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A Kaufmann

Publications and source records attributed to A Kaufmann.

At least 19 recordsLinked to original sources
PubMed1999

Differential expression of monocyte chemotactic protein-1 (MCP-1) in transforming rat hepatic stellate cells.

BACKGROUNDS/AIMS: Hepatic stellate cells and infiltrating leukocytes play a key role in the pathogenesis of liver fibrosis. The chronic phase of liver inflammation is characterized by immigrating mononuclear cells. To understand the underlying mechanisms responsible for the attraction of mononuclear cells in the pathogenesis of liver fibrosis, we investigated the inducible production of chemotactic activities in hepatic stellate cells. METHODS: Cultured hepatic stellate cells of different transformation grades and after in vitro transformation to myofibroblast-like cells were stimulated with tumor necrosis factor-a or bacterial lipopolysaccharide. Mononuclear cell attracting chemotactic activities were evaluated by chemotaxis assays, ELISA, and Northern blot analysis. RESULTS: We observed a transformation grade-dependent differential responsiveness of hepatic stellate cells and myofibroblast-like cells. Monocyte chemotactic protein-1 was inducible by tumor necrosis factor-alpha in non-transformed hepatic stellate cells. In contrast, monocyte chemotactic protein-1 was not inducible by bacterial lipopolysaccharide until the cells were fully transformed into myofibroblast-like cells. Despite a delayed onset, the bacterial lipopolysaccharide-inducible monocyte chemotactic protein-1 expression did not depend on an endogenous production of tumor necrosis factor-alpha. CONCLUSIONS: Our results indicate that the tumor necrosis factor-alpha and bacterial lipopolysaccharide-inducible production of chemokines plays a central role in the pathogenesis of liver fibrosis. These data suggest that when hepatic stellate cells have been transformed to a myofibroblast-like cells phenotype, e.g. by chronic injury, the cells become more sensitive to bacterial lipopolysaccharide, which may potentiate the production of chemotactic and fibrogenic mediators. A strong secretion of monocyte chemotactic protein-1 may contribute to the maintenance of an inflammatory infiltrate dominated by mononuclear cells.

Animals↗
PubMed1998

Differential mononuclear leukocyte attracting chemokine production after stimulation with active and inactivated influenza A virus.

After influenza A virus infection of human monocytes, we found a rapid and marked release of the mononuclear cell attracting chemokines MCP-1, MIP-1 alpha, and IP-10, with corresponding gene expression patterns as determined by Northern blot analysis. In striking contrast, the expression and release of the neutrophil chemoattractant IL-8 was not inducible. To determine the underlying mechanisms responsible for the induction of this differential chemokine pattern, we stimulated monocytes with UV- and heat-inactivated (56 degrees C and 100 degrees C) influenza A virus. In comparison with fully infectious influenza A, 56 degrees C-inactivated virus induced a strong production of MCP-1, MIP-1 alpha, and IP-10, while the release of MIP-1 alpha and IP-10 was substantially lower after exposure to UV-inactivated virus. No chemokine expression was found after stimulation with 100 degrees C-inactivated influenza A virus. Our data indicate that, contingent upon the chemokine examined, the maximal induction depends on the unrestricted infectivity of the virus, the unaltered hemagglutinin molecule, or the intact viral RNA. This diversified chemokine production may enable the infected host to mount an efficient antiviral response against infective and noninfective virus particles.

Animals↗
PubMed1998

[Status febrilis and jaundice].

A 49 year old patient was admitted with fever and jaundice. Laboratory evaluation showed leukocytosis, elevated values for C-reactive protein, transaminases, bilirubin and alkaline phosphatase. Ultrasound and subsequent CT-scan revealed multiple liver lesions and a liquid mass in the bursa omentalis. A CT-guided catheter-drainage was performed and streptococcus milleri isolated from the abscess and one blood culture. After empiric broad spectrum treatment with Piperacillin/Tazobactam (Tazobac) and Netilmicin (Netromycin), Ceftriaxon (Rocephin) was given for a total treatment time of 10 days. Further evaluation revealed a perforated peptic ulcer as possible etiology for the described localized infectious complication.

