Toward understanding the role of cartilage particulates in synovial inflammation.

OBJECTIVE: Arthroscopy with lavage and synovectomy can remove tissue debris from the joint space and the synovial lining to provide pain relief to patients with osteoarthritis (OA). Here, we developed an in vitro model to study the interaction of cartilage wear particles with fibroblast-like synoviocytes (FLS) to better understand the interplay of cartilage particulates with cytokines on cells of the synovium. METHOD: In this study sub-10 µm cartilage particles or 1 µm latex particles were co-cultured with FLS ±10 ng/mL interleukin-1α (IL-1α) or tumor necrosis factor-α (TNF-α). Samples were analyzed for DNA, glycosaminoglycan (GAG), and collagen, and media samples were analyzed for media GAG, nitric oxide (NO) and prostaglandin-E2 (PGE2). The nature of the physical interaction between the particles and FLS was determined by microscopy. RESULTS: Both latex and cartilage particles could be phagocytosed by FLS. Cartilage particles were internalized and attached to the surface of both dense monolayers and individual cells. Co-culture of FLS with cartilage particulates resulted in a significant increase in cell sheet DNA and collagen content as well as NO and PGE2 synthesis compared to control and latex treated groups. CONCLUSION: The proliferative response of FLS to cartilage wear particles resulted in an overall increase in extracellular matrix (ECM) content, analogous to the thickening of the synovial lining observed in OA patients. Understanding how cartilage particles interface with the synovium may provide insight into how this interaction contributes to OA progression and may guide the role of lavage and synovectomy for degenerative disease.

m-Iodobenzoic acid complexes with selected metals: molecular structure and antimicrobial activity.

Complexes of lithium, sodium, potassium, rubidium, cesium, magnesium, calcium, manganese, and zinc with m-iodobenzoic acid were studied. The FT-IR and FT-Raman spectra of the mentioned compounds in the solid state and water solutions were recorded and analyzed. Principal component analysis (PCA) was performed on the wavenumbers of selected bands (eight bands) occurring in the vibrational spectra. The numbers obtained as a result of this procedure characterize the electronic properties of the molecule of each complex. The antimicrobial activity of the studied compounds against selected bacteria (Escherichia coli and Bacillus subtilis) and yeast (Saccharomyces cerevisiae and Hansenula anomala) was estimated. The relationship between the chemical properties (as characterized by PCA of the IR spectra) and antimicrobial properties of the compounds was examined, and a good correlation between the two factors was found.

[Evaluation of surfactant administration in neonates with respiratory distress syndrome. III. The role of intra-uterine stimulation on lung maturity]. / Ocena leczenia surfaktantem noworodków z zespolem zaburzen oddychania. III. Rola wewnatrzmacicznej stymulacji dojrzalosci pluc.

Prenatal corticosteroids and exogenous surfactant therapy independently reduce the adds of neonatal death by about 40%. It is not clear however if babies who develop RDS despite prenatal corticosteroid therapy behave differently when later given exogenous surfactant. This paper presents an evaluation of the outcome of babies treated with Curosurf (a porcine surfactant) for RDS depending upon whether they received prenatal corticosteroids (16 babies) or not (11 babies). Although not randomised these two groups of babies appear to be similar in respect of gestational age. However, lower mortality was found in the group treated with prenatal corticosteroids (12.5% vs. 27.2%).

Induction of anti-human immunodeficiency virus (HIV) neutralizing antibodies in rabbits immunized with recombinant HIV--hepatitis B surface antigen particles.

Fragments of the human immunodeficiency virus (HIV) envelope coding region have been fused with the hepatitis B virus envelope middle protein. In this system, HIV antigenic determinants are exposed at the surface of a highly antigenic structure, the hepatitis B surface antigen particle. Immunization of rabbits with these particles elicited antibodies directed against both parts of the hybrid protein. One of the rabbit antisera not only exhibited a neutralizing effect on the original HIV1 isolate but also on a divergent Zairian isolate. The HIV sequence in this recombinant is 84 amino acids long and contains conserved and variable domains and a region critical for interaction with the CD4 receptor. Such recombinant antigens could be primary elements in the design of a polyvalent vaccine.

Presentation of two epitopes of the preS2 region of hepatitis B virus on live recombinant bacteria.

Having developed a genetic procedure to expose a foreign epitope at the surface of Escherichia coli by using the outer membrane LamB protein as a carrier, we apply this procedure to express two distinct portions of the preS2 region of hepatitis B virus: region A, residues 132-145, and region B, residues 153-171. The resulting hybrid proteins (LamB-preS2 A and LamB-preS2 B) were normally expressed, stable, and still kept most biologic functions of LamB. The corresponding bacterial strains were used directly as immunogens in rabbits and mice. Both viral sequences were found to be immunogenic in the two animal species. With LamB-preS2 A, antibodies induced were able to react with the viral particles and the immobilized peptide. With LamB-preS2 B, the antibodies raised were not able to recognize the immobilized peptide. However, the results suggest that the B epitope, inserted in LamB, was at least as efficient as the corresponding synthetic peptide in raising antiviral antibodies. Thus, epitope presentation with LamB may present advantages for immunization. We also have shown that peptide A is an essential part of the polymerized human serum albumin receptor. These results, which validate further the LamB vector system for epitope presentation, provide information on the two hepatitis B regions expressed.

