Branka Wraber

Submerged cultivation of Ganoderma lucidum biomass and immunostimulatory effects of fungal polysaccharides

Original Ganoderma lucidum strain MZKI G97 isolated from Slovenian forests was cultivated in a liquid substrate based on potato dextrose and olive oil. The influences of inoculum and oxygen partial pressure in batch and fed batch cultivation in a 10-l laboratory stirred tank reactor were studied. Fungal biomass was found to be oxygen and shear sensible. Using a 17% (wet weight) 6 days old vegetative inoculum, 9.6 g l(-1) of dry biomass in batch cultivation and 15.2 g l(-1) in fed batch process were obtained. Extracellular (9.6 g l(-1)) and intracellular (6.3 g l(-1)) polysaccharide fractions were isolated. Extracellular polysaccharide fraction and four intracellular polysaccharide fractions were obtained. Polysaccharides were further separated by ion-exchange, gel and affinity chromatography. The isolated polysaccharides were mainly beta-D-glucanes. Immunostimulatory effects of isolates were tested on induction of cytokine (tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma)) synthesis in primary cultures of human peripheral blood mononuclear cells (PBMC) isolated from a buffy coat. The TNF-alpha inducing activity is comparable with romurtide, which has been used as a supporting therapy in cancer patients treated with radiotherapy and/or chemotherapy.

Interacting Roles of Immune Mechanisms and Viral Load in the Pathogenesis of Crimean-Congo Hemorrhagic Fever

ABSTRACT Until now, the pathogenesis of Crimean-Congo hemorrhagic fever (CCHF) has not been well described. However, it has been hypothesized that it could be a result of the direct injury of virus-infected tissues in combination with the indirect effects of host immune responses, including cytokines. To shed more light on the role of viral load and cytokines, differential influences of CCHF virus (CCHFV) RNA load, antibody response, and cytokine production on severity and outcome of the disease were studied in sera of 46 patients with confirmed acute CCHF from Kosovo. In this study, viral load proved to be strongly related to the severity and outcome of the disease, with higher viral loads detected in patients with fatal outcomes than in surviving patients. Also, patients with fatal outcome had on average a weaker antibody response, if one was present at all. High levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) were associated with poor outcome, since detected concentrations were highest in patients with fatal outcome and lowest in patients with moderate disease course. Additionally, a positive linear dependence between viral load and these cytokines was observed. Interestingly, reduced levels of IL-12 were detected in all CCHF patients. Our study favors the hypothesis that CCHF could be a result of a delayed and downregulated immune response caused by IL-10, which leads to an increased replication and spread of CCHFV throughout the body. This consequently triggers increased production of IFN-γ and TNF-α, cytokines mediating vascular dysfunction, disseminated intravascular coagulation, organ failure, and shock.

Serum levels of inflammatory and regulatory cytokines in patients with hemorrhagic fever with renal syndrome

BackgroundHantaviruses are the causative agents of two zoonotic diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS). The pathogenesis of HFRS is poorly understood. However, it has been suggested that immune mechanisms, including cytokines, might have an important role in HFRS pathogenesis. Thus, the aim of our study was to investigate cytokine profiles in serum samples of HFRS patients from Slovenia and explore a possible correlation between cytokine levels and disease severity.MethodsAcute-phase serum samples from 52 patients, diagnosed with DOBV infection, and 61 patients, diagnosed with PUUV infection, were included in this study. Patients were divided into two groups - severe or mild - based on disease severity. Levels of IL-10, IL-12, INF-γ and TNF-α were measured in the serum samples with commercial ELISA tests.ResultsIncreased levels of IL-10, INF-γ, and TNF-α were found in almost all the serum samples tested. On average, higher concentrations were detected in patients infected with DOBV than PUUV. Furthermore, significantly higher levels of IL-10 (P = 0.001) and TNF-α (P = 0.003) were found in patients with a more severe clinical course of disease. The same association between IL-10 (P < 0.001) and TNF-α (P = 0.021), and the severity of the disease was observed also when only patients infected with DOBV were considered. No differences in cytokine concentrations according to disease severity were observed in patients infected with PUUV. Concentrations of serum IL-12 in HFRS patients were in the normal range, however, higher levels were detected in patients infected with PUUV than in patients infected with DOBV.ConclusionsWe suggest that imbalance in production of proinflammatory and regulatory cytokines might be in part responsible for a more severe course of HFRS.

Murine model of pregnancy‐associated Listeria monocytogenes infection

Listeria monocytogenes has been recognized as a significant pathogen, occurring worldwide, capable of causing animal and human infections. In its most severe form, listeriosis is an invasive disease that affects immunocompromised patients. Additionally, pregnant women represent a high-risk group for L. monocytogenes infection. Abortion, stillbirth or severe neonatal infection can be the serious outcome of such an infection. In an experimental murine model of pregnancy-associated listeriosis we studied the impact of L. monocytogenes on the maternal immune response and pregnancy outcome. In comparison to virgin animals, pregnant mice mounted lower levels of protective cytokines and were unable to eliminate the pathogen. The impaired maternal immune response that has been found both on the systemic and local level, facilitated bacterial multiplication in the liver, placenta and ultimately in the fetal tissues. This resulted in severe necrotizing hemorrhagic hepatitis and Listeria-induced placental necrosis, increasing the incidence of postimplantation loss and poor pregnancy outcome.

