Platelet p110ß mediates platelet-leukocyte interaction and curtails bacterial dissemination in pneumococcal pneumonia.

Phosphatidylinositol 3-kinase catalytic subunit p110ß is involved in tumorigenesis and hemostasis. However, it remains unclear if p110ß also regulates platelet-mediated immune responses, which could have important consequences for immune modulation during anti-cancer treatment with p110ß inhibitors. Thus, we investigate how platelet p110ß affects inflammation and infection. Using a mouse model of Streptococcus pneumoniae-induced pneumonia, we find that both platelet-specific p110ß deficiency and pharmacologic inhibition of p110ß with TGX-221 exacerbate disease pathogenesis by preventing platelet-monocyte and neutrophil interactions, diminishing their infiltration and enhancing bacterial dissemination. Platelet p110ß mediates neutrophil phagocytosis of S. pneumoniae in vitro and curtails bacteremia in vivo. Genetic deficiency or inhibition of platelet p110ß also impairs macrophage recruitment in an independent model of sterile peritonitis. Our results demonstrate that platelet p110ß dysfunction exacerbates pulmonary infection by impeding leukocyte functions. Thereby, our findings provide important insights into the immunomodulatory potential of PI3K inhibitors in bacterial infection.

Radiotherapy for cervical cancer - impact on the vaginal epithelium and sexual function.

BACKGROUND: Women who have been treated for cervical cancer have persistent changes in their sexual function, which result in considerable distress. The aim of this study was to investigate the morphology of the vaginal epithelium in cervical cancer survivors treated with radiotherapy and its correlation to serum levels of sex steroid hormones and sexual function. MATERIAL AND METHODS: We included 34 patients treated for cervical cancer with radiotherapy and 37 healthy age-matched control women scheduled for benign gynecological surgery. After inspection and grading of vaginal atrophy, vaginal biopsies were taken. Epithelial structures were analyzed by measuring epithelial thickness as well as the number, height and width of the dermal papillae and the dermal papillae distance. Sex steroid hormone levels were analyzed and a questionnaire designed to assess sexual function was filled out. RESULTS: In the cervical cancer survivors treated with radiotherapy, the vaginal epithelium volume was reduced compared to control women. Longer distance between the dermal papillae (p < .001) and a shorter distance from basal layer to epithelial surface (p < .05) were measured. Mucosal atrophy was observed in 91% of the survivors. There was no difference in serum estradiol between cancer survivors and control women, implying that the cancer survivors were sufficiently substituted. The epithelial thickness correlated to serum levels of estradiol. The cervical cancer survivors reported more physical sexual symptoms. The highest relative risk (RR) was found for insufficient vaginal lubrication (RR 12.6), vaginal inelasticity (RR 6.5), reduced genital swelling when sexually aroused (RR 5.9), and for reduction of vaginal length during intercourse (RR 3.9). CONCLUSION: We found that cervical cancer treatment including radiotherapy is associated with vaginal epithelial atrophy and sexual dysfunction. To hamper the atrophic process affecting the sexual function, an early start of local estrogen after therapy might be of importance.

Radiotherapy-induced vaginal fibrosis in cervical cancer survivors.

