Distinguishing Intrapulmonary Immune Cells from Intravascular Immune Cell Populations: the Intrajugular Approach.

Circadian rhythms refer to oscillations in various biological process that occur with a 24 h period. At the molecular level, such rhythms are comprised of a web of transcriptional-translational feedback loops (TTFL) of core clock genes. Individual tissues and organ systems, including the immune system, have their own clock. In the systemic circulation, various members of the CD45+ population oscillate across the day; however, many of these rhythms are not identical or even similar in the tissue resident CD45+ leukocyte population. When studying the role of circadian regulation of lung inflammation, CD45+ within the lung may need to be investigated. However, despite optimized perfusion methods, leukocytes trapped from the circulation persist in the lungs. The goal in designing this protocol was to distinguish between intravascular and intraparenchymal leukocytes. Towards this end, mice are injected with a fluorescent tagged CD45 antibody intrajugularly shortly before lung harvest. Thereafter, the lung is digested using a customized lung digestion technique to obtain a single cell suspension. The sample is stained for the regular panel of antibodies for intraparenchymal immune cells (including another CD45 antibody). Flowcytometric analyses shows a clear elucidation of the populations. Thus, the method of labeling and defining intrapulmonary CD45+ cells will be particularly important where the behavior of intrapulmonary and circulating immune cells are numerically and functionally distinct.

Anti-inflammatory and anti-proliferative effects of CBS3830 in arterialized vein grafts in rats.

OBJECTIVE: To investigated whether CBS3830, a highly selectively inhibitor of p38MAPK, could ameliorate inflammation and intimal hyperplasia in arterialized vein grafts (AVGs). METHODS: Sixty male Sprague-Dawley rats underwent a reversed right jugular vein to common carotid artery interposition graft and were randomly treatment with vehicle (control) or single-dose (3 mg/kg, preoperative) or double-dose (3 mg/kg, preoperative and 4 d postoperative) CBS3830. Twenty rats underwent sham operation. The levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6) were determined by ELISA. Vein grafts were analyzed by intimal/medial morphometry, proliferating cell nuclear antigen (PCNA) expression, and p38MAPK phosphorylation. RESULTS: TNF-α, IL-1ß, and IL-6 gradually increased then slowly decreased in AVG rats. However, at 4 d and 7 d, TNF-α levels decreased by 37.5% and 29.5% (p = 0.003, 0.05, respectively) in the single-dose CBS3830 group, and by 37.6% and 32.5%, respectively (both p = 0.003) in the double-dose group compared with those of control. IL-1ß levels significantly reduced at 4 d and 14 d in both dosage groups. IL-6 levels significantly reduced at 7 d in both groups. Intima and medial thickening were significantly reduced in both dosage treated groups at 7, 14, and 28 d (all p = 0.000) compared to the controls. Further study showed CBS3830 inhibited p38MAPK phosphorylation and decreased PCNA expression. CONCLUSIONS: CBS3830 significantly decreases inflammation and intimal hyperplasia in AVGs.

Inflammation modulates murine venous thrombosis resolution in vivo: assessment by multimodal fluorescence molecular imaging.

OBJECTIVE: Assessment of thrombus inflammation in vivo could provide new insights into deep vein thrombosis (DVT) resolution. Here, we develop and evaluate 2 integrated fluorescence molecular-structural imaging strategies to quantify DVT-related inflammation and architecture and to assess the effect of thrombus inflammation on subsequent DVT resolution in vivo. METHODS AND RESULTS: Murine DVT were created with topical 5% FeCl(3) application to thigh or jugular veins (n=35). On day 3, mice received macrophage and matrix metalloproteinase activity fluorescence imaging agents. On day 4, integrated assessment of DVT inflammation and architecture was performed using confocal fluorescence intravital microscopy. Day 4 analyses showed robust relationships among in vivo thrombus macrophages, matrix metalloproteinase activity, and fluorescein isothiocyanate-dextran deposition (r>0.70; P<0.01). In a serial 2-time point study, mice with DVT underwent intravital microscopy at day 4 and day 6. Analyses revealed that the intensity of thrombus inflammation at day 4 predicted the magnitude of DVT resolution at day 6 (P<0.05). In a second approach, noninvasive fluorescence molecular tomography-computed tomography was used and detected macrophages within jugular DVT (P<0.05 versus sham controls). CONCLUSIONS: Integrated fluorescence molecular-structural imaging demonstrates that the DVT-induced inflammatory response can be readily assessed in vivo and can inform the magnitude of thrombus resolution.

