Biomedical subjects

U Lösch

Publications and source records attributed to U Lösch.

At least 37 records · Page 2Linked to original sources

PubMed1992

Development of a direct and indirect chemiluminescence immunoassay for the detection of an organophosphorus compound.

The following study describes the development of two immunoassays for the semiquantitative determination of the organophosphorus compound methyl phosphonic acid, p-aminophenyl 1,2,2-trimethyl-propyl diester (MATP). These assays are a direct competitive, labelled-hapten, enzyme immunoassay and a competitive enzyme immunoassay with indirectly labelled antibody, both with luminescence determination of peroxidase. A camera luminometer was used to determine final signal strength. A constant light reaction was reached using luminol (5-amino-2,3-dihydro-1,4-phalazinedione), and by adding coumaric acid as an enhancer. A five minute interval was selected as exposure time. This phototechnical system makes a semiquantitative detection of MATP possible. The detection limits for both assays were at 10(-6)mol/l MATP. With the help of a microtiter luminometer (reader) the detection limit was reduced to 5 x 10(-8) mol/l MATP, by measuring the real intensity of the chemiluminescence signal.

Antibodies, Monoclonal↗

PubMed1992

The influence of various adjuvants on antibody synthesis following immunization with an hapten.

For the production of specific antibodies to the hapten MATP (4-Amino-1,2,2-trimethyl-phenylphosphonate) in Balb/c mice various non-toxic adjuvants were compared to Freund's complete adjuvant (FCA). For immunization the hapten MATP was coupled to the carrier human serum albumin (HSA). The immunostimulating effect of the synthetic lipopeptides Pam3Cys-OH, Pam3Cys-Ser-Ser-Asn-Ala and different concentrations of the lipohexapeptide Pam3Cys-Ser-(Lys)4 (Pam3Cys = S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-N- palmitoyl-(R)-cysteine as well as of aluminium hydroxide were tested. IgG antibody titers in serum were determined in ELISA. In dose-response studies 50 micrograms Pam3Cys-Ser-(Lys)4 per mouse was the most effective dose with a long period of high antibody levels after the second booster. Pam3Cys-Ser-Ser-Asn-Ala provoked only low antibody titers. Immunostimulation with Pam3Cys--OH did not result in an increased production of specific antibodies. Compared to the control group an enhanced antibody synthesis could be provoked with aluminium hydroxide. However, the increase was much smaller than by using FCA. The lipopeptide Pam3Cys-Ser-(Lys)4 turned out to be a very potent adjuvant. One week after booster injection into mice 50 micrograms of this substance helped to elicit a higher antibody titer than FCA. Hence, as far as the degree of antibody production is concerned, Pam3Cys-Ser--(Lys)4 represents an alternative adjuvant to FCA.

Adjuvants, Immunologic↗

PubMed1991

T cells expressing the V beta 1 T-cell receptor are required for IgA production in the chicken.

While alpha beta T cells in mammals may express one of many variable (V) gene families in the beta locus, chickens have only two V beta gene families. The avian V beta 2+ T cells are recognized by the T-cell receptor 3 (TCR3) monoclonal antibody and V beta 1+ T cells are recognized by the TCR2 antibody, which we used to selectively suppress development of V beta 1+ T cells in order to examine their functional role. Suppression was accomplished by multiple injections of anti-TCR2 antibodies beginning in embryonic life and perpetuated by thymectomy 8 days after hatching. Young birds thus depleted of V beta 1+ T cells had greater than normal numbers of V beta 2+ T cells and appeared as healthy as thymectomized and untreated controls. While production of IgM and IgG antibodies was unimpaired, IgA antibody production was severely compromised in the V beta 1-depleted birds. The levels of secretory IgA in bile and lung lavage fluid were reduced 1000- to 10,000-fold and secretory IgA antibodies were not produced in response to mucosal immunization. B-cell production of IgA antibodies thus appears to require T cells expressing the V beta 1 genes, whereas T cells that express the V beta 2 genes lack this capacity.