BACKGROUND: Cervical cancer survivors treated with radiotherapy report vaginal inelasticity and decreased lubrication that may affect their sexual health, but it is unknown which normal tissue reactions mediate these symptoms. The aim of this study was to investigate the morphology of the connective tissue of the vaginal wall in cervical cancer survivors treated with radiotherapy. MATERIAL AND METHODS: We recruited 34 cervical cancer survivors treated with radiotherapy and 37 age-matched controls. Via clinical examination the degree of vaginal atrophy and pelvic fibrosis were estimated. We collected vaginal biopsies, which underwent morphometric analysis focused on elastin and collagen. Additionally, radiation dose at biopsy site were calculated and correlated to the clinical and morphological findings. RESULTS: The survivors had marked morphological vaginal changes, most prominent in the survivors that had received the highest radiation dose at the biopsy site. Mucosal atrophy was observed in 91% and pelvic fibrosis in 97%. A shorter vagina was measured; 7.0 cm versus 10.3 cm in controls (p < 0.001). The area fraction of elastin was greater in survivors; 10.0% (range 5.8-12.9), compared with controls; 3.4% (range 1.8-5.8), p < 0.001. The survivors had signs of elastosis with thick aggregated elastin fibers irregularly scattered throughout the connective tissue, while the controls had elastin fibers in a thin sub-epithelial layer. The area fraction of high density collagen in the connective tissue was larger among the survivors (p < 0.001). The collagen with the highest density (fibrosis) was more common in the group of cancer survivors that had received external radiation. CONCLUSIONS: We found drastic differences in the vaginal wall between the irradiated cervical cancer survivors and the controls, indicating that radiotherapy-induced vaginal symptoms are mediated by connective tissue fibrosis and elastosis. Our results also support that patients treated with external radiation have the highest risk of developing vaginal fibrosis with impairment of their sexual health.

NETosis promotes cancer-associated arterial microthrombosis presenting as ischemic stroke with troponin elevation.

INTRODUCTION: Large elevations of high sensitive Troponin T (hsTnT) in ischemic stroke patients is associated with a poor outcome. In a pilot study we found a high prevalence of malignancies among these patients. Since neutrophil extracellular traps (NETs) have been linked to cancer-associated thrombosis, we hypothesized that the concomitant cerebral and myocardial ischemia could be the result of a NET-induced hypercoagulable state. MATERIALS AND METHODS: Clinical assessments, plasma analyses and autopsies with histopathology (in cases of in-hospital mortality) were performed on ischemic stroke patients with high elevations of hsTnT (N=12) and normal hsTnT (N=19). RESULTS: Patients with hsTnT elevation had an unexpectedly higher prevalence of cancer (p=0.002), half of which were diagnosed post-mortem. Autopsies of these patients revealed widespread myocardial, cerebral and pulmonary microthrombosis with H3Cit in thrombi. A pro-coagulant state and an increase of the NET specific marker citrullinated histone H3 (H3Cit) was found in plasma of patients with elevated hsTnT compared to patients with normal levels (p<0.001). Plasma analyses in cancer patients showed even higher H3Cit levels (p<0.001), and an increase in granulocyte colony-stimulating factor, known to prime neutrophils towards NETosis. H3Cit correlated positively with thrombin-antithrombin complex (p=0.004) and soluble P-selectin (p<0.001), further linking NETosis to the pro-thrombotic state. CONCLUSIONS: The high prevalence of known or occult cancer in our study suggests that cancer-associated arterial microthrombosis may be underestimated. By linking the thrombosis to NETs, we suggest markers of NETosis that could aid in revealing cancer in arterial microthrombosis as well as arterial microthrombosis in cancer.

Trousseau's Syndrome, a Previously Unrecognized Condition in Acute Ischemic Stroke Associated With Myocardial Injury.

Trousseau's syndrome is a well-known malignancy associated hypercoagulative state leading to venous or arterial thrombosis. The pathophysiology is however poorly understood, although multiple mechanisms are believed to be involved. We report a case of Trousseau's syndrome resulting in concomitant cerebral and myocardial microthrombosis, presenting with acute ischemic stroke and markedly elevated plasma troponin T levels suggesting myocardial injury. Without any previous medical history, the patient developed multiple cerebral infarctions and died within 11 days of admission. The patient was postmortem diagnosed with an advanced metastatic adenocarcinoma of the prostate with disseminated cerebral, pulmonary, and myocardial microthrombosis. Further analyses revealed, to the best of our knowledge for the first time in stroke patients, circulating microvesicles positive for the epithelial tumor marker CK18 and citrullinated histone H3 in thrombi, markers of the recently described cancer-associated procoagulant DNA-based neutrophil extracellular traps. We also found tissue factor, the main in vivo initiator of coagulation, both in thrombi and in metastases. Troponin elevation in acute ischemic stroke is common and has repeatedly been associated with an increased risk of mortality. The underlying pathophysiology is however not fully clarified, although a number of possible explanations have been proposed. We now suggest that unexplainable high levels of troponin in acute ischemic stroke deserve special attention in terms of possible occult malignancy.

