Mikael Maksimow

Distribution and Medical Impact of Loss-of-Function Variants in the Finnish Founder Population.

Exome sequencing studies in complex diseases are challenged by the allelic heterogeneity, large number and modest effect sizes of associated variants on disease risk and the presence of large numbers of neutral variants, even in phenotypically relevant genes. Isolated populations with recent bottlenecks offer advantages for studying rare variants in complex diseases as they have deleterious variants that are present at higher frequencies as well as a substantial reduction in rare neutral variation. To explore the potential of the Finnish founder population for studying low-frequency (0.5–5%) variants in complex diseases, we compared exome sequence data on 3,000 Finns to the same number of non-Finnish Europeans and discovered that, despite having fewer variable sites overall, the average Finn has more low-frequency loss-of-function variants and complete gene knockouts. We then used several well-characterized Finnish population cohorts to study the phenotypic effects of 83 enriched loss-of-function variants across 60 phenotypes in 36,262 Finns. Using a deep set of quantitative traits collected on these cohorts, we show 5 associations (p<5×10−8) including splice variants in LPA that lowered plasma lipoprotein(a) levels (P = 1.5×10−117). Through accessing the national medical records of these participants, we evaluate the LPA finding via Mendelian randomization and confirm that these splice variants confer protection from cardiovascular disease (OR = 0.84, P = 3×10−4), demonstrating for the first time the correlation between very low levels of LPA in humans with potential therapeutic implications for cardiovascular diseases. More generally, this study articulates substantial advantages for studying the role of rare variation in complex phenotypes in founder populations like the Finns and by combining a unique population genetic history with data from large population cohorts and centralized research access to National Health Registers.

Siglec-9 is a novel leukocyte ligand for vascular adhesion protein-1 and can be used in PET imaging of inflammation and cancer

Leukocyte migration to sites of inflammation is regulated by several endothelial adhesion molecules. Vascular adhesion protein-1 (VAP-1) is unique among the homing-associated molecules as it is both an enzyme that oxidizes primary amines and an adhesin. Although granulocytes can bind to endothelium via a VAP-1-dependent manner, the counter-receptor(s) on this leukocyte population is(are) not known. Here we used a phage display approach and identified Siglec-9 as a candidate ligand on granulocytes. The binding between Siglec-9 and VAP-1 was confirmed by in vitro and ex vivo adhesion assays. The interaction sites between VAP-1 and Siglec-9 were identified by molecular modeling and confirmed by further binding assays with mutated proteins. Although the binding takes place in the enzymatic groove of VAP-1, it is only partially dependent on the enzymatic activity of VAP-1. In positron emission tomography, the ⁶⁸Gallium-labeled peptide of Siglec-9 specifically detected VAP-1 in vasculature at sites of inflammation and cancer. Thus, the peptide binding to the enzymatic groove of VAP-1 can be used for imaging conditions, such as inflammation and cancer.

Clever‐1/Stabilin‐1 regulates lymphocyte migration within lymphatics and leukocyte entrance to sites of inflammation

Clever‐1/Stabilin‐1 is a scavenger receptor present on lymphatic and sinusoidal endothelium as well as on a subset of type II macrophages. It is also induced on vasculature at sites of inflammation. However, its in vivo function has remained practically unknown and this work addresses those unknown aspects. We demonstrate using in vivo models that Clever‐1/Stabilin‐1 mediates migration of T and B lymphocytes to the draining lymph nodes in vivo and identify the adhesive epitope of the Clever‐1/Stabilin‐1 molecule responsible for the interaction between lymphocytes and lymphatic endothelium. Moreover, we demonstrate that Ab blocking of Clever‐1/Stabilin‐1 efficiently inhibits peritonitis in mice by decreasing the entrance of granulocytes by 50%, while migration of monocytes and lymphocytes into the inflamed peritoneum is prevented almost completely. Despite efficient anti‐inflammatory activity the Ab therapy does not dramatically dampen immune responses against the bacterial and foreign protein Ag tested and bacterial clearance. These results indicate that anti‐Clever‐1/Stabilin‐1 treatment can target two different arms of the vasculature – traffic via lymphatics and inflamed blood vessels.

