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An M2 streptococcal isolate and isogenic mutants in which either the emm or mrp gene was insertionally inactivated were tested for virulence using either a mouse model or a chicken embryo model. The results of the studies using the mouse model demonstrated that neither the emm nor mrp gene products had a significant effect on virulence when mice were challenged via the i.p. route. However, when the bacteria were injected into the skin the emm gene product was identified as a virulence factor. In parallel studies in the chicken embryo model the mrp gene product was found to be a major virulence factor, while a minor contribution to virulence could also be attributed to the emm gene product. The importance of these gene products to virulence was noted when the chicken embryo were injected either i.v or when the bacteria were placed on top of the chorioallantoic membrane. The direct comparison of a single wild type group A organism and its paired isogenic mutants in two animal models suggests that different combinations of bacterial factors are required to overcome host defense strategies associated with different animal species., (Copyright 1997 Academic Press Limited.)

The M and M-like proteins of Streptococcus pyogenes are fibrous cell surface proteins. They have multiple binding sites for several human proteins and are composed of the C-terminal anchor domain, the alpha-helical coiled-coil domain, and the N-terminal non-coiled-coil domain. The coiled-coil domain of the M1 protein consists of repeat units called B, C, and D and a spacer unit S between B and C. Recombinant fragments A-B-S-C-D, A-B-S, B-S-C, S-C, S-C-D, C-D, and C of the coiled-coil domain were studied by analyzing their secondary structures and binding affinities to human serum albumin (HSA). As shown by circular dichroism, all fragments are in an alpha-helical conformation. C-D and S-C-D form coiled coils at room temperature and bind below 37 degrees C with high affinity to HSA. C-D and S-C-D unfold in two steps with Tm values of approximately 31 and approximately 65 degrees C; complex formation with HSA increases the unfolding temperatures. B-S-C has a lower alpha-helical content, a less pronounced coiled-coil conformation, and a reduced thermal stability, binds HSA weaker, and is only slightly stabilized by HSA binding in comparison to C-D and S-C-D. C and S-C are less stable than the other fragments and are not organized as coiled coils showing some features of alpha-helical single strands only below 20 degrees C, and binding of HSA was not observed. The results indicate that the formation of coiled-coil structures, supported by flanking D regions and, to a lesser extent also B regions, is essential for the binding of C repeat units to HSA.

The vir regulon of group A streptococci (GAS) organizes the expression of several bacterial virulence factors under the control of the Mga regulator. Previously, the genes encoding the Mga regulator (mga), M and M-related proteins (emm, mrp, enn) and C5a peptidase (scpA) were reported to be clustered on the streptococcal genome in a core vir regulon. In the present study, the genomic regions of a serotype M49 strain upstream of mga and downstream of scpA were sequenced to assess the boundaries of the vir regulon. In the upstream region, an operon was identified that may be potentially involved in substrate transport and is independent from Mga regulation. In the downstream region, another Mga-controlled, scpA-cotranscribed gene was detected. This gene termed orfX encoded a 385-amino acid (aa) potential surface protein of unknown function. No binding of serum proteins to a recombinant ORFX was detectable and phagocytosis resistance of an orfX mutant remained unchanged. Downstream of orfX, another Mga-independent gene determined the 3' end of the core vir regulon. Utilizing the M49 wild type, a mga- mutant and comparative Northern blot hybridization, genes encoding the capsule synthesis machinery, streptokinase and streptolysin O, as well as erythrogenic toxin A and DNase C were found to be Mga independent. In contrast, expression of the genes encoding the cysteine protease SpeB, streptococcin A and the oligopeptide permease was reduced in the mga- mutant. This indicated that in addition to the core vir regulon, Mga directly or indirectly controls a number of genes dispersed throughout the GAS genome.

Bacterial oligopeptide permeases are membrane-associated complexes of five proteins belonging to the ABC-transporter family, which have been found to be involved in obtaining nutrients, cell-wall metabolism, competence, and adherence to host cells. A lambda library of the strain CS101 group A streptococcal (GAS) genome was used to sequence 10,192 bp containing the five genes oppA to oppF of the GAS opp operon. The deduced amino acid sequences exhibited 50-84% homology to pneumococcal AmiA to AmiF sequences. The operon organization of the five genes was confirmed by transcriptional analysis and an additional shorter oppA transcript was detected. Insertional inactivation was used to create serotype M49 strains which did not express either the oppA gene or the ATPase genes, oppD and oppF. The mutation in oppA confirmed that the additional shorter oppA transcript originated from the opp operon and was probably due to an intra-operon transcription terminator site located downstream of oppA. While growth kinetics, binding of serum proteins, and attachment to eukaryotic cells were unaffected, the oppD/F mutants showed reduced production of the cysteine protease, SpeB, and a change in the pattern of secreted proteins. Thus, the GAS opp operon appears to contribute to both protease production and export/processing of secreted proteins.

