Small protein blockers of human IL-6 receptor alpha inhibit proliferation and migration of cancer cells.

BACKGROUND: Interleukin-6 (IL-6) is a multifunctional cytokine that controls the immune response, and its role has been described in the development of autoimmune diseases. Signaling via its cognate IL-6 receptor (IL-6R) complex is critical in tumor progression and, therefore, IL-6R represents an important therapeutic target. METHODS: An albumin-binding domain-derived highly complex combinatorial library was used to select IL-6R alpha (IL-6Rα)-targeted small protein binders using ribosome display. Large-scale screening of bacterial lysates of individual clones was performed using ELISA, and their IL-6Rα blocking potential was verified by competition ELISA. The binding of proteins to cells was monitored by flow cytometry and confocal microscopy on HEK293T-transfected cells, and inhibition of signaling function was examined using HEK-Blue IL-6 reporter cells. Protein binding kinetics to living cells was measured by LigandTracer, cell proliferation and toxicity by iCELLigence and Incucyte, cell migration by the scratch wound healing assay, and prediction of binding poses using molecular modeling by docking. RESULTS: We demonstrated a collection of protein variants called NEF ligands, selected from an albumin-binding domain scaffold-derived combinatorial library, and showed their binding specificity to human IL-6Rα and antagonistic effect in HEK-Blue IL-6 reporter cells. The three most promising NEF108, NEF163, and NEF172 variants inhibited cell proliferation of malignant melanoma (G361 and A2058) and pancreatic (PaTu and MiaPaCa) cancer cells, and suppressed migration of malignant melanoma (A2058), pancreatic carcinoma (PaTu), and glioblastoma (GAMG) cells in vitro. The NEF binders also recognized maturation-induced IL-6Rα expression and interfered with IL-6-induced differentiation in primary human B cells. CONCLUSION: We report on the generation of small protein blockers of human IL-6Rα using directed evolution. NEF proteins represent a promising class of non-toxic anti-tumor agents with migrastatic potential.

Antigenicity and immunogenicity of different morphological forms of Borrelia burgdorferi sensu lato spirochetes.

Borrelia burgdorferi sensu lato is a species complex of pleomorphic spirochetes, including species that cause Lyme disease (LD) in humans. In addition to classic spiral forms, these bacteria are capable of creating morphological forms referred to as round bodies and aggregates. The subject of discussion is their possible contribution to the persistence of infection or post-infection symptoms in LD. This study investigates the immunological properties of these forms by monitoring reactivity with early (n = 30) and late stage (n = 30) LD patient sera and evaluating the immune response induced by vaccination of mice. In patient sera, we found a quantitative difference in reactivity with individual morphotypes, when aggregates were recognized most intensively, but the difference was statistically significant in only half of the tested strains. In post-vaccination mouse sera, we observed a statistically significant higher reactivity with antigens p83 and p25 (OspC) in mice vaccinated with aggregates compared to mice vaccinated with spiral forms. The importance of the particulate nature of the antigen for the induction of a Th1-directed response has also been demonstrated. In any of morphological forms, the possibility of inducing antibodies cross-reacting with human nuclear and myositis specific/associated autoantigens was not confirmed by vaccination of mice.

Concurrent Infection of the Human Brain with Multiple Borrelia Species.

Lyme disease (LD) spirochetes are well known to be able to disseminate into the tissues of infected hosts, including humans. The diverse strategies used by spirochetes to avoid the host immune system and persist in the host include active immune suppression, induction of immune tolerance, phase and antigenic variation, intracellular seclusion, changing of morphological and physiological state in varying environments, formation of biofilms and persistent forms, and, importantly, incursion into immune-privileged sites such as the brain. Invasion of immune-privileged sites allows the spirochetes to not only escape from the host immune system but can also reduce the efficacy of antibiotic therapy. Here we present a case of the detection of spirochetal DNA in multiple loci in a LD patient's post-mortem brain. The presence of co-infection with Borrelia burgdorferi sensu stricto and Borrelia garinii in this LD patient's brain was confirmed by PCR. Even though both spirochete species were simultaneously present in human brain tissue, the brain regions where the two species were detected were different and non-overlapping. The presence of atypical spirochete morphology was noted by immunohistochemistry of the brain samples. Atypical morphology was also found in the tissues of experimentally infected mice, which were used as a control.

