Identity of the fungal species present in the homes of asthmatic children.

BACKGROUND: Fungal exposures are believed to play an important role in the development of asthma and atopy, accounting for increased asthmatic symptoms and severe asthma exacerbation. Indoor fungal species vary both in taxa and concentration in different residences and in different regions. OBJECTIVES: We explored the fungal species spectrum in 88 homes with at least one asthmatic child in the Middle West region of the United States mostly during late spring and fall season in comparison with 85 homes that did not contain an asthmatic child during flu season. METHODS: The average fungal spore counts per cubic metre of air in the bedroom of the enrolled child, the main living spaces and outdoor environments, and the culturable fungal colony-forming units per cubic metre of air samples in the main living space from each home were measured. RESULTS: The results indicated that Cladosporium, Penicillium, Aspergillus, Basidiospores, Epicoccum and Pithomyces were found in more asthmatic homes than in homes without an asthmatic child or existed in higher concentration in asthmatic homes than in homes without an asthmatic child even after adjusting outdoor spore concentration. The results for culturable fungal species confirmed most of these findings even after adjusting for seasonal factors. Although Alternaria was commonly found in both kinds of homes, there was no significant difference in detection rate or concentration of Alternaria between asthmatic homes and homes without an asthmatic child by either spore counting or culturable airborne detection. CONCLUSION AND CLINICAL RELEVANCE: Since many allergens have been identified in these fungal species, identifying and controlling these fungal species in asthmatic homes might be expected to improve asthma care and benefit asthmatic children.

Phase I study of paclitaxel in combination with cyclophosphamide and granulocyte colony-stimulating factor in metastatic breast cancer patients.

PURPOSE: In vitro data suggest that prolonged exposure to paclitaxel enhances breast cancer cytotoxicity. Our objective in this phase I study was to determine the tolerability of paclitaxel administered by 72-hour continuous intravenous (i.v.) infusion (CIVI) in combination with high-dose cyclophosphamide and granulocyte colony-stimulating factor (G-CSF) in the ambulatory setting to metastatic breast cancer patients. PATIENTS AND METHODS: Paclitaxel was administered over 72 hours by CIVI and cyclophosphamide was given daily by i.v. bolus on days 1, 2, and 3, followed by G-CSF every 21 days. The availability of ambulatory infusion pumps and paclitaxel-compatible tubing permitted outpatient administration. RESULTS: Fifty-five patients with metastatic breast cancer who had been previously treated with a median of two prior chemotherapy regimens were entered onto the study. Dose-limiting toxicity of grade 4 neutropenia for longer than 5 days and grade 4 thrombocytopenia occurred in three of five patients treated with paclitaxel 160 mg/m2 CIVI and cyclophosphamide 3,300 mg/m2 followed by G-CSF. The maximum-tolerated dose (MTD) was paclitaxel 160 mg/m2 CIVI and cyclophosphamide 2,700 mg/m2 in divided doses with G-CSF. Nonhematologic toxicities were moderate and included diarrhea, mucositis, and arthalgias. Although hemorrhagic cystitis developed in six patients, recurrence was prevented with i.v. and oral mesna, which permitted continued outpatient delivery. One hundred seventy-four cycles were safely administered in the ambulatory setting using infusional pumps and tubing. Objective responses occurred in 23 (one complete and 22 partial) of 42 patients with bidimensionally measurable disease (55%; 95% confidence interval, 38% to 70%), with a response rate of 73% (11 of 15) seen at the highest dose levels. CONCLUSION: Paclitaxel by 72-hour CIVI with daily cyclophosphamide followed by G-CSF can be administered safely in the ambulatory setting, has acceptable toxicity, and is an active regimen in the treatment of metastatic breast cancer.

Phase I crossover study of paclitaxel with r-verapamil in patients with metastatic breast cancer.