Purpose: Liposomes and collagen corneal shields (CCS) have been used as ophthalmic drug delivery devices. With regard to a possibly combined application, we studied the effects of surface charge and bilayer fluidity of liposomes on their uptake and release by CCS., Methods: 12-hours-CCS were soaked in large unilamellar liposomes, which had been labelled with 4,5-carboxyfluorescein (CF) and N-(lissamine rhodamine B sulfonyl)-diacyl-phosphatidylethanolamine (PE-RhB) in the aqueous space and in the liposome bilayer, respectively. Released fluorophores were determined fluorometrically in the elution buffer at intervals from 1 to 240 min after immersion., Results: The CF concentration in the CCS soaked in a CF solution was two to seven times higher than immersion in the liposome suspensions. Among those, the negatively charged, cholesterol-containing preparation led to the highest CF concentration in the CCS. The PE-RhB concentration was highest after soaking the CCS in neutral, cholesterol-free liposomes. All types of liposomes were found inside the CCS by freeze fracture electron microscopy. The release kinetics data indicate a first order release. More than 90% of CF was released by the CCS within the first 30 min. This was equal after soaking the CCS in the CF solution or in liposomes. With DOPC-liposomes, the maximal release was already attained after 10 min. In general, the differences in the release kinetics of both hydrophilic and lipophilic markers, obtained by the various liposome types were small., Conclusions: Our results indicate that surface charge and bilayer fluidity are of minor importance for the interaction with collagen corneal shields. However, since the release kinetics of a liposome-encapsulated hydrophilic or lipophilic substance are similar to the release of a non-encapsulated drug, the combination of liposomes with collagen shields may be useful mainly with respect to the encapsulation of drugs which do not penetrate the ocular surface as well as to prolong corneal contact time of the liposomes.

The human pathogenic microorganism Streptococcus pyogenes can resist against phagocytic attack of human granulocytes. Streptococcal M protein and hyaluronic acid were identified as virulence factors involved in this protection. So far, no experiments have been reported which describe the contribution of both components together in one system. We used the chicken embryo as an in vivo phagocytosis model to investigate the role of both components on the virulence of streptococci. For this, isogeneic mutants of group A streptococcal strains (GAS) which lack hyaluronic acid capsule (cap-) or M protein (M-) expression were used for infection and their virulence was compared with laboratory strains which had lost their ability to produce one or both virulence factors after long-time laboratory passages on blood agar. The experiments revealed that strains producing both M protein and hyaluronic capsule were highly virulent. Only 1-10 colony-forming units were enough to cause a 50% lethality of 12-day-old chicken embryos. Those strains lacking one of these components showed a significant decrease in virulence. Finally, strains which failed to express either hyaluronic acid or M protein showed an additional tenfold decrease in virulence. This indicates a partial contribution of both M protein and hyaluronic acid to the virulence of GAS in the chicken embryo.

M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein.

Amphotericin B remains an important antifungal agent in the treatment of ocular mycosis. Since topical ocular application is limited because of ocular irritation and poor penetration, we studied the pharmacokinetics of amphotericin B encapsulated in unilamellar liposomes (AmBisome). One drop (20 microliters) of AmBisome or an equivalent concentration of amphotericin B was applied to rabbit eyes. Drug concentrations were measured 15, 60, 120 and 240 min following administration of the agents by HPLC in cornea and aqueous humor. The effect of intact (group A) and debrided corneal epithelium (group B) was also studied. Corneal amphotericin B levels were significantly higher (P < 0.01) after 15 min in animals receiving amphotericin B as compared to AmBisome in group A. At later time points no differences in the corneal drug levels were found, and the drug levels following AmBisome application were remarkably stable. Epithelial removal resulted in increased corneal drug levels following application of both amphotericin B preparations. Significantly higher drug levels were observed after free amphotericin B treatment at 15-60 min (P < 0.01). Drug levels in the aqueous humor did not differ between the two amphotericin B preparations and remained below therapeutically effective concentrations. These results suggest that topically delivered AmBisome provides stable corneal drug levels, but has the potential benefit of lowered ocular toxicity.

