Marker profile for the evaluation of human umbilical artery smooth muscle cell quality obtained by different isolation and culture methods.

Even though umbilical cord arteries are a common source of vascular smooth muscle cells, the lack of reliable marker profiles have not facilitated the isolation of human umbilical artery smooth muscle cells (HUASMC). For accurate characterization of HUASMC and cells in their environment, the expression of smooth muscle and mesenchymal markers was analyzed in umbilical cord tissue sections. The resulting marker profile was then used to evaluate the quality of HUASMC isolation and culture methods. HUASMC and perivascular-Wharton's jelly stromal cells (pv-WJSC) showed positive staining for α-smooth muscle actin (α-SMA), smooth muscle myosin heavy chain (SM-MHC), desmin, vimentin and CD90. Anti-CD10 stained only pv-WJSC. Consequently, HUASMC could be characterized as α-SMA+ , SM-MHC+ , CD10- cells, which are additionally negative for endothelial markers (CD31 and CD34). Enzymatic isolation provided primary HUASMC batches with 90-99 % purity, yet, under standard culture conditions, contaminant CD10+ cells rapidly constituted more than 80 % of the total cell population. Contamination was mainly due to the poor adhesion of HUASMC to cell culture plates, regardless of the different protein coatings (fibronectin, collagen I or gelatin). HUASMC showed strong attachment and long-term viability only in 3D matrices. The explant isolation method achieved cultures with only 13-40 % purity with considerable contamination by CD10+ cells. CD10+ cells showed spindle-like morphology and up-regulated expression of α-SMA and SM-MHC upon culture in smooth muscle differentiation medium. Considering the high contamination risk of HUASMC cultures by CD10+ neighboring cells and their phenotypic similarities, precise characterization is mandatory to avoid misleading results.

Comparison of specific adsorbents for tumor necrosis factor-alpha and therapeutic anti-tumor necrosis factor-alpha antibodies: an in vitro sepsis model.

BACKGROUND: Tumor necrosis factor alpha (TNF-alpha), as a key mediator, represents a major point of attack in sepsis. Since it has been shown that systemic anti-TNF-alpha antibodies do not improve the situation of septic patients, the use of specific adsorption technology in the treatment of sepsis could have beneficial effects. METHODS: Magnetic beads coated with polyclonal or with monoclonal anti-TNF-alpha antibodies were investigated in vitro in order to analyze their ability to prevent TNF-alpha induced adhesion of peripheral blood mononuclear cells (PBMCs) at human umbilical vein endothelial cells (HUVECs). Additionally, therapeutical monoclonal anti-TNF-alpha antibodies were proofed for inhibitory effects of TNF-alpha mediated activation of HUVECs. RESULTS: We have shown, in vitro, that beads coated with polyclonal or monoclonal anti-TNF-alpha antibodies were able to significantly reduce monocyte adhesion. It was possible to decrease monocyte adhesion from nearly 9% to 3% within 2 hours and from 18% to 2% within 6 hours of TNF-alpha treatment by the simultaneous use of beads coated with polyclonal anti-TNF-alpha antibodies. Beads coated with monoclonal anti-TNF-alpha antibodies could even prevent monocyte adhesion within the first 2 hours, and reduced monocyte adhesion to 2% during 6 hours of incubation with TNF-alpha. On the other hand, application of therapeutic anti-TNF-alpha antibodies showed no significant difference compared to the measured monocyte adhesion values of activated endothelial cells. CONCLUSION: Adsorption techniques using specific adsorbents, possibly used in MDS (Microspheres-Based Detoxification System), are efficient in specific reduction of TNF-alpha and pathophysiological consequences, since monocyte adhesion at activated HUVECs was shown to be reduced.

Improved antigen and nucleic acid detection in a bovine virus diarrhoea eradication program.

