Biomolecular phenotyping and heterogeneity assessment of mesenchymal stromal cells using label-free Raman spectroscopy.

Easy, quantitative measures of biomolecular heterogeneity and high-stratified phenotyping are needed to identify and characterise complex disease processes at the single-cell level, as well as to predict cell fate. Here, we demonstrate how Raman spectroscopy can be used in the difficult-to-assess case of clonal, bone-derived mesenchymal stromal cells (MSCs) to identify MSC lines and group these according to biological function (e.g., differentiation capacity). Biomolecular stratification is achieved using high-precision measures obtained from representative statistical sampling that also enable quantified heterogeneity assessment. Application to primary MSCs and human dermal fibroblasts shows use of these measures as a label-free assay to classify cell sub-types within complex heterogeneous cell populations, thus demonstrating the potential for therapeutic translation, and broad application to the phenotypic characterisation of other cells.

Core-shell patterning of synthetic hydrogels via interfacial bioorthogonal chemistry for spatial control of stem cell behavior.

A new technique is described for the patterning of cell-guidance cues in synthetic extracellular matrices (ECM) for tissue engineering applications. Using s-tetrazine modified hyaluronic acid (HA), bis-trans-cyclooctene (TCO) crosslinkers and monofunctional TCO conjugates, interfacial bioorthogonal crosslinking was used to covalently functionalize hydrogels as they were synthesized at the liquid-gel interface. Through temporally controlled introduction of TCO conjugates during the crosslinking process, the enzymatic degradability, cell adhesivity, and mechanical properties of the synthetic microenvironment can be tuned with spatial precision. Using human mesenchymal stem cells (hMSCs) and hydrogels with a core-shell structure, we demonstrated the ability of the synthetic ECM with spatially defined guidance cues to modulate cell morphology in a biomimetic fashion. This new method for the spatially resolved introduction of cell-guidance cues for the establishment of functional tissue constructs complements existing methods that require UV-light or specialized equipment.

Ultra-slow inactivation of the ionic currents through the membrane of myelinated nerve.

(1) Voltage-clamp experiments were performed with myelinated fibres isolated from the sciatic nerve of the frog to study slow changes of the specific sodium and potassium currents as a function of membrane (holding) potential and time. (2) The level of the peak sodium current depends on holding potential VH. This dependence can be described by a sigmoidal function uinfinity(VH). The underlying process is called "ultra-slow sodium inactivation" and is different and separable from the short time steady-state inactivation, hinfinity(V), and from the slow inactivation depending on the extracellular potassium concentration (Adelman, Jr., W. J. and Palti, Y. (1969), J Gen. Physiol. 54, 589-606; Peganov, E. M., Khodorov, B.I. and Shishkova, L. D. (1973), Bull. Exp. Biol. Med. 25, 15-19; Khodorov, B. I. Shishkova, L. D. and Peganov, E. M. (1974), Bull. Exp. Biol. Med. 3, 10-14). (3) After a sudden change of the holding potential the sodium current reaches a new steady-state level (due to the transition of uinfinity(VH) to the corresponding value) within approx. 4 min. The kinetics of the transition cannot be described by a single exponential function. (4) A corresponding voltage- and time-dependent process of ultra-slow inactivation exists for the potassium current in the node of Ranvier. The kinetics are faster than those of the sodium system.

Kinetics of the slow variation of peak sodium current in the membrane of myelinated nerve following changes of holding potential or extracellular pH.

(1) Changes of the holding potential applied to the membrane of myelinated nerve fibres induced slow variations of the peak sodium current, which are super-imposed on the effect of sodium inactivation. (2) These slow variations are transitions between various steady levels of available sodium conductance. Their time course can be described by the function erfc (square root t/tau) where tau is the time and erfc the error function complement. The characteristic time tau lies in the range 2-4 min and depends on the membrane potential. (3) Changes of extracellular pH cause a rapid change of the peak sodium current followed by a slow variation as observed after changes of the holding potential. This slow variation can be prevented by applying simultaneously an appropriate change of the holding potential, e.g. the effect of changing pH from 7.3 to 5.3 is balanced by changing the potential from --70 to --55 mV. (4) The results are interpreted by postulating charged components diffusion slowly within the nodal membrane. Their transverse distribution controls the number of sodium channels available at a given membrane potential. The equivalence between change of pH and voltage is explained by assuming negative fixed charges at the outer surface of the membrane, which are protonated at low pH and thus affect the intrinsic membrane potential. (5) It is concluded that effects which are ascribed to the action of agents on individual sodium channels have to be corrected for variations in the number of available channels if these agents influence the intrinsic membrane potential, e.g. changes of extracellular pH.

