Plasmodium falciparum, the parasite responsible for the most severe form of malaria, has highlighted two related polymorphic genes called dblmsp and dblmsp2, which encode Duffy binding-like (DBL) domain-containing proteins located on the merozoite surface but whose function remains unknown. Using recombinant proteins and transgenic parasites, we show that DBLMSP and DBLMSP2 directly and avidly bind human IgM via their DBL domains. We used whole genome sequence data from over 400 African and Asian P. falciparum isolates to show that dblmsp and dblmsp2 exhibit extreme protein polymorphism in their DBL domain, with multiple variants of two major allelic classes present in every population tested. Despite this variability, the IgM binding function was retained across diverse sequence representatives. Although this interaction did not seem to have an effect on the ability of the parasite to invade red blood cells, binding of DBLMSP and DBLMSP2 to IgM inhibited the overall immunoreactivity of these proteins to IgG from patients who had been exposed to the parasite. This suggests that IgM binding might mask these proteins from the host humoral immune system.Diversity at pathogen genetic loci can be driven by host adaptive immune selection pressure and may reveal proteins important for parasite biology. Population-based genome sequencing of  Plasmodium causing malaria in humans, Plasmodium falciparum is responsible for the highest burden of disease. The clinical symptoms of malaria are associated with the blood stage of the infection, when the merozoite stage of the parasite recognizes, invades, and develops within human erythrocytes  are two members of the P. falciparum msp3 family that comprises a cluster of eight paralogous genes on chromosome 10. The msp3 family encodes proteins, which are characterized by the presence of an NLR(K/N)(A/G/N) motif at their N terminus (secreted polymorphic antigen associated with merozoites) domain  domain . DBLMSP ) domain . DBL domd EBA140 –15 and m EBA140 –. Sequencomain 17–. Clearlyeudogene . DespiteP. falciparum bind avidly and directly to human IgM. Using population-based genome sequencing and bespoke assembly tools, we revealed widespread genetic diversity focused on the DBL domain, with two major allelic forms of both dblmsp and dblmsp2 observed in populations from Africa and Southeast Asia. Despite their diversity, binding of the DBL domains from different DBLMSP and DBLMSP2 sequence variants to human IgM was conserved, suggesting an important role in the parasite biology.Here, we used recombinant proteins produced in mammalian cells and a knock-out parasite line to show that the DBL domains of DBLMSP and DBLMSP2 from the 3D7 strain of P. falciparum 3D7 strain using a recently developed expression system based on mammalian cells, which has been shown to produce natively folded P. falciparum proteins (A), and their immunoreactivity to sera from malaria-exposed and unexposed control individuals was quantified. To our surprise, whereas other MSP3 family members  and DBL-containing merozoite surface proteins  reacted, as expected, with only the exposed sera, DBLMSP and DBLMSP2 showed equally strong immunoreactivity to both the unexposed control and exposed sera (B). This suggested that recombinant DBLMSP and DBLMSP2 bound immunoglobulins present in normal serum from individuals without prior exposure to the malaria parasite. Indeed, when each protein was exposed to purified human immunoglobulin isotypes, strong binding was observed to IgM, but not to IgA, IgE, or IgG (C). This binding was specific to human IgM because they did not bind immunoglobulins from other mammalian species including goat, rabbit, guinea pig, and cow, or purified mouse IgM (D). To confirm these observations, we showed that DBLMSP-coated beads but not control beads incubated in normal human serum purified bands with masses there were consistent with the heavy and light chain of IgM (E), and their identities were subsequently confirmed by mass spectrometry.To gain insight into the functional role of DBLMSP and DBLMSP2 during malaria pathogenesis, we first expressed the entire coding region of both proteins from the proteins . Both prproteins A, and thsed sera B. This s, or IgG C. This bouse IgM D. To conn of IgM E, and thF).IgM adopts a flat, planar structure in solution but can change to a “staple” conformation upon antigen binding, thereby permitting interaction with other proteins such as C1q . To deteFig. 14 domain, inhibited the interaction of DBLMSP and DBLMSP2 with human IgM, whereas antibodies IX11 and HB57, with epitopes located in the Cμ1 and Cμ2 domains, respectively, or the Cμ3-specific antibodies 196.6b and 5D7 did not (A). To confirm these observations, we also made use of chimeric recombinant antibodies in which domains of the human μ chain constant region replace domains from the human α or γ chain .To identify the region on IgM that bound DBLMSP and DBLMSP2, we used mouse monoclonal antibodies that bind to specific domains of the human μ chain constant region , 24. Ant did not A. To con γ chain , 26. In  domains B.C).To determine which regions of DBLMSP and DBLMSP2 were involved in IgM binding, we individually expressed either the DBL domain or the SPAM fragment. The SPAM fragments did not bind IgM, whereas the isolated DBL domains bound IgM as efficiently as the full-length proteins in this assay, indicating that the DBL domain encompassed all IgM binding activity C.A). We then resolved purified proteins encompassing the DBL domain and SPAM fragment by size exclusion chromatography. Although the DBL domain of DBLMSP2 eluted as a single monodisperse peak consistent with it being a monomer, the SPAM fragment elution profile was polydisperse, with peaks consistent with tetrameric and other higher order complexes and only a minor fraction consistent with a monomeric form (B). A similar analysis of DBLMSP gave comparable results, suggesting that both DBLMSP and DBLMSP2 are able to form higher order oligomers by interactions through their C terminus.To determine whether DBLMSP and DBLMSP2 could, like MSP3, form oligomers, we first used size exclusion chromatography on each full-length protein. In both cases, polydisperse peaks were observed, suggesting the formation of higher order oligomers composed exclusively of the proteins of interest A. We theric form B. A simiin vivo. IgM binding to both parasite proteins was clearly saturable, demonstrating the specificity of the interactions, with equilibrium binding constants (DK) of 0.3 and 1.1 nm for DBLMSP and DBLMSP2, respectively (C). Given the multimeric nature of both IgM and the two parasite proteins studied, these measurements reflect overall binding avidity and are not easily comparable with other monomeric protein interactions. We therefore immobilized human IgM on the chip and used the monomeric DBL domains as analytes. Using this approach, the affinity for human IgM was 200–550-fold weaker, reflecting the loss of avidity but nonetheless still strong, with an equilibrium binding affinity of 60 nm for DBLMSP versus 600 nm for DBLMSP2 (D). The physiological concentration of IgM in human serum is ∼1–2 mg/ml (1–2 μm), which is at least 3 orders of magnitude higher than the measured binding constants and implies that all serum-exposed DBLMSP and DBLMSP2 would be rapidly and irreversibly complexed with IgM.To quantify the interaction between IgM and both DBLMSPs and demonstrate that the proteins interacted directly, we used surface plasmon resonance. Binding of soluble, purified human IgM was tested against immobilized full-length DBLMSP or DBLMSP2, mimicking the orientation of the proteins that would be expected ectively C. Given  DBLMSP2 D. The phdblmsp P. falciparum merozoites in which the dblmsp gene has been targeted (A) and confirmed this by Southern blotting (B). Using an antibody raised against the SPAM region of DBLMSP, we showed that the Δdblmsp parasites no longer expressed DBLMSP protein (C) and that it was absent from the surface of merozoites (D). We then used immunohistochemistry to test for the presence of human IgM at the surface of 3D7 wild-type or Δdblmsp parasites. In wild-type