PURPOSE: We conducted a phase I crossover study of escalating doses of both paclitaxel (Taxol; Bristol-Myers, Squibb, Princeton, NJ) and r-verapamil, the less cardiotoxic stereoisomer, in heavily pretreated patients with metastatic breast cancer. PATIENTS AND METHODS: Twenty-nine patients refractory to paclitaxel by 3-hour infusion were treated orally with r-verapamil every 4 hours starting 24 hours before the same-dose 3-hour paclitaxel infusion and continuing for a total of 12 doses. Once the maximum-tolerated dose (MTD) of the combination was determined, seven additional patients who had not been treated with either drug were evaluated to determine whether the addition of r-verapamil altered the pharmacokinetics of paclitaxel. Consenting patients had tumor biopsies for P-glycoprotein (Pgp) expression before receiving paclitaxel and after becoming refractory to paclitaxel therapy. RESULTS: The MTD of the combination was 225 mg/m2 of r-verapamil every 4 hours with paclitaxel 200 mg/m2 by 3-hour infusion. Dose-limiting hypotension and bradycardia were observed in three of five patients treated at 250 mg/m2 r-verapamil. Fourteen patients received 32 cycles of r-verapamil at the MTD as outpatient therapy without developing cardiac toxicity. The median peak and trough serum verapamil concentrations at the MTD were 5.1 micromol/L (range, 1.9 to 6.3), respectively, which are within the range necessary for in vitro modulation of Pgp-mediated multidrug resistance (MDR). Increased serum verapamil concentrations and cardiac toxicity were observed more frequently in patients with elevated hepatic transaminases and bilirubin levels. Hematologic toxicity from combined paclitaxel and r-verapamil was significantly worse compared with the previous cycle of paclitxel without r-verapamil. In the pharmacokinetic analysis, r-verapamil delayed mean paclitaxel clearance and increased mean peak paclitaxel concentrations. CONCLUSION: r-Verapamil at 225 mg/m2 orally every 4 hours can be given safely with paclitaxel 200 mg/m2 by 3-hour infusion as outpatient therapy and is associated with serum levels considered active for Pgp inhibition. The addition of r-verapamil significantly alters the toxicity and pharmacokinetics of paclitaxel.

High-frequency attenuation of the cone ERG and ON-response deficits in X-linked retinoschisis.

PURPOSE: Individuals with X-linked retinoschisis (XLRS) show a comparatively greater reduction of the ON response than the OFF response of the electroretinogram (ERG) of the cone system. At high temporal frequencies, they also show a marked attenuation of the flicker ERG that has been attributed to an abnormal cone photoreceptor response. The purpose of this study was to determine whether the high-frequency response attenuation contributes to the abnormal ERG ON response in XLRS. METHODS: Light-adapted ERGs were recorded from three patients with XLRS and from three control subjects, by using rapid-on and rapid-off sawtooth flicker to emphasize ON and OFF responses, respectively, and by using low-pass sawtooth flicker, from which the high temporal frequencies had been removed to mimic the high-frequency attenuation in XLRS. RESULTS: For the control subjects, removing the high stimulus frequencies reduced the amplitude of the b-wave component of the ON response but had little effect on the amplitude of the d-wave component of the OFF response. In the patients with XLRS, the b-wave component of the ON response was already diminished using the full sawtooth stimulus, and removing the higher stimulus frequencies had no further effect. Patients' ERG responses to the 16-Hz stimulus fundamental alone were also abnormal, in that an initial response component normally present in the ERG was absent. CONCLUSIONS: The overall pattern of findings indicates that two factors contribute to the preferential ON-response deficit in XLRS: first, a high-frequency attenuation of the cone photoreceptor response that effectively produces a low-pass stimulus for the postreceptoral pathway and that affects the ON response more than the OFF response and, second, a relatively greater attenuation of the ON- than of the OFF-bipolar cell response that is evident in the aberrant response to the sawtooth fundamental.

On-response deficit in the electroretinogram of the cone system in X-linked retinoschisis.