Do caffeine-containing analgesics promote dependence? A review and evaluation.

OBJECTIVE: Debates about the suspected association between kidney disease and use of analgesics have led to concern about whether caffeine could stimulate an undesirable overuse of phenacetin-free combined analgesics. A committee was asked to critically review the pertinent literature and to suggest guides for clinical practice and for consideration of international regulatory authorities. PARTICIPANTS: A group of international scientists, jointly selected by the regulatory authorities of Germany, Switzerland, and Austria and the pharmaceutical industry. EVIDENCE: All invited experts evaluated relevant literature and reports and added further information and comments. CONCLUSIONS: Caffeine has a synergistic effectiveness with analgesics. Although caffeine has a dependence potential, the potential is low. Experimental data regarding dependence potential for caffeine alone may not correspond to the conditions in patients with pain. Withdrawal is not likely to cause stimulation or sustainment of analgesic intake. For drug-induced headache, no single or combined analgesic was consistently identified as causative, and no evidence exists for a special role of caffeine. Strong dependence behavior was observed only in patients using phenacetin-containing preparations, coformulated with antipyretics/analgesics and caffeine. This finding may have led to the impression that caffeine stimulates overuse of analgesics. SUMMARY: Although more experimental and long-term data would be desirable to show possible mechanisms of dependence and to offer unequivocal proof of safety, the committee concluded that the available evidence does not support the claim that analgesics coformulated with caffeine, in the absence of phenacetin, stimulate or sustain overuse.

Actinomycin D-associated lesions mimicking DNA-DNA interstrand crosslinks detected by alkaline elution in cultured mammalian cells.

Cultured human fibroblasts were exposed to various concentrations of actinomycin D, a DNA intercalating agent, and studied by various alkaline elution techniques for the presence of DNA lesions. DNA-protein crosslinks increased proportionately with increasing actinomycin D, with 1.53 crosslinks/10(6) nucleotides after 5 micrograms/ml exposure. However, in the single-strand DNA break assay, elution of DNA initially increased as expected with increasing actinomycin D but thereafter decreased, with only 0.09 breaks/10(6) nucleotides detected after 25 micrograms/ml exposure, suggesting the presence of DNA crosslinking. A standard alkaline elution assay for DNA-DNA crosslinking was performed, and lesions which mimicked such crosslinks were detected, with a relative crosslink frequency of 2.30 after 5 micrograms/ml exposure. These actinomycin D-associated lesions disappeared when the alkaline elution procedure was modified to include additional proteinase digestion and use of the detergent sodium dodecyl sulfate (SDS) in the elution buffer, suggesting that they represented undigested DNA-protein crosslinks or nonspecific protein on the filters inhibiting DNA elution. Greater than ten times as many DNA-protein crosslinks were detected in fibroblasts than the number of single-strand DNA breaks after cellular exposure to actinomycin, even after determining breaks using the modified methodology for decreasing cellular protein interference. The data suggest that the actinomycin-DNA complex is associated with the formation of DNA-protein crosslinks which represent lesions other than endonuclease-associated DNA strand scission.

Paramyxovirus infection in lung transplant recipients.

