Search

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection. © Copyright © 2020 Ruetten, Steinmetz, Thiersch, Kik, Vaughan, Altamura, Muckenthaler and Gassmann.

The BH3-only protein Bad is a proapoptotic Bcl-2 family member that acts as a sensitizer in intrinsic apoptosis by inactivating antiapoptotic members through heterodimer formation. Bad has been shown to contribute to tumorigenesis, including lymphoma formation in humans and mice, through alteration in expression or functional status. Here, its immunohistochemical expression was analyzed in canine nonneoplastic and lymphoma tissues using tissue microarrays. Bad was expressed in the cytoplasm of a wide range of nonneoplastic tissues, especially epithelial cells. Nonneoplastic lymph nodes displayed weak immunostaining in the follicular germinal centers only. Immunoblotting supported these observations but also revealed presence of nonspecific labeling in some organs. Of 81 lymphomas, 29 (35.8%) displayed moderate to strong immunohistochemical Bad labeling, and a significant expression increase was found in lymphomas (especially B cell and double negative) compared to nonneoplastic lymph nodes. These findings warrant further investigations of the functional status, the involvement of partner proteins, and a possible impact of Bad on prognosis in canine lymphoma.

The teaching of pathology within the veterinary medical curriculum extends through the entire training period and has a dual role. General pathology deals with principles of disease processes as a basis for understanding the reactions of a multi-cellular organism to adverse effects; organ pathology builds on the principles of general pathology and explains the malfunctions of individual organs. Pathology is heavily image dependent and best taught in a highly interactive manner. The Institute of Veterinary Pathology, University of Zurich (IVPZ), has been actively developing concepts for teaching pathology in the new veterinary curriculum, which demands more active participation from students, with 20% of their study time devoted to individual study using traditional materials (books, articles, etc.) and e-learning modules. The Swiss Virtual Animal Pathologist is designed to cover and support the central elements of the Veterinary Pathology curriculum of the Vetsuisse Faculty. The needs of students and staff of the participating institutions for an interactive learning platform to supplement the existing face-to-face lectures and tutorials are the highest priority of this initiative.

Bacteria belonging to the genus Cronobacter spp. have been recognized as causative agents of life-threatening systemic infections, primarily in premature, low-birth weight and/or immune-compromised neonates. Knowledge remains scarce regarding the underlying molecular mechanisms of disease development. In this study, we evaluated the use of a zebrafish model to study the pathogenesis of Cronobacter turicensis LMG 23827(T), a clinical isolate responsible for two fatal sepsis cases in neonates. Here, the microinjection of approximately 50 colony forming units (CFUs) into the yolk sac resulted in the rapid multiplication of bacteria and dissemination into the blood stream at 24 h post infection (hpi), followed by the development of a severe bacteremia and larval death within 3 days. In contrast, the innate immune response of the embryos was sufficiently developed to control infection after the intravenous injection of up to 10(4) CFUs of bacteria. Infection studies using an isogenic mutant devoid of surviving and replicating in human macrophages (ΔfkpA) showed that this strain was highly attenuated in its ability to kill the larvae. In addition, the suitability of the zebrafish model system to study the effectiveness of antibiotics to treat Cronobacter infections in zebrafish embryos was examined. Our data indicate that the zebrafish model represents an excellent vertebrate model to study virulence-related aspects of this opportunistic pathogen in vivo.

Superparamagnetic iron oxide nanoparticles (SPION) were coated with either Polyvinyl alcohol (PVA) or Vinyl alcohol/vinyl amine copolymer and further functionalized with the fluorochromes Cy3.5 or Texas Red. A colloidally stable suspension of nanoparticles was incubated on sheep synovial cells in vitro for 3, 24, 72, and 120 hours. Nanoparticle internalization into synoviocytes as well as biocompatibility was visualized using light, fluorescence and confocal microscopy and fluorochrome labeled cells were quantified by flow cytometry. Data were analyzed by ANOVA factorial tests. Amino-PVA-SPION alone was detectable in cytoplasmic endosome-like structures after 3 hours of incubation but resulted in early cell death after 24 hours. Although amino-PVA-Cy3.5- SPION and PVA-TexasRed-SPION were taken up more slowly and less intensely, both labeled more than 80% of the cells in culture, but did not significantly change cell morphology or vitality at any time of evaluation in comparison to control cells. Results indicate that functionalized amino PVA- coated SPION are biocompatible, were successfully internalized by synoviocytes and hold promise for future biomedical applications utilizing magnetic drug targeting in joint disease.