parasites, DBLMSP is expressed at the surface of merozoites in all schizonts, whereas DBLMSP2 is present in only a very small subset of schizonts (1% or less) (dblmsp-deficient merozoites (A). These observations were further confirmed by immunoelectron microscopy of purified wild-type and knock-out merozoites incubated in the presence of purified human IgM, which showed regular punctate labeling at the surface of wild-type but not Δdblmsp merozoites (B). These data demonstrate that native DBLMSP binds IgM and is the sole IgM-binding protein at the surface of most merozoites. Δdblmsp knock-out mutants grown in the presence of medium containing a serum alternative that does not contain human IgM did not show any invasion phenotype when compared with the wild type. To determine whether the presence of human IgM on the merozoite surface had any effect on the ability of the wild-type parasite to invade red blood cells, we also performed invasion assays with parasites grown in either serum alternative alone or serum alternative supplemented with 1 mg/ml human IgM. No difference in invasion efficiency was observed, suggesting that the presence of human IgM on the merozoite surface does not affect erythrocyte invasion in vitro (C).To demonstrate that native DBLMSP could bind human IgM, we generated Δtargeted A and conblotting B. Using  protein C and tharozoites D. We theor less) . Followirozoites A. These rozoites B. These in vitro C.P. falciparum parasite are able to bind human IgM. Sequencing of some P. falciparum isolates from Africa has, however, shown sequence variability in dblmsp and dblmsp2  as reported elsewhere , and in particular around the MSP3 gene family. Using this approach, the highest peak was observed at msp3 whose two diverged haplotypes, within which recombination is reduced, have been very well studied in the literature and whose divergence precedes the P. reichenowi/P. falciparum split  and shared sequence. As a result of this, together with the unknown multiplicity of infection and lack of phasing information with the short Illumina reads, we took a conservative approach and did not attempt to genotype the implied long multiallelic variants. Applying these approaches, we established that dblmsp and dblmsp2 each contained one major cluster of long non-recombining haplotypes (15 haplotypes in dblmsp and 41 in dblmsp2), with differing lengths (∼570 bp in dblmsp and 1350 bp in dblmsp2) and all overlapping the region encoding the DBL domain. In dblmsp, these 15 haplotypes grouped into two major allelic forms that we termed class 1 (3 haplotypes) and 2 (12 haplotypes) (B and C). In dblmsp2, we found two major allelic forms, class 1 (6 haplotypes) and class 2 (35 haplotypes), which we subdivided into two subclasses (c2.1 and c2.2) on the basis of multiple sequence alignments (B and D). To quantify the divergence between haplotypes, we designated d as the mean number of SNP and indel differences between classes c1 and c2. For dblmsp, d = 1.77 and d = 22.5, whereas d = 185. Similarly for dblmsp2, d = 2.9, d = 34.2, d = 7.0, and d = 61, whereas d = 239 and d = 194, demonstrating that for both genes, the two main allelic classes were deeply diverged.After close examination of the variant calls within lotypes) , B and Cignments , B and DE). Haplotype classes in all 434 samples were clearly identified in the region encoding the DBL domain, with SNP and indel sites present on either side. Outside the DBL domain, a strong long range linkage disequilibrium structure was observed, but all four countries showed different patterns. Within the DBL domain, however, samples from all countries showed both allelic classes at appreciable frequencies, a powerful signal of balancing selection. Although this had been shown previously in Africa, to our knowledge this is the first time it has been demonstrated in Southeast Asia. In the case of dblmsp, one of the two haplotype classes was very close to the P. reichenowi sequence (C) and is a strong example of balancing selection. Until further samples of P. reichenowi are sequenced, we will not know whether the selection pressure is present in P. falciparum only or whether this is a trans-species polymorphism (dblmsp showed much higher levels of mixed infection than expected (E), from which we inferred the existence of a dblmsp paralog, as has been suggested before . Except for the DBL domains from 7G8 and 028, IgM binding was conserved across all variants, including the most divergent (384 and P. reichenowi) (A). Similarly for DBLMSP2, seven representative DBL variants with sequence identities ranging from 62 to 98% were selected . Although variant 082 repeatedly did not produce protein, all the other six bound human IgM (A). Interestingly, whereas the 7G8 and 028 DBLMSP DBL domains could not bind human IgM, their shared DBLMSP2 DBL sequence was able to do so.Because the genetic variability within analyses , 19. Forchenowi) A. Similauman IgM A. Interedblmsp sequence is identical to that of FCR3) grown in the presence of human serum. Following SDS-PAGE and incubation with an anti-DBLMSP antibody, a band corresponding to the DBLMSP protein was identified in the immunoprecipitates from parasite cultures but not from the negative control supernatant, in which no parasite was grown (B). In summary, despite their high level of sequence polymorphism, binding of the DBL domains to human IgM was conserved across most of the DBLMSP and DBLMSP2 DBL domain variants tested.To further confirm that binding to IgM was conserved among sequence variants, we performed IgM pulldown from long term parasite culture supernatants from the var1 and var13 substrains of the IT4 strain . Consistent with the large number of variants observed in P. falciparum populations, almost no binding of purified hyperimmune IgG was observed to either of the two DBL domains. To confirm this was not merely because the sera were collected from individuals who had not been exposed to the 3D7 variants used in this assay, we also tested the immunoreactivity of other DBL variants for DBLMSP and DBLMSP2 and confirmed that they were generally not immunoreactive (B).The fact that the DBL domains of DBLMSP and DBLMSP2 retain IgM binding despite their extraordinary sequence diversity suggests that DBLMSP and DBLMSP2 are targeted by the host immune system and that IgM binding promotes parasite survival. To determine whether individuals regularly exposed to e adults . As compe adults , 34–36. dults 34–A. Consisreactive B.P. falciparum PfEMP1 variants expressed at the surface of infected erythrocytes, which are associated with rosetting or placental malaria (A). Interestingly, the presence of IgM had almost no effect on the already very limited immunoreactivity of the DBL domains. This suggests that binding of IgM to DBLMSP and DBLMSP2 masks antibody epitopes that are located in other regions of the proteins.Non-immune human IgM is also known to bind to some  malaria , 37. Alt malaria , 39. To  malaria A. Interedblmsp and dblmsp2 across P. falciparum populations suggests that they are under extremely strong selection and must have important biological functions. One possibility is that they are direct targets of the host immune response and are therefore potential targets for therapeutic intervention, but their function has remained unknown. Here, we have demonstrated that the DBL domains in both proteins bind directly and avidly to host IgM and that this function is conserved between the different sequence variants identified in P. falciparum isolates.The deeply diverged lineages preserved in dblmsp and dblmsp2 reported here extends earlier findings that both genes exhibit a strong allelic dimorphism with limited interallelic recombination across parasite isolates from all populations sampled. Strikingly, we have now shown that both allelic classes in dblmsp and dblmsp2 are present at relatively high frequency in populations from West Africa  and Southeast Asia (Cambodia), with recombination absent (or selected against) within a specific block of the DBL domain. The pattern of balancing selection preserving two deeply diverged haplotypes with reduced recombination has