Homogeneous Antigen Research Articles

Development of a monoclonal antibody-based lateral flow immunoassay for the detection of benzoic acid in liquid food.

To monitor benzoic acid (BA) residues in liquid food samples, a monoclonal antibody (mAb)-based lateral flow immunoassay (LFA) was developed in this study. First, 2-aminobenzoic acid (2-AA), 3-aminobenzoic acid (3-AA), and 4-aminobenzoic acid (4-AA) were conjugated to BSA and used as immunogens. After cell fusion, mAb 6D8 from 4-AA-BSA performed best with an IC50 value of 0.21 mg L-1 using 3-AA-OVA as a heterogeneous antigen, which represented a 3.4-fold improvement compared with the homogeneous antigen 4-AA-BSA. Subsequently, eight kinds of CGNPs with sizes varying from 20.94 nm to 90.00 nm were synthesized for screening the suitable size to develop a sensitive LFA. Finally, a sensitive LFA based on colloidal gold (23.27 nm) nanoparticles was developed for screening BA with a cut-off value of 4 mg L-1, which could meet the requirement of BA detection in milk, Fanta, Sprite, Coca-Cola, and Smart samples.

SARS-CoV-2 receptor binding domain (RBD) designs for homogeneous monomeric antigen preparations

SARS-CoV-2 receptor binding domain (RBD) designs for homogeneous monomeric antigen preparations

Heat Shock Protein Upregulation Supplemental to Complex mRNA Alterations in Autoimmune Glaucoma.

Glaucomatous optic neuropathy is a common cause for blindness. An elevated intraocular pressure is the main risk factor, but also a contribution of the immune system seems likely. In the experimental autoimmune glaucoma model used here, systemic immunization with an optic nerve homogenate antigen (ONA) leads to retinal ganglion cell (RGC) and optic nerve degeneration. We processed retinae for quantitative real-time PCR and immunohistology 28 days after immunization. Furthermore, we performed mRNA profiling in this model for the first time. We detected a significant RGC loss in the ONA retinae. This was accompanied by an upregulation of mRNA expression of genes belonging to the heat shock protein family. Furthermore, mRNA expression levels of the genes of the immune system, such as C1qa, C1qb, Il18, and Nfkb1, were upregulated in ONA animals. After laser microdissection, inner retinal layers were used for mRNA microarrays. Nine of these probes were significantly upregulated in ONA animals (p < 0.05), including Hba-a1 and Cxcl10, while fifteen probes were significantly downregulated in ONA animals (p < 0.05), such as Gdf15 and Wwox. Taken together, these findings provide further insights into the pivotal role of the immune response in glaucomatous optic neuropathy and could help to identify novel diagnostic or therapeutic strategies.

Structure based design of effective HtpG-derived vaccine antigens against M. tuberculosis.

Vaccine development against Tuberculosis is a strong need, given the low efficacy of the sole vaccine hitherto used, the Bacillus Calmette–Guérin (BCG) vaccine. The chaperone-like protein HtpGMtb of M. tuberculosis is a large dimeric and multi-domain protein with promising antigenic properties. We here used biophysical and biochemical studies to improve our understanding of the structural basis of HtpGMtb functional role and immunogenicity, a precious information to engineer improved antigens. We showed that HtpGMtb is a dimeric nucleotide-binding protein and identified the dimerisation interface on the C-terminal domain of the protein. We also showed that the most immunoreactive regions of the molecule are located on the C-terminal and middle domains of the protein, whereas no role is played by the catalytic N-terminal domain in the elicitation of the immune response. Based on these observations, we experimentally validated our predictions in mice, using a plethora of immunological assays. As an outcome, we designed vaccine antigens with enhanced biophysical properties and ease of production, albeit conserved or enhanced antigenic properties. Our results prove the efficacy of structural vaccinology approaches in improving our understanding of the structural basis of immunogenicity, a precious information to engineer more stable, homogeneous, efficiently produced, and effective vaccine antigens.