Circulating cytokines in predicting development of severe acute pancreatitis

IntroductionSevere acute pancreatitis (AP) is associated with high morbidity and mortality. Early prediction of severe AP is needed to improve patient outcomes. The aim of the present study was to find novel cytokines or combinations of cytokines that can be used for the early identification of patients with AP at risk for severe disease.MethodsWe performed a prospective study of 163 nonconsecutive patients with AP, of whom 25 had severe AP according to the revised Atlanta criteria. Admission serum levels of 48 cytokines and growth factors were determined using Bio-Plex Pro Human Cytokine Assay 21-plex and 27-plex magnetic bead suspension panels. Admission plasma levels of C-reactive protein (CRP), creatinine and calcium were measured for comparison. In subgroup analyses, we assessed the cytokine profiles of patients with severe AP (n = 14) who did not have organ dysfunction (OD) upon admission (modified Marshall score <2).ResultsOf 14 cytokines elevated in the severe AP group, interleukin 6 (IL-6) and hepatocyte growth factor (HGF) levels were independent prognostic markers of severe AP. IL-6, HGF and a combination of them predicted severe AP with sensitivities of 56.0%, 60.0% and 72.0%, respectively, and specificities of 90.6%, 92.8% and 89.9%, respectively. The corresponding positive likelihood ratio (LR+) values were 5.9, 8.3 and 7.1, respectively. The predictive values of CRP, creatinine and calcium were comparable to those of the cytokines. In subgroup analyses of patients with severe AP and without OD upon admission, we found that IL-8, HGF and granulocyte colony-stimulating factor (G-CSF) levels predicted the development of severe AP, with G-CSF being the most accurate cytokine at a sensitivity of 35.7%, a specificity of 96.1% and a LR+ of 9.1.ConclusionsIL-6 and HGF levels upon admission have prognostic value for severe AP which is similar to levels of CRP, creatinine and calcium. Although IL-6 and HGF, as either single or combined markers, were not perfect in identifying patients at risk for severe AP, the possibility that combining them with novel prognostic markers other than cytokines might improve prognostic accuracy needs to be studied. The accuracy of IL-8, HGF and G-CSF levels in predicting severe AP in patients without clinical signs of OD upon admission warrants larger studies.

Ly6C supports preferential homing of central memory CD8(+) T cells into lymph nodes

Ly6C is a murine cell‐surface antigen expressed by plasma cells, subsets of myeloid cells and many T cells, including memory T cells. We previously documented that Ly6C crosslinking induces LFA‐1 clustering on naïve CD8+ T cells. Here, we show that in vitro and in vivo differentiation of naïve CD8+ T cells into central (Tcm) but not effector (Tem) memory T cells enhances Ly6C expression, and its crosslinking induces strong LFA‐1 clustering on Tcm. Blocking Ly6C function inhibits in vivo Tcm homing to LNs as efficiently as blocking L‐selectin but it does not potentiate the inhibition provided by blocking either L‐selectin or LFA‐1 function. Thus, Ly6C, L‐selectin and LFA‐1 all appear to be part of a common homing pathway. In vitro, Ly6C crosslinking enhances Tcm adherence to ICAM‐1 in the presence of CCL21. In summary, Tcm homing involves Ly6C, in addition to L‐selectin and LFA‐1, and appears to potentiate firm adhesion of Tcm to ICAM‐1 in synergy with a chemokine. We propose that Ly6C augments Tcm compartmentalization into LNs during their homing.

