Sustainable aquaculture through the One Health lens.

Aquaculture is predicted to supply the majority of aquatic dietary protein by 2050. For aquaculture to deliver significantly enhanced volumes of food in a sustainable manner, appropriate account needs to be taken of its impacts on environmental integrity, farmed organism health and welfare, and human health. Here, we explore increased aquaculture production through the One Health lens and define a set of success metrics - underpinned by evidence, policy and legislation - that must be embedded into aquaculture sustainability. We provide a framework for defining, monitoring and averting potential negative impacts of enhanced production - and consider interactions with land-based food systems. These metrics will inform national and international science and policy strategies to support improved aquatic food system design.

Siblings of patients with severe chronic obstructive pulmonary disease have a significant risk of airflow obstruction.

Although familial clustering has been described, few studies have quantified the risk of airflow obstruction in siblings of patients with chronic obstructive pulmonary disease (COPD). One hundred fifty-two subjects with airflow obstruction and a low gas transfer factor (but without PiZ alpha(1)-antitrypsin deficiency) were identified and 150 were enrolled in the study. Complete data were obtained from 173 of 221 siblings of these subjects. Forty-four of 126 current or ex-smoking siblings had airflow obstruction (FEV(1)/FVC < 0.7) and 36 also had a FEV(1) < 80% predicted, in keeping with COPD. One hundred eleven current or ex-smoking siblings were matched for age, sex, and smoking history with 419 subjects, without a known family history of COPD, from the European Prospective Investigation of Cancer (EPIC)-Norfolk cohort. The prevalence of COPD was much lower in the EPIC group (9.3%) when compared with the siblings (31.5%; odds ratio, 4.70; 95% confidence interval, 2.63 to 8.41). The odds ratio for COPD in siblings with less than a 30 pack-year smoking history was 5.39 (95% confidence interval, 2.49 to 11.67) when compared with matched control subjects. Taken together these results demonstrate a significant familial risk of airflow obstruction in smoking siblings of patients with severe COPD.

The pig analogue of CD59 protects transgenic mouse hearts from injury by human complement.

BACKGROUND: It has been proposed that hyperacute rejection (HAR) of pig-to-primate vascularized xenografts is due in large part to ineffective regulation of recipient complement by pig complement regulatory proteins (CRPs), and indeed transgenic expression of human CRPs in pigs can prevent hyperacute rejection. However, at least one pig CRP (CD59) efficiently regulates human complement in vitro, suggesting that it is the level of expression of a particular CRP(s) rather than cross-species incompatibility that explains the HAR of porcine xenografts. We investigated the relative effectiveness of transgenically expressed pig and human CD59 in providing protection of mouse hearts from human complement in an ex vivo setting. METHODS: Transgenic mice expressing pig CD59 or human CD59 under the control of the human ICAM-2 promoter, which restricts expression in tissues to vascular endothelium, were used. Hearts from mice expressing similar levels of pig CD59 or human CD59 were perfused ex vivo with 10% human plasma and heart function was monitored for 60 min. Sections of perfused hearts were examined for deposition of the membrane attack complex (MAC). RESULTS: Control nontransgenic hearts (n=5) were rapidly affected by the addition of human plasma, with mean function falling to less than 10% of the initial level within 15 min. In contrast, hearts expressing either pig CD59 (n=6) or human CD59 (n=8) were protected from plasma-induced injury, maintaining 31 and 35% function, respectively, after 60 min of perfusion. MAC deposition was markedly reduced in both pig CD59 and human CD59 transgenic hearts compared to nontransgenic control hearts. CONCLUSIONS: When highly expressed on endothelium in transgenic mice, pig CD59 provided equivalent protection to human CD59 in a model of human complement-mediated xenograft rejection. Thus supranormal expression of endogenous porcine CRPs may be a feasible alternative to the expression of human CRPs in preventing HAR of pig-to-primate xenografts.

Pigs express multiple forms of decay-accelerating factor (CD55), all of which contain only three short consensus repeats.

We report the cloning of cDNAs encoding multiple isoforms of the pig analogue of human decay-accelerating factor (DAF; CD55). Screening of a pig muscle cDNA library using a human DAF probe identified a single clone that encoded a DAF-like molecule comprising three short consensus repeats (SCR) homologous with the amino-terminal three SCR in human DAF, a serine/threonine-rich (ST) region, and sequence compatible with a transmembrane domain and cytoplasmic tail. Northern blot and RT-PCR analysis showed that pig DAF was expressed in a wide range of tissues. Additional isoforms of DAF were sought using RT-PCR and 3'-rapid amplification of cDNA ends followed by sequencing. Isoforms containing a GPI anchor and with differing lengths of ST region were identified; no isoform containing a fourth SCR was found. Cloning of the GPI-anchored isoform from granulocytes confirmed that it was identical with the original transmembrane isoform through the three SCR and first portion of ST and was derived from a frame shift caused by splicing out 176 bp of sequence. A panel of mAbs was generated and used to analyze the distribution and anchoring of pig DAF in circulating cells. Pig DAF was expressed on all circulating cells and was transmembrane anchored on erythrocytes, but completely or partially GPI anchored on granulocytes and mononuclear cells. The transmembrane isoform of pig DAF was expressed on Chinese hamster ovary cells and was shown to affect regulatory activity for the classical pathway of human complement, but was only marginally active against pig serum.

Identification of mutations in rat CD59 that increase the complement regulatory activity.

