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Purpose: An incisional hernia occurs frequently after a midline incision with an incidence of 12.8%. The choice in suture material used for abdominal wall closure is not straightforward and the conflicting literature focuses on clinical outcomes. This study compares a non-absorbable, slow-absorbable and fast-absorbable suture in a rat model, focusing on histological outcomes predicting better fascia healing., Methods: 33 male Wistar rats, divided over three groups, each received two separate 1 cm incisions closed with either Prolene 4/0, PDS 4/0 or Vicryl 4/0. At 7 days and 21 days, one of the incisions was explanted. Tissue was semi-quantitatively scored regarding inflammatory cells and collagen fibres present. Using qPCR macrophage polarisation, fibroblast activity and vascularisation were evaluated. Data were analysed by Kruskal-Wallis test with Mann-Whitney U post hoc test. A p value of 0.017 was considered significant after Bonferroni correction., Results: All animals recovered without complications and completed the 21 days of follow-up. The Vicryl group showed a higher presence of macrophages after 21 days in comparison with Prolene (p = 0.003) and PDS (p = 0.006) and more foreign body giant cells compared to Prolene at 7 days (p = 0.010) and PDS at 21 days (p < 0.001). qPCR showed 2.5-fold higher expression of clec10A in PDS compared to Prolene after 7 days (p = 0.007)., Conclusions: The results of this study carefully support the use of PDS suture, compared to Prolene and Vicryl, in abdominal wall closure based on a favourable macrophage response. The heterogeneity and variability in the data might be explained by the spectrum of the macrophage subtype paradigm.

Background: Promoter methylation of N-myc Downstream-Regulated Gene 4 (NDRG4) in fecal DNA is an established early detection marker for colorectal cancer (CRC). Despite its connection to CRC, NDRG4 is predominantly studied in brain and heart, with little to no knowledge about its expression or role in other organs. In this study, we aimed to determine the whole-body expression of NDRG4, with a focus on the intestinal tract., Methods: We investigated NDRG4 expression throughout the body by immunohistochemistry, Western Blotting and in situ mRNA hybridization using tissues from NDRG4 wild-type, heterozygous and knockout mice and humans. In addition, we explored cell-specific expression of NDRG4 in murine whole-mount gut preparations using immunofluorescence and confocal microscopy., Key Results: NDRG4 is specifically expressed within nervous system structures throughout the body. In the intestinal tract of both mouse and man, NDRG4 immunoreactivity was restricted to the enteric nervous system (ENS), where it labeled cell bodies of the myenteric and submucosal plexuses and interconnecting nerve fibers. More precisely, NDRG4 expression was limited to neurons, as NDRG4 always co-localized with HuC/D (pan-neuronal marker) but never with GFAP (an enteric glial cell marker). Furthermore, NDRG4 was expressed in various neuropeptide Y positive neurons, but was only found in a minority (~10%) of neurons expressing neuronal nitric oxide synthase., Conclusions and Inferences: NDRG4 is exclusively expressed by central, peripheral and enteric neurons/nerves, suggesting a neuronal-specific role of this protein. Our findings raise the question whether NDRG4, via the ENS, an understudied component of the tumor microenvironment, supports CRC development and/or progression., (© 2017 John Wiley & Sons Ltd.)

Background: Anastomotic leakage (AL) is the most dreaded complication after colorectal surgery, causing high morbidity and mortality. Mucus is a first line of defence against external factors in the gastrointestinal tract. In this study, the structural mucus protein Muc2 was depleted in genetically engineered mice and the effect on healing of colonic anastomoses studied in an experimental model., Methods: Mice of different Muc2 genotypes were used in a proximal colonic AL model. Tissues were scored histologically for inflammation, bacterial translocation was determined by quantitative PCR of bacterial 16S ribosomal DNA, and epithelial cell damage was determined by assessing serum levels of intestinal fatty acid-binding protein., Results: Of 22 Muc2-deficient (Muc2 -/- ) mice, 20 developed AL, compared with seven of 22 control animals (P < 0·001). Control mice showed normal healing, whereas Muc2 -/- mice had more inflammation with less collagen deposition and neoangiogenesis. A tendency towards higher bacterial translocation was seen in mesenteric lymph nodes and spleen in Muc2 -/- mice. Intestinal fatty acid-binding protein levels were significantly higher in Muc2 -/- mice compared with controls (P = 0·011)., Conclusion: A functional mucous layer facilitates the healing of colonic anastomoses. Clinical relevance Colorectal anastomotic leakage remains the most dreaded complication after colorectal surgery. It is known that the aetiology of anastomotic leakage is multifactorial, and a role is suggested for the interaction between intraluminal content and mucosa. In this murine model of proximal colonic anastomotic leakage, the authors investigated the mucous layer at the intestinal mucosa, as the first line of defence, and found that a normal, functioning mucous layer is essential in the healing process of colonic anastomoses. Further research on anastomotic healing should focus on positively influencing the mucous layer to promote better postoperative recovery., (© 2017 BJS Society Ltd Published by John Wiley & Sons Ltd.)

Introduction: Despite extensive research, anastomotic leakage (AL) remains one of the most dreaded complications after colorectal surgery. Since butyrate enemas are known to enhance anastomotic healing, several administration routes have been explored in this study., Methods: Three intraluminal approaches involving butyrate were investigated: (1) butyrin-elucidating patch, (2) a single injection of hyaluronan-butyrate (HA-But) prior to construction of the proximal anastomosis and (3) rectal hyaluronan-butyrate (HA-But) enemas designed for distal anastomoses. The main outcome was AL and secondary outcomes were bursting pressure, histological analysis of the anastomosis, zymography to detect MMP activity and qPCR for gene expression of MMP2, MMP9, MUC2 and TFF3., Results: Neither the patches nor the injections led to a reduction of AL in experiments 1 and 2. In experiment 3, a significant reduction of AL was accomplished with the (HA-But) enema compared to the control group together with a higher bursting pressure. Histological analysis detected only an increased inflammation in experiment 2 in the hyaluronan injection group compared to the control group. No other differences were found regarding wound healing. Zymography identified a decreased proenzyme of MMP9 when HA-But was administered as a rectal enema. qPCR did not show any significant differences between groups in any experiment., Conclusion: Butyrate enemas are effective in the enhancement of colonic anastomosis. Enhanced butyrate-based approaches designed to reduce AL in animal models for both proximal and distal anastomoses were not more effective than were butyrate enemas alone. Further research should focus on how exogenous butyrate can improve anastomotic healing after gastrointestinal surgery.