Animals↗

PubMed1991

Lack of alloantigen-specific cytotoxic T cell activity in the TCR-gamma delta T cell subpopulation of alloantigen-immune chickens.

We examined the in vivo generation of alloantigen-specific cytotoxic T lymphocytes in chicken TCR-alpha beta and TCR-gamma delta T cells. The TCR-alpha beta and TCR-gamma delta subpopulations were separated from spleen of alloantigen-immune chickens using a negative selection method. The separated cells were examined for alloantigen-specific CTL activity in a 51Cr release assay. While negatively selected TCR-alpha beta cells exerted alloantigen-specific CTL activity, no cytotoxic activity could be detected with TCR-gamma delta cells.

Animals↗

PubMed1991

Influence of various adjuvants on the synthesis of specific antibodies of chicken, sheep and rabbit following immunization with an hapten.

In order to obtain high amounts of specific antibodies against the hapten methylphosphoric acid, para-aminophenyl-1,2,2-trimethyl-propyldiester (MATP) different animals were immunized with MATP coupled to the carrier protein human serum albumin (MATP12-HSA) using several adjuvants. The best specific immune response in sheep, rabbit and chicken was reached with Freund's complete adjuvant with animal specific differences being tested by an ELISA. The adjuvants aluminium hydroxide (5% and 10%) and diphosphoryl lipid A showed no significant difference compared to the control group (NaCl with MATP12-HSA). In rabbits and chickens MATP12-HSA can be used to reach an immune response without the help of an adjuvant.

Adjuvants, Immunologic↗

PubMed1991

Effect of perinatal anti-TCR2 treatment and thymectomy on serum immunoglobulin levels in the chicken.

Treatment of chickens as embryos and in the first six days after hatching with mg amounts of anti-TCR2 (alpha beta T cell antigen receptor) antibody plus thymectomy 6 to 8 days after hatching suppressed by more than 90% the TCR2 and TCR1 (TCR gamma delta) subpopulations in the blood. Thymectomy alone resulted only in a profound reduction (greater than 90%) of the absolute number of TCR1 cells, while the absolute number of TCR2 cells was reduced only by 65%. We were interested in the regulation of the immunoglobulin synthesis in these T cell deficient chickens. We determined serum IgG, IgM and IgA levels and found a profound selective reduction (by about 90% on average) of the serum IgA level in anti-TCR2 antibody treated plus thymectomized chickens. In chickens which had been thymectomized only, the serum IgA level was reduced on average only by about 60%. Serum IgG and IgM levels were not reduced in any group. Thus, there was a positive correlation of the serum IgA level and the absolute number of TCR2 cells in all groups. These results indicate that T cells which play a role in IgA specific isotype switching or in IgA B cell differentiation belong to the TCR2 subpopulation.

Animals↗

PubMed1991

Distribution of immunoglobulins during embryogenesis in the chicken.

Whereas the yolk of freshly-laid eggs contains only IgG, apart from traces of IgA, we were able to measure, on average, 0.145 mg/ml IgM and 0.207 mg/ml IgA in the yolk sac contents of 21-day-old chicken embryos. Up to the 14th embryonic day, IgG is exclusively contained in the yolk sac contents. It was not possible to demonstrate an increase in the amount of immunoglobulins present by comparing the amounts of IgM and IgA in freshly-laid eggs and in the yolk sac contents of 21-day-old embryos. The offspring of hens with experimentally-induced agammaglobulinemia did not begin with the production of the immunoglobulin isotypes IgG, IgM and IgA until after hatching. From these results the conclusion can be drawn that the immunoglobulins IgM and IgA are transferred from the egg white to the yolk sac contents during the last third of embryonic development. Embryonic synthesis of these immunoglobulins can be discounted as a result of this study. Synthesis was found to be initiated between the 2nd and 7th day of life for IgG, between the 2nd and 4th day of life for IgM and between the 6th and 13th day of life for IgA.

