PAF increases vascular permeability without increasing pulmonary arterial pressure in the rat.

In vivo pulmonary arterial catheterization was used to determine the mechanism by which platelet-activating factor (PAF) produces pulmonary edema in rats. PAF induces pulmonary edema by increasing pulmonary microvascular permeability (PMP) without changing the pulmonary pressure gradient. Rats were cannulated for measurement of pulmonary arterial pressure (Ppa) and mean arterial pressure. PMP was determined by using either in vivo fluorescent videomicroscopy or the ex vivo Evans blue dye technique. WEB 2086 was administered intravenously (IV) to antagonize specific PAF effects. Three experiments were performed: 1) IV PAF, 2) topical PAF, and 3) Escherichia coli bacteremia. IV PAF induced systemic hypotension with a decrease in Ppa. PMP increased after IV PAF in a dose-related manner. Topical PAF increased PMP but decreased Ppa only at high doses. Both PMP (88 +/- 5%) and Ppa (50 +/- 3%) increased during E. coli bacteremia. PAF-receptor blockade prevents changes in Ppa and PMP after both topical PAF and E. coli bacteremia. PAF, which has been shown to mediate pulmonary edema in prior studies, appears to act in the lung by primarily increasing microvascular permeability. The presence of PAF might be prerequisite for pulmonary vascular constriction during gram-negative bacteremia.

Utility of intraoperative frozen section analysis of sentinel lymph nodes in breast cancer.

BACKGROUND: Intraoperative frozen section pathologic analysis of sentinel lymph node (SLN) may guide immediate (single-stage) completion axillary dissection for patients with nodal metastases. METHODS: The results of 203 consecutive patients undergoing SLN biopsy who had intraoperative pathology consultation between January 1998 and September 2000 were reviewed. SLN were analyzed by standard frozen section procedures. Final pathologic analysis included hematoxylin and eosin (H&E) staining of serial sections at 2-mm intervals. RESULTS: Frozen section analysis correctly identified a positive or negative result in 185 of 203 cases (overall accuracy 91%). In 17 of 53 cases, the SLNs were negative for tumor by frozen section, but positive on permanent section analysis (sensitivity 68%). The mean size of the nodal metastases was 6.2 mm and 1.5 mm in patients found to have true positive and false negative results, respectively (P <0.003). A single false positive SLN is reported. CONCLUSIONS: Two thirds of the patients were spared the need for reoperative axillary lymphadenectomy.

Inhibitors of HER2/neu (erbB-2) signal transduction.

Signaling by the HER2 proto-oncogene product results in the activation of several biochemical pathways that in turn modulate the expression and function of molecules involved in cell proliferation and survival. It is well established that forced overexpression of HER2 results in transformation of nontumor cells, and that high levels of HER2 in tumors are associated with a more aggressive biological behavior. It is also clear that a subset of HER2-overexpressing tumors is dependent on HER2 function for proliferation and/or survival. Over the last few years, several elegant studies have dissected the biochemical mechanisms of HER2 signaling. This research has provided information about critical functional domains in HER2 that can be targeted with rational molecular approaches, some of which are already being implemented at the bedside. This report will focus on one of these anti-HER2 signaling strategies.

Platelet-activating factor and bacteremia-induced pulmonary hypertension.