Introduction: Mesh-related adhesions are a significant clinical problem following intraperitoneal mesh placement. In this study, we evaluated adhesion formation to three relatively new meshes for intraperitoneal use., Methods: Three new meshes for intraperitoneal use (Omyra(®) mesh, Physiomesh(®), and Hi-Tex Endo-IP(®)) were implanted intraperitoneally in rats and compared with a polypropylene control mesh (Parietene(®)) after 7 or 90 days. Adhesion formation, incorporation (tensile strength), shrinkage, and foreign body reaction were scored., Results: Hi-Tex Endo-IP and Physiomesh(®) showed significantly less adhesion formation when compared to Parietene at both time points (p < 0.05). Shrinkage was highest in Omyra mesh after 90 days, which was significantly more compared to Parietene(®) (p < 0.001). Physiomesh(®) only showed a significant reduction in craniocaudal mesh length, compared to Parietene and Hi-Tex Endo-IP (p < 0.05). After 90 days, Hi-Tex Endo-IP(®) showed significantly higher and Physiomesh(®) significantly lower incorporation strengths compared to all other groups (p < 0.05). Microscopic evaluation revealed massive foreign body reaction to Hi-Tex Endo-IP(®), leading to an extensive and thick collagenous scar adherent to the abdominal wall. Fractioning of the Physiomesh(®) coating over time led to an increase in interfilamentary granuloma formation, leading to scar plate formation, but with only minimal to no abdominal wall adherence. Both Parietene(®) and Omyra(®) showed a mild foreign body response., Conclusion: Although clear distinctions can be made between meshes and some meshes excel, none of the meshes are superior in all aspects required for effective and safe incisional hernia repair.

Background: Immune processes contribute to the development of obesity and its complications, such as insulin resistance, type 2 diabetes mellitus and cardiovascular disease. Approaches that target the inflammatory response are promising therapeutic strategies for obesity. In this context, we recently demonstrated that the interaction between the costimulatory protein CD40 and its downstream adaptor protein tumor necrosis factor receptor-associated factor 6 (TRAF6) promotes adipose tissue inflammation, insulin resistance and hepatic steatosis in mice in the course of diet-induced obesity (DIO)., Methods: Here we evaluated the effects of a small-molecule inhibitor (SMI) of the CD40-TRAF6 interaction, SMI 6860766, on the development of obesity and its complications in mice that were subjected to DIO., Results: Treatment with SMI 6860766 did not result in differences in weight gain, but improved glucose tolerance. Moreover, SMI 6860766 treatment reduced the amount of CD45(+) leucocytes in the epididymal adipose tissue by 69%. Especially, the number of adipose tissue CD4(+) and CD8(+) T cells, as well as macrophages, was significantly decreased., Conclusions: Our results indicate that small-molecule-mediated inhibition of the CD40-TRAF6 interaction is a promising therapeutic strategy for the treatment of metabolic complications of obesity by improving glucose tolerance, by reducing the accumulation of immune cells to the adipose tissue and by skewing of the immune response towards a more anti-inflammatory profile.

Purpose: Intraperitoneal mesh fixation for hernia repair is associated with adhesion formation. In this experimental study, adhesions against absorbable and non-absorbable fixation methods were compared., Methods: Six commercially available fixation methods were placed intraperitoneally in rats with a small pore polypropylene mesh coated on one side with ePTFE (Intramesh T1(®)). Two non-absorbable fixation methods: Prolene(®) (polypropylene) sutures and Protack(®) (titanium) tackers. Four absorbable methods: Vicryl(®) sutures (polyglactin), Absorbatack(®) and Permasorb(®) tackers (both mixes of lactic and glycolic acids) and Tisseel Duo(®) (fibrin glue). Adhesions and histology were studied at 7 and 90 days follow-up. In addition, fixation methods were placed without mesh, in order to study the reaction to the fixation method per se., Results: No adhesion formation, but also inadequate mesh fixation was found with Tisseel Duo(®), which had been completely resorbed at 7 days follow-up. Vicryl(®) sutures could no longer be detected at 90 days follow-up and were associated with a favorable adhesion profile. All other fixation methods were still intact 90 days after implantation. When placed without mesh, adhesion formation was significantly less than placed with a mesh (18 vs. 93 %, P < 0.001). Without mesh, adhesions were worst with Permasorb(®) tackers., Conclusions: Absorbable fixation methods such as polyglactin sutures and fibrin glue show a favorable adhesion profile compared to longer-term absorbable or non-absorbable fixation methods. However, before using fibrin glue as a single fixation method more research is required.

Antenatal exposure of the fetus to inflammation may alter postnatal organ development. In our previous work, we demonstrated that the fetal liver is involved in the systemic inflammation associated with chorioamnionitis, leading to metabolic changes. On the basis of these findings, we hypothesized that chorioamnionitis can lead to postnatal inflammation-related liver injury and disturbed lipid metabolism. Chorioamnionitis was induced in sheep by intra-amniotic injection of lipopolysaccharide (LPS) or saline at 90, 100 and 110 days of gestation. Liver homeostasis and lipid metabolism were analyzed at term and at 7 weeks of age. At term, hepatic T-lymphocytes and apoptotic hepatocytes were increased. In addition, hepatic cholesterol and triglyceride levels were decreased in LPS-exposed animals compared with controls. At 7 weeks of age, no hepatic inflammation could be detected. However, liver triglycerides and plasma cholesterol levels were increased in LPS-exposed animals relative to controls. The changes in lipid levels at 7 weeks of age were associated with increased leptin receptor mRNA levels, increased lipid peroxidation, increased expression of cytochrome c oxidase subunit 4 as a marker for mitochondrial function and increased circulating ceramide levels. These findings demonstrate that chorioamnionitis-mediated antenatal inflammation-related liver disturbances have long-lasting postnatal effects on lipid metabolism.

Background: In laparoscopic ventral hernia repair a mesh is placed in direct contact with the viscera, often leading to substantial adhesions. In this experimental study the ability of different coated and uncoated meshes to attenuate adhesion formation was examined., Methods: Six commercially available meshes were placed intraperitoneally against a closed peritoneum in rats: Prolene (polypropylene), Timesh and Ultrapro (polypropylene composites with titanium and polyglecaprone respectively), Proceed and Parietex Composite (polypropylene and polyester meshes coated with a layer of cellulose and collagen respectively) and C-Qur (polypropylene mesh coated with a layer of omega-3 fatty acids). Adhesions and incorporation were evaluated macroscopically and microscopically after 7 and 30 days., Results: Parietex Composite and C-Qur significantly reduced adhesion formation at 7 days' follow-up compared with all other meshes. By 30 days, this effect had diminished as a significant increase in adhesions together with phagocytosis of the coating was seen for all meshes with layered coatings (Proceed, Parietex Composite and C-Qur. Incorporation was insufficient for all meshes., Conclusion: The absorbable layers of Parietex Composite and C-Qur reduce adhesion formation to intraperitoneal mesh in the short term, but the effect diminishes and phagocytosis of absorbable coatings may contribute to adhesion formation.