Tesaglitazar, a dual PPAR-α/γ agonist, hamster carcinogenicity, investigative animal and clinical studies.

Tesaglitazar was developed as a dual peroxisome proliferator-activated receptor (PPARα/γ). To support the clinical program, a hamster carcinogenicity study was performed. The only neoplastic findings possibly related to treatment with tesaglitazar were low incidences of hemangioma and hemangiosarcoma in the liver of male animals. A high-power, two-year investigative study with interim necropsies was performed to further elucidate these findings. Treatment with tesaglitazar resulted in changes typical for exaggerated PPARα pharmacology in rodents, such as hepatocellular hypertrophy and hepatocellular carcinoma, but not an increased frequency of hemangiosarcomas. At the highest dose level, there was an increased incidence of sinusoidal dilatation and hemangiomas. No increased endothelial cell (EC) proliferation was detected in vivo, which was confirmed by in vitro administration to ECs. Immunohistochemistry and gene expression analyses indicated increased cellular stress and vascular endothelial growth factor (VEGF) expression in the liver, which may have contributed to the sinusoidal dilatation. A two-fold increase in the level of circulating VEGF was detected in the hamster at all dose levels, whereas no effect on VEGF was observed in patients treated with tesaglitazar. In conclusion, investigations have demonstrated that tesaglitazar does not produce hemangiosarcomas in hamster despite a slight effect on vascular morphology in the liver.

Proliferative and molecular effects of the dual PPARalpha/gamma agonist tesaglitazar in rat adipose tissues: relevance for induction of fibrosarcoma.

The dual peroxisome-proliferator-activated receptor (PPAR) α/γ agonist tesaglitazar has been shown to produce fibrosarcomas in rats. Here, the authors studied morphology, proliferation, differentiation, and inflammation markers in adipose tissue from rats exposed to 1, 3, or 10 µmol/kg tesaglitazar for 2 or 12 weeks, including recovery groups (12 weeks treatment followed by 12 weeks recovery), and 3 or 10 µmol/kg tesaglitazar for 24 weeks. Subcutaneous white and brown fat revealed reversible dose-related histopathological alterations and after 12 and 24 weeks developed areas of thickened skin (fatty lumps). There was a dose-dependent increase in proliferation of interstitial cells in white and brown fat as shown by increased mitotic index in all dose groups after 2 weeks. This was limited to the high dose after 12 and 24 weeks in white fat. Gene expression analyses showed that while tesaglitazar induced differentiation of adipose tissue characterized with a switch in cyclin D1 and D3 mRNA by 12 weeks, longer exposure at high doses reversed this differentiation concurrent with a reappearance of early adipocyte and inflammatory markers. These data suggest that sustained increased turnover of mesenchymal cells in adipose tissues, concomitant with onset of inflammation and fibrosis, drives development of fibrosarcomas in rats treated with tesaglitazar.

Delivering horseradish peroxidase as a respirable powder to the isolated, perfused, and ventilated lung of the rat: the pulmonary disposition of an inhaled model biopharmaceutical.