Fas costimulation of naïve CD4 T cells is controlled by NF-κB signaling and caspase activity

Fas ligation induces apoptosis of activated T cells via the caspase cascade but can also mediate costimulatory signals to naïve T cells at the time of activation. We have previously shown that Fas ligation of naïve CD4 T cells activated by dendritic cells induces death or accelerates their proliferation and increases interferon‐γ (IFN‐γ) production. To understand this costimulation, we investigated the roles of caspases and nuclear factor (NF)‐κB in survival and proliferation of responding T cells. Fas ligation increased caspase‐3 and ‐8 activities during T cell activation, irrespective of cell fate. The accelerated proliferation induced by Fas ligation could be reduced by selective inhibition of both caspases. Inhibition of NF‐κB simultaneously with Fas ligation inhibited the increased IFN‐γ production and caused uniform death of all responding T cells. Thus, Fas‐mediated costimulation of naïve CD4 T cells is driven by active caspases, and NF‐κB acts as a dominant survival‐supporting factor of Fas‐costimulated cells containing high levels of activated caspase‐8 and ‐3.

Soluble Vascular Adhesion Protein-1 Correlates With Cardiovascular Risk Factors and Early Atherosclerotic Manifestations

Objective—Vascular adhesion protein-1 is an endothelial enzyme that regulates leukocyte traffic and contributes to vascular damage in animal models. The relations of soluble vascular adhesion protein-1 (sVAP-1) with cardiovascular risk factors and markers of subclinical atherosclerosis at a population level have not been studied. Methods and Results—We developed a new high-throughput method and measured sVAP-1 activities in serum of 2183 persons (The Cardiovascular Risk in Young Finns Study). In women, sVAP-1 activity correlated indirectly with body mass index (r=−0.15, P<0.0001), triglycerides (r=−0.13, P<0.0001), C-reactive protein (r=−0.23; P<0.0001), and brachial artery flow-mediated vasodilatation (r=−0.076, P=0.0089) and directly with carotid plaques (r=0.066, P=0.023). None of these correlations was significant in men. In women, all these univariate correlations remained significant after adjustment for body mass index, and direct correlations with LDL-cholesterol (r=0.094, P=0.0014) and carotid intima-media thickness (r=0.075, P=0.010) became evident. In men, sVAP-1 activity associated directly with glucose (r=0.074, P=0.020), intima-media thickness (r=0.072, P=0.025), metabolic syndrome (P=0.016), and type 1 (P=0.0002) and type 2 (P<0.0001) diabetes. In multivariable analyses, sVAP-1 activity was an independent determinant of carotid intima-media thickness (P=0.0072) and plaques [odds ratio 1.71 (95% confidence interval 1.07–2.72, P=0.025] in women, but not in men. Conclusion—sVAP-1 activity correlates directly with intima-media thickness and carotid plaques in general population and may play a role in the pathophysiology of preclinical atherosclerosis.

Stabilin-1/CLEVER-1, a type 2 macrophage marker, is an adhesion and scavenging molecule on human placental macrophages

Stabilin‐1/common lymphatic endothelial and vascular endothelial receptor‐1 (CLEVER‐1) is a multidomain protein present in lymphatic and vascular endothelial cells and type 2 immunosuppressive macrophages. In adults, stabilin‐1/CLEVER‐1 is a scavenging receptor and an adhesion molecule, but much less is known about its role during development. Here, we studied the expression and functions of macrophage stabilin‐1/CLEVER‐1 in human placenta and during human ontogeny. Using newly generated mAbs, we found that stabilin‐1/CLEVER‐1 is expressed on virtually all macrophages in term placenta, both in the decidua and in the placental villi. Placental stabilin‐1/CLEVER‐1 was involved in the scavenging of Ac‐LDL (acetylated low density lipoprotein) and in the uptake of fluorescently labeled model antigen OVA. siRNA‐mediated suppression of stabilin‐1/CLEVER‐1 altered the cytokine profile produced by placental macrophages. Stabilin‐1/CLEVER‐1 on placental macrophages mediated their adhesion to placental vessels and supported their transmigration through vascular endothelium. Finally, we found that stabilin‐1/CLEVER‐1 is induced very early in fetal macrophages, high endothelial venules, and lymphatic vessels in multiple lymphatic organs. Together, these data suggest that macrophage stabilin‐1/CLEVER‐1 can potentially regulate leukocyte migration and scavenging during the development of the placenta and fetus.