PURPOSE: The purpose of this study was to evaluate the hypothesis that the reduced b-wave to a-wave ratio of the brief-flash electroretinogram (ERG) of the cone system typically observed in X-linked retinoschisis (XLRS) represents a relatively greater deficit in the ON response (response to light onset) than the OFF response (response to light offset). A second purpose was to investigate the use of sawtooth flicker as a stimulus for eliciting ERG ON and OFF responses. METHODS: Light-adapted, full-field ERGs were recorded in six patients with XLRS and six age-similar control subjects in response to 8-Hz rapid-on and rapid-off sawtooth flicker to emphasize ON and OFF responses, respectively. ERG responses were analyzed in terms of the amplitudes and implicit times of the a-wave, b-wave, and d-wave components. RESULTS: There was no significant difference between the patients with XLRS and the control subjects for either the amplitude of the a-wave of the ON response or the amplitude of the d-wave of the OFF response. However, the amplitude of the b-wave of the ON response was reduced significantly in the patients with XLRS, resulting in a significantly reduced b-wave to d-wave ratio. The patients' implicit times were increased significantly for all waveform components. CONCLUSIONS: The reduced b-wave to d-wave ratio of the ERG of the cone system in these patients with XLRS is consistent with a relative dysfunction of the cone ON bipolar cell pathway in this disorder. The results show further that sawtooth flicker is a promising stimulus for eliciting well-defined ERG waveforms that can provide a quantitative assessment of the properties of ON and OFF responses in retinal disease.

Failure of nitric oxide donors to alter arrhythmias induced by acute myocardial ischaemia or reperfusion in anaesthetized rats.

1. The aim of the present studies was to examine the effects of nitric oxide donors on arrhythmias induced by coronary artery occlusion and reperfusion, and on cardiac cyclic nucleotides. Experiments were performed in pentobarbitone-anaesthetized rats prepared for occlusion of the left coronary artery. 2. Sodium nitroprusside (0.1, 0.3 and 1 microgram kg-1 min-1) had no significant effects on the incidence of ventricular tachycardia, total ventricular fibrillation or the mortality resulting from 25 min of acute myocardial ischaemia when compared with values in controls. In addition, there was no alteration in the number of ventricular premature beats that occurred in survivors. 3. 3-Morpholinosydnonimine-N-ethylcarbamide (SIN-1, 10, 20 and 40 micrograms kg-1 min-1) caused marked hypotension but did not alter the incidence or severity of ischaemia-induced arrhythmias. In rats subject to abrupt reperfusion after 5 min of myocardial ischaemia, lower doses of SIN-1 (1, 3 and 10 micrograms kg-1 min-1) still caused significant reductions in systolic and diastolic blood pressure but were devoid of antiarrhythmic activity. 4. In separate experiments in sham-operated rats, sodium nitroprusside (1 microgram kg-1 min-1), isosorbide dinitrate (30 and 60 micrograms kg-1 min-1) and SIN-1 (20 and 40 micrograms kg-1 min-1) had no significant effects on cardiac cyclic GMP content. 5. These results indicate that nitric oxide donors do not alter arrhythmias induced by acute coronary artery occlusion or reperfusion in anaesthetized rats. Although increases in total cardiac cyclic GMP could not be detected, the results suggest that, at least in the rat, cyclic GMP does not influence these arrhythmias.

Chromatic induction with remote chromatic contrast varied in magnitude, spatial frequency, and chromaticity.

Chromatic induction from a surround is attenuated by chromatic contrast within a remote region outside of the surround (Shevell & Wei, 1998, Vision Research, 38, 1561-1566). The present study reports hue-cancellation measurements that show the attenuation depends on the magnitude, spatial frequency and chromaticity of remote chromatic contrast. Spatial-frequency tuning is shown by maximal attenuation of induction with remote contrast elements of the same size as the test. Experiments with various chromaticities of remote contrast show that S-cone stimulation within the remote region has a much weaker effect than L-/M-cone chromatic contrast, and does not depend on whether the S-cone stimulation is uniform or uneven across the region. Overall, the results show that remote L/M contrast affects classical chromatic induction, with its effect depending on the spatial frequency and magnitude of contrast. The influence of remote S-cone stimulation, on the other hand, is relatively weak and depends on only the S-cone spatial average, at least when S-cone stimulation by the test and its immediate surround is minimal (as in all experiments here).

Detection of briefly flashed sine-gratings in dark-adapted vision.