BACKGROUND: Our aim was to investigate the potential of the DustGun aerosol technology integrated with the isolated, perfused, and ventilated lung of the rat (IPL) to study the pulmonary disposition of an inhaled model biopharmaceutical, the 40-kDa protein horseradish peroxidase (HRP). METHOD: The DustGun aerosol technology was used to deliver respirable powder aerosols of HRP (the mass median aerodynamic diameter: 1.7 µm) as an 80-sec bolus to the IPL perfused in a single-pass mode. Lung perfusate was repeatedly sampled for 125 min after the HRP exposure. The amount of active HRP clearing with the perfusate or being retained in the lung was measured enzymatically. RESULTS AND CONCLUSIONS: The total amount of HRP deposited in the lungs was 335 ± 100 µg and 568 ± 47 µg for a low- and high-dose exposure, respectively. After inhalation, the initial appearance of HRP in the perfusate was rapid. However, the total amount of HRP that cleared with the perfusate remained below 0.5% of the deposited dose. The effect of opening the tight junctions between the alveolar epithelial cells on HRP absorption was studied by exposing the IPL to nebulized aerosols of either 0.02, 0.2, or 2% poly-L-Arginine (PLA) (MW 42.5 kDa) in phosphate-buffered saline (PBS) for 5 min, at 40 min after the HRP exposure. Subsequent exposure to 0.02% PLA did not affect HRP absorption. However, exposure to 0.2% PLA increased the absorption rate ninefold, and the total amount of HRP clearing with the perfusate increased to approximately 4% of the deposited dose. No further increase was obtained with 2% PLA, indicating a steep dose-response for the enhancer. It was concluded that the pulmonary absorption of HRP is quite slow, and absorption enhancers affecting tight junctions have a distinctive, yet limited efficiency. The presented inhalation technology can be very useful in studying the pulmonary absorption of biopharmaceuticals.

Expression of angiopoietins 1, 2 and their common receptor tie-2 in relation to the size of endothelial lining gaps and expression of VEGF and VEGF receptors in idiopathic menorrhagia.

OBJECTIVE: To investigate whether idiopathic menorrhagia (IM) is associated with alterations of the vascular expression of angiopoietin-1, angiopoietin-2, and tie-2 receptor. DESIGN: Prospective clinical study. SETTING: University Hospital, Department of Gynecology. PATIENT(S): Twenty-four patients with IM and 18 women with eumenorrhea. INTERVENTION(S): Endometrial samples underwent immunohistochemical staining for CD34, angiopoetin-1, angiopoietin-2, tie-2, and smooth muscle actin-alpha. Previously published data on gap size and expression of vascular endothelial growth factor family members were used. MAIN OUTCOME MEASURE(S): Differences in immunostaining for these markers by computer-assisted stereological analysis. RESULT(S): There was significantly more angiopoetin-1 positive vessels in IM in the secretory phase, but not of angiopoetin-2 and tie-2, compared with controls. Densities of angiopoetin-1 positive vessels correlated significantly to those of angiopoetin-2 and vascular endothelial growth factor receptor 3. Smooth muscle actin-alpha positive pericytes covered the gaps. Double staining for CD34 and tie-2 receptor was partly identical, but gaps were covered by tie-2 stain. CONCLUSION(S): The discrete deregulation observed of the angiopoetin-1 expression before menstruation might affect vascular integrity, thereby contributing to the excessive blood loss in IM.

Histological inflammatory response to transvaginal polypropylene mesh for pelvic reconstructive surgery.

PURPOSE: We prospectively evaluated the histological inflammatory response to the large polypropylene transvaginal mesh used for pelvic organ prolapse surgery. MATERIALS AND METHODS: Ten patients and 8 controls underwent vaginal punch biopsy sampling before surgery and patients also underwent it 1 year after pelvic reconstructive surgery using polypropylene mesh. Foreign body response to the mesh was assessed using a combination of histological, semiquantitative and computerized image based analysis. RESULTS: Compared to preoperative histology there was a significant postoperative increase in macrophage and mast cell counts (p = 0.03 and 0.01) but no significant changes in the count of cells involved primarily in the infectious cell response or collagen density and the elastin area fraction at the mesh-tissue interface (p = 0.2 and 0.3, respectively). Three cases of mild granuloma formation and 2 of mild erosion were observed. There was no significant change in epithelial thickness when comparing preoperative and postoperative samples. CONCLUSIONS: When used for pelvic reconstructive surgery, macroporous monofilament polypropylene mesh induces a mild but persistent foreign body reaction.