Veins are no arteries: even moderate arterial pressure induces significant adhesion molecule expression of vein grafts in an ex vivo circulation model.

AIM: Coronary artery bypass grafting (CABG) is a standard procedure for treatment of coronary heart disease. Eighty percent of all CABGs are performed with venous grafts which then get exposed to an arterial pressure after surgery. This widely used procedure, however, is complicated by the development of alterations in the vein graft wall, leading to a decreased patency rate and graft failure. This study enlightens the influence of an even moderate arterial pressure on the gene expression of adhesion molecules in venous grafts which play a decisive role for the early induction of atherogenesis. METHODS: Segments of porcine vena jugularis and arteria carotis were mounted in a simulated bypass circuit and subjected to pulsatile flow. Vessel segments were examined for adhesion molecule expression with quantitative real-time - polymerase chain reaction (qRT-PCR) and adherence of leukocytes was observed by confocal laser scanning microscopy and scanning electron microscopy. RESULTS: Veins grafts subjected to an even moderate arterial pressure showed a 14-fold increase of ICAM-1 expression already after 4 hours. An arterial pressure of around 100/80 mmHg was enough to stimulate the adhesion molecule expression Furthermore it led to a 9-fold increase of leukocyte adhesion to the venous endothelium, but, in contrast this was not the case in arteries. CONCLUSION: This study showed, that already 100 mmHg upregulates the expression of several adhesion molecules in pig veins followed by increased adhesion of leukocytes. Therefore, our data demonstrate the advantage of arteries for CABG, and that new therapeutic strategies are urgently necessary to protect vein grafts either physically or pharmacologically if arteries are not available for CABG.

Inflammatory responses of tissue-engineered xenografts in a clinical scenario.

Acellular tissue-engineered (ATE) xenografts and homografts are used in clinical cardiovascular surgery. The present study examined the specific role of carbohydrate antigen (α-Gal and T-antigen) in immune response after decellularisation in tissue-engineered xenografts (porcine pulmonary artery and bovine jugular vein). An enzyme-linked immunosorbent assay (ELISA) was used to ascertain whether implantation of bioprostheses, ATE xenografts and mechanical valve replacement result in augmentation of anti-α-Gal IgM antibodies within eight days of surgery (each group, n=6). Kinetics of host inflammatory response on surgically explanted ATE xenografts was also studied. Immunostaining for α-Gal and T-antigen detected the presence of them in the native tissue but they were absent in processed ATE xenografts from the same tissue. A significant increase in the concentration of anti-α-Gal IgM antibodies was observed in the serum of bioprosthetic valve recipients as compared to ATE xenograft recipients (P<0.05). Organised collagen, and decreased inflammatory response with increase in endothelisation and vascularisation was evident beyond one year of surgery as compared to early periods in ATE xenografts. This study demonstrates that decellularisation of xenografts and further processing of these tissues enabled reduction of inflammatory stimulus with autologous recellularisation with no calcification.

Matrix metalloproteinase expression in vein grafts: role of inflammatory mediators and extracellular signal-regulated kinases-1 and -2.

Matrix metalloproteinases (MMPs) play key roles in vascular remodeling. We characterized the role of inflammatory mediators and extracellular signal-regulated kinases (ERKs) in the control of arterialized vein graft expression of MMP-9, MMP-2, and membrane-type 1-MMP (MT1-MMP) and of the tissue inhibitor of metalloproteinases-2 (TIMP-2). For this purpose we used a canine model of jugular vein to carotid artery interposition graft and analyzed the vein grafts at various postoperative times (30 min to 28 days) using the contralateral vein as a control. To study the role of ERK-1/2, veins were incubated with the mitogen-activated protein kinase kinase (MEK-1/2) inhibitor UO126 for 30 min before being grafted. Vein graft extracts were analyzed for MMPs, TIMP-2, tumor necrosis factor-alpha (TNF-alpha), polymorphonuclear neutrophil (PMN) infiltration, myeloperoxidase (MPO), and thrombin activity, and for ERK-1/2 activation. Vein graft arterialization resulted in rapid and sustained (8 h to 28 days) upregulation of vein graft-associated MMP-9, MMP-2, MT1-MMP, thrombin activity, and TNF-alpha levels with concomitant TIMP-2 downregulation. MMP-2 activation preceded MT1-MMP upregulation. PMN infiltration and vein graft-associated MPO activity increased within hours after arterialization, indicating a prompt, local inflammatory response. In cultured smooth muscle cells, both thrombin and TNF-alpha upregulated MT1-MMP expression; however, only thrombin activated MMP-2. Inhibition of ERK-1/2 activation blocked arterialization-induced upregulation of MMP-2, MMP-9, and MT1-MMP. Thus, thrombin, inflammatory mediators, and activation of the ERK-1/2 pathway control MMP and TIMP-2 expression in arterialized vein grafts.

