Inflammatory markers and pulmonary granuloma infiltration in sarcoidosis.

BACKGROUND AND OBJECTIVE: Previous studies have demonstrated increases of inflammatory mediators in sarcoidosis while epidemiological studies have also demonstrated an association with increased fungi exposure. This study measured the level of ß-glucan in the lungs and of inflammatory mediators in serum, and correlated both with the extent of pulmonary granuloma infiltration. METHODS: This is a cross-sectional study of 98 patients with sarcoidosis and 26 controls. ß-glucan, a cell wall constituent of fungi, was measured in bronchoalveolar lavage. Inflammatory mediator levels were determined in serum. The extent of granuloma infiltration was estimated on the chest X-ray. Exposure to fungi at home was determined by taking air samples in bedrooms and analysing for the presence of ß-N-acetylhexosaminidase. RESULTS: Significantly, higher levels of ß-glucan were found in broncho-alveolar lavage in subjects with sarcoidosis as compared with controls. There were significant positive relationships between the extent of granuloma infiltration and the levels of the different inflammatory mediators, except for interleukin-10. Domestic fungal exposure was higher among subjects with sarcoidosis. CONCLUSIONS: This is the first time that a specific agent, previously suspected to be related to the risk of sarcoidosis, has been detected in the lung of subjects with sarcoidosis and related to the levels of inflammatory mediators and the degree of home exposure to fungi. The results suggest that exposure to fungi should be explored when investigating patients with sarcoidosis.

Production of biomass and polysaccharides of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt. :Fr.) P. Karst. (higher Basidiomycetes), by submerged cultivation.

Submerged batch and repeated fed-batch cultivation techniques were used for mycelia cultivation and polysaccharide production of the Lingzhi or Reishi medicinal mushroom Ganoderma lucidum. Although most publications use various Asiatic G. lucidum strains, the growth of the strain Ga.l 4 (Biotechnical Faculty Strain Collection, Ljubljana, Slovenia), originally isolated from the Slovenian forest, is much faster. The results between the batch and repeated fed-batch cultivation are compared with the polysaccharide production in batch cultivation. From the aspect of biomass production, the best results were obtained in repeated fed-batch after 44 days, where 12.4 g/L of dry fungal biomass was obtained.

Production of Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (W.Curt. :Fr.) P. Karst. (higher Basidiomycetes), biomass and polysaccharides by solid state cultivation.

Solid state cultivation of Ganoderma lucidum biomass, strain BFWS Gal 4, originally isolated from the Slovenian forest, was studied in a horizontal stirred tank reactor. Periodic mixing of N = 80 rpm, 2 min/day was used. Production of fungal polysaccharides and fungal biomass on solid substrate based on beech sawdust, olive oil, and mineral salts was studied. Optimal moisture of the solid matrix was in the range of 80% to 74%. When the moisture content dropped below 57%, the growth of the mycelium and polysaccharide production stopped, but it revived when wet air was applied in further processing. Final concentration of biomass was 0.68 mg/g of solid substrate, while proportions of extracellular and intracellular polysaccharides were 4.5 mg/g and 1.05 mg/g, respectively.

Immunomodulating activities of cultivated maitake medicinal mushroom Grifola frondosa (Dicks.: Fr.) S.F. Gray (higher Basidiomycetes) on peripheral blood mononuclear cells.

Grifola frondosa is a culinary-medicinal mushroom that contains several physiologically active compounds, of which polysaccharides, specifically ß-glucans, are known to possess immunomodulating properties. Its extracts are studied for application as adjuncts for chemotherapy, and experiments in animal models support the use of this mushroom for cancer treatment. The effect of extracts obtained from mushrooms cultivated on different substrates and their capacity of inducing the secretion of cytokines from human peripheral blood mononuclear cells were studied. The activity of extracts at concentrations 12.5, 100, and 200 µg/mL on induction of TNF-α, IFN-γ, and IL-12 was screened. Two extracts from substrates fortified with olive oil press cakes showed appreciable activity and induced the secretion of TNF-α, IL-12, and INF-γ. The extracts differed from the others in the amount of sugar, protein, and ß-glucans, which can explain their higher activity. Present results show that different substrates and different source materials can reasonably modify the bioactivity of cultivated G. frondosa.