Steroid receptor expression and morphology in provoked vestibulodynia.

OBJECTIVE: This study was undertaken to survey the steroid receptor expression and morphology in the vulvar vestibular mucosa in women with provoked vestibulodynia. STUDY DESIGN: Fourteen patients and 25 controls without oral contraceptives were included. Vestibular biopsy specimens were obtained and analyzed by using immunohistochemistry, followed by computerized image analysis of estrogen receptors alpha and beta, progesterone receptors A and B, glucocorticoid receptor, androgen receptor, and the proliferation marker Ki67. The morphology was estimated by measuring 4 parameters in the epithelium. RESULTS: There was a significantly higher expression of estrogen receptor alpha in both the epithelium (P = .04) and the stroma (P = .02) in the patient specimens compared with the controls. There were no significant differences in the other analyses performed. CONCLUSION: There is an increased expression of estrogen receptor alpha in the vestibular mucosa but the epithelial morphology seems unaffected in women with provoked vestibulodynia. Further studies regarding plausible associations to neurogenic inflammation are needed.

A novel method for quantification of the folding of elastic laminae in elastic arteries.

A transgenic mouse overexpressing the human form of semicarbazide-sensitive amine oxidase (SSAO) is known to have an abnormal structure of the elastic laminae and the elastic fibres in the aorta. Compared to the non-transgenic littermates, the elastic laminae are less folded. In order to quantify the undulation of this structure, an image analysis program that identified the elastic laminae was developed. The program measures the area fraction in different sectors from a plane parallel to the aorta wall. Images were taken from unstained aorta specimens where the elastic laminae were visualised with phase contrast microscopy. A contextual operation of the images produced a local orientation estimation for every linear structure. The image was then thresholded in eight sectors from 0 degrees to 180 degrees , with different orientation angles. The results show that the area fraction of the elastic laminae was significantly lower for the transgenic mouse in all sectors measured except for two. At 0-25 degrees , no difference was seen. In the sector at 160-180 degrees , parallel to the aorta wall, the area fraction of elastic laminae was instead significantly higher in the transgenic mouse. A novel method is presented, developed for detection and quantification of pathological changes in the elastic laminae in the aorta wall. The method gave reliable results and is considered to be a useful tool for morphometric studies of aorta with this kind of altered morphology concerning the elastic laminae. When compared with tangent count, the control group had a significantly larger mean curvature.

Macrovascular changes in mice overexpressing human semicarbazide-sensitive amine oxidase in smooth muscle cells.

BACKGROUND: The catalytic activity of semicarbazide-sensitive amine oxidase (SSAO) is increased in diabetes, as well as in other disorders of cardiovascular origin. Our hypothesis is that SSAO is involved in the synthesis or maturation of elastin in vascular tissue. An increased SSAO activity can thereby be involved in the development of vascular damage. METHODS: Elastin quantification was performed in aorta of transgenic mice overexpressing the human form of SSAO, using electron microscopy. Furthermore, lung capacity was measured using a spirometry-mimicking method, developed for mice. The effect of vasoactive substances was estimated by measuring mean arterial pressure and pulse pressure under anesthesia. RESULTS: No differences in elastin quantity or lung capacity could be observed between transgenic or nontransgenic littermates. Pulse pressure was higher in transgenic mice, and electron microscopy of aorta showed elastin fibers parallel with the aorta wall (ie, straight fibers instead of folded compared with control mice). No difference in the response to adrenaline or sodium chloride was observed between the transgenic and control mice. The control mice had a clear decrease in blood pressure (BP) with a longer duration as a response to injection of a nitric oxide (NO) donor, sodium nitroprusside, compared with transgenic mice where only a minor response was observed. The SSAO activity in serum of control mice was elevated in response to injection of the NO donor, but not in response to a ganglion blocker. CONCLUSIONS: An elevated pulse pressure, together with an abnormal elastin structure in the aorta, suggests a rigidity of large arteries as a result of an elevated SSAO activity as well as a physiologic role for SSAO in elastin maturation.