previously been seen in msp3, which lacks a DBL domain. We would draw a distinction between this pattern, and the known dimorphism at EBA175, which is simply a single indel, rather than a long preserved haplotype. This unusual genetic architecture raises the question as to how this dimorphism arose and is maintained within the population. Following our demonstration that the DBL domain can bind host IgM, one possibility is that there is a selective advantage to binding the two major IgM species that either contain or lack a J-chain. Dimorphism, however, is also observed in genes encoding other merozoite surface proteins, suggesting selection by a broader dichotomous feature within the immune system of the host population.The population-based sequence analysis of supplemental Fig. S2). These helices could therefore be potential binding sites for human IgM. Although DBLMSP is highly expressed in all mature schizonts in the parasitophorous vacuole and located on the merozoite surface, DBLMSP2 is only expressed in a small percentage of schizonts  were saturated with 20 μg of enzymatically monobiotinylated DBLMSP or a Cd4 tag-alone control, isolated with a magnet, and washed three times with PBS before incubating with 1 ml of filtered human serum (Sigma) for one h at 4 °C. Beads were washed four times with 1 ml of PBS and eluted with 200 μl of 1% SDS. 20 μl were resolved by SDS-PAGE under reducing conditions and stained with SYPRO Orange (Sigma), and the gel image captured on a Typhoon 9400 phosphorimaging device .Anti-human IgM agarose beads (Sigma) were incubated with long term parasite culture supernatants from the IT4 var1 and var13 strains grown in the presence of human serum or control culture medium without parasite for 1 week at 37 °C. After five washes in PBS, the beads were resuspended in loading buffer in the presence or absence of DTT, and eluates were blotted onto nitrocellulose membranes (Amersham Biosciences Protran) followed by blocking in PBS, 0.1% Tween 20, 5% nonfat milk powder) and incubated for 1 h with a rabbit anti-full-length DBLMSP antibody at a 1:100 dilution. After further washes, the membrane was incubated with an anti-rabbit HRP-conjugated IgG secondary antibody  and developed using 3′,3′-diaminobenzidine (DAKO) according to the manufacturer's instructions.P. falciparum parasites (3D7 strain) were grown in vitro in RPMI 1640 medium containing Albumax II or 10% human serum as described previously  and 10 μm ganciclovir (Sigma-Aldrich) and cloned by limiting dilution.eviously  and traneviously . TransfeP. falciparum, a 520-bp 5′ fragment (F1 region) and a 659-bp 3′ fragment (F2 region) comprising ORF and UTR sequences of dblmsp were amplified using primers 348F1for , 348F1rev , 348F2for , and 348F2rev . Amplification products were digested using SacII + BglII (F1 flank) and ClaI + AvrII (F2 flank) and cloned into pHTK vector . Hybridization was carried out at 62 °C overnight before stringent washes and autoradiography.For Southern blotting, the genomic DNA from 6 schizonts was prepared in SDS reducing sample buffer and boiled at 95 °C for 5 min. The lysates were separated on precast 10% Bis Tris NuPAGE polyacrylamide gels (Invitrogen) and transferred to nitrocellulose membranes by electroblotting. The membranes were blocked and incubated with rabbit anti-DBLMSPSPAM (1:2000), rabbit anti-MSP3 (1:2000), mouse anti-MSP2 (1:1000)  , and ratdblmsp parasites were incubated with anti-DBLMSPSPAM (1:2000) and anti-MSP119  antibodies diluted in 3% BSA/PBS and incubated for 1 h at room temperature before washes and incubation with secondary Alexa Fluor-labeled  antibodies at 1:5000.For immunofluorescence analysis, thin smears of schizont and merozoites from wild-type and ΔTo detect IgM binding to merozoites, parasites were tightly synchronized before late stage schizonts were purified using 70% Percoll, put back into culture containing RPMI + Albumax II with the addition of 20% human serum or 0.125 mg/ml purified human IgM (Sigma). IgM was detected using an Alexa Fluor 488 goat anti-human IgM μ chain antibody  at 1:1000. The slides were viewed on a Zeiss Axioplan 2 imaging system with Plan Apochromat 100×/1.4 oil immersion objective. Images were captured using Axiovision 4.6.3 software and edited using Adobe Photoshop.m phosphate buffer at pH 7.4 for 1 h at room temperature, rinsed three times in buffer, and infiltrated with 1% and then 10% gelatin before immersing in 2.3 m sucrose in phosphate buffer overnight at 4 °C for cryoprotection. Frozen samples were prepared by mounting onto aluminum pins and rapidly immersing in liquid nitrogen in preparation for ultrathin 80 nm sectioning on a Leica EM FC6 ultramicrotome. Ultra thin sections were labeled as per Tokuyasu  or without (Gp) the mouse J-chain  were a kP. falciparum proteins were normalized and immobilized on streptavidin-coated plates, preblocked with HBS and 0.1% Tween 20 (HBST), 2% BSA for 30 min. After washing with HBST, serial dilutions of either pooled human sera from 10 malaria-exposed or malaria-naïve individuals, transfectoma cell culture supernatants, or purified primary antibodies were incubated for 90 min. The plates were again washed with HBST, and appropriate alkaline phosphatase-conjugated secondary antibodies were incubated for 1 h. The plates were washed with HBST and once with HBS before adding p-nitrophenyl at 1 mg/ml, and absorbance at 405 nm was quantified on either a PHERAstar Plus or FLUOstar Optima plate reader (BMG Labtech). For domain mapping experiments involving chimeric human antibodies, an HRP-conjugated anti-human secondary antibody was used. Following PBS washes, tetramethylbenzidine substrate was added, and absorbance was quantified at 450 nm. All procedures were performed at room temperature. Primary antibodies used were: anti-Cd4 OX68 ; purified human IgA, IgE, IgM , or IgG ; human, goat, rabbit, guinea pig, or bovine serum ; purified mouse IgM ; or domain-swapped human antibodies at 25 nm  essentially as described . BrieflyEscherichia coli BL21(DE3)pLysS, and expression was induced with 1 mm isopropyl β-d-thiogalactopyranoside for 4 h at 30 °C. Recombinant DBLMSPSPAM protein was purified using nickel-nitrilotriacetic acid affinity chromatography under non-denaturing conditions. Purified rDBLMSPSPAM was used for antibody production in rabbits (Harlan Laboratories Inc.) following standard procedures. Rabbit antibodies were subsequently affinity-purified using rDBLMSPSPAM coupled to CNBr-activated Sepharose 4B  and IgG-selected using protein G-Sepharose (Sigma-Aldrich).A polyclonal antibody against the SPAM fragment of DBLMSP was generated. Briefly, a 619-bp DNA product (corresponding to amino acids 439–633 of DBLMSP) was amplified from 3D7 genomic DNA using primers 348ForPET30 (5′-GGTATTGAGGGTCGCAAAGAATTTAAAGATAACGTTACACTTCTTAAAGC-3′) and 348revbPET30 (5′-AGAGGAGAGTTAGAGCCTTAACTTTGTTGTTGATTACTGAGTTCTTTTTCC-3′) and cloned into pET-30 Xa/LIC expression vector (Novagen). Expression plasmids were sequenced before transformation into Polyclonal antibodies against the full-length 3D7 form of DBLMSP were raised in rabbits . The full-length, His-tagged protein was produced in mammalian cells, purified by nickel-nitrilotriacetic acid chromatography using HisTrap column  and injected every 2 weeks over an 11-week period with the first injection in complete Freund's adjuvant, and the subsequent five injections in incomplete adjuvant. Rabbit antibodies were subsequently affinity-purified using HiTrap protein G columns .Pentamerized, normalized β-lactamase-tagged parasite proteins were incubated with serial dilutions of purified human IgM for 90 min before being transferred to 20 μg/ml purified IgG from Malawian adults  and immodblmsp and wild-type parasites. A. J. P. performed the IgM purification from human serum. G. K., D. G., and G. D. purified the merozoites and performed the immunoelectron microscopy experiments. L. Y. B. and J. C. R. performed and analyzed parasite culture experiments. S. C. M. and R. J. P. performed the mapping experiment on human IgM and the immunoprecipitation of IgM from parasite culture supernatant and built the homology model. G. J. W. conceived and coordinated the study and wrote the manuscript. All authors analyzed the results and approved the final version of the manuscript.C. C. identified and characterized the interactions between human IgM and recombinant DBLMSP proteins, performed the binding and immunoreactivity studies on DBL variants, and wrote the manuscript. Z. I., S. C. M., A. M., and D. P. K. collated the samples, designed, and analyzed the data corresponding to the genetic analysis of field isolates. E. K. and A. A. H. designed, performed, and analyzed the data relating to the generation and analysis of Δ