[Immunogenicity of bovine jugular vein conduits treated with different cross-linking methods].

OBJECTIVE: To evaluate the immunogenicity of bovine jugular vein conduits (BJVCs) treated with different cross-linking methods. METHODS: The BJVCs were treated with glutaradehyde (GA), dye-mediated photooxidation (DMP) and polyepoxy compound (PC) (n = 10). The tissue homogenates obtained from BJVCs treated with PC, GA, DMP, and fresh BJVCs, were mixed with the complete Freand adjavant to form the emulsive antigen, which were used to immunize rabbits correspondently. The antibody concentrations to BJVCs in those rabbits' serum were measured by counter double immuno diffusion. The immunologic responses to the BJVCs in different groups were measured with Western blotting. RESULTS: The positive bands appeared when the sera of rabbits were immunized by fresh BJVCs reacted with antigens of fresh BJVCs, but no bands appeared when the sera of rabbits were immunized by fresh BJVCs reacted with those antigens of the BJVCs treated with GA, DMP, and PC in Western blotting. CONCLUSION: The immunogenicity of BJVCs treated with PC, DMP, and GA can be reduced significantly and meet the clinical standard.

Wall shear modulation of cytokines in early vein grafts.

OBJECTIVE: Pro-inflammatory cytokine-driven mechanisms have been implicated in vein graft failure, though little is known about the effect of hemodynamic factors and anti-inflammatory counter-regulatory mechanisms. We hypothesized that early temporal expression of the pro-inflammatory cytokine interleukin (IL)-1 beta and the anti-inflammatory cytokine IL-10 proceeds by way of wall shear stress-dependent pathways in the arterializing vein graft. METHODS: Rabbits (n = 27) underwent bilateral jugular vein carotid interposition grafts, and simultaneous unilateral distal carotid branch ligation, to produce both low-flow and high-flow grafts in the same animal. Vein grafts were harvested at 1, 3, 7, 14, and 28 days and were assessed for architecture, wall shear stress, and cytokine messenger RNA levels (quantitative real-time two-step reverse transcription polymerase chain reaction). RESULTS: The model resulted in an immediate 90% flow reduction (P <.001, paired t test) in the vein graft on the ligated side, and a 36% increase (P =.01) in contralateral graft flow. This persisted as approximately 15-fold flow differential throughout the 28-day period. The construction yielded a 15-fold differential in wall shear stress between low-flow and high-flow vein grafts (P <.001, two-way repeated measures analysis of variance). Intimal hyperplasia began by day 3, and was 6-fold more in low wall shear grafts by 28 days (230.6 +/- 35.4 microm intimal thickness vs 36.1 +/- 17.6 microm for low shear versus high shear grafts; P =.001). For both cytokines time independently affected mRNA expression (P <.001, global analysis of variance). Exposure of vein grafts to the arterial circulation markedly up-regulated IL-1 beta at 1 day, with significantly more induction in the low shear setting (P =.002). IL-1 beta protein localized to the developing neointima at days 1 and 3. Conversely, IL-10 slowly increased until day 14, with significantly more expression in the high shear grafts (P <.001). CONCLUSIONS: Vein graft adaptation induces early pro-inflammatory cytokine IL-1 beta expression and delayed protective IL-10 expression (most notable under high shear conditions), both of which are modulated by wall shear. These differential temporal windows offer strategies for appropriately timed pro-inflammatory or anti-inflammatory therapies to interrupt pathologic vein graft adaptations. CLINICAL RELEVANCE: Neointimal hyperplasia continues to limit the durability of vein bypass grafts. Emerging evidence suggests that inflammatory mechanisms drive the neointimal hyperplasic response. This study demonstrates that specific hemodynamic forces (altered wall shear stress) differentially affect early pro-inflammatory interleukin (IL)-1 beta and delayed anti-inflammatory IL-10 signaling. These distinct temporal windows for IL-1 beta and IL-10 cytokine expression offer strategies for appropriately timed pro-inflammatory and anti-inflammatory therapies to interrupt pathologic vein graft adaptations.

