cell culture systems from molluscs have significantly contributed to our basic understanding of complex physiological processes occurring within or between tissue-specific cells, yielding information ...unattainable using intact animal models.
cultures of neuronal cells from gastropods show how simplified cell models can inform our understanding of complex networks in intact organisms. Primary cell cultures from marine and freshwater bivalve and gastropod species are used as biomonitors for environmental contaminants, as models for gene transfer technologies, and for studies of innate immunity and neoplastic disease. Despite efforts to isolate proliferative cell lines from molluscs, the snail
Say, 1818 embryonic (Bge) cell line is the only existing cell line originating from any molluscan species. Taking an organ systems approach, this review summarizes efforts to establish molluscan cell cultures and describes the varied applications of primary cell cultures in research. Because of the unique status of the Bge cell line, an account is presented of the establishment of this cell line, and of how these cells have contributed to our understanding of snail host-parasite interactions. Finally, we detail the difficulties commonly encountered in efforts to establish cell lines from molluscs and discuss how these difficulties might be overcome.
ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 ...activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.
Inflammatory stimuli elicit liver synthesis and subsequent release into the plasma of several proteins (positive acute phase proteins, APP) with functions in innate immunity, tissue repair and ...restoration of homeostasis. To expand the basis for evaluating the degree of conservation of the APR in vertebrates and to assess the extent to which genes encoding both cellular and plasma proteins are affected, we profiled transcriptional changes in livers of individual rainbow trout (
Oncorhynchus mykiss) after intraperitoneal injection of
Listonella (
Vibrio)
anguillarum bacterin in Freund's Incomplete Adjuvant. Twenty genes were down-regulated, some unexpectedly such as complement component 3 and α2-macroglobulin. Sixteen up-regulated genes included three encoding proteins involved in iron metabolism (hepcidin, haptoglobin, and intelectin), from which we infer that sequestration of iron is likely to be a major component of the trout APR. Activated genes encoding proteins of unknown functions included precerebellin-like plasma protein, and differentially regulated trout protein which is predicted to be cell surface associated. The only complement component that increased was C7. Genes encoding proteins that are probably not released into plasma included two fatty acid binding proteins, two transport proteins (SEC61 and a Na–Ca exchanger), GAPDH, an amino transferase, and a hydrolase. When microarray data and quantitative RT-PCR analyses were used to evaluate specific transcripts, variations were notable between individual fish, possibly a basis for natural variation in susceptibility to infectious diseases. This study suggests novel hypotheses relating to NFκB, albumin-related protein, pentraxin, hypoferremia and the complement cascade. While the capacity to mount an APR is conserved throughout vertebrate evolution, the responding genes vary from species to species, and considerable variation is observed from individual to individual within a species.
To develop tools for analysis of the acute phase response, we used suppression subtractive hybridization of cDNAs from the livers of trout in an unchallenged state and in the course of a response to ...injection with a
Vibrio bacterin emulsified in Freund's Incomplete Adjuvant. The resulting cDNA library contains 300–600
bp long fragments of 25 or more immune-relevant genes. Fifteen were previously unreported for salmonids, and 12 were not known from any fish species. Known acute phase proteins include serum amyloid A, transferrin and precerebellin-like protein; trout C-polysaccharide-binding protein 1 is probably also an acute phase protein. Components of both the complement system (
n=5) and the clotting system (
n=3), as well as lectins, various binding proteins, a putative antibacterial peptide, a chemotaxin, an anti-oxidant enzyme, as well as some likely cell-surface receptors and metabolic and lysosomal enzymes are represented in the library. One clone closely resembles a group of Toll-like receptors, including the human IL-1 receptor. Three cDNAs appear to represent complete open reading frames.
Undescended testis (UDT) is one of the most common congenital disorders and is associated with infertility and testicular cancer. Multiple guidelines internationally have recommended orchiopexy by 18 ...months. Multiple large retrospective studies published in the last decade have found persistent delay in timing of orchiopexy.
The aim of the study was to determine timing at which UDTs are referred at the tertiary pediatric hospital and assess factors that are associated with delay in UDT referral.