The British Columbia take-home naloxone (BCTHN) program has been in operation since 2012 and has resulted in the successful reversal of over 581 opioid overdoses. The study aims to explore BCTHN program participant perspectives about the program, barriers to participants contacting emergency services  during an overdose, and perspectives of law enforcement officials on naloxone administration by police officers.Two focus groups and four individual interviews were conducted with BCTHN program participants; interviews with two law enforcement officials were also conducted. Qualitative analysis of all transcripts was performed.Positive themes about the BCTHN program from participants included easy to understand training, correcting misperceptions in the community, and positive interactions with emergency services. Potential barriers to contacting emergency services during an overdose include concerns about being arrested for outstanding warrants or for other illegal activities (such as drug possession) and confiscation of kits. Law enforcement officials noted that warrants were complex situational issues, kits would normally not be confiscated, and admitted arrests for drug possession or other activities may not serve the public good in an overdose situation. Law enforcement officials were concerned about legal liability and jurisdictional/authorization issues if naloxone administration privileges were expanded to police.Program participants and law enforcement officials expressed differing perspectives about warrants, kit confiscation, and arrests. Facilitating communication between BCTHN program participants and other stakeholders may address some of the confusion and remove potential barriers to further improving program outcomes. Naloxone administration by law enforcement would require policies to address jurisdiction/authorization and liability issues. Opioid overdose due to illicit drug use and prescription drugs and their associated mortality and morbidity have emerged as a global health issue –4. Globahttp://www.towardtheheart.com as of April 14, 2016, there are 178 locations within BC that dispense naloxone as part of the BCTHN program and 581 overdose reversals with naloxone have been reported, 7416 people have been trained, and 7418 people received naloxone kits [The British Columbia take-home naloxone (BCTHN) program was developed by the British Columbia Centre for Disease Control (BCCDC) and initiated in 2012 . At partone kits . Recentlone kits , creatinFeedback from the BCTHN Community Advisory Board, consisting of THN site coordinators and people who use drugs, have identified some potential concerns related to interactions between program participants and law enforcement. In particular, it was noted from BCTHN records that despite an increased emphasis to call emergency services , nearly a third of those who administered naloxone  did not call 911 . This quEthics approval was received from the University of British Columbia, Interior Health, and Vancouver Coastal Health ethics boards (H12-02557). All participants provided written informed consent before interviews or focus groups were conducted, and all identifiers were removed from the transcripts.The BCTHN program is operated by the BCCDC Harm Reduction Program, which develops training materials; is responsible for enrolling sites ; and distributes naloxone kits to the sites. Enrolled community sites provide training to clients about overdose prevention, recognition, and response and dispense naloxone kits to eligible individuals. Sites report administrative data to BCCDC who also collects details about naloxone administration. BCTHN sites were used to recruit study participants.A qualitative methodology was selected to explore perceptions, practices, and group dynamics that might contribute to the apprehension of calling 911. The study consisted of focus groups conducted with harm reduction clients who had received program training, key informant interviews with experienced BCTHN naloxone administrators, and law enforcement officials. Semi-structured interview guides were adapted from a prior evaluation . In theProgram participants were recruited from THN sites within the Vancouver Coastal Health, Fraser Health, and Interior Health regions. Focus group participants who used opioids, had received program training, and who were at least 19 years old were eligible to participate in the study. Additionally, client key informants must have administered naloxone at least three times. All clients who participated were provided with a $10 honorarium for their time. Law enforcement participants were recruited by reaching out to existing contacts within two police departments.Focus groups and individual interviews with BCTHN naloxone administrators and law enforcement officials took place between April and June 2015. One investigator (GA) conducted individual interviews and focus groups in person and by phone. The interviewer was not otherwise involved in the BCTHN program. Additionally, research assistants were present at the focus groups to assist in the collection of demographic information, to record notes concerning group dynamics and other observable information, and to remunerate participants who left early to avoid disruption of the focus group. Focus groups and interviews took approximately 1 h to complete and were digitally recorded and transcribed verbatim. Paraphrased examples of questions posed in the semi-structured interview include “Did you ever have your naloxone kit confiscated?” or “How did you find out about the training sessions?”Transcripts were anonymized by removing identifying information and imported into NVivo 10 for analysis. All the authors reviewed the transcripts independently to identify codes for qualitative analysis; the codes were discussed and agreed upon through consensus and themes were developed using a grounded theory approach . Using aTwo focus groups involving eight BCTHN program clients each and four individual interviews with BCTHN naloxone administrators were conducted for a total of 20 client interviewees Table . Two indThe study participants (including BCTHN program participants and police officers) were recruited from various locations in southern British Columbia see Fig. : 10 wereProgram participants expressed positive perspectives about the THN program including the ability to save lives, the understandable nature of the educational content, its accessibility to those with limited reading skills, and integration into the community  outstanding warrants; (2) being arrested for illegal activities, such as possession of drugs and breach of conditions of probation; and (3) kit confiscation. We identified no differences in the themes from rural versus urban interviewees.From the interviews with law enforcement officers, a set of complementary themes was identified. For instance, the police officers noted that while they are required to enforce the law and pursue individuals for outstanding warrants, it can depend on the context. Additionally, the law enforcement officials interviewed noted that in general they would not confiscate naloxone kits in contradiction to the assumptions of some of the BCTHN program participants. The participants’ concern regarding being arrested was contradicted by the information provided by law enforcement: it is often a more nuanced situation in which an officer will consult with a commanding official and weigh the medical and public good of pursuing an arrest in the context of the situation. Communicating to BCTHN naloxone administrators that Vancouver