Expression of vascular endothelial growth factor receptor-3 in the endometrium in menorrhagia.

Angiogenesis is essential for endometrial growth and repair, and disruption of this process may lead to common gynecological disorders, including menorrhagia and endometriosis. We have recently shown that expression of vascular endothelial growth factor (VEGF)-A and its two main receptors, VEGFR-1 and -2, is increased in idiopathic menorrhagia (IM). The aim of this study was to determine the expression of VEGFR-3 in normal and IM endometrium. Endometrial biopsies from 24 patients with IM and 18 healthy and fertile women were used for immunohistochemistry assessments and image analyses of VEGFR-3 and CD34-stained endothelial structures. We found that weak to moderate expression of VEGFR-3 was present in stroma and glands throughout the menstrual cycle without differences between patients and controls. Capillaries expressed VEGFR-3 markedly, whereas arterioles and venules stained moderately to markedly. However, we observed that vascular expression of VEGFR-3 in capillaries was 1.6-fold higher in the IM group than in controls, when assessed as the number of stained capillaries per mm(2). There was also a 2.0-fold higher number of arterioles, which were VEGFR-3 positive in the IM group. There was no difference with regard to the menstrual cycle between patients and controls. Thus, human endometrium expresses VEGFR-3, and expression of this receptor is increased in idiopathic menorrhagia. These results indicate that VEGFR-3 may play a role in the abnormal endometrial angiogenesis of IM.

Tesaglitazar, a PPARalpha/gamma agonist, induces interstitial mesenchymal cell DNA synthesis and fibrosarcomas in subcutaneous tissues in rats.

The development of the dual peroxisome proliferator-activated receptor (PPAR) alpha/gamma agonist tesaglitazar as an oral antidiabetic was recently discontinued. Here we present tumor data from a 2-year carcinogenicity study in rats given 0.3, 1, 3, and 10 micromol/kg tesaglitazar is presented with focus on the findings of subcutaneous fibrosarcomas. To investigate the mechanism for induction of fibrosarcomas, replicative DNA synthesis (immunohistochemical detection of BrdU-labeled cells) and expression of PPARgamma (immunohistochemistry and reverse transcription-polymerase chain reaction) in subcutaneous adipose tissues was assessed in rats administered 1 or 10 micromol/kg for 2 weeks or 3 months. Poorly differentiated subcutaneous mesenchymal sarcomas with a predominant spindle cell appearance occurred at the highest dose level of 10 micromol/kg in both sexes, and these tumors were diagnosed as fibrosarcomas. The 10-micromol/kg dose was at or above the maximum tolerated dose and caused considerable cardiovascular mortality. Tesaglitazar stimulated DNA synthesis mainly in subcutaneous interstitial mesenchymal cells. The percentage of BrdU-labeled interstitial cells was increased at 1 and 10 micromol/kg after 2 weeks. The increase in DNA synthesis was still significant at the end of the 12-week treatment at 10 mumol/kg, the dose producing fibrosarcoma. However, at 1 micromol/kg, a dose below the no-observed-effect level for fibrosarcoma, the level of DNA synthesis was similar to control levels at 12 weeks. Immunohistochemical analyses showed no detectable PPARgamma protein in the majority of BrdU-labeled interstitial mesenchymal cells in white and brown fat. This indicates that stimulation of DNA synthesis is not mediated via direct activation of PPARgamma in these cells. The results suggest that the induction of rat fibrosarcoma by tesaglitazar, at exposures 100-fold above the human therapeutic exposure, may involve proliferation of undifferentiated mesenchymal cells in subcutaneous tissues.

Wall discontinuities and increased expression of vascular endothelial growth factor-A and vascular endothelial growth factor receptors 1 and 2 in endometrial blood vessels of women with menorrhagia.

