Vladimir Kotnik

Molecular, genetic, and functional analysis of homozygous C8β-chain deficiency in two siblings

UNLABELLED C8 deficiency is associated with an increased susceptibility to neisserial infections. We present a case of an 11 year old boy who suffered from infection with Neisseria meningitidis. Medical history of the patient and his family (n = 5) did not indicate any previous immunodeficiency symptoms. Results from the analysis of phagocyte and lymphocyte functions were within the normal range. No hemolytic activities of the classical (CH50) and the alternative (APH50) pathways of complement were measurable, and SC5b-9 protein complexes could not be detected in the patients plasma. Further analysis by highly sensitive ELISA and functional assays revealed a complete deficiency of C8. Upon the reconstitution with purified C8 total hemolytic activity could be restored. SDS-PAGE and Western blot analysis established a deficiency of the C8 beta chain. Genetic analysis at the genomic DNA level demonstrated the common C-T mutation in exon 9 of the C8B gene. Family analysis presented the older sister with non-detectable function of C8 in serum, both parents with about half-normal C8 titres, and the younger sister with normal C8 function. The parents and both sisters were asymptomatic, although the older of the sisters presented with the same complete C8 beta-chain deficiency as the patient described. IN CONCLUSION the common C-T mutation in the C8B genes is the genetic basis of C8 beta-chain deficiency in two members of this Bosnian family.

CD69 Expression on CD4+ T Lymphocytes after In Vitro Stimulation with Tuberculin Is an Indicator of Immune Sensitization against Mycobacterium tuberculosis Antigens

ABSTRACT The expression of the CD69 antigen on CD4 T lymphocytes after in vitro stimulation with purified protein derivative (2 tuberculin units) was used to evaluate the tuberculin reactivities of 52 individuals from four experimental groups: Mycobacterium bovis BCG-vaccinated healthy individuals with a negative tuberculin skin test (TST) result (group A), BCG-vaccinated healthy individuals with a positive TST result (group B), patients with active tuberculosis (TB) before treatment (group C), and individuals with clinically inactive TB who had previously completed a prescribed course of chemotherapy (group D). The expression of CD69 on CD4 T lymphocytes was significantly higher in patients with active TB (16.2% ± 7.3%), individuals with clinically inactive TB (10.5% ± 7.4%), and healthy individuals with a positive TST result (15.5% ± 7.2%) than in healthy individuals with a negative TST result (3.8% ± 4.3%) (P < 0.005). We confirmed the correlation between CD69 antigen expression on T lymphocytes after stimulation with tuberculin and the TST induration diameter (Spearman rho = 0.783; P < 0.001), an assay for gamma interferon (the Quantiferon-TB assay; Spearman rho = 0.613; P < 0.001), and the lymphocyte BLAST transformation test (Spearman rho = 0.537; P < 0.001). Our results demonstrate the usefulness of the determination of CD69 on CD4 T lymphocytes after in vitro stimulation with tuberculin as a rapid indicator of immune sensitization against Mycobacterium tuberculosis.

Human peripheral blood lymphocytes sensitised to PPD respond to in vitro stimulation with increased expression of CD69 and CD134 activation antigens and production of Th-1 type cytokines

Abstract Individuals sensitised to Mycobacterium tuberculosis antigens by infection, vaccination or Mantoux test generate specific memory cells. The response to in vitro restimulation with PPD is observed as the lymphoid cell proliferation and production of Th-1 type cytokines. Cell-mediated immune response was measured by Mantoux test, lymphocyte blast transformation test, estimation of IFN-gamma production (Quantiferon, ELISPOT), and expression of CD69 and CD134 molecules on the T-helper lymphocytes (CD4+). All the methods used were compared for parity of the results. According to Mantoux test results, the patients could be distributed into two groups: responder and non-responder group. Induration in Mantoux test after a new contact with PPD in non-responders was smaller than 5 mm, they produced only small amounts of IFN-gamma, lymphocyte blast transformation was poor, and expression of CD69 and CD134 was low. In responders reaction was much more intensive in all tests measured. We conclude that the reactivity of memory cells to M. tuberculosis antigens can be effectively detected by Mantoux test. The same was true also for the in vitro tests presented but in addition the in vitro tests give more information about the mechanism involved in the immune response against M. tuberculosis.

The humoral and cellular immune response to a lipid attenuated pore-forming toxin from the sea anemone Actinia equina L.