Scotopic contrast sensitivity was measured near 20 deg retinal eccentricity for briefly flashed (10 or 20 msec) sine-wave gratings presented in darkness to dark-adapted subjects. For very low spatial frequencies (0.2-0.5 c/deg), curves of contrast sensitivity vs luminous energy show evidence of a low rod plateau and a high scotopic region, with an intervening transition at around -2 to -2.5 log scot td sec. Similar measurements made using long flashed or flickering gratings do not show a plateau. The results suggest that vision in the low rod region is impaired for brief flashes. For the briefly flashed stimuli, curves of contrast sensitivity versus spatial frequency in the low region were best fit by simple Gaussian functions with a variable centre size (sigma c = 0.5----0.25 deg), size decreasing with increasing flash energy. Difference-of-Gaussian functions with constant centre size (sigma c = 0.25 deg) provided the best fit in the high region. Overt input from the cones and grating area artefacts are excluded by appropriate tests. Calculation of photon flux into the receptive field centres suggests that signal compression in P alpha ganglion cells contributes to the low rod plateau.

Contrast discrimination deficits in retinitis pigmentosa are greater for stimuli that favor the magnocellular pathway.

Luminance contrast discrimination was measured in 14 patients with retinitis pigmentosa (RP) and 14 control observers with normal vision, using steady-pedestal and pulsed-pedestal paradigms [Pokorny, J., & Smith, V. C. (1997). Psychophysical signatures associated with magnocellular and parvocellular pathway contrast gain. Journal of the Optical Society of America A, 14, 2477-2486] to bias performance toward the magnocellular (MC) or parvocellular (PC) pathway, respectively. The aim was to determine the relative effects of retinal degeneration on MC- and PC-pathway function in RP. For five of the RP patients, contrast discrimination thresholds were within normal limits for both the steady-pedestal and pulsed-pedestal paradigms. The other nine RP patients showed threshold elevations for the steady-pedestal paradigm (presumed magnocellular mediation), whereas their thresholds for the pulsed-pedestal paradigm (presumed parvocellular mediation) were within normal limits for all but the two patients who had the most extreme threshold elevations using the steady-pedestal paradigm. A control experiment on four of the RP patients, using a greater number of pedestal contrasts, verified that the patients' thresholds for the pulsed-pedestal paradigm showed the pattern expected for contrast discrimination mediated by the PC pathway. The higher threshold elevations for the steady-pedestal paradigm than for the pulsed-pedestal paradigm indicate that the retinal degeneration that occurs in RP predominantly disrupts contrast discrimination under stimulus conditions that favor the MC pathway.

Physician assistant students and diabetes: evaluation of attitudes and beliefs.

PURPOSE: Physician assistants are assuming a greater role in patient care in the US health system. The objective of this study was to examine attitudes and beliefs about diabetes among physician assistant trainees. METHODS: A survey of 3 currently enrolled classes of physician assistant students was conducted using the Diabetes Attitude Survey (DAS, version 3). An additional question was presented to gather information about the level of hyperglycemia at which students would intensify diabetes therapy. RESULTS: On average, students scored high on all subscales, indicating general agreement with the attitudes examined by the DAS. For 3 subscales (seriousness of type 2 diabetes, value of tight glucose control, and patient autonomy), significant differences were seen across year of training. When asked about the level of glucose control at which they would advance therapy, a wide range of responses occurred, with some being out of target. CONCLUSIONS: Physician assistant students had favorable attitudes regarding type 2 diabetes. However, deficits appear to exist in understanding when to advance therapy. More studies on physician assistant students' knowledge of diabetes standards of care are required.

Production of a recombinant protein from Alternaria containing the reported N-terminal of the Alt a1 allergen.

