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T Montag

Publications and source records attributed to T Montag.

At least 19 recordsLinked to original sources
PubMed2006

FACS technology used in a new rapid bacterial detection method.

Culture methods for bacterial detection (BacT/ALERT or Pall eBDS) are currently implemented in blood donor screening procedures in many countries. Experience in the first years after implementation of these detection assays showed that although the analytical sensitivity was extremely high (about 1 CFU mL(-1)), the majority of these platelets were still transfused before a positive screening result was attained. Rapid technologies were developed to more effectively prevent transfusion-transmitted bacterial infection. In this study, a new rapid bacterial detection method based on fluorescence-activated cell sorting (FACS) technology was developed. Bacteria were stained with thiazole orange dye for 5 min and measurement was taken immediately after staining. The entire process took only 30 min. Six transfusion-relevant bacteria strains were tested in a spiking study. Without pre-incubation in a special bacteria growth medium, analytical sensitivity ranged between 10(5) CFU mL(-1) (Klebsiella oxytoca and Serratia marcesens) and 10(3) CFU mL(-1) (Escherichia coli). Sensitivity could be improved to 10(1) CFU mL(-1) for all tested bacteria by adding a pre-incubation step (6 h at 37 degrees C). Although preliminary in nature, results of our study suggest that bacterial detection by FACS technology in conjunction with a pre-incubation step offers a sensitive alternative technology to culture methods. Additionally, it provides the benefit of a rapid test time and the opportunity of preventing bacterial transmitted infections more effectively.

Bacteriological Techniques↗
PubMed2006

Fluorescence quencher improves SCANSYSTEM for rapid bacterial detection.

BACKGROUND AND OBJECTIVES: The optimized scansystem could detect contaminated platelet products within 24 h. However, the system's sensitivity was reduced by a high fluorescence background even in sterile samples, which led to the necessity of a well-trained staff for confirmation of microscope results. MATERIALS AND METHODS: A new protocol of the optimized scansystem with the addition of a fluorescence quencher was evaluated. Pool platelet concentrates contaminated with five transfusion-relevant bacterial strains were tested in a blind study. RESULTS: In conjunction with new analysis software, the new quenching dye was able to reduce significantly unspecific background fluorescence. Sensitivity was best for Bacillus cereus and Escherichia coli (3 CFU/ml). DISCUSSION: The application of a fluorescence quencher enables automated discrimination of positive and negative test results in 60% of all analysed samples.

Bacteriological Techniques↗
PubMed2005

Analysis of leukocyte binding to depletion filters: role of passive binding, interaction with platelets, and plasma components.

Since limited knowledge exists on the mechanisms which regulate cell binding to leukocyte removal filter surfaces, we investigated the binding patterns of leukocytes to individual layers of leukocyte depletion filters. After passage of 1 unit of whole blood, blotting of isolated filter layers on glass slides or elution of cells from filter layers revealed that most leukocytes were located within the first 10 of a total of 28 filter layers, peaking at layers 6 to 8, with granulocytes binding on average to earlier filter layers than lymphocytes. Leukocytes preincubated with inhibitors of actin activation showed unchanged distribution between filter layers, suggesting that cytoskeletal activation does not significantly contribute to their binding. When leukocytes were directly incubated with single filter layers, binding of up to 30% of input cells was recorded in the absence of Ca(2+). Immunohistological analyses showed colocalization of platelets and leukocytes, with co-clustering of platelets and leukocytes. Monocytes and to some degree lymphocytes but not granulocytes competed with platelets for filter binding. Precoating of filter layers with individual plasma components showed that hyaluronic acid, plasma type fibronectin, and fibrinogen all increased the binding of leukocytes compared with albumin coating. In conclusion, leukocytes can bind passively to filters in a process which does not require Ca(2+), which is independent of cytoskeletal activation and which may depend on individual plasma components. These results are of importance when new selective cell enrichment or depletion strategies through specific filters are envisaged.

Blood Cells↗
PubMed2001

Characterization and crystallization of a novel Sarcocystis muris lectin, SML-2.