A bovine viral diarrhoea/mucosal disease (BVD/MD) control and eradication program was introduced in Lower Austria in 1996, according to the Swedish model. An important risk factor for BVD transmission under local conditions is communal grazing where susceptible pregnant cattle from several herds may be mixed with unrecognised persistently infected (PI) animals. A reliable system for identification of PI animals is therefore essential for BVD eradication and steps were taken to improve a commercially available antigen-capture ELISA (Ag-ELISA) by modifying the method for leukocyte preparation and adjusting the negative cut-off value. A single-tube reverse transcriptase-polymerase chain reaction (RT-PCR) employing panpestivirus 324/326 primers targeting the 5'-untranslated region of the virus genome was also simplified and used on pooled blood samples to facilitate larger sample throughputs. RT-PCR positive pools were analysed individually to identify infected animals. Seven hundred eighty-six samples were tested by Ag-ELISA according to the instruction manual and 5324 samples with the modified method. All 6110 samples were retested by RT-PCR. The percentage of RT-PCR positive results with doubtful and negative Ag-ELISA samples significantly diminished using the modified method (from 4.71 to 0.82%). Selected BVD viruses were genetically typed by PCR product sequencing; special attention being paid to RT-PCR amplicons from samples which were negative or doubtful by ELISA. However, no correlation was found between the phylogenetic grouping of the viruses and the Ag-ELISA results.

In vitro cell culture systems as the basis for an extracorporeal blood purification strategy in multiorgan failure treatment.

Multiorgan failure (MOF) based on septic processes is very common but prognostically an extremely severe disease that has to be treated exclusively under intensive care conditions. Extracorporeal blood purification (ECBP) using specific and efficient systems such as the microspheres based detoxification system (MDS) (Artif Organs 1996;20:420) could improve significantly the situation of MOF in terms of the efficient removal of endotoxins as well as key mediators such as tumor necrosis factor alpha (TNF alpha). The purpose of the study was to test the effectiveness of endotoxin and cytokine removal to blunt cellular response. In terms of the in vitro principle methodology, isolated peripheral blood mononuclear cells (PBMC) were incubated with endotoxins and a selective endotoxin adsorbent, which was added at various times (immediately or 30, 60, 120, 240, or 360 min) after the onset of incubation. TNF alpha release of monocytes was measured following a standard procedure after 20 h. Human TNF alpha was incubated with cultured human endothelial cells with and without a specific TNF alpha adsorbent (polyclonal antibodies coated on polystyrene particles). The results showed that after the initial addition of endotoxins, the activation of monocytes can be stopped within 120 min by addition of endotoxin adsorbents. In addition, specific TNF alpha adsorbents are able to prevent intercellular adhesion molecule 1 (ICAM-1) expression of endothelial cells, therefore avoiding activation of endothelial cells. In conclusion, cell culture models are suitable to simulate cell interaction in MOF. Specific adsorbents are able to reduce or block pathophysiologically relevant cell interactions, and the time frame for effective ECBP seems to be very short, and therefore, efficiency must be high.

Rotary cell culture system (RCCS): a new method for cultivating hepatocytes on microcarriers.

The Rotary Cell Culture System (RCCS) is a new technology for growing anchorage dependent or suspension cells in the laboratory. The RCCS is a horizontally rotated, bubble free disposable culture vessel with diffusion gas exchange. The system provides a reproducible, complex 3D in vitro culture system with large cell masses. During cell growing the rotation speed can be adjusted to compensate for increased sedimentation rates. The unique environment of low shear forces, high mass transfer, and microgravity, provides very good cultivating conditions for many cell types, cell aggregates or tissue particles in a standard tissue culture laboratory. The system enables to culture HepG2 cells on Cytodex 3 microcarriers (mcs) to high densities. We inoculated 2 x 10(5)/ml HepG2 cells and 200 mg Cytodex 3 mcs in 50 ml Williams E medium (incl. 10% FCS) allowing them to attach to the mcs in the rotating vessel (rotation rate 14-20 rpm). HepG2 cells readily attached to the mcs while the vessel was rotating. Attachment of HepG2 to the mcs was about 50% after 24 hrs and 100 % within 48 hrs. After 72 hrs of rotary culturing small aggregates of Hep G2 on mcs were built. HepG2 cells and the aggregates rotated with the vessel and did not settle within the vessel or collide with the wall of the vessel. We conclude that this new RCCS is an excellent technology for culturing HepG2 cells on Cytodex 3 mcs. The system is easy to handle and enables to culture anchorage dependent cells to high densities in a short period.