Allergen content of extract derived from Alternaria is somewhat variable. Allergenic molecules from Alternaria that appear as differing molecular size bands on IgE probed immunoblots may have a great deal of sequence homology and differ only in the length of the amino acid chain. One method to study this problem is to produce recombinant proteins from Alternaria. To explore these possibilities, the following experiments were performed. A strain of Alternaria was grown on minimum salts and glucose in a fermentation container with constant stirring and aeration. Rapidly expanding mycelia were removed from the culture and mRNA was extracted. Purified mRNA was reacted with reverse transcriptase and an aliquot of first copy single strand DNA was enriched for the presence of DNA coding for an Alternaria allergen by PCR amplification. Modified DNA was then spliced into lambda gt11 phage and yielded a recombinant library with 10(5) PFU. The library was screened for the presence of allergenic proteins using IgE containing human sera from Alternaria-sensitive patients. Positive plaques were cloned. PCR analysis of positive clones using an oligonucleotide from the reported N-terminal sequence of Alt a1 indicated an insert of 295 base pairs. Sequence analysis yielded a reading frame containing 84 amino acid and confirmed that this segment contained the code for the reported N-terminal amino acid sequence of Alt a1. A computer search for this sequence found no homologous proteins in the Entrez sequences. Northern blotting studies on RNA purified from nine strains of Alternaria with the radiolabeled 247 BP DNA fragment indicated that this sequence was present in all strains. The 247 BP nucleotide was spliced into the Pflag vector and clones containing insert in the proper reading frame were identified. The presence of recombinant protein in the clones was verified by SDSPAGE time studies. Protein produced in time studies was shown by immunoblotting and sandwich EIA to bind human IgE from Alternaria sensitive patients. This recombinant protein, containing amino acid sequence for Alt a1, is bound by human IgE and therefore should be useful as a model for studying allergy to the native Alternaria glycoprotein. Further research should define where this sequence occurs in the Alternaria genome and should determine the sequence of the entire protein.

Dissociation and unfolding of sulfhydryl oxidase in solutions of guanidinium chloride.

Sulfhydryl oxidase was dissociated completely in 5 M guanidinium chloride as indicated by light scattering molecular weight studies giving masses of 80,000 to 90,000 daltons. Concentrations of guanidinium chloride above 2 M resulted in loss of enzymatic activity and caused extensive unfolding of the enzyme as shown by fluorescence measurements. Replacement of the denaturant with physiological buffers restored native fluorescence characteristics, but the enzyme remained partially dissociated, with a molecular weight of 300,000 to 360,000, enzymatic activity was not restored.

ON-pathway dysfunction in a patient with acquired unilateral night blindness.

PURPOSE: To investigate possible functional correlates of an apparent ON-pathway defect observed in the cone electroretinogram of a patient with acquired unilateral night blindness. METHOD: Visual evoked potentials were recorded to the onset of a grid pattern consisting of either incremental or decremental squares. Saccadic eye movements were measured to luminance increments and decrements presented 5 degrees from fixation. The patient's results were compared with normative data. RESULTS: Visual evoked potential latencies were prolonged to incremental stimulation of the patient's affected left eye but were within normal limits for the other three conditions (increments and decrements, right eye; decrements, left eye). A similar pattern of asymmetry between latencies to incremental and decremental stimulation of the affected eye was observed for saccadic eye movements. CONCLUSIONS: The observed predominant delay in response to luminance increments supports the hypothesis of an ON-pathway dysfunction in this patient with acquired unilateral night blindness.

Enhanced sensitivity of immunoblotting with peroxidase-conjugated antibodies using an adsorbed substrate method.

Immunoblots probed with immunoglobulin E (IgE)-containing sera from allergic patients are frequently used in allergy research. Current techniques for detection of specific IgE include radiolabeled and enzyme-linked methods. Although radiolabeled methods are very sensitive, many research groups prefer non-radioactive procedures with equal or greater sensitivity. Alkaline phosphatase (AP) and horse radish peroxidase (HRP) are the most frequently used conjugating enzymes for immunoblotting with the former generally recognized as more sensitive. We describe a method of immunoblot detection using HRP-conjugated immunochemicals with sensitivity equal to and for some systems greater than that of AP conjugates. An adsorbed substrate method for developing immunoblots probed with HRP immunochemical conjugates is compared with traditional AP and HRP methods. The adsorbed substrate system, when used to detect IgE binding to allergic proteins, gives high resolution and delineates bands not otherwise seen. The system has advantages of high sensitivity, rapid development and conservation of immunochemicals. Problems of fading, sensitivity to heat and light, and high background can be solved with increased washing, prompt photography and computer scanning.

Origin of deficits in the flicker electroretinogram of the cone system in X-linked retinoschisis as derived from response nonlinearities.