Monoclonal antibody recognizing pre-S(2) epitope of hepatitis B virus: characterization of pre-S(2) epitope and anti-pre-S(2) antibody.

A hybrid cell line producing monoclonal antibodies recognizing an epitope encoded by the pre-(S)2 region of hepatitis B virus (HBV) genome was obtained by fusion of mouse myeloma cells with lymphocytes from mice immunized with HBV. The monoclonal antibody Mo-F124 secreted from the hybrid line reacted with the pre-S(2) epitope expressed on the surface of both viral and recombinant HBsAg particles--pre-S(2) and S gene product--localised on 34 kD glycoprotein of the viral envelope. The pre-S(2) epitope was sensitive to digestion with V8 protease from Staphylococcus aureus. The enzyme abolished reactivity with Mo-F124 and polymerized human serum albumin (pHSA) binding activity of recombinant particles. Mo-F124 antibody was used to develop highly sensitive radioimmunoassays for determination of pre-S(2) epitope and anti-pre-S(2) antibody in sera of hepatitis B patients. Detection of a pre-S(2) epitope by the monoclonal antibody-based assay in the early phase of acute HBV infection correlated well with the presence of markers of active viral replication (HBeAg, HBV DNA). The appearance of anti-pre-S(2) antibody, usually in the third month after onset of symptoms, was followed by elimination of circulating HBsAg and seroconversion to anti-HBs in all tested cases of uncomplicated acute hepatitis followed by recovery. Anti-pre-S(2) response was not observed in patients with chronic hepatitis B or acute HBV infection progressing to chronic disease. The observed correlation of anti-pre-S(2) response with recovery suggests that the pre-S(2) epitope may represent one of the epitopes inducing antibodies that neutralize the hepatitis B virus.

Expression of the hepatitis B virus large envelope protein in Saccharomyces cerevisiae.

The yeast vector pPV2 has been constructed for inducible expression of non-fused proteins from the PHO5 promoter. Signals of the URA3 gene are used for transcription termination. The 226-amino-acid 'major' and the 389-amino-acid 'large' envelope protein of hepatitis B virus (HBV) have been produced in Saccharomyces cerevisiae following insertion of the S gene or of the entire pre-S region and the S gene, respectively, of HBV into pPV2. Although normally only a minor constituent of the viral envelope, the 'large' protein forms particles with cellular lipids similar to those composed of the 'major' envelope protein. Such particles carry pre-S1, pre-S2, and S-encoded epitopes and, in addition, a receptor for polymerized human serum albumin.

Synthesis in animal cells of hepatitis B surface antigen particles carrying a receptor for polymerized human serum albumin.

A recombinant plasmid (pSVS dhfr) encoding the pre-S region and the S gene of human hepatitis B virus (HBV) and murine dihydrofolate reductase (DHFR) cDNA has been used for the transfection of Chinese hamster ovary (CHO) DHFR- cells. Selection of clones resistant to methotrexate has permitted amplification of HBV sequences and an increase in production of hepatitis B surface antigen (HBsAg). HBV-specific transcripts have been characterized. The HBsAg 22-nm particles contain a receptor for polymerized human serum albumin (pHSA) and elicit in animals the synthesis of antireceptor antibodies. This property is ascribed to a 34,000-dalton polypeptide in the particles, which is most likely encoded by the S gene and part of the pre-S region. Especially because the pHSA receptor is most abundantly present on the virion and because, in hepatitis B infection, the appearance of anti-pHSA receptor antibodies seems to be a highly reliable criterion for viral clearance, the HBsAg particles obtained may constitute a particularly efficient vaccine.

A transformed Vero cell line stably producing the hepatitis B virus surface antigen.

We have constructed plasmids in which transcription of the surface antigen gene of the human hepatitis B virus (HBsAg) is under the control of the SV40 early promoter. These plasmids have been used to analyze the expression of the HBsAg gene, both in mouse cells and in African green monkey kidney (Vero) cells. We have established a Vero cell line continuously expressing HBsAg that is indistinguishable from authentic 22 nm HBsAg particles. Post-transcriptional modification of HBsAg mRNA also appears to occur normally in the monkey cells. These cells might be useful as a source of antigen for the preparation of an antihepatitis vaccine.

[Cellular structure of propionibacteria during their multiplication]. / Untersuchung der Zellstruktur von Propionsäurebakterien während ihrer Vermehrung.

