Natalia Kučić

Metallothioneins and heat shock proteins 70 in marine mussels as sensors of environmental pollution in Northern Adriatic Sea

In an attempt to assess the intensity of environmental pollution in industrial zones of Kvarnerian Bay in Northern Adriatic Sea and the reactivity of Mytilus galloprovincialis to these changes, in this study we estimated the concentration of heavy metals at four locations in both sea-sediment and in the mussels. Further we tried to correlate these changes with seasonal variations in environmental temperature, pH and salinity, as well as with the expression of metallothioneins (MTs) and heat shock proteins (HSPs) in the digestive tract of the mussels. Sampling in vivo was performed monthly, during the year 2008, while under the laboratory conditions the reactivity of acclimated mussels were tested to increasing concentrations of CdCl(2) and to thermal stress. The data have shown that the induction of MTs and HSP isoforms of the 70-kDa size class were highly affected by model agents treatment including contamination of sea-sediment by Pb, Hg and Cd, implying that these stress proteins might be power biomarkers of marine pollution.

Neuroimmunomodulative properties of dipeptidyl peptidase IV/CD26 in a TNBS‐induced model of colitis in mice

Causal connections between dipeptidyl peptidase IV, also known as CD26 molecule (DPP IV/CD26) and inflammatory bowel disease (IBD) have been shown, but mechanisms of these interactions are unclear. Our hypothesis was that DPP IV/CD26 could affect the neuroimmune response during inflammatory events. Therefore, we aimed to evaluate its possible role and the relevance of the gut–brain axis in a model of IBD in mice. Trinitrobenzenesulfonic acid‐induced (TNBS) colitis was induced in CD26‐deficient (CD26−/−) and wild‐type (C57BL/6) mice. Pathohistological and histomorphometrical measurements were done. Concentrations and protein expressions of DPP IV/CD26 substrates neuropeptide Y (NPY) and vasoactive intestinal peptide (VIP) were determined. Concentrations of IL‐6 and IL‐10 were evaluated. Investigations were conducted at systemic and local levels. Acute inflammation induced increased serum NPY concentrations in both mice strains, more enhanced in CD26−/− mice. Increased NPY concentrations were found in colon and brain of C57BL/6 mice, while in CD26−/− animals only in colon. VIP and IL‐6 serum and tissue concentrations were increased in both mice strains in acute inflammation, more pronouncedly in CD26−/− mice. IL‐10 concentrations, after a decrease in serum of both mice strains, increased promptly in CD26−/− mice. Decreased IL‐10 concentration was found in brain of C57BL/6 mice, while it was increased in colon of CD26−/− mice in acute inflammation. DPP IV/CD26 deficiency affects the neuroimmune response at systemic and local levels during colitis development and resolution in mice. Inflammatory changes in the colon reflected on investigated parameters in the brain, suggesting an important role of the gut–brain axis in IBD pathogenesis. J. Cell. Biochem. 112: 3322–3333, 2011.

Constitutive internalization of murine MHC class I molecules

The total number of cell surface glycoprotein molecules at the plasma membrane results from a balance between their constitutive internalization and their egress to the cell surface from intracellular pools and/or biosynthetic pathway. Constitutive internalization is net result of constitutive endocytosis and endocytic recycling. In this study we have compared spontaneous internalization of murine major histocompatibility complex (MHC) class I molecules (Kd, Dd, full Ld, and empty Ld) after depletion of their egress to the cell surface (Cycloheximide [CHX], brefeldin A [BFA]) and internalization after external binding of monoclonal antibody (mAb). MHC class I alleles differ regarding their cell surface stability, kinetics, and in the way of internalization and degradation. Kd and Dd molecules are more stable at the cell surface than Ld molecules and, thus, constitutively internalized more slowly. Although the binding of mAbs to cell surface MHC class I molecules results in faster internalization than depletion of their egress, it is still slow and, thereby, can serve as a model for tracking of MHC class I endocytosis. Internalization of fully conformed MHC class I molecules (Kd, Dd, and Ld) was neither inhibited by chlorpromazine (CP) (inhibitor of clathrin endocytosis), nor with filipin (inhibitor of lipid raft dependent endocytosis), indicating that fully conformed MHC class I molecules are internalized via the bulk pathway. In contrast, internalization of empty Ld molecules was inhibited by filipin, indicating that non‐conformed MHC class I molecules require intact cholesterol‐rich membrane microdomains for their constitutive internalization. Thus, conformed and non‐conformed MHC class I molecules use different endocytic pathways for constitutive internalization. J. Cell. Physiol. 210: 445–455, 2007.

