Impact of Appropriate Empirical Antibiotic Treatment on the Clinical Response of Septic Patients in Intensive Care Unit: A Single-Center Observational Study.

The appropriate antibiotic treatment of patients with bacterial sepsis in the intensive care unit (ICU) remains a challenge. Considering that current international guidelines recommend 7 days of antibiotic therapy as sufficient for most severe infections, our primary outcome was a comparison of clinical response to initial empirical therapy on day 7 and mortality between two groups of septic patients-with appropriate (AEAT) and inappropriate (IEAT) empirical antibiotic therapy according to the in vitro sensitivity of bacteria detected in a blood culture (BC). Adult patients admitted to the ICU between 2020 and 2023, who were diagnosed with sepsis according to the Sequential Organ Failure Assessment (SOFA) score ≥ 2 in association with a suspected or documented infection, were selected for the study. Of the 418 patients, 149 (35.6%) died within 7 days. Although the AEAT group had a lower mortality rate (30.3% vs. 34.2%) and better clinical improvement (52.8% vs. 47.4%) on day 7 after starting empirical antibiotic therapy, there was no significant difference. A causative organism was isolated from BCs in 30% of septic patients, with gram-negative bacteria (GNB) predominating in 60% of cases, and multidrug-resistant (MDR) or extensively drug-resistant (XDR) bacteria predominantly detected in the BCs of the IEAT group. Although the AEAT group had slightly worse clinical characteristics at the onset of sepsis than the IEAT group, the AEAT group showed faster improvement on days 7 and 14 of sepsis. In this retrospective cross-sectional study, the AEAT group was associated with better clinical response at day 7 after sepsis onset and lower mortality, but without a significant difference. Comorbidities and the type of bacterial pathogen should also be taken into account as they can also contribute to the prediction of the final outcome. These results demonstrate the importance of daily assessment of clinical factors to more accurately predict the clinical outcome of a septic patient.

Relationship between Physical Activity and Sedentary Behavior, Spinal Curvatures, Endurance and Balance of the Trunk Muscles-Extended Physical Health Analysis in Young Adults.

BACKGROUND: Physical inactivity and sedentary behavior are associated with poor well-being in young people with adverse effects extending into adulthood. To date, there are many studies investigating the relationship between physical activity (PA) and posture, but there are no data on the relationship between the type and intensity of PA and sedentary behavior, their association with thoracic and lumbar spine angles, and with endurance and balance of the trunk muscles, especially in healthy young adults aged 18-25 years. Moreover, there are no data on the relationship between PA and sedentary behavior and musculoskeletal and cardiopulmonary health, as well as quality of life (QoL) and sleep that would provide a more comprehensive picture of physical health status. AIM: Therefore, the aim of this cross-sectional study was to investigate the extent to which PA and sedentary behavior are associated with each other and with changes in spinal curvatures, endurance and balance of trunk muscles in an extended analysis of physical health status in young adults aged 18-25 years by additionally including measures of body composition, cardiorespiratory capacity, and QoL and sleep. METHODS: A total of 82 students (58% female, 42% male) aged 18-25 years completed all required tests. Primary outcome measures included the following: PA and sedentary behavior calculated from the long form of International PA Questionnaire (IPAQ-LF), spinal curvatures measured by a Spinal Mouse® device, endurance and balance of the trunk muscles measured using trunk endurance tests and their ratio. RESULTS: Overall, 50% of students were classified as minimally active and 50% as health-enhancing PA (HEPA) active. The angles of thoracic kyphosis and lumbar lordosis showed no correlation with PA or time spent sitting. However, students with the lowest PA had significantly higher scores on the trunk extensor endurance test and trunk extensor/flexor endurance test ratio, indicating imbalanced trunk muscles. Moreover, these students spent the most their time sitting. Only PA of vigorous intensity and PA during recreation, leisure, and sports significantly correlated with QoL related to physical health. QoL related to physical and psychosocial health had significantly higher scores when students spent less time sitting. In addition, we found significantly better respiratory performance and SQ at higher PA values, i.e., PA during recreation, leisure, and sport. CONCLUSIONS: Our results suggest that students with low PA levels and more time spent sitting have imbalanced trunk muscles, worse respiratory function, and poorer QoL and sleep. Moreover, these findings in college students may reflect their lifestyle and suggest that more PA needs to be promoted to prevent the development of chronic diseases including musculoskeletal disorders.

