Brucellae through the food chain: the role of sheep, goats and springbok (Antidorcus marsupialis) as sources of human infections in Namibia.

A confirmed case of human brucellosis motivated an investigation into the potential source of infection in Namibia. Since domestic animals are principal sources of Brucella infection in humans, 1692 serum samples were screened from sheep, goats and cattle from 4 presumably at-risk farms and 900 springbok (Antidorcas marsupialis) serum samples from 29 mixed farming units for Brucella antibodies by the Rose-Bengal test (RBT) and positive cases confirmed by complement fixation test (CFT). To assess the prevalence of human brucellosis, 137 abattoir employees were tested for Brucella antibodies using the standard tube agglutination test (STAT) and by enzyme linked immunosorbent assay (ELISA). Cattle and sheep from all 4 farms were negative by RBT and CFT but 2 of the 4 farms (Ba and C) had 26/42 and 12/285 seropositive goats, respectively. Post mortem examination of seropositive goats revealed no gross pathological lesions typical of brucellosis except enlarged mesenteric and iliac lymph nodes seen in a single buck. Culture for brucellae from organs of seropositive animals was negative. None of the wildlife sera tested positive by either RBT or CFT. Interviews revealed that besides the case that prompted the investigation, a family and another person from other farms with confirmed brucellosis shared a common history of consumption of unpasteurised goat milk, home-made goat cheese and coffee with raw milk and prior contact with goats, suggesting goats as the likely source of infection. All 137 abattoir employees tested negative by STAT, but 3 were positive by ELISA. The 3 abattoir workers were clinically normal and lacked historical connections with clinical cases. Although goats are often associated with B. melitensis, these studies could not explicitly implicate this species owing to cross-reactivity with B. abortus, which can also infect goats. Nevertheless, these data reinforce the need for a better National Control Programme for brucellosis in Namibia.

Long-term results of tissue development and cell differentiation on Dacron prostheses seeded with microvascular cells in dogs.

PURPOSE: The purpose of this study was to investigate in vivo the long-term development, differentiation, and proliferation of the subendothelial tissue on Dacron prostheses seeded with microvascular cells (MVC). METHODS: Autologous MVC from omental adipose tissue were seeded on 4 mm Dacron prostheses and the prostheses interposed in the carotid arteries of mongrel dogs for 5, 13, and 26 weeks. RESULTS: Light and electron microscopic evaluation of patent seeded prostheses demonstrated an almost complete monolayer of endothelial cells and well-organized subendothelial tissue, whereas patent control prostheses were mainly covered by red and white thrombi, which were partially replaced by organized tissue with increased implantation time. The measurements of the thickness of the luminal cell layer in seeded and control grafts showed no statistically significant increase between 5 and 26 weeks of implantation. The subendothelial tissue of seeded prostheses demonstrated a time-dependent maturation of highly synthesizing myofibroblasts embedded in a collagen matrix to cells with features of smooth muscle cells located in a collagen-elastin matrix. In control grafts examined after 26 weeks the spontaneous endothelialization was accompanied by a delayed or incomplete maturation of subendothelial tissue. CONCLUSIONS: Our study indicates that MVC seeded onto Dacron prostheses are able to generate a vascular wall that does not continue to proliferate after prolonged implantation and that increasingly resembles the wall of a normal artery in cell differentiation and intercellular matrix.

Quantification of specific DNA O-alkylation products in individual cells by monoclonal antibodies and digital imaging of intensified nuclear fluorescence.