Murine Cytomegalovirus Perturbs Endosomal Trafficking of Major Histocompatibility Complex Class I Molecules in the Early Phase of Infection

ABSTRACT Murine cytomegalovirus (MCMV) functions interfere with protein trafficking in the secretory pathway. In this report we used Δm138-MCMV, a recombinant virus with a deleted viral Fc receptor, to demonstrate that MCMV also perturbs endosomal trafficking in the early phase of infection. This perturbation had a striking impact on cell surface-resident major histocompatibility complex class I (MHC-I) molecules due to the complementary effect of MCMV immunoevasins, which block their egress from the secretory pathway. In infected cells, constitutively endocytosed cell surface-resident MHC-I molecules were arrested and retained in early endosomal antigen 1 (EEA1)-positive and lysobisphosphatidic acid (LBPA)-negative perinuclear endosomes together with clathrin-dependent cargo (transferrin receptor, Lamp1, and epidermal growth factor receptor). Their progression from these endosomes into recycling and degradative routes was inhibited. This arrest was associated with a reduction of the intracellular content of Rab7 and Rab11, small GTPases that are essential for the maturation of recycling and endolysosomal domains of early endosomes. The reduced recycling of MHC-I in Δm138-MCMV-infected cells was accompanied by their accelerated loss from the cell surface. The MCMV function that affects cell surface-resident MHC-I was activated in later stages of the early phase of viral replication, after the expression of known immunoevasins. MCMV without the three immunoevasins (the m04, m06, and m152 proteins) encoded a function that affects endosomal trafficking. This function, however, was not sufficient to reduce the cell surface expression of MHC-I in the absence of the transport block in the secretory pathway.

Early endosomal rerouting of major histocompatibility class I conformers

Major histocompatibility class I (MHC‐I) molecules are present at the cell surface both as fully conformed trimolecular complexes composed of heavy chain (HC), beta‐2‐microglobulin (β2m) and peptide, and various open forms, devoid of peptide and/or β2m (open MHC‐I conformers). Fully conformed MHC‐I complexes and open MHC‐I conformers can be distinguished by well characterized monoclonal antibody reagents that recognize their conformational difference in the extracellular domain. In the present study, we used these tools in order to test whether conformational difference in the extracellular domain determines endocytic and endosomal route of plasma membrane (PM) proteins. We analyzed PM localization, internalization, endosomal trafficking, and recycling of human and murine MHC‐I proteins on various cell lines. We have shown that fully conformed MHC‐I and open MHC‐I conformers segregate at the PM and during endosomal trafficking resulting in the exclusion of open MHC‐I conformers from the recycling route. This segregation is associated with their partitioning into the membranes of different compositions. As a result, the open MHC‐I conformers internalized with higher rate than fully conformed counterparts. Thus, our data suggest the existence of conformation‐based protein sorting mechanism in the endosomal system. J. Cell. Physiol. 227: 2953–2964, 2012.

BMP signaling in rats with TNBS-induced colitis following BMP7 therapy.

Beyond stimulating bone formation, bone morphogenetic proteins (BMPs) are important in development, inflammation, and malignancy of the gut. We have previously shown that BMP7 has a regenerative, anti-inflammatory, and antiproliferative effect on experimental inflammatory bowel disease (IBD) in rats. To further investigate the BMP signaling pathway we monitored the effect of BMP7 therapy on the BMP signaling components in the rat colon during different stages of experimentally induced colitis by 2,4,6-trinitrobenzene sulfonic acid (TNBS). The results showed a significantly decreased BMP7 expression in the acute phase, followed by a significantly increased BMP2 and decreased BMP6 expression during the chronic phase of colitis. BMP7 therapy influenced the expression of several BMPs with the most prominent effect on downregulation of BMP2 and upregulation of BMP4 in the chronic phase of colitis. Importantly, connective tissue growth factor and noggin expression were elevated in the acute stage and significantly decreased upon BMP7 therapy. BMP receptor I expression was unchanged, whereas BMP receptor II was decreased at day 2 and increased at days 14 and 30 of TNBS inflammation. However, an opposite pattern of expression following BMP7 therapy has been observed. BMP7 increased the expression of BR-Smad including Smad3 and Smad4. Inhibitory Smads were increased in colitis and significantly decreased following BMP7 therapy at later stages of the disease. We suggest that BMP signaling was altered during TNBS-induced colitis and was recovered with BMP7 administration, suggesting that IBD is a reversible process.