Borderline SARS-CoV-2 patients: the trace behind.

BACKGROUND: Effective and precise SARS-CoV-2 detection assays are crucial for maintaining regular hospital routines and identifying infected hospital employees and infected patients before hospital admission. Inconclusive PCR test results of potentially infectious borderline SARS-CoV-2 patients can confuse clinicians and delay appropriate infection control. OBJECTIVES AND STUDY DESIGN: In this retrospective study, we followed up borderline SARS-CoV-2 patients who were tested (from the second sample with the same method) at the Clinical Department of Clinical Microbiology. We aimed to determine the positivity conversion ratio within 7 days after inconclusive PCR test results. RESULTS: Out of 247 borderline patients, who were resampled and retested in the same laboratory, 60 patients (29.4%) showed conversion of the borderline viral load (inconclusive RT-PCR test) to a positive RT-PCR test result. CONCLUSIONS: Our results highlight the need for retesting of borderline patients with inconclusive SARS-CoV-2 results. Follow-up testing of inconclusive PCR results within 7 days can identify additional positive results and reduce the potential risk of intrahospital transmission.

Human Decidual CD1a+ Dendritic Cells Undergo Functional Maturation Program Mediated by Gp96.

Heat shock proteins (hsps), in certain circumstances, could shape unique features of decidual dendritic cells (DCs) that play a key role in inducing immunity as well as maintaining tolerance. The aim of the study was to assess the binding of gp96 to Toll-like receptor (TLR) 4 and CD91 receptors on decidual CD1a+ DCs present at the maternal-fetal interface in vitro as well as the influence of CD1a+ DCs maturation status. Immunohistology and immunofluorescence of paraffin-embedded first-trimester decidua tissue sections of normal and pathological (missed abortion MA and blighted ovum BO) pregnancies were performed together with flow cytometry detection of antigens in CD1a+ DCs after gp96 stimulation of decidual mononuclear cells. Gp96 efficiently bound CD91 and TLR4 receptors on decidual CD1a+ DCs in a dose-dependent manner and increased the expression of CD83 and HLA-DR. The highest concentration of gp96 (1000 ng/mL) increased the percentage of Interferon-γ (INF-γ) and IL-15 expressing gp96+ cells. Gp96 binds CD91 and TLR4 on decidual CD1a+ DCs, which causes their maturation and significantly increases INF-γ and IL-15 in the context of Th1 cytokine/chemokine domination, which could support immune response harmful for ongoing pregnancy.

Upper-lower limb and breathing exercise program for improving sleep quality and psychological status in multiple sclerosis: a pilot randomized controlled trial.

PURPOSE: To examine the feasibility and possible effect of an 8-week exercise program on sleep quality, insomnia and psychological distress in individuals with multiple sclerosis (MS). METHODS: Twenty-four individuals with MS were recruited into a controlled pre-post feasibility study and divided into 2 groups: exercise (n = 13; Expanded Disability Status Scale (EDSS): 1.0-7.5) and a related control group with no exercise (n = 11; EDSS: 1.0-7.0). The exercise group performed combined upper limb, lower limb and breathing exercises in a controlled group (2d/week, 60 min/session) for 8 weeks. Participants were administered measures to evaluate sleep quality (Pittsburgh Sleep Quality Index, PSQI), insomnia severity (Insomnia Severity Index, ISI), psychological distress (Clinical Outcomes in Routine Evaluation-Outcome Measure, CORE-OM) and additionally impact of fatigue (Modified Fatigue Impact Scale, MFIS) after 8-weeks. RESULTS: Insomnia severity measured with ISI (F(1;22)=5.95, p = 0.023, η p 2 = 0.213, 90% CI = 0.02-0.42) and psychological distress measured with the CORE-OM (F(1;22)=4.82, p = 0.039, η p 2 = 0.179, 90% CI = 0.01-0.40) showed statistically significant group-by-time interaction. Sleep quality measured with the PSQI showed statistically significant group-by-time interaction only in an aspect of daytime sleep dysfunction (F(1;22)=5.33, p = 0.031, η p 2 = 0.195, 90% CI = 0.01-0.40). The fatigue impact measured with the MFIS showed statistically significant group-by-time interaction in physical (F(1;22)=6.80, p = 0.016, η p 2 = 0.236, 90% CI = 0.02-0.44) and cognitive aspects (F(1;22)=9.12, p = 0.006, η p 2 = 0.293, 90% CI = 0.05-0.49), and total score (F(1;22)=11.29, p = 0.003, η p 2 = 0.339, 90% CI = 0.08-0.52). CONCLUSIONS: Our 8-week program reduced insomnia severity, psychological distress and some aspects of fatigue (physical; cognitive; total), and improved sleep quality in an aspect of daytime sleep dysfunction in a small group of individuals with MS. Good feasibility and significant positive changes from baseline warrant further exploratory work.

