Leukotriene B4-Mediated Neutrophil Recruitment Causes Pulmonary Capillaritis during Lethal Fungal Sepsis.

Candida albicans bloodstream infection causes fungal septicaemia and death in over half of afflicted patients. Polymorphonuclear leukocytes (PMN) mediate defense against invasive candidiasis, but their role in protection versus tissue injury and sepsis is unclear. We observe PMN intravascular swarming and subsequent clustering in response to C. albicans yeast in a lethal septic mouse and human pulmonary circulation model. Live C. albicans sequester to the endothelium and are immediately captured by complement-dependent PMN chemotaxis, which is required for host survival. However, complement activation also leads to Leukotriene B4 (LTB4)-mediated intravascular PMN clustering and occlusion, resulting in capillaritis with pulmonary hemorrhage and hypoxemia. This clustering is unique to fungi and triggered by fungal cell wall components. PMN clustering is absent in mice lacking LTB4-receptor, and capillaritis is attenuated upon pharmacological LTB4 blockade without affecting phagocytosis. Therefore, therapeutically disrupting infection-induced capillaritis may limit organ injury without impairing host defense during fungal sepsis.

Diagnosis of catastrophic anti-phospholipid syndrome in a patient tested negative for conventional tests.

Catastrophic antiphospholipid syndrome (CAPS) is a severe variant of APS, characterised by clinical evidence of multiple organ involvement developing over a very short period of time, histopathological evidence of multiple small vessel occlusions and laboratory confirmation of the presence of aPL (lupus anticoagulant and/or anticardiolipin antibodies and/or anti-Beta2-glcyoprotein I antibodies). Here we report a case of a 39-year-old woman patient who developed a CAPS which was negative to the conventional aPL but positive for aPL in thin layer chromatography immunostaining and vimentin/cardiolipin antibodies by ELISA test. The patient was treated with high doses of glucocorticoids, intravenous immunoglobulins plasma exchange and immunoadsorbent apheresis with a significant improvement of the ischaemic lesions of the hands even though the necrosis of the feet progressively worsened. As a result, the patient underwent partial surgical amputation of the feet. To our knowledge, this is the first ever reported case of CAPS diagnosed by means of thin layer chromatography immunostaining and vimentin/cardiolipin antibody ELISA test.

Elevated levels of soluble CD40 ligand are associated with antiphospholipid antibodies in patients with systemic lupus erythematosus.

OBJECTIVES: The CD40L/CD40 pathway is involved in the pathophysiology of atherothrombotic disease, and elevated levels of soluble CD40L (sCD40L) were reported in SLE patients. However, the clinical implication of sCD40L in SLE remains elusive. METHODS: We measured levels of plasma sCD40L in 241 SLE patients and 37 healthy controls and investigated its association with clinical manifestation and laboratory parameters. RESULTS: Levels of plasma sCD40L in SLE patients were significantly elevated compared with healthy controls (p=0.013) and positively correlated with levels of soluble P-selectin (γ=0.336, p<0.001). SLE patients who experienced arterial thrombosis had a higher level of sCD40L than those who did not (p=0.029). Plasma sCD40L levels were positively correlated with the titers of anti-cardiolipin and anti-ß2 glycoprotein I antibodies (γ=0.338, p<0.001 and γ=0.364, p<0.001, respectively). Its levels were also significantly higher in patients with clinical antiphospholipid syndrome (APS) than in non-APS patients, irrespective of antiphospholipid antibody (aPL) positivity. Of those with arterial thrombosis, sCD40L levels were significantly elevated in patients with positive aPL, compared to those with negative aPL (p=0.011). Multiple regression analysis revealed that the presence of hypertension and positive aPL were independently associated with the occurrence of arterial thrombosis in SLE patients. A parallel analysis showed that sCD40L was also an independent variable for arterial thrombosis; however, this association disappeared when aPL, a strong variable, was included in the model because of collinearity between aPL and sCD40L. CONCLUSIONS: Plasma sCD40L levels were elevated in SLE patients who had positive aPL and experienced arterial thrombosis, suggesting that enhanced release of sCD40L through platelet activation presumably by aPL could contribute to the development of atherothrombotic disease.

Neutrophil/Lymphocyte ratio and association with arch intervention in patients with hypoplastic left heart syndrome undergoing hybrid procedure.