Upper jugular lymph nodes (submuscular recess) in non-squamous-cell cancer of the head and neck: surgical considerations.

Cervical lymphadenectomy of level II encompasses lymph nodes associated with the upper internal jugular vein and the spinal accessory nerve (SAN). Removal of tissue superior to the SAN (submuscular recess-(SMR)) was recently shown to be unwarranted in selected cases of squamous-cell cancer. Thirty-five patients with non-squamous-cell cancer (SCC) of the head and neck treated with cervical lymphadenectomy were prospectively evaluated. Thirty-seven neck dissection specimens were histologically analysed for the number of lymph nodes involved with cancer. At the time of surgery, level II was separated into the supraspinal accessory nerve component (IIa) and the component anterior to the SAN (IIb). Neck dissections were most commonly performed for cancer of the thyroid gland (19) followed in frequency by the parotid gland (seven), skin: melanoma (five), basal-cell cancer (two), and other sites (four). Twenty-five neck dissections were modified-selective procedures and 12 were either radical or modified radical neck dissection. Twenty-nine necks were clinically N+ and eight N0. Histological staging was pathologically N+ in 32 neck dissection specimens. Level IIb contained an average of 12 nodes and the IIa component contained a mean of 5.0 nodes. Level II contained metastatic disease in 28 of 32 histologically node-positive specimens (87 per cent). Level IIa was involved with cancer in six cases (16 per cent), five of which were pre-operatively staged as clinically N+. All cases (100 per cent) with level IIa involvement had level IIb positive nodes. Three of the level IIa positive cases were cancer of the parotid gland comprising 43 per cent of this sub-group of patients. Incidence of involvement of SMR in non-SCC cases is not uncommon. The additional time required and morbidity associated with dissection of the supraspinal accessory nerve component of level II are probably justified when performing neck dissection in cancer of the thyroid gland. The SMR should be excised in cancer of the parotid gland. Large-scale prospective controlled studies with long-term follow-up periods are necessary to support resection of level IIb only.

Influence of species, environmental factors, and tissue cellularity on calcification of porcine aortic wall tissue.

Valve tissue calcification has complex host, implant, and mechanical determinants. We studied the influence of species (rat v sheep), environmental factors (presence v absence of blood contact and arterial stress), and tissue cellularity (normal v acellularized tissue) on porcine aortic wall mineralization. Porcine aortic wall samples underwent standard glutaraldehyde-fixation or combined enzyme-detergent acellularization. Samples were implanted subcutaneously in rats (n = 8) and in juvenile sheep (n = 8). Furthermore, in juvenile sheep, similar samples were implanted into the jugular vein (blood contact) and into the carotid artery (blood contact and arterial stress). After 8 and 12 weeks, tissue was explanted and evaluated by X-ray, light- and electron-microscopy, and calcium content measurement (atomic absorption spectrometry). On the Von Kossa staining, auto-fluorescence of elastic fibers was used to identify the relation between calcific deposits and elastin. Subcutaneously implanted, glutaraldehyde-fixed tissue calcified severely in rat, but much less in sheep (calcium content: 56.2 +/- 13.6 v 9.9 +/- 9.0 microg/mg, respectively; P <.001). In sheep, the presence of blood contact (venous implants) increased wall calcification significantly (36.9 +/- 15.8; P <.001), but hemodynamic stress (arterial implants) had no additional mineralizing effect on the aortic wall (P >.05 v venous implants). Calcification of glutaraldehyde-fixed tissue occurred predominantly at the level of cells and cellular remnants, as confirmed by electron- and fluorescence-microscopy, locating calcific deposits in between elastic fibers. Acellularized tissue calcified significantly less, but an inflammatory response towards the tissue led to fragmentation, lysis, and subsequent calcification of elastic fibers. Results from subcutaneous implantations show large inconsistencies in calcification between the species. In sheep, blood contact increases aortic wall calcification significantly, while arterial stress has no additional effect. The sheep-jugular implantation model can be used as a simplified model for further study of aortic wall calcification and new antimineralization treatments. Calcification of glutaraldehyde-fixed aortic wall tissue is initiated at the level of cellular remnants, with little or no contribution from elastic fibers. Acellularization can avoid this cell-mediated calcification, but an additional treatment (glutaraldehyde, cryopreservation, photo-fixation,.) will be necessary to avoid the inflammation leading to elastolysis and consequent calcification of elastic fibers.