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

BACKGROUND: Hantaviruses 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. METHODS: Acute-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. RESULTS: Increased 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. CONCLUSIONS: We suggest that imbalance in production of proinflammatory and regulatory cytokines might be in part responsible for a more severe course of HFRS.

Anticryptococcal cytotoxicity of murine nonadherent cells is perforin and nonperforin mediated.

The encapsulated fungal pathogen Cryptococcus neoformans is a significant agent of life-threatening infections, particularly in people with suppressed cell-mediated immunity. The cellular cytotoxicity against C. neoformans infection is mainly mediated by NK and T cells, but effector mechanisms are not well understood. The objective of this study was (i) to determine whether prior exposure to the cryptococcal antigens enhances anticryptococcal activity of cytotoxic cells in mice and (ii) the contribution of perforin- and nonperforin-mediated cytotoxicity of NK and T cells in growth inhibition of C. neoformans. Our data showed that in vitro exposure of nonadherent (NA) spleen mononuclear cells from nonimmunized mice to heat-killed C. neoformans strain Cap67 unencapsulated mutant of B3501 (Ag1) or its supernatant (Ag2) demonstrated higher anticryptococcal activity. This effector mechanism can be enhanced further after immunization with either Ag1 or Ag2. There is a synergistic effect of immunization and in vitro incubation of the NA cells with the same antigens. Concanamycin A (CMA) and strontium chloride (SrCl2) inhibition assays were performed to clarify the contribution of perforin- and nonperforin-mediated anticryptococcal cytotoxicity of NA cells in these events. Treatment with these inhibitors demonstrated that anticryptococcal cytotoxicity of nonprimed NA cells was primarily perforin mediated. Anticryptococcal activity of the NA cells obtained from immunized mice after in vitro incubation with cryptococcal antigens was both perforin and nonperforin mediated. Taken together these data demonstrate that in mice a nonperforin-mediated pathway of anticryptococcal cytotoxicity can be induced by immunization. Further research is needed to examine their potential role for human vaccines strategies and/or therapies.

Chitotriosidase activity in sarcoidosis and some other pulmonary diseases.

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.

Influence of conjugated linoleic acid on the porcine immune response and morbidity: a randomized controlled trial.

BACKGROUND: Conjugated linoleic acid (CLA) has diverse influences on the immune response in different experimental models. In the present study we investigated the effect of CLA feeding on inflammatory and immune responses in a piglet model. We studied the duration of this effect and possible detrimental effects of CLA feeding. After 12 weeks of CLA and control supplementation and washout, animals were sacrificed and parenchymal organs were histologically examined. RESULTS: In activated peripheral mononuclear cells interferon-gamma was significantly (p = 0.008) lower in the CLA group by the end of the feeding period. This effect disappeared as soon as supplementation was stopped. No differences were found in the tumour necrosis factor-alpha, interleukin-10 production, serum immunoglobulin-G levels and fat infiltration of the liver, except that fat storage cell infiltration was significantly (p < 0.04) higher in the CLA-fed group. The effect of time for interferon-gamma, interleukin-10 and immunoglobulin-G levels was statistically significant. CONCLUSION: At the end of the feeding period the interferon-gamma response was depressed. However, the maturation of the piglet immune system in our young pig model probably outweighs the impact of CLA feeding on the immune response, even though liver fat storage cell infiltration, which plays an important role in liver regeneration, increased during CLA feeding of the piglets.

Late dendritic cells are still able to evoke a potent alloreactive CTL response.