Characteristic features of the persistent chlamydial developmental cycle, associated with chronic infections in both humans and animals, include the generation of non-replicative, morphologically aberrant bodies which are distinct from normal propagating reticulate bodies. Previous studies have correlated these morphological and metabolic changes with differential expression of diverse functional subsets of chlamydial genes. To further investigate these correlations, we compared mRNA expression of predicted chlamydial signal transduction genes between normal Chlamydophila pneumoniae A-03 infections in HEp-2 cells and those treated with gamma interferon (IFN-gamma) by using real-time RT-PCR. Inspection of the Cp. pneumoniae genome revealed at least 39 candidate signal transduction genes, of which 30 were differentially expressed in Cp. pneumoniae mediated persistence. Functional sub-groups of differentially expressed signal transduction genes include chlamydial GTPases (hflX, ychF, yhbZ and yphC), linked to bacterial cellular processes such as cell cycle control and ribosome assembly and stability. Other up-regulated signal transduction genes sharing similarity to bacterial stress response genes (htrA, surE, lytB and hrcA) were also detected. The transcriptional changes observed for the majority of signal transduction genes appear to be unique for Cp. pneumoniae, as similar changes were not observed in recent whole genomic analysis of C. trachomatis IFN-gamma mediated persistence. These results suggest that chlamydial signal transduction genes play potentially important roles in the establishment and maintenance of Cp. pneumoniae persistence, likely as part of the IFN-gamma response stimulon as described for C. trachomatis, but with considerable differences in the transcriptional profile.

Objective—To detect and characterize the full range of chlamydial infections in cats with ocular disease by use of polymerase chain reaction (PCR) assays, cytologic examination, immunohistochemical analysis, and evaluation of clinical information including status for feline herpesvirus-1 (FeHV-1). Sample Population—DNA extracted from 226 conjunctival samples obtained from cats with clinically diagnosed keratitis or conjunctivitis and 30 conjunctival samples from healthy cats. Procedure—PCR assays for the 16S rRNA gene specific for the order Chlamydiales and a new Chlamydophila felis (formerly Chlamydia psittaci) species-specific 23S rRNA gene were performed. Seventy-four conjunctival samples were prepared with Romanowsky-type stain, grouped on the basis of inflammatory pattern, and screened for chlamydial inclusions by use of immunohistochemical analysis. Clinical information and FeHV-1 status were recorded. Results—26 (12%) specimens had positive results for the only known feline chlamydial pathogen, C felis. Surprisingly, an additional 88 (39%) were positive for non-C felis chlamydial DNA. Identification of non- C felis chlamydial DNA by direct sequencing revealed 16S rRNA gene sequences that were 99% homologous to the sequence for Neochlamydia hartmannellae, an amebic endosymbiont. Chlamydial prevalence was significantly higher in cats with ocular disease. Conclusions and Clinical Relevance—Application of a broad-range detection method resulted in identification of a new agent associated with ocular disease in cats. Finding chlamydia-like agents such as N hartmannellae in coinfections with their obligate amebic host, Hartmannella vermiformis, raises questions about the potential role of these microorganisms in causation or exacerbation of ocular disease in cats. (Am J Vet Res 2003;64:1421–1428)