Police Department members do not routinely attend the overdose scenes may address some concerns. Revisiting the general police policy of recording the names of all present at the overdose scene to determine if there are outstanding warrants would help allay fears of arrest. While there are rare instances in which an arrest is required (such as during the case of an assault occurring at the site of an overdose), clear policies outlining when arrests may or may not occur in key scenarios may help law enforcement officials and BCTHN program participants navigate the legal, ethical, and safety issues that can occur during an overdose.Interestingly, the themes in Table While the law enforcement officials noted that there were major benefits to police administering naloxone, the role of police officers with respect to other first responders emerged as a potential issue of contention. However, the difference in opinion noted in Table Limitations of this study include the potentially limited representativeness of the interviewees, who are predominantly older, long-term drug users (>20 years). Additionally, convenience sampling was used to recruit interviewees which may also make the opinions, themes, and other observations not representative of the program participants as a whole. The researchers involved in the qualitative analysis process attempted to provide an unbiased appraisal of themes and opinions expressed during the interview process. Despite member checking, the role that some of the researchers played in the organizations supporting BCTHN may inadvertently influence results.The qualitative assessment of the BCTHN program identified several strengths of the program and potential concerns that may prevent BCTHN naloxone administrators from contacting emergency services during an overdose, such being arrested for outstanding warrants and illegal activity, and respondent concerns about kit confiscation. Instituting clear lines of communication between THN participants and law enforcement with guidelines concerning warrants and kit confiscations could help address issues identified by both parties. With respect to naloxone administration by the police, law enforcement officials identified the issues of liability and jurisdiction/authorization as potential barriers. Based on experience in other jurisdictions especially in the USA, implementing “Good Samaritan” regulations and educational programs emphasizing the benefits of naloxone to the community could be instrumental for such programs going forward in BC, Canada, and other jurisdictions. Discussion and communication about these changes could be facilitated through the use of online portals and other tools.Individual data from participants were not reported, and full permission to use interviewee data (including quotations) as outlined in the manuscript was obtained.


Salmonella enterica indicate that the members of this species are unable to utilize lactose as a carbon source. However, lactose-fermenting (Lac+) strains of several Salmonella serovars have been isolated from different foodborne outbreaks as well as different geographical regions worldwide. In the present study, we sequenced the genomes of 13 Lac + S. enterica isolates and characterized the lac region, comparing it to the lac region in other enteric bacterial species.Classical bacteriological characteristics of lac operons in the S. enterica genomes revealed that they all contain intact lacI, lacZ, and lacY genes. However, lacA was truncated in all of the S. enterica subsp. enterica isolates, encoding a 56 amino acid peptide rather than the full length 220 amino acid LacA protein. Molecular analyses of the 13 isolates revealed that the lac operon resided on a plasmid in some strains and in others was integrated into the bacterial chromosome. In most cases, an insertion sequence flanked at least one end of the operon. Interestingly, the S. enterica Montevideo and S. enterica Senftenberg isolates were found to harbor a plasmid with a high degree of sequence similarity to a plasmid from Klebsiella pneumoniae strain NK29 that also harbors the lac operon. In addition, two S. enterica Tennessee isolates carried two copies of the lac operon. Phylogenetic analysis based on lacIZY gene sequences determines distinct clusters, and reveals a greater correlation between lacIZY sequence and flanking organization than with either bacterial species or genomic location.Genetic analysis of the lac region is highly mobile among Enterobacteriaceae and demonstrate that the Lac + S. enterica subsp. enterica serovars acquired the lac region through parallel events. The acquisition of the lac operon by several S. enterica serovars may be indicative of environmental adaptation by these bacteria.Our results indicate that the The online version of this article (doi:10.1186/s12866-015-0511-8) contains supplementary material, which is available to authorized users. Salmonella enterica consists of over 2500 serovars, most of which are non-typhoidal and invade the host intestinal epithelium resulting in enterocolitis/diarrhea  encoded in the Klebsiella pneumoniae genome. For three of the S. enterica isolates, 50260, 50261, and 50269, this gene is 39 bp longer and the protein includes an additional oxidoreductase region, resulting in the highest protein sequence identity to a bifunctional oxidoreductase/alginate lyase found in Enterobacteriaceae [GenBank:WP_032414406]. The presence or absence of this additional gene along with the gene length if present is noted in Table Interestingly, characterization of the genome upstream of the lac region were inserted in the S. enterica genomes. The lac regions of several of the S. enterica isolates in this study displayed a high sequence similarity and flanking organization with that of the lac region on plasmid pK29 harbored in K. pneumoniae strain NK29 [GenBank:EF382672]. Plasmid pK29 is 269,674 bp in size and the flanking organization is included in Table S. enterica subsp. enterica isolates in this study were mapped to the plasmid pK29 reference sequence. The mappings demonstrated that seven of the isolates, namely Montevideo isolates 50262 and 50270 and Senftenberg isolates 50263, 50264, 50265, 50271 and 50272, harbor a plasmid with similarity to pK29. The consensus sequences resulting from the mappings revealed 99 % identity to pK29. Furthermore, BLAST comparisons revealed that these seven strains contain the repHI2 and repA genes that encode replication proteins found in the pK29 plasmid sequence. Gaps in the S. enterica plasmid sequences were primarily due to the presence of regions containing a total of six antibiotic resistance genes in pK29. However, isolate 50271 was missing only three of the resistance genes. Although it was determined that the consensus from the mapping was ~20.8 kb shorter than pK29, it is not known whether there are additional insertions in the plasmids harbored by these seven S. enterica isolates.We sought to determine where the mobile elements carrying the lac region of Tennessee isolate 50259 and the lac1 regions of Tennessee isolates 50260 and 50261 exhibited identical flanking organization and sequence with that of the lac region harbored on the chromosome of the S. enterica Tennessee isolate responsible for an outbreak associated with contaminated peanut butter [GenBank:AHUY01000000]. Genome comparisons confirmed that the lac regions harbored in the Tennessee isolates included in this study (lac1 regions for 50260 and 50261) are carried in the chromosome at the same location, within a 72 kb island inserted at pheV, as the Tennessee peanut butter outbreak isolate. Despite the similarity in flanking organization between the lac2 regions harbored in the genomes of isolates 50260 and 50261 with those found on two different plasmids, pKP007 and pENT-22e, carried in other enteric species  displayed a high degree of similarity (99.98 %) with those found on the K. pneumoniae plasmid pK29  homology with those of the S. enterica subsp. enterica serovars and other Enterobacteriaceae. Clearly, the lac gene sequences do not cluster according to plasmid or chromosomal location , ST17 (Indiana), ST316 (Montevideo), and ST319 (Tennessee). The Lac- closed genome strains Enteritidis P125109 (ST11), Typhimurium LT2 (ST19), and Typhi CT18 (ST2) were included in the analysis for reference. Among the S. enterica subsp. diarizonae strains, isolate 50267 was found to possess ST1261, while isolate 50268 has a variant of ST432 due to a single T to C synonymous transition in sucA. The S. enterica subsp. arizonae closed genome strain RSK2980 was also included for reference as it rooted the MLST phylogeny. RSK2980 has a novel ST comprised of previously observed alleles. Its allele profile of aroC65, dnaN25, hemD29, hisD24, purE20, sucA50, and thrA497 is most similar to ST1430, differing by 7 polymorphic sites .MLST analysis of the oci Fig. . IsolateS. enterica are commonly regarded as non-lactose fermenting pathogens; however a small percentage of isolates have acquired the lac region which can lead to confusion from a diagnostic standpoint. The lac operon has been well studied in E. coli and is known to be chromosomally located. However it has been noted some time ago that the lac operon is carried on a plasmid in some Enterobacteriaceae species including S. enterica Typhimurium and Oranienburg isolates  as a query. In most cases the entire lac operon was contained on one contig; otherwise two contigs were bioinformatically joined to obtain the entire lac operon sequence. The joined sequence was then verified by mapping the reads onto the joined sequence using CLC Genomics Workbench (CLC bio). The sequence regions flanking the operons were characterized by BLAST analysis. To determine the location of the lac operons within the genome, the lac region was used as a BLAST query, followed by identification of the flanking genes by BLAST. The locations of lac operon regions in bacterial species with the highest sequence identity were examined for similarity with the S. enterica genomes in this study.The lacIZY region (~5.7 kb) of the operons were combined with those of selected lac + bacterial species from GenBank and aligned with the ClustalW algorithm using the MegAlign module of the Lasergene software package . A pairwise similarity matrix of the lacIZY sequences analyzed is provided as supplemental material. In silico multilocus sequence typing (MLST) was performed on seven conserved housekeeping genes  [Salmonella isolates from GenBank  were concatenated for phylogenetic analysis. Allele and sequence type designations were determined via the Salmonella MLST database website (http://mlst.warwick.ac.uk/mlst/dbs/Senterica). Neighbor-joining trees were constructed using the Kimura two-parameter model of nucleotide substitution with the MEGA3 software [lacIZY and MLST sequences.DNA sequences of the nd thrA)  through software , and theS. enterica strains 50259, 50260, 50261, 50262, 50263, 50264, 50265, 50267, 50268, 50269, 50270, 50271, and 50272 were deposited at DDBJ/EMBL/GenBank under accession nos. JZTJ00000000, JZTK00000000, JZTL00000000, JZTM00000000, JZTN00000000, JZTO00000000, JZTP00000000, JZTQ00000000, JZTR00000000, JZTS00000000, JZTT00000000, JZTU00000000 and JZTV00000000, respectively.The draft genome sequences of the


Anti-glycan antibodies are an abundant subpopulation of serum antibodies with critical functions in many immune processes. Changes in the levels of these antibodies can occur with the onset of disease, exposure to pathogens, or vaccination. As a result, there has been significant interest in exploiting anti-glycan antibodies as biomarkers for many diseases. Serum contains a mixture of anti-glycan antibodies that can recognize the same antigen, and competition for binding can potentially influence the detection of antibody subpopulations that are more relevant to disease processes. The most abundant antibody isotypes in serum are IgG, IgM, and IgA, but little is known regarding how these different isotypes compete for the same glycan antigen. In this study, we developed a multiplexed glycan microarray assay and applied it to evaluate how different isotypes of anti-glycan antibodies  compete for printed glycan antigens. While IgG and IgA antibodies typically outcompete IgM for peptide or protein antigens, we found that IgM outcompete IgG and IgA for many glycan antigens. To illustrate the importance of this effect, we provide evidence that IgM competition can account for the unexpected observation that IgG of certain antigen specificities appear to be preferentially transported from mothers to fetuses. We demonstrate that IgM in maternal sera compete with IgG resulting in lower than expected IgG signals. Since cord blood contains very low levels of IgM, competition only affects maternal IgG signals, making it appear as though certain IgG antibodies are higher in cord blood than matched maternal blood. Taken together, the results highlight the importance of competition for studies involving anti-glycan antibodies. Human serum contains a vast assortment of carbohydrate-binding antibodies that play critical roles in human health and provides a rich reservoir of potential biomarkers for many biomedical applications and diseases. For example, detection of anti-glycan antibodies against blood group A and B antigens provides a simple and reliable strategy to predict which individuals are suitable matches for transfusions and transplants –3. Anti-Detection of serum anti-glycan antibodies is typically carried out by immobilizing a carbohydrate of interest, capturing specific antibodies, and then measuring amounts of bound antibodies. This process is complicated by the fact that serum often contains a mixture of antibodies that recognize the same antigen, and certain antibodies within the mixture may be more relevant to immune protection or disease processes than others. Antibodies to a particular glycan may vary in terms of affinity, specificity, concentration, and/or isotype, but all can compete for binding to the same antigen. As a result, binding of one can influence detection of the others, and it can be challenging to reliably measure a target antibody subpopulation of interest.One of the best examples illustrating the complexities of competition is detection of one antigen-specific antibody isotype in the presence of others. Different isotypes have distinct biological functions, and detection of different isotypes to a given antigen is critical for understanding the immune response and developing diagnostics. The most abundant antibody isotypes in serum are IgG, IgM, and IgA. Due to the affinity maturation and class switching processes, IgG and IgA antibodies typically have substantially higher affinities than IgM antibodies ,19. ConsCarbohydrates are a unique class of antigens with unusual molecular recognition features. Unlike protein antigen, monovalent interactions between a single binding site of an antibody and a single glycan are generally weak. Even for affinity-matured IgG antibodies, monovalent binding dissociation constants for glycans are typically in the range of 100 nM to 100 μM. As a result, avidity effects play a more important role in achieving tight binding. Serum IgM antibodies are pentameric and have 10 binding sites whereas serum IgG and IgA are mainly monomeric with 2 binding sites. With more binding sites, carbohydrate-binding IgM have the potential to outcompete IgG and IgA through enhanced avidity. For the vast majority of serum anti-glycan antibodies, it is not known whether IgG, IgA, or IgM antibodies have a competitive advantage, or if the competitive advantage varies from one antigen-antibody pair to another.Glycan array technology provides a powerful high-throughput tool for studying the interactions between carbohydrates and macromolecules, and to identify anti-glycan antibodies as biomarkers for variety of diseases –31. It aOur approach for evaluating competition involved the use of purified IgG, IgA, and IgM antibodies from pooled human serum. Each polyclonal antibody sample would be profiled on our glycan microarray individually and in the presence of other isotypes. Additionally, changes in IgG and IgM anti-glycan antibody signals would be evaluated in whole serum upon addition of IgG, IgA, and IgM. Although IgD and IgE