SYST-12 D2C7 CAR: A NOVEL CAR T CELL THAT SIMULTANEOUSLY TARGETS WILDTYPE EGFR AND ITS MUTANT ISOFORM EGFRVIII FOR TREATMENT OF GLIOMA AND BRAIN METASTASES

INTRODUCTIONChimeric antigen receptor (CAR) T-cells represent a revolutionary class of immunotherapy, achieving considerable success in hematological cancers but generally failing to control solid tumors, including gliomas, partly due to the lack of a ubiquitously-expressed target antigen. In this study, we engineered a novel CAR T-cell consisting of the D2C7scfv targeting moiety that binds a shared epitope between EGFR and EGFRvIII. EGFR is the most homogeneous antigen on glial brain tumors, and the mutant EGFR variant, EGFRvIII, is present on a considerable subset of high grade gliomas. CAR T-cells targeting EGFRvIII alone fail to treat tumors possessing as few as 5-10% EGFRvIII-negative cell. Thus, D2C7 CAR is expected to be superior to the EGFRvIII CAR. METHODSWe retrovirally transduced T-cells with a vector encoding the D2C7scFv in tandem with intracellular signaling domains of CD28, 4-1BB, and CD3ζ to generate D2C7 CAR. We co-cultured D2C7 CAR or control CAR with fluorescently-tagged tumor cells expressing either EGFRwt or EGFRvIII to validate efficacy and specificity by flow cytometry. To determine in vivo efficacy, EGFRwt or EGFRvIII-expressing tumors were implanted intracranially in immunodeficient NSG mice. 48 hours later, D2C7 CAR, VIII CAR, or Mock CAR were administered intracranially and mice were monitored for survival. RESULTSD2C7 CAR specifically killed tumor cells that expressed either EGFRwt or EGFRvIII, but not cells that lacked EGFR. Intracranial D2C7 CAR administration resulted in significantly prolonged survival of mice bearing EGFRwt or EGFRvIII tumors compared to Mock CAR controls. Importantly, D2C7 CAR significantly benefitted mice bearing a heterogeneous mix of EGFRwt and EGFRvIII tumor cells, a model of tumor heterogeneity. CONCLUSIONSD2C7 CAR is efficacious against EGFRwt/EGFRvIII heterogeneous tumors. Intracranial administration of D2C7 CAR is predicted to safely and effectively treat a large cohort of patients due to the relatively high prevalence of EGFR and/or EGFRvIII-expressing brain tumors.

D2C7 CAR: A novel CAR T cell that simultaneously targets wildtype EGFR and its mutant isoform EGFRvIII for treatment of glioma

Abstract Brain tumors are the leading cause of cancer death in children and a significant cause of morbidity and mortality in adults. Conventional treatments are suboptimal, thus signifying the need for novel therapeutic strategies, such as immunotherapy. Chimeric antigen receptor (CAR) T cells represent a revolutionary class of immunotherapy, achieving considerable success in eliminating hematological cancers but generally failing to control solid tumors in part due to the lack of a suitably-expressed target antigen. Epidermal growth factor receptor (EGFR) is the most ubiquitous and homogeneous antigen on glial brain tumors, and EGFR-directed therapies have been hotly pursued. Moreover, the mutant EGFR variant, EGFRvIII, is present on a subset of pediatric and adult high grade gliomas, representing a targetable, tumor-specific antigen. Unfortunately, CAR T cells targeting EGFRvIII fail to treat tumors possessing as few as 5–10% EGFRvIII-negative cells due to antigen escape. Thus, a CAR T cell that can target both EGFR and EGFRvIII is expected to be superior to a CAR that targets EGFRvIII alone. In this study, we developed a novel third generation CAR T cell consisting of the D2C7scfv targeting moiety that binds a shared epitope between EGFR and EGFRvIII. This D2C7 CAR was able to specifically and potently kill tumor cells expressing wildtype EGFR or EGFRvIII. Importantly, D2C7 CAR significantly prolonged survival of mice bearing EGFR or EGFRvIII-expressing gliomas of both adult (U87) and pediatric (DAOY) origin. Toxicity experiments involving EGFR-expressing human skin grafts provided evidence that D2C7 CAR is safe and effective when administered intracranially to mice bearing intracranial tumors.

