A 'hot clinic' for cold limbs: the benefit of urgent clinics for patients with critical limb ischaemia.

INTRODUCTION: The national reconfiguration of vascular surgery means that arterial centres serve larger populations with increased demand on resources. Emergency general surgery ambulatory clinics facilitate timely review and intervention, avoiding admission; a critical limb ischaemia (CLI) 'hot clinic' (HC) was implemented to achieve similar for vascular patients. The aim of the study was to determine HC efficacy. METHODS: This was a prospective cohort study comparing HC patients with emergency admission (EA) patients between 1 May and 1 December 2017. Age, sex, comorbidities, CLI severity and smoking status were noted. HC patients were provided with satisfaction surveys. Primary outcome measures were freedom from reintervention and major amputation. Secondary outcome measures included time to procedure, length of stay, returns to theatre and 30-day readmission. RESULTS: A total of 147 patients (72 HC, 75 EA) were enrolled in the study. No statistical difference was found in age, sex, smoking status, severity of CLI or prevalence of comorbidities between the groups except that diabetes was more prevalent in EA patients (p=0.028). The median length of stay for the HC cohort was shorter (3 days vs 17 days, p<0.001), with no difference between time to procedure, return to theatre or 30-day readmission. HC patients were nearly 6 times more likely to experience freedom from reintervention (odds ratio: 5.824, p<0.001) and 2.5 times less likely to undergo amputation (odds ratio: 2.616, p=0.043). HC utilisation saved a total of 441 bed days. Over 90% of attendees responded with 100% positive feedback. CONCLUSIONS: A vascular HC facilitates urgent review and revascularisation. It provides comparable in-hospital outcomes and better long-term outcomes, with greater efficiency than hospital admission, demonstrating its value in treating CLI.

Potential for recombinant Babesia bovis antigens to protect against a highly virulent isolate.

Two antigens from Babesia bovis,12D3 and 11C5, were expressed and purified as recombinant proteins in Escherichia coli and used to vaccinate groups of six Babesia-susceptible cattle. These were subsequently challenged with a highly virulent strain of B. bovis. All cattle showed symptoms of disease and most required treatment. Cattle vaccination groups receiving either 12D3 or 11C5 or a combination of both, reduced parasitaemia by approximately fourfold and a number of individual animals appeared to control the parasite infection. Control of parasites correlated with high monocyte numbers late in infection. The results thus confirm the potential usefulness of both antigens but also demonstrate the limitations of current formulations.

Surgery for deep venous incompetence.

BACKGROUND: Chronic deep venous incompetence (DVI) is caused by incompetent vein valves and/or the blockage of large calibre leg veins, with a range of symptoms including recurrent ulcers, pain and swelling. OBJECTIVES: To establish the effectiveness of various surgical procedures for treating DVI. SEARCH STRATEGY: Trials were identified through the Cochrane Peripheral Vascular Diseases Group's trials register, reference lists of relevant studies, and contact with principal investigators of identified trials and world experts in deep venous surgery. SELECTION CRITERIA: Randomised controlled trials of surgical treatment for patients with DVI. DATA COLLECTION AND ANALYSIS: Reviewers extracted data independently. Outcome measures included ambulatory venous pressure (AVP) and venous refill time (VRT). MAIN RESULTS: Three trials were included, one trial was excluded. Two trials compared external valvuloplasty using limited anterior plication (LAP) in combination with ligation (L) of incompetent superficial veins (L+LAP) against ligation only (L). The other trial compared external valvuloplasty and ligation (V+L) of incompetent superficial veins against ligation only (L). Trial participants had primary valvular incompetence with mild to moderate symptoms but no venous ulcers.L+LAP produced significant improvement in AVP: the mean difference between L+LAP and L groups was -15 mm Hg (95% confidence interval (CI) -20.9 to -9.0) at one year and -15 mm Hg (95% CI -21 to -8.9) at ten years.AVP values after surgery remained relatively high. Nine of eleven valves repaired remained competent after two years of follow up. No complications occurred. The overall mean score for clinical outcome was +2 (moderate improvement) in the L+LAP group compared with +1 (mild improvement) in the L group. Patients with deteriorating clinical dynamics over the five years preceding surgery had a significantly higher rate of improvement in clinical condition in V+L compared to L (81% versus 51%; p < 0.05) after seven years follow-up. Patients with stable preoperative clinical dynamics demonstrated a similar rate of improvement in both groups (96% versus 90%; p> 0.1). AVPs were not performed. REVIEWERS' CONCLUSIONS: These results indicate that ligation and valvuloplasty may have produced a moderate and sustained improvement for seven to ten years after surgery, in patients with mild to moderate DVI caused by primary valvular incompetence. However, there is insufficient evidence to recommend the treatment to this subgroup of patients, as the trials were small, used different methods of valvuloplasty and different methods of assessment.