Abdominal Abscess↗
PubMed1998

Interaction between SecA and SecYEG in micellar solution and formation of the membrane-inserted state.

Preprotein translocation in Escherichia coli is mediated by the translocase with SecA as peripheral ATPase and SecY, SecE, and SecG as membrane domain. To facilitate large-scale purification of the SecYEG heterotrimer, SecY was fused at its amino terminus with a hexahistidine tag and co-overexpressed with SecE and SecG. The presence of the His tag allowed purification of homogeneously pure SecYEG complex by a single anion-exchange chromatographic step starting from octyl glucoside-solubilized inner membranes. Endogenous levels of SecD and SecF copurified with the SecYEG protein. Purified SecYEG complex retained a nativelike, alpha-helical conformation in octyl glucoside and in micellar solution binds SecA with high affinity. In the presence of the nonhydrolyzable nucleotide analogue adenosine 5'-(beta, gamma-imidotriphosphate), octyl glucoside-solubilized SecYEG is nearly as effective as the reconstituted enzyme in inducing the formation of a proteinase K-protected 30 kDa fragment of 125I-labeled SecA, while SecYEG is proteolyzed to fragments smaller than 6 kDa. These data demonstrate that the 30-kDa SecA fragment is not protected by the lipid phase nor by SecYEG but rather indicate that it represents a SecYEG- and nucleotide-induced stable conformational state of a SecA domain.

Adenosine Triphosphatases↗
PubMed1998

Hypochlorite-modified LDL: chemotactic potential and chemokine induction in human monocytes.

Within blood vessels the accumulation of monocytes/macrophages at sites of modified lipoproteins is an important feature in atherosclerosis. Recently the presence of LDL and other proteins modified by hypochlorous acid (HOCl-LDL) was demonstrated in human atherosclerotic vessels and human inflammatory kidney disease by immunohistology and protein chemistry. Chemokines contribute to a specific and directed migration of inflammatory cells. IL-8 (alpha-chemokine) attracts mainly neutrophils and distinct T-cell subsets while MCP-1 (beta-chemokine) preferentially acts on monocytes/macrophages. In the present study it was postulated that HOCl-LDL may induce and amplify inflammatory reactions by the induction of chemokine synthesis in local monocytes. After exposure of human monocytes to HOCl-LDL, it was found that mRNA and protein of the chemokine IL-8 was strongly induced, while the chemokine MCP-1 was not. HOCl-LDL itself led to a chemotactic migration of neutrophils. A chemotactic response of human monocytes toward HOCl-LDL was not detectable. We propose that HOCl-LDL may represent a form of LDL modification in the atherosclerotic process which initiates leukocyte infiltration; these mononuclear cells have been observed in the early stages of atherosclerosis.

Arteriosclerosis↗
PubMed1997

Single-chain Fv fusion proteins suitable as coating and detecting reagents in a double antibody sandwich enzyme-linked immunosorbent assay.

A recombinant enzyme-linked immunosorbent assay (ELISA) system, entirely based on antibody fragments, is described here as an attractive alternative to assays using polyclonal antisera or monoclonal antibodies. Two expression vectors have been developed for cloning and production of single-chain Fv (scFv) fusion proteins suitable as coating and detecting reagents, respectively, in a highly sensitive double antibody sandwich ELISA. The coating reagent is produced from the vector pZIP1, as a bivalent miniantibody with a leucine zipper for dimerization. The detection reagent is a fusion protein, in which a modified Escherichia coli alkaline phosphatase with increased specific activity is attached to the carboxy-terminus of the scFv. This conjugate is produced using pDAP2/S as cloning and expression vector. Optimized bacteria expression and simple one-step purification by hexahistidine tag-mediated metal chelate affinity chromatography yielded milligrams of ELISA reagent per liter of bacterial culture in both cases. A double antibody sandwich ELISA for the detection of beet necrotic yellow vein virus, the causal agent of sugarbeet rhizomania, was developed using fusion proteins obtained by means of pZIP1 and pDAP2/S. The plant pathogen was detected with a sensitivity higher than that reached in a conventional ELISA employing polyclonal antisera. Both plasmid vectors are compatible to phage display vectors such as pHEN1, pCOCK, and the pCANTAB series, allowing simple subcloning after isolation of scFv from phage display libraries. It is therefore easy to develop and produce an ELISA entirely by using bacterial recombination and expression techniques.