Superantigens use an elaborate and unique mechanism of T lymphocyte stimulation. Prototype superantigen are the pyrogenic exotoxins produced by Staphylococcus aureus and Streptococcus pyogenes. Many candidate proteins of bacterial, viral and protozoal origin have recently been reported to be superantigens. In most cases the evidence that these proteins are in fact superantigens is highly indirect. In this review the evidence that gram-positive cocci produce superantigens other than the pyrogenic exotoxins is critically discussed. Evidence in described demonstrating that the epidermolytic toxins of Staphylococcus aureus and the pyrogenic exotoxin B and M-proteins of Streptococcus pyrogenes are not superantigens. Criteria are described for acceptance of a candidate as a superantigen.

There have been a number of stages in the development of cholesteatoma surgery. After the introduction of the radical operation the disadvantages of this procedure led to the development of more conservative operative techniques, for example the anterior-posterior approach with preservation of the posterior wall of the external ear canal or the obliteration of the mastoid cavity with muscle flaps or various materials. The assumption that organic fragments of the bone enable certain unspecified mesenchymal cells to form bone led to this investigation. Allogenic bone matrix and gelatin were implanted in the epitympanic recess in 16 rabbits. The animals were sacrificed 2, 4, and 8 weeks after the operation and the temporal bones were examined histologically. Both the implanted substances led to the formation of new bone in the epitympanic recess in rabbits. The newly formed bone bonded itself with the bony wall of the epitympanic recess. The existence of foreign body giant cells could not be verified. Perhaps the difficulties involved in treating cholesteatomas could be solved with osteoinductive substances.

Colonic duplication is a rare, congenital anomaly. A case is presented in order to show the etiology, pathology and clinical importance of colonic duplication.

It has been supposed for many years that group A streptococci may elaborate more than the three well known erythrogenic toxins A, B or C (ETA, ETB, ETC). The analysis of the culture supernatant of streptococcal strain 27297 carrying neither genes for ETA nor ETC revealed mitogenic activity at pH 7.3 in isoelectric focusing. This mitogen of strain 27297 was purified by hydrophobic adsorption to Phenyl-Sepharose following FPLC chromatography on a Mono S column resulting in two proteins with mitogenic activity called AX and BX, respectively. Both differed in only one aminoterminal residue. The mitogenic activity of BX lacking one aminoterminal arginine was found to be about 100 times higher than that of AX. The aminoterminus of BX does not correspond to a predictable cleavage site for signal peptidase. We assume that BX was produced after translation by cleavage of the mature protein or the AX molecule with streptococcal proteinase (ETB) or an arginylaminopeptidase which is detectable on whole cells. The purified proteins BX and AX showed molecular weights of about 27 kDa in SDS electrophoresis and isoelectric points of 8.3 (AX) and 7.3 (BX) in isoelectric focusing, respectively. Both proteins were produced by practically all group A strains tested but not by groups B, C, G or H streptococci. Therefore, AX or BX seem to be proteins characteristic of group A streptococci.

M1 protein and Protein H are surface proteins simultaneously present at the surface of certain strains of Streptococcus pyogenes, important pathogenic bacteria in humans. The present study concerns the structure, protein-binding properties and relationship between these two molecules. The gene encoding M1 protein (emm1) was found immediately upstream of the Protein H gene (sph). Both genes were preceded by a promoter region. Comparison of the sequences revealed a high degree of similarity in the signal peptides, the C repeats located in the central parts of the molecules and in the C-terminal cell-wall-attached regions, whereas the N-terminal sequences showed no significant similarity. Protein H has affinity for the Fc region of IgG antibodies. Also M1 protein, isolated from streptococcal culture supernatants or from Escherichia coli expressing emm1, was found to bind human IgGFc. When tested against polyclonal IgG from eight other mammalian species, M1 protein and Protein H both showed affinity for baboon, rabbit and pig IgG. M1 protein also reacted with guinea-pig IgG, whereas both streptococcal proteins were negative in binding experiments with rat, mouse, bovine and horse IgG. The two proteins were also tested against other members of the immunoglobulin super family: human IgM, IgA, IgD, IgE, beta 2-microglobulin, and major histocompatibility complex (MHC) class-I and class-II antigens. M1 protein showed no affinity for any of these molecules whereas Protein H reacted with MHC class-II antigens. M1 protein is known to bind albumin and fibrinogen also. The binding sites for these two plasma proteins and for IgGFc were mapped to different sites on M1 protein. Thus albumin bound to the C repeats and IgGFc to a region (S) immediately N-terminal of the C repeats. Finally, fibrinogen bound further towards the N-terminus but close to the IgGFc-binding site. On the fibrinogen molecule, fragment D was found to mediate binding to M1 protein. The IgGFc-binding region of M1 protein showed no similarity to that of Protein H. Still, competitive binding experiments demonstrated that the two streptococcal proteins bound to overlapping sites on IgGFc.