Embryonic pathways are often re-expressed in adult pathology. Here we investigated the role of the morphogen hedgehog (hh), which we found to be re-expressed in atherosclerotic plaques. Male ApoE - /- mice were treated for 12 weeks with an anti-hh antibody (5E1) or a control IgG (1E6) starting at the age of 6 or 18 weeks. Inhibition of hh signalling induced a significant increase in total plaque area in the aortic arch, a result of an increase (54% and 36%, respectively) in the area of advanced plaques (atheromata). In mice treated with anti-hh, plaques contained large (18-35% > ctrl), lipid-filled, sometimes multinucleated macrophage foam cells. Plasma cholesterol levels decreased after anti-hh treatment. In bone marrow-derived macrophages, foam cell formation was enhanced after inhibition of hh signalling. Anti-hh treatment caused a 54-75% increase in early oxLDL uptake (10-240 min), which was scavenger receptor-mediated. After 3-24 h of oxLDL incubation, intense Oil red O staining as well as increased amounts of cholesterol esters were present in these macrophages after anti-hh treatment. Activation of the HH-signalling cascade by recombinant Shh induced a decrease in oxLDL uptake. Here we show that the hh-signalling pathway is one of the morphogenic pathways that regulate plasma lipid levels and atherosclerosis development and progression., (Copyright (c) 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Inhibition of CD40-CD40L interactions results in a reduction of innate regulatory T cells (Tregs) in CD40(-/-) mice and induces a stable plaque phenotype in atherosclerosis-prone mouse strains. Here we investigated the effects of leukocyte CD40L on the Treg population and on atherosclerosis. LDLR(-/-) mice were reconstituted with wild-type or CD40L(-/-) bone marrow (BM). These BM chimeras were analysed by flow cytometry for the presence of innate Tregs (CD45RB(low) CD25(+) CD4) in lymphoid organs and peripheral blood. As in CD40(-/-) mice, the CD45RB(high):CD45RB(low) CD4 T cell ratio significantly increased and the CD25(+) CD4(+) subpopulation significantly decreased in LDLR(-/-) mice receiving CD40L(-/-) BM compared to LDLR(-/-) mice receiving wild-type BM. However, atherosclerotic plaque progression and plaque phenotype did not change in LDLR(-/-) mice reconstituted with CD40L(-/-) BM. In conclusion, the present study shows that CD40-CD40L interactions on leukocytes are essential for the size of the CD45RB(low) CD25(+) CD4 Treg subpopulation. Nevertheless, CD40L deficiency on hemopoietic cells did not affect atherosclerosis, implying that CD40L expressing leukocytes alone are not responsible for the stable plaque phenotype observed after total CD40L blockade.

Allogeneic stem cell transplantations (SCT) are currently being used as a therapy for hematological malignancies, some solid tumors and nonmalignant bone marrow deficiencies. Nevertheless, clinical applicability is limited due to toxicity of conditioning regimens, graft-versus-host disease (GVHD) and the scarcity of HLA-identical family donors. New concepts are based on nonmyeloablative conditioning to reduce toxicity, prevention or amelioration of GVHD and the use of haploidentical donors to increase donor availability. To combine these requirements, we have developed a nonmyeloablative conditioning regimen, consisting of low-dose total body irradiation and cyclophosphamide-based chemotherapy. In a haploidentical F1 --> F1 mouse model, this nonmyeloablative transplantation protocol resulted in stable full donor chimerism, but also in the development of severe GVHD. Administration of keratinocyte growth factor (KGF) reduced GVHD, evident as reduced weight loss and a lesser degree of dermatitis, compared to saline-treated controls. KGF preserved plasma citrulline and tumor necrosis factor-alpha levels, both indicative for reduced injury to the gastrointestinal tract. This was confirmed by histological findings. At 6 months after transplantation, survival rates were significantly higher in KGF-treated animals as compared to phosphate buffered saline-treated controls. These results indicate that KGF preserves gut integrity and might therefore contribute substantially to reduction of lethal GVHD in (nonmyeloablative) haploidentical transplantation.

Although many epidemiological studies have shown an association between hyperfibrinogenemia and atherosclerosis, it is not established whether elevated fibrinogen has an etiological role in the pathogenesis or is only a reflection of the ongoing disease. We have studied the contribution of fibrinogen to the development of atherosclerosis in atherosclerosis-prone ApoE*3-Leiden mice that have been cross-bred with transgenic mice overexpressing fibrinogen. Genetic compound offspring were used to evaluate the progression of atherosclerotic lesions after being fed an atherogenic diet for 7 weeks. It was observed that the lesion area of the plaques as well as the severity of the lesions in the aortic valve was comparable in control single transgenic ApoE*3-Leiden mice and in double transgenic apoE*3-Leiden mice overexpressing fibrinogen. No thrombus or fibrin deposition was observed in atherosclerotic lesions in either group of mice. These results indicate that elevated plasma fibrinogen concentrations in ApoE*3-Leiden transgenic mice do not affect the progression of diet-induced atherosclerotic lesions.

Although genes determining lipoprotein homeostasis and atherosclerosis are the subject of intensive investigation, only a subset of these genes is known at present. Hence, we do not have sufficient knowledge to explain the genetic basis of hyperlipidemia in the majority of subjects. Our aim was to identify novel genes and pathways underlying lipoprotein homeostasis by using serial analysis of gene expression. The liver expression profile of mild hyperlipidemic apolipoprotein E3-Leiden (E3L) transgenic mice was compared with that of the wild-type C57BL/6JIco (B6) mice. Over 18 000 liver transcripts of B6 as well as E3L mice were analyzed, representing >9400 unique genes. One hundred seventy-five genes showed altered expression between the strains (P<0.05). Although several of these genes belonged to known metabolic pathways, such as lipoprotein metabolism, detoxification processes, glycolysis, and the acute-phase response, most were novel. Differential gene expression of 8 of 10 genes tested could be confirmed by Northern blot analysis. This inventory of differentially expressed genes will provide a unique basis for detailed studies to gain more insight into their role in lipoprotein homeostasis and atherosclerosis.

It has previously been reported that mice lacking the VLDL receptor (VLDLR-/-) exhibit normal plasma lipid levels and a modest decrease in adipose tissue mass. In the present study, the effect of VLDLR deficiency on profound weight gain was studied in mice. Obesity was induced either by feeding of a high-fat, high-calorie (HFC) diet or by crossbreeding mice onto the genetically obese ob/ob background. After 17 weeks of HFC feeding, VLDLR-/- mice remained lean, whereas their wild-type littermates (VLDLR+/+) became obese. Similarly, the weight gain of ob/ob mice was less profound in the absence of the VLDLR. Moreover, VLDLR deficiency led to increased plasma triglycerides after HFC feeding. The protection from obesity in VLDLR-/- mice involved decreased peripheral uptake of fatty acids, because VLDLR-/- mice exhibited a significant reduction in whole-body free fatty acid uptake, with no clear differences in food intake and fat absorption. These observations were supported by a strong decrease in average adipocyte size in VLDLR-/- mice of both obesity models, implying reduced adipocyte triglyceride storage in the absence of the VLDLR. These results suggest that the VLDLR plays a role in the delivery of VLDL-derived fatty acids into adipose tissue.