Neuroprotective Panel of Olive Polyphenols: Mechanisms of Action, Anti-Demyelination, and Anti-Stroke Properties.

Neurological diseases such as stroke and multiple sclerosis are associated with high morbidity and mortality, long-term disability, and social and economic burden. Therefore, they represent a major challenge for medical treatment. Numerous evidences support the beneficial effects of polyphenols from olive trees, which can alleviate or even prevent demyelination, neurodegeneration, cerebrovascular diseases, and stroke. Polyphenols from olive oils, especially extra virgin olive oil, olive leaves, olive leaf extract, and from other olive tree derivatives, alleviate inflammation and oxidative stress, two major factors in demyelination. In addition, they reduce the risk of stroke due to their multiple anti-stroke effects, such as anti-atherosclerotic, antihypertensive, antioxidant, anti-inflammatory, hypocholesterolemic, hypoglycemic, and anti-thrombotic effects. In addition, olive polyphenols have beneficial effects on the plasma lipid profiles and insulin sensitivity in obese individuals. This review provides an updated version of the beneficial properties and mechanisms of action of olive polyphenols against demyelination in the prevention/mitigation of multiple sclerosis, the most common non-traumatic neurological cause of impairment in younger adults, and against cerebral insult with increasing incidence, that has already reached epidemic proportions.

Alloiococcus otitidis-Cause of Nonspecific Acute Sinusitis: First Case Report and Review of Literature.

Although most sinus infections are viral, potential bacterial pathogens such as Streptococcus pneumoniae, Haemophilus influenza and Moraxella catarrhalis can migrate during a viral respiratory infection from the nasopharynx into the sinus cavity causing sinusitis. Alloiococcus otitidis is a commensal of the external auditory canal and is considered one of the potential middle ear pathogens. Unlike most otopathogens, A. otitidis is rarely found in the nasopharynx of healthy individuals. This difficult-to-culture organism has not previously been described as a causative agent of sinusitis. Here we describe one case of acute sinusitis due to A. otitidis and review previous knowledge of this controversial organism based on recent literature.

Gender-Related Differences in BMP Expression and Adult Hippocampal Neurogenesis within Joint-Hippocampal Axis in a Rat Model of Rheumatoid Arthritis.

BMPs regulate synovial quiescence and adult neurogenesis in the hippocampus in non-stress conditions. However, changes in BMP expression that are induced by inflammation during rheumatoid arthritis (RA) have not yet been reported. Here, we show that signalling with synovial BMPs (BMP-4 and -7) mediates the effect of systemic inflammation on adult neurogenesis in the hippocampus during pristane-induced arthritis (PIA) in Dark Agouti (DA) rats, an animal model of RA. Moreover, we show gender differences in BMP expressions and their antagonists (Noggin and Gremlin) during PIA and their correlations with the clinical course and IL-17A and TNF-α levels in serum. Our results indicate gender differences in the clinical course, where male rats showed earlier onset and earlier recovery but a worse clinical course in the first two phases of the disease (onset and peak), which correlates with the initial increase of serum IL-17A level. The clinical course of the female rats worsened in remission. Their prolonged symptoms could be a reflection of an increased TNF-α level in serum during remission. Synovial inflammation was greater in females in PIA-remission with greater synovial BMP and antagonist expressions. More significant correlations between serum cytokines (IL-17A and TNF-α), and synovial BMPs and their antagonists were found in females than in males. On the other hand, males showed an increase in hippocampal BMP-4 expression during the acute phase, but both genders showed a decrease in antagonist expressions during PIA in general. Both genders showed a decrease in the number of Ki-67+ and SOX-2+ and DCX+ cells and in the ratio of DCX+ to Ki67+ cells in the dentate gyrus during PIA. However, in PIA remission, females showed a faster increase in the number of Ki67+, SOX-2+, and DCX+ cells and a faster increase in the DCX/Ki67 ratio than males. Both genders showed an increase of hippocampal BMP-7 expression during remission, although males constantly showed greater BMP-7 expression at all time points. Our data show that gender differences exist in the BMP expressions in the periphery-hippocampus axis and in the IL-17A and TNF-α levels in serum, which could imply differences in the mechanisms for the onset and progression of the disease, the clinical course severity, and adult neurogenesis with subsequent neurological complications between genders.

