Exposure of juvenile Leghorn chickens to lead acetate enhances antibiotic resistance in enteric bacterial flora.

Heavy metals have been implicated for their ability to increase antibiotic resistance in bacteria collected from polluted waters, independent of antibiotic exposure. Specific-pathogen-free Leghorn chickens were therefore given Pb acetate in the drinking water to expose the enteric bacteria to Pb and to determine if antibiotic resistance changed in these bacteria. Concentrations of Pb used were 0.0, 0.01, 0.1, 1.0, or 10.0 mM; birds given the highest 2 concentrations showed signs of moribundity and dehydration and were removed from the study. Vent culture samples were collected for bacterial cultures on d 0 before Pb exposure, d 7 and 14, and then birds were euthanized by CO2 gas for necropsy on d 14, at which time intestinal contents were also collected for bacterial cultures. Fecal swabs but not intestinal samples from Pb-exposed birds contained isolates that had significantly elevated antibiotic resistance. Some of the isolates contained bacteria that were resistant to up to 20 antibiotics. These results suggest the need for repeated studies in chickens infected with zoonotic pathogens.

Development of a storage-compatible microtiter plate-based technique for lymphocyte proliferation.

The lymphocyte proliferation assay (LPA) is an important tier 1 test to assess non-specific lymphocyte function, in vitro. However, this assay requires fresh preparation, is time consuming, and labor intensive. Developing a plate coating technique for lymphocyte proliferation that is both stable and storage compatible would be useful to the basic and clinical researcher. In this study, we compared the effects of different mitogen plate coating techniques on lymphocyte proliferation to freshly prepared plates. The results show that plates prepared with complete media and stored at -40 degrees C up to 10 days corresponded well to control plates.

Viability and function in lymphocytes cultured from the horse, chicken, and mouse: effects of different leukocyte enrichment techniques.

Methods of lymphocyte enrichment tend to vary across species, with the most common techniques employed being density-gradient separation and erythrocyte lysis buffer enrichment. In this study, we assessed lymphocyte viability and proliferation of avian, equine, and murine lymphocytes enriched by a commercial density-gradient technique and under identical, standardized culture conditions. The results of this study clearly show that, under identical enrichment and culture conditions, lymphocyte viability and function can be quite different among the equine, bird, and mouse species. Secondly, the type of enrichment technique employed in the mouse can impact the quality of the immune data generated.

Intermittent calf and foot compression increases lower extremity blood flow.

PURPOSE: Although foot compression increases foot skin perfusion and calf compression increases popliteal artery blood flow, these compression techniques have not been evaluated in combination. The purpose of this study was to evaluate whether calf and foot compression applied separately and simultaneously increase popliteal artery blood flow and/or foot skin perfusion, and to assess the relative merits of compression in patients with superficial femoral artery occlusion. METHODS: Twenty-two legs from 12 normal volunteers with ankle/brachial indices (ABIs) > 0.96, and 10 legs from 7 claudicator patients with angiographically documented superficial femoral artery (SFA) occlusion and patent popliteal arteries with ABIs < 0.8 were studied in the sitting position. Calf and foot cuffs connected to a rapidly inflating and deflating timed-pressure pump (Art-Assist-AA 1000; ACI Medical Inc., San Marcos, California) were applied to the subject in the sitting position. Skin blood flow of the great toe was measured with a laser doppler (Laserflo model BPM 403A; TSI Inc., St. Paul, Minnesota), and popliteal artery blood flow was measured using duplex ultrasonography (ATL-Ultramark 9; Advanced Tech Laboratory, Bothell, Washington). Foot and calf compression was applied separately and simultaneously at 120 mm Hg pressure, with a 10-second inflation and 20-second deflation cycle. Popliteal artery blood flow and foot skin perfusion were recorded and the mean of 6 cycles calculated. RESULTS: Precompression popliteal artery blood flow (mL/min) for volunteers was 38.86 +/- 3.94, and for patients was 86.30 +/- 14.55 (P = 0.001). Precompression foot skin perfusion (mL/min/ 100/g tissue) for volunteers was 1.67 +/- 0.29, and for patients was 4.00 +/- 0.92 (P = 0.01). With the application of calf, foot, and simultaneous calf and foot compression, the popliteal artery blood flow increased in volunteers by 124%, 54%, and 173%, respectively, and in patients by 76%, 13%, and 50%. Foot skin perfusion increased in volunteers by 260%, 500%, and 328%, respectively, and in patients by 116%, 246%, and 188%. Relative increases in popliteal artery blood flow and foot skin perfusion were higher in volunteers compared with patients during compression; however, the absolute values for foot skin perfusion and popliteal artery blood flow were consistently higher in patients. CONCLUSIONS: Measured in the sitting position, the resting popliteal artery blood flow and foot skin perfusion are greater in patients with SFA occlusion compared with normal volunteers. Following compression, popliteal artery blood flow and foot skin perfusion increased in both groups, but relatively more in volunteers. Increases in popliteal artery blood flow are significantly higher with calf compression than with foot compression for both groups. A patent SFA allows for additive increases in popliteal artery blood flow with simultaneous foot and calf compression in normal persons, whereas this is not observed in patients. However, the increases in foot skin perfusion in patients with an occluded SFA parallel the increases shown in normal volunteers, with separate and simultaneous foot and calf compression.

