Salmonella Typhimurium caused an unprecedentedly large foodborne outbreak in Finland in 2021.

AIMS: Salmonella infections are significant causes of foodborne outbreaks in the European Union. This study investigates a sudden increase in gastroenteritis patients in the hospital district of Central Finland in June 2021. The primary aim was to study the outbreak's magnitude and source of the outbreak. METHODS AND RESULTS: Epidemiological, microbiological, environmental and traceback investigations were conducted. Over 700 persons fell ill during the outbreak caused by Salmonella Typhimurium associated with a daycare lunch. Similar S. Typhimurium was found in the patients and a vegetable mix containing iceberg lettuce, cucumber and peas served during lunch. The traceback investigation revealed that the batch information of vegetables from the wholesaler was not complete. The wholesaler had received quality complaints about the iceberg lettuce from the central kitchen. The manufacturer did not test the suspected batch for Salmonella since the production plant had given a certificate declaring it Salmonella negative. CONCLUSIONS: The most suspect ingredient was one batch of iceberg lettuce due to quality complaints. The lettuce had not been served in two daycare centres without cases. We recommend that in order to enable thorough microbiological investigation, institutional kitchens store the food samples separately as part of the internal quality control and that food items should always be tested when Salmonella contamination in an outbreak is suspected.

Lymphatic vessels are present in human saccular intracranial aneurysms.

Saccular intracranial aneurysm (sIA) rupture leads to subarachnoid haemorrhage and is preceded by chronic inflammation and atherosclerotic changes of the sIA wall. Increased lymphangiogenesis has been detected in atherosclerotic extracranial arteries and in abdominal aortic aneurysms, but the presence of lymphatic vessels in sIAs has remained unexplored. Here we studied the presence of lymphatic vessels in 36 intraoperatively resected sIAs (16 unruptured and 20 ruptured), using immunohistochemical and immunofluorescence stainings for lymphatic endothelial cell (LEC) markers. Of these LEC-markers, both extracellular and intracellular LYVE-1-, podoplanin-, VEGFR-3-, and Prox1-positive stainings were detected in 83%, 94%, 100%, and 72% of the 36 sIA walls, respectively. Lymphatic vessels were identified as ring-shaped structures positive for one or more of the LEC markers. Of the sIAs, 78% contained lymphatic vessels positive for at least one LEC marker. The presence of LECs and lymphatic vessels were associated with the number of CD68+ and CD163+ cells in the sIA walls, and with the expression of inflammation indicators such as serum amyloid A, myeloperoxidase, and cyclo-oxygenase 2, with the presence of a thrombus, and with the sIA wall rupture. Large areas of VEGFR-3 and α-smooth muscle actin (αSMA) double-positive cells were detected in medial parts of the sIA walls. Also, a few podoplanin and αSMA double-positive cells were discovered. In addition, LYVE-1 and CD68 double-positive cells were detected in the sIA walls and in the thrombus revealing that certain CD68+ macrophages are capable of expressing LEC markers. This study demonstrates for the first time the presence of lymphatic vessels in human sIA walls. Further studies are needed to understand the role of lymphatic vessels in the pathogenesis of sIA.

Associations of Sex Hormones and Hormonal Status With Arterial Stiffness in a Female Sample From Reproductive Years to Menopause.