BACKGROUND: Hybrid procedure is an alternative initial palliation for patients with hypoplastic left heart syndrome (HLHS). One major complication with this procedure is stenosis in the aortic arch isthmus possibly due to inflammation from the patent ductus arteriosus (PDA) stent. In adult studies, neutrophil/lymphocyte (N/L) ratio has been used as a marker for increased inflammation and has been associated with increased risk for coronary artery stent stenosis. The goal of this study was to determine if there were differences in N/L ratio between patients with HLHS undergoing hybrid procedure that required an arch intervention (AI-Group) vs. those that did not require an intervention (NAI-Group). METHODS: Retrospective chart review was performed on patients with HLHS undergoing hybrid procedure between July 2002 and January 2013. Complete blood counts as well as differentials were recorded at four time periods: 1 day prehybrid palliation, one day posthybrid palliation, 1 week posthybrid palliation, and 3 weeks posthybrid palliation. RESULTS: One hundred six patients were evaluated (AI-Group = 38, NAI-Group = 68). AI-Group generally had a higher N/L ratio vs. NAI-group and this was significant immediately 1 day posthybrid palliation: AI-Group vs. NAI-Group, prehybrid (2.95 ± 2.62 vs. 2.44 ± 1.71), 1 day posthybrid (5.95 ± 4.16 vs. 4.34 ± 3.87, P < .05), 1 week posthybrid (2.72 ± 3.01 vs. 2.28 ± 2.12), and 3 weeks posthybrid (1.85 ± 1.24 vs. 1.45 ± 1.16), respectively. Lymphocyte percentage was significantly lower in the AI-Group vs. NAI-Group 3 weeks posthybrid palliation (33.00 + 11.30% vs. 40.65 + 16.82%). CONCLUSION: Patients that required an arch intervention after hybrid palliation had a higher N/L ratio immediately after the procedure. This may signify increased inflammatory reaction that places these patients at risk for stenosis. Future studies are needed to determine if N/L ratio is a robust marker to risk stratify patients undergoing the hybrid procedure for arch complications.

Serial analysis of clinical and imaging indices reveals prolonged efficacy of TNF-α and IL-6 receptor targeted therapies in refractory Takayasu arteritis.

OBJECTIVES: We analysed a large cohort of patients with Takayasu arteritis, seeking robust clinical evidence for prolonged responses to tumour necrosis factor-α (TNF-α) and interleukin-6 receptor (IL-6R) antagonists in severe refractory disease. METHODS: Case notes from ninety-eight patients with Takayasu arteritis were retrospectively reviewed. Drug treatment, laboratory and serial non-invasive imaging data were analysed, and the Indian Takayasu arteritis activity (ITAS) and damage scores (TADs) calculated. RESULTS: Nine patients were treated with biologic therapies. All had previously received high dose prednisolone and ≥1 conventional immunosuppressant. Five patients had failed cyclophosphamide. The patients prescribed biologics had more extensive arterial injury than the remainder of the cohort and persistent active disease (ITAS range 2-9, CRP 12-206 mg/L, TADs 3--1). Eight patients were prescribed anti-TNF-α therapy, three IL-6R blockade. The mean duration of anti-TNF-α treatment was 42 months (maximum 8 years). One patient developed new arterial stenoses while receiving anti-TNF-α and subsequently achieved disease remission with tocilizumab. Two patients have now demonstrated sustained responses to IL-6R inhibition at 19 and 20 months. Following introduction of biologic therapy, serial non-invasive imaging has revealed no significant progression in arterial injury. A significant fall in CRP (p<0.01), prednisolone dose (p<0.01) and ITAS (p<0.01) was observed, with no increase in TADs. CONCLUSIONS: We report for the first time sustained responses to both anti-TNF-α and IL6R antagonists in refractory Takayasu arteritis. As 5/9 patients were cyclophosphamide non-responders, we propose that biologics should now be considered ahead of cyclophosphamide in these young patients.

Hospital based prospective longitudinal clinical and immunologic study of 179 patients of primary anti-phospholipid syndrome.