The aim of this study was to identify the origin of a high-frequency attenuation in the flicker electroretinogram (ERG) of patients with X-linked retinoschisis (XLRS) through an analysis of nonlinearities in the ERG response. The ERGs of six patients with XLRS and six age-similar control subjects were recorded in response to stimuli that consisted of pairs of sinusoids that had varying temporal frequencies and that differed by either 8 or 16 Hz. Compared with the control subjects, the patients with XLRS showed a significant reduction in the amplitude of the difference frequency to high-frequency stimuli that paralleled the high-frequency attenuation of their ERG response fundamental. This result indicates that a response attenuation at an initial linear filter, most likely photoreceptoral, was a major determinant of the reduced ERG amplitude of the XLRS patients at high temporal frequencies. Additional analyses of nonlinearities in the ERG responses provided evidence of a postreceptoral component to the flicker ERG deficits of the XLRS patients, as well.

An examination of some reputed antifertility plants.

Solvent extracts prepared from 80 plant species reputed to show antifertility effects on human beings or from species closely related to such plants, showed no significant antifertility activity when fed to rats.

Identification and characterization of IgA and Vicia villosa-binding T cell subsets in rheumatoid arthritis.

Autoimmunity may be due to augmentation of immune responses by human CD8 cells which bind the lectin Vicia villosa (VV). We have investigated T cells in rheumatoid arthritis (RA) by double immunofluorescence flow cytometry, in order to assess VV-binding CD8 and CD4 cells from the peripheral blood and synovial fluid. A significant increase in CD8+VV adherent (P less than 0.0001) and CD4+VV adherent cells (P less than 0.001) was found in synovial fluid, as compared with peripheral blood from patients with RA. A significant increase in VV-binding CD8+ or CD4+ cells was, however, not found in the blood of patients with RA, as compared with controls. We suggest that the lack of VV-binding T cells separated from blood, in contrast to those from synovial fluid, may be due to an inhibiting agent expressing N-acetyl D-galactosamine. Indeed, IgA1 is rich in N-acetyl D-galactosamine, it inhibits VV binding to T cells and is significantly bound to CD8 cells (P less than 0.001). The IgA1 was then characterized and in about half the patients J chains and secretory component was found, suggesting that the IgA1 is of the polymeric and secretory variety. IgA bound to the T cells engaged the Fc alpha receptors and a significant decrease in the Fc alpha receptors was found in CD8 cells (P less than 0.0001) and CD4 cells (P less than 0.01). Desorption studies were then carried out on CD8 and CD4 cells which showed that a loss of cell-bound IgA1 was associated with an increase in VV binding. Conversely, adsorption of IgA to T cells was associated with a loss in VV binding. The results suggest that the failure of VV binding to CD8+ and CD4+ cells from peripheral blood of patients with RA can be ascribed to cell-bound IgA1. Cytophilic IgA1 may inhibit the function of CD8+VV binding cells, thereby preventing augmentation of the systemic immune response, consistent with the lack of extra-articular disease in these patients with RA.

Saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion, decreases platelet aggregation and intimal hyperplasia in a rat carotid endarterectomy model.

