Determination of equine serum inhibitors for poliovirus by the gel-adsorption technique

Summary Inhibitors found in certain equine sera active against poliovirus type 1 have been determined by the gel-adsorption method using aluminiumhydroxide-gel in the absence of cells. The inhibitor belongs to the 19S-class of macroglobulins (IgM), as revealed by gel-filtration with Sephadex G 200, by DEAE-cellulose-chromatography, and by sucrose density centrifugation. It is bound to the viral surface in-vitro in the absence of tissue cells. The specific complex may be precipitated with anti-equine globulin from rabbits. The inhibitor is destroyed by papain and by 2-mercapto-ethanol. The residual infectivity (10–25%) has been found bound to the inhibitor in-vitro. Its behavior in the gel-adsorption system is not altered if the virus-inhibitor complexes have previously been diluted. — Nonsensitive mutants and double mutants have been selected. There must exist at least three different combining sites for equine inhibitors on the surface of poliovirus type 1, strain Mahoney. Equine sera may be grouped according to the specificity of their inhibitory activity. The specific binding capacity is lost if the virus is heated at 50° C for 30 min.Supported by the Deutsche Forschungsgemeinschaft, Unit Medizinische Virologie; partly presented at the 5th Viruscolloquium of the Deutsche Forschungsgemeinschaft, Marburg Sept. 1965.The authors wish to acknowledge the excellent technical assistance of MissSigrid Bonk.     

A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes

CD8(hi+) cytotoxic T lymphocytes (CTL) are major players in immune defense. In addition, they contribute to the maintenance of immune homeostasis. We now describe a hitherto unavailable, but simple assay to determine ex vivo lytic granule-based cytotoxic functions of human CD8(hi+) CTL subgroups in a clinical setting, under target cell free conditions. Ficoll-isolated peripheral blood lymphocytes from 17 healthy volunteers were stimulated either by phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin or by antibody mediated crosslinking of the CD3 molecule on the T cell surface. Using perforin as a marker for lytic granules, the reduction of CTL granules over time intervals up to 120 min was quantified by FACScan flow cytometry. The kinetics of perforin reduction were compared to the kinetics of NA-CBZ-L-lysine-thiobenzyl ester hydrochloride (BLT)-esterase release and of CD63 upregulation. The reduction in the perforin(+) portion of CD8(hi+) CTLs was correlated inversely with BLT-esterase release and CD63 upregulation. At 30 and 120 min after PMA/ionomycin stimulation, 55 +/- 14% and 42 +/- 14%, respectively, of CD8(hi+) CTLs still stained perforin(+) (time point 0 min = 100%). Perforin-granule release induced by CD3-crosslinking occurred as fast within 30 min (55 +/- 17%), but over the 120 min time interval it was not as complete when compared to PMA/ionomycin-stimulated perforin-reduction. Thus, the combination of an established degranulation assay with the power of immuno flow cytometry allows one to investigate the cytotoxic capability of CTL-subtypes and the kinetics of perforin-granule release. In addition, the assay may prove useful in the elucidation of intracellular signaling cascades governing the perforin-granule release process.     

Quantitation of cytomegalovirus (hCMV) DNA and β-actin DNA by duplex real-time fluorescence PCR in solid organ (liver) transplant recipients

Even now rare human cytomegalovirus (hCMV) reactivation is still a life-threatening complication after solid organ transplantation. Although PCR techniques are regarded as the most sensitive detection methods for hCMV, their accuracy and reproducibility are limited. This is a major disadvantage with quantitative PCR assays, which are thought to provide valuable information about hCMV latency or active viral replication in transplant patients. To enhance the diagnostic safety of quantitative hCMV PCR, we developed a duplex real-time fluorescence PCR that is capable of quantifying hCMV DNA and beta-actin DNA as internal control simultaneously within one reaction. By the use of 6-carboxyfluorescein and hexa-chloro-6-carboxyfluorescein as reporter fluorophores and 4-(4'-dimethylamino-phenylazo) benzoic acid as dark quencher dye, hCMV DNA and beta-actin DNA could be quantified in parallel in a wide linear range from 10(1) to 10(7) copies, each. To test the clinical applicability of this approach, we investigated hCMV DNA kinetics in peripheral leukocytes of three hCMV antigen-positive and four antigen-negative patients after liver transplantation, as assessed by intracellular hCMV pp65 alkaline phosphate-anti-alkaline phosphate (APAAP) complex. While all APAAP-negative individuals remained PCR negative, kinetics of HCMV DNA in leukocyte DNA samples of APAAP-positive patients correlated closely with hCMV antigen tests. Here, comparison of separate and simultaneous target quantitation revealed identical results. It is of interest that, while single hCMV antigen positivity is commonly not regarded as a reliable parameter of viral reactivation, in our study a viral load greater than 10(4) copies/2x10(5) beta-actin DNA copies clearly indicated a subsequent increase in APAAP-positive leukocytes. We conclude that with the presented method the reliability of hCMV quantitation via real-time PCR can be substantially increased and may be used to monitor hCMV kinetics in vivo.     