AIM: To study the clinical and immunological features of primary antiphospholipid syndrome (APS), and to analyze the differences between primary APS and APS associated with autoimmune rheumatic disease (ARD/APS). METHODS: This prospective, longitudinal study, carried out from December 2004 to July 2011 included 179 patients with primary APS and 52 patients of ARD/APS diagnosed as per modified 2006 Sapporo's Criteria. RESULTS: Out of 179 patients of primary APS, 12 were male and 167 were female. The mean age at the time of study entry was 27 ± 4.33 years. Venous thrombosis was noted in 33 (18.43%) patients. Seventeen patients had deep vein thrombosis and 11 (7.19%) had cortical vein and/or cortical sinus thrombosis. Arterial thrombosis was noted in 19 (10.61%) patients, out of which nine had intracranial arterial thrombosis. Thirty-two (17.85%) had recurrent early fetal losses (< 10 weeks) and 97 (54.18%) had late fetal loss (> 10 weeks). Immunoglobulin G (IgG) and IgM aCLA were present in 141 (78.77%) and 32 (17.87%) patients respectively, whereas lupus anticoagulant was present in 99 (55.3%) patients. In patients with bad obstetric outcome, lupus anticoagulant positivity was significantly more prevalent (P < 0.05) than aCLA positivity. Both venous and arterial thrombosis were significantly more common (P < 0.05) in ARD/APS. However, late fetal loss was significantly more prevalent (P < 0.001) in primary APS. CONCLUSION: Primary APS may lead to a variety of clinical manifestations due to venous and/or arterial thrombosis, which at times may be lethal. It is also an important cause of early and late pregnancy loss(es) and other pregnancy morbidities.

Castleman's disease and arterial thrombosis: result of excessively elevated interleukin-6 plasma level?

Morbus Castleman is a benign non-clonal lymphoproliverative disorder. Immunomodulatory and antiproliferative drugs are used to treat this plasma cell disorder. We report the case of a 46-year old female patient with multicentric Castleman's disease and limb ischemia. Thrombotic occlusions of the popliteal and tibioperoneal arteries were treated by percutaneous thrombus aspiration. We discuss the role of increased interleukin-6 plasma levels during therapy with Tocilizumab, an antibody to interleukin-6 receptor, as a potential cause for arterial thrombosis.

Annexin A5 prevents post-interventional accelerated atherosclerosis development in a dose-dependent fashion in mice.

BACKGROUND: Activated cells in atherosclerotic lesions expose phosphatidylserine (PS) on their surface. Annexin A5 (AnxA5) binds to PS and is used for imaging atherosclerotic lesions. Recently, AnxA5 was shown to inhibit vascular inflammatory processes after vein grafting. Here, we report a therapeutic role for AnxA5 in post-interventional vascular remodeling in a mouse model mimicking percutaneous coronary intervention (PCI). METHODS AND RESULTS: Associations between the rs4833229 (OR = 1.29 (CI 95%), p(allelic) = 0.011) and rs6830321 (OR = 1.35 (CI 95%), p(allelic) = 0.003) SNPs in the AnxA5 gene and increased restenosis-risk in patients undergoing PCI were found in the GENDER study. To evaluate AnxA5 effects on post-interventional vascular remodeling and accelerated atherosclerosis development in vivo, hypercholesterolemic ApoE(-/-) mice underwent femoral arterial cuff placement to induce intimal thickening. Dose-dependent effects were investigated after 3 days (effects on inflammation and leukocyte recruitment) or 14 days (effects on remodeling) after cuff placement. Systemically administered AnxA5 in doses of 0.1, 0.3 and 1.0mg/kg compared to vehicle reduced early leukocyte and macrophage adherence up to 48.3% (p = 0.001) and diminished atherosclerosis development by 71.2% (p = 0.012) with a reduction in macrophage/foam cell presence. Moreover, it reduced the expression of the endoplasmic reticulum stress marker GRP78/BiP, indicating lower inflammatory activity of the cells present. CONCLUSIONS: AnxA5 SNPs could serve as markers for restenosis after PCI and AnxA5 therapeutically prevents vascular remodeling in a dose-dependent fashion, together indicating clinical potential for AnxA5 against post-interventional remodeling.

Ulnar artery occlusion is predictive of digital ulcers in SSc: a duplex sonography study.