OBJECTIVE: To investigate whether the structure or regulation of the growth of endometrial blood vessels might be abnormal in women with idiopathic menorrhagia (IM). Perturbation of angiogenesis is associated with IM. DESIGN: Prospective, clinical study. SETTING: Department of gynecology at a university hospital. PATIENT(S): Twenty-four patients with IM, and 18 women with eumenorrhea. INTERVENTION(S): Endometrial biopsy samples underwent immunohistochemical staining for CD34, CD31, von Willebrand factor, vascular endothelial growth factor (VEGF)-A, and VEGF receptors 1 and 2. MAIN OUTCOME MEASURE(S): Differences in immunostaining for these markers by computer-assisted stereological analysis. RESULT(S): Endometrial vessels in patients and controls manifested focal discontinuities, or gaps, in endothelial staining for CD34, CD31, and von Willebrand factor. Electron and confocal microscopy revealed that perivascular cells, probably pericytes, covered these gaps in the vessel wall. The relative size of the gaps was significantly greater in patients with IM than in controls. Vessel circumference was also larger, and more vessels were positive for VEGF-A and for VEGF receptors 1 and 2, in patients than in controls. Gap size was significantly correlated with the number of vessels expressing VEGF-A or VEGF receptor 1. CONCLUSION(S): Endometrial blood vessels possess a discrete morphology that is characterized by endothelial gaps, and these gaps [1] are more pronounced in women with IM, [2] are related to overexpression of VEGF-A and VEGF receptor 1, and [3] might contribute to IM, e.g., by destablizing vessels.

Increasing exposure levels cause an abrupt change in the absorption and metabolism of acutely inhaled benzo(a)pyrene in the isolated, ventilated, and perfused lung of the rat.

The carcinogenic polycyclic aromatic hydrocarbons (PAHs) are active primarily at the site of entry to the body. Lung cancer following inhalation of PAH-containing aerosols such as tobacco smoke is one likely example. A suggested mechanism for this site preference is a slow passage of the highly lipophilic PAHs through the thicker epithelia of the conducting airways, accompanied by substantial local metabolism in airway epithelium. However, it is likely that the airway epithelium will become saturated with PAHs at surprisingly low exposure levels. The purpose of this research was to quantify the level of saturation for inhaled benzo(a)pyrene (BaP) in the isolated, perfused lung (IPL) of the rat. BaP was coated onto carrier particles of silica 3.5 microm diameter at three different levels. The DustGun aerosol generator was then used to deliver respectively 2.2, 36, and 8400 ng of BaP to the IPL with the carrier particles in less than 1 min. For 77 min after the exposure, single-pass perfusate was collected from the lungs. Lungs were then removed and, with the perfusate, analyzed for BaP and metabolites. Results show that the absorption and metabolism of inhaled BaP in the lungs was highly dose dependent. At low exposure levels absorption of BaP in the mucosa was proportional to the concentration in the air/blood barrier and proceeded with substantial local metabolism. At higher exposure levels the capacity of the epithelium to dissolve and metabolize BaP became saturated, and the absorption rate remained constant until crystalline BaP had dissolved, and the process proceeded with much smaller fractions of BaP metabolites produced in the mucosa. This phenomenon may explain the well-known difficulties of inducing lung cancer in laboratory animals with inhalants containing carcinogenic PAHs, where similar lifespan exposures are used as humans may experience but with much higher dose rates.

Microvascular density, vascular endothelial growth factor A, and its receptors in endometrial blood vessels in patients with menorrhagia.