Synthesis, Characterization, and in vivo Evaluation of a Novel Potent Autotaxin-Inhibitor.

The autotaxin-lysophosphatidic acid (ATX-LPA) signaling pathway plays a role in a variety of autoimmune diseases, such as rheumatoid arthritis or neurodegeneration. A link to the pathogenesis of glaucoma is suggested by an overactive ATX-LPA axis in aqueous humor samples of glaucoma patients. Analysis of such samples suggests that the ATX-LPA axis contributes to the fibrogenic activity and resistance to aqueous humor outflow through the trabecular meshwork. In order to inhibit or modulate this pathway, we developed a new series of ATX-inhibitors containing novel bicyclic and spirocyclic structural motifs. A potent lead compound (IC50 against ATX: 6 nM) with good in vivo PK, favorable in vitro property, and safety profile was generated. This compound leads to lowered LPA levels in vivo after oral administration. Hence, it was suitable for chronic oral treatment in two rodent models of glaucoma, the experimental autoimmune glaucoma (EAG) and the ischemia/reperfusion models. In the EAG model, rats were immunized with an optic nerve antigen homogenate, while controls received sodium chloride. Retinal ischemia/reperfusion (I/R) was induced by elevating the intraocular pressure (IOP) in one eye to 140 mmHg for 60 min, followed by reperfusion, while the other untreated eye served as control. Retinae and optic nerves were evaluated 28 days after EAG or 7 and 14 days after I/R induction. Oral treatment with the optimized ATX-inhibitor lead to reduced retinal ganglion cell (RGC) loss in both glaucoma models. In the optic nerve, the protective effect of ATX inhibition was less effective compared to the retina and only a trend to a weakened neurofilament distortion was detectable. Taken together, these results provide evidence that the dysregulation of the ATX-LPA axis in the aqueous humor of glaucoma patients, in addition to the postulated outflow impairment, might also contribute to RGC loss. The observation that ATX-inhibitor treatment in both glaucoma models did not result in significant IOP increases or decreases after oral treatment indicates that protection from RGC loss due to inhibition of the ATX-LPA axis is independent of an IOP lowering effect.

Macroevolutionary foundations of a recently evolved innate immune defense.

Antagonistic interactions between hosts and parasites may drive the evolution of novel host defenses, or new parasite strategies. Host immunity is therefore one of the fastest evolving traits. But where do the novel immune traits come from? Here, we test for phylogenetic conservation in a rapidly evolving immune trait—peritoneal fibrosis. Peritoneal fibrosis is a costly defense against a specialist tapeworm, Schistocephalus solidus (Cestoda), expressed in some freshwater populations of threespine stickleback fish (Gasterosteus aculeatus, Perciformes). We asked whether stickleback fibrosis is a derived species‐specific trait or an ancestral immune response that was widely distributed across ray‐finned fish (Actinopterygii) only to be employed by threespine stickleback against the specialist parasite. We combined literature review on peritoneal fibrosis with a comparative experiment using either parasite‐specific, or nonspecific, immune challenge in deliberately selected species across fish tree of life. We show that ray‐finned fish are broadly, but not universally, able to induce peritoneal fibrosis when challenged with a generic stimulus (Alum adjuvant). The experimental species were, however, largely indifferent to the tapeworm antigen homogenate. Peritoneal fibrosis, thus, appears to be a common and deeply conserved fish immune response that was co‐opted by stickleback to adapt to a new selective challenge.

Natural and synthetic carbohydrate-based vaccine adjuvants and their mechanisms of action.