Descriptions for tenascin-C distribution are largely restricted to epithelial tumours. The present study utilized newly developed and characterized monoclonal (hT191) and polyclonal antibodies to investigate the distribution pattern of tenascin-C in a panel of mesenchymal tumours, which was contrasted with normal tissue. The specific antibodies recognized the distinctive star-like hexabrachion protein isolated from transformed cell-culture medium and serum from normal individuals. In normal tissues, a strong tenascin-C expression in the extracellular matrix was largely restricted to basement-membrane regions of epithelium and tonsilar sinusoids, pericellularly within smooth-muscle bundles, associated with perimysial, -chondrial, -neurial and -tendon surfaces, and diffusely within vascular adventitia. It was found in the corresponding tumours of the neural sheath (schwannoma) and smooth muscle (leiomyosarcoma), and was abundantly present around certain blood vessels of mesenchymal tumours. Although not detected in normal muscle, or in adipose or fibrous connective tissue, neo-expression of tenascin-C was shown in more than half of the rhabdomyosarcomas, fibromas and liposarcomas, with an increased positive percentage in variably malignant myxoid liposarcomas compared with lipoma-like sarcomas. Tenascin-C was typically found in the extracellular matrix of soft-tissue tumours, but was notably absent from the epithelial-cell components of mixed epithelial/mesenchymal tumours. Its apparently enhanced expression in soft-tissue tumours differs from that of most other large extracellular-matrix proteins, suggesting possible functional involvement of the cell-adhesion molecule, tenascin-C, in the neoplastic phenotype. Int. J. Cancer 72:217–224, 1997. © 1997 Wiley-Liss, Inc.

Chlamydia are obligate intracellular bacteria and important pathogens of humans and animals. Chlamydia-related bacteria are also major fish pathogens, infecting epithelial cells of the gills and skin to cause the disease epitheliocystis. Given the wide distribution, ancient origins and spectacular diversity of bony fishes, this group offers a rich resource for the identification and isolation of novel Chlamydia. The broad-nosed pipefish (Syngnathus typhle) is a widely distributed and genetically diverse temperate fish species, susceptible to epitheliocystis across much of its range. We describe here a new bacterial species, Candidatus Syngnamydia venezia; epitheliocystis agent of S. typhle and close relative to other chlamydial pathogens which are known to infect diverse hosts ranging from invertebrates to humans. ISSN:1932-6203

A particularly intriguing role in communication within the nervous system is played by glycosyl phosphatidylinositol (GPI)-anchored glycoproteins. Although our understanding of how neuronal GPI-linked molecules transduce signals is still in its infancy, it is now possible to assemble a tentative model to describe their functioning. Shared features of these otherwise diverse receptors will be outlined, with particular emphasis given to the exclusively GPI-anchored contactin/F11 subgroup of the immunoglobulin superfamily. Contactin/F11 itself, with five extracellular ligands and an intracellular pathway involving Fyn, serves to illustrate the wide range of signalling possible through this group of receptors.

The neural immunoglobulin-like cell adhesion molecule contactin/F11 and the extracellular matrix glycoprotein tenascin-C are prominent molecules in the developing nervous system which interact in in vitro assays (Zisch et al., J. Cell Biol. 119, 203-213). To determine their potential role in neural development, the distribution of tenascin-C and contactin/F11 was examined in the developing chick retina. The onset of both tenascin-C and contactin/F11 expression coincides with the appearance of ganglion cell dendrides and neurites from bipolar and amacrine cells in the inner layer (IPL) at E8, and the extension of bipolar and horizontal cell processes in the outer plexiform layer (OPL) at E9. Contactin/F11 expression is co-ordinately upregulated with the TN190 and TN200 tenascin-C isoforms between embryonic day 8 (E8) and E17, while little, if any, of the TN220 isoform, which does not bind contactin/F11, is detected. In situ hybridization reveals that tenascin-C and contactin/F11 mRNAs are synthesized by different neuronal types. Tenascin-C mRNA probes hybridize to amacrine and displaced amacrine neurons, and horizontal neurons. In cultured retinal cells, tenascin-C is also present on process-bearing neurofilament-positive cells. Contactin/F11 mRNA is detected in bipolar cells or their precursors from E8-9, and later in horizontal and ganglion neurons. The highest levels and greatest overlap in the synaptic IPL and OPL are reached at E17, when the stratification of the retina is nearly complete. These results are consistent with a putative role for contactin/F11-tenascin-C interactions in the establishment of synaptic layers in the retina.