Paradoxical cerebral embolisation. An explanation for fat embolism syndrome.

Fat embolism occurs following fractures of a long bone or arthroplasty. We investigated whether paradoxical embolisation through a venous-to-arterial circulation shunt (v-a) could lead to cerebral embolisation during elective hip or knee arthroplasty. Transcranial Doppler ultrasound (TCD), following the intravenous injection of microbubble contrast, identified the presence of a shunt in 41 patients undergoing hip (n = 20) or knee (n = 21) arthroplasty. Intra-operative cerebral embolism was detected during continuous TCD monitoring. Of the 41 patients, 34 had a v-a shunt of whom 18 had an embolism and embolism only occurred in patients with a shunt (p = 0.012). Spontaneous and larger shunts were associated with a greater number of emboli (rs = 0.67 and rs = 0.71 respectively, p < 0.01). Observations in two patients with large spontaneous shunts revealed 368 and 203 emboli and unexplained post-operative confusion and pancreatitis. Paradoxical cerebral embolisation only occurred in patients with a shunt and may explain both postoperative confusion and fat embolism syndrome following surgery.

Carotid endarterectomy improves cognitive function in patients with exhausted cerebrovascular reserve.

OBJECTIVE: To investigate changes in cognitive function following carotid endarterectomy (CEA). DESIGN: Prospective study with controls. METHODS: CEA patients (n=159) were compared to a urology surgery control group (n=20). In CEA patients cerebrovascular reserve (CVR) was measured preoperatively. During surgery emboli and blood flow velocity in the middle cerebral artery were measured by transcranial Doppler (TCD) and cerebral oxygen saturation (CsO2) by near infrared spectroscopy. Cognitive function was measured preoperatively and at 5 days and 8 weeks postoperatively using a standardised computer battery of tests. RESULTS: Only 8% of patients had normal CVR bilaterally. The median number of emboli during CEA was 12 (range 0-181). On carotid clamping, TCD velocity fell a median of 41% and cerebral oxygen saturation by 5%. Attention deteriorated compared to controls 5 days following CEA (p=0.003) and this deterioration was related to the rise in TCD velocity on declamping (r=-0.3, p=0.002). Median attention reaction times improved significantly by 8 weeks (p=0.001) especially in patients' with severely impaired CVR before surgery (p=0.02). CONCLUSIONS: Attention improved at 2 months following CEA in patients with impaired CVR. CEA may offer more than reduced stroke risk to patients with impaired CVR.

Identification of an immuno-protective mucin-like protein, peritrophin-55, from the peritrophic matrix of Lucilia cuprina larvae.

A mucin-like glycoprotein, peritrophin-55 was isolated and purified from the peritrophic matrix of Lucilia cuprina larvae. When injected into sheep, peritrophin-55 induced an immune response that inhibited larval growth by 51-66% when larvae subsequently fed on sera from the vaccinated sheep. The protein may have potential use as an immunogen probably accompanying other antigens to protect sheep from the cutaneous myiasis caused by these larvae. Peritrophin-55 was uniformly distributed throughout the peritrophic matrix where it probably lubricates the surface of the peritrophic matrix and protects the midgut from invasion by bacteria. The protein consists of an 8-cysteine amino-terminal domain (peritrophin-B domain) and a carboxy-terminal proline and threonine-rich domain with high probability for extensive O-linked glycosylation. The gene consists of two exons separated by a small intron. Peritrophin-55 mRNA was only detected in the larval cardia and midgut and to a minor extent in the hindgut. Sequence upstream of the transcriptional start site contained a putative promoter region, sequence similar to an ecdysone response element, sequence related to the Drosophila transposon S element and a tetranucleotide repeat region. A putative Drosophila melanogaster ortholog or paralog of peritrophin-55 (CG7714) was located within a 3458 bp intron of the Cha gene (choline-O-acetyltransferase), but on the opposite strand. Comparison of the putative promoter regions of the peritrophin-55 and CG7714 genes revealed little similarity except for a small semi-conserved sequence that is suggestive of a common transcription factor-binding site possibly contributing to the highly restricted developmental and tissue-specific expression patterns of these genes.