Modern subunit vaccines based on homogeneous antigens offer more precise targeting and improved safety compared with traditional whole-pathogen vaccines. However, they are also less immunogenic and require an adjuvant to increase the immunogenicity of the antigen and potentiate the immune response. Unfortunately, few adjuvants have sufficient potency and low enough toxicity for clinical use, highlighting the urgent need for new, potent and safe adjuvants. Notably, a number of natural and synthetic carbohydrate structures have been used as adjuvants in clinical trials, and two have recently been approved in human vaccines. However, naturally derived carbohydrate adjuvants are heterogeneous, difficult to obtain and, in some cases, unstable. In addition, their molecular mechanisms of action are generally not fully understood, partly owing to the lack of tools to elucidate their immune-potentiating effects, thus hampering the rational development of optimized adjuvants. To address these challenges, modification of the natural product structure using synthetic chemistry emerges as an attractive approach to develop well-defined, improved carbohydrate-containing adjuvants and chemical probes for mechanistic investigation. This Review describes selected examples of natural and synthetic carbohydrate-based adjuvants and their application in synthetic self-adjuvanting vaccines, while also discussing current understanding of their molecular mechanisms of action.

Loss of the Extracellular Matrix Molecule Tenascin-C Leads to Absence of Reactive Gliosis and Promotes Anti-inflammatory Cytokine Expression in an Autoimmune Glaucoma Mouse Model.

Previous studies demonstrated that retinal damage correlates with a massive remodeling of extracellular matrix (ECM) molecules and reactive gliosis. However, the functional significance of the ECM in retinal neurodegeneration is still unknown. In the present study, we used an intraocular pressure (IOP) independent experimental autoimmune glaucoma (EAG) mouse model to examine the role of the ECM glycoprotein tenascin-C (Tnc). Wild type (WT ONA) and Tnc knockout (KO ONA) mice were immunized with an optic nerve antigen (ONA) homogenate and control groups (CO) obtained sodium chloride (WT CO, KO CO). IOP was measured weekly and electroretinographies were recorded at the end of the study. Ten weeks after immunization, we analyzed retinal ganglion cells (RGCs), glial cells, and the expression of different cytokines in retina and optic nerve tissue in all four groups. IOP and retinal function were comparable in all groups. Although RGC loss was less severe in KO ONA, WT as well as KO mice displayed a significant cell loss after immunization. Compared to KO ONA, less βIII-tubulin+ axons, and downregulated oligodendrocyte markers were noted in WT ONA optic nerves. In retina and optic nerve, we found an enhanced GFAP+ staining area of astrocytes in immunized WT. A significantly higher number of retinal Iba1+ microglia was found in WT ONA, while a lower number of Iba1+ cells was observed in KO ONA. Furthermore, an increased expression of the glial markers Gfap, Iba1, Nos2, and Cd68 was detected in retinal and optic nerve tissue of WT ONA, whereas comparable levels were observed in KO ONA. In addition, pro-inflammatory Tnfa expression was upregulated in WT ONA, but downregulated in KO ONA. Vice versa, a significantly increased anti-inflammatory Tgfb1 expression was measured in KO ONA animals. We conclude that Tnc plays an important role in glial and inflammatory response during retinal neurodegeneration. Our results provide evidence that Tnc is involved in glaucomatous damage by regulating retinal glial activation and cytokine release. Thus, this transgenic EAG mouse model for the first time offers the possibility to investigate IOP-independent glaucomatous damage in direct relation to ECM remodeling.