Animals↗

PubMed1991

Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. V. In vivo studies on protective effects against Escherichia coli diarrhea in pigs.

A field study and a controlled infection trial showed the protective effect of egg yolk lyophilisate and whole egg lyophilisate against enterotoxic E. coli germs. The lyophilisates were gained from eggs of hens immunized against pilus antigen of porcine-enterotoxic E. coli. In a first field study using egg yolk antibodies, 92% of 299 diarrhea affected piglets were cured. In a further field study diarrhea affected piglets were cured after 3 days by application of egg yolk lyophilisate from immunized hens. Piglets treated only with egg yolk of not immunized hens showed no signs of recovery. The infection trial showed, that whole egg lyophilisate of immunized hens was as successful as a common antibiotic therapy in curing piglets, orally infected with 5 x 10(10) E. coli/feeding and animal. The present data show that chicken egg antibodies can be used for treatment of infectious diarrheal diseases in young animals. So far they represent a good alternative to the common used antibiotic therapy.

Animals↗

PubMed1991

Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. IV. In vitro studies on protective effects against adhesion of enterotoxogenic Escherichia coli to isolated enterocytes.

The pathogenic effect of enterotoxogenic E. coli is mainly to be found in the jejunum. Hence, an effective immunoprophylaxis should be focussed at this site. This can be done effectively by an oral application of specific antibodies. The present study describes the effectiveness of specific antibodies against E. coli. The antibodies were gained from the yolk of E. coli immunized hens and tested for their in vitro activity. The tests showed a significantly reduced adhesion of enterotoxogenic E. coli onto isolated pig enterocytes as long as the bacteria had been incubated with the specific yolk antibodies beforehand. For this reason, yolk immunoglobulins from immunized hens prove to be an effective and handy source of specific antibodies which offers good protection against adhesion of E. coli onto pig enterocytes.

Adhesins, Escherichia coli↗

PubMed1990

Detection of the organophosphorus nerve agent soman by an ELISA using monoclonal antibodies.

The development of a specific and sensitive immunologic ELISA detection system for methylphosphonoflouridic acid. 1,2,2-trimethylpropylester (soman) by the use of monoclonal antibodies (MAbs) is described. The monoclonal antibodies F71D7, F71H10, F71B12 and F71H9 originally produced against the soman derivative methyl phosphonic acid, p-aminophenyl 1,2,2-trimethylpropyldiester (MATP) also reacted with soman in a previously developed, direct competitive ELISA. After optimizing the ELISA system by varying the reaction mixture and the solvents for the organophosphate, 5.0 x 10(-7) mol/l soman (80% purity), e.g. 2.5 ng or 2 ng pure soman per 25 microliters test buffer, could be detected after a total test duration of 40 min. A shortening of the incubation time to 10 min resulted in a drop of sensitivity to 1.8 x 10(-6) mol/l soman. Various alcohols which may be used as extraction media for soman from various materials (isopropanol, ethanol and methanol) were shown to inhibit peroxidase activity and thereby reduce the sensitivity of the test. However, the influence of alcohols decreased with the shortening of incubation time. All monoclonal antibodies showed little cross reactivity to sarin and no cross reactivity to tabun and VX. Judging on the reactivity of the MAbs with MATP and soman oxidazed by 1,2-dihydrobenzol, some reactivity with some other (non-toxic) soman analogues containing the same pinacolyl group can be expected. There was no evidence for stereoselectivity of the MAbs tested. Finally, soman could be detected in different biological samples like human serum, goat serum, rabbit serum, chicken serum, milk, and tap water in concentrations between 1.3 x 10(-6) and 2.0 x 10(-6) mol/l.

Alcohols↗

PubMed1990

Tissue distribution and appearance in ontogeny of alpha/beta T cell receptor (TCR2) in chicken.