OBJECTIVE: To analyze the expression of vascular endothelial growth factor A (VEGF-A) and receptors (VEGFR-1 and VEGFR-2) in endometrial blood vessels, as well as microvascular density (MVD), in endometrial biopsy samples from idiopathic menorrhagia patients. DESIGN: Prospective clinical study. SETTING: University hospital, unit of gynecology. PATIENT(S): Twenty-four patients with idiopathic menorrhagia and 18 healthy fertile women. INTERVENTION(S): Blood sampling for hormone measurement, hysteroscopy, and endometrial biopsy sampling. Endometrial biopsy samples were used for immunohistochemistry assessments and image analysis of stained endothelial structures for VEGF-A, VEGFR-1, VEGFR-2, and CD34. MAIN OUTCOME MEASURE(S): Appearance of the endometrial vascular immunoreactivity for VEGF-A, VEGFR-1, and VEGFR-2, MVD and computer-assisted stereological analysis of immunoassayed blood vessels. RESULT(S): Although the MVD did not differ between patients and controls, we observed that vascular expression of VEGF-A, VEGFR-1, and VEGFR-2 in capillaries was 1.8-fold, 1.8-fold, and 2.0-fold higher, respectively, in the menorrhagia group when assessed as the number of stained capillaries per unit area. There were also a twofold higher number of arterioles, which were VEGFR-2 positive in the menorrhagia group. CONCLUSION(S): Up-regulation of VEGF-A and receptors VEGFR-1 and VEGFR-2 in capillaries in menorrhagia could be involved in abnormal endometrial vascular structure and permeability.

Phenytoin teratogenicity: hypoxia marker and effects on embryonic heart rhythm suggest an hERG-related mechanism.

BACKGROUND: The antiepileptic drug phenytoin (PHT) is a human and animal teratogen. The teratogenicity has been linked to PHT-induced embryonic cardiac arrhythmia and hypoxic damage during a period when regulation of embryonic heart rhythm is highly dependent on a specific K(+) ion current (I(Kr)). PHT has been shown to inhibit I(Kr). The aims of this study were to investigate whether teratogenic doses cause embryonic hypoxia during and after the I(Kr) susceptible period and to further characterize PHT effects on embryonic heart rhythm. METHODS: Pregnant C57BL mice were administered the hypoxia marker pimonidazole followed by PHT or saline (controls) on GD 10 or GD 15. The embryos were fixed and sectioned, and the immunostained sections were analyzed with a computer assisted image analysis. Effects of PHT (0-250 microM) on heart rhythm in GD 10 embryos cultured in vitro were videotaped and then analyzed by using a digitalization technique. RESULTS: PHT dose-dependently increased the hypoxia staining (6- and 11-fold after maternal dosing of 100 and 150 mg/kg, respectively) during the period I(Kr) is expressed and functional (GD 10). In contrast, there were no differences between the PHT doses in hypoxia staining, and much less pronounced hypoxia after this period (GD 15). With increasing PHT concentrations, increased length of the interval (bradycardia) and large variations in length between individual heartbeats (arrhythmia) were recorded. CONCLUSIONS: PHT induced bradycardia/arrhythmia and severe embryonic hypoxia during the I(Kr) susceptible period, supporting the idea of an I(Kr)-arrhythmia-hypoxia-related teratogenic mechanism.

Clinical and histological safety assessment of rectocele repair using collagen mesh.

AIM: To clinically and histologically evaluate inflammatory response following rectocele repair using porcine collagen mesh. METHODS: Seventeen patients underwent rectocele repair using porcine collagen mesh. Inflammatory response was assessed by clinical and histological inflammatory grading pre- and postoperatively. Postoperative body temperature, complications and hospital stay was compared with 15 patients undergoing posterior colporraphy. RESULTS: Postoperative clinical examination did not demonstrate any inflammatory reaction. There were no significant changes in fibroblast count (P = 0.43), connective tissue density grading (P = 0.54), macrophage count (P = 0.20), inflammatory cell count (P = 0.48), total cell count (P = 0.51), or inflammatory grading (P = 0.87) postoperatively compared with preoperative values. Body temperature was significantly elevated for both the study and control group, although higher for the study group, postoperatively day 1 (P < 0.001). There were no significant differences in hospital stay and postoperative complications. CONCLUSION: Porcine collagen mesh was not associated with an adverse inflammatory response at clinical or histological evaluation and appears to be a safe material when used for rectocele repair.