter application that these concentrations remain elevated. This long-term study was conducted to measure NH4-N and DRP in surface runoff from grasslands fertilized with broiler litter. Six 0.75-ha, fescue (Festuca arundinacea Schreb.-)bermudagrass [Cynodon dactylon (L.) Pers.] paddocks received broiler litter applications in the spring and fall of 1995-1996 and only inorganic fertilizer N in the spring of 1997-1998. Surface runoff from each paddock was measured and analyzed for NH4-N and DRP. Broiler litter increased flow-weighted NH4-N and DRP concentrations from background values of 0.5 and 0.4 mg L(-1), respectively, to values > 18 mg L(-1) in a runoff event that took place immediately after the third application. Ammonium concentrations decreased rapidly after an application and were not strongly related to time after application or runoff volume. In contrast, DRP concentrations tended to decrease more slowly, reaching values near 1 mg L(-1) by 19 mo after the last application. Dissolved reactive P concentrations decreased linearly with the natural logarithm of days after application (p<0.03), and increased linearly with the natural logarithm of runoff volume (p<0.0001).

Phosphorus losses from grasslands fertilized with broiler litter: EPIC simulations.

Broiler litter, a mixture of poultry excreta and bedding material, is commonly used to fertilize grasslands in the southeastern USA. Previous work has shown that under certain situations, application of broiler (Gallus gallus domesticus) litter to grasslands may lead to elevated levels of phosphorus (P) in surface runoff. The EPIC simulation model may be a useful tool to identify those situations. This work was conducted to evaluate EPIC's ability to simulate event and annual runoff volume and losses of dissolved reactive phosphorus (DRP) from tall fescue (Festuca arundinacea Schreb.)-bermudagrass [Cynodon dactylon (L.) Pers.] paddocks fertilized with broiler litter. The EPIC simulations of event runoff volume showed a trend toward underestimation, particularly for runoff events >30 mm. On an annual basis, EPIC also tended to underestimate runoff, especially at runoff volumes > 100 mm. Both event and annual runoff estimations were strongly associated with observed values, indicating that model calibration could improve the simulation of surface runoff volume. The relationship between simulated and observed values of DRP loss was relatively poor on an event basis (r=0.65), but was stronger (r=0.75) on an annual basis. In general, EPIC tended to underestimate annual DRP losses. This underestimation was apparently caused by the lack of an explicit mechanism to model broiler litter on the soil surface. These results suggested that additional work on the EPIC P submodel would be warranted to improve its simulation of surface application of broiler litter to grasslands.

Pharmacokinetic profiles in rats after intravenous, oral, or dermal administration of dapsone.

Dapsone is a potent anti-inflammatory and antibacterial agent that has been used extensively in the oral treatment of leprosy and dermatitis herpetiformis. This study compared the pharmacokinetic profile of dapsone in rats given a single oral or i.v. 12 mg/kg dose (n = 8/group) or a single dermal application of 12 or 60 mg/kg (n = 12/group) in an aqueous gel application medium containing 10 or 25% diethylene glycol monoethyl ether (DGME). Blood samples (200 microl) were collected via tail vein from each rat and pooled at intervals up to the 24-h period. A terminal blood sample was collected by cardiac puncture from each animal. Plasma concentrations of dapsone were determined by liquid chromatography atmospheric pressure ionization tandem mass spectroscopy. There was no treatment-related overt toxicity observed in any of the animals. Peak levels were reached 1 h after oral dosing (4890 ng/ml), and 6 to 8 h after dermal application, with Cmax values of 1.62, 5.56, and 12.8 ng/ml, for 12 mg/kg at 10 or 25% DGME, and for 60 mg/kg at 25% DGME, respectively. Bioavailability was calculated at 78% after oral dosing and <1% after dermal application. Apparent elimination half-lives (t(1/2))s were similar after i.v. and oral dosing. Both the calculated area under the plasma concentration versus time curve up to 24 h and Cmax values were 3- to 4-fold higher in the dermal application group administered 12 mg/kg dapsone in 25 versus 10% DGME gel, whereas the calculated area under the plasma concentration versus time curve up to 24 h and Cmax values for the 60 mg/kg group were only 3.3- and 2.3-fold greater than those obtained after application of 12 mg/kg in 25% DGME. These results show that both systemic exposure and peak plasma concentrations of dapsone are minimized by dermal versus oral administration of the compound.

Suppression of Epstein-Barr virus reactivation in lymphoblastoid cells cultured in simulated microgravity.