On exposure to maturation stimuli, immature dendritic cells (DCs) undergo changes that turn them into potent amplifiers of innate immunity and into antigen-presenting cells (APCs) able to prime naïve T cells. However, their progression through the maturation process is very rapid and finally ends in apoptosis. The aim of our study was to investigate the importance of the maturation stage of DCs, defined by morphology, expression of surface markers and IL-12 production, for their immunostimulatory capacity. DCs were matured with LPS, monocyte-conditioned medium (MCM) or TNF-alpha, sampled several times during a 3-day long maturation period and used as stimulators of allogeneic T cells over a wide range of DC/T cell ratios. T-cell response was assessed by cell proliferation, CTL generation and IFN-gamma production. Our results indicate that the in vitro T cell response is determined mainly by the level of expression of co-stimulatory molecules on DCs and the DC/T cell ratio in the culture. Thus, DCs matured for over 20h, with high expression of co-stimulatory molecules, can still induce a potent CTL response at DC/T cell ratios of 1:10 and 1:20, although their IL-12 production, as well as their ability to induce IFN-gamma production by T cells, are both decreased. In contrast, the CTL response at DC/T cell ratios of 1:2 and 1:5 can be profoundly decreased. Notably, the proportion of proliferating CD4+ T cells in these cultures is reduced. This could well be the reason for the absence of CTL response, since we showed that, even in the case of high expression of co-stimulatory molecules on DCs, generation of CTLs still depends on CD4+ T cells. Our study emphasizes the importance of strong expression of co-stimulatory molecules on DCs and of their ability to activate CD8+ and CD4+ T cells concomitantly in order to initiate a potent cell-mediated immune response. We therefore suggest that a combination of early DCs, which are strong producers of cytokines, and late DCs, which have high expression of co-stimulatory molecules, could prove beneficial in the attempt to initiate in vitro and in vivo cell-mediated immune responses for therapeutic purposes.

Could cytokines levels or adhesion molecules expression be predictor of IFN-beta treatment response in multiple sclerosis patients?

OBJECTIVE: Some patients with relapsing-remitting multiple sclerosis (RRMS) do not respond to treatment with interferon-beta and continue to have relapses and new enhancing lesions on MRI. The markers which would predict the treatment response are still not known. The objectives of the study were to compare cytokines levels (IFN-gamma, IL-4, IL-6, IL-10) and expression of adhesion molecules before and during treatment in responders and nonresponders to IFN-beta treatment. METHODS: Twenty-nine patients with RRMS were enrolled in the study. Cytokine levels were evaluated by ELISA in supernatants of IONO/PMA activated PBMC cultures (IFN-gamma, IL-4, IL-6, IL-10), and by flow cytometry (intracellular IFN-gamma, IL-4 and IL-2R expression) before and during treatment. Expression of adhesion molecules (VLA-4, ICAM-1) was evaluated by flow cytometry (CD49+ and CD54+) before and during treatment. RESULTS: Only 9 of 29 patients were responders to treatment according to definition (no relapse in the first 2 years of treatment). We found significant differences in the expression of IL-2R after 1 month of treatment, intracellular IFN-gamma after 6 months of treatment and IL-10 level in nonactivated PBMC cultures after 1 week of treatment. CONCLUSION: We concluded that the differences we had found between responders and nonresponders are most probably incidental and not predictive of treatment response. Our study shows that cytokines levels and expression of adhesion molecules cannot be used as markers for treatment response.

Intestinal permeability and cytokine inflammatory response in multiply injured patients.

In experimental settings, the increased intestinal permeability (IP) following severe trauma is associated with increased serum concentrations of cytokines. Multiply injured patients are susceptible to the development of multiple organ failure (MOF). The aim of this study was to determine if altered IP after trauma was associated with upregulation of cytokines and if cytokines and IP influenced the development of MOF. In 30 multiply injured patients, IP was measured on days 2 and 4 after injury using the lactulose-mannitol (L-M) test, and the levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-8 were determined simultaneously. The L-M ratio increased significantly from 0.049 (0.017-0.133) on day 2 to 0.150 (0.059-0.339) on day 4 (p < 0.02) On day 4, a significant correlation was also found between the L-M ratio and IL-6 (r = 0.43, p < 0.03). The IL-6 level on days 2 and 4 was significantly (p < 0.01 and p < 0.03, respectively) higher in MOF patients than in those without MOF, as was the TNF-alpha level on day 4 significantly higher (p < 0.04) in MOF patients. IP increases following multiple trauma, and on day 4 it correlates with the IL-6 level. However, in patients who develop MOF only cytokines are invariably increased, with IL-6 alone being significantly increased on both measurements in these patients.

Interleukin-10, T-lymphocytes, and cardiac output in children after ventricular septal defect repair: a pilot study.