Cell proliferation and cell death (either necrosis or apoptosis) are key processes in the progression of atherosclerosis. The tumor suppressor gene p53 is an essential gene in cell proliferation and cell death and is upregulated in human atherosclerotic plaques, both in smooth muscle cells and in macrophages. In the present study, we investigated the importance of macrophage p53 in the progression of atherosclerosis using bone marrow transplantation in APOE*3-Leiden transgenic mice, an animal model for human-like atherosclerosis. APOE*3-Leiden mice were lethally irradiated and reconstituted with bone marrow derived from either p53-deficient (p53(-/-)) or control (p53(+/+)) donor mice. Reconstitution of mice with p53(-/-) bone marrow did not result in any hemopoietic abnormalities as compared with p53(+/+) transplanted mice. After 12 weeks on an atherogenic diet, APOE*3-Leiden mice reconstituted with p53(-/-) bone marrow showed a significant (P=0.006) 2.3-fold increase in total atherosclerotic lesion area as compared with mice reconstituted with p53(+/+) bone marrow. Although likely a secondary effect of the increased lesion area, p53(-/-) transplanted mice also showed significantly more lesion necrosis (necrotic index, 1.1+/-1.3 versus 0.2+/-0.7; P=0.04) and lesion macrophages (macrophage area, 79.9+/-40.0 versus 39.7+/-27.3x10(3) micrometer(2) per section; P=0.02). These observations coincided with a tendency toward decreased apoptosis (terminal deoxynucleotidyl transferase end-labeling [TUNEL]-positive nuclei going from 0.42+/-0.39 to 0.14+/-0.15%, P=0.071), whereas the number of proliferating cells (5'-bromo-2'-deoxyuridine-positive nuclei) was not affected (3.75+/-0.98 versus 4.77+/-2.30%; P=0.59). These studies indicate that macrophage p53 is important in suppressing the progression of atherosclerosis and identify a novel therapeutic target for regulating plaque stability.

Background: The present study investigated whether the ACAT inhibitor avasimibe can reduce atherogenesis independently of its cholesterol-lowering effect in ApoE*3-Leiden mice., Methods and Results: Two groups of 15 female ApoE*3-Leiden mice were put on a high-cholesterol (HC) diet; 1 group received 0.01% (wt/wt) avasimibe mixed into the diet. The HC diet resulted in a plasma cholesterol concentration of 18.7+/-2.6 mmol/L. Addition of avasimibe lowered plasma cholesterol by 56% to 8.1+/-1.2 mmol/L, caused mainly by a reduction of and composition change in VLDL and LDL. In a separate low-cholesterol (LC) control group, plasma cholesterol was titrated to a level comparable to that of the avasimibe group (10.3+/-1.4 mmol/L) by lowering the amount of dietary cholesterol. After 22 weeks of intervention, atherosclerosis in the aortic root area was quantified. Treatment with avasimibe resulted in a 92% reduction of lesion area compared with the HC control group. Compared with the LC control, avasimibe reduced lesion area by 78%. After correction for the slight difference in cholesterol exposure between the LC control and avasimibe groups, the effect of avasimibe on lesion area (73% reduction) remained highly significant. In addition, monocyte adherence to the endothelium, free cholesterol accumulation, and lesion severity were reduced by avasimibe treatment., Conclusions: Treatment with avasimibe potently lowered plasma cholesterol levels in ApoE*3-Leiden mice and considerably reduced atherosclerotic lesion area in addition to its cholesterol-lowering effect. Because monocyte adherence to the endothelium and lesion severity were also reduced by avasimibe, treatment with avasimibe may result in higher plaque stability and therefore a reduced risk of plaque rupture.

Chronic proliferative dermatitis (cpd) is a spontaneous mutation in C57BL/Ka mice (cpdm/cpdm). The dermatitis is characterized by redness, hairloss, scaling, pruritus and histologically by epithelial hyperproliferation, infiltration of eosinophils, macrophages and mast cells. Lesions similar to those in the skin occur in the esophagus and forestomach. In this paper, we describe the effect of drug treatments directed against epidermal hyperproliferation (calcipotriene and etretinate), against inflammation (corticosteroids and dapsone) and against pruritus (loratidine and capsaicin). The criteria used to objectively estimate the effect of the treatment were 1) macroscopic evaluation of the lesions (cpd score), 2) degree of epithelial hyperproliferation assessed by BrdU incorporation and epithelial thickness, and 3) microscopic evaluation of the inflammatory cells in the skin samples. Treatment of the cpdm/cpdm mice with calcipotriene (5 microg/day for 3 weeks) inhibited epidermal proliferation and the number of eosinophils. Systemic etretinate treatment (30 microg/g/day for 3 weeks) was not very effective. Topical corticosteroids (0.05 microg/day, for 3 weeks) exerted a therapeutic effect on the hyperproliferation and the number of eosinophils. Oral dapsone treatment (34 microg/g/day, for 5 weeks) reduced the BrdU incorporation in the skin and the epithelial thickness in the esophagus. The anti-histamine loratidine (orally, 1.7 microg/ g/day, for 4 weeks) reduced the severity of the lesions macroscopically, probably by suppressing the pruritus. Capsaicin (topically, 30 mM, for 5 weeks) also reduced the severity of the macroscopic observable lesions. Moreover, capsaicin reduced the dorsal and ventral epidermal thickness. The results from this and previous studies indicate that steroids (topically and systemically) and less strongly calcipotriene are the most effective treatments for the lesions observed in the cpdm/cpdm mice, since both hyperproliferation and the influx of eosinophils are reduced. Although the pathogenesis of the cpd lesions remains to be determined, our results indicate that the cpdm/cpdm mouse can be used to investigate new drugs for their possible application in chronic dermatitis.