BACKGROUND: Acute lung injury is a common complication of gram-negative sepsis. Pulmonary hypertension and increased lung vascular permeability are central features of lung injury following experimental bacteremia. Platelet-activating factor is a prominent proinflammatory mediator during bacterial sepsis. Our previous studies have demonstrated that exogenous administration of platelet-activating factor (PAF) induces pulmonary edema without causing pulmonary hypertension. Interestingly, inhibition of PAF activity during Escherichia coli bacteremia prevents the development of both pulmonary hypertension and pulmonary edema. These data suggest that PAF contributes to pulmonary hypertension during sepsis, but that this is unlikely to be a direct vascular effect of PAF. The goal of the present study was to investigate the mechanism by which acute E. coli bacteremia induces pulmonary injury and to define the role that PAF plays in this injury. We hypothesized that the effects of PAF on pulmonary hypertension during bacteremia are due to the effects of PAF on other vascular mediators. Several studies suggest that PAF induces the expression of endothelin-1 (ET), a potent peptide vasoconstrictor. Further, our previous studies have implicated ET as a central mediator of systemic vasoconstriction during bacteremia. We therefore sought to assess whether ET is modulated by PAF. E. coli has also been demonstrated to increase endothelial production of nitric oxide (NO), which contributes to maintenance of basal vascular tone in the pulmonary circulation. We hypothesized that PAF might increase pulmonary vascular resistance during bacteremia by activating neutrophils, increasing expression of ET, and decreasing the tonic release of NO. Furthermore, we hypothesized that hypoxic vasoconstriction did not contribute to pulmonary vasoconstriction during the first 120 min of E. coli bacteremia. METHODS: Pulmonary artery pressure (PAP), blood pressure (BP), heart rate (HR), and arterial blood gases (ABG) were measured in anesthetized spontaneously breathing adult male Sprague-Dawley rats. E. coli (10(9) CFU/100 g body wt) was injected at t = 0, and hemodynamic data were obtained at 10-min intervals and ABG data at 30-min intervals for a total of 120 min. Sham animals were treated equally but received normal saline in place of E. coli. In treatment groups, a 2.5 mg/kg dose of WEB 2086, a PAF receptor antagonist, was administered intravenously 15 min prior to the onset of sepsis or sham sepsis. The groups were (1) intravenous E. coli (n = 5); (2) intravenous WEB 2086 pretreatment + intravenous E. coli (n = 5); (3) intravenous WEB 2086 alone (n = 5); and (4) intravenous normal saline (n = 6). Nitric oxide metabolites (NOx) and ET concentrations were assayed from arterial serum samples obtained at the end of the protocol. Lung tissue was harvested for measurement of myeloperoxidase (MPO) activity and pulmonary histology. RESULTS: E. coli bacteremia increased HR, PAP, and respiratory rate early during sepsis (within 20 min), while hypoxemia, hypotension, and hemoconcentration were not manifest until the second hour. Pretreatment with WEB 2086 completely abrogated all of these changes. E. coli bacteremia increased the activity of serum ET, lung MPO, and neutrophil sequestration in the lung parenchyma via a PAF-dependent mechanism. However, the mechanism of increased production of NO appears to be PAF independent. CONCLUSIONS: These data support the hypothesis that E. coli bacteremia rapidly induces pulmonary hypertension stimulated by PAF and mediated at least in part by endothelin-1 and neutrophil activation and sequestration in the lung. Microvascular injury with leak is also mediated by PAF during E. coli bacteremia, but the time course of resultant hypoxemia and hemoconcentration is slower than that of pulmonary hypertension. The contribution of hypoxic vasoconstriction in exacerbating pulmonary hypertension in gram-negative sepsis is probably a late

Platelet-activating factor mediates pulmonary macromolecular leak following intestinal ischemia-reperfusion.

Platelet-activating factor (PAF) causes hypotension, cardiac dysfunction, increased vascular permeability, intestinal necrosis, and pulmonary microvascular injury when administered experimentally. Receptor antagonism attenuates or abolishes many of these effects in animal models of bacteremia, endotoxemia, and intestinal ischemia/reperfusion (I/R). The purpose of this study was to further examine the role of PAF in intestinal I/R-induced pulmonary injury using the PAF receptor antagonist WEB 2086. Sprague-Dawley rats were anesthetized and cannulated for measurement of mean arterial pressure, heart rate, and cardiac output. Laparotomy and thoracotomy were performed and the superior mesenteric artery was occluded for 45 min and reperfused for 120 min. Sham animals were treated similarly but without I/R. In the treatment groups, iv WEB 2086 (20 mg/kg/l cc NS) was administered as a bolus 15 min prior to reperfusion. Hemodynamic and videomicroscopic data were obtained before and during ischemia, and after reperfusion at 30-min intervals. Alveolar leak index was calculated offline via computer analysis of videomicroscopic images. Intestinal I/R caused pulmonary macromolecular leakage and hemodynamic instability. Treatment with WEB 2086 attenuated the pulmonary leak during the entire reperfusion period but improved cardiac output only during the first 30 min of reperfusion and had no effect on other hemodynamic variables. These data suggest that PAF is an important, but not the exclusive, mediator of pulmonary injury after intestinal I/R. PAF appears to play a minor role in the hemodynamic derangements observed after rat intestinal I/R.

Pentoxifylline attenuates pulmonary macromolecular leakage after intestinal ischemia-reperfusion.