G-CSF regulates macrophage phenotype and associates with poor overall survival in human triple-negative breast cancer

ABSTRACT Tumor-associated macrophages (TAMs) have been implicated in the promotion of breast cancer growth and metastasis, and a strong infiltration by TAMs has been associated with estrogen receptor (ER)-negative tumors and poor prognosis. However, the molecular mechanisms behind these observations are unclear. We investigated macrophage activation in response to co-culture with several breast cancer cell lines (T47D, MCF-7, BT-474, SKBR-3, Cal-51 and MDA-MB-231) and found that high granulocyte colony-stimulating factor (G-CSF) secretion by the triple-negative breast cancer (TNBC) cell line MDA-MB-231 gave rise to immunosuppressive HLA-DRlo macrophages that promoted migration of breast cancer cells via secretion of TGF-α. In human breast cancer samples (n = 548), G-CSF was highly expressed in TNBC (p < 0.001) and associated with CD163+ macrophages (p < 0.0001), poorer overall survival (OS) (p = 0.021) and significantly increased numbers of TGF-α+ cells. While G-CSF blockade in the 4T1 mammary tumor model promoted maturation of MHCIIhi blood monocytes and TAMs and significantly reduced lung metastasis, anti-CSF-1R treatment promoted MHCIIloF4/80hiMRhi anti-inflammatory TAMs and enhanced lung metastasis in the presence of high G-CSF levels. Combined anti-G-CSF and anti-CSF-1R therapy significantly increased lymph node metastases, possibly via depletion of the so-called “gate-keeper” subcapsular sinus macrophages. These results indicate that G-CSF promotes the anti-inflammatory phenotype of tumor-induced macrophages when CSF-1R is inhibited and therefore caution against the use of M-CSF/CSF-1R targeting agents in tumors with high G-CSF expression.

Early prediction of persistent organ failure by soluble CD73 in patients with acute pancreatitis

Objective:New biomarkers are needed to better predict the severity of acute pancreatitis. CD73/ecto-5’-nucleotidase is an enzyme that generates adenosine, which dampens inflammation and improves vascular barrier function in several disease models. CD73 also circulates in a soluble form in the blood. We studied whether levels of soluble form of CD73 predict the development of organ failure in acute pancreatitis. Design:A prospective cohort study of patients with acute pancreatitis from 2003 to 2007. Setting:Admissions to the biggest tertiary care hospital in Finland. Patients:One hundred sixty-one patients with acute pancreatitis, of which 107 were subclassified according to the revised Atlanta criteria into mild, 29 into moderately severe and 25 into severe. Interventions:None. Measurements and Main Results:Serum and blood cell samples were collected at admission. Protein levels of soluble form of CD73 in serum were determined using a novel enzyme-linked immunosorbent assay, activity of soluble form of CD73 using radioactive enzyme assays, and CD73 messenger RNA levels from leukocytes using quantitative polymerase chain reaction. Activity and protein concentration of soluble form of CD73, and messenger RNA level of CD73 all decreased along with the disease severity (p ⩽ 0.01 for all). The activity of soluble form of CD73 at admission predicted the development of the severe pancreatitis in different groups of the patients. The area under the receiver-operating characteristic curve value for activity of soluble form of CD73 was 0.65 (95% CI, 0.51-0.80) among a subgroup of patients comprising moderately severe and severe disease, 0.79 (95% CI, 0.69-0.88) among all patients including mild pancreatitis, and 0.75 (95% CI, 0.60-0.89) among patients who had no signs of organ failure (modified Marshall score < 2) at admission. Especially, in the last-mentioned group, activity of soluble form of CD73 was better than C-reactive protein or creatinine in predicting the severe pancreat Conclusions:Activity of soluble form of CD73 at admission to hospital has prognostic value in predicting the development of the severe form of acute pancreatitis.