Complement in skin diseases.

Complement is one of the most important mechanisms of natural resistance preventing infections in humans and animals. It is actively involved in the pathogenesis of several diseases, including skin diseases, characterized by the presence of autoantibodies, foreign microorganisms, altered tissue cells, and the presence of mannan. Complement is intended to kill invading microorganisms but it can also destroy the organism's own damaged or altered cells. It is characterized by vigorous activity and is also potentially harmful for the host if triggered in its own body. This review discusses the significance of complement activation for emerging skin diseases and highlights the importance of serological laboratory tests for the detection of complement system activity alterations in skin diseases such as pemphigus vulgaris, bullous pemphigoid, herpes gestationis, dermatitis herpetiformis, porphyria, urticaria, angioedema, cutaneous vasculitis, systemic lupus erythematosus, partial lipodystrophy, lichen planus, xeroderma pigmentosum, psoriasis, and recurrent cutaneous infections. Finally, we draw attention to the current potential for treating these diseases with complement inhibitors.

Intrathecal antitreponemal antibody synthesis determination using the INNO-LIA Syphilis Score.

BACKGROUND: Laboratory detection of intrathecal synthesis of specific antitreponemal antibodies remains a challenge. Traditional syphilis serology is unable to provide a satisfactory result; therefore, several other diagnostic procedures were used to demonstrate central nervous system (CNS) involvement in this disease. The introduction of molecular methods makes today's laboratory testing easier. OBJECTIVE: Our study used a new commercially available test, the INNO-LIA Syphilis Score, intended for use on serum samples, to detect specific antitreponemal antibodies in the cerebrospinal fluid (CSF) of patients with the tertiary stage of syphilis. PATIENTS AND METHODS: We tested 26 patients suspected of neurological complications of late syphilis with conventional immunological tests such as VDRL-RPR, TPHA, FTA-ABS IgG, FTA-ABS-IgM, and the molecular INNO-LI Syphilis Score test for the presence of nontreponemal and treponemal antibodies. All tests were performed simultaneously in serum and CSF. The test results were evaluated with descriptive statistics and the probability was tested with an ANOVA test. RESULTS: All 26 samples of serum were LIA-S (line immune assay in serum) positive and presented anticardiolipin and antitreponemal antibodies in high titer. Seventeen samples of CSF were LIA-L (line immune assay in liquor) positive and nine were LIA-L negative. Anticardiolipin and antitreponemal antibodies were detected only in the group of LIA-L positive samples. Anticardiolipin antibodies were present in two cases, antitreponemal (TPHA) in five cases, specific IgG (FTA-ABS IgG) in six cases, and specific IgM (FTA-ABS IgM) in one case. Six patients with antitreponemal antibodies in CSF presented with pathologic albumin index, two with a milder form, and four with a severe form. Two had a pathological IgG index and four a pathological IgM index. Altogether, two of the patients had laboratory signs of neurosyphilis. CONCLUSIONS: Detecting anticardiolipin and antitreponemal antibodies in CSF in patients with a late form of syphilis is laborious. Using the new INNO-LIA Syphilis Score molecular test, we were able to identify patients with silent neurosyphilis together with patients with active intrathecal synthesis of IgG antibodies. The development of a new generation of tests for the detection of specific antitreponemal antibodies in CSF offers a valuable tool for discovering possible CNS involvement in syphilis.

Basiliximab and mycophenolate mofetil in combination with low-dose cyclosporine and methylprednisolone effectively prevent acute rejection in kidney transplant recipients.

The aim of the study was to analyze whether immunosuppressive treatment with basiliximab and mycophenolate mofetil (MMF), allowed a reduction in methylprednisolone and cyclosporine dosages without increasing the incidence of acute rejection episodes, reducing 1-year graft and patient survivals, or increasing the rates of infections and malignancy in the first year. The two groups were group A (n = 72), treated with methylprednisolone and cyclosporine and in the first 2 weeks with azathioprine; group B (n = 72), treated with basiliximab, MMF, and low-dose cyclosporine and methylprednisolone. The patients were followed for 1 year. The incidence of acute rejection episodes in the first year was significantly lower in group B (2.8%) than group A (12.5%; P < .05). The cumulative methylprednisolone dose, the daily dose, and the average cyclosporine trough blood level were significantly lower in group B (P < .001). One-year serum creatinine was significantly lower in group B (112 +/- 45 micromol/L) than group A (138 +/- 51 micromol/L; P < .01). One-year graft survival was 91.7% in group A and 98.6% in group B. One-year patient survival was 98.6% in group A and 100% in group B. The groups did not differ significantly in the incidence of bacterial, viral, or fungal infections. Immunosuppression with basiliximab and MMF allowed a reduction in cyclosporine and methylprednisolone dosages and was associated with significantly lower incidences of acute rejections episodes with better graft function in the first year as opposed to immunosuppression with higher doses of cyclosporine and methylprednisolone alone. Both immunosuppressive regimens showed the same infection rates and did not differ significantly in the occurrence of malignant diseases within the first year.

Reduced blood flow and oxygenation in SA-1 tumours after electrochemotherapy with cisplatin.