Only limited information is available on the presence of Chlamydiaceae in wildlife, a deficit that is particularly acute concerning mammalian wildlife in Africa. In a retrospective analysis of organ material from an earlier study on wild mammals from the Seregenti National Park, 521 samples from 54 animals of 14 mammalian species were investigated. The presence of Chlamydiaceae was analyzed using molecular methods and immunohistochemistry. Chlamydial DNA was detected by real-time polymerase chain reaction in formalin-fixed and paraffin-embedded tissues from large ruminants (African buffaloes, Syncerus caffer, n=4) and a large predator (spotted hyena, Crocuta crocuta, n=1). Microarray results revealed Chlamydia abortus in all cases, confirmed by sequencing of selected samples, and a mixed infection with Chlamydia abortus and Chlamydia pneumoniae in an African buffalo. This is the first report of Chlamydiaceae in African wildlife of the Serengeti area.

The stringent stress response is vital for bacterial survival under adverse environmental conditions. Obligate intracellular Chlamydia lack key stringent response proteins, but nevertheless can interrupt the cell cycle and enter stasis or persistence upon amino acid starvation. A possible key protein retained is YhbZ, a homologue of the ObgE guanosine triphosphatase (GTPase) superfamily connecting the stringent stress response to ribosome maturation. Curiously, chlamydial YhbZ lacks the ObgE C-terminal domain thought to be essential for binding the large ribosomal subunit. We expressed recombinant Chlamydia abortus YhbZ and showed it to be a functional GTPase, with similar activity to other Obg GTPase family members. As Chlamydia are resistant to genetic manipulation, we performed heterologous expression and gradient centrifugation experiments in Escherichia coli and found that, despite the missing C-terminal domain, C. abortus YhbZ co-fractionates with the E. coli 50S large ribosomal subunit. In addition, overexpression of chlamydial YhbZ in E. coli leads to growth defects and elongation, as reported for other Obg members. YhbZ did not complement an E. coli obgE temperature-sensitive mutant, indicating the C-terminal acidic domain may have an additional role. This data supports a role for YhbZ linking the chlamydial stress response to ribosome function and cellular growth.

Growing evidence suggests that a novel member of the Chlamydiales order, Waddlia chondrophila, is a potential agent of miscarriage in humans and abortion in ruminants. Due to the lack of genetic tools to manipulate chlamydia, genomic analysis is proving to be the most incisive tool in stimulating investigations into the biology of these obligate intracellular bacteria. 454/Roche and Solexa/Illumina technologies were thus used to sequence and assemble de novo the full genome of the first representative of the Waddliaceae family, W. chondrophila. The bacteria possesses a 2'116'312 bp chromosome and a 15'593 bp low-copy number plasmid that might integrate into the bacterial chromosome. The Waddlia genome displays numerous repeated sequences indicating different genome dynamics from classical chlamydia which almost completely lack repetitive elements. Moreover, W. chondrophila exhibits many virulence factors also present in classical chlamydia, including a functional type III secretion system, but also a large complement of specific factors for resistance to host or environmental stresses. Large families of outer membrane proteins were identified indicating that these highly immunogenic proteins are not Chlamydiaceae specific and might have been present in their last common ancestor. Enhanced metabolic capability for the synthesis of nucleotides, amino acids, lipids and other co-factors suggests that the common ancestor of the modern Chlamydiales may have been less dependent on their eukaryotic host. The fine-detailed analysis of biosynthetic pathways brings us closer to possibly developing a synthetic medium to grow W. chondrophila, a critical step in the development of genetic tools. As a whole, the availability of the W. chondrophila genome opens new possibilities in Chlamydiales research, providing new insights into the evolution of members of the order Chlamydiales and the biology of the Waddliaceae.

