In vitro and in vivo reactivity to fungal cell wall agents in sarcoidosis.

Sarcoidosis is an inflammatory disease. Epidemiological and treatment studies suggest that fungi play a part in the pathogenesis. The aim of this work was to study the effect of fungal cell wall agents (FCWA) on the in vitro secretion of cytokines from peripheral blood monocytes from subjects with sarcoidosis and relate the results to fungal exposure at home and clinical findings. Subjects with sarcoidosis (n=22) and controls (n=20) participated. Peripheral blood mononuclear cells were stimulated with soluble or particulate ß-glucan (S-glucan, P-glucan), chitin or lipopolysaccharide (LPS), whereafter tumour necrosis factor (TNF)-α, interleukin (IL)-6, IL-10 and IL-12 were measured. The severity of sarcoidosis was determined using a chest X-ray-based score. Serum cytokines (IL-2R, IL-6, IL-10 and IL-12) were determined. To measure domestic fungal exposure, air in the bedrooms was sampled on filters. N-acetylhexosaminidase (NAHA) on the filters was measured as a marker of fungal cell biomass. The induced secretion of cytokines was higher from peripheral blood mononuclear cells (PBMC) from subjects with sarcoidosis. P-glucan was more potent than S-glucan inducing a secretion. Chitin had a small effect. Among subjects with sarcoidosis there was a significant relation between the spontaneous PBMC production of IL-6, IL-10 and IL-12 and the NAHA levels at home. The P-glucan induced secretion of IL-12 was related to the duration of symptoms at the time of diagnosis. Their X-ray scores were related to an increased secretion of cytokines after stimulation with LPS or P-glucan. Subjects with sarcoidosis have a higher reactivity to FCWA in vitro and to home exposure. The influence of FCWA on inflammatory cells and their interference with the inflammatory defense mechanisms in terms of cytokine secretion could be important factors for the development of sarcoidosis.

Influence of desmuramylpeptides from LK-409 series on the cytokine production in the mouse spleen cells.

LK-409 (7-oxooctanoyl-L-Ala-D-iGln) was found to modulate immune response. To study the impact of minimal structural changes in the LK-409 molecule on the immunomodulatory activity a series of LK-409 analogues was prepared and their activities were evaluated. After the treatment of NmRI mice at a dose of 25 microg daily for three consecutive days the isolated spleen cells were stimulated with concanavaline A and phorbol 12-myristate 13-acetate (PMA) or ionomycin and PMA, and the production of IL-2, IL-4,-IL-6, IL-10 and IFN-gamma studied as a function of the different LK-409 analogues. All the compounds modulate the Th1/Th2 cytokine response, especially in the presence of ConA&PMA activation. The minimal structure modification of LK-409 strongly influences the regulation of the cytokine production.

An effective tumor vaccine against malignant melanoma: irradiated autologous tumor cells admixed with MVE-2.

The aim of this study was to develop as effective as possible autologous tumor vaccine which would be at the same time easy to produce, highly controllable, and without undesired side effects on normal tissue. Therefore, irradiated autologous - syngeneic B-16 tumor cells admixed with a non-specific immunomodulator MVE-2 (a polymer fraction of 1,2-co-polymer of divinyl ether and maleic anhydride) were used for subcutaneous (s.c.). or intraperitoneal (i.p.) prevaccination of experimental mice. Compared to the control mice, a statistically significant delay in tumor development of s.c. tumors was achieved in prevaccinated mice (p<0.05). An even better effect was observed in mice challenged i.p. with viable tumor cells. Using a single prevaccination complete protection was obtained in between 40-85% of the experimental mice. When the survivors from the groups injected once with the tumor vaccine were rechallenged with viable tumor cells (101 day after the first tumor challenge, no additional prevaccination), 15.7% remained free of tumor, while the survivors from the groups injected with the tumor vaccine twice and 101 day later rechallenged with viable tumor cells remained free of tumor in 60% of the cases. Based on these results we can postulate that our vaccine is effective for prevention of tumor development. The achieved protection can be augmented with serial vaccinations and can be maintained for a longer period of time.

Construction of an expression cassette with hTNF-alpha gene for transient expression of the gene in mammalian cells.