We have performed immunoperoxidase staining on cryostat tissue sections and immunofluorescence analysis on cell suspensions to identify cells expressing the alpha/beta T cell antigen receptor during ontogeny and adult life in chickens. We used the mouse monoclonal antibody, TCR2, which was previously shown to recognize the alpha/beta TCR in chickens. TCR2+ cells were observed in thymic cortex and medulla and in T-dependent areas of spleen, intestine, and cecal tonsils of young adult chickens. Some TCR2+ cells were found in the cortex of bursal follicles and in liver. The first TCR2+ cells appear in thymus on Day 13 of the embryonic life and it is only after hatching that TCR2+ cells begin to migrate to the periphery.

Age Factors↗

PubMed1990

Chicken egg antibodies for prophylaxis and therapy of infectious intestinal diseases. III. In vivo tenacity test in piglets with artificial jejunal fistula.

Large quantities of specific egg antibodies can be produced with little effort by immunization of laying hens. Several antibody containing egg preparations were subjected to a tenacity test against digestive activity taking piglets with artificial jejunum fistulas as a model. Even with high doses of orally administered yolk lyophilisate no antibody activity could be found in the distal jejunum. The additional feeding of egg white, however, showed a significant protective effect on the yolks antibodies during the digestive act. No difference between egg white of immunized hens and egg white of unimmunized hens could be detected. Buffering the acidic gastric environment with sodium bicarbonate increased the antibody activity in the intestine by an average of 40%. Eggs administered in hot (70 degrees C) beef stock had higher antibody titers than eggs that were boiled for 4 minutes. Tenacity in antibodies fed in pellets was significantly lower than in powdered feed.

Animals↗

PubMed1990

Porcine granulocyte functions: evaluation and modulation.

Functions of porcine polymorphonuclear neutrophils are evaluated with in vitro test systems. Results are compared with those from human PMN and the relevance for in vivo conditions is discussed. Ethanol was inhibitory to all porcine PMN functions investigated here. Influex, a combination product, containing extracts of Echinacea, Aconitum, Apis and Lachesis stimulated adherence, chemotaxis, and phagocytosis, but inhibited chemiluminescence. These results suggest an effect of the product in the generation of reactive oxygen species.

Animals↗

PubMed1990

Complement-dependence of polymorphonuclear yeast cell phagocytosis: a comparison between man and various domestic animals.

The complement-dependence of polymorphonuclear yeast cell phagocytosis of sheep, goat, cattle, horse, dog, pig and man is determined by comparing the opsonizing abilities of untreated sera with complement-inactivated autologous sera at a serum concentration of 2.5% and at different phagocytosis periods. The performed phagocytosis assays only detects incorporated particles and allows for the differentiation of granulocytes with different quantities of phagocytosed particles. In sheep, horse and man the addition of heat-inactivated serum reduces the phagocytosis index to less than -80% of the value obtained at untreated serum addition at a phagocytosis period of 60 min. The other species show a smaller reduction ranging from -66.9% (dog) to 41.4% (cattle). The evaluation of the distribution pattern of granulocytes incorporating a certain number of yeasts offers significant differences in the investigated species.

Animals↗

PubMed1989

Analysis of the first two waves of thymus homing stem cells and their T cell progeny in chick-quail chimeras.

Chick-quail chimeras were used to study precursor/progeny relationships of hemopoietic stem cells (SC) that enter the embryonic thymus in waves to give rise sequentially to the TCR-1+, TCR-2+, and TCR-3+ lineages of T cells. The first wave of SC and their progeny were examined by grafting thymus from 9-d chick embryos (E9) into E3 quails. mAbs specific for chick T cell antigens were used to trace the development of T cells in the recipients. All three lineages of TCR-bearing cells were generated from the first wave of SC. The cortico-medullary transit time was several day shorter for the TCR-1 subpopulation than for the TCR-2 subpopulation, and the peripheral seeding of TCR-2 cells also occurred later in development. The duration of thymocyte production from the first wave of SC that entered the thymus was approximately 3 wk, during which gradual cortical to medullary replacement by second wave SC progeny occurred. When the latter was examined, after transplantation of E7 quail thymus into E3 chick embryos, a sequential generation pattern for the TCR-1 and TCR-2 cell progeny was not evident. Finally, recirculation of T cells to the thymus medulla was defined in this avian model.