Ocular infections by chlamydiae are associated with ocular disease manifestations such as conjunctivitis and keratitis in humans and animals. Limited evidence exists that members of the order Chlamydiales can also cause ocular disease in sheep. In the current study, the prevalence of chlamydiae in the eyes of sheep was investigated by using PCR methods. Data obtained in sheep by broad-range 16S rRNA order Chlamydiales-specific PCR were compared to the prevalence of antibodies against chlamydiae detected by a competitive enzyme-linked immunosorbent assay (cELISA). Flocks tested included a clinically healthy flock and two flocks suffering from ocular disease and with histories of Ovine Enzootic Abortion (OEA). PCR detected DNA of Chlamydophila (Cp.) abortus and Cp. pecorum in the eyes of both healthy and sick animals but also identified Chlamydia (C.) suis and a variety of uncultured chlamydia-like organisms. Good correlation was found between the presence of Cp. abortus DNA in sheep conjunctival samples and seropositivity detected by cELISA. Despite these findings, no association was found between the presence of chlamydial DNA in the sheep conjunctival samples and the onset of clinical disease. These results suggest that the biodiversity of chlamydiae in the eyes of sheep is greater than that previously thought. Further investigations are needed to determine whether a causal relationship between infection by chlamydiae and ocular disease exists in these animals.

*This article is free to read on the publisher's website* Predicted members of the HflX subfamily of phosphate-binding-loop guanosine triphosphatases (GTPases) are widely distributed in the bacterial kingdom but remain virtually uncharacterized. In an attempt to understand mechanisms used for regulation of growth and development in the chlamydiae, obligate intracellular and developmentally complex bacteria, we have begun investigations into chlamydial GTPases; we report here what appears to be the first analysis of a HflX family GTPase using a predicted homologue from Chlamydophila pneumoniae. In agreement with phylogenetic predictions for members of this GTPase family, purified recombinant Cp. pneumoniae HflX was specific for guanine nucleotides and exhibited a slow intrinsic GTPase activity when incubated with [gamma-(32)P]GTP. Using HflX-specific monoclonal antibodies, HflX could be detected by Western blotting and high-resolution confocal microscopy throughout the vegetative growth cycle of Cp. pneumoniae and, at early time points, appeared to partly localize to the membrane. Ectopic expression of Cp. pneumoniae HflX in Escherichia coli revealed co-sedimentation of HflX with the E. coli 50S large ribosomal subunit. The results of this work open up some intriguing possibilities for the role of GTPases belonging to this previously uncharacterized family of bacterial GTPases. Ribosome association is a feature shared by other important conserved GTPase families and more detailed investigations will be required to delineate the role of HflX in bacterial ribosome function.

A total of 210 frog samples originating either from a mass mortality (1991/1992) or from routine postmortem investigations of the years 1990 to 2004 were examined retrospectively for a possible involvement of Chlamydiae. For a prevalence study of Chlamydia in a selected Swiss amphibian population, 403 samples from free-ranging Rana temporaria were examined. Histopathology, immunohistochemistry using a monoclonal antibody against chlamydial lipopolysaccharide, and a 16S rRNA polymerase chain reaction (PCR) followed by DNA sequencing were performed on the formalin-fixed and paraffin-embedded tissues. Using PCR, 8 of 54 (14.8%) frog samples from the mass mortality (1991/1992) were positive for Chlamydia suis S45. A control group of healthy Xenopus laevis had 3 of 38 positive samples, sequenced as C suis S45 (2/3) and an endosymbiont of Acanthamoeba species UWE1 (1/ 3). Chlamydophila pneumoniae TW-183 was detected from exotic frogs kept in a zoo. Of the frogs collected for the prevalence study, 6 of 238 (2.5%) tested positive, 1 each for C suis S45, Cp pneumoniae TW-183, and uncultured Chlamydiales CRG22, and the remaining 3 revealed Chlamydophila abortus S26/3. In immunohistochemistry, there were 2 positive labeling reactions, 1 in intestine and the other in the epithelium coating the body cavity, both testing positive for Cp pneumoniae TW-183 in PCR. Histologically there were no lesions recorded being characteristic for Chlamydia. Although there is a prevalence of Chlamydia in Swiss frogs, no connection to a mass mortality (1991/1992) could be established. For the first time, C suis S45 and Cp abortus S26/3 were detected in frog material.