OBJECTIVES: To assess the prevalence and risk factors of ulnar artery occlusion (UAO) in an unselected SSc patient cohort and to determine whether UAO is associated with digital ulcers (DUs). METHODS: A total of 79 SSc patients and 40 'healthy' controls underwent colour duplex sonography of the radial and ulnar artery to compare blood flow velocity, resistive indices (RIs) and presence of occlusion and were followed for a mean of 53 months. RESULTS: In both, radial and ulnar arteries, peak systolic velocity (PSV) and end-diastolic velocity (EDV) were significantly lower and RI higher in SSc patients compared with controls (PSVrad: 40.1 vs 48.6 cm/s; PSVuln 38.2 vs 56.6 cm/s; EDVrad 3.8 vs 10.4 cm/s; EDVuln 3.0 vs 13.0 cm/s; RIrad 0.91 vs 0.82; RIuln 0.92 vs 0.80; all P < 0.01). Seventeen (21.5%) SSc patients had UAO (11 patients bilateral) compared with none in the control subjects. Patients with UAO had a significantly longer disease duration (170 vs 66 months, P < 0.001). At baseline, the prevalence of DU was not different in upper extremities with UAO [8/28 (28.6%)] compared with upper extremities without UAO [36/129 (27.9%)]. However, during follow-up new or recurrent DU occurred more often in upper extremities with UAO than in those without UAO [14/28 (50%) vs 24/113 (21.2%); relative risk (RR) = 2.4; 95% CI 1.4, 3.7; P = 0.002]. CONCLUSION: Blood flow is significantly decreased in radial and ulnar arteries in SSc. UAO is frequent and an important risk factor for the development of DUs in patients with SSc.

Microarray applications in occlusive vascular disease.

Interventional cardiology procedures and drug therapy have been widely applied for the treatment of occlusive vascular disease. However, there remains a critical lack of understanding of the disease process at a molecular level. Microarray technology has the unique advantage in the ability to analyze thousands of genes simultaneously. So far, several studies based on microarray analysis have already provided valuable expression data in diseases such as atherosclerosis and in-stent stenosis. This review summarizes: a) latest microarray research indentifying gene-expression profiles; b) the methodological analysis of the available microarray studies; c) generation of biological processes or pathways; d) detection of better diagnostic and therapeutic targets in atherosclerosis and in-stent stenosis. Further improvements in microarray interpretation as well as in study design, combined with definition and evaluation in the clinical arena, will enhance our understanding of the causes and mechanisms contributing to occlusive vascular diseases, and therefore will help to improve treatment of patients suffering from these diseases.

Targeted deletion of the inhibitory NF-kappaB p50 subunit in bone marrow-derived cells improves collateral growth after arterial occlusion.

AIMS: Adaptive collateral artery growth (arteriogenesis) is an important mechanism to maintain tissue perfusion upon arterial obstruction. Leucocytes and inflammatory mediators play a crucial role in this process. Depletion of the nuclear factor kappa B (NF-κB) p50 subunit modulates inflammatory processes in cardiovascular disease. We hypothesized that NF-κB p50 is a regulator of the inflammatory response after arterial occlusion and subsequent collateral perfusion. METHODS AND RESULTS: Unilateral femoral artery ligation was performed in NF-κB p50-/- and wild-type (Wt, B6/129PF2) mice. Seven days after arterial occlusion, tissue perfusion restoration was significantly enhanced in NF-κB p50-/- mice compared with Wt mice (42.9 ± 3.9 vs. 32.0 ± 2.6% perfusion recovery, P = 0.04). Transplantation of NF-κB p50-/- bone marrow (bm) into Wt mice and vice versa showed that the effect of p50 subunit depletion can be predominantly attributed to the bone marrow-derived circulating cells (NF-κB p50-/- bm in Wt mice 42.1 ± 1.5%, Wt bm in NF-κB p50-/- mice 35.4±1.5% perfusion recovery). Histological analyses revealed a more elaborate extravasation of monocytes in hindlimb tissue of NF-κB p50-/- mice. Chemotaxis assays confirmed the increased migration ability of NF-κB p50-/- monocytes, which may be due to an observed increased integrin expression. Upon stimulation of blood from NF-κB p50-/- and Wt mice more interleukin-6 was produced, confirming the pro-inflammatory phenotype in absence of the p50 subunit. CONCLUSION: Depletion of the NF-κB p50 subunit enhances collateral artery growth. Its absence in circulating cells improves tissue perfusion restoration after femoral artery ligation by increasing macrophage influx into the growing collateral vessels.

