Reinhard Burger

Communicable Diseases Prioritized for Surveillance and Epidemiological Research: Results of a Standardized Prioritization Procedure in Germany, 2011

Introduction To establish strategic priorities for the German national public health institute (RKI) and guide the institutes mid-term strategic decisions, we prioritized infectious pathogens in accordance with their importance for national surveillance and epidemiological research. Methods We used the Delphi process with internal (RKI) and external experts and a metric-consensus approach to score pathogens according to ten three-tiered criteria. Additional experts were invited to weight each criterion, leading to the calculation of a median weight by which each score was multiplied. We ranked the pathogens according to the total weighted score and divided them into four priority groups. Results 127 pathogens were scored. Eighty-six experts participated in the weighting; “Case fatality rate” was rated as the most important criterion. Twenty-six pathogens were ranked in the highest priority group; among those were pathogens with internationally recognised importance (e.g., Human Immunodeficiency Virus, Mycobacterium tuberculosis, Influenza virus, Hepatitis C virus, Neisseria meningitides), pathogens frequently causing large outbreaks (e.g., Campylobacter spp.), and nosocomial pathogens associated with antimicrobial resistance. Other pathogens in the highest priority group included Helicobacter pylori, Respiratory Syncytial Virus, Varicella zoster virus and Hantavirus. Discussion While several pathogens from the highest priority group already have a high profile in national and international health policy documents, high scores for other pathogens (e.g., Helicobacter pylori, Respiratory syncytial virus or Hantavirus) indicate a possible under-recognised importance within the current German public health framework. A process to strengthen respective surveillance systems and research has been started. The prioritization methodology has worked well; its modular structure makes it potentially useful for other settings.

Monoclonal antibodies against complement 3 neoantigens for detection of immune complexes and complement activation. Relationship between immune complex levels, state of C3, and numbers of receptors for C3b.

C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogrens syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.

A highly sensitive cytotoxicity assay based on the release of reporter enzymes, from stably transfected cell lines

The well-established methods of generating stably transfected cell lines, and the detection of nanomolar amounts of an enzyme in a fast and reproducible assay, were utilised to establish non-radiometric cytotoxicity assays. In these assay systems, the detection of released enzymes was used to quantitate the leakage of intracellular proteins after membrane disintegration. Target cell lines were transfected with a luciferase reporter gene under the control of a strong eucaryotic promoter. Release of the intracellular expressed enzyme into the culture supernatant occurred after membrane perforation and was measured as an indicator of cellular death. The quantitation of released enzyme was a reliable indicator of cell death initiated either by complement-mediated killing, or by cell-mediated cytotoxicity. This system was initially established with P815 mastocytoma cells as an example of a target cell line. Transfection with the firefly luciferase gene provided an intracellular enzyme absent in mammalian cells. In a parallel approach, P815 and BW5147 target cells were transfected with bacterial beta-galactosidase to provide a similar cytotoxicity system. This enzyme, however, has a considerably longer half life in tissue culture medium than luciferase. In a direct comparison between the standard 51Cr release and beta-galactosidase release, the enzyme release showed a much higher signal-to-noise ratio, i.e., low background and high induced release if spontaneous release and detergent induced maximal lysis were measured. Since a wide range of human and murine cell lines can be stably transfected and several reporter genes are available, the system should provide an alternative for conventional cytotoxicity assays. The detection of released enzymes by colorimetric or luminometric methods makes this cytotoxicity assay independent of radionuclides. The sensitivity of luminometric enzyme detection systems should also permit the measurement of apoptotic processes and might make in vivo studies of cellular death using transgenic animals feasible.

Is there a risk of prion-like disease transmission by Alzheimer- or Parkinson-associated protein particles?

The misfolding and aggregation of endogenous proteins in the central nervous system is a neuropathological hallmark of Alzheimer’s disease (AD), Parkinson’s disease (PD), as well as prion diseases. A molecular mechanism referred to as “nucleation-dependent aggregation” is thought to underlie this neuropathological phenomenon. According to this concept, disease-associated protein particles act as nuclei, or seeds, that recruit cellular proteins and incorporate them, in a misfolded form, into their growing aggregate structure. Experimental studies have shown that the aggregation of the AD-associated proteins amyloid-β (Aβ) and tau, and of the PD-associated protein α-synuclein, can be stimulated in laboratory animal models by intracerebral (i.c.) injection of inocula containing aggregated species of the respective proteins. This has raised the question of whether AD or PD can be transmitted, like certain human prion diseases, between individuals by self-propagating protein particles potentially present on medical instruments or in blood or blood products. While the i.c. injection of inocula containing AD- or PD-associated protein aggregates was found to cause neuronal damage and clinical abnormalities (e.g., motor impairments) in some animal models, none of the studies published so far provided evidence for a transmission of severe or even fatal disease. In addition, available epidemiological data do not indicate a transmissibility of AD or PD between humans. The findings published so far on the effects of experimentally transmitted AD- or PD-associated protein seeds do not suggest specific precautionary measures in the context of hemotherapy, but call for vigilance in transfusion medicine and other medical areas.