Reduced oviposition of Boophilus microplus feeding on sheep vaccinated with vitellin.

The most abundant protein present in Boophilus microplus eggs, vitellin, was isolated and purified as a non-covalent complex of six glyco-polypeptides of Mr 44-107kDa. The protein complex bound haem. Immuno-blots demonstrated that antibodies raised to vitellin recognised a 200kDa polypeptide in the haemolymph of adult female ticks. This is consistent with the general proposal that in arthropods vitellin is derived by proteolytic processing from a large precursor protein, vitellogenin. In parallel with this study, an 80kDa glycoprotein (GP80) was independently purified from larvae of B. microplus using efficacy in vaccination trials as an assay. Antibodies to GP80 also recognised a 200kDa protein in the haemolymph of ticks and a major 87kDa polypeptide present in the vitellin complex. Conversely, antibodies to purified vitellin recognised GP80. The amino-terminal amino acid sequences of the 87kDa vitellin polypeptide and GP80 were identical for at least the first 11 residues and internal peptide sequences from both polypeptides were co-located in a single but incomplete deduced amino sequence of B. microplus vitellogenin. Thus, GP80 is a processed product from vitellogenin and highly related to but not completely identical with the 87kDa vitellin polypeptide. Vaccination trials in the model host sheep were performed with purified vitellin and GP80. Sheep vaccinated with either purified vitellin or GP80 returned significantly reduced numbers of engorged female ticks with decreased weights and reduced oviposition. In contrast, sheep vaccinated with recombinant hexahis-GP80, which was incorrectly folded and not glycosylated showed no significant effects on ticks. It was concluded that vitellin and GP80 could induce immune responses that partially protect sheep from the tick, B. microplus. However, critical protective epitopes are associated with the folding of the protein and/or the oligosaccharides attached to it.

A novel family of chitin-binding proteins from insect type 2 peritrophic matrix. cDNA sequences, chitin binding activity, and cellular localization.

The peritrophic matrix is a prominent feature of the digestive tract of most insects, but its function, formation, and even its composition remain contentious. This matrix is a molecular sieve whose toughness and elasticity are generated by glycoproteins, proteoglycans, and chitin fibrils. We now describe a small, highly conserved protein, peritrophin-15, which is an abundant component of the larval peritrophic matrices of the Old World screwworm fly, Chrysomya bezziana, and sheep blowfly, Lucilia cuprina. Their deduced amino acid sequences code for a 8-kDa secreted protein characterized by a highly conserved and novel register of six cysteines. Two Drosophila homologues have also been identified from unannotated genomic sequences. Recombinant peritrophin-15 binds strongly and specifically to chitin; however, the stoichiometry of binding is low (1:10,000 N-acetyl glucosamine). We propose that peritrophin-15 caps the ends of the chitin polymer. Immunogold studies localized peritrophin-15 to the peritrophic matrix and specific vesicles in cells of the cardia, the small organ of the foregut responsible for peritrophic matrix synthesis. The vesicular contents are disgorged at the base of microvilli underlying the newly formed peritrophic matrix. This is the first time that the process of synthesis and integration of a peritrophic matrix protein into the nascent peritrophic matrix has been observed.

Vaccination against the Old World screwworm fly (Chrysomya bezziana).

Chrysomya bezziana is an endemic pest of livestock or a threat to livestock production in large areas of Africa, the Middle East, southern and south-east Asia and Australia. Its control is difficult. The feasibility of vaccinating against this pest has now been explored. In-vitro and in-vivo assays have been established. Using these assays, it has been shown that first instar larvae, third instar peritrophic membrane and cardia are all sources of material able to induce immunological reactions in sheep which lead to significant reductions in larval growth. In-vitro assays following vaccination with peritrophic membrane also show larval mortality. Taken together, these effects lead to an 82% reduction in the weight of recovered larvae in vitro and 45% reduction in vivo. Preliminary evidence suggests that the mechanism of protection may be complex.