Alkaline Phosphatase↗
PubMed1997

Susceptibility of mononuclear phagocytes to influenza A virus infection and possible role in the antiviral response.

Among leukocytes, only monocytes and macrophages were found to be highly susceptible to an infection by influenza A virus. After infection, de novo viral protein synthesis was initiated but then interrupted after 4-6 h. Most macrophages died by apoptosis within 25-30 h. Before cell death, however, macrophages responded to influenza A virus with a high cytokine gene transcription and subsequent release of tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-6, interferon (IFN)-alpha/beta, and CC-chemokines. The basic mechanisms of virus-induced cytokine expression are still unknown and appear to involve transcription factors such as nuclear factor-kappaB and AP-1 which, however, were only activated for 2 h and declined below control values thereafter. After influenza A virus infection, only the mononuclear cell attracting CC-chemokines macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and RANTES were produced while the prototype neutrophil CXC-chemoattractants IL-8 and GRO-alpha were entirely suppressed. This selective induction of CC-chemokines may explain the preferential influx of mononuclear leukocytes into virus-infected tissue. Our data show that monocytes and macrophages represent a primary target for an influenza A virus infection. Thus, the mononuclear phagocyte response leads to a rapid proinflammatory reaction and an enhanced immigration of mononuclear leukocytes, which may condition the infected host for the subsequent virus antigen-specific defense.

Apoptosis↗
PubMed1997

Induction of neutrophil-attracting chemokines in transforming rat hepatic stellate cells.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a key role in the pathogenesis of liver fibrosis. Immigrating leukocytes can potentiate the progression of liver fibrosis by release of fibrogenic mediators and cytotoxic actions. The inducible production of neutrophil chemotactic activities in HSCs was investigated to understand the underlying mechanisms responsible for the attraction of leukocytes in the pathogenesis of liver fibrosis. METHODS: Cultured HSCs of different transformation grades and after transformation to myofibroblasts (MFBs) were stimulated with tumor necrosis factor (TNF)-alpha and lipopolysaccharide (LPS), respectively. Induced leukocyte chemotactic activities were evaluated by chemotaxis assays, enzyme-linked immunosorbent assay, and Northern blot analysis. RESULTS: A transformation grade-dependent differential responsiveness of HSCs and MFBs was observed. TNF-alpha-inducible production of chemotactic mediators increased substantially with advancing transformation. Only transformed MFBs were LPS responsive. Macrophage inflammatory protein 2 was identified as one of the inducible chemokines. CONCLUSIONS: The results suggest that chemokines play an important role in the pathogenesis of liver fibrosis. Proinflammatory cytokines can initiate the production of chemotactic activities. The more HSCs are transformed to MFBs, e.g., by chronic injury, the more sensitive the cells become to LPS, which may lead to a vicious circle of enhanced fibrogenic and chemotactic mediator production.

Animals↗
PubMed1997

Beet soil-borne virus RNA 2: similarities and dissimilarities to the coat protein gene-carrying RNAs of other furoviruses.