are also present in serum at low concentrations and capable of competing, this study focused on the highest abundance antibodies in serum.1-IgG4)  of purified serum antibodies  were spotted onto nitrocellulose membranes and were detected by their corresponding secondary antibodies. The relative signals obtained for the IgM secondary were 20–30% and 45–55% higher than that of IgG and IgA, respectively : IgG 9.9 (6.4–13.5) mg/mL, IgA 1.7 (0.7–3.1) mg/mL, and IgM 1.6 (0.6–3.5) mg/mL]. For thePurified IgG, IgA, and IgM were first profiled individually at various concentrations in the absence of other isotypes. IgM provided high signals to numerous glycans on the array  in the presence of 4 serum equivalents of IgM  upon adding 200 μg/mL of IgA. Even with the addition of 400 μg/mL of IgA, only 2 IgG signals (1.5%) decreased by about 2-fold.Purified IgG was profiled at 200 μg/mL  in presence of varying amounts of added IgM or IgA. IgG signals decreased significantly (p > 0.3). Upon the addition of 400 μg/mL IgA, 6 IgM signals (2.5%) decreased by about 2-fold.Purified IgM was profiled at 50 μg/mL  in presence of varying amounts of added IgG or IgA. Interestingly, addition of IgG or IgA had little or no effect on the IgM signals . For exaIt is important to note that the data showed no evidence for interference by rheumatoid factors (RF) for our samples. If IgM-class RF are present, one can observe false positives  or general increases in IgM signal upon addition of IgG due to the detection of both anti-glycan IgM and IgM-class RF that bind anti-glycan IgGs. The measured IgM signals were the same in the absence or presence of IgG or IgA antibodies, indicating that our purified IgM sample does not contain IgM-class RF. Moreover, none of the glycans gave rise to IgM signals when profiling purified IgG, indicating the absence of IgM-class RF in the purified IgG sample.p < 1.0 x 10−9) in the presence of 4 serum equivalent of IgM (p = 0.004) but to a lesser degree than IgM. Upon addition of four “serum equivalents” (800 μg/mL) of IgG, about 1/4 of all the IgA signals that were at least 4-fold above the floor decreased by 2-fold or greater  in presence of varying amounts of added IgG or IgM. Similar to IgG, IgA signals decreased significantly . In contrast, the IgM signals were relatively unchanged upon the addition of IgG or IgA. Even upon addition of up to 500 μg/mL of IgG and 200 μg/mL IgA, we did not observe significant changes in the overall IgM signals (p > 0.4). Only about 14% of IgM signals that were at least 4-fold above the floor decreased by 2-fold or greater upon the addition of 1000 μg/mL IgG. Similarly, About 12% of IgM signals decreased by 2-fold or greater upon the addition of 400 μg/mL IgA. Taken together, these results demonstrate that many anti-glycan IgM outcompete IgG in the context of human serum.Considering the findings that IgM anti-glycan antibodies appeared to inhibit IgG and IgA reactivity toward printed glycan antigens, we sought to verify these changes in pooled reference serum sample. We profiled IgG and IgM anti-glycan antibodies in our “reference” serum (1:50) in the presence or absence of varying amounts of added IgG, IgA, or IgM. As illustrated in Although fetuses produce little or no IgG, fetal IgG levels and diversity in serum at birth are nearly equivalent with maternal IgGs due to active transport of maternal IgG to the fetus . MaternaIn an ongoing project within our group, we have been comparing anti-glycan antibody profiles of matched cord and maternal sera. Like previous reports, we found that IgG antibodies to certain glycans were detected at higher levels in cord blood relative to maternal blood. Although preferential transport could account for this difference, we noticed that many of the “enriched” IgG antibodies recognized glycans that are also bound well by IgM. This observation led us to postulate an alternative explanation as to why certain IgG signals are higher in cord versus maternal blood. Rather than preferential transport, we hypothesized that IgG levels are approximately equivalent in cord and maternal sera and that competition with IgM in maternal blood results in reduced IgG signals. Since fetal serum has very low levels of IgM (IgM and IgA are not transported), the reduction in IgG signals only occurs in maternal blood. As IgM levels and avidities vary from antigen-to-antigen, certain IgG signals are strongly inhibited while others are not affected making it appear as though some IgG antibodies are selectively enriched in fetuses.To test this hypothesis, we compared matched cord and maternal sera from 3 mother-baby pairs with and without depletion of IgM. We anticipated that removal of serum IgM would restore IgG binding leading to similar IgG signals in cord-maternal pairs. Cord and maternal serum samples were either treated with anti-IgM resin or control resin and then signals were compared. Samples treated with control resin had a number of antibody signals higher in cord than maternal blood. For these three serum pairs, there were a combined total of 102 IgG signals  that were at least 4-fold higher in cord relative to maternal sera, and 33 IgG signals that were at least 10-fold higher in cord sera. Interestingly, many of the signals that were higher in cord sera were antibodies to non-human glycans . More specifically, 37 of the 102 signals that were 4-fold higher in cord and 20 of the 33 signals that were 10-fold higher in cord were to non-human glycans. Adult humans have especially high IgM signals to non-human glycans, which could result in high levels of competition for this family of antibodies.Next we evaluated IgM and IgG signals after treatment with anti-IgM resin. Depletion of IgM was verified by comparing anti-glycan IgM signals for anti-IgM resin-treated and control resin-treated samples. As expected, treatment of sera with anti-IgM resin resulted in a large decrease in all IgM signals ; most (>80%) glycans had no measurable IgM signal over background. Interestingly, when we compared IgG signals for cord/maternal pairs after IgM depletion, none of the cord IgG signals were >4-fold higher than maternal IgG (as compared to 102 for the control resin). The absence of differences between cord and maternal IgG profiles after IgM depletion was due to increases in various maternal IgG signals, rather than decreases in cord IgG signals. It is important to note that anti-IgM resin treatment had little to no effect on serum IgG signals for cord samples, indicating that the resin does not directly affect IgG antibodies. Since depletion of IgM abolished the “enriched” IgG signals in cord blood, we believe the observed effect is due to IgM competition, at least for the anti-glycan antibodies evaluated in this study.There is growing interest in studying the biological roles of anti-glycan antibodies and exploiting this family of antibodies as biomarkers. Recently, glycan microarray technology has emerged as a powerful high-throughput tool for profiling serum anti-glycan antibodies  with essentially no effect on IgM signals. Therefore, the proportion of IgM that are glycan-binders would have to be >16 times higher than the proportion of IgG glycan binders to account for the competitive advantage of IgM. Alternatively, IgM antibodies may have higher overall binding avidity toward glycan antigens than IgG and IgA. In other studies we have found that monoclonal IgM antibodies can bind very tightly to glycans on our array surface, with apparent Kd values in the low nanomolar to subnanomolar range ,75. AddiIn summary, we have demonstrated that anti-glycan IgM can outcompete IgG and IgA for surface-bound carbohydrate antigen in a microarray assay. Accurate measures of glycan-binding IgG and IgA are critical for studying immune responses to carbohydrate antigens and for identifying serum antibody subpopulations with utility as biomarkers. By accounting for IgM competition, one can obtain a more true measure of anti-glycan IgG and IgA levels, thus facilitating both basic research and clinical applications in the field of glyco-immunology. Finally, our results highlight the unique aspects of antibody-carbohydrate interactions