Identification and characterisation of the excreted/secreted serine proteases of larvae of the old world screwworm fly, Chrysomya bezziana.

Serine proteases are the major proteolytic activity excreted or secreted from Chrysomya bezziana larvae as demonstrated by gelatin gel analyses and the use of specific substrates, benzoyl-Arg-p-nitroanilide and succinyl-Ala-Ala-Pro-Phe-p-nitroanilide. Serine proteases were identified through their inhibition by 4-(2-aminoethyl)-benzene sulphonyl fluoride and classified as trypsin- and chymotrypsin-like on the basis of inhibition by tosyl-L-lysine chloromethyl ketone and tosyl-L-phenylalanine chloromethyl ketone, respectively. Like most insect serine proteases, the C. bezziana enzymes were active over broad pH range from mildly acidic to alkaline. The excreted or secreted serine proteases were purified by affinity chromatography using soybean trypsin inhibitor. A different subset of the serine proteases was isolated by salt elution from washed larval peritrophic matrices. Amino-terminal sequencing identified both trypsin and chymotrypsin-like sequences in the excreted or secreted pool with the latter being the dominant protease, whereas trypsin was the dominant species in the peritrophic matrix eluant. These results suggest that trypsin was possibly preferably adsorbed by the peritrophic matrix and may act as a final proteolytic processing stage as partially digested and ingested polypeptides pass through the peritrophic matrix. Immunoblot analysis on dissected gut tissues indicated that the anterior and posterior midguts were the main source of the serine proteases, although a novel species of 32 kDa was predominantly associated with the peritrophic matrix. Proteases are a target for a partially protective immune response and understanding the complexity of the secreted and digestive proteases is a necessary part of understanding the mechanism of the host's immunological defence against the parasite.

Vaccination of cattle against the Boophilus microplus using a mucin-like membrane glycoprotein.

An antigen, BMA7, which induced partial immunity against tick infestation has been isolated from Boophilus microplus using two different protein fractionation protocols, accompanied by vaccination and parasite challenge trails. The antigen is a 63 kDa glycoprotein isolated from semi-engorged adult female ticks. Though significant, the induced immunity is less striking than that previously reported for antigen Bm86 from the same parasite. However, co-vaccination with Bm86 and BMA7 can enhance immunity over that seen with a commercial vaccine based on Bm86 alone. Limited peptide sequence information shows significant variation in the BMA7 protein occurs. The antigen has approximately 36 kDa of glycosylation, in both N-linked and O-linked oligosaccharides. There is evidence that both polypeptide and oligosaccharide are antigenic, but the chemical nature of the protective antigenic sites is not clear. There is little or no immunological response to the antigen during natural infestation with parasites, suggesting the antigen is 'concealed' and protective immunity dependent on artificial vaccination. The antigen has some similarities with the vertebrate mucins. It is widely distributed in tick tissues and membrane bound but its function is currently unknown.

cDNA and deduced amino acid sequences of a peritrophic membrane glycoprotein, 'peritrophin-48', from the larvae of Lucilia cuprina.

The gut of most insects is lined with a semi-permeable peritrophic membrane (or peritrophic matrix) composed of chitin, proteoglycans and proteins. Despite the probable importance of the peritrophic membrane in facilitating the digestive process and protecting insects from invasion by micro-organisms and parasites, there has been little characterization of the specific components and their interactions within this acellular structure. Here we report the characterization of an integral peritrophic membrane glycoprotein, peritrophin-48, from the larvae of the fly Lucilia cuprina, a primary agent of cutaneous myiasis in sheep. Peritrophin-48 was purified from peritrophic membrane obtained by larval culture and its location within the peritrophic membrane determined by immuno-fluorescence and immuno-gold localizations. The cDNA coding for peritrophin-48 was cloned and sequenced. The deduced amino acid sequence codes for a protein of 375 amino acids containing an amino-terminal signal sequence followed by five similar, but non-identical domains, each approximately 65-70 amino acids in length and characterised by a specific register of six cysteines. The deduced amino acid sequence shows significant similarity to two other peritrophic membrane proteins, peritrophin-95 and peritrophin-44, from the same species. A reverse transcriptase-PCR approach indicated that there are several highly related peritrophin-48 genes expressed in each individual. Reverse transcriptase-PCR also demonstrated the expression of peritrophin-48 in all three larval instars and adults but not pupae or eggs. Peritrophin-48 was expressed only by the cardia and by the larval midgut. A simple structural model of a basic unit of a type 2 peritrophic membrane is presented.