OBJECTIVE: To evaluate the acute inflammatory response and cardiac output in children after surgery for ventricular septal defect. DESIGN AND SETTING: Prospective, observational study in a level III multidisciplinary neonatal and pediatric intensive care unit. PATIENTS: Ten children undergoing open-heart surgery for ventricular septal defect. INTERVENTIONS: All children received methylprednisolone (30 mg/kg) in cardiopulmonary bypass (CPB) prime. MEASUREMENTS AND RESULTS: Before and after cardiopulmonary bypass, plasma interleukin-10 and tumor necrosis factor alpha were measured by enzyme-linked immunosorbent assay, and lymphocyte subsets in peripheral blood by flow cytometry. Relative values (post-/pre-CPB) of interleukin-10 and tumor necrosis factor alpha were calculated. The cardiac index (CI) was measured continuously beat-to-beat by a pulse contour analysis (PiCCO). Children above the cutoff value (median cardiac index value 3.0 l min(-1) m(-2)) were designated as the normal CI group and those below this value as the low CI group. In the normal CI group the relative values of interleukin-10 remained almost seven times higher than pre-CPB values at 24 h while in the low CI group they decreased almost to pre-CPB values. Furthermore, the normal CI group, but not the low CI group, exhibited more than threefold decrease in T-lymphocytes (lymphocyte T-cells, T-helper cells, and cytotoxic T-cells) 24 h after CPB. CONCLUSIONS: Children operated on for ventricular septal defect developed either a normal or low CI. The higher relative values of interleukin-10 and lower counts of lymphocyte T-cells, T-helper and cytotoxic T-cells differentiated the normal CI group from the low CI group at 24 h after cardiopulmonary bypass.

Structure of a synthetic fragment of the lipopolysaccharide (LPS) binding protein when bound to LPS and design of a peptidic LPS inhibitor.

Peptidic lipopolysaccharide (LPS) antagonists are the subject of intensive research. We report an NMR and modeling study of LBP-14 (RVQGRWKVRASFFK), a synthetic fragment of the LPS binding protein (LBP). In a mixture with LPS we observed the transferred nuclear Overhauser effect and determined the LPS-bound structure of LBP-14 that was used for docking calculations to LPS. The derived complex was used to design a peptide that displayed more than 50% increase in LPS inhibition in vitro.

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.

Shift from Th2 to Th1 response in immunotherapy with venoms.

Cytokines released from T lymphocytes regulate antibody production by B lymphocytes and releasability of mast cells and basophils. Immune tolerance in specific allergen (Ag) immunotherapy (SIT) might be a consequence of decreased Th2 or increased Th1 response of Ag specific T lymphocytes. We compared function of T lymphocytes in 11 patients with Hymenoptera venom allergy and in 7 healthy volunteers. We followed function of T lymphocytes in 6 patients during SIT. We measured released IL-2, IFN-γ, IL-4 and IL-5 from Ag and mitogens (either with phorbol-miristate-acetate (PMA) and ionomycine or anti-CD3 antibodies) stimulated peripheral blood mononuclear cells (PBMC). After stimulation with Ag PBMC of patients released 86 ± 133 pg/ml IFN-γ and 19.7 ± 20.1 pg/ml IL-4 (Th2 response), PBMCs of healthy controls released 184 ± 116 pg/ml IFN-γ and 4.8 ± 8 pg/ml IL-4 (Th1 response). Spontaneous release of IFN-γ in cultures of PBMC of patients increased after rush SIT (before: 25 ± 31 pg/ml, after 241 ±281 pg/ml, p<0.05). After 6 months of SIT response of PBMCs of patients became similar to response of PBMCs of healthy controls. Time pattern of cytokines secreted from PBMCs after stimulation with PMA and ionomycine was different than after stimulation through T-cell receptor/CD3 complex.

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

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.

Changes in Th1/Th2 cytokine balance in Graves' desease.