BACKGROUND: Respiratory infections are common after lung transplantation. The significance of respiratory paramyxoviruses, respiratory syncytial virus, and parainfluenza virus in lung transplant recipients has not been determined. METHODS: In a retrospective fashion, we examined the incidence and clinical characteristics of paramyxovirus infection in 84 consecutive lung transplant recipients at the University of Minnesota Hospital and Clinics from 1986 through 1993. RESULTS: We identified 19 cases of paramyxovirus infection in 18 patients (21% of all transplant recipients). All patients had symptoms with lower respiratory tract involvement, and nine (47%) had coexisting upper respiratory involvement. Symptom onset was 24 to 2056 days after transplantation (median = 260 days). Respiratory syncytial virus infection was seasonal (January through June), but parainfluenza virus infection occurred throughout the year. Six patients showed a decline in spirometry (26% +/- 2.8% decrease in forced expiratory volume in 1 second); four returned to baseline. Diagnosis was made by bronchoalveolar lavage in 15 cases, nasopharyngeal swab in three cases, and sputum in one case. Most patients (74%) were treated with ribavirin, and all but one treated patient recovered fully. In untreated patients, respiratory syncytial virus contributed to one death and one parainfluenza virus infection resulted in a persistent reduction in spirometry. Age was the strongest predictor of infection, with a higher incidence in patients under 18 years old (57%, p < 0.05). Preexisting obliterative bronchiolitis did not correlate with an increased incidence of paramyxovirus infection (20% with obliterative bronchiolitis, 22% without obliterative bronchiolitis; p > 0.05). CONCLUSIONS: Lower respiratory tract infection with paramyxovirus is common in lung transplant recipients and capable of causing death or a permanent reduction in pulmonary function.

Randomized trial of daily versus three-times-weekly prophylactic ganciclovir after lung and heart-lung transplantation.

BACKGROUND: Cytomegalovirus is an important cause of morbidity and mortality risk after lung transplantation. Ganciclovir, when given for a period of up to 3 months after lung transplantation, has been shown to reduce the incidence and severity of cytomegalovirus. However, daily prophylaxis is associated with considerable expense, inconvenience, and morbidity risk. The goal of this study was to determine whether 3-times-weekly dosage is as effective as daily prophylaxis with ganciclovir in preventing cytomegalovirus disease. METHODS: Seventy-two consecutive subjects who had either donor or recipient cytomegalovirus seropositivity were randomized to the daily group (n = 35) or the 3-times-weekly group (n = 37). All subjects received twice-daily ganciclovir treatment for 2 weeks. Thereafter, subjects received either daily or 3-times-weekly ganciclovir dosing until 90 days after transplantation. Subjects were then monitored for 28 +/- 13 months to identify outcomes and complications. RESULTS: There were no significant differences between the daily and 3-times-weekly groups with respect to survival free from cytomegalovirus infection or survival free from cytomegalovirus disease. In both groups, cytomegalovirus infection and disease frequently emerged after the termination of prophylaxis. However, in most cases the cytomegalovirus syndromes observed were mild and in many cases could be treated on an outpatient basis. There was no significant difference between the groups in the incidence of obliterative bronchiolitis or time to onset of grade 2 bronchiolitis obliterans syndrome. Overall patient survival was better in the daily group, but the survival advantage did not appear to be related to a reduction in cytomegalovirus-related disease. Complications of ganciclovir prophylaxis included leukopenia in 2 subjects in the 3-times-weekly group and catheter-related sepsis in 6 subjects from each group. CONCLUSIONS: We conclude that intravenous ganciclovir given 3 times weekly for 3 months after transplantation is as effective as daily ganciclovir given for a similar time period. The 3-times-weekly dosing regimen did not result in increased infection, disease, or sequelae of cytomegalovirus infection when compared with the daily regimen.

Single lung transplantation for severe emphysema.

UNLABELLED: Lung transplantation is effective therapy for patients with severe obstructive lung disease. We reviewed seven patients with severe emphysema (age, 48 +/- 5 years; forced expiratory volume in 1 second [FEV1] 0.76 +/- 0.26 liters) who received single-lung transplants (SLT) at our institution between August 1989 and September 1990. Studies to assess the adequacy of cardiac function before transplantation showed moderately reduced right ventricular function (by multiple gated acquisition, 34 +/- 6%), moderately elevated pulmonary artery pressure (25 +/- 3 mm Hg), and normal left ventricular function (by multiple gated acquisition 65% +/- 12%) and coronary arteriograms. Time on the waiting list before transplantation was reduced compared with heart-lung transplant (HLT) recipients (waiting time, 2.9 +/- 1.5 months for SLT, 9.6 +/- 10.2 months for HLT). Six of the SLT recipients are currently alive (after transplantation interval, 17 +/- 5 months); the remaining recipient died of pulmonary embolism 21 days after SLT. Number of ventilator days, intensive care unit days, and days to hospital discharge after transplantation did not differ significantly from HLT recipients. Cardiopulmonary bypass was necessary in four SLT recipients. Pulmonary function was markedly improved after SLT (FEV1, 1.78 +/- 0.73 L/min after SLT versus 0.75 +/- 0.3 L/min before SLT; p less than 0.01), and functional status is correspondingly improved. CONCLUSIONS: SLT constitutes effective therapy for patients with severe emphysema, including those with moderate reduction of right ventricular function; and SLT offers distinct advantages over HLT, including decreased waiting time before transplantation, improved donor organ utilization, and less frequent need for cardiopulmonary bypass.