Olive Leaf Polyphenols (OLPs) Stimulate GLUT4 Expression and Translocation in the Skeletal Muscle of Diabetic Rats.

Skeletal muscles are high-insulin tissues responsible for disposing of glucose via the highly regulated process of facilitated glucose transporter 4 (GLUT4). Impaired insulin action in diabetes, as well as disorders of GLUT4 vesicle trafficking in the muscle, are involved in defects in insulin-stimulated GLUT4 translocation. Since the Rab GTPases are the main regulators of vesicular membrane transport in exo- and endo-cytosis, in the present work, we studied the effect of olive leaf polyphenols (OLPs) on Rab8A, Rab13, and Rab14 proteins of the rat soleus muscle in a model of streptozotocin (SZT)-induced diabetes (DM) in a dose-dependent manner. Glucose, cholesterol, and triglyceride levels were determined in the blood, morphological changes of the muscle tissue were captured by hematoxylin and eosin histological staining, and expression of GLUT4, Rab8A, Rab13, and Rab14 proteins were analyzed in the rat soleus muscle by the immunofluorescence staining and immunoblotting. OLPs significantly reduced blood glucose level in all treated groups. Furthermore, significantly reduced blood triglycerides were found in the groups with the lowest and highest OLPs treatment. The dynamics of activation of Rab8A, Rab13, and Rab14 was OLPs dose-dependent and more effective at higher OLP doses. Thus, these results indicate a beneficial role of phenolic compounds from the olive leaf in the regulation of glucose homeostasis in the skeletal muscle.

Age-Related Changes and Sex-Related Differences in Brain Iron Metabolism.

Iron is an essential element that participates in numerous cellular processes. Any disruption of iron homeostasis leads to either iron deficiency or iron overload, which can be detrimental for humans' health, especially in elderly. Each of these changes contributes to the faster development of many neurological disorders or stimulates progression of already present diseases. Age-related cellular and molecular alterations in iron metabolism can also lead to iron dyshomeostasis and deposition. Iron deposits can contribute to the development of inflammation, abnormal protein aggregation, and degeneration in the central nervous system (CNS), leading to the progressive decline in cognitive processes, contributing to pathophysiology of stroke and dysfunctions of body metabolism. Besides, since iron plays an important role in both neuroprotection and neurodegeneration, dietary iron homeostasis should be considered with caution. Recently, there has been increased interest in sex-related differences in iron metabolism and iron homeostasis. These differences have not yet been fully elucidated. In this review we will discuss the latest discoveries in iron metabolism, age-related changes, along with the sex differences in iron content in serum and brain, within the healthy aging population and in neurological disorders such as multiple sclerosis, Parkinson's disease, Alzheimer's disease, and stroke.

Olive Leaf Polyphenols Attenuate the Clinical Course of Experimental Autoimmune Encephalomyelitis and Provide Neuroprotection by Reducing Oxidative Stress, Regulating Microglia and SIRT1, and Preserving Myelin Integrity.