Rotating-wall vessels allow for the growth of cells in simulated microgravity. Lymphoblastoid cells cultured in rotating-wall vessels exhibited significant differences in the expression of both early and late Epstein-Barr Virus (EBV) antigens. Viral protein expression (as measured by indirect immunofluorescence) was significantly suppressed in cells cultured in simulated microgravity. A significantly greater percentage of P3HR-1 cells and Daudi cells were positive for the expression of BamH1-Z-DNA fragment of Epstein-Barr replication activator (ZEBRA), early antigen restricted (EA-R), and viral capsid antigen (VCA) in cells cultured in static tissue culture flasks as compared to cells cultured in rotating-wall vessels. We observed a 7, 11, and 25-fold reduction, respectively, for EA-R, VCA, and ZEBRA protein in P3HR-1 cells cultured in simulated microgravity. Additionally, suspension cultures of P3HR-1 cells exhibited significantly greater ZEBRA antigen expression than cells cultured in rotating-wall vessels. As an independent confirmation of the reduction in ZEBRA-protein production in simulated microgravity in P3HR-1 cells, ZEBRA-mRNA was quantitated by reverse transcription polymerase chain reaction. We observed between a 4 to 10-fold reduction in ZEBRA-mRNA in cells cultured in simulated microgravity as compared to cells cultured at 1 x g in tissue culture flasks. Rotating-wall vessels, by virtue of providing a simple culture environment triggering marked differences in viral activation, provide a model whereby both host and viral factors involved in regulating the maintenance of EBV latency can be examined.

Rhinovirus replication in HeLa cells cultured under conditions of simulated microgravity.

BACKGROUND: Rotating-wall vessels (RWVs) allow for the growth of cells under conditions of simulated microgravity. Information about the replication of viruses in simulated microgravity using RWVs has not been reported. Cells grown in RWVs are subjected to low shear motion, and the replication of certain viruses such as rhinoviruses has been reported to be enhanced by motion. HYPOTHESIS: Our research was based on the hypothesis that rhinovirus replication would be enhanced under conditions of simulated microgravity. METHODS: HeLa cells were cultured in three-dimensional cultures on microcarrier beads in simulated microgravity using RWVs and in sealed Teflon roller bottles. Two-dimensional cultures of HeLa cells were also grown in tissue culture flasks (T-150s). Viral infections for all cultures were carried out under standardized conditions at 1 x g. The amount of new virus released during the first viral replication cycle and the total viral yields obtained from multiple viral replication cycles were determined. RESULTS: Viral quantitation during the first viral replication cycle showed that after 10-13 h RWV and Teflon roller bottle supernatants contained significantly more virus than the supernatants from T-150 cultures. After multiple viral replication cycles (at 24, 48, 72, and 96 h following infection), total viral samples (both free and cell-associated virus) from RWV cultures contained significantly more virus than Teflon roller bottle cultures. CONCLUSIONS: The rhinovirus replication cycle was enhanced in cultures grown in the presence of motion (Teflon roller bottle cultures and RWV cultures). Additionally, multiple rounds of rhinovirus replication yielded more virus in simulated microgravity conditions. Viral transmission in cell cultures in RWVs was efficient and was similar to or better than what occurred in the Teflon roller bottles. The cultivation of cells in simulated microgravity possibly affected the rate of viral adsorption/uptake, the viral replication cycle, and/or the viral yield. RWVs provide an effective means for culturing human rhinoviruses.

Membrane insertion, glycosylation, and oligomerization of inositol trisphosphate receptors in a cell-free translation system.

In order to study the membrane topology, processing, and oligomerization of inositol trisphosphate receptor (IP3R) isoforms, we have utilized RNA templates encoding putative transmembrane domains to program a cell-free translation system of rabbit reticulocyte lysates supplemented with canine pancreas microsomes. In the absence of microsomes, translation of the RNA templates encoding all the putative transmembrane domains present in the C-terminal segment of the type I (1TM) and type III (3TM) IP3R isoforms resulted in a 62- and 59-kDa polypeptide, respectively. In both cases, an additional band approximately 3 kDa larger was observed upon the addition of microsomes. Both bands in the translation doublet were integrated into microsomal membranes and were full-length translation products, as shown by sedimentation through a sucrose cushion and immunoprecipitation with C-terminal isoform-specific antibodies. With both isoforms, N-glycopeptidase F digestion indicates that the upper band in the doublet corresponds to a glycosylated translation product. A 17-kDa protected fragment was observed after proteinase-K digestion of 1TM translated in the presence of microsomes. The pattern and size of protected fragments was consistent with the current six-transmembrane domain model of IP3R topology. Cotranslation of both 1TM and 3TM RNA templates in the presence of microsomes followed by immunoprecipitation with isoform specific antibodies revealed coimmunoprecipitation of translation products. This was not observed when the isoforms were translated separately and then mixed, suggesting that heteroligomerization occurs cotranslationally. A construct encoding only the first putative transmembrane domain of the type I isoform was found to be sufficient for integration into membranes but was unable to oligomerize with either 1TM or 3TM. Cotranslation experiments using additional constructs indicate that the major structural determinant for homoligomerization lies between putative transmembrane domain 5 and the C terminus. A second oligomerization domain involved in stabilization of heteroligomers is present within the first four transmembrane domains.