Monocytes and monocyte chemoattractant protein 1 (MCP-1) as early predictors of disease outcome in patients with cerebral ischemic stroke.

SummaryIn this study to identify prognostic biomarkers for ischemic stroke (IS) outcome, we monitored monocyte number and monocyte chemoattractant protein (MCP-1) concentration in peripheral blood of 44 patients with IS during the week following IS. According to the severity of IS, patients were allocated to three groups: patients with transient ischemic attack (TIA), patients with National Institute of Health Stroke Scale (NIHSS) score ≤ 12, and patients with NIHSS > 12. In patients with NIHSS > 12 statistically significant increased number of monocytes was observed on day 7. MCP-1 plasma concentration initially increased, decreased at day 3 in patients with NIHSS > 12 and increased and restored on day 7. A negative correlation between the number of monocytes and MCP-1 concentration was observed on day 3 after IS. Higher day-7 MCP-1 level was associated with higher modified Rankin Scale (mRS) value (indicating worse outcome) at 90 days post-IS in patients with NIHSS > 12. Our findings suggest that number of monocytes and plasma MCP-1 level could be clinical prognostic biomarkers as early predictors of disease outcome in patients with IS.

Influence of CD26/dipeptidyl peptidase IV deficiency on immunophenotypic changes during colitis development and resolution

A lot of evidence for the importance of CD26/dipeptidyl peptidase IV (CD26/DPP IV) in immunoactivation has been reported; however, its involvement in colitis remains unclear. The aim of this study was to investigate the influence of CD26/DPP IV deficiency on immunophenotypic changes associated with dextran sulfate sodium (DSS)-induced colitis in wild-type (WT) and CD26-deficient mice. Development of clinical symptoms of colitis and animal health status parameters were assessed; the expression of the nuclear factor (NF)-κB p65 subunit was measured by quantitative real-time PCR, while cell characterization was determined by flow cytometry and immunohistochemical staining. DSS treatment induced loss of body weight and colon length shortening in both mouse strains. An increase of myeloperoxidase activity in CD26-deficient mice was more intensive than in WT mice, in spite of similar histopathological changes. Furthermore, a significant increase in the expression of NF-κB p65 subunit in the colon of CD26-deficient mice was determined. The percentage of splenic CD4+ and CD8+ cells in the acute phase of colitis was significantly decreased in WT mice, while in the same period, an increase in the percentage of splenic CD8+ cells was present in CD26-deficient mice. Development of colitis was accompanied by a significant increase in the percentage of intrahepatic NKT cells in both mouse strains, but their percentage in spleen was increased only in CD26-deficient mice. CD26 deficiency was associated with a heightened response to DSS accompanied by increased expression of NF-κB p65 subunit and distinct changes in leukocyte trafficking. These results provide new insights into the role of CD26/DPP IV during the development of colitis.

Distinct Pathways for Constitutive Endocytosis of Fully Conformed and Non‐conformed Ld Molecules

PROBLEM: To characterize the constitutive internalization of major histocompatibility complex (MHC) class I molecules, we have studied the expression of completely conformed (full) and unconformed (empty) Ld molecules on non‐polarized murine P815 cells.

Cortical gray matter loss in schizophrenia: Could microglia be the culprit?

Cortical gray matter loss in schizophrenia remains a great therapeutic difficulty. Each psychotic episode causes irreversible cortical gray matter loss, that causes the patients to never regain their previous state of functioning. Microglial cells are part of the innate immune system and their functions, among others, include phagocytosis and release of neurotrophic factors. They have a key impact on developmental and plasticity-induced removal of neuronal precursors, live-but-stressed neurons and synapses, while also stimulating synaptic growth and development. We hypothesize that microglia are the culprit for the cortical gray matter loss in schizophrenia through abnormal synaptic pruning, phagocytosis of stressed neurons and lacking neurotrophic factor release. Furthermore, we propose a research that could validate the hypotheses using serum samples of first-episode early-onset patients. By measuring the serum levels of milk fat globule-EGF factor 8 (MFG-E8), subcomponent in the classical pathway of complement activation (C1q), brain-derived neurotrophic factor (BDNF), interleukin-6 (IL-6) and interleukin-10 (IL-10), we could gain an insight into the state of microglial activation during various stages of the disease. If this hypothesis is valid, new targeted drugs could be developed in order to reduce the deterioration of cortical gray matter, thereby possibly improving negative symptoms and cognitive deficits.