Degenerative Osteoarthropathy in Laboratory Housed Xenopus (Silurana) tropicalis

In this case study, 15 adult laboratory Xenopus (Silurana) tropicalis (7 adult males and 8 adult females) were examined for nodular enlargements of the clawed digits (digits 0, I, II, and III) on the hind feet. Radiographs showed smoothly margined, rounded, peripherally mineralized lesions arising from the distal phalanges of digits 0-III with osteoproductive and osteolytic components in all frogs. Micro computed tomography (microCT) scans further revealed interphalangeal (IP), metacarpophalangeal (MCP), and metatarsophalangeal (MTP) joint osteoarthritis characterized by periarticular new bone formation, rounded mineral foci both peripherally and centrally within the joints, and more rarely, linear mineralization palmar/plantar to the joints in the flexor tendons. In the nonclawed digits, the shape of the distal phalanx was variably distorted and both subluxation and malangulation of IP joints were identified. Histologically, nodules corresponded to a peripheral rim of mature cortical bone surrounding central adipose tissue, scattered hematopoietic elements, and residual bone of the distal phalanx. Occasionally, the peripheral rim of cortical bone extended proximally to encompass the distal aspect of adjacent phalanx. MCP, MTP and IP joint spaces of most digits exhibited widespread osteoarthritis characterized by periarticular cartilaginous or osseous metaplasia, bony remodeling, and less frequently, granulomatous osteomyelitis. Nutritional analyses of the feed did not indicate imbalances nor were the lesions consistent with metabolic bone disease. The exact etiopathogenesis of these lesions is unknown; however, we hypothesize that the osteoarthritic changes are due to a combination of the frogs’ mature age, the unique structure of the Xenopus spp. claw, genetics and biomechanical forces on the digits and distal phalanges of the hind feet.

Xenopus spp. are a well-established model in biomedical research and are commonly used in the fields of embryology and vertebrate developmental biology, endocrinology, toxicology, cancer, and genomics.^{1-3,5,10,11,13,16,17,19,20,22} In research laboratory environments, X. tropicalis are typically housed in either static or recirculating aquatic systems in water maintained at 25 to 28 °C, with a pH between 6.8 and 8.5, and a hardness between 100 and 300 mg/L CaCo₃.⁷ Housing tanks range in size from 1 L or more, depending on the needs of the laboratory and the type and configuration of the housing system. Stocking density ranges from approximately 1-5 adult frogs/L per tank.⁷ Laboratory X. tropicalis are commonly fed commercially available complete and balanced pelleted diets 3 times per week, or as appropriate for their different life stages.^7,18,21 With stringent husbandry and disease surveillance protocols in place, laboratory-reared and housed Xenopus spp. generally remain healthy well through adulthood and their prime egg-producing years and can live 10 y or longer in captivity.⁷To date, most reports describing disease in laboratory or wild Xenopus spp. are attributed to infectious agents (bacterial, viral, or parasitic) and/or conditions related to water quality aberrations or pollutants.⁷ Naturally occurring bone diseases due to nutritional appear to be rare in laboratory Xenopus spp., although they have been described in other frogs and amphibians.^6,12,23 Reports of other spontaneous skeletal diseases in laboratory Xenopus are particularly rare. One group²⁵ recently described axial skeletal deformities in genetically engineered and wild type laboratory Xenopus spp. Here we report spontaneously occurring appendicular skeletal degenerative osteoarthropathy with hindlimb distal phalangeal nodules (digits 0-III) in mature, laboratory X. tropicalis, as confirmed by radiography, computed tomography and histopathology.     

Influence of thrombin on proliferation and prostaglandin production in cultured bovine endometrial cells