Myositis Autoantibodies in Patients with Suspected Post-Treatment Lyme Disease Syndrome.

Most patients suffering from Lyme disease are effectively treated with antibiotics. In some patients, however, problems persist for a long time despite appropriate therapy. The term post-treatment Lyme disease syndrome (PTLDS) is currently used for this condition in scientific literature. The pathogenesis is still not precisely known, but the involvement of immunopathological mechanisms is assumed. In our study, we analyzed the presence of autoantibodies including myositis-specific (MSA) and myositis-associated autoantibodies (MAA) in patients with laboratory proven history of Lyme disease and with clinical symptoms of PTLDS. A total of 59 patients meeting the criteria for PTLDS were enrolled in this study. The control group consisted of 40 patients undergoing differential diagnosis of neurological disorders without clinical and/or laboratory-proven history of Lyme disease. The presence of autoantibodies was determined by immunoblot methods and positive samples were further tested for serum creatine kinase (CK) and myoglobin levels. The presence of myositis autoantibodies was detected in 18 subjects with suspected PTLDS (30.5%), but only in 5% of control subjects exhibiting no evidence of Lyme disease history. The difference was statistically significant (p = 0.002). The subsequent biochemical analysis of muscle-damage markers in positive subjects found a mild elevation in six MSA/MAA-positive PTLDS patients. The study detected raised MSA/MAA autoantibodies formation in the group of PTLDS patients raising the question about their involvement in the pathogenesis of this syndrome.

Seroprevalence of Antibodies against Tick-Borne Pathogens in Czech Patients with Suspected Post-Treatment Lyme Disease Syndrome.

The hypothesized importance of coinfections in the pathogenesis of post-treatment Lyme disease syndrome (PTLDS) leads to the use of combined, ongoing antimicrobial treatment in many cases despite the absence of symptoms typical of the presence of infection with specific pathogens. Serum samples from 103 patients with suspected post-treatment Lyme disease syndrome were tested for the presence of antibodies to the major tick-borne pathogens Anaplasma phagocytophilum, Bartonella henselae/Bartonella quinatana, and Babesia microti. Although the presence of anti-Anaplasma antibodies was detected in 12.6% of the samples and anti-Bartonella antibodies in 9.7% of the samples, the presence of antibodies against both pathogens in the same samples or anti-Babesia antibodies in the selected group of patients could not be confirmed. However, we were able to detect autoantibodies, mostly antinuclear, in 11.6% of the patients studied. Our results are in good agreement with previously published studies showing the presence of a wide spectrum of autoantibodies in some patients with complicated forms of Lyme disease and post-treatment Lyme disease syndrome, but they do not reveal a significant influence of co-infections on the development of PTLDS in the studied group of patients.

Preparation and properties of recombinant Clostridium ramosum IgA proteinase. Isolation of Fc-SC and Fab fragments of human secretory IgA.

Immunoglobulin A (IgA) proteinase from Clostridium ramosum is the enzyme which cleaves IgA of both subclasses; in contrast, the other bacterial proteinases cleave only IgA1 proteins. Previous reports characterized the activity of proteinase naturally secreted by C. ramosum specific for the normal human serum IgA of IgA1 and IgA2m(1) subclasses and also for secretory IgA (SIgA). Its amino acid sequence was determined, and the recombinant proteinase which cleaved IgA of both subclasses was prepared. Here we report the optimized expression, purification, storage conditions and activity testing against purified human milk SIgA. The recombinant C. ramosum IgA proteinase isolated in the high degree of purity exhibited almost complete cleavage of SIgA of both subclasses. The proteinase remained active upon storage for more than 10 month at -20 °C without substantial loss of enzymatic activity. Purified SIgA fragments are suitable for studies of all antigen-binding and Fc-dependent functions of SIgA involved in the protection against infections with mucosal pathogens.