Viremia in human Cowpox virus infection.

BACKGROUND: Several poxviruses can infect humans and cause diseases of varying severity. Besides the eradicated Variola virus that induced high mortality rates, numerous further human pathogenic orthopoxviruses are potentially fatal but generally cause less severe infections. While infection-related viremia was described for Variola virus and seems to be rare for Monkeypox virus, it is still debated for Vaccinia virus. So far, viremia in Cowpox virus-infected humans has not been reported. OBJECTIVES: To estimate the potential risk of Cowpox virus to disseminate and develop severe infections, two Cowpox virus patients were examined for viremia. STUDY DESIGN: Whole blood, serum and fluid from virus-induced lesions were analyzed by serology or quantitative real-time PCR. RESULTS: Real-time PCR and sequence analysis of the hemagglutinin gene confirmed Cowpox virus in the lesions of both patients. Serology performed on serum obtained at the same time as the lesion specimens demonstrated orthopoxvirus-specific IgG and IgM antibodies, indicating a recent orthopoxvirus infection. In addition, Cowpox virus DNA was detectable in whole blood, but not in serum, as late as week 4 post-infection. CONCLUSIONS: In contrast to observations following vaccination with Vaccinia virus, DNAemia in patients with localized symptoms of a Cowpox virus infection does not seem to be a rare event. However, its relevance for Cowpox virus pathogenicity has to be elucidated.     

Immunohistochemistry (S 100, KL 1) and human papillomavirus DNA hybridization on Morbus Bowen and bowenoid papulosis

Summary In this study 55 paraffin embedded samples defined as Bowen's disease or bowenoid papulosis were investigated with antibodies against S 100 protein and keratins (KL 1). S 100-positive cells were quantified and related to defined section area of the epidermal compartment by computer-assisted image analysis. The density of S 100-positive cells was compared with normal skin and was particularly related to growth patterns and keratinization of the different lesions under study. S 100-positive dendritic cells were found to be reduced overall in bowenoid lesions when compared with normal skin. Lesions with high counts of S 100-positive dendritic cells most frequentty showed a solitary growth pattern with highly conserved architecture and differentiation and no tendency to stromal invasion. In contrast, cases with low counts of S 100-positive cells very often showed multifocal development, a high degree of architectural disturbance and dedifferentiation. In this group, stromal invasion (cases of invasive carcinoma associated with Bowen's disease) was seen more often. Interestingly, this latter group of cases also revealed a peculiar keratin pattern. Frequently, the basal cell layer was decorated with KL 1 antibody, which usually recognizes only suprabasaly located keratinocytes. No differences between Bowen's disease and bowenoid papulosis were found in terms of densities of S 100-positive dendritic cells and keratin pattern. In our experience, extragenital Bowen's disease and genital Bowen's disease can not be distinguished on purely morphological grounds or with the immunocytochemical approach presented here. Interestingly, when employing in situ hybridization with HPV 16 probes three of seven samples of genital Bowen's disease harboured HPV 16 DNA, whereas six cases of extragenital disease were negative.Supported by the Deutsche Forschungsgemeinschaft (Lo 285/2-4)     

Iduronate-2-sulfatase gene mutations in 16 patients with mucopolysaccharidosis type II (Hunter syndrome)