Preservation of optic nerve structure by complement inhibition in experimental glaucoma

Glaucoma is characterized by a progressive damage of the retina and the optic nerve. Despite a huge research interest, the exact pathomechanisms are still unknown. In the experimental autoimmune glaucoma model, rats develop glaucoma-like damage of the retina and the optic nerve after immunization with an optic nerve antigen homogenate (ONA). An early activation of the complement system, even before optic nerve degeneration, was reported in this model. Here, we investigated the effects of a monoclonal antibody against complement factor C5 on optic nerves. Rats were immunized with ONA and compared to controls. In one eye of some ONA animals, the antibody against C5 was intravitreally injected (15 μmol: ONA + C5-I or 25 μmol: ONA + C5-II) before immunization and then every 2 weeks. After 6 weeks, optic nerves were processed for histology (n = 6/group). These analyses demonstrated that the intravitreal therapy reduced the depositions of the membrane attack complex compared to ONA animals (ONA + C5-I: p = 0.005; ONA + C5-II: p = 0.002). Cellular infiltration was significantly reduced in the ONA + C5-I group (p = 0.003), but not in ONA + C5-II tissues (p = 0.41). Furthermore, SMI-32 staining revealed that neurofilament was preserved in both treatment groups compared to ONA optic nerves (both p = 0.002). A decreased amount of microglia was found in treated animals in comparison to the ONA group (ONA + C5-I: p = 0.03; ONA + C5-II: p = 0.009). We observed, for the first time, that a complement system inhibition could prevent optic nerve damage in an autoimmune glaucoma model. Therefore, complement inhibition could serve as a new therapeutic tool for glaucoma.

Axenic Caenorhabditis elegans antigen protects against development of type-1 diabetes in NOD mice

Studies in humans and animals have demonstrated that infection with helminths (parasitic worms) is protective against a range of hyperinflammatory diseases. A number of factors limit translation into clinical use, including: potential contamination of helminths obtained from infected humans or animals, lack of batch to batch stability, and potential pathological risks derived from live worm infections. To overcome these limitations we tested whether an antigen homogenate of the non-pathogenic nematode Caenorhabditis elegans confers protection against type 1 diabetes mellitus (T1D) using the Non Obese Diabetic (NOD) mouse model.Our study demonstrates that twice weekly intraperitoneal injections of axenically cultured C. elegans antigen (aCeAg) confers substantial protection against type 1 diabetes in NOD mice. Whereas 80% of control mice (PBS-injected) developed clinical disease, only 10% of aCeAg-treated mice became diabetic. Additionally, aCeAg treated mice had significantly greater numbers of insulin-producing pancreatic islets and greater numbers of islets negative for lymphocyte infiltration. Immunological changes observed in aCeAg treated mice included increases in total IgE and total IgG1, consistent with induction of a type 2 immune response similar to that typically seen in parasitic worm infection. Although evidence suggests that helminth infections induce strong immunoregulatory signals, we did not observe significant changes in regulatory T cell numbers or in production of the regulatory cytokines TGFβ and IL-10. The lack of a regulatory response may be due to our time point of observation, or perhaps the mechanism of aCeAg efficacy may differ from that of helminth infection.Discovery that antigens obtained from a non-parasitic environmental nematode replicate the protective phenotype induced by parasitic worm infections may accelerate our ability to develop nematode-derived therapies for allergy and autoimmune diseases.

Intravitreal Therapy Against the Complement Factor C5 Prevents Retinal Degeneration in an Experimental Autoimmune Glaucoma Model.