Outcome measures in spasticity management.

Development of validated and reliable outcome measures for spasticity rehabilitation has been hampered by the difficulty of quantifying functionally important parameters such as pain, ease of care, and mobility. Nonetheless, a combination of measures designed to assess technical and functional outcomes, patient satisfaction, and the cost effectiveness of treatment can be used together to evaluate status and track change in spasticity management, including treatment programs involving botulinum toxin. While double-blind, placebo-controlled studies remain the gold standard for clinical testing, the single-subject design is a useful alternative in many treatment protocols. Because no single tool can measure the many types of changes possible with treatment, the choice of assessment tools must be based on the functional changes expected from the treatment. A wide range of assessment tools are critically reviewed for their sensitivity, reliability, validity, and ease of administration.

Botulinum toxin A in the treatment of spasticity: functional implications and patient selection.

OBJECTIVE: To explore the range of functional indications and benefit of botulinum toxin A (BTA) in spastic patients. DESIGN: Case report of a series of patients selected for BTA treatment. Clinical information was collected in a prospective fashion on each patient. SETTING: Freestanding acute rehabilitation hospital. PATIENTS: 39 consecutive patients with 40 limbs with acquired spasticity. INTERVENTION: All 39 patients received BTA injections into muscles targeted for treatment based on functional indications. MAIN OUTCOME MEASURES: Objective evaluation of outcome was measured by Ashworth Scale, goniometry, ambulation score, and brace wear scale. Subjective measures included patient self report of improvement and pain relief. RESULTS: Mean BTA dose per limb was 180 units, mean number of muscles injected per limb was 2. Twenty-nine patients had subjective and/or objective improvement with treatment. Mean Ashworth Scale improvement was one point. Mean gain in active range of motion (AROM) was 17.0 degrees, and in passive range of motion (PROM) 18.4 degrees. Brace tolerance improved in 14 of 22 patients and pain relief occurred in 10 of 13 patients. There were no adverse effects, and there was no difference in duration of effect compared to dystonia patients. CONCLUSION: BTA is a useful intervention in the treatment of spasticity, with the majority of patients demonstrating improvement on objective measures of tone and function, and reporting improvement on subjective measures. Careful patient selection will maximize functional benefit.

Heteroligomers of type-I and type-III inositol trisphosphate receptors in WB rat liver epithelial cells.

We have previously shown that a 222-kDa polypeptide co-immunoprecipitates together with the type-I myoinositol 1,4,5-trisphosphate receptor (IP3R) in WB rat liver epithelial cell extracts, when the immunoprecipitation is carried out with a type-I isoform specific antibody (Joseph, S. K. (1994) J. Biol. Chem. 269, 5673-5679). Utilizing isoform-specific antibodies raised to unique sequences within the COOH-terminal region of IP3 receptors, we now report that the co-immunoprecipitating 222-kDa polypeptide is the type-III IP3R isoform and that type-III IP3R antibodies (Abs) can co-immunoprecipitate the type-I IP3R isoform. Co-immunoprecipitation of IP3R isoforms was not due to cross-reactivity of the antibodies for the following reasons: (a) on immunoblots the type-III antibodies did not cross-react with type-I IP3R and vice versa; (b) inclusion of the COOH-terminal type-III peptide had no effect on the ability of type-I IP3R Ab to co-immunoprecipitate the type-III IP3R but blocked the ability of type-III IP3R Ab to coimmunoprecipitate the type-I isoform; and (c) crude hepatocyte lysates contain undetectable amounts of type-III IP3R, and immunoprecipitation with type-III IP3R Ab does not co-immunoprecipitate any other isoforms. However, type-I and type-II IP3R isoforms were co-immunoprecipitated by their respective antibodies in hepatocyte lysates. Sucrose density gradient analysis of WB cell lysates indicated that the co-immunoprecipitating fraction is exclusively located at the density expected for tetrameric receptors, suggesting that co-immunoprecipitation was not a reflection of the nonspecific aggregation of IP3R isoforms. Phosphorylation of either type-I or type-III immunoprecipitates by protein kinase A indicated that only the type-I IP3R could be phosphorylated in vitro. Fractionation of WB cell membranes and immunofluorescence studies showed that the type-I and type-III isoforms have very similar sub-cellular localizations. We conclude that the WB cell contains both type-I and type-III IP3R isoforms and that a proportion of these receptors exist as heterotetramers.

Trypsin digestion of the inositol trisphosphate receptor: implications for the conformation and domain organization of the protein.