Several in vivo studies have been performed on the role of the macrophage scavenger receptor class A (SR-A) in atherosclerosis using SR-A knockout mice. The results indicate both an antiatherogenic and a proatherogenic role of SR-A, depending on the nature of the animal model serving as the athero-susceptible background. To study the role of SR-A in a different model, we generated a transgenic mouse model with high level expression of the human SR-A gene using a 180 Kb yeast artificial chromosome (MSR1 transgenic mice). These mice show increased expression of SR-A according to the natural expression pattern. The MSR1 transgenic mice were crossed onto a low-density lipoprotein receptor deficient background and were fed a high fat diet for 10 weeks. After this period, the size of the atherosclerotic lesions in the proximal aorta was measured. Surprisingly, atherosclerosis was significantly reduced in the MSR1 transgenic mice. In a second study, the effect of SR-A was examined in APOE-3 Leiden mice providing a different athero-susceptible background. To exclude nonmacrophage effects, bone marrow was transplanted from MSR1 mice and wild-type littermates to APOE-3 Leiden transgenic mice. After 8 weeks on a high fat diet, atherosclerosis in the mice that had received MSR1 bone marrow was reduced compared with mice that had received wild-type bone marrow. This difference reached statistical significance when individual cholesterol exposure of the mice was taken into account. Both experiments indicated an antiatherogenic role of the SR-A. This observation cannot be explained easily by SR-A function in foam cell formation because in MSR1 macrophages in vitro foam cell formation is increased. Alternatively, however, SR-A may affect the activation of macrophages. Hence the response to lipopolysaccharide was measured in MSR1-transgenic macrophages. These macrophages showed a reduction in their activation in response to lipopolysaccharide, as measured by nitric oxide production. These data show that an elevated level of SR-A expression reduces atherosclerosis, potentially by modifying the response of macrophages to activation signals in the plaque.

Macrophage scavenger receptors class A (MSR) are thought to play an important role in atherogenesis by mediating the unrestricted uptake of modified lipoproteins by macrophages in the vessel wall leading to foam cell formation. To investigate the in vivo role of the MSR in this process, a transgenic mouse model expressing both isoforms of the human MSR was generated. A 180-kb yeast artificial chromosome (YAC) containing the human MSR gene (MSR1) with 60- and 40-kb flanking sequence at the 5' and 3' end, respectively, was obtained by reducing the size of a 1050-kb YAC by homologous recombination. This 180-kb YAC was microinjected into mouse oocytes. In the resulting transgenic mice, high levels of mRNA for both type I and type II human MSR1 were detected in peritoneal macrophages and trace levels in other organs, known to contain macrophage-derived cells. Using an antibody against the human MSR, the Kupffer cells in the liver were shown to contain the MSR protein. In vivo clearance of acetyl-LDL was not changed in the MSR1-transgenic mice. However, in vitro studies using peritoneal macrophages from the transgenic mice showed a two-fold increased degradation of acetyl-LDL and cholesterolester accumulation concomitant with a four-fold increase in foam cell formation, as compared to wild-type macrophages. Thus, macrophage specific overexpression of the MSR may lead to increased foam cell formation, which is one of the initial and crucial steps in atherogenesis.

Apolipoprotein (apo) E3Leiden is a dysfunctional apo E variant associated with familial dysbetalipoproteinemia in humans. Transgenic mice carrying the APOE3Leiden gene develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. An early step in atherosclerosis is foam cell formation, which is thought to result from the unrestricted uptake of modified lipoproteins by macrophages. To investigate the role of the macrophage scavenger receptor type I and II (MSR-A) in this process, APOE3Leiden transgenic mice were crossed onto a MSR-A deficient background and the development of atherosclerosis was examined. In view of recent results with apo E deficient mice (Suzuki H et al., A role for the macrophage scavenger receptors in atherosclerosis. Nature 1997; 386(6622):292-296), absence of the MSR-A in APOE3Leiden mice was expected to lead to a reduction of atherosclerosis. In our study we compared APOE3Leiden/MSR-A deficient mice (E3L MSR-A -/-) to APOE3Leiden/MSR-A wild-type mice (E3L MSR-A +/+). These animals were fed an atherogenic diet for 10 weeks. Quantification of the lesion area showed no significant difference between E3L MSR-A -/- and E3L MSR-A +/+ mice although there was a trend towards the development of larger lesions in the E3L MSR-A -/- mice. All lesions were typed according to their cellular composition. In both male and female E3L MSR-A -/- mice, significantly more severe lesions developed as compared to E3L MSR-A +/+ mice. These results indicate that the effect of MSR-A deficiency on atherogenesis may depend on the presence or absence of apo E.

Apolipoprotein E3-Leiden (APOE3-Leiden) transgenic mice develop hyperlipidemia and are highly susceptible to diet-induced atherosclerosis. We have studied the progression and regression of atherosclerosis using immunohistochemistry. Female transgenic mice were fed a moderate fat diet to study atherosclerosis over a longer time period. Fatty streaks arose in the intima and consisted of lipid filled macrophages which differed in origin. All macrophages expressed the macrophage scavenger receptor while two thirds expressed sialoadhesin and were positive for an antibody recognizing marginal zone macrophages (MOMA-1). All macrophages were negative for the scavenger receptor MARCO and 50% were positive for CD4. Small fatty streaks contained CD-3 positive T-lymphocytes which were for more than 70% CD4-positive. ICAM-1 was positive both in atherosclerotic and control mice. In early plaques, fibrosis was observed on the luminal and medial site of the foam cells while smooth muscle cells were only observed in the fibrous cap. To study regression, we used a high fat, high cholesterol diet to rapidly induce atherosclerosis (14 weeks). The animals were then fed normal chow. Subsequently, atherosclerosis was assayed over time (4, 8, 16 weeks). Cholesterol levels dropped in 4 weeks to control levels. The animals did not show a significantly decrease in plaque size over time. but the percentage macrophages was significantly smaller in the animals after 4 weeks. In conclusion, the APOE3-Leiden mouse is a useful model to study the progression and regression of atherosclerosis.

Transgenic mice were generated with different levels of human apolipoprotein C1 (APOC1) expression in liver and skin. At 2 mo of age, serum levels of cholesterol, triglycerides (TG), and FFA were strongly elevated in APOC1 transgenic mice compared with wild-type mice. These elevated levels of serum cholesterol and TG were due mainly to an accumulation of VLDL particles in the circulation. In addition to hyperlipidemia, APOC1 transgenic mice developed dry and scaly skin with loss of hair, dependent on the amount of APOC1 expression in the skin. Since these skin abnormalities appeared in two independent founder lines, a mutation related to the specific insertion site of the human APOC1 gene as the cause for the phenotype can be excluded. Histopathological analysis of high expressor APOC1 transgenic mice revealed a disorder of the skin consisting of epidermal hyperplasia and hyperkeratosis, and atrophic sebaceous glands lacking sebum. In line with these results, epidermal lipid analysis showed that the relative amounts of the sebum components TG and wax diesters in the epidermis of high expressor APOC1 transgenic mice were reduced by 60 and 45%, respectively. In addition to atrophic sebaceous glands, the meibomian glands were also found to be severely atrophic in APOC1 transgenic mice. High expressor APOC1 transgenic mice also exhibited diminished abdominal adipose tissue stores (a 60% decrease compared with wild-type mice) and a complete deficiency of subcutaneous fat. These results indicate that, in addition to the previously reported inhibitory role of apoC1 on hepatic remnant uptake, overexpression of apoC1 affects lipid synthesis in the sebaceous gland and/or epidermis as well as adipose tissue formation. These APOC1 transgenic mice may serve as an interesting in vivo model for the investigation of lipid homeostasis in the skin.