Formation of the membrane attack complex (MAC) of complement on host cells is inhibited by the glycosylphosphatidylinositol- (GPI-) anchored glycoprotein CD59. Published data on the active site of human CD59 are confusing. To clarify these data, we set out to elucidate the active site of a nonprimate CD59 molecule by site-directed mutagenesis. We also undertook to investigate a region of potential species selectivity, and to this end rat CD59 was chosen for all mutations. Our investigations confirmed the proposal that the active site of CD59 is the major hydrophobic groove, with mutations Y36A, W40A, and L54A ablating complement inhibitory function of CD59. Other mutations reducing the function of rat CD59 were I56E, D24A, and D24R. Importantly, mutations at one residue increased the function of rat CD59. The K48E mutation significantly increased function against human rat or rabbit serum, whereas the K48A mutation increased function against human serum alone. A similar mutation in human CD59 (N48E) had no effect on activity against human or rat serum but completely abolished all activity against rabbit serum. These findings suggest that the alpha-helix of human CD59, adjacent to the hydrophobic groove, influences the interaction between human CD59 and rabbit C8, C9, or both.

Molecular cloning, expression and characterization of the rat analogue of human membrane cofactor protein (MCP/CD46).

In humans, host cells are protected from homologous complement by membrane proteins encoded in the regulators of complement activation (RCA) gene cluster. These include complement receptor 1 (CR1), decay-accelerating factor (DAF, CD55) and membrane cofactor protein (MCP, CD46). In mouse and rat a single membrane inhibitor, Crry, appeared to perform the functions of both DAF and MCP and was proposed to be the functional analogue of both. Recently, however, murine homologues of DAF and MCP have been identified, prompting a search for the rat counterparts. We have described the identification of rat DAF and here describe the cloning of rat MCP from cDNA and genomic libraries, using a probe based on the mouse MCP cDNA sequence. The domain structure for rat MCP was identical to that of mouse MCP with four short consensus repeats (SCRs) followed by a STP domain, transmembrane segment and cytoplasmic tail. Overall identity of rat and mouse MCP was 77% at the amino acid level and 88% at the nucleotide level. Northern blot analysis from a range of tissues indicated that high-level expression was limited to the testis, although expression in other tissues was detected using reverse transcription-polymerase chain reaction. Rat MCP mRNA localized to Sertoli cells and spermatogonia in seminiferous tubules by in situ hybridization, but was absent in mature sperm. In cofactor assays utilizing human factor I, a recombinant soluble form of rat MCP catalysed cleavage of human C3ma.

Molecular cloning and functional characterization of the rat analogue of human decay-accelerating factor (CD55).

We report here the cloning of cDNAs encoding two forms of the rat analogue of human decay-accelerating factor (DAF; CD55). Screening of a rat kidney cDNA library using a mouse DAF probe identified a partial cDNA encoding the 3' end of rat DAF. The 5' end of the cDNA was cloned using the rapid amplification of cDNA ends (RACE) technique. A second form of rat DAF was identified using 3'RACE. Cloning and sequencing of full length cDNAs for both forms showed that they were identical up to nucleotide 1143 except for a 51-bp insert in the ST-rich region of the second form. After nucleotide 1143, the two sequences diverged; the cDNA cloned from the library encoded a unique 112-amino acid "tail," whereas the second form, identified by 3'RACE, encoded an 18-amino acid hydrophobic stretch, which was predicted to be a glycosylphosphatidylinositol (GPI) anchor addition signal. Expression in the NIH-3T3 mouse fibroblast cell line confirmed that the short tail did encode a GPI-addition signal, whereas the longer tail caused the protein to be secreted. Northern blot analysis identified two distinct transcripts for the GPI form, as well as a variability in expression levels of the different transcripts in the panel of tissues screened. Southern blot analysis showed that both the GPI and secreted forms of rat DAF were expressed in a wide range of tissues. The GPI-linked form of rat DAF stably expressed in a murine fibroblast cell line reduced C3 deposition and conferred protection from lysis by rat serum.

Molecular cloning and functional characterization of the pig analogue of CD59: relevance to xenotransplantation.

In this work, we report the cloning of the cDNA for the porcine analogue of human CD59. Degenerate primers, derived from the N-terminal sequence of pig erythrocyte CD59, were used to obtain the corresponding cDNA sequence. From this sequence, gene-specific primers were designed and used to amplify the 3' and 5' ends of the cDNA using the rapid amplification of cDNA ends (RACE) method. The complete 768-bp cDNA so obtained consisted of a 84-bp 5' untranslated region, a 26-amino-acid NH2-signal peptide, a 98-amino-acid coding region, including putative N-glycosylation sites and a glycosylphosphatidylinositol-anchoring signal, and a 312-bp 3' untranslated region. The mature protein sequence was 48% identical to human CD59 at the amino acid level. Northern blot analysis revealed several distinct CD59 transcripts, and a variability in expression levels of the different transcripts in the panel of tissues screened. Stable expression of pig CD59 in a CD59-negative human cell line conferred protection against lysis by complement from pig and several other species. Separate expression of pig and human CD59 at similar levels in the same cell line allowed a direct functional comparison between these two analogues. Pig CD59 and human CD59 showed similar activity in inhibiting lysis by complement from all species tested; in particular, expressed pig CD59 efficiently inhibited lysis by human complement. The relevance of these data to current work in the engineering of pig organs for xenotransplantation is discussed.