OBJECTIVE: To assess the effects of pentoxifylline posttreatment on hemodynamic variables and acute pulmonary injury in the rat intestinal ischemia-reperfusion (I-R) model, using a recently developed method of fluorescent intravital pulmonary videomicroscopy. DESIGN: Anesthetized male Sprague-Dawley rats were cannulated for measurement of mean arterial pressure, heart rate, cardiac output, arterial blood gas values, and hematocrit. Rats underwent isolation of the superior mesenteric artery for intestinal I-R (45 minutes of ischemia, 120 minutes of reperfusion) and right lateral thoracotomy for pulmonary videomicroscopy. Epi-illumination fluorescent videomicroscopy was used to quantitate leakage of intravascular fluorescently labeled albumin into alveoli, while hemodynamic variables were simultaneously recorded. In the treatment groups, pentoxifylline was administered after 30 minutes of intestinal ischemia. Data (mean +/- SEM) were recorded before and during intestinal ischemia and after reperfusion at 30-minute intervals. MAIN OUTCOME MEASURE: The appearance of fluorescently labeled albumin into alveolar airspaces was quantitated off-line by computer and reported as the alveolar leak index. RESULTS: Intestinal I-R caused alveolar macromolecular leakage, marked by a 300% +/- 48% increase from baseline (P < .05) in the alveolar leak index. Intestinal I-R also produced systemic hemodynamic instability demonstrated by a decrease in the mean arterial blood pressure (-36% +/- 5% vs baseline, P < .05) and cardiac output (-42% +/- 6% vs baseline, P < .05), metabolic acidosis (final arterial pH of 7.17, P < .05 vs initial pH), and a 2.3-fold increase in the intravenous fluid requirement when compared with that in sham animals (P < .05). Treatment with pentoxifylline 30 minutes after intestinal ischemia attenuated pulmonary macromolecular leakage (P < .05 vs nontreated I-R) and reduced the decrease in cardiac output (-15% +/- 7% vs baseline, not statistically significant). Pentoxifylline treatment had no effect on the mean arterial blood pressure, heart rate, metabolic acidosis, or intravenous fluid requirement. CONCLUSIONS: Pentoxifylline reduces alveolar capillary membrane injury and subsequent protein leakage and improves cardiac output when administered after 30 minutes of intestinal ischemia. These data suggest that pentoxifylline may be a possible candidate as a future therapy for acute pulmonary dysfunction. Further studies in human patients are necessary.

Pulmonary subpleural arteriolar diameters during intestinal ischemia/reperfusion.

Adult respiratory distress syndrome (ARDS) often occurs in response to sepsis, shock, or ischemia/reperfusion (I/R) of a remote organ and is a frequent cause of mortality in the ICU patient. Pulmonary vascular resistance (PVR) increases during ARDS, yet direct observations of the pulmonary microcirculation are needed to characterize the vascular response. The purpose of this study was to quantitate the changes in hemodynamic variables, subpleural arteriolar diameters (AD), and alveolar cross-sectional areas (ACSA) during intestinal I/R-induced lung injury in rats, using a new method of in vivo videomicroscopy. Sprague-Dawley rats were anesthetized and cannulated, and superior mesenteric arteries were looped. A thoracotomy was performed with animals ventilated with air with 1 cm PEEP. Hemodynamic and videomicroscopic data were obtained before and during 45 min of SMA occlusion and after reperfusion, up to 120 min. Maximal vessel dilation was measured using topical 10(-5) M nitroprusside. The ability of vessels to constrict was confirmed by applying topical 10(-6) M endothelin-1. Intestinal I/R produced decreases in arterial pH, mean arterial pressure, and cardiac output. Despite these alterations, subpleural AD remained maximally dilated. Arterioles maintained the ability to constrict as demonstrated by the response to topical endothelin-1. ACSA did not change, indicating a uniform inflation of the lung. Using a unique method of in vivo pulmonary videomicroscopy, we have shown that AD do not change following 120 min of intestinal I/R, despite systemic hemodynamic instability. It appears that pulmonary arteriolar vasoconstriction does not contribute to increased PVR during the early phase of lung injury.

Proline metabolism in N2-fixing root nodules: energy transfer and regulation of purine synthesis.

N2-fixing root nodules of soybean (Glycine max L. Merr.) convert atmospheric N2 to ammonia(um) in an energy-intensive enzymatic reaction. These nodules synthesize large quantities of purines because nitrogen fixed by bacteria contained within this tissue is transferred to the shoots in the form of ureides, which are degradation products of purines. In animal systems, it has been proposed that proline biosynthesis by pyrroline-5-carboxylate reductase (P5CR) is used to generate the NADP+ required for the synthesis of the purine precursor ribose 5-phosphate. We have examined the levels, properties, and location of P5CR and proline dehydrogenase (ProDH) in soybean nodules. Nodule P5CR was found in the plant cytosol. Its activity was substantially higher than that reported for other animal and plant tissues and is 4-fold higher than in pea (Pisum sativum) nodules (which export amides). The Km for NADPH was lower by a factor of 25 than the Km for NADH, while the Vmax with NADPH was one-third of that with NADH. P5CR activity was diminished by NADP+ but not by proline. These characteristics are consistent with a role for P5CR in supporting nodule purine biosynthesis rather than in producing proline for incorporation into protein. ProDH activity was divided between the bacteroids and plant cytosol, but less than 2% was in the mitochondria-rich fractions. The specific activity of ProDH in soybean nodule bacteroids was comparable to that in rat liver mitochondria. In addition, we propose that some of the proline synthesized in the plant cytosol by P5CR is catabolized within the bacteroids by ProDH and that this represents a novel mechanism for transferring energy from the plant to its endosymbiont.