Antibody-mediated inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host.

Many insects feed on blood or tissue from mammalian hosts. One potential strategy for the control of these insects is to vaccinate the host with antigens derived from the insect. The larvae of the fly Lucilia cuprina feed on ovine tissue and tissue fluids causing a cutaneous myiasis associated with considerable host morbidity and mortality. A candidate vaccine antigen, peritrophin 95, was purified from the peritrophic membrane, which lines the gut of these larvae. Serum from sheep vaccinated with peritrophin 95 inhibited growth of first-instar L. cuprina larvae that fed on this serum. Growth inhibition was probably caused by antibody-mediated blockage of the normally semipermeable peritrophic membrane and the subsequent development of an impervious layer of undefined composition on the gut lumen side of the peritrophic membrane that restricted access of nutrients to the larvae. The amino acid sequence of peritrophin 95 was determined by cloning the DNA complementary to its mRNA. The deduced amino acid sequence codes for a secreted protein containing a distinct Cys-rich domain of 317 amino acids followed by a mucin-like domain of 139 amino acids. The Cys-rich domain may be involved in binding chitin. This report describes a novel immunological strategy for the potential control of L. cuprina larvae that may have general application to the control of other insect pests.

Characterization of a major peritrophic membrane protein, peritrophin-44, from the larvae of Lucilia cuprina. cDNA and deduced amino acid sequences.

The peritrophic membrane is a semi-permeable chitinous matrix lining the gut of most insects and is thought to have important roles in the maintenance of insect gut structure, facilitation of digestion, and protection from invasion by microrganisms and parasites. Proteins are integral components of this matrix, although the structures and functions of these proteins have not been characterized in any detail. The peritrophic membrane from the larvae of the fly Lucilia cuprina, the primary agent of cutaneous myiasis in sheep, was shown to contain six major integral peritrophic membrane proteins. Two of these proteins, a 44-kDa glycoprotein (peritrophin-44) and a 48-kDa protein (peritrophin-48) together represent >70% of the total mass of the integral peritrophic membrane proteins. Peritrophin-44 was purified and its complete amino acid sequence was determined by cloning and sequencing the DNA complementary to its mRNA. The deduced amino acid sequence codes for a protein of 356 amino acids containing an amino-terminal signal sequence followed by five similar but nonidentical domains, each of approximately 70 amino acids and characterized by a specific register of 6 cysteines. One of these domains was also present in the noncatalytic regions of chitinases from Brugia malayi, Manduca sexta, and Chelonus. Peritrophin-44 has a uniform distribution throughout the larval peritrophic membrane. Reverse transcriptase-polymerase chain reaction detected the expression of peritrophin-44 in all three larval instars but only trace levels in adult L. cuprina. The protein binds specifically to tri-N-acetyl chitotriose and reacetylated chitosan in vitro. It is concluded that the multiple cysteine-rich domains in peritrophin-44 are responsible for binding to chitin, the major constituent of peritrophic membrane. Peritrophin-44 probably has roles in the maintenance of peritrophic membrane structure and in the determination of the porosity of the peritrophic membrane. This report represents the first characterization of an insect peritrophic membrane protein.

Cloning and characterisation of angiotensin-converting enzyme from the dipteran species, Haematobia irritans exigua, and its expression in the maturing male reproductive system.