A novel lectin (SML-2) consisting of 138 amino acids was isolated from cyst merozoites of Sarcocystis muris and sequenced by Edman degradation and mass spectrometry. All 12 cysteinyl residues are involved in disulfide bridges, four of which are attributed to a characteristic pattern of cysteines as found in the so-called PAN-module superfamily. Crystals of SML-2 diffracting to 2.1 A resolution at a synchrotron were grown by the hanging-drop vapour-diffusion technique. They belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 53.6, b = 128.8, c = 158.2 A and eight molecules in the asymmetric unit. SML-2 cocrystallized with Au galactose results in two different crystal forms. The first form is isomorphous with the native crystals and the second form adopts space group C222(1), with unit-cell parameters a = 74.7, b = 82.0, c = 131.0 A, and diffracts to 2.4 A at a rotating-anode X-ray generator.

Amino Acid Sequence↗
PubMed2000

Growth hormone treatment of breeding bulls used for artificial insemination improves fertilization rates.

To evaluate new therapeutical concepts for male subfertility, we tested the effects of exogenous recombinant bovine growth hormone (rbGH) on various endocrine and metabolic parameters both in blood and in seminal plasma of bulls. Sperm quality was assessed morphometrically and by monitoring the number of successful artificial inseminations (AIs) defined as non-return rates (NRR). Aliquots of 450 semen samples were used from each bull and each experimental period (4 wk before, 14 weeks during and 6 wk after treatment). Six out of ten sires (average age 8.4 years) were treated every two weeks with 640-mg depot formulated rbGH (Eli Lilly). Four bulls received vehicle only. Blood plasma bGH, IGF-I, insulin and glucose concentrations were increased with rbGH treatment. In seminal plasma there was no effect of rbGH treatment on fructose and citrate or on testosterone concentrations. With one exception, rbGH-treated bulls had greater IGFBP-3 concentrations in seminal plasma. Motility of spermatozoa after freezing and thawing was increased compared with pretreatment rates. Most interestingly, the number of successful AIs was increased by an average of 6.0% NRR when ejaculates from rbGH-treated bulls were used.

Animals↗
PubMed1998

Expression, purification, and biochemical characterization of a recombinant lectin of Sarcocystis muris (Apicomplexa) cyst merozoites.

The mature major microneme protein of Sarcocystis muris cyst merozoites, which is known as a dimeric lectin with high affinity to galactose and some of its derivatives, was expressed in Escherichia colias a histidine-tagged fusion protein. The recombinant polypeptide, which was recognized by a monoclonal antibody directed against the native lectin, was purified from inclusion bodies after solubilization and refolding, using a combination of metal chelate and lactose affinity chromatography. The apparent molecular mass of the refolded polypeptide as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoreses was 16 kDa, whereas gel filtration chromatography clearly demonstrated that the recombinant protein, like its native counterpart, exists as a homodimer of two non-covalently associated subunits. Inhibition of haemagglutination suggests that the combining site of the recombinant lectin recognizes N-acetyl-galactosamine as the dominant sugar, thus confirming the correct folding of the monosaccharide combining site in the renatured lectin. To the best of our knowledge, this work represents the first reported detailed characterization of a recombinant lectin from apicomplexan parasites, and may contribute to a better understanding of the process of host cell recognition and invasion by these obligate intracellular protozoa.

Animals↗
PubMed1996

Characterization of indole-negative Bacteroides fragilis group species with use of polymerase chain reaction fingerprinting and resistance profiles.

Biochemical tests alone do not adequately differentiate the various Bacteroides species, groups, and antimicrobial-resistant variants. Consequently, we used a polymerase chain reaction (PCR) fingerprinting technique, with either a single nonspecific primer derived from the t-DNA intergenic spacer region (T3B) or a single primer that anneals to minisatellite DNA sequences (M13 core), to identify and characterize 58 clinical isolates of Bacteroides fragilis group species (B. fragilis, B. distasonis, and B. caccae). In addition to species- and subspecies-specific differences, 4 strains of B. fragilis, 1 of B. distasonis, and 3 of B. caccae that showed increased resistance to imipenem, ampicillin, and ampicillin/sulbactam also produced unique PCR fingerprint profiles. Analysis by the clinical source of isolation (i.e. blood or intraabdominal, skin, or soft-tissue infection) indicated that no particular PCR fingerprint type was associated with greater pathogenicity of any individual clinical source. The PCR fingerprinting technique proves to be a useful tool for species identification and taxonomic studies, as well as for epidemiological studies of Bacteroides species.

Bacterial Typing Techniques↗
PubMed1996

[Comparison of standard methods for the preparation of egg yolk antibodies].