Free flap failure in an anticardiolipin antibody-positive patient with neoplasm--a case report.

We present herein a case of massive arterial thrombosis of a free rectus abdominal musculocutaneous flap used for reconstructive surgery of gingival carcinoma that could not be rescued. A 54-year-old woman underwent the operation. She had experienced two miscarriages in her 20s, but medical history was otherwise uneventful. Intraoperatively, the anastomosed artery often showed massive arterial thrombosis, and the flaps had become necrotic after bilateral flaps were used. Laboratory findings, 7 days postoperatively, showed high levels of immunoglobulin G anticardiolipin antibody. This value normalized by 2 months postoperatively after using chemotherapy. This case does not match the criteria for antiphospholipid syndrome, but some English-language reports have shown rising antiphospholipid antibody levels, particularly anticardiolipin antibodies, in patients with neoplasm. In those cases, levels have normalized after successful therapy. Antiphospholipid antibody levels should be examined before surgery to identify risks of hypercoagulability.

Is obstetric antiphospholipid syndrome a primary nonthrombotic, proinflammatory, complement-mediated disorder related to antiphospholipid antibodies?

UNLABELLED: Pregnancy loss is the main obstetrical complication of the obstetric antiphospholipid syndrome. Classically, such losses have been attributed to placental thrombosis and infarcts, although in many cases there is no evidence of decidual thrombosis or placental vasculopathy, and instead inflammatory signs are present. In addition, the prevalence of systemic thrombosis is low in obstetric antiphospholipid syndrome, suggesting an alternative pathogenesis. There is evidence that antiphospholipid antibodies, mainly beta2-glycoprotein-I/anti-beta2-glycoproteina-I complexes, activate both classical and alternative complement pathways. Complement proteins may injure trophoblast cells, recruiting and activating monocytes and neutrophils. Free radicals and proteolytic enzymes could also attack trophoblastic cells, and amplification of the causal loop between tissue factor, inflammatory cells, and complement proteins could also be a factor. Overall, these diverse mechanisms may explain both inflammatory and thrombotic placental alterations. The role played by certain pro-inflammatory cytokines, mainly tumor necrosis factor-alpha, and the altered balance between angiogenic and anti-angiogenic factors remains to be clarified. In the end, obstetric antiphospholipid syndrome seems to be a clinical subset of classical APS. In these women, systemic thrombotic risk seems to be low. Current knowledge about inflammatory pathway involvement in obstetric antiphospholipid syndrome will permit us to modify the time to start heparin treatment, currently recommended to begin it as soon as possible after pregnancy confirmation. TARGET AUDIENCE: Obstetricians & Gynecologists, Family Physicians. LEARNING OBJECTIVES: After completion of this article, the reader will be able to recall manifestations of obstetric antiphospholipid syndrome, describe nonthrombotic mechanisms that may affect obstetric outcomes in women with antiphospholipid syndrome, and predict changes in the evaluation and treatment of obstetric patients with antiphospholipid syndrome should inflammatory factors prove to be an important feature of antiphospholipid syndrome.

Autoantibodies to heat shock protein 60 promote thrombus formation in a murine model of arterial thrombosis.

BACKGROUND AND OBJECTIVES: Anti-heat shock protein (HSP)60 autoantibodies are associated with atherosclerosis and are known to affect endothelial cells in vitro. However, their role in thrombus formation remains unclear. We hypothesized that anti-HSP60 autoantibodies could potentiate thrombosis, and evaluated the effect of anti-murine HSP60 antibodies in a ferric chloride (FeCl3)-induced murine model of carotid artery injury. METHODS: Anti-HSP60, or control, IgG was administered to BALB/c mice 48 h prior to inducing carotid artery injury, and blood flow was monitored using an ultrasound probe. RESULTS: Thrombus formation was more rapid and stable in anti-HSP60 IGG-treated mice than in controls (blood flow=1.7%+/-0.6% vs. 34%+/-12.6%, P=0.0157). Occlusion was complete in all anti-HSP60 IgG-treated mice (13/13), with no reperfusion being observed. In contrast, 64% (9/14) of control mice had complete occlusion, with reperfusion occurring in 6/9 mice. Thrombi were significantly larger in anti-HSP60 IgG-treated mice (P=0.0001), and contained four-fold more inflammatory cells (P=0.0281) than in controls. Non-injured contralateral arteries of anti-HSP60 IgG-treated mice were also affected, exhibiting abnormal endothelial cell morphology and significantly greater von Willebrand factor (VWF) and P-selectin expression than control mice (P=0.0024 and P=0.001, respectively). CONCLUSIONS: In summary, the presence of circulating anti-HSP60 autoantibodies resulted in increased P-selectin and VWF expression and altered cell morphology in endothelial cells lining uninjured carotid arteries, and promoted thrombosis and inflammatory cell recruitment in FeCl3-injured carotid arteries. These findings suggest that anti-HSP60 autoantibodies may constitute an important prothrombotic risk factor in cardiovascular disease in human vascular disease.