Objective: Loss of sex hormones has been suggested to underlie menopause-associated increment in cardiovascular risk. We investigated associations of sex hormones with arterial stiffness in 19-58-years-old women. We also studied associations of specific hormonal stages, including natural menstrual cycle, cycle with combined oral contraceptives (COC) and menopausal status with or without hormone therapy (HT), with arterial stiffness. Methods: This study includes repeated measurements of 65 healthy women representing reproductive (n=16 natural, n=10 COC-users) and menopause (n=5 perimenopausal, n=26 postmenopausal, n=8 HT-users) stages. Arterial stiffness outcomes were aortic pulse wave velocity (PWVao) and augmentation index (AIx%) assessed using Arteriograph-device. Generalized estimating equation models were constructed to investigate associations of each hormone (wide age-range models) or hormonal stage (age-group focused models) with arterial stiffness. PWVao models with cross-sectional approach, were adjusted for age, relative fitness, fat mass and mean arterial pressure, while models with longitudinal approach were adjusted for mean arterial pressure. AIx% models used the same approach for adjustments and were also adjusted for heart rate. Results: Negative and positive associations with arterial stiffness variables were observed for estradiol and follicle-stimulating hormone, respectively, until adjustment for confounding effect of age. In naturally menstruating women, AIx% was higher at ovulation (B=3.63, p<0.001) compared to the early follicular phase. In COC-users, PWVao was lower during active (B=-0.33 - -0.57, p<0.05) than inactive pills. In menopausal women, HT-users had higher PWVao (B=1.43, p=0.03) than postmenopausal non-HT-users. Conclusions: When using wide age-range assessments covering reproductive to menopausal lifespan it is difficult to differentiate age- and hormone-mediated associations, because age-mediated influence on arterial stiffness seemed to overrule potential hormone-mediated influences. However, hormonal status associated differentially with arterial stiffness in age-group focused analyses. Thus, the role of sex hormones cannot be excluded. Further research is warranted to resolve potential hormone-mediated mechanisms affecting arterial elasticity.

Serum Amyloid A Is Present in Human Saccular Intracranial Aneurysm Walls and Associates With Aneurysm Rupture.

Saccular intracranial aneurysm (sIA) rupture leads to a disabling subarachnoid hemorrhage. Chronic inflammation and lipid accumulation in the sIA wall contribute to wall degenerative remodeling that precedes its rupture. A better understanding of the pathobiological process is essential for improved future treatment of patients carrying sIAs. Serum amyloid A (SAA) is an acute-phase protein produced in response to acute and chronic inflammation and tissue damage. Here, we studied the presence and the potential role of SAA in 36 intraoperatively resected sIAs (16 unruptured and 20 ruptured), that had previously been studied by histology and immunohistochemistry. SAA was present in all sIAs, but the extent of immunopositivity varied greatly. SAA immunopositivity correlated with wall degeneration (p = 0.028) and rupture (p = 0.004), with numbers of CD163-positive and CD68-positive macrophages and CD3-positive T lymphocytes (all p < 0.001), and with the expression of myeloperoxidase, matrix metalloproteinase-9, prostaglandin E-2 receptor, and cyclo-oxygenase 2 in the sIA wall. Moreover, SAA positivity correlated with the accumulation of apolipoproteins A-1 and B-100. In conclusion, SAA occurs in the sIA wall and, as an inflammation-related factor, may contribute to the development of a rupture-prone sIA.

Triglyceride-Rich Lipoproteins as a Source of Proinflammatory Lipids in the Arterial Wall.

Apolipoprotein B -containing lipoproteins include triglyceride-rich lipoproteins (chylomicrons and their remnants, and very low-density lipoproteins and their remnants) and cholesterol-rich low-density lipoprotein particles. Of these, lipoproteins having sizes below 70-80 nm may enter the arterial wall, where they accumulate and induce the formation of atherosclerotic lesions. The processes that lead to accumulation of lipoprotein-derived lipids in the arterial wall have been largely studied with a focus on the low-density lipoprotein particles. However, recent observational and genetic studies have discovered that the triglyceriderich lipoproteins and their remnants are linked with cardiovascular disease risk. In this review, we describe the potential mechanisms by which the triglyceride-rich remnant lipoproteins can contribute to the development of atherosclerotic lesions, and highlight the differences in the atherogenicity between low-density lipoproteins and the remnant lipoproteins.

Macrophage Infiltration in the Saccular Intracranial Aneurysm Wall as a Response to Locally Lysed Erythrocytes That Promote Degeneration.