We report the establishment of a standardized, monoclonal antibody (Mab)-based immunocytological assay (quantitative ICA) for the visualization and quantification of low levels of specific DNA O-alkylation products in individual cells by electronically intensified, indirect or direct immunofluorescence. In terms of specific binding to alkali-denatured nuclear DNA and low background noise, 10 Mabs from a collection of 154 Mabs specific for O6-methyl-2'-deoxyguanosine (O6-MedGuo), O6-ethyl-2'-deoxyguanosine (O6-EtdGuo), O6-n-butyl-2'-deoxyguanosine (O6-BudGuo) and O4-ethyl-2'-deoxythymidine (O4-EtdThd) with antibody affinity constants ranging between 1.0 x 10(6)-3.0 x 10(10) l/mol, were found to be best suited for ICA. At present, > or = 200 O6-EtdGuo residues (corresponding to an O6-EtdGuo/dGuo molar ratio in DNA of > or = 8.4 x 10(-8)), > or = 400 O6-BudGuo residues (O6-BudGuo/dGuo, > or = 1.7 x 10(-7)), > or = 1800 O4-EtdThd residues (O4-EtdThd/dThd, > or = 7.5 x 10(-7)) and > or = 4800 O6-MedGuo residues (O6-MedGuo/dGuo, > or = 2.0 x 10(-6)), can be quantified per diploid genome. Using a SIT video camera in combination with multiparameter image digital analysis, DNA adduct-specific rhodamine fluorescence signals are measured relative to nuclear DNA content (DAPI fluorescence). Adduct-specific fluorescence recordings in three different rat cell lines (BT3Ca, Fao and NO) were in excellent agreement with the data obtained by competitive radioimmunoassay (RIA) for hydrolysates of DNA isolated from the respective cells exposed in parallel to the same alkylating carcinogens (N-methyl-, N-ethyl- and N-[n-butyl]-N-nitrosourea). Accordingly, the kinetics of O6-EtdGuo repair, as determined by ICA and RIA, respectively, were superimposable. Cell-specific, quantitative ICA can, therefore, be used for the quantification of specific, stable DNA adducts induced by alkylating carcinogens or chemotherapeutic agents and for DNA repair measurements in individual (e.g. human) cells. Work is currently underway to extend the spectrum of carcinogen--DNA adduct-specific Mabs suited for quantitative ICA.

Immunoaffinity purification and gas chromatography-mass spectrometric quantification of 3-alkyladenines in urine: metabolism studies and basal excretion levels in man.

Immunoaffinity gels were prepared by coupling monoclonal antibody (Mab) EM-6-47 to protein A-Sepharose, and were used to make small columns retaining 3-alkyladenines (3-alkAde) of diverse structure. An analytical procedure for determination of 3-methyladenine (3-MeAde), 3-ethyladenine (3-EtAde), 3-(2-hydroxyethyl)adenine (3-HOEtAde) and 3-benzyladenine (3-BzAde) was developed. Deuterated internal standards (d3-3-MeAde, d5-3-EtAde, d4-3-HOEtAde and d7-3-BzAde) were synthesized and added to urine samples prior to immunoaffinity purification. 3-alkAde were separated and quantitated as tert-butyl-dimethylsilyl (TBDMS) derivatives by capillary gas chromatography-low resolution mass spectrometry (GC-MS). Detection limits for 3-MeAde, 3-EtAde and 3-HOEtAde were 0.2 pmol/ml urine and for 3-BzAde, 1 pmol/ml urine. Studies in two volunteers showed that 3-MeAde and 3-HOEtAde were excreted almost quantitatively (> 90%) within 24 h, that 3-EtAde was less well excreted (67-74%) and that 3-BzAde was poorly excreted (21-25%). Studies on basal levels of 3-alkAde urinary excretion in three volunteers showed that 3-MeAde was > 90% derived from the diet as the preformed product. 3-HOEtAde was present at approximately 10 nmol/day and was reduced to approximately 1 nmol/day when the diet was standardized suggesting that it is also dietary in origin. 3-BzAde was not detected in human urine. 3-EtAde was not only excreted at low levels (< 1 nmol/day) but was also only very slightly affected by diet. This general and sensitive method will be useful in biomonitoring studies in subjects exposed to alkylating agents of diverse structure.

Developmental regulation and tissue distribution of the liver transcription factor LFB1 (HNF1) in Xenopus laevis.

The transcription factor LFB1 (HNF1) was initially identified as a regulator of liver-specific gene expression in mammals. It interacts with the promoter element HP1, which is functionally conserved between mammals and amphibians, suggesting that a homologous factor, XLFB1, also exists in Xenopus laevis. To study the role of LFB1 in early development, we isolated two groups of cDNAs coding for this factor from a Xenopus liver cDNA library by using a rat LFB1 cDNA probe. A comparison of the primary structures of the Xenopus and mammalian proteins shows that the myosin-like dimerization helix, the POU-A-related domain, the homeo-domain-related region, and the serine/threonine-rich activation domain are conserved between X. laevis and mammals, suggesting that all these features typical for LFB1 are essential for function. Using monoclonal antibodies, we demonstrate that XLFB1 is present not only in the liver but also in the stomach, intestine, colon, and kidney. In an analysis of the expression of XLFB1 in the developing Xenopus embryo, XLFB1 transcripts appear at the gastrula stage. The XLFB1 protein can be identified in regions of the embryo in which the liver diverticulum, stomach, gut, and pronephros are localized. The early appearance of XLFB1 expression during embryogenesis suggests that the tissue-specific transcription factor XLFB1 is involved in the determination and/or differentiation of specific cell types during organogenesis.