In glaucoma, studies revealed an involvement of the complement system. In an experimental autoimmune glaucoma model, immunization with an optic nerve homogenate antigen (ONA) led to retinal ganglion cell (RGC) loss, while intraocular pressure (IOP) remained unchanged. Here, we investigated the therapeutic effect of a complement system inhibition in this model. Hence, rats were immunized with ONA and compared to controls. In one eye of the ONA animals, an antibody against complement factor C5 was intravitreally injected (15 μmol: ONA+C5-I or 25 μmol: ONA+C5-II) before immunization and then every two weeks. IOP was measured weekly. After 6 weeks, spectral-domain optical coherence tomographies (SD-OCT), electroretinograms (ERG), immunohistochemistry, and quantitative real-time PCR analyses were performed. IOP and retinal thickness remained unchanged within all groups. The a-wave amplitudes were not altered in the ONA and ONA+C5-I groups, whereas a decrease was noted in ONA+C5-II animals (p < 0.05). ONA immunization provoked a significant decrease of the b-wave amplitude (p < 0.05), which could be preserved in ONA+C5-I, but not in ONA+C5-II animals. ONA animals showed a loss of RGCs (p = 0.001), while ONA+C5-I and ONA+C5-II retinae had similar cell counts as controls. A significant downregulation of apoptotic Bax/Bcl2 mRNA was noted in ONA+C5-I retinae (p = 0.02). Significantly more C3+ and MAC+ cells were observed in ONA animals (p < 0.001). The amount of C3+ cells in both treatment groups was significantly increased (p < 0.01), while the number of MAC+ cells in the treated retinas did not differ from controls. The number of activated microglia cells remained unchanged in ONA animals, but was increased in the treatment groups (p < 0.05). Recoverin+ cells were diminished in ONA animals (p = 0.049), but not in treated ones. Rho mRNA was downregulated in ONA and in ONA+C5-II retinas (both p = 0.014). Less opsin+ cones were observed in ONA animals (p = 0.009), but not in the treated groups. Our results indicate that the C5 antibody inhibits activation of the complement system, preventing the loss of retinal function as well as RGC, cone bipolar, and photoreceptor loss. Therefore, this approach might be a suitable new treatment for glaucoma patients, in which immune dysregulation plays an important factor for the development and progression of glaucoma.

Transfer of the Experimental Autoimmune Glaucoma Model from Rats to Mice-New Options to Study Glaucoma Disease.

Studies have suggested an involvement of the immune system in glaucoma. Hence, a rat experimental autoimmune glaucoma model (EAG) was developed to investigate the role of the immune response. Here, we transferred this model into mice. Either 0.8 mg/mL of the optic nerve antigen homogenate (ONA; ONA 0.8) or 1.0 mg/mL ONA (ONA 1.0) were injected in 129/Sv mice. Controls received sodium chloride. Before and 6 weeks after immunization, the intraocular pressure (IOP) was measured. At 6 weeks, retinal neurons, glia cells, and synapses were analyzed via immunohistology and quantitative real-time PCR (RT-qPCR). Additionally, optic nerves were examined. The IOP stayed in the normal physiological range throughout the study (p > 0.05). A significant reduction of retinal ganglion cells (RGCs) was noted in both immunized groups (p < 0.001). Remodeling of glutamatergic and GABAergic synapses was seen in ONA 1.0 retinas. Furthermore, both ONA groups revealed optic nerve degeneration and macrogliosis (all: p < 0.001). An increase of activated microglia was noted in ONA retinas and optic nerves (p < 0.05). Both ONA concentrations led to RGC loss and optic nerve degeneration. Therefore, the EAG model was successfully transferred from rats to mice. In further studies, transgenic knockout mice can be used to investigate the pathomechanisms of glaucoma more precisely.

Hyaluronan-based dissolving microneedles with high antigen content for intradermal vaccination: Formulation, physicochemical characterization and immunogenicity assessment

The purpose of this study was to optimize the manufacturing of dissolving microneedles (dMNs) and to increase the antigen loading in dMNs to investigate the effect on their physicochemical properties. To achieve this, a novel single-array wells polydimethylsiloxane mold was designed, minimizing antigen wastage during fabrication and achieving homogeneous antigen distribution among the dMN arrays. Using this mold, hyaluronan (HA)-based dMNs were fabricated and tested for maximal ovalbumin (OVA) content. dMNs could be fabricated with an OVA:HA ratio as high as 1:1 (w/w), without compromising their properties such as shape and penetration into the ex vivo human skin, even after storage at high humidity and temperature. High antigen loading did not induce protein aggregation during dMN fabrication as demonstrated by complementary analytical methods. However, the dissolution rate in ex vivo human skin decreased with increasing antigen loading. About 2.7 µg OVA could be delivered in mice by using a single array with an OVA:HA ratio of 1:3 (w/w). Intradermal vaccination with dMNs induced an immune response similar as subcutaneous injection and faster than after hollow microneedle injection. In conclusion, results suggest that (i) the polydimethylsiloxane mold design has an impact on the manufacturing of dMNs, (ii) the increase in antigen loading in dMNs affects the microneedle dissolution and (iii) dMNs are a valid alternative for vaccine administration over conventional injection.