Erythrogenic toxin type C (ETC) from different streptococcal group A strains was successively purified by absorption on phenylsepharose, acidic dialysis of the eluate at 40% saturated ammonium sulphate solution, CM-Sepharose chromatography, finally by immunoaffinity chromatography on monoclonal antibodies. Second, after growing of bacteria in the presence of [32P]orthophosphate to phosphorylate ETC, the ETC was purified with phenylsepharose following immunoaffinity chromatography. The occurrence of phosphoamino acids in the purified ETC was investigated by an immunoassay. No phosphoamino acids could be detected in the ETC molecule. Also after radiolabelling with 32P it was not possible to demonstrate a radioactive signal. The treatment with alkaline phosphatase has no influence on the mitogenicity or position of ETC in isoelectric focusing. The results obtained led to the conclusion that in contrast to the literature, ETC is not a phosphorylated protein.

Protein H, a molecule expressed at the surface of some strains of Streptococcus pyogenes, has affinity for the constant (IgGFc) region of immunoglobulin (Ig) G. In absorption experiments with human plasma, protein H-sepharose could absorb not only IgG but also albumin from plasma. The affinity constant for the reaction between albumin and protein H was 7.8 x 10(9) M-1, which is higher than the affinity between IgG and protein H (Ka = 1.6 x 10(9) M-1). Fragments of protein H were generated with deletion plasmids and polymerase chain reaction (PCR) technology. Using these fragments in various protein-protein interaction assays, the binding of albumin was mapped to three repeats (C1-C3) in the C-terminal half of protein H. On the albumin molecule, the binding site for protein H was found to overlap the site for protein G, another albumin- and IgGFc-binding bacterial surface protein. Also IgGFc-binding could be mapped with the protein H fragments and the region was found N-terminally of the C repeats. A synthetic peptide (25 amino acid residues long) based on a sequence in this region was shown to inhibit the binding of protein H to immobilized IgG or IgGFc. This sequence was not found in previously described IgGFc-binding proteins. However, two other cell surface proteins of S. pyogenes exhibited highly homologous regions. The results identify IgGFc- and albumin-binding regions of protein H and further define and emphasize the convergent evolution among bacterial surface proteins interacting with human plasma proteins.

It is well-established that three types of erythrogenic toxins (ETA, ETB, ETC) are produced by Streptococcus pyogenes (group A streptococci) strains. Culture filtrate concentrates from Streptococcus pyogenes strains T19P (T19, ETA+, ETB+, ETC-), 27337 (T12, B3264, ETA-, ETB+, ETC+), 27252 (T4, ETA-, ETB+, ETC+) and 27195 (T8, ETA-, ETB+, ETC-) were analyzed by preparative isoelectric focusing. These concentrates and the purified erythrogenic toxin type B (ETB) isolated by ion exchange chromatography had mitogenic and pyrogenic activity. Now, it has been found that the mitogenic activity and the pyrogenic activity of this ETB can be separated by preparative isoelectric focusing in Sephadex gels. This means that ETB is not a superantigen as described in literature. The mitogenic and biological activity is caused by traces of ETA (strain T19P), ETC (strains 27252 and 27337) and/or by unknown mitogen(s) (MX, strain 27195) which preferentially stimulate V beta 8+ T cells. The differentiation between ETA (stimulating V beta 12+ but not V beta 8+ or V beta 2+), ETC (stimulating V beta 2+ but not V beta 8+), and MX (stimulating V beta 8+) was done using established leukemic cell lines.

We investigated the ability of liposomes to deliver the immunosuppressive agent cyclosporine A (CsA) to the cornea, anterior sclera, aqueous humor, and vitreous in rabbit eyes. One drop (10 microliters) of liposome-encapsulated CsA (CsA-LIP) or olive oil drops containing an equivalent concentration of CsA (CsA-DR) were administered at 15-minute intervals within the first hour and then one hourly over a 6-hour period. In addition, collagen shields soaked for 30 minutes in the liposome preparation (CsA-LIP-CS) were tested in vitro and in vivo as a new drug delivery approach. CsA levels were measured by fluorescence-immunoassay after 1, 3, or 6 hours of drug administration. CsA levels in this study were highest in cornea and anterior sclera. In animals receiving either CsA-DR or CsA-LIP, CsA levels generally increased from 1 to 6 hours. In animals receiving a single application of CsA-CS-LIP, CsA levels peaked at 3 hours and declined at 6 hours in cornea and sclera. CsA-LIP and CsA-CS-LIP delivered significantly higher levels of CsA to the cornea and sclera at 1 and 3 hours than CsA-DR. In aqueous and vitreous humor, CsA levels increased from 1 to 6 hours in animals receiving either CsA-DR or CsA-LIP. On the other hand, animals receiving a single application of CsA-CS-LIP had lower levels of CsA at 6 hours than at the earlier time points. Animals receiving CsA-LIP or CsA-CS-LIP had significantly higher levels of CsA in aqueous and vitreous humor at 1, 3, and 6 hours than animals receiving CsA-DR.(ABSTRACT TRUNCATED AT 250 WORDS)