Second-harmonic generation with partially poled polymers.

The poling of polymers leads in general to inhomogeneous polarization distributions within the polymer film. These polarization distributions affect any nonlinear-optic experiment based on second-order nonlinearities. Second-harmonic generation measurements are presented for a partially poled ferroelectric polyvinylidene fluoride film. By partial poling, we mean that the film is polarized only within a part of the film thickness. The measured angle-dependent second-harmonic signal is explained only if the polarization distribution as measured by the piezoelectric pressure pulse technique is taken into account.

Highly sensitive, specific detection of O6-methylguanine, O4-methylthymine, and O4-ethylthymine by the combination of high-performance liquid chromatography prefractionation, 32P postlabeling, and immunoprecipitation.

A highly sensitive and specific method for the detection of O6-methylguanine (O6-meG), O4-methylthymine (O4-meT), and O4-ethylthymine (O4-etT) has been established by combining prefractionation by high-performance liquid chromatography (HPLC), 32P postlabeling, and immunoprecipitation by monoclonal antibodies (PREPI method). DNA was enzymatically hydrolyzed to 2'-deoxynucleoside-3'-monophosphates (3'-dNps). Each alkyl 3'dNp was separated by reverse-phase HPLC, radiolabeled at the 5' position with [gamma-32P]ATP and polynucleotide kinase. After removing 3'-phosphate for better recognition by the antibodies, the resulting alkyl nucleotides were further fractionated by HPLC and finally precipitated specifically with respective antibodies. The detection limits were 1 fmol for all the alkyl nucleotides analyzed, so that one adduct in 10(8) of its normal counterpart nucleotide can be determined using approximately 100 micrograms (O4-meT and O4-etT) or approximately 150 micrograms (O6-meG) of DNA, i.e., 3-5 x 10(7) cells corresponding to approximately 10 ml of peripheral blood or a few hundred milligrams of tissue. By the use of the PREPI method, three leukocyte and three liver DNA samples from Japanese living in the Tokyo area were analyzed with respect to O-alkyl adduct content. O6-meG was detected in all three of the leukocyte samples (O6-meG:G molar ratio, 1.1 x, 0.8 x, and 1.6 x 10(-8) as molar ratios to guanine). Neither O4-meT nor O4-etT was detected (detection limit, O4-alkylT:thymine molar ratio less than 0.5 x 10(-8)). Among the liver samples analyzed, two cases showed positive O6-meG values (4.2 x and 1.1 x 10(-7) O6-meG:guanine molar ratios). Contrary to the leukocyte DNA, O4-meT (3.9 x, 4.3 x, and 7.5 x 10(-8) as O4-meT:thymine) and O4-etT (1.9 x, 4.9 x, and 8.7 x 10(-8) as O4-etT:thymine) were detected in all the liver samples. These results indicate the validity of the PREPI method for molecular epidemiological studies on DNA alkylation products.

E-cadherin-mediated cell-cell adhesion prevents invasiveness of human carcinoma cells.

The ability of carcinomas to invade and to metastasize largely depends on the degree of epithelial differentiation within the tumors, i.e., poorly differentiated being more invasive than well-differentiated carcinomas. Here we confirmed this correlation by examining various human cell lines derived from bladder, breast, lung, and pancreas carcinomas. We found that carcinoma cell lines with an epithelioid phenotype were noninvasive and expressed the epithelium-specific cell-cell adhesion molecule E-cadherin (also known as Arc-1, uvomorulin, and cell-CAM 120/80), as visualized by immunofluorescence microscopy and by Western and Northern blotting, whereas carcinoma cell lines with a fibroblastoid phenotype were invasive and had lost E-cadherin expression. Invasiveness of these latter cells could be prevented by transfection with E-cadherin cDNA and was again induced by treatment of the transfected cells with anti-E-cadherin mAbs. These findings indicate that the selective loss of E-cadherin expression can generate dedifferentiation and invasiveness of human carcinoma cells, and they suggest further that E-cadherin acts as an invasion suppressor.