Numerous evidences suggest that plant polyphenols may have therapeutic benefits in regulating oxidative stress and providing neuroprotection in many neurodegenerative diseases, including multiple sclerosis (MS). However, these mechanisms are not yet completely understood. In this study, we investigated the effect of olive leaf polyphenols on oxidative stress through oxidation marker level and activity (TBARS, SOD, and GPX) and their protein expression (SOD1, SOD2, and GPX1), as well as the protein expression of Sirtuin 1 (SIRT1) and microglia markers (Iba-1, CD206, and iNOS) and myelin integrity (proteolipid protein expression) in the brain of rats with induced experimental autoimmune encephalomyelitis (EAE) and subjected to olive leaf therapy. Experiments were performed in male EAE DA rats, which were randomly divided into 2 main groups: EAE groups treated with the therapy of olive leaf (EAE+TOL) and untreated EAE control groups. The EAE treated groups consumed olive leaf tea instead of drinking water (ad libitum) from the beginning to the end of the experiment. In addition, olive leaf extract was injected intraperitoneally (i.p.) for the 10 continuous days and started on the 8th day after EAE induction. The clinical course was monitored in both groups until the 30th day after EAE induction. Our results demonstrated that TOL attenuated the clinical course of EAE; reduced the oxidative stress (by decreasing the concentration of MDA); upregulated antioxidant enzymes (SOD1, SOD2, and GPX1), SIRT1 (overall and microglial), and anti-inflammatory M2 microglia; downregulated proinflammatory M1 type; and preserved myelin integrity. These data support the idea that TOL may be an effective therapeutic approach for treating MS and other neurodegenerative diseases.

Combined upper limb and breathing exercise programme for pain management in ambulatory and non-ambulatory multiple sclerosis individuals: part II analyses from feasibility study.

PURPOSE: The present small semi-controlled feasibility study investigated a possible efficacy of a combined upper limb and breathing exercise programme in managing pain in ambulatory and non-ambulatory patients with EDSS from 0.0-8.0. METHOD: People with MS (N = 19) were enrolled in this single-blind randomized controlled study and divided into 2 groups: exercise group (5 ambulatory, 5 non-ambulatory; Expanded Disability Status Scale (EDSS), 1.0-8.0) and related control group that performed no exercise (4 ambulatory, 5 non-ambulatory; EDSS, 1.0-7.5). The exercise group performed combined upper limb and breathing exercises in a group led by a physiotherapist (2 days/week, 60 min/session) accompanied by independent home exercises (3 days/week, ≥ 20 min/session). Participants underwent measures of pain level (visual analogue scale) for physical pain, functional independence of daily activities (Barthel index) and handgrip strength (HGS) for dominant (D) and non-dominant (ND) hand evaluated by a dynamometer before and after the 4-week period by the blinded assessor. RESULTS: The VAS for pain showed statistically significant group-by-time interaction only in non-ambulatory (p = .049) individuals, but with large intervention effects on both subgroups (ambulatory, p = .159; non-ambulatory, d = 0.97). Functional independence in daily activities (Barthel index) showed statistically non-significant group-by-time interaction in ambulatory (p = .195, d = 0.89) and non-ambulatory (p = .102, d = 1.64) individuals, but despite the absence of statistical significance, there were large intervention effects. Handgrip strength was significantly improved for both hands in ambulatory (D, p = .012; d = 2.07; ND, p = .025, d = 1.77) and only non-dominant hand in non-ambulatory individuals (D, p = .288, d = 0.83; ND, p = .012, d = 2.21). CONCLUSION: This small pilot study provides preliminary proof-of-concept data supporting low-intensity upper limb and breathing exercise programme for potential reduction of pain and improvement of functional independence in both ambulatory and non-ambulatory individuals with MS in a larger sample and that strengthening the upper limbs might be an additional pain relief mechanism. TRIAL REGISTRATION: NTC03222596.

Cryptozoospermia after treatment with clomiphene citrate following long-term use of intramuscular testosterone undecanoate depot injection (Nebido®).