Expression, purification, characterization and immunogenicity of human immunodeficiency virus-1 glycoprotein gp120 derived from insect cells

Objective To establish an efficient baculovirus-insect cell expression system for the production of human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 and to evaluate the physiochemical properties, antigenicity and immunogenicity of the recombinant protein. Methods The gene encoding HIV-1 NL4-3 gp120 was cloned into the downstream of pH promoter of the baculovirus transfer vector pAcgp67B to construct the recombinant transfer vector pAc-gp120. Expression of the protein of interest was induced in baculovirus-infected High Five™ insect cells. ELISA, analytical ultracentrifugation and size-exclusion chromatography were carried out to characterize physicochemical properties of the expressed gp120 protein. Immunogenicity of the recombinant gp120 protein was analyzed by HIV neutralization assay after immunizing BALB/c mice with it. Results The recombinant HIV-1 gp120 protein was successfully obtained from the established insect cell expression system with a purity of more than 90% and a mean yield of 13 mg/L in four batches. That recombinant HIV-1 gp120 protein was characterized by homogeneity in solution and possessed a good immunoreactivity to neutralizing antibodies and antisera against HIV. Immunogenicity analysis in BALB/c mice demonstrated that the recombinant gp120 protein could induce effective immune responses against HIV-1 NL4-3. Conclusion A simple and scalable approach to obtain homogeneous and immunogenic HIV-1 gp120 antigen is successfully established, which will promote further investigation of HIV vaccine candidates. Key words: Human immunodeficiency virus-1; Insect cell expression system; Glycoprotein; gp120; Immune response

Simultaneous Complement Response via Lectin Pathway in Retina and Optic Nerve in an Experimental Autoimmune Glaucoma Model.

Glaucoma is a multifactorial disease and especially mechanisms occurring independently from an elevated intraocular pressure (IOP) are still unknown. Likely, the immune system contributes to the glaucoma pathogenesis. Previously, IgG antibody depositions and retinal ganglion cell (RGC) loss were found in an IOP-independent autoimmune glaucoma model. Therefore, we investigated the possible participation of the complement system in this model. Here, rats were immunized with bovine optic nerve homogenate antigen (ONA), while controls (Co) received sodium chloride (n = 5–6/group). After 14 days, RGC density was quantified on flatmounts. No changes in the number of RGCs could be observed at this point in time. Longitudinal optic nerve sections were stained against the myelin basic protein (MBP). We could note few signs of degeneration processes. In order to detect distinct complement components, retinas and optic nerves were labeled with complement markers at 3, 7, 14, and 28 days and analyzed. Significantly more C3 and MAC depositions were found in retinas and optic nerves of the ONA group. These were already present at day 7, before RGC loss and demyelination occurred. Additionally, an upregulation of C3 protein was noted via Western Blot at this time. After 14 days, quantitative real-time PCR revealed significantly more C3 mRNA in the ONA retinas. An upregulation of the lectin pathway-associated mannose-serine-protease-2 (MASP2) was observed in the retinas as well as in the optic nerves of the ONA group after 7 days. Significantly more MASP2 in retinas could also be observed via Western Blot analyses at this point in time. No effect was noted in regard to C1q. Therefore, we assume that the immunization led to an activation of the complement system via the lectin pathway in retinas and optic nerves at an early stage in this glaucoma model. This activation seems to be an early response, which then triggers degeneration. These findings can help to develop novel therapy strategies for glaucoma patients.