2-5A Synthetase and 2-5A phosphodiesterase were determined by analytical capillary isotachophoresis in comparison to radioenzymatic methods. By means of isotachophoretic analysis, a frequently used radioenzymatic 2-5A synthetase assay was optimized and the results of both assays were compared. Using the isotachophoretic assay the influence of interferon-related cytokines (tumor necrosis factor-alpha and interleukin-2) on 2-5A synthetase induction in neuroblastoma cells was estimated. In contrast to mononuclear blood cells, the tumor necrosis factor induced 2-5A synthetase in these cells. 2-5A Phosphodiesterase was determined using an isotachophoretic assay and a radioenzymatic method. Degradation of A2'p5'A2'p5'A (trimeric form of 2-5A core) was measured by isotachophoresis whereas degradation of a mixture of phosphorus-32 labeled 2-5A cores was registered by radioenzymatic assay. Activity of 2-5A phosphodiesterase was only insignificantly enhanced by interferon in mononuclear blood and neuroblastoma cells. In contrast to the radioenzymatic assays, an accurate determination of 2-5A synthetase as well as of 2-5A phosphodiesterase is possible using the isotachophoretic method because the reactions are followed by measuring the substrates ATP and A2'p5'A2'p5'A, respectively.

Binding of 125I-heparan sulphate to the cell surface of Streptococcus pyogenes is mediated by proteins, that could be released from the streptococcal cell wall by using alkaline buffer. SDS-electrophoresis revealed two bands with molecular weights of 63 and 58 kDa. Binding of the 125I-labelled heparan sulphate probe to streptococci seems to be due to charge interactions, as the same probe was displaced by unlabelled heparan sulphate, other negatively charged molecules such as heparin, dextran sulphate, dermatan sulphate or by high ionic strength. The interaction was also strongly influenced by pH. The binding constant at pH 7.2 was estimated to be 9.8 x 10(6) mol/l, suggesting a moderate affinity. The presence of collagen of different types enhanced binding of 125I-labelled heparan sulphate to streptococci, whereas fibronectin and vitronectin had an inhibitory effect. The cooperation between heparan sulphate and collagen could be important for the adhesion of streptococci to connective tissue.

One problem in investigating group A streptococcal infections and virulence is the lack of appropriate in vivo models. In this study we introduce the chicken embryo model for determining virulence of Streptococcus pyogenes. We found that M protein positive strains, if administered intravenously, were highly virulent for 12-day-old chicken embryos. The LD50 of the strains tested could be correlated directly with the amount of cell wall exposed M protein, which has been determined by the capacity of streptococci to bind fibrinogen and by the ability of streptococci to survive in fresh normal human blood. The number of colony forming units (cfu) of M+ strains necessary to kill 50% of embryonated eggs was significantly lower (< 10(2) cfu) than for M-variants (> 10(4) cfu). Albumin and/or IgG binding streptococcal cells, which can also take place in proteins of the M protein family which do not bind to fibrinogen, did not show that clear correlation to the virulence in chicken embryos that did fibrinogen binding. Application of anti-streptococcal M protein antisera from chicken and rabbit reduced the lethality of the chicken embryos. In contrast, no correlation was found between lethality of chicken embryos and the in vitro production of erythrogenic toxins by the administered strains. Thus the results indicate that the presence of M-protein with its fibrinogen binding activity on the streptococcal cell surface is necessary for virulence of group A streptococci in the chicken embryo model.

Determination methods for purine and pyrimidine bases, nucleosides, nucleotides and related compounds using analytical capillary isotachophoresis are reviewed. First, the isotachophoretic characterization of these compounds, as well as methods for sample preparation prior to analysis, and the different ways of detecting unknown substances in complex biological systems are described. Then applications of isotachophoretic analysis in medical diagnosis and biomedical research are reviewed. In particular, the analysis of purines, pyrimidines, nucleosides and related compounds in blood and serum for the diagnosis and treatment of inherited diseases and cancer is described. Selected applications of nucleotide analysis in biomedical research using different tissue extracts are also reviewed, and some examples of nucleotide-dependent enzymic reactions, which were performed by means of analytical isotachophoresis, are presented.