Saccular intracranial aneurysm (sIA) rupture is often fatal. Rupture-prone sIA walls are infiltrated by macrophages expressing hemoglobin-receptor CD163, suggesting a role for erythrocyte lysis in the degenerative remodeling predisposing to rupture. We therefore studied erythrocyte remnants in 16 unruptured and 20 ruptured sIA walls using histology and immunohistochemistry. Glycophorin A (GPA), an erythrocyte membrane protein, was present in 34/36 (94%) sIA walls and correlated with loss of αSMA+ cells, reflecting loss of mural smooth muscle cells ([SMCs]; r = -0.592, p < 0.001), wall degeneration (p = 0.008), and rupture (p = 0.005). GPA correlated with high numbers of CD163+ and CD68+ phagocytes (r = 0.65 and r = 0.54, p ≤ 0.001 for both). CD163+ phagocytes were mostly HLA-DR-. Interestingly, single SMCs expressed HLA-DR and also CD163 was expressed in sporadic SMCs, which may reflect their response to hemoglobin accumulation. GPA associated with iron (p = 0.014) was detectable by MRI. An additional 11 sIAs were therefore imaged ex vivo with a 4.7 T MRI prior to histology. In the sIA walls, high GPA and iron accumulation associated with signal intensity in T1-weighted gradient echo MRI. We conclude that accumulation of lysed erythrocytes is a potential driver of inflammatory response in the sIA walls and is associated with the degenerative wall remodeling, thereby predisposing to rupture.

Myeloperoxidase Associates With Degenerative Remodeling and Rupture of the Saccular Intracranial Aneurysm Wall.

Rupture of a saccular intracranial aneurysm (sIA) is often fatal. Thus, early detection of rupture-prone sIAs is vital. Myeloperoxidase (MPO), derived mainly from neutrophils, associates with sIA rupture, and therefore its role in sIA pathogenesis warrants further studies. We analyzed MPO and its association with other histological markers in 36 (16 unruptured and 20 ruptured) sIA samples by immunohistochemistry. MPO was present in all studied sIAs, and its expression associated with wall inflammatory cell infiltrations (r = 0.50, 0.63, and 0.75, all p ≤ 0.002), degenerative remodeling (p = 0.002) and rupture (p = 0.003). MPO associated strongly with the presence of organized luminal thrombi (p < 0.001), which also stained positive for MPO. Polymorphonuclear MPO+ cells were detected in the sIA walls, indicating neutrophils as MPO-source. MPO correlated strongly with accumulation of oxidized lipids (r = 0.67, p < 0.001) and loss of smooth muscle cells (r = -0.68, p < 0.001), suggesting that MPO is a relevant source of oxidative stress leading to cell death in the sIA wall. Furthermore, MPO associated with erythrocyte fragmentation (r = 0.74, p < 0.001) and iron deposition (p = 0.041), 2 outcomes known to amplify MPO-dependent oxidative stress. Taken together, these results suggest that MPO associates with degenerative remodeling predisposing to sIA wall rupture and may serve as a biomarker of a rupture-prone sIA wall.

Extracellular Lipids Accumulate in Human Carotid Arteries as Distinct Three-Dimensional Structures and Have Proinflammatory Properties.

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

Smooth Muscle Cell Foam Cell Formation, Apolipoproteins, and ABCA1 in Intracranial Aneurysms: Implications for Lipid Accumulation as a Promoter of Aneurysm Wall Rupture.