Quantitation of the anaphylatoxin C3a in the presence of C3 by a novel sandwich ELISA using monoclonal antibody to a C3a neoepitope

C3a levels in plasma are usually measured by a competitive inhibition radioimmunoassay (RIA) using 125I-labelled C3a-desArg and antibodies to C3a capable of detecting C3a determinants which are also present on the native C3. Therefore, prior to the assay native, non-cleaved C3 has to be removed completely from the C3a-containing sample by precipitation. We developed a new rapid two-site sandwich ELISA system for the quantitation of C3a-desArg in plasma. This immunoassay uses a monoclonal antibody (mAb H466) reacting with C3a-desArg but not with C3. The reactivity of mAb H466 with a neoantigenic determinant of C3a-desArg permitted the direct quantitation of C3a-desArg without removal of C3 from the sample. The mAb H466 was used as a capture antibody and bound C3a-desArg was detected with a second peroxidase-labelled anti-C3a mAb. The lower limit of detection of C3a-desArg in this ELISA was 1 ng/ml. The C3a-desArg levels measured in the plasma samples of various patients were found to differ over a wide range. A good correlation was observed between the results obtained in the RIA and those obtained in the ELISA (r = 0.95). High levels of C3a-desArg were detected in plasma from patients with multiple trauma and patients undergoing haemodialysis. The C3a-desArg assay described should facilitate the routine quantitation of C3a in samples of plasma.

Immune reactions of CD4- and CD8-positive T cell subpopulations in spleen and lymph nodes of guinea pigs after vaccination with Bacillus Calmette Guerin.

Vaccination of guinea pigs with Mycobacterium bovis BCG confers partial resistance against infection with Mycobacterium tuberculosis. Induction of immunity is associated with a strong T cell response. The reactions of the cytotoxic and helper T lymphocyte subsets after BCG vaccination were analyzed by cytofluorometry and in functional tests. The relative number of CD8(+) T cells in the spleen increased substantially after injection of BCG. In vitro restimulation after immunization induced a strong proliferative response but no cytotoxic reactions of CD8(+) T cells against BCG-infected macrophages. A specific induction of IFN-gamma and RANTES mRNA was observed after vaccination particularly in CD8(+) but not in CD4(+) T cells of the lymph nodes.

Tools for cellular immunology and vaccine research the in the guinea pig: Monoclonal antibodies to cell surface antigens and cell lines

The use of monoclonal antibodies directed against membrane proteins of leukocytes has greatly contributed to our understanding of the function and development of the immune system. Meanwhile these reagents provide valuable tools in many fields of research, stretching far beyond immunology and hematology. For guinea pigs only a limited number of such reagents have been described, and the information about availability and specificity is scattered over many years and journals. We provide an overview on the monoclonal antibodies produced since the technique was applied first in this species, with a focus on those reagents which have been characterized in more detail, and which should still be available either commercially or directly from the labs that created them.

Epidemiological Data – an Important Part of the Hemovigilance System

Epidemiological data are essential for monitoring trends and outbreaks of infectious diseases in the general population. The reporting system pursuant to the Infection Protection Act in Germany results in a very good quality of timely nationwide data on all reportable diseases including those relevant for the blood supply: HIV, hepatitis C, hepatitis B and syphilis. Notifications of acute hepatitis B and first-time diagnosed hepatitis C infections in the general population showed a declining trend in the past years, but the number of reports of HIV and syphilis infections increased until 2007 especially among men who have sex with men. New preventive strategies should also address changes in sexual behavior. The specific surveillance of blood donors is an important part of the hemovigilance system. The highly effective donor selection process results in a small number of confirmed infections among donors in Germany. The surveillance data enable us to identify specific trends that might challenge blood safety like the increase in HIV infections among repeat donors. Specific evaluations are performed when needed. These additional studies can be used to modify guidelines or recommendations and to (re)evaluate the need for or the effect of further testing.

T-lymphocyte responses in guinea pigs vaccinated with foot-and-mouth disease virus

The guinea pig provides an alternative experimental model for analysis of the immune response against foot-and-mouth disease virus (FMDV). The cellular immune response against FMDV in this experimental animal is unknown and was analyzed by in vivo and in vitro studies. In guinea pigs immunized with an FMDV A5 vaccine, a marked change in T-lymphocyte count appeared. For analyzing which functional T-cell compartment was affected, immunofluorescence studies, using monoclonal antibodies directed against differentiation antigens on guinea pig lymphoid cells, were performed. The proliferating T-cells were predominantly CD4-positive and, therefore, helper cells. T-cells from these animals were re-stimulated in vitro with homologous inactivated virus. The antigen-specific proliferative response of the T-cells in vitro was measured using the thymidine incorporation assay. A proliferative response to FMDV was observed that depended on the dose of the antigen. High concentration of virus had an inhibitory effect on T-cell proliferation. These data indicate that the guinea pig is a useful model for analysis of T-cell mediated mechanisms in the pathogenesis and immunity of foot-and-mouth disease.

Analysis of guinea pig leukocyte antigens using interspecies T cell hybrids

In the guinea pig system, there are no T lymphocyte tumors available. In addition, in this species the production of T cell lines in vitro for analysis of T cell markers or function proved to be difficult. As an alternative approach, guinea pig X mouse and guinea pig X rat T cell hybridomas were obtained by fusion of guinea pig T cells with HAT-sensitive mouse or rat thymomas. In ten fusions a total of 578 hybrids were produced and kept in culture for periods ranging from weeks up to several months. The highest yield was obtained when the Mb C 12 line was used as tumor parent. BW 5147 and the rat thymoma W/Fu(C58NT)D gave slightly lower fusion efficiencies. The yield depended also on the pretreatment of the parental cells. Activation by MLR proved to be most effective. A number of interspecies hybrids expressed guinea pig T cell differentiation antigens as detected by a cell ELISA. The positive hybrids were recloned several times and exhibited a stable expression of these markers, even after continuous culture for more than 2 months. Western blot analysis was used to confirm antigen expression at the protein level by comparing the hybrids with both parental cells. The hybrids expressed the proteins of the guinea pig lineage. No reaction was observed with the murine or rat thymomas. Therefore, the application of interspecies T cell hybrids provides an alternative for studies of guinea pig T cells. The cells are easy to grow and to clone and may be stored frozen in liquid nitrogen. The hybrids should permit analysis at the clonal level also for functional studies.