Objective To illustrate the importance of treatment duration with intramuscular testosterone undecanoate (Nebido®) for the final spermatogenesis recovery after treatment cessation. Also, to show a subsequent poor efficacy of the selective estrogen receptor modulator (SERM) clomiphene citrate (CC) in treating steroid-induced azoospermia following Nebido® cessation and describe that initial oligozoospermia, existing before starting Nebido®, largely contributes to that treatment outcome. Methodology Setting: Department of Human Reproduction and Department of Endocrinology, Clinical Hospital Center Rijeka, Rijeka, and Department of Endocrinology, Clinical Hospital Center Sestre milosrdnice, Zagreb, Croatia. PATIENT: A male patient having been diagnosed with primary hypogonadotropic hypogonadism, oligozoospermia and low testosterone (T) level, was treated with intramuscular testosterone undecanoate (TU) depot 1 g (Nebido®) to prevent further progression of testosterone deficiency symptoms (low mood, energy and concentration, fatigue, muscle weakness). INTERVENTIONS: Stopping Nebido® and treatment with CC 50 mg per day 5 days per week for 3-6 month to recover spermatogenesis. MAIN OUTCOME MEASURES: T levels and semen analyses. Results Semen analyses did not return to values before taking Nebido® 1 year after cessation nor after 3 months of treatment with CC. Values of T, follicle stimulating hormone (FSH) and luteinizing hormone (LH) dropped even more than before starting Nebido®, after 1 year of cessation. Conclusions Here we describe a case of initially idiopathic gonadal failure with subsequent secondary gonadal failure and infertility resulting from testosterone replacement therapy (TRT) treatment, and poor spermatogenesis recovery outcome of CC used post Nebido® cessation.

Exploring the feasibility of a mild and short 4-week combined upper limb and breathing exercise program as a possible home base program to decrease fatigue and improve quality of life in ambulatory and non-ambulatory multiple sclerosis individuals.

PURPOSE: To evaluate the feasibility of a combined upper limb and breathing exercise for a home-based program and to explore its effect on primary fatigue and quality of life in ambulatory and non-ambulatory individuals with multiple sclerosis (MS) in a short time. METHOD: Nineteen individuals with MS were assigned into semi-controlled pre-post feasibility study based on Expanded Disability Status Scale (EDSS) status and divided into two groups: exercise (five ambulatory, five non-ambulatory; EDSS 1.0-8.0) and related control with no exercise (four ambulatory, five non-ambulatory; EDSS 1.0-7.5). Exercise group performed combined upper limb and breathing exercise in a controlled group (2 days/week, 60 min/session) accompanied by independent home exercise (3 days/week, ≥ 20 min/session). Participants underwent measures of fatigue impact (Modified Fatigue Impact Scale (MFIS) and quality of life (RAND Medical outcomes study 36-item short-form health survey (SF-36)) before and after a 4-week period. RESULTS: The MFIS (physical, psychosocial, total) showed statistically significant group-by-time interaction in ambulatory (p = 0.033, d = 1.60; p = 0.039, d = 1.59; p = 0.033, d = 1.62) and non-ambulatory individuals (p = 0.009, d = 2.42; p = 0.018, d = 1.96; p = 0.0008, d = 3.92). Physical functioning (SF-36) showed statistically significant group-by-time interaction in ambulatory (p = 0.014, d = 2.14) but no significance in non-ambulatory (p = 0.368, d = 0.68) individuals. Despite the absent statistical significance, there were large intervention effects on MFIS cognitive scores for ambulatory (d = 1.28) and non-ambulatory (d = 1.47), and on other SF-36 scores for ambulatory (general health: d = 1.76 and pain: d = 1.02) and non-ambulatory (physical limitation: d = 1.03 and emotional well-being: d = 0.94) individuals. CONCLUSION: Our 4-week program reduced some aspects of fatigue and improved some aspects of quality of life in a small group of ambulatory and non-ambulatory individuals with MS. Good feasibility and significant positive changes from baseline warrant further exploratory work. TRIAL REGISTRATION: Name of the registry: The Impact of Exercise Training on Living Quality in Multiple Sclerosis. Registration: The study was registered at www.clinicaltrial.gov on July 14, 2017. First participant enrollment: August 28, 2017. URL: 602-01/17-01-147; Trial registration ID: NTC03222596.

Expression Profiles of Metallothionein I/II and Megalin in Cuprizone Model of De- and Remyelination.

