Partial purification and characterization of acetylcholinesterase isozymes from adult bovine filarial parasite Setaria cervi

379-385Filariasis is a major health problem, affecting millions of people in tropical and sub-tropical regions of the world. The isolation and characterization of parasite-specific enzyme targets is essential for developing effective control measures against filariasis. Acetylcholinesterase (AchE, E.C. 3.1.1.7), an important enzyme of neuromuscular transmission is found in a number of helminths including filarial parasites and may be playing a role in host-parasite interactions. Earlier, we demonstrated the presence of two isozymes of AchE, different from the host enzyme in the human (Brugia malayi) and bovine (Setaria cervi) filarial parasites. In the present study, two isozymes of AchE (pAchE1 and pAchE2) were isolated from S. cervi adults and characterized biochemically and immunochemically. The AchE was partially purified on Con-A Sepharose column and then subjected to preparative polyacrylamide gel electrophoresis (PAGE) for separation of the isozymes. The AchE activity was localized by the staining of gel and the isozymes were isolated from the PAGE strips by electroelution. Both isozymes preferentially utilized acetylcholine iodide as substrate and were strongly inhibited by the true AchE inhibitor (BW284c51), suggesting that they were true AchE. The polyclonal antibodies produced against the isozymes showed significant cross-reactivity with B. malayi AchE, but not against the host enzyme. These findings suggested that both the isozymes were biochemically (in terms of their substrate specificity and inhibitor sensitivity) and immunochemically similar, but different from the host enzyme

Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite surface protein-1

429-436Plasmodium vivax is one of the most widely distributed human malaria parasites and due to drug-resistant strains, its incidence and prevalence has increased, thus an effective vaccine against the parasites is urgently needed. One of the major constraints in developing P. vivax vaccine is the lack of suitable in vivo models for testing the protective efficacy of the vaccine. P. vivax and P. cynomolgi bastianelli are the two closely related malaria parasites and share a similar clinical course of infection in their respective hosts. The merozoite surface protein-1 (MSP-1) of these parasites has found to be protective in a wide range of host-parasite systems. P. vivax MSP-1 is synthesized as 200 kDa polypeptide and processed just prior to merozoite release from the erythrocytes into smaller fragments. The C- terminal 42 kDa cleavage product of MSP-1 (MSP-1₄₂) is present on the surface of merozoites and a major candidate for blood stage malaria vaccine. In the present study, we have biochemically and immunologically characterized the soluble and refolded 42 kDa fragment of MSP-1 of P. vivax (PvMSP-1₄₂) and P. cynomolgi B (PcMSP-1₄₂). SDS-PAGE analysis showed that both soluble and refolded E. coli expressed P. vivax and P. cynomolgi B MSP-1₄₂ proteins were homogenous in nature. The soluble and refolded MSP-1₄₂ antigens of both parasites showed high reactivity with protective monkey sera and conformation-specific monoclonal antibodies against P. cynomolgi B and P. vivax MSP-1₄₂ antigens. Immunization of BALB/c mice with these antigens resulted in the production of high titres of cross-reactive antibodies primarily against the conformational epitopes of MSP-1₄₂ protein. The immune sera from rhesus monkeys, immunized with soluble and refolded MSP-142 antigens of both parasites also showed high titered cross-reactive antibodies against MSP-1₄₂ conformational epitopes. These results suggested that the soluble and refolded forms of E. coli expressed P. vivax MSP-1₄₂ antigens were highly immunogenic and thus a viable candidate for vaccine studies

Merozoite Surface Protein 1 of Plasmodium vivax Induces a Protective Response against Plasmodium cynomolgi Challenge in Rhesus Monkeys

The 42-kDa fragment of the merozoite surface protein 1 (MSP-1(42)) is a leading candidate for the development of a vaccine to control malaria. We previously reported a method for the production of Plasmodium vivax MSP-1(42) (PvMSP-1(42)) as a soluble protein (S. Dutta, L. W. Ware, A. Barbosa, C. F. Ockenhouse, and D. E. Lanar, Infect. Immun. 69:5464-5470, 2001). We report here a process to manufacture the same PvMSP-1(42) protein but as an insoluble inclusion body-derived protein which was then refolded in vitro. We compared the immunogenicity and protective efficacy of the soluble and refolded forms of PvMSP-1(42) protein by using a heterologous but closely related P. cynomolgi-rhesus monkey challenge model. As comparative controls we also expressed, purified, and immunized rhesus with the soluble and refolded forms of the P. cynomolgi MSP-1(42) (PcMSP-1(42)) proteins. All proteins induced equally high-titer, cross-reacting antibodies. Upon challenge with P. cynomolgi, none of the MSP-1(42)-vaccinated groups demonstrated sterile protection or a delay in the prepatent period. However, following an initial rise in parasitemia, all MSP-1-vaccinated animals had significantly lower parasite burdens as indicated by lower cumulative parasitemia, lower peak parasitemia, lower secondary peak parasitemia, and lower average daily parasitemia compared to the adjuvant control group (P < 0.05). Except the soluble PcMSP-1(42) group, monkeys in all other groups had fewer numbers of days with parasitemia of >10,000 parasites mm(−3). Interestingly, there was no significant difference in the level of partial protection observed in the homologous and heterologous groups in this challenge model. The soluble and refolded forms of PcMSP-1(42) and PvMSP-1(42) proteins also appeared to have a similar partially protective effect

Pancreatic surgery outcomes: multicentre prospective snapshot study in 67 countries

Background: Pancreatic surgery remains associated with high morbidity rates. Although postoperative mortality appears to have improved with specialization, the outcomes reported in the literature reflect the activity of highly specialized centres. The aim of this study was to evaluate the outcomes following pancreatic surgery worldwide.Methods: This was an international, prospective, multicentre, cross-sectional snapshot study of consecutive patients undergoing pancreatic operations worldwide in a 3-month interval in 2021. The primary outcome was postoperative mortality within 90 days of surgery. Multivariable logistic regression was used to explore relationships with Human Development Index (HDI) and other parameters.Results: A total of 4223 patients from 67 countries were analysed. A complication of any severity was detected in 68.7 percent of patients (2901 of 4223). Major complication rates (Clavien-Dindo grade at least IIIa) were 24, 18, and 27 percent, and mortality rates were 10, 5, and 5 per cent in low-to-middle-, high-, and very high-HDI countries respectively. The 90-day postoperative mortality rate was 5.4 per cent (229 of 4223) overall, but was significantly higher in the low-to-middle-HDI group (adjusted OR 2.88, 95 per cent c.i. 1.80 to 4.48). The overall failure-to-rescue rate was 21 percent; however, it was 41 per cent in low-to-middle-compared with 19 per cent in very high-HDI countries.Conclusion: Excess mortality in low-to-middle-HDI countries could be attributable to failure to rescue of patients from severe complications. The authors call for a collaborative response from international and regional associations of pancreatic surgeons to address management related to death from postoperative complications to tackle the global disparities in the outcomes of pancreatic surgery (NCT04652271; ISRCTN95140761)