Electrochemotherapy is an antitumour treatment that utilises locally delivered electric pulses to increase cytotoxicity of chemotherapeutic drugs. Besides increased drug delivery, application of electric pulses affects tumour blood flow. The aim of this study was to determine tumour blood flow modifying effects of electrochemotherapy with cisplatin, its effects on tumour oxygenation and to determine their relation to antitumour effectiveness. Electrochemotherapy of SA-1 subcutaneous tumours was performed by application of electric pulses to the tumours, following administration of cisplatin. Tumour blood flow modifying effects of electrochemotherapy were determined by measurement of tumour perfusion using the Patent blue staining technique, determination of tumour blood volume, and microvascular permeability using contrast enhanced magnetic resonance imaging, and tumour oxygenation using electron paramagnetic resonance oximetry. Antitumour effectiveness was determined by tumour growth delay and the extent of tumour necrosis and apoptosis. Tumour treatment by electrochemotherapy induced 9.4 days tumour growth delay. Tumour blood flow was reduced instantaneously and persisted for several days. This reduction in tumour blood flow was reflected in reduced tumour oxygenation. The maximal reduction in partial oxygen pressure (pO2) levels was observed at 2 h after the treatment, with steady recovery to the pretreatment level within 48 h. The reduced tumour blood flow and oxygenation correlated well with the extent of tumour necrosis and tumour cells apoptosis induced by electrochemotherapy with cisplatin. Therefore, the data indicate that antitumour effectiveness of electrochemotherapy is not only due to increased cytotoxicity of cisplatin due to electroporation of tumour cells, but also due to anti-vascular effect of electrochemotherapy, which resulted in reduced tumour blood flow and oxygenation.

Cell-mediated immune response to high-passage Borrelia spirochetes in C57bl/6 mice is strictly dependent on antigen specificity.

Inbred C57bl/6 mice were challenged with high-passage Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen specific T-cell response in vitro. Sonicated preparations of washed spirochetes were potent cell activators, capable of stimulating polyclonal proliferation after 72h of culture while increasing the incubation time up to 120h provoked specific cell-mediated response. Isolated murine spleocytes previously sensitized to B. burgdorferi sensu lato but not those from control mice could be induced for antigen-specific proliferation in vitro, as revealed by [3H]thymidine incorporation assay, Moreover, in mice presensitized to B. burgdorferi sensu lato, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies. The current study emphasises that the B. burgdorferi antigen-specific response may also be expected in different genospecies infections in men.

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.

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.

Murine monoclonal antibodies directed against human recombinant Macrophage Migration Inhibitory Factor.

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 M1 inhibited MIF activity in the test, which was performed in the 48 well Boyden chamber system. It is presumed that M1 MAb could be used as a potential therapeutic agent.

The cell-mediated immune response to Borrelia afzelii, garinii and burgdorferi in C57BL/6 mice is dependent on antigen specificity.

Inbred C57BL/6 mice were challenged with Borrelia afzelii, Borrelia garinii and Borrelia burgdorferi sensu stricto and tested for antigen-specific T-cell response in vitro. The sonicated preparations of in vitro grown spirochetes were capable of stimulating polyclonal proliferation and specific cell-mediated response, depending on duration of the cell culture. Murine splenocytes previously sensitized to B. burgdorferi sensu lato (s.l. ), but not those from control mice, could be induced for antigen-specific proliferation in vitro. Moreover, detectable cell-mediated response could be induced only with antigen preparations derived from a corresponding strain but not with those obtained from other Borrelia genospecies as revealed by the [(3)H]thymidine incorporation assay. The current study considers that the strict B. burgdorferi s.l. antigen-specific response may also be expected in infections in humans and contributes to the explanation of the frequently poor antibody- and cell-mediated immune response observed in patients diagnosed with Lyme disease.

N-[trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl]-L-alanyl-D-glutamic acid: a novel immunologically active carbocyclic muramyl dipeptide analogue.

A novel non-pyrogenic carbocyclic muramyl dipeptide (MDP) analogue, N-¿trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl¿-L-alanyl-D-glutamic acid, was obtained by replacement of the N-acetylmuramic acid part and the D-isoglutamine residue of the MDP molecule by a trans-2-[[2'-(acetylamino)cyclohexyl]oxy]acetyl moiety and D-glutamic acid, respectively. The title compound was selected as a promising candidate for further evaluation among several related analogues on the basis of an immunorestoration test in mice. This novel nor-MDP analogue protects mice against the immunosuppressive effect of cyclophosphamide and increases the nonspecific resistance of mice against fungal infection. It is an immunomodulator which enhances the maturation of lymphocytes B to plasma cells and increases the activity of lymphocytes B and lymphocytes T as well as that of macrophages but does not alter the number of these cells.

Local changes in membrane potential intensify neutrophil oxidative burst.