Copper chelator cuprizone (CPZ) is neurotoxicant, which selectively disrupts oligodendroglial respiratory chain, leading to oxidative stress and subsequent apoptosis. Demyelination is, however, followed by spontaneous remyelination owing to the activation of intrinsic CNS repair mechanisms. To explore the participation of metallothioneins (MTs) in these processes, in this study we analyzed the expression profiles of MT-I/II and their receptor megalin (low-density lipoprotein receptor related protein-2) in the brain of mice subjected to different protocols of CPZ feeding. Experiments were performed in female C57BL/6 mice fed with 0.25% CPZ during 1, 3 and 5 weeks. They were sacrificed immediately after feeding with CPZ or 2 weeks after the withdrawal of CPZ. The data showed that CPZ-induced demyelination was followed by high astrogliosis and enhanced expression of MTs and megalin in white (corpus callosum and internal capsule) and gray matter of the brain (cortex, hippocampus, and cerebellum). Moreover, in numerous cortical neurons and progenitor cells the signs of MT/megalin interactions and Akt1 phosphorylation was found supporting the hypothesis that MTs secreted from the astrocytes might directly affect the neuronal differentiation and survival. Furthermore, in mice treated with CPZ for 5 weeks the prominent MTs and megalin immunoreactivities were found on several neural stem cells and oligodendrocyte progenitors in subgranular zone of dentate gyrus and subventricular zone of lateral ventricles pointing to high modulatory effect of MTs on adult neuro- and oligodendrogenesis. The data show that MT I/II perform important cytoprotective and growth-regulating functions in remyelinating processes activated after toxic demyelinating insults.

Hepcidin and metallothioneins as molecular base for sex-dependent differences in clinical course of experimental autoimmune encephalomyelitis in chronic iron overload.

Multiple sclerosis is a chronic demyelinating disease of the central nervous system characterised by inflammatory and degenerative changes. It is considered that disease arises from the influence of environmental factors on genetically susceptible individuals. Recent researches, using magnetic resonance imaging, connected iron deposits in different brain regions with demyelinating process in multiple sclerosis patients. Although iron is an essential trace element important for many biological functions it could be harmful because iron excess can induce the production of reactive oxygen species, development of oxidative stress and lipid peroxidation which leads to demyelination. In experimental autoimmune encephalomyelitis model, the most common experimental animal model for multiple sclerosis, we recently found that chronic iron overload influences the clinical course of disease in Dark Agouti rats. In female rats iron overload accelerated the onset of disease, while in male rats it accelerated the progression of disease and increased mortality rate. We hypothesize that those differences arise on molecular level in different expression of stress response proteins hepcidin and metallothioneins in male and female iron overloaded rats. They are both upregulated by metal ions in both sexes. Hepcidin is additionally upregulated by estrogen in female rats and therefore causes higher degradation of iron exporter ferroportin and sequestration of iron in the cells, lowering the possibility for the development of oxidative stress. Antioxidative effect of metallothioneins could be increased in female rats because of their ability to reversibly exchange metal ions with the estrogen receptor. In case of iron excess metallothioneins release zinc, which is normally bound to them. Zinc binds to estrogen receptor and leaves metallothioneins binding domains free for iron, causing at least provisional cytoprotective effect. To test this hypothesis, we propose to determine and compare serum levels of hepcidin and estrogen using ELISA essay as well as expression and distribution of acute stress response proteins hepcidin and metallothioneins, iron and estrogen receptor in the brain and spinal cord tissue using immunohistochemistry in control and chronic iron overloaded male and female rats in experimental autoimmune encephalomyelitis model. It would be also possible to perform the same immunohistochemistry in the brain tissue of multiple sclerosis patients post mortem. The results of experiments could contribute to better understanding of cytoprotective mechanisms in chronic iron overload that could have possible therapeutic applications in iron disturbances. In order to elucidate whether common measure of systemic iron status, like ferritin, haemoglobin concentration and transferrin saturation levels, may be used to distinguish physiologic from potentially harmful iron levels in local disease, for example multiple sclerosis and Still's disease, well-designed clinical trials would be of great interest.

Osteopontin-metallothionein I/II interactions in experimental autoimmune encephalomyelitis.