[Effect of hemodynamic conditions and prosthesis structure on endothelialization of clinical and experimental small lumen vascular prostheses]. / Einfluss der hämodynamischen Bedingungen und der Prothesenstruktur auf die Endothelialisierung von klinischen und experimentellen kleinlumigen Gefässprothesen.

Seeding of small-diameter vascular prostheses (ID less than or equal to 6 mm) with autologous microvascular cells (AMVC) results in a complete endothelial cell layer on the luminal surface. The purpose of this study was to examine the influence of the blood flow velocity (due to 4 or 6 mm ID) and the structure of inner graft surface (crimped, uncrimped) on the endothelialization. AMVC were harvested from omental adipose tissue (mean: 0.56 X 10(6) cells/g tissue) from 10 mongrel dogs (mean: 27.9 kg). During preclotting, the 4 mm uncrimped and the 6 mm crimped double velour Dacron prostheses (Meadox Medicals, Inc.) were seeded with 1.0 X 10(6) cells/cm2 graft surface. Grafts were implanted into the carotid arteries (N = 5 in each group). The animals received antiplatelet therapy. After five weeks, all seeded prostheses were patent. The thrombus free surface (TFS) of seeded prostheses was 99.9% (4 mm) and 90.5% (6 mm). Scanning electron microscopy revealed an athrombogenic layer of endothelial cells on a smooth surface. -It is concluded that in canine experiments endothelialization of 4 and 6 mm grafts after seeding with AMVC is not affected by blood flow velocity or graft structure.

Repair of O6-ethylguanine in DNA protects rat 208F cells from tumorigenic conversion by N-ethyl-N-nitrosourea.

O6-Ethylguanine (O6-EtGua) is one of about a dozen different alkylation products formed in the DNA of cells exposed to the alkylating N-nitroso carcinogen N-ethyl-N-nitrosourea (EtNU). We have evaluated selectively the relative capacity of cells for the specific enzymatic repair of O6-EtGua as a determinant for the probability of malignant conversion. Eleven O6-EtGua-repair-proficient (R+) variant subclones were isolated from the O6-EtGua-repair-deficient (R-) clonal rat fibroblast line 208F by selection for resistance to 1,3-bis-(2-chloroethyl)-1-nitrosourea (frequency, approximately equal to 10(-5). Contrary to the 208F wild-type cells, all variants expressed O6-methylguanine-DNA methyltransferase activity, while both kinds of cells were deficient for repair of the DNA ethylation products O2- and O4-ethylthymine. After exposure to EtNU (less than or equal to 500 micrograms/ml; 20 min), cells were analyzed for the formation of piled-up foci in monolayer culture and of anchorage-independent colonies in semisolid agar medium. Depending on the EtNU concentration, the frequencies of piled-up foci and agar colonies, respectively, in the R+ variants were as low as 1/28th and 1/56th of those in the R- wild type. Contrasting with the cells from R+ variant-derived agar colonies, cells from 208F (R-) agar colonies gave rise to highly malignant tumors when implanted subcutaneously into syngeneic rats. No significant differences in the frequencies of piled-up foci were found between wild-type and variant cells after exposure to the major reactive metabolite of benzo[a]pyrene, (+)-7 beta, 8 alpha-dihydroxy-9,10 alpha-epoxy-7,8,9,10 alpha-tetrahydrobenzo[a] pyrene, for which stable binding to guanine O6 in cellular DNA has not been observed. The relative capacity of cells for repair of O6-alkylguanine is, therefore, a critical determinant for their risk of malignant conversion by N-nitroso carcinogens.

Monoclonal antibodies for the specific detection of 3-alkyladenines in nucleic acids and body fluids.