A retrospective study on reptile tissues presenting with granulomatous inflammation was performed to detect the possible presence of mycobacteria and chlamydiae in these lesions. Ninety cases including 48 snakes, 27 chelonians, and 15 lizards were selected. Mycobacteria were detected by Ziehl-Neelsen (ZN) staining and a broad-range polymerase chain reaction (PCR) followed by DNA sequencing. To detect chlamydiae, immunohistochemistry with monoclonal antibodies against chlamydial lipopolysaccharide (LPS) and a Chlamydiales order-specific PCR and sequencing were applied. Acid-fast bacilli were found in 14 cases (15.6%) by ZN staining and in 23 cases (25.6%) by PCR. Sequence analysis revealed the presence of Mycobacteria other than Mycobacterium tuberculosis complex (MOTT). Chlamydial LPS antigen was observed within granulomas from five samples (5.6%), whereas the PCR screen revealed 58 positive cases (64.4%). Of these, 9 cases (10%) showed 98–99% similarity to Chlamydophila ( Cp.) pneumoniae and 49 cases (54.4%) displayed a high similarity (88–97%) to the newly described “Chlamydia-like” microorganisms Parachlamydia acanthamoebae and Simkania negevensis. Results from this study confirm, on the one hand, that MOTT are probably the most important infectious etiology for granulomatous inflammation in reptiles. On the other hand, they indicate that chlamydia infects reptiles and that Cp. pneumoniae should be considered an etiological agent of granulomatous lesions of reptiles. Because both MOTT and Cp. pneumoniae are human pathogens, the potential of zoonotic transmission from reptiles to humans has to be considered. In contrast, the significance of Chlamydia-like isolates remains completely open, and further studies are needed to evaluate their role.

Chondroitin sulfate proteoglycans have been implicated in the regulation of cell migration and pattern formation in the developing peripheral nervous system. To identify whether the large aggregating proteoglycan versican might be mediating these processes, we prepared monospecific antibodies against a recombinant core protein fragment of chick versican. The purified antibodies recognize the pre-dominant versican splice-variants V0 and V1. Using these antibodies, we revealed a close correlation between the spacio-temporal expression of versican and the formation of molecular boundaries flanking or transiently blocking the migration pathways of neural crest cells or motor and sensory axons. Versican is present in the caudal sclerotome, the early dorsolateral tissue underneath the ectoderm, the pelvic girdle precursor and to a certain extent in the perinotochordal mesenchyme. Versican is completely absent from tissues invaded by neural crest cells and extending axons. Upon completion of neural crest cell migration and axon outgrowth, versican expression is shifted to pre-chondrogenic areas. Since versican inhibits cellular interactions with fibronectin, laminin and collagen I in vitro, the selective expression of versican within barrier tissues may be linked to a functional role of versican in the guidance of migratory neural crest cells and outgrowing axons.