The immunogenicity of a pore-forming polypeptide, equinatoxin II, from the sea anemone Actinia equina was studied after attenuation of the toxins lethal and cytolytic activity by autologous polar lipids. In BALB/c mice, the lipid-inactivated toxin was used to raise specific antibodies and cellular immunity, resulting in in vivo protection. In vitro, haemolytic activity could be diminished by both normal and immune serum, the latter being more efficient. Purified specific IgG1 and IgG2 did not or only poorly neutralized the haemolytic activity, therefore implying the marked role of serum lipoproteins in the toxin attenuation. In response at the cellular level, equinatoxin II activated specific splenocytes. Increased concanavalin A stimulation of specific splenocytes was observed in the absence of antigen.

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 ofN-acteylmuramyl-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; Jernes 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.

Pulsed electric current enhances the phorbol ester induced oxidative burst in human neutrophils

Oxidative burst (OB) response in human neutrophils, measured with chemiluminescence (CL), has been used to determine whether pulsed electric current (PEC) might induce a functional response in these electrically nonexcitable cells, and also whether it might modify cellular response to tumor‐promoting phorbol ester (PMA). Five minutes of PEC treatment caused no significant changes in neutrophil CL levels in HBSS (1.2 mM Ca2+ concentration) as well as in HBSS‐EGTA, where the extracellular Ca2+ concentration was reduced to less than 30 nM. The CL level of PMA‐activated neutrophils in HBSS was 52% higher than in HBSS‐EGTA. In HBSS the CL level, after the combined PMA and PEC treatment, was 53% higher than in PMA‐alone‐treated neutrophils. Activation of the OB in HBSS‐EGTA with PMA and PEC was 13% higher than in solely PMA treated neutrophils. The results suggest that in neutrophil OB response, the PEC effect is closely related with cellular calcium mobilization, since depletion of extracellular Ca2+ decreased the PEC effect.

Murine monoclonal antibodies directed against human recombinant Macrophage Migration Inhibitory Factor

Abstract Macrophage Migration Inhibitory Factor (MIF) is a crucial component of the immune system acting together with glucocorticosteroids to regulate immunity and inflammation. Understanding of its many putative functions and action mechanisms is still ambiguous. Due to the newest findings that a local MIF expression is up regulated in allograft rejection and in glomerulonephritis, an interest in MIF research is increasing and is focused on possibilities of anti-MIF treatment.In the present work new murine monoclonal antibodies (MAbs) directed against human recombinant MIF (hrMIF) are described. hrMIF protein used for the immunisation was tested for its biological activity and has evident macrophage migration inhibitory activity. The selected MAbs were purified and further characterised. They recognised MIF in a Western blot experiment after a native IEF. Anti-MIF MAb designated as Ml inhibited MIF activity in the test, which was performed in the 48 well Boyden chamber system. It is presumed that Ml MAb could be used as a potential therapeutic agent.

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 non-primed 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 non-perforin 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.

Therapeutic electric stimulation does not affect immune status in healthy individuals - a preliminary report

BackgroundNeuromuscular electric stimulation is widely used for muscle strengthening in clinical practice and for preventative purposes. However, there are few reports on the effects of electric stimulation on the immune response of the organism, and even those mainly describe the changes observed immediately after the electrotherapeutic procedures. The objective of our study was to examine the possible immunological consequences of moderate low-frequency transcutaneous neuromuscular electric stimulation for quadriceps muscle strengthening in healthy individuals.MethodsThe study included 10 healthy volunteers (5 males, 5 females, mean age 37.5 years). At the beginning and after a two-week electric stimulation program, muscle strength was measured and peripheral blood was collected to analyse white blood cells by flow cytometry for the expression of cell surface antigens (CD3, CD19, CD4, CD8, CD4/8, DR/3, NK, Th reg, CD25 + CD3+, CD25 + CD4+, CD25 + CD8+, CD69 + CD3+, CD69 + CD4+, CD69 + CD8+) and phagocytosis/oxidative killing function.ResultsMuscle strength slightly increased after the program on the dominant and the nondominant side. No statistically or clinically significant difference was found in any of the measured blood and immune cells parameters as well as phagocytosis and oxidative burst function of neutrophil granulocytes and monocytes one day after the program.ConclusionsThe program of transcutaneous low-frequency electric stimulation slightly strengthened the quadriceps femoris muscle while producing no changes in measured immunological parameters. Hence, therapeutic low-frequency electric stimulation appears not to be affecting the immune response of healthy persons.

Immunological tests and the detection of Helicobacter pylori infection.

Background: Herewith we present the results of four-year testing, and more than ten years of experience in laboratory determination and interpretation of Helicobacter pylori (Hp) infection. An analysis was performed to draw attention of the physicians to the value and importance of immunological testing for the recognition of Hp infection.