Mutations of the iduronate-2-sulfatase gene were identifiedin 16 patients with mucopolysaccharidosis type II (Hunter syndrome).Together with another 10 cases reported by us earlier it emergesthat about 20% of the patients have deletions of the whole geneor other major structural alterations. One, two or three basepair deletions are found in about 23% of the cases while theremaining about 57% carry point mutations predicting amlno acidreplacement, premature termination of translation, or aberrantsplicing. Molecular analysis of mRNA in splice site mutantsshowed that these latter defects frequently resulted in useof cryptic splice sites in exons or introns. 62% of the smalldeletions and point mutations have occurred in 3 of the 9 iduronate-2-sulfatasegene exons. Knowledge of the primary genetic defect allows fastand reliable carrier detection and prenatal diagnosis as wellas insight into the relationship between genotype and phenotype.     

Cellular and developmental patterns of expression of Ret and glial cell line-derived neurotrophic factor receptor alpha mRNAs

 Glial cell line-derived neurotrophic factor (GDNF) has recently been shown to signal by binding to GDNF receptor-alpha (GDNFR-α), after which the GDNF-GDNFR-α associates with and activates the tyrosine kinase receptor Ret. We have localized Ret messenger RNA (mRNA) in the developing and adult rodent and compared with to the expression of GDNF and GDNFR-α mRNA. Ret mRNA is strongly expressed in dopamine neurons and α-motorneurons as well as in thalamus, ruber and occlumotor nuclei, the habenular complex, septum, cerebellum, and brain stem nuclei. Ret mRNA was also found in several sensory systems, in ganglia, and in nonneuronal tissues such as teeth and vibrissae. Very strong Ret mRNA signals are present in kidney and the gastrointestinal tract, where Ret and GDNF mRNA expression patterns are precisely complementary. The presence of Ret protein was confirmed in adult dopamine neurons using immunohistochemistry. GDNFR-α mRNA was strongly expressed in the developing and adult dopamine neurons. It was also found in neurons in deep layers of cortex cerebri, in hippocampus, septum, the dentate gyrus, tectum, and the developing spinal cord. In the kidney and the gastrointestinal tract, GDNFR-α mRNA and Ret mRNA distribution overlapped. Dorsal root ganglia, cranial ganglia, and developing peripheral nerves were also positive. GDNFR-α was additionally found in sensory areas and in developing teeth. Sensory areas included inner ear, eye, olfactory epithelium, and the vomeronasal organ, as well as developing tongue papillae. The temporospatial pattern of expression of GDNFR-α mRNA did not always match that of Ret mRNA. For instance, GDNFR-α mRNA was also found in the developing ventral striatum, including the olfactory tubercle, and in hippocampus. These areas seemed devoid of Ret mRNA, suggesting that GDNFR-α might also have functions unrelated to Ret. Received: 2 January 1997 / Accepted: 26 February 1997     

Detection of fish antigens aerosolized during fish processing using newly developed immunoassays

BACKGROUND: Aerosolization of fish proteins during seafood processing has been identified as a potential route for allergic sensitization and occupational asthma among workers involved in high-risk activities. The aim of this study was to develop immunological assays for the quantification of aerosolized fish antigens in a fish-processing factory. METHODS: Polyclonal antibodies to the main fish species processed in the factory (anchovy and pilchard) were generated in rabbits and compared by ELISA inhibition assay and immunoblotting. These antisera were utilized to develop ELISA assays for the detection of fish antigens. The ELISA inhibition assays were evaluated by analyzing environmental air samples collected from three areas in a fish-processing factory: pilchard canning, fish meal production and lobster processing. RESULTS: By immunoblotting, the rabbit polyclonal antibodies demonstrated IgG antibody binding patterns comparable with IgE antibodies of fish-sensitized patients, particularly in regard to the major fish allergens parvalbumins. The sensitivity of the fish-specific ELISA assays developed was 0.5 microg/ml. The ELISA inhibition assays were able to differentiate between the two different fish species of interest but did not recognize a crustacean species. Notable differences in exposure levels to canned pilchard and anchovy antigens were demonstrated in the three different working areas of the factory, with assays having a detection limit as low as 105 ng/m(3). CONCLUSION: These ELISA-based assays are sensitive and specific to quantify differential exposure levels to fish antigens produced during fish processing, making it possible to investigate exposure-disease response relationships among workers in this industry.