The three isoforms of chicken tenascin, an extracellular matrix glycoprotein, are generated by alternatively spliced fibronectin type III domains. The resulting proteins migrate as bands of MT 220000 (ten220), Mr 200000 (ten200) and Mr 190000 (tenl90) on SDS-PAGE. We describe here two monoclonal antibodies, one specific for ten220 (mAb T17) and another that recognizes all isoforms (mAb T16). These were used to examine the differential expression of isoforms during development. Most impressive is the close correlation between ten220 expression and cell migration in the embryonic cornea. Initially (stage 18), tenl90/200 can be detected within the corneal epithelium and along the basement membranes of the lens and sclera. Ten220 appears within the primary stroma immediately prior to the invasion by neural-crest-derived cells. This expression is maintained during the subsequent migration of fibro blasts from the conjunctiva into the primary stroma. With the completion of migration and the marked increase in matrix synthesis by corneal fibroblasts, ten220 disappears. Tenl90/200 remains in the region adjoining the endothelium, the Bowman’s membrane and the adjacent stroma. The cell-migration-associated isoform is isolated from extracts of embryonic tissues as a homohexamer. Low molecular weight forms appeared absent but a new tenascin band of Mr 210 000 could be detected in brain extracts which may be a new isoform. We conclude that the synthesis of tenascin isoforms is under tight developmental control and speculate that a function of the additional domains is to facilitate cell migration.

Human α-1-antitrypsin (AT) was reacted with hydroxylamine at pH 9.0 giving cleavage at an Asn-Gly bond. A fragment of molecular weight 8,500 was released and this was isolated and sequenced. The fragment had the same carboxy terminal amino acid sequence as intact AT. The 80 residue polypeptide contained the Z variant mutation site and a portion of sequence identical to that found by others for the reactive site, inferring the presence in AT of two active sites. This sequence combined with prviously published work gives a continuous sequence of 152 amino acid residues from the carboxy terminal end of the AT molecule, including the mutation site of the S variant. The sequence shows strong homology with human antithrombin III.

α1-Antitrypsin has a microheterogeneity evident on isoelectric focusing as three major and several minor bands. We have identified the carbohydrate structures of the major bands; band 6 (isoform I) has three bi-antennary sidechains, band 4 (isoform II) has two bi- and one tri-antennary and band 2 (isoform III) has one bi- and two tri-antennary sidechains. The identity of the isoforms with the bands permitted their measurement in plasma by photometric scanning of the electrofocused gels. In healthy controls the levels of isoforms I, II and III were relatively constant and in the proportions of 5, 4 and 1, respectively. A marked change occured during inflammation and oestrogen stress with isoforms II and III accounting for most of the increase in α1-antitrypsin. One possible consequence of the changed proportions was shown to be the increased catabolism of the partia!!y desialylated tri-antennary isoforms compared to that of the predominant bi-antennary form of the healthy individual.

Type IX collagen was isolated as a native protein from chicken embryo sternal cartilages and purified to homogeneity. Chondroitin and/or dermatan sulfate were bound covalently to one of the three polypeptide chains present in this protein containing collagenous and noncollagenous domains. Type IX collagen could be metabolically labeled with both radioactive sulfate and glycine. The protein containing either of these labels was sensitive to digestion by bacterial collagenase as well as chondroitinase ABC. Besides the glycosaminoglycans, type IX collagen contains asparagine-linked carbohydrate chains because the protein could be labeled with radioactive mannose and no glycosaminoglycans other than those mentioned above were present. The melting curve indicated that, in contrast to interstitial collagens, this molecule contains at least two disulfide-bonded collagenous domains with distinct thermal stabilities.

It has recently become apparent that collagen fibrils may be composed of more than one kind of macromolecule. To explore this possibility, we developed a procedure to purify fibril fragments from 17-d embryonic chicken sternal cartilage. The fibril population obtained shows, after negative staining, a uniformity in the banding pattern and diameter similar to the fibrils in situ. Pepsin digestion of this fibril preparation releases collagen types II, IX, and XI in the proportion of 8:1:1. Rotary shadowing of the fibrils reveals a d-periodic distribution of 35-40-nm long projections, each capped with a globular domain, which resemble in form and dimensions the aminoterminal globular and collagenous domains, NC4 and COL3, of type IX collagen. The monoclonal antibody (4D6) specific for an epitope close to the amino terminal of the COL3 domain of type IX collagen bound to these projections, thus confirming their identity. Type IX collagen is therefore distributed in a regular d-periodic arrangement along cartilage fibrils, with the chondroitin sulfate chain of type IX collagen in intimate contact with the fibril.