An analysis of error rates for disc agar-diffusion testing of Pseudomonas aeruginosa versus aminoglycosides.

Five hundred thirty-five recent clinical isolates of Pseudomonas aeruginosa were tested in parallel by the standardized disc agar-diffusion test and a micro-broth dilution test to evaluate the error rates associated with the use of both fixed indeterminate-zone breakpoints and floating indeterminate-zone breakpoints for assessing susceptibility to amikacin, gentamicin, and tobramycin. Error rate-bound analysis showed that unacceptably high rates of error were associated with the use of all fixed breakpoints. Error rates were improved when the floating indeterminate-zone principle was used, but rates still remained unacceptably high. These findings indicate the disc agar-diffusion test should not be used for testing the susceptibility of P. aeruginosa to the aminoglycosides.

Comparison of minimum inhibitory concentration values determined by three antimicrobic dilution methods for Pseudomonas aeruginosa.

This investigation compares minimum inhibitory concentration measurements by three antimicrobic dilution methods for Pseudomonas aeruginosa versus seven antimicrobics. Minimum inhibitory concentrations were measured for 650 P. aeruginosa clinical isolates and for repeated tests with P. aeruginosa ATCC 27853 versus gentamicin, tobramycin, amikacin, netilmicin, sisomicin, carbenicillin, and ticarcillin, using the macro-broth, micro-broth, and agar dilution methods. For all antimicrobics, it was found that the micro-broth and agar dilution methods produced comparable minimum inhibitory concentration measurements, which were found to lie 1 to 2 double dilution steps below those determined by the macro-broth method. Acceptably replicability was found for both the macro-broth and the agar dilution methods. The micro-broth method showed less replicability, with 4.7% of minimum inhibitory concentration values lying +/- 2 or more double dilution steps from the modal value. It is important to recognize such differences if micro-broth or agar dilution methods are substituted for the macro-broth method.

An evaluation of burn wound quantitative microbiology. I. Quantitative eschar cultures.

The reliability of quantitative data from burn wound biopsy cultures was investigated. This was done by comparing the recovery of microorganisms from a series of burn wound eschar biopsy specimens that were each divided into two approximately equal portions and cultured in parallel. The results indicate that a microorganism present in the burn wound site in any quantity has at least a 25% chance of being missed by a single quantitative eschar culture. For recovery levels corresponding to quantitative breakpoints that have been proposed to be predictive of burn wound sepsis, only 38% of paired quantitative results agreed within the same log10 unit, and 44% differed by +/- 2 log10 units or more. These findings indicate that quantitative results derived from burn wound biopsy cultures are unreliable and may be significantly misleading when used for decision-making relative to patient care.

Inability of the API-20E system to speciate the fluorescent group of pseudomonads.

One thousand randomly selected clinical isolates of the fluorescent group of Pseudomonas were speciated in parallel by the API-20E system and by a classic microbiologic 17-test battery. The classic battery identified the isolates as 993 Pseudomonas aeruginosa, five Pseudomonas putida, and two Pseudomonas fluorescens. To augment the P. putida and P. fluorescens data, 52 reference isolates were also tested in parallel. API-20E was found to identify 99% of P. aeruginosa as P. aeruginosa. However, the 59 P. putida and P. fluorescens isolates were not accurately identified and were designated 58% P. aeruginosa, 24% P. stutzeri, 16% ambiguous categories, and only 2% in agreement with the classic designation. These findings indicated that the API-20E system does not accurately identify the non-P. aeruginosa fluorescent pseudomonads and that errors in P. aeruginosa designations will increase as the percentages of P. putida and P. fluorescens increase in the population sample.

Catalytic asymmetric vinylation of ketone enolates.

[see reaction]. A protocol for the catalytic asymmetric vinylation of ketone enolates has been developed. Key to the success of this process was the development of new electron-rich chiral monodentate ligands.