Age Factors↗

PubMed1989

Development of an ELISA for detection of an organophosphorus compound using monoclonal antibodies.

This paper describes a specific and highly sensitive ELISA system using monoclonal antibodies in order to assay an organophosphorus compound. The soman derivative methyl phosphonic acid, p-aminophenyl 1,2,2,-trimethyl-propyl diester (MATP) served as model substance. In order to obtain antibody-producing hybridomas BALB/c mice were immunized with MATP linked onto human serum albumin (HSA). The spleen cells of immunized mice were fused with syngenic plasmacytomas of the non-producer-line X63Ag8.653 with the aid of polyethylene glycol. To eliminate undesirable cross-reaction, common screening procedures were modified by directly coating the ELISA plates with hapten. Five out of 15 positive cell-lines were cloned by limiting dilution and further propagated. The respective immunoglobulin class and subclass of the obtained monoclonal antibodies was determined. Four of which were identified as IgG1, the other as IgG2a. After enrichment of antibodies in ascites and their isolation by protein A-sepharose, the affinity of various monoclonal antibodies was estimated in competitive inhibition enzyme immunoassay (CIEIA) by measuring the IC50 rates of free MATP. The rates were found to lie between 2.5 x 10(-6) mol/l and 4.3 x 10(-4) mol/l MATP. The IC10 rate for detectable MATP concentration was 5.4 x 10(-7) mol/l MATP. Test duration was 280 min. The reactivity of the monoclonal antibodies with structurally related substances was used to check their specificity. Cross-reaction turned out to be negative. In order to develop a direct competitive ELISA, MATP was linked to horse radish peroxidase (HRPO) by adding a spacer. This helped to reduce total duration to 40 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

TCR3: a third T-cell receptor in the chicken.

Avian homologues of mammalian gamma delta and alpha beta T-cell antigen receptors, TCR1 and TCR2, have been identified with monoclonal antibodies. These TCR isotypes are associated with the avian CD3 proteins on the T-cell surface. During chick development, T-cell subpopulations bearing CD3/TCR1 or CD3/TCR2 receptor complexes are generated sequentially in the thymus and seeded to the periphery in the same order. In this study, we used two-color immunofluorescence to identify the subsequent development of a third subpopulation of T cells, provisionally named TCR3. These CD3+ cells, which expressed neither TCR1 nor TCR2, were first detected in the blood 1 week after hatching and increased numerically as a function of age to account for approximately 15% of the circulating T-cell pool in adults. Most (greater than 80%) of the TCR3 cells expressed the CD4 accessory molecule. The relative incidence of the TCR3 subpopulation increased dramatically as a consequence of embryonic treatment with anti-TCR2 antibody and thymectomy after hatching. Two disulfide-linked polypeptides, of Mr 48,000 and 40,000, were associated with the CD3 complex on the TCR3 cells. Examination of the TCR protein backbones and peptide mapping of the TCR chains after partial proteolysis indicated that the TCR3 heterodimer differs from both TCR1 and TCR2. These results suggest the existence of a third class of T-cell receptors in birds.

Animals↗

PubMed1989

Quantitative characterization of yeast cell incorporation in phagocytes: microscopic methods for differentiation between adherent and ingested particles.

A light- and a fluorescence-microscopic method for quantitative assessment of yeast cell incorporation in phagocytes were developed. The light-microscopic method offers methylene blue prestained yeast cells as phagocytosis particles and counterstains nonincorporated yeasts with eosine. The fluorescence-microscopic method works by acridine orange staining of phagocytosis assays. Fluorescence of nonincorporated yeast cells is suppressed by addition of methylene blue. Different ways of evaluating the results of microscopic quantitation of phagocytosis are discussed.

Acridine Orange↗