Limited digestion of rat cerebellum microsomal vesicles with trypsin resulted in the proteolysis of the 240 kDa inositol 1,4,5-trisphosphate receptor (IP3R) and the formation of a 94 kDa species that remained membrane-bound and retained immunoreactivity to an antibody raised against the C-terminal sequence of this protein. The appearance of the 94 kDa species was associated with a loss of [3H]IP3 binding sites in the membrane and the appearance of [3H]IP3 binding sites in the soluble fraction. The 94 kDa fragment retained reactivity to biotinylated concanavalin A. In vitro phosphorylation of the IP3R in membranes with cyclic AMP-dependent protein kinase and [gamma-32P]ATP produced an unlabelled 94 kDa fragment after tryptic digestion. According to current models of the cerebellar IP3R this would place the proteolytic site between the phosphorylation site at serine-1755 and the first transmembrane segment of the IP3R. A second antibody raised to amino acids 401-414 in the N-terminal region of the receptor recognizes a 68 kDa fragment released into the soluble fraction after trypsin treatment. The time course of release of the 68 kDa fragment was correlated with the appearance of soluble binding sites, and the fragment was bound by IP3-Affigel resin. A large proportion of the 68 kDa fragment remained associated with the membrane fraction and could be specifically immunoprecipitated from detergent extracts of digested membranes by anti-C-terminus antibody. Our results provide experimental evidence to further localize the ligand binding domain and suggest that regions of the N-terminus and C-terminus may be non-covalently associated.

The effect of mersalyl on inositol trisphosphate receptor binding and ion channel function.

A number of thiol-reactive agents induce repetitive Ca2+ spiking in cells by a mechanism thought to involve sensitization of the inositol 1,4,5-trisphosphate receptor (IP3R). To further define the basis of this interaction, we have studied the effect of several thiol-reactive agents on [3H]IP3 binding, IP3-gated channel activity, and conformation of the IP3R in membranes from hepatocytes, cultured WB rat liver epithelial cells, and cerebellum microsomes. At 4 degrees C, the organomercurial thiol-reactive agent mersalyl markedly stimulates (3-4fold) [3H]IP3 binding to permeabilized hepatocytes. The closely related molecule, thimerosal, has only a small stimulatory effect under these conditions, and GSSG or N-ethylmaleimide are without effect. The stimulatory effect of mersalyl was associated with a decrease in Kd of the IP3R with no change in Bmax. Mersalyl was without effect on detergent-solubilized hepatocyte binding sites or on the [3H]IP3 binding activity of cerebellum microsomes. In contrast to thimerosal, which potentiates IP3-mediated Ca2+ release, mersalyl blocked IP3-gated Ca2+ channels. Mersalyl pretreatment of WB membranes altered the pattern of immunoreactive receptor fragments generated upon subsequent cleavage of the receptor with proteinase K. This effect was not reproduced by thimerosal and was also not observed in experiments on cerebellum microsomes. We conclude that the WB cell and brain IP3 receptors are differently regulated by modification of thiol groups. Reaction of the WB cell IP3 receptor with mersalyl alters its conformation and modifies the accessibility of sites on the protein that are cleaved by proteinase K. In the presence of mersalyl, the receptor has high affinity for IP3 but is inactive as a Ca2+ channel. This contrasts with the high affinity receptor/active Ca2+ channel induced by thimerosal, suggesting that even closely related thiol agents may interact at different thiol groups.

Rapid restoration of B-cell function in XID mice by intravenous transfer of peritoneal cavity B cells.

The primary method employed to correct immune deficiency is bone marrow transfer. Depending upon the exact nature of the immune deficiency, however, alternative cell sources may be used to provide a more rapid reconstitution of immune function. In this report, peritoneal cavity (PerC) B cells are shown to be effective in the rapid emendation of the B-cell defect exhibited by XID mice. Restoration of normal numbers of splenic IgM antibody-secreting cells (ASC) and serum IgM levels were observed 4 and 7 days, respectively, after the i.v. transfer of 3 x 10(6) PerC. This regimen also restored responsiveness to thymus-independent type 2 (TI-2) antigens in XID recipients. Transfer of 30 x 10(6) spleen (SP) cells restored these functions in XID recipients but at a considerably slower rate. The data indicate that introducing a small number of PerC B cells into systemic circulation results in the rapid restoration of serum IgM levels in unirradiated XID mice.

Treatment of stiff-man syndrome with intravenous immune globulin.

OBJECTIVE: To evaluate the efficacy of intravenous immune globulin (IVIG) in the treatment of stiff-man syndrome (SMS). METHODS: An open, unblinded study of 3 patients with active disease and/or disease refractory to treatment with diazepam and/or corticosteroids. RESULTS: All 3 bedridden patients improved substantially shortly after infusion with IVIG and regained function. CONCLUSION: IVIG may be useful for the treatment of SMS.