M proteins are major virulence factors of group A streptococci which enable the bacteria to resist phagocytic attack. Their binding capacity for different plasma proteins seems to be one reason for the antiphagocytic activity of M protein. In the present study we demonstrate that M3 protein, isolated from the streptococcal culture supernatant of strain 4/55, and the recombinant form (rM3), purified from an E. coli lysate after cloning in phage lambda-EMBL3, show a multiple binding to fibrinogen, albumin and fibronectin in Western blot and dot binding assays. Binding of M3 protein to the multifunctional extracellular matrix and plasma protein fibronectin may not only influence phagocytosis but may also contribute to the adherence of these bacteria to endothelial and epithelial cells.

Purpose: To study the effects of different formulations of topical cyclosporine (Cyclosporin A [CsA]) on corneal allograft rejection in a rat model., Methods: Female Lewis rats received penetrating keratoplasties from female Wistar-Furth donors. A total of 78 allogeneic grafts were performed. An additional 15 syngeneic grafts (Lewis) were used as technical controls. Two CsA preparations with equivalent drug concentrations (2.1 mg/ml) were applied as drops: CsA encapsulated in large unilamellar liposomes (CsA-LIP) and CsA dissolved in olive oil (CsA-DR). Allogeneic grafts were randomly assigned to receive CsA-LIP or CsA-DR beginning on the day of surgery five times daily for 10 days. Animals without any treatment or receiving empty liposomes (EM-LIP) were used as treatment controls. Grafts were graded three times weekly and a rejection index was generated based on graft clarity, neovascularization, and vessel size., Results: All syngeneic grafts remained clear over the observation period of 60 days. Rejected allogeneic grafts without any treatment and those receiving EM-LIP or CsA-DR showed a mean survival time (+/- standard deviation) of 14 +/- 4, 14 +/- 5, and 14 +/- 4 days, respectively. There was no significant difference in mean survival time between the grafts without any treatment and those in CsA-DR or EM-LIP treatment groups. The mean survival time of rejected grafts in animals receiving CsA-LIP was prolonged to 20 +/- 4 days. There was a significant difference in the mean survival time between the CsA-LIP treatment group and groups receiving CsA-DR, EM-LIP, or no treatment (P < or = 0.05). The Kaplan-Meier survival curve of the CsA-LIP treatment group was significantly different from the other experimental groups. The graft survival rate in the CsA-LIP group was 77%, whereas the rate was 37% in the non-treated group, 45% in the CsA-DR group, and 36% in the EM-LIP group., Conclusion: Encapsulation of CsA in liposomes might be a promising formulation for use in the prevention of corneal graft rejection.

Analytical capillary isotachophoresis was used to determine ascorbic acid (AA) in different matrices (cell-free system, neuroblastoma cell extracts and urine). The system for purging bone marrow of neuroblastoma cells, including 6-hydroxydopamine (6-OHDA) and AA, was analysed with regard to the interaction of AA with 6-OHDA and its autoxidation product, hydrogen peroxide. Furthermore, analyses concerning the uptake of AA into neuroblastoma cells as well as its excretion in urine after uptake of large amounts were carried out.

The effect of high frequent electromagnetic radiation pulse (PHEE) on the resorption of postoperative hematomas was investigated in 38 patients who underwent rhinoseptoplasty. 18 patients were treated twice a day using a diapulse equipment, whereas the patients in the control group did not get radiation. In the radiated patients, measurements of the infraorbital hematoma showed accelerated resorption-contrary to the course of the supraorbital hematoma. Diapulse radiation also prevented the enlargement of hematomas that usually proceeds until the third postoperative day. Results of undiminished resorption in the upper eyelid may be caused by inadequate penetration of PHEE or a methodical mistake in the analysis. Additionally, patients treated with PHEE needed a shorter course of postoperative antiphlogistic treatment when compared to the control group. The treatment might further be improved by preoperative use of PHEE or additional radiation of the liver, spleen, and adrenal gland. The results of our investigation showed the improvement of wound healing by using PHEE.

A purified bone-inducing protein complex (BIC), isolated from bovine bone and causing de novo bone formation in vivo, induces defined effects on rat mesenchymal cells in vitro. Spindle-like mesenchymal cells growing in monolayers change to polygonal cells, forming a multilayered growth pattern. The mesenchymal cells acquire alkaline phosphatase activity. Upon culture with BIC, the typical collagen Type III deposition of these mesenchymal cells is remarkably reduced whereas the collagen Type I expression remains unaffected. All these in vitro effects are consistent with the strong bone-forming capacity of BIC in vivo. A combination of two cytokines, transforming growth factor beta 1 (TGF beta 1) and epidermal growth factor (EGF), shows a similar activity to BIC. Neutralizing anti-TGF beta antibodies interfere with all in vitro effects of BIC. The neutralization of BIC and the inductive capacity of the combination of TGF beta 1 plus EGF point to the substantial role of TGF beta or TGF beta-like molecules in BIC; whether the active polypeptides are identical to TGF beta or somewhat structurally homologous to TGF beta remains to be elucidated.