The aim of the present study was to determine the structure of bacterial cells from Propionibacterium genus as well as their structure during the cellular division. On the basis of the observations made in the electron transmission microscope, in uranyl-acetates-tained preparations of ultra-thin specimens of bacteria, it was stated that propionic bacteria appeared in a shape of short rods, possessing regular profiles of cell walls as opposed to Gram-negative bacteria with a very creased edge line. Besides, it was observed that division of cells had place by formation of septum, most probably preceded by the division of mezosome, which is a signal for creating the divisional wall. In the conducted studies, the following phenomena were started: presence of membraneous structure of mezosomes, which is linked with the chain of circular DNA in bacterial cell, appearance of numerous ribosomes in the regions of tangled threads of nucleic acids, and existence of other undefinite elements. Mezosome present in the cell of propionic bacteria is probably linked with the cell wall at least in two places and on the surface of external cell wall at the site of its linking; it causes the change in electronic density, demonstrated by the undefined holes or scars in cell wall. This finding gives the possibility of distinguishing this genus of Propionibacterium, in the respect of morphology, from other bacteria what, in the opinion of the authors, is a new achievement in the studies on the structure of propionic bacteria.

[Morphology of bacteria belonging to Propionibacterium genus in scanning and transmission electron microscopy]. / Morphologie von Bakterien der Gattung Propionibacterium im Scanning- und Transmissionselektronenmikroskop.

Bacteria belonging to Propionibacterium genus reveal a characteristic shape of short rods. Their length is from 0.8 micrometer to 1.5 micrometer and their width reaches from 0.3 micrometer to 0.5 micrometer. Most bacteria of this genus have a constant ratio of cell length and their diameter is 2: 1. Their surface is smooth and has no cilia. Certain species of this genus, grown on liquid media, demonstrate the capacity of developing involution forms and of linking the cells into greater complexes which may appear in a form of flocculus. The characteristic feature, distinguishing this genus of bacteria from other ones, is that on their surface, the undefined scars and not-stated till now, structural formations were visible.

Proteoglycan populations of baboon (Papio papio) cartilages from different anatomical sites: gel electrophoretic analysis of dissociated proteoglycans and of fractions obtained by density gradient centrifugation.

Proteoglycans extracted by 4 M guanidinium chloride from different cartilages and vertebral discs of young baboons (Papio papio) were purified by ion-exchange chromatography and assessed by gel electrophoresis. Proteoglycans fractionated by equilibrium density gradient centrifugation under 'dissociative' conditions were similarly purified and assessed. (1) Non-fractionated proteoglycans from articular, nasal, laryngeal, tracheal, costal, vertebral plate and ear cartilage and from anulus fibrosus gave three metachromatic bands on gel electrophoresis: two broad, closely running bands (I and II) and a faster, more discrete band (III). The migrations rates of corresponding bands of various cartilages were similar, with minor differences in staining intensity. Gel electrophoresis of material fractionated on the gradient indicated the presence of bands with the same migration and appearance as those found for the unfractionated proteoglycans and also partial separation of bands (buoyant density I greater than II greater than III). (2) Non-fractionated proteoglycans from growth cartillage yielded bands I and II only. Small amounts of material corresponding to band III were detected in the upper fractions of the gradients. (3) Proteoglycans from nucleus pulposus migrated as two diffuse bands corresponding to bands I and II. The two bands were partially separated in the gradients and an additional very slow band was formed in the middle fractions of the gradients. (4) Proteoglycans from knee menisci contained a densely stained band of corresponding mobility to band III. Faint bands corresponding to band I and II were also detected. The bands were partially separated in the gradients.

Proteoglycan populations of baboon (Papio papio) articular cartilage.

1. Gel electrophoresis of proteoglycans extracted with use of 4 M-guanidinium chloride from baboon (Papio papio) articular cartilage and purified on DEAE-cellulose in 8 M-urea yielded three bands on electrophoresis in polyacrylamide/agarose gels: two wide bands close together (I and II) and a third, thinner and more rapidly moving band (III). 2. Gel electrophoresis of fractions from direct 'dissociative' gradients showed that these bands were partially separated (buoyant density of I greater than II greater than III). 3. Reduction and alkylation of proteoglycans did not alter either the gel-electrophoretic pattern or the distribution of the bands in the fractions of the gradient. 4. Band III was found in the upper third of 'associative' gradients but not in the bottom fraction, which yielded after dissociation only bands I and II. 5. The third band was completely extracted for 24h with an iso-osmotic solution, but was contaminated with bands I and II. The second extraction step with 4M-guanidinium chloride yielded only bands I and II. 6. The data strongly suggest the presence in the articular cartilage of several populations of dissociated proteoglycans differing in gel-electrophoretic migration, buoyant density and aggregation capacity.

Age-dependent change in the gel-electrophoretic pattern of proteoglycans of human growth cartilage.

The gel-electrophoretic pattern of dissociated proteoglycans was studied in 7 fetuses, 5 premature newborns, 4 term newborns, 5 infants and 5 children. The tibial growth cartilage was extracted with 4 M guanidinium chloride. After dialysis against 8 M urea at pH 7 the proteoglycans were obtained by ion chromatography in urea on DEAE cellulose and submitted to gel electrophoresis on polyacrylamide agarose gels. Gel electrophoresis of proteoglycans showed a different pattern in fetuses from that found in children. The change occurs in the first months of extrauterine life.