The structural basis for the interaction between tenascin-C and the neuronal cell adhesion molecule, contactin/F11, was investigated using plasmon surface resonance technology. The binding site on tenascin-C for contactin/F11 is shown to span the two fibronectin type III homology domains 5 and 6. Either domain alone is insufficient for binding. Heparin, heparan sulfate and dermatan sulfate inhibit this interaction through binding to a conserved heparin-binding site on domain 5. In contrast, chondroitin sulfates A and C have no such effect.

The distribution of collagen XI in fibril fragments from 17-d chick embryo sternal cartilage was determined by immunoelectron microscopy using specific polyclonal antibodies. The protein was distributed throughout the fibril fragments but was antigenically masked due to the tight packing of collagen molecules and could be identified only at sites where the fibril structure was partially disrupted. Collagens II and IX were also distributed uniformly along fibrils but, in contrast to collagen XI, were accessible to the antibodies in intact fibrils. Therefore, cartilage fibrils are heterotypically assembled from collagens II, IX, and XI. This implies that collagen XI is an integral component of the cartilage fibrillar network and homogeneously distributed throughout the tissue. This was confirmed by immunofluorescence.

In this paper we present strong evidence that the aberrations in keratoconus corneas are not directly related to alterations in collagen composition and distribution. This conclusion is based on comparative studies of collagen types I, III, IV, V and the recently described collagen types VI and VII in keratoconus and normal corneas. The data are derived from biochemical analysis of collagen fractions sequentially extracted with pepsin and sodium-dodecylsulphate, from amino acid analysis of hydrolysates of entire corneal tissues as well as from immunoblotting of the extracted collagens with specific antibodies. These antibodies were also used to examine the distribution of the collagens in immunofluorescence experiments on corneal sections. The yields of the collagen extractions were demonstrated to be age dependent but were not altered in keratoconus samples. Apart from one case associated with osteogenesis imperfecta type I, comparative studies of keratoconus and normal corneas showed no differences in collagen composition of the extracts. This was confirmed by amino acid analysis of tissue-hydrolysates. The distributions of collagen types I, III, IV, V, VI and VII in corneal sections were in general unchanged in keratoconus corneas, the only differences being in scar tissues observed in the Bowman layer of some keratoconus samples.

This study evaluated in vivo gene delivery and subsequent gene expression within cells of the synovium in the presence of static and pulsating magnetic field application following intraarticular injection of superparamagnetic iron oxide nanoparticles linked to plasmids containing reporter genes encoding for fluorescent proteins. Plasmids encoding genes for either green fluorescent protein or red fluorescent protein were bound to superparamagnetic nanoparticles coated with polyethyleneimine. Larger (200–250 nm) and smaller (50 nm) nanoparticles were compared to evaluate the effects of size on transfection efficiency as well as any associated intraarticular reaction. Comparisons between groups were evaluated at 24, 72, and 120 h time periods. Inflammatory response was mild to moderate for all injected particles, but was present in the majority of synovial membrane samples evaluated. Larger particles tended to be associated with more inflammation than smaller ones. Nevertheless, intraarticular application of both experimental and control nanoparticles were well tolerated clinically. Gene expression as determined by observation of either green or red intracellular fluorescence was difficult to assess by both epifluorescent light, and confocal microscopy. An insufficient concentration of nanoparticles in relation to joint volume likely resulted in a limited number of samples with positive evidence of iron staining and with suspected positive evidence of cells expressing fluorescent proteins. Our results indicate that intraarticular administration of functionalized superparamagnetic iron oxide nanoparticles resulted in a mild to moderate synovitis and there was in conclusive evidence of gene expression. Further research is warranted to determine the best and most effective reporter assay for assessment of the in vivo gene delivery into the joints. In addition, the best suited concentration and size of nanoparticles, which will optimize gene delivery and expression, while minimizing intraarticular inflammation, needs to be determined.