Equine lentivirus, comparative studies on four serological tests for the diagnosis of equine infectious anaemia.

Serological diagnosis of equine infectious anemia is of necessity group-reactive, i.e. based on viral core protein p26, because viral envelope components as well as the host's immune response to them undergo rapid antigenic change. Since 1970 the agar gel-immunodiffusion test ("Coggins-test") has been the diagnostic method of choice. Recently, ELISA tests have been introduced for faster and theoretically more sensitive serodiagnosis, while Western blots have been used to clarify doubtful results obtained in Coggins-tests. A commercial competitive ELISA was found to give practically equivalent results to the Coggins-test. The sensitivity of this market product is intentionally kept marginal in order to avoid false-positive "reactor horses". Another commercial ELISA, non-competitive, gave inconsistent results, creating great turmoil among horse owners when falsely positive. Caution is also indicated when interpreting Western blots. Sera of strongly positive horses gave as many as eleven bands, of medium positives fewer bands, and of the weakest reactors solely the p26 band. Single p26 banding was, however, also encountered in 5% healthy horses, in two of them consistently over time, which are accordingly considered non-specific. In order to be interpreted as positive, a Western blot for this equine lentivirus must band with its core protein plus at least one glycoprotein, similar to the recommended criterion for a positive reading of serum samples from AIDS patients.

Attempts to immunoprotect adult horses, specifically pregnant mares, with commercial vaccines against clinical disease induced by equine herpesvirus-1.

In a project lasting 4 years more than 300 Lipizzans, around 180 of them adults, were vaccinated systematically against Equine Herpesvirus-1 (EHV-1) and representative groups thereof were serologically controlled for their antibody responses. In part, vaccination intervals recommended on packing slips were followed, in part other intervals, implicated by intermediary results, were used. A live virus vaccine proved ineffective if humoral antibodies were present. An oil-adjuvanted vaccine proved of highest antiviral immunogenicity, but after repeated revaccinations caused severe local reactions so frequently that we had to discontinue its use in adults. Fetal calf serum originating from the cell cultures used for viral propagation and not eliminated from the marketed product, was accused of being responsible for the incompatibilities. An inactivated mixed virus vaccine was of weak antigenicity regarding its EHV-1 component (whereas good regarding the influenza viruses) so that it proved unsatisfactory for primary immunization. It was, however, potent enough, and clinically well tolerated, to maintain suitable antibody levels in horses which had been initially primed by the oil-adjuvanted vaccine. Consequently, optimal humoral immunity as well as clinical acceptability resulted when two different vaccines were used, one for induction, the other for maintenance of protection. Vaccination intervals different from those on the packing slips are recommended for the mixed vaccine.

Comparative evaluation of the agar gel immunodiffusion test and two commercial ELISA kits for the serodiagnosis of equine infectious anemia.

Selected sets of serum samples of horses were tested blindly in a comparative investigation for antibodies against Equine Infectious Anemia (EIA) virus. Three commercial kits were used, a well-established agar-gel immuno-diffusion kit which our laboratory has been using routinely for 14 years on one hand, a competitive ELISA kit (CELISA) and a non-competitive ELISA kit on the other hand. The American EIA Reference Laboratory in Ames cotested 56 serum samples with the same 3 products, with highest-level correlation, thereby ascertaining full dependability of our own results. Five EIA experts supplied us critically weak or doubtfully reacting serum samples of experimentally infected horses together with their own test results, by necessity limited to the then available AGID in most instances. A high degree of correlation was found between our and their AGID results. In our own laboratory good correlation was found between the AGID test and one lot of the CELISA product. Time of seroconversion was coincident in some experimentally infected horses, partly AGID, partly CELISA proved more sensitive. Another lot of the CELISA product deteriorated completely long before the warranted validity, an unpleasant finding experienced by many other laboratories alike. The non-competitive ELISA product showed unacceptable inter-lot differences, oscillation between positive and negative results on consecutive samples of one and the same horse, never reacted with the weak positive International Reference Serum, and one lot deteriorated well beyond its expiration date. We discuss our results with the background: high sensitivity versus false-positive horses and advocate to maintain at their present sensitivity levels the AGID and the CELISA tests and not to push them further, as would be technically possible.