The complete sequence of the 3454 nt of RNA 2 of the Ahlum isolate of beet soil-borne furovirus (BSBV) has been determined starting with two short stretches of cloned cDNA. Unknown parts of the sequence were amplified by means of RT-PCR techniques using combinations of specific and random primers. BSBV RNA 2 is more similar in its genetic organization to potato mop top virus (PMTV) RNA 3 than to any other furoviral RNA, although it is more than 1100 nt longer. Its 3'-end, unlike that of PMTV RNA 3, has the potential to fold into a tRNA-like structure. It contains one large open reading frame for a readthrough protein with a molecular mass of 104 kDa (104K protein) which is interrupted internally by an amber stop codon terminating the coding region for a protein of 19 kDa (19K), most likely the viral coat protein (CP). The readthrough domain of the 104K protein is much larger than that of PMTV, but the N- and C-proximal portions of these domains are similar for the two viruses. No serological relationships were found between the particles of the two viruses, although more than 50% of the amino acid sequences of the putative CPs are identical.

Amino Acid Sequence↗
PubMed1997

Borrelia burgdorferi induces chemokines in human monocytes.

Lyme disease is clinically and histologically characterized by strong inflammatory reactions that contrast the paucity of spirochetes at lesional sites, indicating that borreliae induce mechanisms that amplify the inflammatory response. To reveal the underlying mechanisms of chemoattraction and activation of responding leukocytes, we investigated the induction of chemokines in human monocytes exposed to Borrelia burgdorferi by a dose-response and kinetic analysis. Lipopolysaccharide (LPS) derived from Escherichia coli was used as a positive control stimulus. The release of the CXC chemokines interleukin-8 (IL-8) and GRO-alpha and the CC chemokines MIP-1alpha, MCP-1, and RANTES was determined by specific enzyme-linked immunosorbent assays, and the corresponding gene expression patterns were determined by Northern blot analysis. The results showed a rapid and strong borrelia-inducible gene expression which was followed by the release of chemokines with peak levels after 12 to 16 h. Spirochetes and LPS were comparably effective in stimulating IL-8, GRO-alpha, MCP-1, and RANTES expression, whereas MIP-1alpha production preceded and exceeded chemokine levels induced by LPS. Unlike other bacteria, the spirochetes themselves did not bear or release factors with intrinsic chemotactic activity for monocytes or neutrophils. Thus, B. burgdorferi appears to be a strong inducer of chemokines which may, by the attraction and activation of phagocytic leukocytes, significantly contribute to inflammation and tissue damage observed in Lyme disease.

Borrelia burgdorferi Group↗
PubMed1997

[Investigations on the endoparasitic fauna of domestic cats in eastern Brandenburg].

The internal organs of 155 cats originating from 3 different territories in the eastern part of Brandenburg state were examined for parasites between 1993 and 1995. Parasites were found in 85% of cats. Parasites with the highest prevalence were Toxocara mystax (45%), Hydatigera taeniaeformis (22%), Ancylostoma tubaeforme (17%) and Opisthorchis felineus (16%). Metorchis bilis, Mesocestoides spp., Taenia pisiformis, Capillaria plica, Capillaria sp., Ollulanus tricuspis, Aelurostrongylus abstrusus, Cystoisospora spp. and Sarcocystis spp. were diagnosed with a lower frequency. The occurrence of H. taeniaeforme, A. tubaeformis und O. felineus was depending on the origin of the cats.

Animals↗
PubMed1996

Selective induction of monocyte and not neutrophil-attracting chemokines after influenza A virus infection.

It is characteristic for virus infections that monocytes/macrophages and lymphocytes infiltrate infected tissue while neutrophils are absent. To understand the mechanisms selectively attracting mononuclear cells in viral diseases, we examined in an influenza A virus model the expression and regulation of chemokines as candidate molecules responsible for the immigration of leukocytes into inflamed tissue. After influenza A virus infection of human monocytes, a rapid expression of the mononuclear cell attracting CC-chemokine genes MIP-1, MCP-1, and RANTES occurred which was followed by the release of chemokine proteins. In striking contrast to CC-chemokines, the expression of the prototype neutrophil CXC-chemoattractants IL-8 and GRO-alpha was completely suppressed after influenza A infection. The release of other neutrophil chemotactic factors was excluded by microchemotaxis assays. These results suggest that the virus-specific induction of mononuclear cell-attracting chemokines accounts for the preferential influx of mononuclear leukocytes into virus-infected tissue.