The effect of flecainide acetate, a new antiarrhythmic, on plasma digoxin levels.

The possible effect of oral flecainide acetate on steady-state digoxin levels was assessed in 15 healthy men. Each volunteer received digoxin 0.25 mg daily (8 AM) for 22 consecutive days and flecainide 200 mg bid (8 AM and 8 PM) on days 11 through 15. Plasma digoxin and flecainide levels were measured by radioimmunoassay and gas-liquid chromatography methods, respectively. Flecainide levels were within the range associated with suppression of premature ventricular contractions in patients. Mean plasma digoxin levels just before the 8 AM dose were 0.46 ng/mL on days 9 and 10 (baseline), 0.57 ng/mL (P less than .05) on day 13, and 0.49 ng/mL (not significant [NS]) on day 15. Compared with a mean six-hour postdose baseline digoxin level of 0.58 ng/mL, postdose levels were 0.62 ng/mL (NS) and 0.65 ng/mL (P less than .05) on days 13 and 15, respectively. On an average for each subject, predose and six-hour postdose digoxin levels increased by 24 +/- 35% and 13 +/- 19%, respectively, during coadministration. The changes in electrocardiographic intervals and vital signs that occurred during concomitant drug administration were not clinically significant although a slight prolongation of the PR interval was noted in some subjects. Unless plasma digoxin levels are in the upper end of the therapeutic range, changes in magnitude as observed in this study should be clinically inconsequential for most patients.

Oral flecainide pharmacokinetics in patients with impaired renal function.

The pharmacokinetics of flecainide acetate were studied in 20 patients with varying degrees of renal impairment following a single oral dose. The patients were divided into two groups, on the basis of renal creatinine clearance (CLCR), for statistical and kinetic analysis. Patients with a CLCR between 4 and 41 mL/min/m2 were designated group 1 and those below 4 mL/min/m2 or unmeasurable because of lack of urine output were designated group 2. In both groups peak plasma flecainide concentrations, time to peak concentrations, and apparent volume of distribution (Vd) were similar to those reported in healthy subjects with normal renal function. The mean flecainide plasma elimination half-lives from both groups 1 and 2 were longer than those previously reported by several investigators in normal subjects. Nine patients in group 1 and seven patients in group 2 had half-lives within the range reported in healthy subjects. Therefore, CLCR alone is not a good predictor of plasma elimination half-life following a single oral dose of flecainide. Although renal clearance of flecainide is significantly reduced in end-stage renal disease (ESRD), total plasma clearance of flecainide (CLflec) was not reduced to the same degree, although there was a significant, modest correlation with CLCR. Less than 1% of the administered oral dose of flecainide was removed during hemodialysis. The relationship between dosage and plasma elimination half-life in patients with ESRD needs further study to evaluate possible dose-dependent kinetics.

The action of thiamine and its di- and triphosphates on the slow exponential decline of the ionic currents in the node of Ranvier.

Sodium and potassium currents in the node of Ranvier decrease exponentially with time during long lasting voltage clamp experiments. This decline is strongly dependent on temperature (Q10 approximately 3). Thiamine and, particularly, its diand triphosphoric acid esters are shown to prevent this exponential decline of the ionic currents. Thiamine acts from the outside and from the inside of the nodal membrane, but more potently from the inside. Thiamine diphosphate prevents the exponential decline of the ionic currents only when applied internally. Thiamine triphosphate, the most effective thiamine derivative was tested form the inside only. Bacterial thiaminases applied externally were not effective, presumably because they do not permeate the nodal membrane. Tetrodotoxin, that has been shown by other investigators to induce a release of thiamine from nerve membranes, does not alter the action of thiamine on the exponential decline of current and vice versa. It is concluded that: (1) thiamine diphosphate or thiamine triphosphate are the active thiamine compounds in nerve membranes; (2) the site of action is located at the internal suface of the membrane; (3) the reduction of the thiamine concentration in the membrane or in the axoplasm could cause the exponetial decline of currents; (4) the release of thiamine from nerve membranes induced by tetrodotoxin is interpreted as a side effect not even related to the mechanism by which tetrodotoxin blocks the sodium channels; (5) thiamine polyphosphates appear to stabilise the intrinsic electric field strength of the nodal membrane in the resing state. Threfore, as a working hypothesis, it is suggested that the thiamine derivatives control the number of functioning ionic channels by stabilising the density of negative surface charges at the inner side of the nerve membrane.