Lead at 2.5 and 5.0 µM induced aberrant MH-II surface expression through increased MII exocytosis and increased autophagosome formation in Raw 267.4 cells.

Aberrant major histocompatibility complex class II (MHC-II) surface expression on antigen presenting cells (APCs) is associated with dysregulated immune homeostasis. Lead (Pb) is known to increase MHC-II surface expression on murine peritoneal macrophages ex vivo at concentrations exceeding 25 µM. Little data exist examining this effect at physiologically relevant concentrations. To address this deficit, we examined the effects of Pb on MHC-II surface expression, secondary T-cell activation markers (CD80, CD86, CD40), cell viability, cellular metabolic activity, and ß-hexosaminidase activity in RAW 267.4 macrophage cell lines, with changes in cell ultrastructure evaluated by electron and confocal microscopy. Pb induced an increase in MHC-II, CD86, and lysosome-associated LAMP-1 and LAMP-2 surface mean expression during one doubling cycle (17 h), which was mirrored by increased ß-hexosaminidase activity. Although cell viability was unaffected, cellular metabolism was inhibited. Electron microscopy revealed evidence of lipid vacuolization, macroautophagy and myelin figure formation in cells cultured with either Pb or LPS. Confocal microscopy with antibodies against LC3B showed a punctate pattern consistent with the presence of mature autophagosomes. Collectively, these data suggest that 2.5-5.0 µM Pb increased MHC-II surface expression by inhibiting metabolic activity, inducing autophagy, and increasing MHC-II trafficking in a macrophage cell line.

Is venous duplex imaging an appropriate initial screening test for patients with suspected pulmonary embolism?

Noninvasive tests for deep venous thrombosis (DVT) are helpful in evaluating patients with suspected pulmonary embolism (PE) who have non-high-probability ventilation/perfusion (V/Q) lung scans. Based on the enthusiasm for these noninvasive tests, venous duplex imaging (VDI) has evolved as the initial screening test for patients with clinically suspected PE in some centers. This study evaluates the utility of VDI as the initial test in a diagnostic algorithm for patients with suspected PE. A total of 306 consecutive patients who underwent VDI as the initial screening test for clinically suspected PE during the past 24 months were reviewed; 121 patients were subsequently evaluated with V/Q scans and 20 underwent pulmonary arteriography. VDI demonstrated DVT in 10% (23/216), with 7% (22/306) having proximal DVT and 3% (9/306) having isolated calf DVT. In 25 patients with unilateral leg symptoms, DVT was found in 40% (10/25); however, among the 281 without unilateral leg symptoms, results of VDI were abnormal in only 5% (15/281). V/Q scans were obtained in 40% (121/306), with only 16% (19/121) of scans showing a high probability of PE. DVT was found in 25% (5/19) of patients with high-probability V/Q scans and in 25% (26/102) with non-high-probability scans. In patients with clinically suspected PE the incidence of detectable infrainguinal DVT is low. VDI appears to be a reasonable initial screening test in patients with clinically suspected PE and unilateral leg symptoms. However, in patients without unilateral leg symptoms, the diagnostic yield is low and an alternative diagnostic approach appears justified.

Variability of venous-hemodynamics with daily activity.