Chicken egg yolk antibodies (lgY) play an increasing role as alternative to mammalian polyclonal antibodies. They are widely used in biomedical research, for diagnostics, prophylaxis, and therapy of diseases. The extraction steps of IgY from egg yolk must be simple, with high output of purified antibodies. The aim of the present study was a comparison of different purification methods of egg yolk antibodies. The results of eight extraction methods of IgY and method combinations were investigated by PAGE and densitometric analysis. It has been demonstrated, that the IgY preparation with dextran sulfate is very effective, quick and simple to perform. It is well-suited in combination with other methods, e.g. ammonium sulfate precipitation.

Animals↗
PubMed1995

Identification and strain differentiation of 'Bacteroides fragilis group' species and Prevotella bivia by PCR fingerprinting.

Using single consensus primers of genomic nucleotide sequences, PCR-generated fingerprints were used for identification and differentiation of the Bacteroides fragilis group (B. fragilis, B. thetaiotaomicron, B. ovatus, B. distasonis, B. vulgatus) and Prevotella bivia (B. bivius) by comparing the DNA profiles with those of reference strains from the American Type Culture Collection and German Culture Collection. When primed by a single primer phage M13 core sequence, intra-species specific differences and species-specific bands were detected. Using primers derived from the evolutionarily conserved tRNA gene sequence, species-specific patterns were produced. A computer program, GelManager, was used to analyze the profiles and generate dendrograms. The correlation coefficients determined from the DNA fingerprint profiles of the clinical isolates (using the M13 core primer) fell within a narrow range, reflecting a high level of homology within the species. Based on the dendrograms, strains of one species were clearly differentiated from strains of other species. For comparison, SDS-PAGE analysis of whole cell extracts was also performed to obtain protein band patterns of various strains. Because of the simplicity of the PCR fingerprinting method and the ease of performance of computerized evaluation of data, this technique is a useful method for both species and strain differentiation, as well as for characterization of Bacteroides species and Prevotella bivia.

Journal Article↗
PubMed1991

Activation of human CD4+ and CD8+ cells by Sarcocystis gigantea lectin.

As recently reported, Sarcocystis gigantea lectin (SGL) is a powerful mitogen and a polyclonal activator (Syn. S. ovifelis) of human peripheral B-cells. In the present study we investigated the reactivity of human T-helper (CD4) and T-suppressor (CD8) cells to SGL. Mononuclear cells (MNCs) from five newborns and six adults were examined cytofluorometrically for the expression of cell-surface differentiation and activation antigens using a set of seven monoclonal antibodies. In all, 96% of cord-blood and 81% of adult CD4 cells expressed receptors for interleukin-2 (Tac+) after 64 and 164 h microgram/ml). The percentages of neonatal and adult Tac+ CD8 cells amounted to 67% and 59%, respectively. Major histocompatibility complex (MHC) class II antigens identified using the monoclonal anti-human leukocyte antigen (HLA)-DR antibody L243 were expressed on 30% (CD4) and 44% (CD8) of adult T-cells. Neonatal HLA-DR+ T-lymphocytes were not detectable. In parallel, functional tests were performed to examine cell proliferation and MNC antibody production.

Adult↗
PubMed1990

Sarcocystis gigantea lectin--mitogen and polyclonal B-cell activator.

The present study further examined the in vitro response of human mononuclear cells (MNC) to the Sarcocystis gigantea lectin (SGL). The results confirm our previous report that SGL is mitogenic for human MNC. We now report that SGL is not only a potent mitogen but also a polyclonal activator for human peripheral B cells. As was true for pokeweed mitogen (PWM, 2 micrograms/ml), the addition of SGL (25 micrograms protein/ml) to cultures of MNC caused lymphocyte proliferation and B-cell maturation, indicated by a marked increase in IgG and IgM production. As measured by the [3H]-thymidine incorporation assay, SGL induced significantly higher proliferative responses than PWM (P less than 0.01, n = 24). The values obtained by SGL and PWM for IgG and IgM synthesis were essentially identical. As opposed to SGL, the sarcotoxin-containing fraction (SGTF) did not induce antibody formation or proliferative responses in human MNC.

Animals↗
PubMed1990

Suppression of blastogenic transformation of lymphocytes by Bacteroides fragilis in vitro and in vivo.