Chronic proliferative dermatitis is a spontaneous mutation in C57BL/Ka mice (cpdm/cpdm), showing alopecia, epithelial hyperproliferation, infiltration by eosinophils and macrophages, and vascular dilatation. To further elucidate its pathogenesis, organs of 1-, 2-, 3-, 4-, 5-, and 6-week-old cpdm/cpdm mice were examined. At 4 weeks, the epidermal thickness was increased, whereas already at 3 weeks, the bromodeoxyuridine incorporation was increased in the basal keratinocytes. However, already at the age of 1 week, skin, lungs, and lymph nodes were infiltrated by eosinophils although no macroscopic lesions were present. Compared with control animals, 6-week-old cpdm/cpdm mice had decreased serum IgE levels and increased numbers of mast cells. From the age of 1 week these mast cells became increasingly IgE positive. In contrast, the mast cells of the control animals remained IgE negative. Mast cells of control and cpdm/cpdm mice were interleukin-4 and tumor necrosis factor-alpha positive. A likely explanation for the tissue infiltration of eosinophils could be the release of interleukin-4 and tumor necrosis factor-alpha from activated mast cells. Tumor necrosis factor-alpha may lead to the expression of E-selectin on endothelial cells, facilitating interleukin-4-mediated eosinophil transendothelial migration. Although various pathogenetic aspects of the cpdm/cpdm mouse need further elucidation, this model can be a tool to study eosinophil infiltration, leukocyte-endothelial cell interactions, and mast cell proliferation. Furthermore, the cpdm/cpdm mouse can be used to study chronic inflammatory skin disease because of the severe epidermal proliferation.

Chronic proliferative dermatitis is a spontaneous mutation in C57BL/Ka mice (cpdm/cpdm) and is characterized by epithelial hyperproliferation, infiltration by eosinophils and macrophages, and vascular dilatation. To elucidate whether these pathologic features are the result of a local (skin) process or a consequence of a systemic disorder, transplantations were performed of full-thickness grafts of affected skin from cpdm/cpdm mice and normal skin from control (C57BL/Ka) mice on the back of cpdm/cpdm, C57BL/Ka and athymic nude mice. After 3 months, the grafts maintained the histologic phenotype of the donor animal. Intercellular adhesion molecule-1 continued to be expressed by basal keratinocytes of the cpdm/cpdm grafts after transplantation. In contrast, the basal keratinocytes of the C57BL/Ka grafts onto cpdm/cpdm mice remained negative for intercellular adhesion molecule-1 3 months after transplantation. An increased number of proliferating keratinocytes was present in the cpdm/cpdm skin-graft transplanted to nudes or to C57BL/Ka mice based on short-term bromodeoxyuridine labeling. The bromodeoxyuridine incorporation in the keratinocytes of the control C57BL/Ka skin grafts transplanted to cpdm/cpdm, nude, or C57BL/Ka mice was the same as in the keratinocytes of normal C57BL/Ka mice. This study demonstrates that the pathologic features found in the cpdm/cpdm mice are the result of a disorder in the epidermis or dermis and not due to a systemic defect.

C57BL/Ka mice with chronic proliferative dermatitis (cpdm/cpdm) develop chronic persistent skin lesions characterized by epidermal hyperplasia, infiltration by granulocytes and macrophages, and vascular dilatation. Similar lesions are present in other orthokeratotic epithelia in affected mice, in particular the esophagus and forestomach. Here, we report on further characterization of epidermal hyperplasia and the granulocytes. Keratinocytes of lesional skin, but not of normal skin, show round and electron-dense mitochondrial inclusions that are present in all layers of the epidermis. Similar inclusions are also present in the esophagus and forestomach of affected mice. There appears to be a direct relation between the presence of intramitochondrial inclusions and epidermal hyperplasia in the mouse. Furthermore, the presence of keratinocyte-derived apoptotic bodies in the epidermis, esophagus, and forestomach was frequently observed in the lesions, which is consistent with previous light microscopic observations of single cell death of keratinocytes. The granulocytes present in the skin, esophagus, and forestomach were mainly eosinophils. There were widespread gaps observed in the lamina densa in the epidermis that were mostly directly associated with dermal or epidermal eosinophils. This type of gap is also observed in psoriasiform diseases in humans. This electron microscopic study demonstrated that this mouse model should be useful to screen potential therapeutic strategies for psoriasiform and other inflammatory skin disorders.

Apolipoprotein (apo) E-deficient mice were fed a hypercholesterolemic diet for 14 weeks. Mean serum cholesterol levels rose to 37.5 mM. Upon complete necroscopy, massive xanthomatous lesions were noticed in various tissues, with a predilection for subcutaneous and peritendinous tissues, while control animals on the same diet (3.4 mM serum cholesterol) and apo E-deficient mice on a regular chow diet (20 mM serum cholesterol) did not show such lesions. Also, apo E3-Leiden transgenic mice fed a high fat diet, with 60 mM of serum cholesterol, did not exhibit any xanthomatosis. The xanthomatous lesions found in the Apoe knock-out mouse clearly differed in location from xanthomas previously found in low density lipoprotein receptor-deficient mice. We conclude that the lack of apo E results in atypical disseminated xanthomatosis, suggesting that apo E has an important role in determining the tissue distribution of cholesterol deposition.

The murine chronic proliferative dermatitis mutation (cpdm/cpdm) is characterized by epidermal hyperplasia and hyperproliferation of ventral and dorsal skin sites. The expression of endothelium-associated adhesion molecules was studied in combination with the binding capacity of various cell types on frozen sections of the affected skin. In correlation with the relative absence of lymphocytes in the cpdm/cpdm skin no lymphocyte binding could be observed, but avid adhesion of neutrophils was seen. Binding of neutrophils could be blocked with antibodies against L-selectin, LFA-1, CR3 and anti ICAM-1. No expression of vascular addressins or E-selectin on endothelium in the dermis was found. The cpdm/cpdm mutation has therefore characteristics of a psoriasis-like as well as a more generalized inflammatory skin condition.