Plasma cytokine response in mice with bacterial infection

BACKGROUND: Exposure to microorganisms elicts the production of cytokines. These soluble factors enhance several innate immune functions and regulate the ensuing specific immune response aimed at limiting the spread of infection. AIM: This study was undertaken to quantify the plasma levels of pro-inflammatory cytokines during the course of primary Listeria monocytogenes and Campylobacter jejuni infection. Using an in vivo infection the relationship between endogenous cytokines and the bacterial number in the liver of infected animals was examined. METHODS: C57BL/6 mice were infected by the intraperitoneal route. At different time points we determined the number of colony-forming units of bacteria in the liver of infected animals and paralled these with the plasma levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) measured by enzyme immunoassays. RESULTS: L. monocytogenes infection lasted 10-11 days. IFN-gamma production occurred in the early phase but was more pronounced after day 4, following the appearance of specific immunity. The duration of experimental campylobacteriosis was 15 days. Early IFN-gamma production was not significant but a progressive rise of this cytokine in plasma was seen during the second week post infection. Mice produced measurable amounts of plasma TNF-alpha immediately after being given viable L. monocytogenes, peaking on day 2-3 when the greatest number of bacteria was present in the examined organs. During C. jejuni infection plasma TNF-alpha was produced in a similar manner, but the highest concentrations were found a few days later than in listeriosis, in correlation with the different course of campylobacteriosis. The quantity of IL-6 increased and decreased in concordance with clearance of L monocytogenes and the clinical status of the animals. C. jejuni did not promote the induction of this cytokine. This is to some extent an unusual finding. With respect to the role of IL-6 in Th2 responses and antibody production, the appearance of this cytokine in campylobacteriosis was more expected. DISCUSSION: During systemic bacterial infection, a network of pro-inflammatory cytokines is activated and blood levels of these cytokines are elevated, albeit inconsistently, with large individual variations and depending on microbial characteristics and structure.

IFN-β1a and IFN-β1b have different patterns of influence on cytokines

Abstract Multiple sclerosis is characterized by elevated levels of proinflammatory cytokines produced by Th1 cells and decreased levels of anti-inflammatory cytokines produced by Th2 cells. IFN-β treatment shifts the immune response from the Th1 to Th2 pattern, thus enhancing the production of anti-inflammatory Th2 cytokines such as IL-4, IL-10, and decreasing the production of proinflammatory Th1 cytokines such as IFN-γ. To determine which IFN-β has the stronger immunomodulatory effect we compared the levels of IL-4, IL-10, and IFN-γ of 12 relapsing-remiting MS patients treated with IFN-β1b (Betaferon®) with those of 10 patients treated with IFN-β1a (Avonex®). There were no statistically significant differences in duration of disease, number of relapses before and during treatment, and in EDSS after 2 years of treatment. After 1 year of treatment the concentration of IFN-γ was significantly lower in the Betaferon® group, and concentrations of IL-4 and IL-10 were significantly higher in the Avonex® group. It appears that IFN-β1b has a downregulatory effect on both Th1 and Th2 cytokines, while IFN-β1a causes a shift of the cytokine profile toward the Th2 phenotype. These two IFN have different influences on the pattern of cytokines in MS: IFN-β1a enhances the production of anti-inflammatory cytokines IL-4 and IL-10 and IFN-β1b decreases the production of the proinflammatory cytokine IFN-γ.

lnterleukin-8 and procalcitonin in early diagnosis of early severe bacterial infection in critically ill neonates

Abstract We studied the value of serum interleukin-8 (IL-8) and procalcitonin (PCT) in the early diagnosis of early severe bacterial infection in 58 critically ill ventilated neonates. ELISA was used for determining IL-8 and immunoluminometric assay for PCT. IL-8 and PCT were compared with routinely used serum C-reactive protein (CRP). Neonates were divided into four groups: Ia - proven severe bacterial infection (n = 9), Ib - clinical sepsis (n = 16), II - respiratory distress without bacterial infection (n = 12), and III - various types of neonatal distress (n = 21). Sera were collected on admission, at 24 h and 48 h after admission. There was no significant difference between groups Ia and Ib for either parameter at any time interval. Significant difference was found between group Ia+b (septic neonates) and group II for PCT and CRP at 24 and 48 h, but not for IL-8. There was no difference between group Ia+b and group III except for CRP at 24 h. Diagnostic accuracy was best for PCT on admission and for CRP at 24 h. Serum PCT and IL-8 are not specific markers for early severe bacterial infection in critically ill neonates and are not better than CRP.