Chemokine CCL2↗
PubMed1996

Beet soil-borne virus RNA 3--a further example of the heterogeneity of the gene content of furovirus genomes and of triple gene block-carrying RNAs.

The complete nucleotide sequence of RNA 3 of the Ahlum isolate of beet soil-borne virus (BSBV) was determined from cDNAs obtained with immunocaptured virus particles and denatured preparations of dsRNA. BSBV RNA 3 is unique among the plant virus RNAs studied so far in containing apparently only the coding sequences of a triple gene block (TGB). The derived amino acid sequences of the three putative TGB-encoded proteins showed the highest level of sequence similarities with those of the corresponding proteins of potato mop top furovirus (PMTV) followed by those of peanut clump furovirus and barley stripe mosaic hordeivirus. Progressively fewer similarities were found with the TGB-encoded proteins of beet necrotic yellow vein virus (uncertain classification), potato X potexvirus, and potato M carlavirus. The 3'-terminal 78 nucleotides of BSBV RNA 3 can be folded into a tRNA-like structure and a high degree of sequence similarity exists between the 122 3'-terminal nucleotides of BSBV RNA 3 and PMTV RNA 2. In other regions, however, no pronounced sequence similarities were found between the two RNAs, and PMTV RNA 2 contains an additional putative gene for a cysteine-rich protein downstream of the TGB. The two viruses are unrelated serologically. BSBV RNA 3 adds a further variant to the heterogeneity of the gene content of furovirus genomes and of triple gene block-carrying RNAs.

Amino Acid Sequence↗
PubMed1996

Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana.

The coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli. For expression in higher plants the scFv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from the constructs. The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants. The scFv were not secreted into the culture fluids of suspension cultures, but were retained in the cells. The amount of expression of scFv in the best expressing plants was at least as high as in bacterial culture supernatants. In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E. coli or from plants. The majority of the 21 plants expressing cp-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots.

Antibodies, Viral↗
PubMed1996

Effect of water deprivation on eating patterns of lactating cows fed grass and corn pellets ad lib.

The present study investigated the effect of 48 h of water deprivation on eating behavior of 12 lactating cows fed grass and corn pellets ad lib. Water deprivation reduced grass and corn pellet intake significantly, with corn pellet intake decreasing earlier than grass intake. The reduction of feed intake was entirely due to a reduction of meal size for both grass and corn-pellet meals. The size of the first meal of the water deprivation period was already reduced by about 30%. Grass-meal frequency increased, whereas corn-pellet meal frequency decreased during water deprivation. Water deprivation also decreased live weight and milk yield by about 12 and 30%, respectively. All parameters returned to baseline values with rehydration. With ad lib access to feed and water, 77% of drafts occurred in relation to meals, but the number of meals clearly exceeded the number of drafts. The results demonstrate that water deprivation rapidly leads to premature meal termination. This is in line with the assumption that an enhanced prandial increase in ruminal fluid osmotality contributes to dehydration-induced hypophagia. Yet, further studies are necessary to prove this assumption and to better understand the complex relationships between eating and drinking in ruminants.

Animals↗
PubMed1995

Feeding patterns of lactating cows of three different breeds fed hay, corn silage, and grass silage.