Apolipoprotein E3-Leiden (APOE*3-Leiden) transgenic mice have been used to study the effect of different cholesterol-containing diets on the remnant lipoprotein levels and composition and on the possible concurrent development of atherosclerotic plaques. On high fat/cholesterol (HFC) diet, the high expressing lines 2 and 181 developed severe hypercholesterolemia (up to 40 and 60 mmol/liter, respectively), whereas triglyceride levels remained almost normal when compared with regular mouse diet. The addition of cholate increased the hypercholesterolemic effect of this diet. In lines 2 and 181, serum levels of apo E3-Leiden also increased dramatically upon cholesterol feeding (up to 107 and 300 mg/dl, respectively). In these high expressing APOE*3-Leiden transgenic mice, the increase in both serum cholesterol and apo E3-Leiden occurred mainly in the VLDL/LDL-sized fractions, whereas a considerable increase in large, apo E-rich HDL particles also occurred. In contrast to the high expressing lines, the low expressing line 195 reacted only mildly upon HFC diet. On HFC diets, the high expresser APOE*3-Leiden mice developed atherosclerotic lesions in the aortic arch, the descending aorta, and the carotid arteries, varying from fatty streaks containing foam cells to severe atherosclerotic plaques containing cholesterol crystals, fibrosis, and necrotic calcified tissue. Quantitative evaluation revealed that the atherogenesis is positively correlated with the serum level of cholesterol-rich VLDL/LDL particles. In conclusion, with APOE*3-Leiden transgenic mice, factors can be studied that influence the metabolism of remnant VLDL and the development of atherosclerosis.

Chronic proliferative dermatitis is a new spontaneous mutation in C57BL/Ka mice. Breeding results suggest an autosomal recessive mode of inheritance. Mutant mice develop skin lesions at the age of 5 to 6 weeks. The lesions occur in the ventral and dorsal skin of the body, whereas ears, footpads, and tail are not involved. The lesions are characterized by epidermal hyperplasia, hyper- and parakeratosis, and single cell necrosis of keratinocytes. The dermis and epidermis are infiltrated by granulocytes and macrophages, and occasionally subcorneal and intracorneal microabscesses are formed. The number of mast cells in the dermis progressively increases with age. There is dilatation and proliferation of dermal capillaries. Similar lesions develop in the mouth, esophagus, and forestomach, which, in the mouse, are all lined by orthokeratinizing stratified squamous cell epithelium. Studies with bromodeoxyuridine confirm the increased rate of epithelial cell proliferation. Most inflammatory cells in the affected skin express Mac-1, and few express the T lymphocyte marker CD3. There is increased expression of intracellular adhesion molecule-1 on keratinocytes and endothelial cells. Infiltration of neutrophils and macrophages are also seen in the liver, lung, and several joints. The disease could not be transferred by bone marrow or spleen transplants into irradiated normal syngeneic hosts. Treatment of the mice with triamcinolone, a long-acting corticosteroid, resulted in nearly complete regression of the lesions over a period of 4 weeks, whereas systemic cyclosporin A treatment was ineffective.

Normal epithelial cell differentiation is characterized by the production of distinct cytokeratin proteins. It is well known that epithelia of several organs show squamous metaplasia in a vitamin A-deficient status. It is not yet known whether these histological changes are concomitant with a change in cytokeratin expression. Therefore, 3-week-old female rats (BN/BiRij) were fed a vitamin A-deficient diet for 8 weeks. The cytokeratin expression in epithelia of various organs was monitored immunohistochemically during the induction of vitamin A deficiency. Therefore, monoclonal antibodies specific for human cytokeratin 4, 5, 5 + 8, 7, 10, 14, 18 and 19 were used. In a normal vitamin A status, the distributional pattern for the different cytokeratins in rats was similar to that reported for human tissue. No change in cytokeratin expression was seen in trachea, skin, liver and colon at any time point studied. Squamous metaplasia in urinary bladder and salivary glands was observed after six weeks on the vitamin A-deficient diet. This was concomitant with a substitution of cytokeratins 4, 5 + 8, 7, 18 and 19 by cytokeratin 10. The latter cytokeratin is specific for keratinized squamous epithelium. A change in cytokeratin expression was observed in bladder, ureter, kidney, salivary glands, uterus and conjunctiva before histological alterations appeared. In conclusion, the changes in cytokeratin expression observed under vitamin A deficiency in epithelia in vivo are in agreement with those described in other studies for epithelial cells in vitro. The changes in cytokeratin expression and the subsequent differentiation into squamous cells occurs in basal cells of the bladder but not in transitional cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of vitamin A deficiency on the mitogen response of splenic B and T lymphocytes was determined in adult vitamin A-deficient rats. Female weanling Brown Norway/Billingham-Rijswijk (BN/BiRij) and Sprague-Dawley rats were fed a semipurified, essentially vitamin A-free diet, which resulted in clinical symptoms of vitamin A deficiency and severely decreased plasma retinol contents at the age of about 17 and 41 wk for BN/BiRij and Sprague-Dawley rats, respectively. A lower B cell proliferative response after stimulation with lipopolysaccharide in combination with dextran sulfate was observed in vitamin A-deficient rats of both strains, but the T cell proliferative response after concanavalin A stimulation was unchanged. The lower B cell mitogen response was not associated with changes in the cellular composition of the spleen (as analyzed with monoclonal antibodies specific for the various subsets of T and B cells and of macrophages). We suggest that the age at which clinical symptoms of vitamin A deficiency are induced may be an important determinant for the immunological variables affected.

Archival paraffin embedded material was used to examine whether additional quantitative criteria would be helpful to discriminate between histologically benign and malignant rat mammary tumours. To this end nuclear DNA content expressed as DNA ploidy index (DI) was measured using flow cytometry (FCM). A total of 63 benign and malignant mammary tumours were investigated. Thirteen out of 38 (34%) mammary carcinomas were DNA aneuploid against 0 out of 25 benign mammary tumours. Aneuploidy was not significantly increased in tumours showing histological signs of greater malignancy such as cribriform-comedo type or invasive growth. In addition to DI other quantitative criteria indicative for malignancy, such as mitotic count and nuclear morphometric characteristics, were estimated in 24 benign and malignant tubulopapillary tumours, a category where the histological classification may be difficult. It appeared that five out of nine noninvasive tubulopapillary carcinomas and six out of seven invasive carcinomas had abnormal values for either DI, mitotic count or nuclear area or for a combination of these parameters. Each single parameter however was abnormal only in a minority of the malignant tumours. In this respect our data are in accordance with the fact that rat mammary carcinomas are clinically and histologically less malignant than their human counterparts.

Copper uptake and retention were studied in primary cultures of liver parenchymal cells isolated from copper-deficient rats. Male Sprague-Dawley rats were fed a copper-deficient diet (less than 1 mg Cu/kg) for 10 wk. Copper-deficient rats were characterized by low copper concentrations in plasma and liver, anemia, low plasma ceruloplasmin oxidase activity and increased 64Cu whole-body retention. Freshly isolated liver parenchymal cells from copper-deficient rats showed a higher 64Cu influx, which was associated with a higher apparent Vmax of 45 +/- 4 pmol Cu.mg protein-1.min-1 as compared with 30 +/- 3 pmol Cu.mg protein-1.min-1 for cells isolated from copper-sufficient rats. No significant difference in the apparent Km (approximately 30 mumol/L) was observed. Relative 64Cu efflux from cells from copper-deficient rats was significantly smaller than the efflux from cells from copper-sufficient rats after prelabeling as determined by 2-h efflux experiments. Analysis of the medium after efflux from cells from copper-deficient rats showed elevated protein-associated 64Cu, suggesting a higher incorporation of radioactive copper during metalloprotein synthesis. Effects of copper deficiency persist in primary cultures of parenchymal cells derived from copper-deficient rats, and short-term cultures of these cells offer a prospect for the study of cell biological aspects of the metabolic adaptation of the liver to copper deficiency.