The control of cell proliferation by thrombin was studied in vitro in cultured epithelial and stromal cells of the endometrium. The effect of thrombin was studied after chronic treatment (72 hr) in medium containing 10% fetal bovine serum (FBS) combined or not with sex steroids. Thrombin inhibited slightly the proliferation (based on DNA measurements) only in epithelial cells (P < 0.05). 17β-estradiol (E) and progesterone (P4) had no mitogenic effects. The presence of functional thrombin receptors was estimated by stimulation of second messenger generation in response to increasing doses of thrombin (0-1,500 ng/ml). In confluent cultures of epithelial cells, the addition of thrombin for 10 min stimulated cAMP production by 50% with a maximal response at 500 ng/ml (P < 0.05). Similarly, in stromal cells, thrombin stimulated cAMP production in a dose-dependent manner (P < 0.01). Generation of inositol-phosphates was also stimulated by 50% in epithelial cells (P < 0.03), with a maximal response at 500 ng/ml, and by 45% in stromal cells (P < 0.01), with a maximal response at 50 ng/ml. The effect of thrombin on cell proliferation was investigated by ³H-thymidine incorporation in serum-free medium for 24 hr. Thrombin inhibited incorporation in epithelial cells (P < 0.0001) in a dose-dependent manner. Conversely, thrombin stimulated significantly incorporation of stromal cells (P < 0.05) at 50 ng/ml. The effect of sex steroids was also evaluated and it was found that E had no effect on cell proliferation, while P4 inhibited the incorporation in both epithelial (P < 0.001) and stromal cells (P < 0.001). The effect of a combined treatment with thrombin and E inhibited both epithelial (P < 0.001) and stromal cell (P < 0.001) growth, but a combination of thrombin and P4 had no additional effect on growth compared to P4 alone. Further investigation of the role of thrombin has been carried out by measuring prostaglandin (PG) responses. Addition of thrombin for 24 hr inhibited PGF_2α production by epithelial cells (P < 0.0001) but had no effect on PGE₂ production by stromal cells. Therefore, functional receptors for thrombin appear to be present in epithelial and stromal cells of the bovine endometrium. The minimal effect of thrombin alone or in combination with sex steroids on endometrial cell proliferation in vitro combined with the evidence of functional thrombin receptor in these cells, suggest that: (1) the effect of sex steroids in cultured endometrial cells is not modulated by the presence of thrombin, and (2) other factors are necessary for the full expression of mitogenic responses to sex steroids in vitro. © 1996 Wiley-Liss, Inc.     

Incorporation of Wild-Type and C-Terminally Truncated Human Epidermal Growth Factor Receptor into Human Immunodeficiency Virus-Like Particles: Insight into the Processes Governing Glycoprotein Incorporation into Retroviral Particles

Previous results have indicated that incorporation of surface glycoprotein into retroviral particles is not a specific process and that many heterologous viral and cellular glycoproteins can be incorporated as long as they do not have long cytoplasmic C-terminal regions which were presumed to be sterically inhibitory. In this study, this concept has been directly examined by analyzing the incorporation of the wild-type human epidermal growth factor receptor (Wt-EGFR) and of a C-terminally truncated mutant of Wt-EGFR (Tr-EGFR) into human immunodeficiency virus (HIV)-like particles. Incorporation was directly analyzed at the protein level and by immunogold labelling of enriched HIV-like particles. In agreement with the above concept, Tr-EGFR, with only 7 C-terminal amino acids (aa), was efficiently incorporated into HIV-like particles. Incorporation of the Wt-EGFR species, with 542 C-terminal cytoplasmic aa, was reduced by a factor of about 5 in comparison to that of the Tr-EGFR species. However, the Wt-EGFR species was still very significantly present in the HIV-like particles. A series of control experiments verified that this represents genuine incorporation of Wt-EGFR into the membrane of HIV-like particles. These observations allow further speculation as to the processes governing glycoprotein incorporation into retroviral particles and indicate that the internal virus structure of HIV (in particular the matrix layer [MA]) can accommodate much larger heterologous cytoplasmic domains in incorporated glycoproteins than previously assumed.     

The protective effect of breast feeding in relation to sudden infant death syndrome (SIDS): I. The effect of human milk and infant formula preparations on binding of toxigenic Staphylococcus aureus to epithelial cells