Based on clinical observations and previous data, a series of clinical and socio-economic variables were constructed to design a prospective database. All patients who underwent orchiopexy for UDT from March 1, 2017, to August 31, 2018, were reviewed for demographic and clinical data. Referral appointments after 18 months were considered delayed. Factors associated with delay in UDT referral were analyzed using univariate and multivariate analysis with logistic regression.
One hundred seventy-eight patients underwent orchiopexy for UDT. The median age was 44 months, and 64% of them had delay in referral. On univariate analysis, normal birth testicular examination, diagnosis of ‘retractile testicle,’ long gap without seeing pediatrician, diagnosis by a new physician, and primary language non-English were associated with delayed UDT referral. On multivariate analysis, delayed referral was associated with normal testicular examination at birth, history of ‘retractile testis,’ diagnosis not by the regular primary care provider, and other health or social issues that may have led to delay.
This is the first prospective study analyzing timing of referral for boys with cryptorchidism. It was found that timing of treatment of UDT with orchiopexy has not improved over the last decade. Major causes in delay in referral may be due to poor of education of families and lack of routine testicular examinations by referring providers. Secondary ascent may account a significant number of delayed orchiopexy cases.
Most patients at Doernbecher had delayed referral of cryptorchidism. Factors associated with delay were determined. To improve treatment of cryptorchidism, quality-based interventions and the importance of education and routine testicular examinations need to be focused on. Display omitted
The fight-or-flight response prepares an animal for coping with alarming situations and their potential consequences, which include injury. The possible involvement of innate components of immunity ...in the response has received little attention. We determined plasma concentrations of stress hormones and lysozyme activity before and after a 10 min handling stressor. Rainbow trout (
Oncorhynchus mykiss) were anesthetized in their home tanks, bled, revived, and then stressed by being held in the air in a net for 30 s and placed in a shallow bucket of water for 10 min. Fish were then captured, concussed (in one of two experiments) and bled again. Control fish were also bled twice, but were kept anesthetized in their holding tanks between bleedings. Following the stressor, plasma cortisol, adrenaline and lysozyme activity were significantly increased. The experiment was repeated 4 months later with a similar outcome. While chronic stress is eventually immunosuppressive, acute stress/ trauma may help enhance both cellular and humoral components of innate defenses at times of likely need.
The Gram-negative bacterium Pasteurella multocida causes pneumonic and systemic pasteurellosis in bovids for which vaccines are either unavailable or inadequate. The work assessed whether an ...intranasal P. multocida challenge in mice might provide a model of infection for future vaccine development work. Clinical, pathological and biochemical responses were compared in seven strains of mice challenged with a virulent bovine pneumonic isolate of P. multocida A:3. Six mouse strains (Porton, CD-1, BALB/c, VM, C57BL/10 and C57BL/6) developed clinical signs of pneumonic disease and variable pneumonic lesions 41–70h post-infection. In contrast, mouse strain RIII became septicaemic within 36h post-infection. Concentrations of plasma acute phase proteins and serum lipopolysaccharide increased in all mice after infection, and the main or interaction effect of mouse strain and infection status was statistically significant (P<0.05). Responses in C57BL/10 mice showed close similarity to bovine pneumonic and in RIII mice to bovine systemic pasteurellosis.