BACKGROUND: Tumor vaccines, which are created by the insertion of cDNA encoding different cytokines into the tumor cells, are capable of inducing a very complex immune reaction including activation of CD8 T cells, granulocytes, macrophages, the triggering of cytokine cascades and antibody production. Aiming to create genetically modified tumor cells which could produce and secrete Human Tumor Necrosis Factor-alpha (hTNF-alpha), we constructed the expression cassette containing hTNF-alpha gene in pcDNA3 plasmid vector. MATERIALS AND METHODS: The successful ligation of cDNA encoding for hTNF-alpha into pcDNA3 plasmid vector was confirmed by PCR, restriction mapping and sequence determination. The constructed expression cassette in pcDNA3 vector was than transferred in vitro into malignant melanoma B16 tumor cells by the method of Receptor Mediated Gene Transfer (RMGT). RESULTS: Measurable amounts of hTNF-alpha protein detected in the medium of transfected cells proved that tumor cells modified in this manner became producers of hTNF-alpha protein. CONCLUSION: The expression of the transferred gene was transient and the produced protein was biologically active. Furthermore, the production of hTNF-alpha protein was also observed in sub-lethally irradiated tumor cells, showing that the expression cassette was preserved during the irradiation and that the cells were potentially applicable as a tumor vaccine.

Flow cytometric analysis of lymphocytes isolated from nasal polyps.

We analyzed by flow cytometry the expression of IL-2 receptors (alpha subunit-CD25) and ICAM-1 adhesion molecules (CD54) on T cells and subsets (CD4, CD8) isolated from nasal polyp tissue in allergic and non-allergic patients. We found a significant increase in IL-2 receptor and ICAM-1 molecule expression on T cells isolated from nasal polyp tissue compared to peripheral blood lymphocytes. We also found a significantly increased expression of ICAM-1 molecules on CD8+ cells in non-allergic compared to allergic patients. The latter may reflect a difference in cytotoxic immune response between allergic and non-allergic patients, but the result should be confirmed in a more extensive study including cytokine and immunoglobulin analysis. We hope that it would enable us to obtain a deeper insight into the local immune events and further to clarify the etiology and pathogenesis of nasal polyps and their relation to allergy.

Synthesis and modulation of cytokine production by two new adamantane substituted acyclic desmuramyldipeptide analogs.

Two new adamantyl-desmuramyldipeptides LK 415 and LK 517 with 1-adamantylcarboxamido moiety as a replacement for muramyldipeptide's N-acetylglucosamine fragment were synthesized. Their efficacy to modulate the production of cytokines was measured in vitro in ionomycin and phorbol-12-myristate-13-acetate (PMA) activated cultures of human peripheral blood mononuclear cells (PBMC), co-incubated with the substances tested. The results were compared with the activity of muramyldipeptide (MDP). All three substances are strong up-regulators of IL-12 synthesis and hence of the IFN gamma synthesis as well. While MDP and LK 415 are relatively ineffective in modulation of IL-2, IL-4 and IL-10 production in vitro, the synthesis of all three cytokines is considerably up-regulated when peripheral blood mononuclear cells are co-incubated with LK 517. It seems likely that the introduction of the diethyl phosphonate moiety into LK 517 is of great importance for the augmented T-cell cytokine production.

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-gamma, IL-4 and IL-5 from Ag and mitogens (either with phorbol-myristate-acetate (PMA) and ionomycin or anti-CD3 antibodies) stimulated peripheral blood mononuclear cells (PBMC). After stimulation with Ag PBMC of patients released 86 +/- 133 pg/ml IFN-gamma and 19.7 +/- 20.1 pg/ml IL-4 (Th2 response), PBMCs of healthy controls released 184 +/- 116 pg/ml IFN-gamma and 4.8 +/- 8 pg/ml IL-4 (Th1 response). Spontaneous release of IFN-gamma 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 ionomycin was different than after stimulation through T-cell receptor/CD3 complex.

In vivo administration of azithromycin affects lymphocyte activity in vitro.

A therapeutic dose of azithromycin was administered to test subjects and then the following lymphocyte functions were examined in vitro: proliferative lymphocyte response to stimulation with pokeweed mitogen, levels of immunoglobulins G, A, and M in serum, and the amount of the soluble interleukin 2 receptors in supernatants of mononuclear cell cultures stimulated with phytohemagglutinin and phorbol myristate acetate. The study was performed as a controlled clinical trial comparing an azithromycin-treated group (n = 21) and a placebo-treated control group (n = 10). Healthy female volunteers were placed into one of the two groups, and the study was performed as a double-blind trial. Although the findings of the present study showed that azithromycin significantly increased the proliferative lymphocyte response to pokeweed mitogen, the results could have been due to experimental variation. However, impairment of the lymphocyte function was not observed, which could represent valuable information. Likewise, no effect of azithromycin on levels of the immunoglobulins in serum was observed. The most marked effect of azithromycin on the lymphocyte function was demonstrated by an elevation in the amount of soluble interleukin 2 receptor production in mononuclear cell cultures. The lack of impairment or, perhaps, even a beneficial influence on the immunodefense system may be an important property of azithromycin, especially in immunocompromised individuals.