We describe an immunoanalytical procedure for the detection and quantitation of 3-alkyladenines in biological samples with the use of anti-(3-alkyladenine) monoclonal antibodies (Mab). A new hapten-protein conjugate, 3-ethyl-8-(3-carboxypropyl)-adenine, was used for immunization of BALB/c mice after conjugation to carrier proteins via the carboxyl group. Eighty-nine hybridomas were established which secrete anti-(3-alkyladenine) Mab with antibody affinity constants ranging from 1 x 10(7) to 5 x 10(9) l/mol for 3-ethyladenine (3-EtAde). One of these Mab (EM-6-47) had detection limits of 30 fmol for 3-EtAde, 17 fmol for 3-n-butyladenine (3-BuAde) and 475 fmol for 3-methyladenine (3-MeAde) respectively, at 25% inhibition of tracer-antibody binding. The binding pattern of Mab EM-6-47 revealed high specificity for adenine substituted at N-3 with different alkyl residues and no, or very low, cross-reactivity with other alkylated or unmodified nucleic acid components or structurally related compounds. 3-MeAde and 3-EtAde can be well separated from nucleic acids, and from rat and human urine samples, using HPLC with two successive stationary phases. Using Mab EM-6-47 in conjunction with a competitive RIA, both 3-MeAde and 3-EtAde were detected in the range of 100-300 ng (3-MeAde) and 2-10 ng (3-EtAde) in urine samples (10 +/- 2 ml) of untreated rats collected over a 24 h period. Only 3-MeAde (range 1.3-24.20 micrograms) was found in human urine samples. The concentration of 3-EtAde in rat urine increased significantly during the 24 h following a single i.v. application of N-ethyl-N-nitrosourea. After i.p. application of known amounts of 3-MeAde and 3-EtAde, greater than 90% of 3-MeAde and greater than 70% of 3-EtAde were excreted in rat urine within the subsequent 24 h. The concentration of 3-alkyladenines in body fluids (urine) may thus provide a useful indicator of environmental exposure to nucleic acid-reactive agents, and the immunoanalytical procedure described here permits the sensitive determination of adenines carrying different substituents at N-3.

[Endothelialization of 6 mm vascular prostheses with microvascular endothelial cells from fatty tissue of the omentum]. / Endothelialisierung von 6-mm-Gefässprothesen mit mikrovaskulären Endothelzellen aus dem Fettgewebe des Omentums.

The seeding of 6 mm Dacron prostheses with microvascular endothelial cells from omental adipose tissue leads to endothelialized prostheses, 5 weeks later with a complete coverage of functional and thrombus free endothelium. The method is ready for clinical trials.

Late neutrophil infiltration of canine endothelial cell seeded Dacron grafts.

Neutrophil infiltration is known to affect the endothelial monolayer of seeded vascular grafts. The aim of this study was to develop an in vitro system allowing the monitoring of neutrophil (PMN) adherence after graft implantation. Dacron prostheses were seeded with autologous canine microvascular cells from omental adipose tissue and implanted for 35 days. In vitro, mature monolayers of canine homologous venous endothelial cells (CHVENC) were exposed to heparinized whole blood samples taken at days one and four postoperatively, followed by weekly tests. PMNs adherent to the CHVENC were counted per culture area. Results showed the feasibility of PMN monitoring, and demonstrated a late PMN adhesion, reaching its maximum about 20 days after implantation and decreasing to normal values after five weeks. It is concluded that in vitro tests can be used for noninvasive studies of host plasma factors and leukocyte activation.

Investigations on the relationship between DNA ethenobase adduct levels in several organs of vinyl chloride-exposed rats and cancer susceptibility.

The levels of 1,N6-ethenodeoxyadenosine (epsilon dAdo) and 3,N4-ethenodeoxycytidine (epsilon dCyd) were measured in DNA of several target organs of vinyl chloride (VC)-exposed rats. Seven-day-old (group I) and 13-week-old (group II) BD VI rats were exposed during 2 weeks to 500 ppm VC in air (7 hr per day and 7 days per week). epsilon dAdo and epsilon dCyd were measured by a combination of prepurification of DNA hydrolysates by HPLC and competitive radioimmunoassay using specific murine monoclonal antibodies. Both ethenodeoxynucleosides were detected in liver, lungs and brain (levels ranging from 0.6 x 10(-7) to 1.3 x 10(-7) for epsilon dAdo/2'-deoxyadenosine and from 1.95 x 10(-7) to 4.92 x 10(-7) for epsilon dCyd/2'-deoxycytidine) but not in kidneys of group I rats. In group II rats, only liver DNA was analysed and the levels of each adduct were six times lower than in young (group II) rats. These findings are in good agreement with the organotropism and the age-related sensitivity of VC-induced carcinogenesis in rodents.