Saccular intracranial aneurysm (sIA) aneurysm causes intracranial hemorrhages that are associated with high mortality. Lipid accumulation and chronic inflammation occur in the sIA wall. A major mechanism for lipid clearance from arteries is adenosine triphosphate-binding cassette A1 (ABCA1)-mediated lipid efflux from foam cells to apolipoprotein A-I (apoA-I). We investigated the association of wall degeneration, inflammation, and lipid-related parameters in tissue samples of 16 unruptured and 20 ruptured sIAs using histology and immunohistochemistry. Intracellular lipid accumulation was associated with wall remodeling (p = 0.005) and rupture (p = 0.020). Foam cell formation was observed in smooth muscle cells, in addition to CD68- and CD163-positive macrophages. Macrophage infiltration correlated with intracellular lipid accumulation and apolipoproteins, including apoA-I. ApoA-I correlated with markers of lipid accumulation and wall degeneration (p = 0.01). ApoA-I-positive staining colocalized with ABCA1-positive cells particularly in sIAs with high number of smooth muscle cells (p = 0.003); absence of such colocalization was associated with wall degeneration (p = 0.017). Known clinical risk factors for sIA rupture correlated inversely with apoA-I. We conclude that lipid accumulation associates with sIA wall degeneration and risk of rupture, possibly via formation of foam cells and subsequent loss of mural cells. Reduced removal of lipids from the sIA wall via ABCA1-apoA-I pathway may contribute to this process.

Spatial distributions of lipids in atherosclerosis of human coronary arteries studied by time-of-flight secondary ion mass spectrometry.

The accurate spatial distribution of various lipid species during atherogenesis has remained unexplored. Herein, we used time-of-flight secondary ion mass spectrometry (TOF-SIMS) to analyze the lipid distribution in human coronary artery cryosections. The images from the TOF-SIMS allowed visualization of ions derived from individual species of cholesterol esters, phospholipids, and triacylglycerols in the context of lesion characteristics and severity. In addition, cholesterol-containing crystal-like structures were seen in high-resolution images of advanced lesions. The ratio of cholesterol fragment ions (m/z 385:m/z 369) was found to differentiate unesterified cholesterol from cholesterol esters. This ratio changed during atherogenesis and in different areas of the lesions, reflecting differences in the accumulation of the two forms of cholesterol. Thus, atheromas were characterized by accumulation of cholesterol esters with apolipoprotein B near the intima-media border, whereas in the complicated lesions, unesterified cholesterol dominated in neovessel-containing areas enriched in glycophorin A. Interestingly, triacylglycerols were found in areas surrounding neovessels and lacking either form of cholesterol. The lipid composition of the tunica media reflected the alterations observed in the intimal lipids, yet being more subtle. The detailed molecular information obtained by TOF-SIMS revealed unanticipated differences in the type and composition of the accumulating lipids in different stages of atherogenesis, notably the spatial segregation of cholesterol and triglycerides in the advancing lesions.

Mast cells, neovascularization, and microhemorrhages are associated with saccular intracranial artery aneurysm wall remodeling.

Chronic inflammation contributes to remodeling, degeneration, and rupture of saccular intracranial artery aneurysms. Mast cells are important proinflammatory and proangiogenic cells in chronic inflammatory vascular diseases. Here we studied mast cells and neovascularization in 36 intraoperatively resected aneurysms using histology and immunohistochemistry and analyzed the clinical characteristics of the aneurysms according to bleeding status (unruptured vs ruptured). Among the 36 aneurysms, 9 contained mast cells (tryptase-positive cells) and 15 contained neovessels (CD34- and CD31-positive capillarylike structures). The density of neovessels was significantly higher in aneurysm walls containing mast cells than in walls not containing them. In particular, wall areas with abundant mast cells and neovessels also contained iron deposits, indicating damage of newly formed endothelium with ensuing microhemorrhages. Walls with the highest neovessel density and the greatest iron deposition also showed evidence of degeneration. Finally, none of the mast cell-containing aneurysms showed an intact luminal endothelium. Thus, mast cells may adversely affect both neovascular and luminal endothelia. The novel association of mast cells with neovessels and injurious microhemorrhages, as well as with luminal endothelial erosion, suggests that mast cells contribute to remodeling and degeneration of saccular intracranial artery aneurysms.

Modified lipoprotein-derived lipid particles accumulate in human stenotic aortic valves.