Bacteroides species and Enterobacteriaceae are known to cause synergistic infections. However, the mechanisms behind this synergy are not completely understood. Several authors have shown that Bacteroides species may inhibit the phagocytosis of Enterobacteriaceae by polymorphonuclear leukocytes as well as by macrophages. With the present study we have addressed the question of whether Bacteroides fragilis (BF) is also capable of suppressing specific immune functions. When incubated together with murine lymphocytes, BF significantly inhibited the blastogenic transformation of these cells stimulated by Escherichia coli-lipopolysaccharide (LPS) or concanavalin A. This effect was dose dependent and was not mediated by prostaglandins. Other bacteria such as E. coli or Listeria monocytogenes did not show such an extensive suppression, while Streptococcus pneumoniae was equally active. BF also inhibited the pokeweed mitogen induced blastogenic transformation of human lymphocytes. Moreover, lymphocytes from BF-injected animals obtained 3 to 12 hours after infection proved to be partly refractory for LPS-stimulation. Finally, BF injections also affected T-cell dependent immunity as judged from the aggravation of an experimental listeriosis in mice.

Animals↗
PubMed1990

Development of a DNA-adsorbent for the specific removal of anti-DNA autoantibodies in systemic lupus erythematosus (SLE).

Autoantibodies against DNA are of primary importance for the diagnosis and pathogenesis of systemic lupus erythematousus (SLE). The level of anti-DNA antibodies correlates well with the disease activity and renal involvement. In such patients the removal of anti-DNA antibodies from plasma may lead to a clinical improvement. For this reason an adsorbent was made by covalent coupling of calf thymus DNA to a solid support based on ethylene dimethacrylate cross-linked hydroxethyl methacrylate. Up to 2.5 mg of DNA were immobolized to 1 ml of the support activated chemically by aminosilane and glutaraldehyde. The incubation of 40 ml of SLE plasma with 1 ml of the adsorbent resulted in a 50% decline in anti-DNA activity. There was no release of immobilized P-32-DNA into the plasma. Biocompatibility, sterilisation, and reapplication (without loss of binding capacity) of the adsorbent could be demonstrated. We concluded that the adsorbent may be suitable for treatment.

Autoantibodies↗
PubMed1990

Interactions between group B streptococci and human cord blood lymphocytes.

Group B streptococci (Streptococcus agalactiae) of various serotypes (Ib/c, III/x, III/R, and B variant) and different sources of isolation were analyzed for their to interact with human mononuclear cells (MNC). Type I streptococci were obtained from a neonate dead of an invasive group B streptococcal infection. Examining the attachment of these bacteria to human peripheral lymphocytes and their ability to influence the proliferation of cultured adult and neonatal MNC, the following results were obtained: (1) Type Ib/c, III/x, and B variant strains adhered spontaneously to neonatal cord blood lymphocytes. The bacteria were bound by large lymphoid cells not detectable in the peripheral blood of adults. (2) Addition of group B streptococci to neonatal MNC cultures increased lymphocyte proliferation approximately 8 fold (n = 32) above the background. The reactivity of neonatal lymphocytes was highly reproducible with a mean peak proliferation close to day 5 of culture and a ratio of 60 CFU MNC. The control cell proliferation induced by Pokeweed mitogen (2 micrograms/ml) gave a mean stimulation index of 58. In contrast to cord blood cells, adult MNC responded only slightly with either proliferation of streptococcal binding. Clinical type I strain induced mean peak [3H]thymidine incorporation into neonatal MNC was approximately 16 times higher than into that of adults. Streptococcal adherence and lymphocyte activation did not depend on serotype specificity and clinical origin of the strains investigated. Therefore, the ability to interact with human cord blood lymphocytes seems to be a common property of group B streptococci.

Adult↗
PubMed1990

[Determination of serum- and phagocyte resistance of E. coli using bioluminescence].

Resistance to serum and phagocytosis belong to the most important virulence markers of the bacteria. These properties enable the microorganism to have some selective advantage by overcoming of host defence, thus increasing the invasiveness of the bacteria. Determination of these properties make it possible to evaluate better the virulence of the facultative-pathogenic microorganisms and can therefore be used for microbiological diagnosis. The method used until now to test these characteristics are very time consuming. For these reason we have employed bioluminescence to determine the number of viable cells. This method is very suitable for this purpose.

Blood Bactericidal Activity↗