1,N6-etheno-2'-deoxyadenosine and 3,N4-etheno-2'-deoxycytidine detected by monoclonal antibodies in lung and liver DNA of rats exposed to vinyl chloride.

1,N6-Etheno-2'-deoxyadenosine (epsilon dAdo) and 3,N4-etheno-2'-deoxycytidine (epsilon dCyd) are formed in vitro by reaction of DNA with the electrophilic metabolites of vinyl chloride (VC), chloroethylene oxide and chloroacetaldehyde. To detect and quantitate these DNA adducts in vivo, we have raised a series of specific monoclonal antibodies (Mab). Among those, Mab EM-A-1 and Mab EM-C-1, respectively, were used for detection of epsilon dAdo and epsilon dCyd by competitive radioimmunoassay (RIA), following pre-separation of the etheno adducts from DNA hydrolysates by high performance liquid chromatography. At 50% inhibition of tracer-antibody binding, both Mab had a detection limit of 187 fmol and antibody affinity constants (K) of 2 x 10(9) l/mol. The levels of epsilon dAdo and epsilon dCyd were quantitated in the DNA of lung and liver tissue of young Sprague-Dawley rats exposed to 2000 p.p.m. of VC for 10 days. The epsilon dAdo/2'-deoxyadenosine and epsilon dCyd/2'-deoxycytidine molar ratios were 1.3 x 10(-7) and 3.3 x 10(-7), respectively, in lung DNA, and 5.0 x 10(-8) and 1.6 x 10(-7) in liver DNA. When hydrolysates of 3 mg of DNA were analyzed by RIA at 25% inhibition of tracer-antibody binding, epsilon dAdo and epsilon dCyd were not detected in liver DNA from untreated rats above the limiting epsilon dAdo/2'-deoxyadenosine and epsilon dCyd/2'-deoxycytidine molar ratios of 2.2 x 10(-8) and 3.1 x 10(-8), respectively.

Monoclonal antibody-based immunoanalytical methods for detection of carcinogen-modified DNA components.

Hybridoma cell lines secreting monoclonal antibodies (Mab) directed against the products formed by reaction of alkylating N-nitroso carcinogens with DNA have been established by fusion of rat or mouse myeloma cells, respectively, with spleen cells of rats or mice immunized either with conjugates of various alkyl-ribonucleosides with suitable carrier proteins, or with alkylated DNA electrostatically complexed to carrier proteins. Due to their high affinity and specificity, some of these Mab detect very low amounts of the respective alkyl-deoxynucleosides (e.g., O6-methyl-2'-deoxyguanosine, O6-ethyl-2'-deoxyguanosine, O6-n-butyl-2'-deoxyguanosine, O6-isopropyl-2'-deoxyguanosine, O4-methyl-2'deoxythymidine, O4-ethyl-2'-deoxythymidine) and can be used in various types of immunoassays. With a competitive radioimmunoassay (RIA), specific DNA alkylation products can be quantitated in hydrolysates of cellular DNA, in body fluids, or in urine. The RIA is routinely applicable, reproducible, and sufficiently sensitive to permit the quantitation of femtomole amounts of modified nucleosides in small samples of DNA. When the alkyl-deoxynucleosides in question are separated from bulk DNA by high-performance liquid chromatography prior to analysis by RIA, very low levels of modification in DNA can be detected. The immuno-slot-blot (ISB), a noncompetitive solid-phase immunoassay, is more sensitive than the RIA. For analysis by ISB, alkylated DNA is heat-denatured and immobilized on nitrocellulose filters prior to exposure to the respective Mab and subsequent binding of a second (125I-labelled or biotinylated) antibody. In immunocytological analysis (ICA), the binding of Mab to alkyl-deoxynucleosides is visualized in individual cells by immunostaining of denatured nuclear DNA in situ (direct immunofluorescence; peroxidase-staining).(ABSTRACT TRUNCATED AT 250 WORDS)