The angiotensin-converting enzymes (ACE) are involved in the regulation of the specific maturation or degradation of a number of mammalian bioactive peptides. A carboxydipeptidase similar to mammalian ACE has now been identified in the adult stage of the haematophagous fly, Haematobia irritans exigua (buffalo fly), a close relative of the horn fly of North America. The enzyme was purified by lectin-affinity chromatography and ion-exchange chromatography and migrated as a doublet of 70 kDa upon reducing SDS/PAGE. Unlike mammalian ACE, the fly carboxydipeptidase (HieACE) is not membrane bound. The amino acid sequence of an internal peptide from HieACE and a conserved amino acid region present in all mammalian ACE were used to design degenerate oligonucleotide primers suitable for PCR. A DNA fragment amplified from adult buffalo fly cDNA was used to identify a cDNA clone that encoded the enzyme. The cDNA sequence encodes a carboxydipeptidase with 41-42% amino acid identity to the mammalian testicular ACE. The active-site regions of mammalian ACE are conserved in the deduced amino acid sequence of HieACE. Enzymatically, HieACE is very similar to its mammalian counterparts, with comparable Km and V(max) values for the synthetic substrate, benzoylglycylglycylglycine, and similar patterns of inhibition by EDTA, ACE inhibitor peptide and captopril. HieACE also specifically activates angiotensin I to angiotensin II and degrades other mammalian ACE substrates such as bradykinin, substance P and cholecystokinin-8. In the adult fly, HieACE is expressed in the compound ganglion and in the posterior region of the midgut. Similar to the mammalian system, expression of this enzyme is induced in the maturing male reproductive system, which suggests conservation of ACE function in these species.

Carboxydipeptidase from Boophilus microplus: a "concealed" antigen with similarity to angiotensin-converting enzyme.

A protein, Bm91, which was first identified as a protective vaccine antigen from the tick Boophilus microplus, has regions of very strong amino acid sequence similarity to mammalian carboxydipeptidases or angiotensin converting enzymes (ACE; E.C. 3.4.15.1). This protein is now shown to share many biochemical and enzymatic properties with mammalian carboxydipeptidases. It is enzymatically active in a conventional assay for ACE using hippuryl-Gly-Gly as substrate. The hydrolysis of the C-terminal nonapeptide of the insulin B chain proceeds by sequential removal of carboxy-terminal dipeptides. The similarities extend to the dependence of activity on pH and added salt. Bm91 is inhibited by two well-characterized inhibitors of the mammalian enzymes, the drug Captopril and a nonapeptide, and the inhibition occurs in similar concentration ranges to those effective with the mammalian enzymes. However, the natural substrates of the tick enzyme are unknown. Angiotensin I itself is a poor substrate and the enzyme's natural substrates are likely to be one or more of the pharmacologically active peptides occurring in insects and arthropods.

A protective "concealed" antigen from Boophilus microplus. Purification, localization, and possible function.

A membrane protein that can be used successfully to vaccinate cattle against the tick Boophilus microplus has been purified and characterized. The mature protein, which is referred to as Bm91, has an apparent m.w. of 86,000, an isoelectric point of 4.8 to 5.2, and is glycosylated, with an affinity for lentil lectin. Bm91 is of relatively low abundance, with approximately 300 to 400 micrograms being recovered from 1 kg of semiengorged adult female ticks. The protein is located largely in the salivary gland and gut of these ticks. Partial amino acid sequence data for the protein show striking similarities to that of mammalian angiotensin-converting enzyme, suggesting that the Ag may have an enzymatic function. The protein seems not to be recognized by sera from cattle with extensive exposure to ticks under natural conditions. The immunity induced by vaccination, therefore, represents another example of vaccination against a hematophagous parasite with "concealed" Ags.

Excretory/secretory chymotrypsin from Lucilia cuprina: purification, enzymatic specificity and amino acid sequence deduced from mRNA.

Two chymotrypsin-like proteases were purified from the secretory and excretory material of first-instar larvae of Lucilia cuprina. The hydrolysis of N-succinyl-L-phenylalanine-nitroanilide was used to monitor the purification of these proteases which was achieved by affinity chromatography on soybean trypsin inhibitor-Sepharose followed by anion exchange and hydrophobic interaction chromatographies. The enzymatic specificity of the most abundant protease (Lucilia chymotrypsin b; LCTb) was further defined by determining the amino acid sequence of peptides released from insulin B chain after incubation with LCTb. Peptide amino acid sequences obtained from LCTb were used to design degenerate oligonucleotide primers which, in conjunction with the polymerase chain reaction, enabled cDNA coding for LCTb to be cloned and sequenced. The deduced amino acid sequence of LCTb showed many of the structural features of serine proteases as well as significant amino acid sequence homology with chymotrypsins from a diverse range of species. It is probable that LCTb plays an important role in establishing the myiasis-causing larvae of L. cuprina on host skin as well as providing nutrients for the rapidly growing larvae.