Epidemiological studies indicate that breast-fed infants are at a decreased risk of sudden infant death syndrome (SIDS) compared to formula-fed infants. Increasing evidence suggests that infectious agents might be involved in some of these deaths, in particular bacteria which colonise mucosal surfaces and produce superantigenic toxins. One species implicated in recent studies of SIDS infants is Staphylococcus aureus. We tested the hypothesis that in comparison to infant formula, human milk might be a better inhibitor of binding of S. aureus to epithelial cells. In this study, two protocols were used for the binding assays which were assessed by flow cytometry: the in vitro method in which bacteria were treated with milk or formula, washed and added to epithelial cells; and a method more closely reflecting the competitive interactions in vivo in which cells, bacteria, and milk or infant formula were added at the same time. With the in vivo method, breast milk caused enhancement of bacterial binding to cells whilst infant formula caused inhibition; however, for the in vitro method, both human milk and infant formula caused consistent enhancement of binding. Flow cytometry and light microscopy studies indicated that the enhancement was due to the formation of bacterial aggregates. Human milk and infant formula preparations were also compared for components (antibodies or oligosaccharides) that could inhibit binding of S. aureus using the in vitro method. Human milk contained both IgA and IgG. Neither human milk nor infant formula contained oligosaccharides reactive with the Ulex europaeus lectin but both contained components that bound monoclonal antibodies to Lewis(a) and Lewis(b) antigens which can act as receptors for S. aureus. With both methods, synthetic Lewis(a) and Lewis(b) inhibited S. aureus binding in a dose-dependent manner. With human milk, however, the only component which showed a significant correlation with inhibition of binding was the IgA specific for the staphylococcal surface component that binds Lewis(a). Both human milk and infant formula contain components which could potentially inhibit bacterial binding but only breast milk contains the IgA specific for the bacterial adhesin that binds Lewis(a). Studies using the in vivo method suggest that protection associated with breast feeding in relation to SIDS could be due mainly to the formation of bacterial aggregates. The studies have implications for further research into constituents of infant formula.     

The protective effect of breast feeding in relation to sudden infant death syndrome (SIDS): II. The effect of human milk and infant formula preparations on binding of Clostridium perfringens to epithelial cells

Breast feeding is known to protect an infant against gastrointestinal pathogens and epidemiological studies indicate that compared to breast fed infants, formula fed infants are at a greater risk of dying from sudden infant death syndrome (SIDS). Many SIDS infants have symptoms of gastrointestinal infections prior to death and one gastrointestinal pathogen associated with SIDS is Clostridium perfringens. Studies have found that a significantly higher number of formula fed SIDS infants have C perfringens and its enterotoxin in their faeces compared to breast fed infants. The aim of the study was to compare the effects of human milk and infant formula on binding of C perfringens to epithelial cells. Two protocols were used to assess the effect of human milk and infant formula to inhibit binding of C perfringens to epithelial cells. Binding was assessed by flow cytometry. For the in vivo protocol which more closely represents interactions on the mucosal surface, breast milk enhanced bacterial binding but infant formula caused inhibition of binding; however for the in vitro method, both human milk and infant formula resulted in consistent enhancement of binding. Flow cytometry studies indicated that enhancement of binding was due to the formation of bacterial aggregates. Lewis(a) and Lewis(b) antigens, found in both breast milk and infant formula, inhibited C. perfringens binding in a dose dependent manner. The Lewis(a) and Lewis(b) antigens in human milk and infant formula can inhibit C. perfringens binding to epithelial cells. While infant formula reduced binding of C. perfringens to epithelial cells in the experiments carried out with the in vivo protocol, the protective effects of breast feeding in relation to colonisation with C. perfringens are more likely to be due to formation of bacterial aggregates. These findings have implications for improving infant formula preparations.     

The protective effect of breast feeding in relation to sudden infant death syndrome (SIDS): III. Detection of IgA antibodies in human milk that bind to bacterial toxins implicated in SIDS

Two toxin-producing bacteria implicated in sudden infant death syndrome (SIDS) are Staphylococcus aureus and Clostridium perfringens. Epidemiological studies have shown that breast feeding reduces an infant's risk of SIDS. This protective effect could be due partly to IgA antibodies to these toxins in human milk. The aim of this work was to use a quantitative ELISA to determine levels of IgA antibodies that bound to toxic shock syndrome toxin (TSST-1), staphylococcal enterotoxin C (SEC) and C. perfringens enterotoxin A (CEA) in individual samples of human milk. All samples of milk tested contained IgA antibodies that bound to the bacterial toxins. For individual samples, IgA bound to TSST-1, SEC and CEA were in the range of 900-3100 ng ml(-1), 1000-3600 ng ml(-1) and 1000-4300 ng ml(-1) respectively. Isolation of S. aureus from mothers donating breast milk samples was used to determine if the presence of bacteria affected IgA levels which bound TSST-1 and SEC. For 3/5 samples with levels above the upper limit of the standard deviation (2375 ng ml(-1)) for IgA bound to TSST-1, S. aureus was isolated from the mother whilst 4/5 samples found to contain levels above the upper limit of the standard deviation (2627 ng ml(-1)) for IgA bound to SEC, had S. aureus isolated from the mother. In conclusion, if bacterial toxins do play a role in precipitating a SIDS death, the presence of IgA antibodies to toxins in breast milk, but not in infant formula, might contribute to the protective effect of breast feeding in relation to SIDS.     