In aortic stenosis plasma lipoprotein-derived lipids accumulate in aortic valves. Here, we first compared the lipid compositions of stenotic aortic valves and atherosclerotic plaque cores. Both pathological tissues were found to be enriched in cholesteryl linoleate, a marker of extracellularly accumulated lipoproteins. In addition, a large proportion of the phospholipids were found to contain arachidonic acid, the common precursor of a number of proinflammatory lipid mediators. Next, we isolated and characterized extracellular lipid particles from human stenotic and non-stenotic control valves, and compared them to plasma lipoproteins from the same subjects. The extracellular valvular lipid particles were isolated from 15 stenotic and 14 non-stenotic aortic valves. Significantly more apoB-100-containing lipid particles were found in the stenotic than in the non-stenotic valves. The majority of the lipid particles isolated from the non-stenotic valves had sizes (23±6.2 nm in diameter) similar to those of plasma low density lipoprotein (LDL) (22±1.5 nm), while the lipid particles from stenotic valves were not of uniform size, their sizes ranging from 18 to more than 500 nm. The lipid particles showed signs of oxidative modifications, and when compared to isolated plasma LDL particles, the lipid particles isolated from the stenotic valves had a higher sphingomyelin/phosphatidylcholine -ratio, and also higher contents of lysophosphatidylcholine and unesterified cholesterol. The findings of the present study reveal, for the first time, that in stenotic human aortic valves, infiltrated plasma lipoproteins have undergone oxidative and lipolytic modifications, and become fused and aggregated. The generated large lipid particles may contribute to the pathogenesis of human aortic stenosis.

Vascular cell kinetics in response to intimal injury ex vivo.

BACKGROUND: Recent studies indicate that the smooth muscle-like cells contributing to neointimal hyperplasia after vascular injury derive from circulating precursor cells. Here, we define the time course of precursor cell influx, the roles of separate vascular layers, and the relative role of migration versus proliferation to intimal hyperplasia. METHODS AND RESULTS: After rat aortic denudation injury the neointimal cell number increased several 100-fold between days 4 and 28, preceded by a 5-fold increase in the number of adventitial cells and a 4-fold increase in the number of adventitial microvessels. The influx, migration, and maturation of neointimal cells were quantitated by culturing whole vessel explants at different time points after injury. Explant outgrowth increased 14-fold, and cell migration 3.5-fold on days 2-14 after injury. Cell proliferation increased less than 2-fold. The frequency of precursors to outgrowing cells, determined using limiting dilution analysis, increased 8-fold between days 2 and 4 after injury. Many outgrowing cells displayed characteristics of undifferentiated cells. CONCLUSIONS: Adventitial activation precedes development of the neointima, and precursor cell influx occurs on days 2-14 after injury. Cell migration, more than proliferation, contributes to fibrointimal dysplasia. These findings underline the importance of early therapeutic intervention with antimigratory compounds to prevent neointimal hyperplasia.

Caveolar transport through nasal epithelium of birch pollen allergen Bet v 1 in allergic patients.

BACKGROUND: Previous work in type I pollen allergies has focused on aberrant immunoresponses. OBJECTIVE: Our systems-level analyses explore the role of epithelium in early pathogenesis of type I allergic reactions. METHODS: We began top-down analyses of differences in human nasal epithelial cells and biopsy specimens obtained from patients with birch allergy and healthy control subjects in the resting state and after intranasal in vivo birch pollen challenges. Immunohistochemistry, immunotransmission electron microscopy, mass spectrometry, transcriptomics, and integration of data to a pathway were conducted. RESULTS: Bet v 1 allergen bound to epithelium immediately after in vivo birch pollen challenge during winter only in allergic individuals. It also travelled through epithelium with caveolae to mast cells. Sixteen unique proteins were found to bind to the Bet v 1 column only in lysates from allergic epithelial cells; 6 of these were caveolar and 6 were cytoskeletal proteins. The nasal epithelial transcriptome analysis from allergic and healthy subjects differed during the winter season, and these subjects also responded differentially to birch pollen challenge. Within this pollen-induced response, the gene ontology categories of cytoskeleton and actin cytoskeleton were decreased in allergic patients, whereas the actin-binding category was enriched in healthy subjects. Integration of microscopic, mass spectrometric, and transcriptomic data to a common protein-protein binding network showed how these were connected to each other. CONCLUSION: We propose a hypothesis of caveolae-dependent uptake and transport of birch pollen allergen in the epithelium of allergic patients only. Application of discovery-driven methodologies can provide new hypotheses worth further analysis of complex multifactorial diseases, such as type I allergy.