Chitotriosidase activity in sarcoidosis and some other pulmonary diseases

Abstract Background: Patients with sarcoidosis have elevated levels of several markers of inflammation. Particularly high levels have been reported for chitotriosidase. In this study, we evaluate whether determining chitotriosidase in serum would be useful in the diagnosis and clinical management of patients with sarcoidosis. Methods: Patients with newly diagnosed sarcoidosis and patients with asthma, fibrosis, asbestosis, lung cancer or chronic obstructive pulmonary disease (n=190) were recruited from an outpatient department. Individuals with no disease (n=26) served as controls. An X-ray was taken, diffusion capacity was measured and blood samples were taken for analysis of chitotriosidase, soluble receptor for interleukin-2, tumour necrosis factor alpha and angiotensin converting enzyme. In most patients with sarcoidosis, the analyses were done before and after regular treatment with corticosteroids over 6 months. Results: Some patients with sarcoidosis had markedly high activities of chitotriosidase, but activities above controls were also found among patients with asbestos, fibrosis and lung cancer. There were significant relationships between chitotriosidase and interleukin-2 receptor and angiotensin-converting enzyme. After treatment, chitotriosidase activity decreased in 52 of 69 patients. Conclusions: The results confirm that chitotriosidase activity is markedly increased in some cases of sarcoidosis. As increased activities are also found in other diseases, chitotriosidase cannot be considered a specific marker of sarcoidosis. In cases of sarcoidosis where high CTO activities are found, this enzyme could serve as a useful marker supporting the diagnosis of sarcoidosis when following the effects of treatment and in surveillance for recurrence of the disease.

Submerged cultivation of Ganoderma lucidum and the effects of its polysaccharides on the production of human cytokines TNF-α, IL-12, IFN-γ, IL-2, IL-4, IL-10 and IL-17.

An original strain of Ganoderma lucidum (W.Curt.:Fr.) Lloyd, MZKI G97 isolated from Slovenian habitats was grown by a submerged liquid substrate cultivation in a laboratory stirred tank reactor. Five fractions of extracellular and cell-wall polysaccharides were obtained by extraction, ethanol precipitation, and purification by ion-exchange, gel and affinity chromatography. The capacity of isolated polysaccharide fractions to induce innate inflammatory cytokines, and to modulate cytokine responses of activated lymphocytes was investigated. Human peripheral blood mononuclear cells (PBMC) were activated in vitro with polysaccharide fractions, in order to induce innate inflammatory cytokines: tumor necrosis factor alpha (TNF-α), interleukin (IL) 12 and interferon gamma (IFN-γ). For the immunomodulation capacity, polysaccharide fractions were cultured with ionomycine and phorbol myristate acetate (IONO+PMA) activated PBMC, and the concentrations of induced IL-2, IL-4, IFN-γ, IL-10 and IL-17 were measured. The results showed that polysaccharides from G. lucidum induced moderate to high amounts of innate inflammatory cytokines. Fungal cell-wall polysaccharides were stronger innate inflammatory cytokines inducers, while extracellular polysaccharides demonstrated a higher capacity to modulate cytokine responses of IONO+PMA induced production of IL-17. The results indicate that G. lucidum polysaccharides enhance Th1 response with high levels of IFN-γ and IL-2, and display low to no impact on IL-4 production. A similar pattern was observed at regulatory cytokine IL-10. All of the polysaccharide fractions tested induced IL-17 production at different concentration levels.

Intrauterine inflammation and the onset of peri-intraventricular hemorrhage in premature infants.

Background: Peri-intraventricular hemorrhage (P/IVH) is a common neonatal morbidity among premature infants. The aim of the study was to examine the association between placental and/or fetal inflammation and the onset of P/IVH in premature infants. Methods: A prospective study included 125 infants with gestational age 23–29 weeks. Placentas were examined for the presence of chorioamnionitis and funisitis, cord blood was sampled for the measurement of cytokines (IL-6 and IL-8). Fetal inflammation was defined as levels of IL-6 higher than 7.6 pg/ml. P/IVH was defined as early if diagnosed within the 1st day after birth; thereafter P/IVH was defined as late. Results: Adjusted for the influence of gestational age, early-onset sepsis (OR 3.2, p = 0.045) and no or incomplete antenatal steroid course (OR 6.0, p = 0.001) significantly predicted early P/IVH. Funisitis (OR 1.6, p = 0.06) and fetal inflammation (OR 2.6, p = 0.06) were only partially associated with early hemorrhage. Contrary to that, respiratory distress syndrome (OR 3.4, p = 0.04), mechanical ventilation (OR 5.9, p = 0.008), low blood pressure (OR 3.5, p = 0.02), and vasopressors (OR 5.7, p = 0.002) were associated with late P/IVH. In multivariate analysis no or incomplete steroid course remained independent predictors for early and use of vasopressors for late P/IVH. The interaction of fetal inflammation and vaginal delivery with no or incomplete steroid course increased the risk of early P/IVH. Conclusions: These results indicate different risk factors for early and late P/IVH. Neither funisitis nor fetal inflammation independently predicts the onset of P/IVH. However, the interaction of fetal inflammation and vaginal delivery with no or incomplete antenatal steroid course increase the risk of early but not also late P/IVH.