The aim of this paper is to study the effects of the pulsed electrical field/current alone or combined with ionomycin, fMLP and PMA, the chemical stimuli that operate through distinctly different activation pathways, on the time course of the oxidative burst response in human neutrophils. Neither the control groups nor the neutrophils treated with electrical field alone showed any increase in oxidative burst activity measured by the luminol-enhanced chemiluminescence technique. It was found that electrical treatment potentiates chemically induced activation with either of the chemical stimulators used. The integrated oxidative burst response--which represents a cumulative amount of oxygen metabolites produced during whole response--was 87% higher in neutrophils treated with a combination of ionomycin and an electric field than in solely ionomycin treated cells, while the peak level of the response was 114% higher. In neutrophils stimulated with fMLP the electrochemical treatment caused a 32% higher integral response as well as a 22% higher peak level compared to the neutrophils treated with chemical stimulant alone. The integrated oxidative burst response in the combined PMA and electric treatment was only 4.7% higher than in the cells treated with PMA alone, and no significant difference in the peak level was found. The results suggest that electric field treatment preferentially stimulates calcium-induced activation with ionomycin rather than calcium-dependent activation with fMLP or PMA.

Molecular, genetic, and functional analysis of homozygous C8 beta-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 patient's 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.

Electrochemotherapy with bleomycin in SA-1 tumor-bearing mice--natural resistance and immune responsiveness.

Electrochemotherapy is an antitumor treatment that utilizes locally delivered electric pulses to increase the effectiveness of chemotherapeutic drugs in cells and tissues. Electric pulses permeabilize tumor cells to allow nonpermeant drugs such as bleomycin to enter the cells. Although preclinical data indicate that immune responsiveness of the organism is important for obtaining cures of the tumors after electrochemotherapy with bleomycin, it is not known how electrochemotherapy affects the immune system of the organism. The aim of the study was to determine the effects of electrochemotherapy with bleomycin on natural resistance and immune responsiveness. Natural resistance was evaluated by phagocytic and intracellular killing activity (oxidative burst) in monocytes and polymorphonuclear granulocytes from venous blood, and immune responsiveness by blast transformation of spleen mononuclear cells to mitogens. The percentage of monocytes in venous blood able to elicit oxidative burst was significantly increased 7 days after the electrochemotherapy and returned to normal values after 14 days. In addition, increased blast transformation of spleen mononuclear cells by stimulation with concanavalin A (T lymphocytes activity) was found 14 days after electrochemotherapy treatment. The results of our study demonstrate that electrochemotherapy with bleomycin affects the immune system of the organism

Potential therapeutic indications for synthetic desmuramyl MDP analogue (LK-409).

Synthetic desmuramyl analogues are potentially immunostimulating drugs. Until today a lot of different molecules were constructed, some with interesting augmenting properties, but most of them with unpleasant pyrogenic side effects. LK-409, 7-oxooctanoyl-L-alanyl-D-isoglutamine, synthesised at the Pharmaceutical faculty of Ljubljana, is an apyrogenic derivative with promising immunostimulating activity. LK-409 and the control substance romurtide were tested for more than 50 parameters. All the immune response levels were included in the study; the development and proliferation of stem cells from bone marrow, the natural defence mechanisms with special regard to the function of phagocytic cells, the humoral immune response with special regard to the development of plaque forming cells and the cellular immune response with regard to the proliferating ability of the immune cells and production of regulating and differentiating cytokines, in vitro and in vivo. Results of LK-409 and romurtide activity were sorted in six groups, and evaluated. The analysis shows that LK-409 affects the measured parameters more efficiently than does romurtide. The most pronounced effect was stimulation of the immune response in drug and tumor induced immunosuppression.

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 toxin's 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.

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.

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.

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.

The killing activity of microwaves on some non-sporogenic and sporogenic medically important bacterial strains.

The killing activity of microwaves of 2450 MHz frequency and 325 W, 650 W and 1400 W power on some bacterial strains was investigated. Vegetative strains of Staphylococcus aureus, Streptococcus pyogenes Group A, Escherichia coli, Pseudomonas aeruginosa and Enterococcus faecalis and spores of Bacillus subtilis and Bacillis stearothermophilus in aqueous suspensions were exposed to 325 W and 650 W waves for different lengths of time. Enterococcus faecalis and spores of B. subtilis and B. stearothermophilus were exposed additionally to 1400 W waves in aqueous and 'dried' suspensions. Vegetative bacteria were promptly killed in 5 min or less, E. faecalis being slightly more resistant. Bacterial spores were only killed in aqueous suspension when a 1400 W setting was used for 10 to 20 min. Bacterial spores adhering to the tube walls after the aqueous suspension was poured out were reduced in number. We assume that the conventional microwave ovens available on the market may be used for a high level of disinfection but not for sterilization, and only then if sufficient water is present.

A simple and rapid method to determine hematopoietic growth factor activity.

A rapid and simple colorimetric microassay method for the determination of hematopoietic growth factor activities was established. The assay was used to detect CSF-1, GM-CSF, and IL-3 activities. The assay was based on the metabolism of the tetrazolium dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by metabolically active cells. Results obtained with the colorimetric microassay are comparable with those obtained with the soft agarose assay. Advantages of the colorimetric microassay include the conservation of reagents, the shorter incubation time for the experiment, the shorter assay time, and the ability to evaluate large numbers of samples.