Osteopontin (OPN), an extracellular matrix (ECM) glyco-phosphoprotein, plays an important role in autoimmune-mediated demyelinating diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). As an integrin and CD44 binding protein it participates in bidirectional communication between the ECM and target cells and affects transduction pathways that maintain neuronal and immune cell homeostasis. Its biological activity is also heavily influenced by microenvironment, which stimulates the cleavage of OPN and changes its functions. In this study we estimated the expression profile of OPN in neural tissues of DA rats during the first relapse of chronic relapsing EAE and investigated the relationship of OPN to metallothionein I+II (MTs), which play pivotal role in zinc-related cell homeostasis and in protection of CNS against cytokine-induced injury. The data showed that in EAE rats OPN mRNA and protein levels increased concurrently with the transcription of MTs and that within the spinal cord (SC) lysates EAE-afflicted rats had a higher content of OPN fragments of low molecular weight than untreated and CFA-treated rats. The expression of OPN and MTs was upregulated on ependymal, lymphoid and astroglial cells and on multiple αvß3+ neurons in SC and in the brain (cortex, white matter, hippocampus, and cerebellum). Besides, multiple cells co-expressed OPN and MTs. Granular OPN signals were detected in secretory vesicles of Golgy (αvß3 neurons) and in patches adjacent to the plasma membrane (subventricular zone). The findings imply that in demyelinating lesions are generated proteolytic OPN fragments and that OPN/MT interactions contribute to tissue remodeling during an autoimmune attack.

Hippocampal expressions of metallothionein I/II and glycoprotein 96 in EAE-prone and EAE-resistant strains of rats.

Inflammatory demyelinating diseases such as multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) are often followed by cognitive deficits associated with the neuronal injury, synaptic loss and altered neurogenesis within the hippocampus. Changes depend on the genetic and epigenetic factors that ensure the cellular and environmental homeostasis and regulate the interactions of immunocompetent, glial and neural cells. Owing to high impact of stress proteins on these processes, in this study we compared the protein content of interleukin-6, transforming growth factor-ß1, metallothioneins I/II (MTs) and glycoprotein 96 (gp96) in the hippocampus of DA and AO rats that differ in the susceptibility to the induction of EAE, and tested the relationship of MTs and gp96 to granule neurons, glial cells and neural progenitors in different subfields of dentate gyrus. Rats were immunized with bovine brain homogenate emulsified in complete Freund's adjuvant or only with CFA. The data showed that acute attack of EAE in DA rats was followed by accumulation of IL-6, TGF-ß1 and MTs proteins, by increased expression of MTs in molecular and granular cell layer, by reduced expression of gp96/granular cell, by apoptosis and by microgliosis with appearance of Iba-1+ cells, co-expressing MT I/II and gp96. Furthermore, in subgranular zone (SGZ) of DA rats an augmented number of GFAP+ precursors, but decreased number of doublecortin (DCX)+ neuroblasts and immature NeuN+ neurons were found, implying that in DA rats the neurogenesis was delayed or reduced. Besides, in SGZ of both strains several DCX+ and NeuN+ cells co-expressing gp96 and MT I/II were found.

Metallothionein I+II expression as an early sign of chronic relapsing experimental autoimmune encephalomyelitis in rats.

Metallothioneins (MTs) are small, cysteine-rich proteins which have been implicated in various forms of stress providing cytoprotective action against oxidative injury, DNA damage and apoptosis. Owing to their high affinity for physiological metals, such as zinc and copper MTs are also critical components of regulatory proteins involved in cell growth and multiplication, as well as in the maintenance of immune homeostasis. To elucidate the role of MTs in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MT I+II proteins and the content of free Zn ions in the brain, spinal cord and in the liver early in the course of chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) pathogenesis, i.e. before the onset of any clinical symptoms. Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control animals were treated with CFA alone. The data, obtained by immuno-histochemistry and in situ fluorescent labeling of free zinc ions, have shown that in the presymptomatic phase of CR-EAE (on the seventh postimmunization day) MTs I+II were markedly upregulated in the cells that form blood-brain and blood-cerebrospinal fluid barriers, as well as in the cerebellar parenchyma and hippocampal dentate gyri. Furthermore, we found that the liver also becomes a site of extensive MTs I+II synthesis shortly after immunization. Simultaneously, tissue content of free zinc ions increased at the sites of MTs induction, reflecting their antioxidative activity. The data, described in this paper point to regulatory and neuroprotective role of MTs in the pathogenesis of CR-EAE.