The thorax of WAG/Rij rats was irradiated with fractionated doses of X rays. Irradiation schedules were designed either to allow virtually complete repair of sublethal damage between subsequent fractions by fractionating at 6-h intervals, or to result in incomplete repair by allowing only 1-h intervals between subsequent fractions. Combination of the data from both experimental series permitted the calculation of alpha/beta ratios and values for the repair halftime T1/2. The animals were monitored by assessment of the breathing frequency and by recording deaths. At the end of the experiments, 18 months after treatment, the hydroxyproline content of the lung tissue was determined as a biochemical indicator of radiation-induced fibrosis, and an histopathological analysis was performed. Early endpoints, indicative of radiation-induced pneumonitis, resulted in an alpha/beta ratio of 3.5 Gy and a T1/2 value of 0.95 h. Late endpoints were presumed to be indicative of radiation-induced fibrosis. Based on the combined analysis of data from three different late endpoints, the mean alpha/beta ratio was 2.3 Gy, and the T1/2 value was 1.13 h. The difference in alpha/beta ratio and T1/2 value between early and late endpoints was not significant, since the 95% confidence limits were overlapping. For each individual early or late endpoint as well as for the two early or the three late endpoints combined, there was a trend for lower alpha/beta ratios and higher T1/2 values associated with low doses per fraction. However, widely overlapping confidence limits indicated that again the differences were not significant.

The composition of the phthalide mixture of the essential oil from roots of Levisticum officinale Koch was investigated. The phthalide mixture was analyzed by combining separation methods - GLC, LSC and TLC - and subsequently using spectroscopic methods - IR, MS and NMR. E- and Z-butylidenephthalide, E- and Z-ligustilide, senkyunolide and validene-4,5-dihydrophthalide were found to be present; isosenkyunolide and propylidenephthalide were tentatively identified. The influence of the isolation procedure on the composition of the phthalide mixture was tentatively studied by comparison of mixtures isolated by solvent extraction and hydrodistillation respectively.

In petroleum ether and diethyl ether extracts from roots of Opopanax chironium (L.) Koch five phthalides were identified: Z-butylidenephthalide, butylphthalide, cnidilide, Z-ligustilide and senkyunolide. The compounds were analyzed by TLC, GLC and GC-MS.

The long-term pulmonary morphologic and biochemical changes induced by a single, sublethal dose of O,O,S-trimethyl phosphorothioate (OOS-TMP), an impurity present in organophosphorus insecticides, was examined in female WAG/Rij rats. Animals received either corn oil or a corn oil solution of OOS-TMP at a dose of 40 mg/kg body weight by gavage and were studied at the following time intervals after treatment: 10 days, 30 days, 90 days, 6 months, and 1 year. No control or OOS-TMP-treated animal died spontaneously. Significant increases in wet and dry lung weights occurred at all times examined in OOS-TMP-treated rats compared to those in age-matched controls. No difference was observed in percentage lung water content between treated and control rats. OOS-TMP treatment resulted in hypertrophy and hyperplasia of type II alveolar epithelial cells having morphologic alterations in the size and number of osmiophilic lamellar bodies. Interstitial changes were characterized as increased numbers of fibroblasts accompanied by increased amounts of collagen fibrils and basement membrane alterations. There was also a significant increase in pulmonary hydroxyproline content in OOS-TMP-treated animals compared to that in controls at all time periods. Collagen deposition was predominantly associated with the interalveolar septa rather than being oriented around airways. The results of this study indicate that a single, sublethal dose of OOS-TMP induces long-term structural and biochemical changes in the rat lung.

The (1)H- and (13)C-NMR spectra of several naturally occurring phthalides are reported; among others, the new natural compound 3,3alpha- CIS-neocnidilide isolated from APIUM GRAVEOLENS L. (and also found to be present in ANETHUM GRAVEOLENS L.). For some compounds, data are compared with previously published data.

In extracts from roots of CAPNOPHYLLUM PEREGRINUM Z-ligustilide was found to be present and in similar extracts of PEUCEDANUM OSTRUTHIUM cnidilide, Z-ligustilide and senkyunolide were detected. The compounds were analyzed by TLC, GLC and GCMS.

The toxicity of ozone and nitrogen dioxide is generally ascribed to their oxidative potential. In this study their toxic mechanism of action was compared using an intact cell model. Rat alveolar macrophages were exposed by means of gas diffusion through a Teflon film. In this in vitro system, ozone appeared to be 10 times more toxic than nitrogen dioxide. alpha-Tocopherol protected equally well against ozone and nitrogen dioxide. It was demonstrated that alpha-tocopherol provided its protection by its action as a radical scavenger and not by its stabilizing structural membrane effect, as (1) concentrations of alpha-tocopherol that already provided optimal protection against ozone and nitrogen dioxide did not influence the membrane fluidity of alveolar macrophages and (2) neither one of the structural alpha-tocopherol analogs tested (phytol and the methyl ether of alpha-tocopherol) could provide a protection against ozone or nitrogen dioxide comparable to the one provided by alpha-tocopherol. It was concluded that reactive intermediates scavenged by alpha-tocopherol are important in the toxic mechanism of both ozone and nitrogen dioxide induced cell damage. However, further results presented strongly confirmed that the kind of radicals and/or reactive intermediates, and thus the toxic reaction mechanism involved, must be different in ozone- and nitrogen dioxide-induced cell damage. This was concluded from the observations that showed that (1) vitamin C provided significantly better protection against nitrogen dioxide than against an equally toxic dose of ozone, (2) glutathione depletion affected the cellular sensitivity toward ozone to a significantly greater extent than the sensitivity towards nitrogen dioxide, and (3) the scavenging action of alpha-tocopherol was accompanied by a significantly greater reduction in its cellular level during nitrogen dioxide exposure than during exposure to ozone. One of the possibilities compatible with the results presented in this study might be that lipid (peroxyl) free radicals formed in a radical-mediated peroxidative pathway, resulting in a substantial breakdown of cellular alpha-tocopherol, are involved in nitrogen dioxide-induced cell damage, and that lipid ozonides, scavenged by alpha-tocopherol as well, are involved in ozone-induced cell damage.