PURPOSE: This study was designed to evaluate changes in venous hemodynamics that occur in normal, symptom-free male and female volunteers, as a consequence of daily activity. METHODS: Each leg of 25 symptom-free volunteers was prospectively studied twice in the early morning and twice in the late afternoon on 2 days. Air plethysmography was used to evaluate venous volumes, venous valvular function, calf muscle pump function, and the noninvasive equivalent of ambulatory venous pressure. RESULTS: There was significant change in venous valvular function (venous filling index) indicating progressive insufficiency in the late afternoon compared with the results of the morning studies (p = 0.039). This was demonstrated by a shortened venous filling time (p = 0.033) but not a change in venous volume (p = 0.794). Calf muscle pump function and ambulatory venous pressures remained constant. Although there were significant leg volume and ejection volume differences at baseline in male volunteers compared with female volunteers, no gender differences were evident as a result of daily activity. Five of 25 (20%) volunteers and seven of 50 (14%) extremities had normal venous refill times and venous function index in the morning, which became abnormal in the afternoon, indicating deterioration of venous valve function. CONCLUSIONS: Venous hemodynamic changes occur normally as a consequence of daily activity and seem to result from valvular dysfunction. This occurs in men and women and can alter diagnostic conclusions in 20% of otherwise normal patients. These findings have important implications for venous testing and accurate patient evaluation. However, extrapolation of these data to patients with established venous disease should not be made.

The piezoelectric pulse sensor device: a prospective evaluation.

The goal of this prospective study of the piezoelectric pulse sensor device was to determine its technical applications and its ability to detect lower extremity occlusive arterial disease. Ten extremities (five volunteers) were evaluated to assess the ability to place the sensor in the correct anatomic position on a foot without a palpable pulse during cuff occlusion so that pulsatile flow would be detected following cuff deflation; its sensitivity as an end-point detector for pulsatile perfusion; and whether there is a linear qualitative pulse wave response with increasing perfusion pressures. Forty extremities (20 patients) with suspected occlusive arterial disease were studied to evaluate its capability of detecting perfusion as compared with the presence of a palpable pulse, an audible Doppler signal, and a foot volume waveform. The placement of the sensor on 10 normal limbs with temporary arterial occlusion resulted in a recordable waveform following cuff deflation in 100% of the dorsalis pedis arteries and in 10% of the posterior tibial arteries. The piezoelectric pulse sensor was as sensitive for detecting pulsatile perfusion as an audible Doppler signal and demonstrated a linear change in the waveform's amplitude and shape with incremental changes in perfusion pressure. In the 40 extremities with ankle/brachial indices ranging from 0.00 to 1.35, there was uniform agreement between pulse volume and Pulse Check waveforms. The piezoelectric pulse sensor is a sensitive method for monitoring lower extremity arterial perfusion when supplied by the dorsalis pedis artery; however, it is inadequate for the posterior tibial artery.(ABSTRACT TRUNCATED AT 250 WORDS)

Hemodynamic deterioration in chronic venous disease.

Clinical deterioration of patients with chronic venous disease (CVD) has been well described and a standardized classification has been proposed. The progressive hemodynamic deterioration producing these clinical findings is less well appreciated. This study examines and correlates venous hemodynamics with clinical severity in patients with CVD. Two hundred seventy-four extremities from 149 patients with varying degrees of CVD and 56 extremities from 28 symptom-free volunteers were evaluated clinically and hemodynamically. Each limb was assessed for functional venous volume, degree of valvular insufficiency, efficiency of the calf muscle pump, and noninvasive estimate of ambulatory venous pressure. In addition, exercise venous pressures were recorded in 56 extremities from 36 patients and 9 extremities from 6 volunteers. As CVD progresses from class 0 to class 2, venous volume expands, valvular function deteriorates, the calf muscle pump becomes inefficient, and ambulatory venous hypertension develops. However, once extremities develop brawny edema or hyperpigmentation, further deterioration of limb hemodynamics does not occur. Patients with deep venous obstruction have more severe valvular insufficiency, calf muscle pump dysfunction, and ambulatory venous hypertension than have patients without evidence of obstruction. Residual volume fraction offers a reliable noninvasive estimate of ambulatory venous pressure (r = 0.76), although its correlation was significantly better for patients without venous obstruction (r = 0.86) than for those with obstruction (r = 0.40; p < 0.05). Deterioration in venous hemodynamics parallels clinical severity through class 2. Once brawny edema and hyperpigmentation occur, ulceration develops without additional deterioration of venous hemodynamics.

The hemodynamics of venous ulceration.