Interleukin-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.

Interleukin-6 (IL-6) and soluble receptors for interleukin-2 (sIL-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.

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

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-gamma, 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.

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.

Methimazole upregulates T-cell-derived cytokines without improving the existing Th1/Th2 imbalance in Graves' disease.

There is probably a systemic shift of cytokine production in patients with Graves' disease (GD) toward the Th2 cytokine response. Methimazole (MMI) is the first choice for patients with GD and presumably has some direct immunomodulatory action. The aim of this study was to evaluate the balance shift in Th1/Th2 cytokines in patients with GD after 1 yr of MMI treatment, when compared to the same balance in patients with newly diagnosed GD before treatment and in healthy controls. Peripheral blood mononuclear cells (PBMC) were isolated from 17 healthy volunteers, from 18 patients with newly diagnosed GD before treatment and from 15 euthyroid patients with GD after 1 yr of MMI treatment. The PBMC were activated with ionomycin and phorbol 12-myristate 13-acetate (PMA). The concentrations of Th1/Th2 related cytokines [interferon (IFN)-gamma, interleukin (IL)-12 vs IL-4, IL-10] in the culture supernatants were measured by ELISA. PBMC from patients with GD after treatment produced significantly more IFN-gamma and IL-4 than PBMC from patients with GD before treatment, but there were no significant differences in calculated ratios of Th1 against Th2 cytokines between these two groups. When compared to PBMC from healthy controls, PBMC from patients with GD after treatment produced significantly more IL-4 and significantly less IL-12. The calculated IL-12/IL-4 ratio after treatment was significantly lower than the same ratio from healthy controls. In conclusion, our results show no significant change in the ratio between Th1 and Th2 cytokines produced by PBMC from patients with GD after 1 yr of MMI treatment, when compared to the ratio before treatment. The ongoing prevalence of the Th2 immune response after treatment speaks against the immunomodulatory action of the drug on the systemic level.

Modulation of tumour necrosis factor production with desmuramyldipeptide analogues.

Some synthetic analogues of the immunomodulatory agent muramyl dipeptide (MDP), i.e. phthalimido- (LK-511, LK-413, LK-512, LK-423, LK-508), adamantyl- (LK-415, LK-517), 7-oxoalkyl-(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.

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-gamma 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.

Immune responses and cytokine induction in the development of severe hepatitis during acute infections with murine cytomegalovirus.

Salivary gland-derived murine cytomegalovirus (SGV) infections of mice have been widely used as models of human cytomegalovirus infections and in the study of CMV biology. Still, many aspects of SGV pathogenesis are not clearly defined. Fatal and non-fatal SGV infections were investigated to characterize pathogenetic correlates of mortality and to assess the role of the immune response in disease progression. Suppression of immune responses was observed in both lethal and sublethal infections. Depletion of immune cell populations in spleen, however, correlated with severe CMV-induced hepatitis and mortality. In addition, T cell depletion studies indicated a requirement for this immune cell subset in control of liver damage and survival of infected mice. Examination of cytokine responses revealed a previously undescribed shock-like syndrome in lethally-infected mice characterized by high levels of tumor necrosis factor alpha and interferon gamma. Furthermore, the sites of tumor necrosis factor alpha gene induction did not strictly correlate with either viral load or the sites of tissue damage during infection. Taken together, these findings define the pathogenetic progression of disease as it relates to disease outcome and suggests that organ-specific differences in cytokine induction play a significant role in the late stages of acute lethal MCMV infections.

Apyrogenic synthetic desmuramyldipeptide, LK-409, with immunomodulatory properties.

The synthesis and some immunological characteristics of a new desmuramyl dipeptide 7-oxooctanoyl-L-alanyl-D-isoglutamine (LK-409) are presented. The effects of this compound were compared with those of N-acetylmuramyl-L-alanyl-D-isoglutamine (MDP). The influence of LK-409 on the number of B and T cells in spleen and the number of peritoneal macrophages was studied; Jerne's plaque forming cells assay was performed to monitor the effect of B cell differentiation. The blast transformation of T cells stimulated with concanavalin A was used to detect the influences on T lymphocytes. The activation of macrophages was studied as well. In contrast to MDP, LK-409 was apyrogenic in the doses applied but had similar immunomodulatory properties. Tested immunological properties and the absence of pyrogenicity and low toxicity make LK-409 a candidate for an immunomodulatory drug and a model molecule suitable for studying and understanding the dual activity of the MDP and its analogues.