The time of onset and subsequent degree and progression of clinical signs, bacterial colonization and tissue pathology during experimental disease induced by intratracheal inoculation of either a UK ...or USA isolate of Pasteurella multocida serotype A recovered from clinical cases of bovine pneumonia were determined. Calves aged 8 weeks were challenged with 300 ml phosphate buffered saline (PBS) alone (group 1, n = 3, negative control) or containing 7.1 × 108 colony forming units (cfu) of UK isolate (group 2, n = 8) or 5.8 × 108 cfu of USA isolate (group 3, n = 8). Bronchoalveolar lavage (BAL) at 0, 1 and 4 days post challenge (dpc) and at the time of necropsy examination (7–8 dpc) showed no significant differences between groups 2 and 3 in bacterial numbers recovered. No P. multocida were recovered from group 1 animals. No clinical disease was present in group 1 calves and in group 3 was limited to scour in 1 calf at 1 dpc. All calves in group 2 had reduced food intake at 4–5 dpc, five had periods of dullness, three a mild nasal discharge at 1 dpc, four had mild to substantial respiratory stridor and one was killed at 6 dpc for humane reasons. Rectal temperatures remained about 39°C in group 1 calves, but increased in P. multocida-challenged calves to 40–41°C within 8–12 h of challenge. Significantly (P = 0.01) greater percentages of lung surface area were consolidated in group 2 (mean ± SD, 21 ± 10.1) compared with group 3 (7 ± 8.6) calves. Significantly more extensive and severe histological lesions were present in the lung lobes (P = 0.006) and lymph nodes (P = 0.02) of group 2 compared with group 3 calves. Pleurisy was present in group 2 calves only and no pathology was present in group 1. Pulsed-field gel electrophoresis (PFGE) produced 11 (group 2, UK isolate) or 10 (group 3, USA isolate) bands with differences in banding patterns. Results overall showed that two isolates, distinct geographically and genetically (by PFGE), caused pneumonic pasteurellosis in a single host with significantly different severity of pathology. This information is relevant to the development of novel vaccine control and interpretation of diagnostic results.
Histamine exerts its numerous physiological functions through interaction with G protein-coupled receptors. Three such receptors have been defined at both the pharmacological and molecular level, ...while pharmacological evidence hints at the existence of further subtypes. We report here the cloning and characterization of a fourth histamine receptor subtype. Initially discovered in an expressed-sequence tag database, the full coding sequence (SP9144) was subsequently identified in chromosome 18 genomic sequence. This virtual coding sequence exhibited highest homology to the H(3) histamine receptor and was used to generate a full-length clone by polymerase chain reaction (PCR). The distribution of mRNA encoding SP9144 was restricted to cells of the immune system as determined by quantitative PCR. HEK-293 cells transiently transfected with SP9144 and a chimeric G protein alpha-subunit (Galpha(q/i1,2)) exhibited increases in intracellular Ca(2+) in response to histamine but not other biogenic amines. SP9144-transfected cells exhibited saturable, specific, high-affinity binding of (3)Hhistamine, which was potently inhibited by H(3) receptor-selective compounds. The rank order and potency of these compounds at SP9144 differed from the rank order at the H(3) receptor. Although SP9144 apparently coupled to Galpha(i), HEK-293 cells stably transfected with SP9144 did not exhibit histamine-mediated inhibition of forskolin-stimulated cAMP levels. However, both (35)SGTPgammaS binding and phosphorylation of mitogen-activated protein kinase were stimulated by histamine via SP9144 activation. In both of these assays, SP9144 exhibited evidence of constitutive activation. Taken together, these data demonstrate that SP9144 is a unique, fourth histamine receptor subtype.
The fate of Schistosoma mansoni (Trematoda) sporocysts in its molluscan host Biomphalaria glabrata (Gastropoda) is determined by circulating phagocytes (hemocytes). When the parasite invades a ...resistant snail, it is attacked and destroyed by hemocytes, whereas in a susceptible host it remains unaffected. We used 3 inbred strains of B. glabrata: 13-16-R1 and 10-R2, which are resistant to the PR-1 strain of S. mansoni, and M-line Oregon (MO), which is susceptible to PR-1. In an in vitro killing assay using plasma-free hemocytes from these strains, the rate of parasite killing corresponded closely to the rate by which S. mansoni sporocysts are killed in vivo. Hemocytes from resistant snails killed more than 80% of S. mansoni sporocysts within 48 hr, whereas sporocyst mortality in the presence of hemocytes from susceptible snails was <10%. Using this in vitro assay, we assessed the involvement of reactive oxygen species (ROS) produced by resistant hemocytes, during killing of S. mansoni sporocysts. Inhibition of NADPH oxidase significantly reduced sporocyst killing by 13-16-R1 hemocytes, indicating that ROS play an important role in normal killing. Reduction of hydrogen peroxide (H2O2) by including catalase in the killing assay increased parasite viability. Reduction of superoxide (O2−), however, by addition of superoxide dismutase or scavenging of hydroxyl radicals (·OH) and hypochlorous acid (HOCl) by addition of hypotaurine did not alter the rate of sporocyst killing by resistant hemocytes. We conclude that H2O2 is the ROS mainly responsible for killing.
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