Systemic ocular antigen immunization leads only to a minor secondary immune response

The immunization with optic nerve homogenate antigen (ONA) or S100 induced retinal degeneration. Since many neurological diseases are reinforced or initiated by immune cells, leucocytes were analyzed. CD3+ T-cells in the retina increased slightly in ONA rats, but not in S100 treated retinas. No CD45R+ B-cells and granulocytes could be detected in the retinas. At early stages, CD3+ cells, Iba1+ macrophages and granulocytes of the secondary lymphoid organs were not affected. Yet, the sole injection of pertussis toxin led to a shift to fewer CD45R+ cells and more granulocytes in spleens. Later, splenic Iba1+ macrophages were increased in both groups. We conclude that the retinal infiltration of lymphocytes is not crucial for the degeneration process and rather an epiphenomenon.

Diagnostics, treatment, and prevention of hemorrhagic fever caused by the Ebola and Marburg

Ebola virus haemorrhagic fever is an acute infectious highly contagious diseases with high morbidity and mortality rate. Rapid development of clinical symptoms, severity of the disease and the mortality rate of up to 90 % require taking urgent medical measures already during the initial identification of the patient, of the quarantine measures and the development of preventive measures aimed at limiting spreading of the disease. Infectious agent is a virus of Ebolavirus of the Filoviridae family. First cases of the disease were revealed in 1976 in the west of Equatorial Province of Sudan with further in other countries of Africa. Bats and primates are natural reservoirs of the Ebola virus. The virus is transmitted from person to person from the first day of the onset of clinical symptoms. The patient remains contagious for several weeks after the onset of the disease. The virus has wide diversity of ways of transmission – through blood, nasopharyngeal mucus, urine, vomit, mucus of the genital tract of the patient. The incubation period is 3 to 21 days. The onset of the disease is acute: fever, chills, headache, muscle pain, maculopapular rash; in severe forms – the development of multiple organ failure. The death is caused with bleeding and / or shock. Diagnostics is based upon the analysis of epidemiological, clinical, and laboratory data. The combination of the analyses that determine the antigen or RNA and IgM or IgG antibodies is usually used. Polymerase chain reaction in real time or ELISA may be used to determint the antigen in blood, serum or homogenates of organs (presence of IgM antibodies indicates recent infection). No specific therapy has been developed as of the present moment. Therapeutic events boil down to pathogenetic and symptomatic treatment. No specific vaccine has been developed yet. The most important method of Ebola disease control is conducting control activities within the epidemic focus and beyond its borders to limit the spread of infection.

Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes

Genotyping in closed tube is commonly performed using polymerase chain reaction (PCR) amplification and allele-specific oligonucleotide probes using fluorescence resonance energy transfer (FRET). Here we introduce a homogeneous human leukocyte antigen (HLA)–DQA1∗05 end-point PCR assay based on switchable lanthanide luminescence probe technology and a simple dried blood sample preparation. The switchable probe technology is based on two non-luminescent oligonucleotide probes: one carrying a non-luminescent lanthanide chelate and the other carrying a light-absorbing antenna ligand. Hybridization of the probes in adjacent positions to the target DNA leads to the formation of a highly luminescent lanthanide chelate complex by self-assembly of the reporter molecules. Performance of the HLA–DQA1∗05 assay was evaluated by testing blood samples collected on sample collection cards and was prepared by lysing the punched samples (3-mm discs) using alkaline reaction conditions and high temperature. Testing of 147 blood samples yielded 100% correlation to the heterogeneous DELFIA technology-based reference assay. Genotyping requires carefully designed probe sequences able to discriminate matched and mismatched target sequences by hybridization. Furthermore, definite genotype discrimination was achieved because inherently non-luminescent switchable probes together with time-resolved measurement mode led to very low background signal level and, therefore, very high signal differences averaging 54-fold between DQA1∗05 and other alleles.