The superantigenic properties of M protein type 5 of Streptococcus pyogenes have been implicated as an important pathogenicity factor in streptococcal autoimmune diseases. Here we show that after a single purification step by affinity chromatography on immobilized albumin or fibrinogen, M protein has no mitogenic activity for T cells. We demonstrate that the superantigenicity of M proteins of type 5 and type 1 is due to contamination with the highly potent pyrogenic exotoxins of S. pyogenes in the range of 0.1 to 0.01%. These results raise a general caveat for work with these extremely active T-cell mitogens, because the mitogenicity of other streptococcal or staphylococcal proteins could be due to similar minute contamination with potent superantigens that are undetectable by any biochemical method but extremely effective in stimulating sensitive T cells.

Binding of 125I-labelled fibronectin and vitronectin to streptococci of group A (S. pyogenes), group B (S. agalactiae) and group C (S. dysgalactiae and S. zooepidemicus) isolated from various human infections and bovine mastitis, and S. uberis bovine isolates, was studied. Binding of vitronectin and fibronectin was common among both human groups A and C, and bovine group C streptococci. S. agalactiae strains of human and bovine origin as well as S. uberis bovine isolates bound low levels of both proteins. The binding of radiolabelled fibronectin and vitronectin to selected groups A and C streptococcal strains was specific, time-dependent and occurred with both live and heat-killed (80 degrees C for 15 min) cells. Binding declined rapidly after treatment of cells with trypsin or proteinase K, while pepsin digestion at pH 5.5 affected vitronectin but not fibronectin binding.

The streptococcal streptokinase gene truncated at its 5' end was fused to regions of the staphylococcal protein A gene encoding the Fc-binding domains A and B. The resultant fusion gene, when expressed in the Escherichia coli lacPO system or under the speA expression/secretion signals in S. sanguis, specified a bifunctional hybrid protein, SPA-SKC, capable of Fc binding and plasminogen activation. When used in immunoassays designed to titrate antisera raised against bovine chymosin, human serum albumin and fibrinogen, the assay using SPA-SKC compared well with that using a commercial SPA-enzyme conjugate. The simple preparative method together with its efficacy and ease of use, make SPA-SKC a potentially valuable detector reagent in quantitative immunology.

Together with 2-5A synthetase and ribonuclease L, 2-5A phosphodiesterase belongs to the 2-5A system, which plays an important role in the action of interferon. Analytical capillary isotachophoresis was used for the determination of 2-5A phosphodiesterase activity. Enzyme assay was optimized using snake venom phosphodiesterase as a source of 2-5A phosphodiesterase activity. The 2-5A trimer core was used as a substrate. Enzyme activity was determined in time- and concentration-dependent reactions. In addition, 2-5A phosphodiesterase activity was determined in lysates of mononuclear blood cells.

The results of local application of monoclonal antibodies (mAb) in rabbit eyes are presented. To improve intraocular access of the high-molecular-weight protein it was entrapped in large (0.2 microns) unilamellar, negatively charged liposomes. Concentrations of the free or encapsulated drug were measured by ELISA in different eye compartments following repetitive drop administration or single subconjunctival injection. Although mAb became measurable in specimens of conjunctiva and cornea, it was not detectable (less than 0.5 ng/ml) in aqueous humor, lens or the vitreous body. In contrast, concentrations of the liposome-encapsulated drug were measurable as little as 30 min after topical application in the aqueous humor.

An extracellular protein (eMP) of Streptococcus pyogenes M type 1 was isolated by affinity chromatography on albumin- and IgG-Sepharose. The protein was found to bind to the human plasma proteins, fibrinogen, IgG, and albumin. Analysis of eMP by Western blotting demonstrated a major band with a molecular weight of 49 kD which was responsible for binding of the three plasma proteins. The purified protein was found to bind selectively to human and primate polyclonal IgG, human and mouse albumin, as well as human fibrinogen which has been the only fibrinogen tested. Serological investigations revealed a close relation of eMP to streptococcal M1 protein. It showed a reaction of identity with cell-extracted M1 protein in immunodiffusion. Moreover, the 49 kD peptide responsible for binding, was recognized with an antiserum directed against the 20 amino acids comprising synthetic peptide (42VAL-61GLU) of the N-terminal part of the M1 protein sequence. The affinity of M protein to plasma proteins other than fibrinogen opens new approaches to its purification by affinity chromatography.