Functional evaluation of the pes anserinus transfer by electromyography and gait analysis.

The effectiveness of the pes anserinus transfer, proposed by Slocum and Larson to control anteromedial rotatory instability of the knee, depends on the pes anserinus muscles contracting at times when the instability threatens the patient's function. To examine the activity of the transferred pes anserinus muscles, quantitated dynamic electromyography, isometric testing of muscle strength, and gait-analysis techniques were employed. Seven normal subjects and nine patients who had undergone pes anserinus transfer for anteromedial rotatory instability were tested. In contrast to the normal subjects, who demonstrated pes anserinus muscle activity primarily during swing phase, the patients displayed conspicuous activity of the semitendinosus, gracilis, and sartorius tendons during stance phase. Quantitated electromyography indicated that the pes anserinus muscles worked harder in the patients than in the normal subjects, and more effort was required from the muscles of patients with poor results than from those of patients with good results. All of the patients also demonstrated reduction of single-limb support time, as well as a shorter stride length and reduced gait velocity. We concluded that the pes anserinus transfer is kinetically sound. Since instability of the knee is a functional defect that is present during stance, the finding of marked activity of the pes anserinus muscles during stance phase in the patients but not in the normal subjects suggests that these muscles were being used to control the instability.

Arthroscopy--"no-problem surgery". An analysis of complications in two thousand six hundred and forty cases.

In a retrospective review of 3,261 arthroscopic procedures on the knee, 2,640 met the criteria for inclusion in this analysis. The patients' ages ranged from eight to eighty-three years. There were 1,541 male and 1,099 female patients. Eight hundred and ninety-five of the injuries were work-related. A tourniquet was used in 1,175 procedures and the average tourniquet time was thirty minutes. There were 216 complications over-all (8.2 per cent), 126 being designated as major and ninety-seven, as minor. The major complications that were evaluated were infections, hemarthrosis, adhesions, effusions, cardiovascular, neurological, reflex sympathetic dystrophy, and instrument breakage, and the minor complications were difficulties with wound-healing and ecchymosis. Chi-square analysis showed the following factors to be significant (p less than 0.05). Patients with an industrial injury had a higher rate of neurological complications and reflex sympathetic dystrophy. Diagnostic arthroscopy had the lowest over-all complication rate. Partial medical meniscectomy was associated with a higher over-all complication rate and the highest hemarthrosis rate, and partial lateral meniscectomy was associated with the highest rate of instrument breakage. Abrasion arthroplasty had the highest rate of complications of wound-healing, and subcutaneous lateral release was associated with the most adhesions. The sex of the patient and whether or not a tourniquet had been used had no effect on complications. The experience of the surgeon with arthroscopic procedures also had no correlation with the complication rate. Multiple regression analysis showed that two factors (age and, if a tourniquet was used, the tourniquet time) were dominant predictors of complications. From these data, a model was devised for predicting which patients were at risk for complications and their relative levels of risk. Certain complications may be preventable, and for others the risk factors can be reduced. The high-risk patients in our series were fifty years old or older and had a tourniquet time of sixty minutes or longer.

Accuracy of diagnoses from magnetic resonance imaging of the knee. A multi-center analysis of one thousand and fourteen patients.

Magnetic resonance images of the knee were made for 1014 patients, and the diagnosis was subsequently confirmed arthroscopically. The accuracy of the diagnoses from the imaging was 89 per cent for the medial meniscus, 88 per cent for the lateral meniscus, 93 per cent for the anterior cruciate ligament, and 99 per cent for the posterior cruciate ligament. The magnetic resonance examinations were done at several centers, and the results varied substantially among centers. The accuracy ranged from 64 to 95 per cent for the medial meniscus, from 83 to 94 per cent for the lateral meniscus, and from 78 to 97 per cent for the anterior cruciate ligament. The results from different magnetic-resonance units were also compared, and the findings suggested increased accuracy for the units that had a stronger magnetic field. Of the menisci for which the magnetic resonance signal was reported to be Grade II (a linear intrameniscal signal not extending to the superior or inferior meniscal surface), 17 per cent were found to be torn at arthroscopy.