PURPOSE: Post-carotid endarterectomy, thrombosis, and intimal hyperplasia may be decreased by the inhibition of platelet adhesion and activation. In this study, a novel agent, saratin, was used to inhibit platelet-to-collagen adhesion in a rat carotid endarterectomy model. Saratin is a recombinant protein isolated from the saliva of the medicinal leech Hirudo medicinalis, which is thought to act by binding to collagen, and inhibits von Willebrand factor-collagen interaction under conditions of increased shear and therefore, the adherence and activation of platelets at the vessel wall. Saratin has the advantage of being a nonsystemic, site-specific topical application. METHODS: A rat carotid endarterectomy model was used in which an open technique with arteriotomy and intimectomy was used. Saratin was applied to the endarterectomized surface of the carotid artery before arterial closure. End point measurements included platelet adhesion, thrombosis rate, intimal hyperplasia development, bleeding times, and platelet counts. Electron micrographs of carotid arteries were used for quantitative analysis of platelet aggregation and platelet counts. Intimal hyperplasia and thrombosis were assessed with computer-assisted morphometric analysis of elastin-stained carotid artery sections with direct measurement of the intimal hyperplasia area. RESULTS: The topical application of saratin significantly decreased platelet adhesion compared with controls at 3 hours after carotid endarterectomy (64 +/- 17 vs 155 +/- 33 platelets per grid, P = .05), and 24 hours after carotid endarterectomy (35 +/- 11 vs 149 +/- 37 platelets per grid, P = .0110), respectively. A percent luminal stenosis, as a measure of intimal hyperplasia, was significantly decreased with saratin application compared with controls (10.9% +/- 1.8% vs 29.8% +/- 6.8%, P = .0042). This decrease in intimal hyperplasia formation correlated with the inhibition of platelet adhesion. Thirty-three percent of control arteries were found to be thrombosed 2 weeks after carotid endarterectomy compared with a 0% thrombosis rate in the saratin-treated group (P = .0156). No increased bleeding was encountered along the arterial suture line in the saratin group. Bleeding times and systemic platelet counts were not found to change significantly in the saratin-treated rats compared with control rats at 3 and 24 hours after endarterectomy. CONCLUSION: Saratin significantly decreased platelet adhesion, intimal hyperplasia, luminal stenosis, and thrombosis after carotid endarterectomy in rats. Saratin did not increase suture line bleeding or bleeding times, and did not decrease platelet counts. Saratin may serve as a topical agent to be used for the site-specific inhibition of thrombosis and intimal hyperplasia after vascular manipulation.

Production and characterization of saratin, an inhibitor of von Willebrand factor-dependent platelet adhesion to collagen.

Platelets tether to collagen in both a von Willebrand factor (vWF)-dependent and a vWF-independent manner. We have recently characterized a recombinant protein, saratin, isolated from the saliva of the leech Hirudo medicinalis, expressed it in Hansenula polymorpha, and studied its effect on direct and indirect platelet-collagen interactions. Saratin dose dependently inhibited the binding of purified human vWF to human type I and III collagens (IC(50)= 0.23 +/- 0.004 and 0.81 +/- 0.04 microg mL(-1), respectively) and to calf skin collagen (IC(50)= 0.44 +/- 0.008 microg mL(-1)). Furthermore, saratin showed a similar inhibitory potency against the binding of human, rodent, and porcine plasma vWF to these collagens. In a flow chamber under conditions of elevated shear (2700 s(-1)), saratin dose dependently and potently inhibited platelet aggregate formation on a collagen-coated surface (IC(50)= 0.96 +/- 0.25 microg mL(-1)), but at reduced shear (1300 s(-1)) a rightward shift in the dose-response curve was noted (IC(50)= 5.2 +/- 1.4 microg mL(-1)). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (K(d) 5 x 10(-8) M and 2 x 10(-6) M, respectively). Although low concentrations of saratin, which inhibited platelet adhesion under increased shear (i.e., saturation of high-affinity binding sites), had no effect on vWF-independent collagen-induced platelet aggregation, high concentrations (i.e., saturation of low-affinity binding sites) were found to inhibit platelet aggregation. These data demonstrate that saratin is a potent inhibitor of vWF-dependent platelet adhesion to collagen and hence may have therapeutic potential as an antithrombotic agent.

Clinical inertia.

Medicine has traditionally focused on relieving patient symptoms. However, in developed countries, maintaining good health increasingly involves management of such problems as hypertension, dyslipidemia, and diabetes, which often have no symptoms. Moreover, abnormal blood pressure, lipid, and glucose values are generally sufficient to warrant treatment without further diagnostic maneuvers. Limitations in managing such problems are often due to clinical inertia-failure of health care providers to initiate or intensify therapy when indicated. Clinical inertia is due to at least three problems: overestimation of care provided; use of "soft" reasons to avoid intensification of therapy; and lack of education, training, and practice organization aimed at achieving therapeutic goals. Strategies to overcome clinical inertia must focus on medical students, residents, and practicing physicians. Revised education programs should lead to assimilation of three concepts: the benefits of treating to therapeutic targets, the practical complexity of treating to target for different disorders, and the need to structure routine practice to facilitate effective management of disorders for which resolution of patient symptoms is not sufficient to guide care. Physicians will need to build into their practice a system of reminders and performance feedback to ensure necessary care.