Formation of new bone is suggested to be caused by interaction of a set of chemical factors with mesenchymal target cells. A specific assembly of factors, i.e. chemotaxis-, adhesion-, proliferation- and differentiation-factors, as well as macromolecular structure components, essential for formation of large amounts of ectopic bone was termed "osteopoetin" (OP). In the present study a partially purified osteopoetin containing bovine bone extract (OCBE) was used to induce ectopic bone formation. In order to reduce the amount of OCBE necessary for bone induction, OCBE was seeded onto different commercially available collagens or poly-L-lactic acids (PL). Solid collagens in a sponge-like form were used for the first time to function as an attachment system for an osteoinductive substance. The test substances were implanted into abdominal muscle pouches of male Wistar rats. After 21 days the implants were harvested and evaluated histologically. OCBE resulted in the formation of large ossicles containing hematopoietic bone marrow. The minimal amount of OCBE to elicit ectopic bone formation can be reduced by a factor of 10 when attached to quickly resorbable collagens, but not when attached to slowly resorbable PL. These results suggest that collagens are suitable OCBE attachment systems and useful for clinical application.

The viscosity of suspensions of hemoglobin-containing liposomes (Hb liposomes) and of liposomes without Hb was determined as a function of the shear rate. Liposomes of varying lipid composition were prepared by detergent dialysis. All liposome suspensions showed non-Newtonian viscous behavior, as did erythrocyte suspensions. The viscosity of suspensions of Hb liposomes with a bilayer consisting of egg yolk lecithin and phosphatidylserine was almost identical to erythrocyte suspension viscosity. When cholesterol was included in the lipid component of the Hb liposomes, the dependence of viscosity on the shear rate decreased (flattening of the curve), but overall suspension viscosity became higher than that of erythrocyte suspensions. Evidence is provided for the lipid concentration at which liposomes can be applied intravenously.

48 essential hypertensives were randomly assigned to (1) a behavioral treatment (n = 20), (2) a pharmacological treatment (n = 20), or (3) self recordings of BP (n = 8). Group 1 and 2 showed during 1 year of therapy comparable significant decreases of systolic and diastolic BP, whilst group 3 showed significant increases after 3 months of therapy.

Selected M-positive and M-negative group-A streptococcal strains were investigated with respect to their selective absorption of plasmin fibrinogen degradation products (FDP) in a simple batch technique. After incubation of killed streptococci with the FDP mixture, the centrifuged supernatants were investigated by SDS-electrophoresis and the binding capacity of the strains was calculated by evaluation of the scanning curves of stained gels. It was found that there is a specific uptake of the C-terminal fragment D by both M-positive and M-negative strains. Although the M-positive strains bound more fragment D (30%-80%) than did the M-negative strains (10%-15%), it could be clearly shown that the loss of M-protein was not necessarily linked with a total disappearance of fibrinogen binding activity. Fragment D blocked the agglutination of streptococci by fibrinogen. Washing the streptococci preincubated with FDP with a 0.1 M citric acid, 6 M urea buffer, pH 3.0, restored agglutination. Treatment of FDP-incubated bacteria with this buffer was found to be a means of recovering pure fragment D from streptococcal cells. It is suggested that M-positive and M-negative streptococci have qualitatively similar binding sites. These receptors might be reduced in the M-negative streptococci. Human serum albumin and Tween 20 did not influence the interaction between streptococcus and fibrinogen.

Cell surface hydrophobicity of group A, B, C, D and G streptococcal strains has been studied and compared in a new test based on the fact that the degree of bacterial aggregation in ammonium sulphate depends on amphiphilic surface antigens. M-positive group A strains showing good growth in normal human blood aggregated in the standard salt aggregation test at very low concentrations of ammonium sulphate, while M-negative strains, which were killed in normal human blood, usually aggregated at high salt concentrations. Agents such as 2 M-KSCN, 2 M-guanidine. HC1 or 2 M-urea decreased the aggregation of the M-positive strains in the salt aggregation test while non ionic detergents such as Tween 20 (1%, w/v) and ethylene glycol (2 M) did not affect cell aggregation. Binding of fibrinogen and albumin resulted in a decrease of surface hydrophobicity of the group A M-positive strains. Group B strains possess a hydrophilic surface character and did not aggregate, while group C and G strains behaved in the salt aggregation test like M-negative strains of group A streptococci. Group D strains did not aggregate even at high ammonium salt concentrations. The results are discussed in relation to the influence of lipoteichoic acid and other surface antigens on strains of the various groups, and it is suggested that M protein and possibly also other surface proteins contribute to the high surface hydrophobicity of group A strains.