Graves' disease (GD) is characterised by hyperthyroidism, caused by stimulatory thyrotropin receptor (TSHR) antibodies. Recent research shows that an important factor in the pathogenesis of autoimmune diseases is the change in the balance between Th1 cytokines, which promote cell mediated immunity, and Th2 cytokines, which promote humoral immunity. There are contradictory data about this balance shift in GD. Our objective was to determine the Th1/Th2 cytokine balance shift in patients with newly diagnosed GD, when compared to the same balance in healthy controls. We isolated mononuclear cells (MNC) from the peripheral blood of healthy donors and from patients with newly diagnosed GD before treatment. The MNC were activated with ionomycin in combination with phorbol 12-myristate 13-acetate (PMA). After 40-hour incubation, the concentrations of the cytokines produced (IFN-γ, IL-4, IL-10, IL-12) in the culture supernatants were measured by ELISA (Endogen, USA). The MNC cultures from patients with GD produced significantly less IL-12 and significantly more IL-10 and IL-4 than MNC cultures from healthy controls. All calculated ratios of Th1 against Th2 cytokines in MNC cultures from patients with GD were significantly lower than in MNC cultures from healthy controls. Our results show a systemic shift of cytokine production in patients with GD toward the Th2 cytokine response, thus confirming the key role of TSHR antibodies and humoral immunity in the pathogenesis of GD.

Modulation of tumour necrosis factor production with desmuramyldipeptide analogues.

Some synthetic analogues of the immuno-modulatory agent muramyl dipeptide (MDP), i.e. phthalimido- (LK-511, LK-413, LK-512, LK-423, LK-508), adamantyl- (LK-415, LK-517), 7-oxoaIkyl-(LK-409) desmuramylpeptides were assessed for the tumour necrosis factor (TNF) inducing activity and the ability to modulate TNF production in in vitro phorbol 12-myristate 13-acetate (PMA) & ionomycin stimulated cultures of human peripheral blood mononuclear cells. A kinetic study over a 40-hour period indicated that desmuramyldipeptides were weak TNF inducers compared to romurtide, PMA & ionomycin or lipopolysaccharide. By contrast, they showed the potential to up- or down-regulate the production of TNF evoked by PMA & ionomycin, which was strongly dependent on the time of the stimulation. After 4h of stimulation, the TNF secretion was augmented by LK-508, LK-409 and LK-511, after 18 h by LK-409 and LK-423, and after 40 h by LK-423, LK-511, LK-415 and LK-512. However, LK-517 and LK-512 inhibited the secretion of TNF after the 18-h period.

lnterleukin-6 (IL-6) and soluble receptors for interleukin-2 (slL-2R) in the diagnosis of early severe infection in the critically ill newborns.

The aim of our study was to evaluate the diagnostic accuracy of serial determination of interleukin-6 (IL-6) and soluble receptors of interleukin-2 (sIL-2R) in the diagnosis of early infection in the critically ill newborns and compare it with the routinely used C-reactive protein (CRP). Fourty-six critically ill newborns (median age 8 h, range 1-96 h), treated at the multidisciplinary intensive care unit, Division for Paediatric Surgery and Intensive Care, University Medical Centre Ljubljana, were included in the study. Newborns were divided into three groups: group I microbiologically confirmed severe infection (n = 14), group II suspected but not confirmed infection (n = 12) and group III respiratory distress syndrome without laboratory signs of infection. Serum concentrations of IL-6, sIL-2R and CRP were determined on admission and at 12 and 24 h after admission. On admission the concentrations of IL-6 and sIL-2R were significantly higher in group I than in group III, but there was no difference between groups I and II. On admission area under receiver operating characteristic (ROC) curve for IL-6 was 0.756, for IL-2R 0.821 and for CRP 0.799. Repeated determination at 12 h improved diagnostic accuracy for sIL-R and CRP but not for IL-6.

Immune response in lymphocyte cultures stimulated by oral bacteria preparations.

Lymphocyte cultures were used as an in vitro experimental model to get a deeper insight into immune response to oral bacteria in periapical granulomas. Lymphocytes isolated from leucocyte concentrate were in lymphocyte cultures stimulated by antigen preparations of oral bacteria. Lymphocyte subsets that have developed in lymphocyte cultures after a week of stimulation were analysed by flow cytometry. A significant increase in expression of INF-γ molecules in CD3+ cells stimulated by antigen preparations of oral streptococci was found, compared with negative control. On the other hand we observed a significant increase in expression of IL-4 in CD3+ cells stimulated by antigens of anaerobic bacteria, compared with negative control. Our results show that antigens of oral streptococci in in vitro lymphocyte cultures induce the differentiation of T helper cells into Th2 cells and that antigen preparations of anaerobic bacteria induce the differentiation of T helper cells into Th1 cells. Furthermore, an increased expression of HLA-DR molecules on CD8+ T cells stimulated by antigens of oral streptococci was found, compared with negative control.