Mechanisms behind the synergistic effect of sirolimus and imatinib in preventing restenosis after intimal injury.

BACKGROUND: We have shown that the combination of sirolimus and imatinib synergistically inhibits denudation-induced neointimal hyperplasia in rats. We have now dissected the mechanisms behind this synergy and evaluated its long-term efficacy. METHODS: After aortic denudation injury, rats received established submaximal doses of sirolimus (1.0 mg/kg/day), imatinib (10.0 mg/kg/day), the combination of these, or vehicle per os from 3 days before the operation until 14 days after injury. Vessel histology and complete blood counts were monitored until 90 days after injury. Neointimal cell outgrowth, migration and proliferation were evaluated in ex vivo vessel cultures. Quantitative real-time polymerase chain reaction and immunohistochemistry were used for gene and protein expression analysis. RESULTS: The combination therapy caused a synergistic decrease in the number of neointimal nuclei and area throughout the observation period. It also prevented postinjury thrombocytosis and leukocytosis, and almost abolished neointimal cell outgrowth and migration. Furthermore, the combination therapy resulted in upregulation of smooth muscle cell (SMC) markers SM22alpha and cysteine and glycine-rich protein 2, and of the anti-apoptotic BCL2 mRNA. CONCLUSIONS: Combination therapy confers superior long-term vasculoprotection, possibly by inhibition of postoperative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury site and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation.

Synergistic suppression of rat neointimal hyperplasia by rapamycin and imatinib mesylate: implications for the prevention of accelerated arteriosclerosis.

BACKGROUND: Accelerated arteriosclerosis remains a major limitation to therapeutic interventions such as angioplasty, stent deployment, and solid organ transplantation. Rapamycin, a powerful new immunosuppressant set to replace calcineurin inhibitors in the transplant setting, and imatinib mesylate, a receptor tyrosine kinase inhibitor, are both angioprotective. Here, we explored the pharmacological and therapeutic interactions of these two agents in a rat model of neointimal hyperplasia. METHODS: Wistar rats, subjected to balloon catheter-induced aortic injury, received daily drug treatment until postoperative day 14 and were subsequently sacrificed or followed up to day 40 without further treatment. Development of neointimal lesions was assessed histologically and immunohistochemically. Steady-state rapamycin levels in whole blood were determined by HPLC-UV. RESULTS: Rapamycin and imatinib, administered individually or in combination, produced no signs of overt toxicity. Continuous postoperative therapy with either rapamycin (0.5-1.5 mg/kg/day) or imatinib (2- 50 mg/kg/day) dose-dependently suppressed neointimal hyperplasia on day 14. Combined treatment (0.5 or 1 + 10 mg/kg/day, respectively) showed a trend towards synergistic action on day 14. Withdrawal of medication on day 14 nullified the early therapeutic effect of either agent by day 40. In contrast, early combination therapy (1 + 10 mg/kg/day) achieved long-term suppression of neointimal hyperplasia by approximately 81%. Notably, coadministration of imatinib appeared to reduce exposure to rapamycin, although this finding did not reach statistical significance. CONCLUSIONS: Short-term combination therapy with rapamycin and imatinib is well tolerated and produces synergistic, sustained suppression of neointimal hyperplasia in rats. Subject to clinical evaluation, this new drug regimen may afford definitive prophylaxis against accelerated arteriosclerosis.