Feeding behavior of 35 lactating cows of three different breeds (Holstein-Friesian = HF, Simmental = SI, and Jersey = JER) was compared in the present study. The cows were kept in a loose housing system and fed hay, corn silage (CS) and grass silage (GS) ad lib. Within 7 consecutive days, 2918 meals were recorded and analyzed: 2503 meals were eaten during the light phase (0430-2200), and 57% of these diurnal meals consisted of only one feedstuff (43% hay, 9% CS, 5% GS). During the dark phase, only 23% of the 415 meals consisted of hay alone, but 16% of CS and 21% of GS alone. This circadian distribution showed no breed differences, yet meal patterns did: HF and SI cows ate 11 meals per day, and JER cows ate more than 13 meals/day. Meal size was 9.8MJ, 7.9MJ, and 5.8MJ for HF, SI, and JER cows, respectively. HF cows had the highest 24 h energy intake and JER cows the lowest, but the latter spent the most time feeding. During the light phase all three breeds obtained the most energy from hay, followed by CS and GS. In contrast, during the dark phase most energy was derived from GS, and hay contributed the least. A significant premeal correlation was observed for cows of all breeds, in particular during the time with the highest feeding activity (0800-1300). During the dark phase no premeal correlation could be detected; instead a significant postmeal correlation appeared, in particular in HF and SI cows.(ABSTRACT TRUNCATED AT 250 WORDS)

Animal Feed↗
PubMed1995

Nicotine-induced exocytotic norepinephrine release in guinea-pig heart, human atrium and bovine adrenal chromaffin cells: modulation by single components of ischaemia.

The influence of single components of myocardial ischaemia, such as anoxia, substrate withdrawal, hyperkalemia and extracellular acidosis, on nicotine-induced norepinephrine (NE) release was investigated in the isolated perfused guinea-pig heart, in incubated human atrial tissue and in cultured bovine adrenal chromaffin cells (BCC). In normoxia, nicotine (1-1000 mumol/l) evoked a concentration-dependent release of NE (determined by high pressure liquid chromatography and electrochemical detection) from guinea-pig heart and human atrium. In contrast to selective anoxia (Po2 < 5 mmHg) or glucose withdrawal, respectively, anoxia in combination with glucose withdrawal (5-40 min) markedly potentiated nicotine-induced NE release both in guinea-pig heart and human atrium. The sensitization of cardiac sympathetic nerve endings to nicotine was characterized by a lower threshold concentration and an approximate two-fold increase of maximum NE release, peaking after 10 min of anoxia and glucose withdrawal. Cyanide intoxication (1 mmol/l) combined with glucose withdrawal resulted in a similar increase of nicotine-induced sympathetic transmitter release both in guinea-pig heart and human atrium. In contrast, the nicotine-induced (10 mumol/l) NE overflow was only slightly potentiated by 10 min of global ischaemia in guinea-pig heart. Both hyperkalemia ([K+] 16 mmol/l) and acidosis (pH 6.8-6.0) distinctly attenuated the stimulatory effect of nicotine in guinea-pig heart and human atrium under normoxic conditions. Consistent with an exocytotic release mechanism, NE release was dependent on the presence of extracellular calcium under all conditions tested. Furthermore, NE overflow from guinea-pig heart was accompanied by a release of the exocytosis marker neuropeptide Y (NPY; determined by radioimmunoassay). In BCC, nicotine (1-10 mumol/l) evoked a release of NE and NPY and a transient rise of [Ca2+]i (determined with fura-2) during normoxia which were both dependent on the presence of extracellular calcium. Both hyperkalemia and acidosis markedly reduced the exocytotic release of sympathetic transmitters and the corresponding [Ca2+]i-transients. These data demonstrate that nicotine-induced cardiac exocytotic NE release is markedly potentiated during short-term anoxia in combination with glucose withdrawal. In contrast, a brief period of ischaemia causes only a slight sensitization of cardiac sympathetic nerve endings to nicotine. This discrepancy may be due to an attentuation of nicotine-evoked NE release by hyperkalemia and by acidosis. The protective effect of these factors against anoxia-induced sensitization to nicotine appears to be related to the inhibition of nicotine-evoked [Ca2+]i-transients.

Adrenal Medulla↗