Small genes/gene-products in Escherichia coli K-12

Forty-two protein spots of observed M_r 6–15 kDa were resolved by two-dimensional gel electrophoresis, stained by Coomassie blue and subjected to Edman microsequencing. All of the proteins could be related back to their encoding open reading frames, thereby vindicating the bioinformatic tools currently utilised in their identification. However, only 14/42 gene-products were expressed as annotated. Translation was confirmed for 14 open reading frames with no attributed function (EcoGene Y-entries), while N-terminal sequence allowed the start codon to be accurately annotated for the genes yjgF, yccU, yqiC, ynfD, and yeeX. The methionine start codon was cleaved in 11 gene-products (AtpE, Hns, RpoZ, RplL, CspC, YccJ, YggX, YjgF, HimA, InfA, RpsQ) and a further five showed loss of a signal peptide (PspE, HdeB, HdeA, YnfD, YkfE). Internal (Tig, AtpA, TufA) and N-terminal fragmentation (CspD, RpsF, AtcU) of much larger proteins was also detected, which may have resulted from physiological or translational processes. M_r and pI isoforms were detected respectively for PtsH and GatB, each being phosphoproteins, as well as RplY which manifested differences with respect to predicted M_r and pI. In addition, YjgF was shown to belong to a small gene family of unknown function with ancient conserved regions across procaryotes and eucaryotes. YgiN was revealed to have a paralogue and orthologues in Bacillus subtilis, Synechocystis sp., Mycobacterium tuberculosis, Neisseria gonorrhoea, and Rhodococcus erythropolis. Orthologues are also reported for YihD, YccU and YeeX. Of the 14 Y-genes, only YkfE possessed no detectable orthologues. These results highlight the need to complement genomic analysis with detailed proteomics in order to gain a better understanding of cellular molecular biology, while the confirmation of the open reading frame start codon using Edman degradation protein microsequencing has yet to be superseded by recent advances in mass spectrometry.     

ApoA-I mimetics reduce systemic and gut inflammation in chronic treated HIV

Novel therapeutic strategies are needed to attenuate increased systemic and gut inflammation that contribute to morbidity and mortality in chronic HIV infection despite potent antiretroviral therapy (ART). The goal of this study is to use preclinical models of chronic treated HIV to determine whether the antioxidant and anti-inflammatory apoA-I mimetic peptides 6F and 4F attenuate systemic and gut inflammation in chronic HIV. We used two humanized murine models of HIV infection and gut explants from 10 uninfected and 10 HIV infected persons on potent ART, to determine the in vivo and ex vivo impact of apoA-I mimetics on systemic and intestinal inflammation in HIV. When compared to HIV infected humanized mice treated with ART alone, mice on oral apoA-I mimetic peptide 6F with ART had consistently reduced plasma and gut tissue cytokines (TNF-α, IL-6) and chemokines (CX3CL1) that are products of ADAM17 sheddase activity. Oral 6F attenuated gut protein levels of ADAM17 that were increased in HIV-1 infected mice on potent ART compared to uninfected mice. Adding oxidized lipoproteins and endotoxin (LPS) ex vivo to gut explants from HIV infected persons increased levels of ADAM17 in myeloid and intestinal cells, which increased TNF-α and CX3CL1. Both 4F and 6F attenuated these changes. Our preclinical data suggest that apoA-I mimetic peptides provide a novel therapeutic strategy that can target increased protein levels of ADAM17 and its sheddase activity that contribute to intestinal and systemic inflammation in treated HIV. The large repertoire of inflammatory mediators involved in ADAM17 sheddase activity places it as a pivotal orchestrator of several inflammatory pathways associated with morbidity in chronic treated HIV that make it an attractive therapeutic target.