Anticardiolipin antibodies associated with alcoholic cardiomyopathy.

Anticardiolipin antibodies are associated with arterial and venous thrombosis, and repetitive miscarriages. The involvement of the heart has been described frequently and can evolve into cardiomyopathy. It has been known for some decades that chronic alcoholism can lead to alcoholic cardiomyopathy (ACM). The objective of this study was to evaluate whether anticardiolipin antibodies represent a worse prognosis for patients with ACM. The authors present a case of a chronic alcoholic patient (30 y of alcoholism) who died at 44 y of age, and who was considered positive for anticardiolipin antibodies. The patient developed deep vein thrombosis, and peripheral arterial and pulmonary embolism. The presence of another risk factor seems to represent a worse prognosis for patients with ACM.

The role of chemokines in transplant graft arterial disease.

Despite the development of effective immunosuppressive therapy, transplant graft arterial disease (GAD) remains the major limitation to long-term graft survival. Multiple immune and nonimmune risk factors contribute to this vasculopathic intimal hyperplastic process. Thus, initial interplay between host inflammatory cells and donor endothelial cells triggers alloimmune responses, whereas alloantigen-independent factors such as prolonged ischemia, surgical manipulation, ischemia-reperfusion injury, and hyperlipidemia enhance the antigen-dependent events. Intrinsic to all stages of this process are chemokines, a family of 8- to 10-kDa proteins mediating directional migration of immune cells to sites of inflammation and injury. Beyond their role in immune-cell chemotaxis, chemokines also contribute to cellular activation, vascular remodeling, and angiogenesis. Expression of chemokines and their cognate receptors in allografts correlates with acute organ rejection, as well as GAD. Moreover, chemokine or chemokine receptor blockade prolongs graft survival and attenuates GAD in experimental models. Further studies will likely confirm a substantial utility for antichemokine therapy in human organ transplantation.

Deficiency of tumour necrosis factor-alpha and interferon-gamma in bone marrow cells synergistically inhibits neointimal formation following vascular injury.

AIMS: Neointimal formation after percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since it is now known that vascular injury involves an inflammatory response, we examined the role of tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in the neointimal formation after injury. METHODS AND RESULTS: Control (BALB/c), TNF-alpha-deficient (Tnf(-/-)), IFN-gamma-deficient (Ifng(-/-)), or double-deficient (Tnf(-/-)Ifng(-/-)) mice were subjected to wire-mediated vascular injury of the right femoral artery. Neointimal formation after injury was significantly reduced after the injury in the Tnf(-/-)Ifng(-/-) mice, compared to that in the control, Tnf(-/-), and Ifng(-/-) mice. Immunohistochemical analysis showed that TNF-alpha and IFN-gamma were expressed in neointimal lesions in the control mice, but not in mice with deficiency of the corresponding cytokine. No significant difference in re-endothelialization was observed among these groups. The number of proliferating cell nuclear antigen in the neointimal lesions was significantly decreased in the Tnf(-/-)Ifng(-/-) mice. Bone marrow transplantation experiments revealed that deficiency of TNF-alpha and IFN-gamma specifically in bone marrow cells significantly inhibited neointimal formation after vascular injury. CONCLUSION: The absence of TNF-alpha and IFN-gamma in bone marrow cells synergistically inhibits neointimal formation following vascular injury, and thus, may provide new insights into the mechanisms underlying restenosis after PCI.