Venous ulceration is the result of progressive chronic venous insufficiency, the pathophysiology of which is complex and incompletely understood. Ambulatory venous hypertension in this disease has been well-documented; however, relatively little attention has been directed toward other parameters of venous function. This study evaluates a spectrum of hemodynamic variables and the degree to which they are altered in patients with venous ulceration, and correlates ambulatory venous pressure (AVP) with the noninvasive estimate of this parameter. Air-plethysmography was used to evaluate 36 ulcerated extremities from 30 patients with chronic venous disease and 80 asymptomatic extremities from 54 patients. This technique measures the functional venous volume (VV), assesses valvular function [Venous Filling Index (VFI)], evaluates the efficiency of the calf muscle-pump [Ejection Fraction (EF)], and provides an estimation of ambulatory venous pressure [Residual Volume Fraction (RVF)]. In addition, AVP's were recorded in 13 asymptomatic extremities from 10 patients and 16 ulcerated extremities from 14 patients with chronic venous disease. Significant differences existed between the two groups for all of the hemodynamic parameters. Ulcerated extremities had greater venous volumes, displayed marked deterioration in valvular competence and calf muscle-pump function, and showed significant ambulatory venous hypertension compared to the asymptomatic group. Additionally, the relationship between RVF and AVP appeared linear, with a correlation coefficient of 0.87. Air-plethysmography currently provides the most complete evaluation of venous hemodynamics and should improve our understanding of the pathophysiology of chronic-venous disease.

Duplex venous imaging: role for a comprehensive lower extremity examination.

Real-time compression ultrasound (CU) along with venous duplex imaging is the most commonly performed noninvasive vascular examination. It has become the definitive diagnostic test for most patients with deep venous thrombosis (DVT). Some practioners have recommended that CU alone of the common femoral vein (CFV) and of the popliteal vein (PV) are all that is required since a complete examination is time consuming and calf veins are difficult to visualize. However, if only the CFV and PV are examined, all patients with isolated superficial femoral vein (SFV) and calf DVT remain undiagnosed. The purpose of this study is to establish the value of a comprehensive venous duplex examination compared to CFV and PV compression alone for detecting both proximal and infrapopliteal DVT. From January 1996 through December 1997, the initial venous duplex examinations of 5767 extremities in 3067 patients were reviewed and results tabulated according to presence and location of clot. The ATL 3000 with a 7-14 mHz probe was utilized. Studies were interpreted as normal, proximal DVT (popliteal and above, with or without calf DVT), isolated calf, or isolated SFV deep venous thrombosis. If only the CFV and PV had been examined, 30.3% (isolated SFV + isolated calf vein DVT) of all DVT and 4.5% of proximal DVT would have been missed. A complete venous duplex examination altered the care in 288 (30.3%) of all patients examined who had DVT, and is therefore recommended as the standard noninvasive examination when evaluating patients for acute DVT.

Is transcranial Doppler a worthwhile addition to screening tests for cerebrovascular disease?

PURPOSE: Carotid duplex imaging has become the standard diagnostic evaluation for patients with suspected cerebrovascular disease. Transcranial Doppler ultrasonography expands the noninvasive diagnostic capabilities to the intracranial circulation. The purpose of this study was to evaluate the results of routine transcranial Doppler studies on patients referred for noninvasive cerebrovascular evaluation. METHODS: A total of 670 patients had routine transcranial Doppler examinations as part of their noninvasive cerebrovascular evaluation. Patients were categorized clinically and according to their severity of extracranial internal carotid artery stenosis (< 50%, 50% to 79%, 80% to 99%, occlusion). Transcranial Doppler examinations were classified as normal or abnormal (intracranial stenosis, collateral pathway, > 30% velocity difference normal or abnormal (intracranial stenosis, collateral pathway, > 30% velocity difference between sides, flow reversal, and velocities +/- SD from normal). RESULTS: Forty-eight percent of the patients were women, and 52% were men. The average age was 65.5 years. Fifty-four percent of the patients were white, 42% were black, 3% were Hispanic, and 1% were other. Forty-eight percent presented with hemispheric symptoms, 34% had no symptoms, and 18% had nonhemispheric symptoms. Forty-five percent (304 of 670) had an interpretable transcranial Doppler examination. The ability to insonate the basal cerebral arteries through the temporal bone was significantly reduced in women (p < 0.0001), black patients (p < 0.0001), and older patients (p < 0.0001). The results of forty-four percent of interpretable examinations were normal, 19% demonstrated side-to-side velocity differences, 13% showed collateral pathways, 11% showed velocities +/- 2 SD, 10% showed an intracranial stenosis, and 4% showed reversed flow pattern. Although 56% of the patients had notable findings, no patient had their diagnostic or therapeutic plan altered by the transcranial Doppler results. CONCLUSION: Less than 50% of the patients referred for first-time cerebrovascular examination had access for an interpretable transcranial Doppler examination. Though the number of positive findings is reasonably high, no material impact on diagnostic or treatment plans was seen in the patients in this series. These results indicate that selection criteria for examination of the intracranial arteries should be refined and that transcranial Doppler scanning should not be incorporated as part of the "routine" noninvasive cerebrovascular examination.