and emphasize the need to consider these interactions separately from antibody-protein interactions.1 I5154, IgG2 I5404, IgG3 I5654, IgG4 I4639) were purchased from Sigma-Aldrich . The pooled reference serum was purchased from Valley Biomedical Products and Services . Samples were tested in accordance with FDA regulations and found to be negative for HBSAG, HIV, and HCV. Sera of three matched cord-maternal pairs were purchased from ProMedDx .Purified serum antibodies  and all the different IgG subclasses (IgGPurified serum antibodies were heat denatured in 50% Laemmli sample buffer (Bio-Rad #161-0737) and 2.5% β-mercaptoethanol. The denatured proteins and molecular weight markers were loaded into a Novex 4–20% Tris-Glycine gel. The proteins were separated by gel electrophoresis using 1X NuPAGE MES SDS running buffer at constant voltage (125 V) for 1.5 hrs. The separated proteins were transferred to nitrocellulose membrane and blocked with StartingBlock blocking buffer (Thermo Scientific 37538) for 2hrs. The membrane was rinsed 3 times with PBST (PBS with 0.05% (v/v) Tween 20) and then incubated for 1hr with the proper goat anti-human detection reagent  diluted 1:500 in the blocking buffer. The membrane was washed 6 times with PBST and imaged with ImageQuant LAS 400.The diluted working concentrations from all samples were prepared in phosphate-buffered saline . From the diluted solutions 2.0 μL were spotted in duplicate onto a nitrocellulose membrane and were allowed to dry at room temperature. The membrane was block with StartingBlock blocking buffer for 2hrs. The membrane was rinsed 3 times with PBST and then incubated for 1hr with the proper goat anti-human detection reagent  diluted 1:500 in the blocking buffer. The membrane was washed 6 times with PBST, imaged with ImageQuant LAS 400, and analyzed with Genepix Pro 6.0 software to obtain the fluorescence intensity signals.Glycan arrays were fabricated as previously reported  except fSlides were scanned at 10 μm resolution with a Genepix 4000B microarray scanner (Molecular Devices Corporation) and analyzed with Genepix Pro 6.0 software as previously reported . The spoAnti-human IgM agarose (Sigma A9935) was used for IgM depletion and Sepharose 4B  was used as control resin. Both resins were equilibrated on column cartridges, washed with 5× PBS buffer and then mixed with equal column volume of PBS buffer. For IgM depletion assays, 110 uL anti-human IgM agarose solution was mixed with 110 uL cord or maternal serum samples . In the control assays, 110 uL Sepharose 4B was mixed with 110 uL diluted sera. These solutions were incubated at room temperature for 1.5 h with gentle agitation (150 rpm). The resins were centrifuged at 10,000×g for 5 min, and the serum supernatant (∼130 μL/sample) was collected for antibody profiling the glycan microarray. Both cord and maternal sera were treated using the same method.Each sample was evaluated on a glycan microarray in duplicate as described above with the following differences: the volume of serum samples was 60 μL/well, the slides were scanned with a GenePix 4000B fluorescent scanner (Molecular Devices), and fluorescent intensity for each array component was quantified using GenePix Pro 7.0 software (Molecular Devices). Since these samples were evaluated at a dilution of 1:8, any signals lower than 900 RFU were adjusted to 900 (floor).S1 File(DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


In 2007, a non-contributory pension program was launched in rural areas of Mexico. The program consisted in a non-conditional cash transfer of US$40 monthly to all older adults (OA) aged 70 and over. We evaluate the effect of the program on mental well-being of its beneficiaries.Quantitative and qualitative methods were used. For the quantitative component, we used the selection criteria established by the program  to form the Intervention  and Control groups . Baseline data collection was conducted in 2007 where 5,465 OA were interviewed. The follow-up survey was conducted in 2008, and it was possible to interview 5,270 OA, with a response rate of 96%. A difference-in-difference linear probability model with individual fixed effect was used to estimate the impact of the program on mental well-being indicators. In 2009 a qualitative component was designed to explore possible causal pathways of such effect.95% −0.12; −0.01) and an increase in empowerment indicators: OA participated in important household decisions ; and OA participated in household decisions pertaining to expenses . Qualitative analysis found a strong trend showing a reduction of sadness, and feeling of increasing empowerment.After a year of exposure, the program had a significant effect on reduction of depressive symptoms (β = −0.06, CIThese results suggest that a non-conditional transfer in older ages have an impact beyond the economic sphere, impacting even the mental well-being. This effect could be explained because the pension produces feelings of safety and welfare. It is recommendable that governments should invest efforts towards universalizing the non-contributory pension programs in order to ensure a basic income for the elderly. One of the most important demographic challenges expected for low and middle-income countries in the 21st century will be the increase of the number of older adults (OA) and the pressure this will have on social security systems, available medical assistance, and service demand for elderly care. Moreover, estimates of health problems and disability suggest OA in these countries are aging with more functional limitations and worse health conditions than OA in developed countries Aging process in low and middle-income countries is characterized by the presence of poverty and inequality. In these countries, poverty among OA (60 years and over) is higher than that for the entire population Poverty in old age is also an important problem in low and middle-income countries. Studies in 15 low-income countries in sub-Saharan Africa report households with an elderly individual had higher levels of poverty that general population In terms of income distribution, Latin America is considered the world's most imbalanced region The majority of uninsured and retired individuals lives and works in low and middle-income countries. In Latin America, over 30% of the OA do not report retirement, pension, or employment income The rapid growth of the OA population in low and middle-income countries and the low coverage of social security reinforce the need to adapt social protection systems at a faster rate than the developed countries did The most distinctive characteristics of noncontributory social pensions are that they are designed to be accessible as a right to all those who meet the requirements of the program and that the grant conditions are unrelated to work experience or the history of the market The results from the evaluation studies suggest that noncontributory social pension programs have had a major impact in reducing poverty in old age, as well as the incidence of extreme poverty, and a positive effect on reducing indigence 70 y más on mental well-being of its beneficiaries.Given the currently gap in the evaluation literature regarding the impact of economic transfer programs on the OA in other areas more than just economic effects, our objective was to estimate the impact of the non-contributory pension program 70 y más was aimed at improving the living conditions among adults aged 70 years and older by boosting their social protection through policy mechanisms. Centered on two components, 70 y más pursues a twofold objective: (1) to raise the income of the elderly and (2) to improve the social protection of the elderly. At the start of the program in 2007, the program had enrolled a total of one million beneficiaries and had a total annual budget of 6,250 million Mexican pesos (approximately US$595 million). In 2009 the number of beneficiaries had grown to 1.8 million, and the total budget had more than doubled to 13,000 million Mexican pesos  Implemented nationally throughout Mexico, Program Under the first program objective, OA receive a direct unconditional cash transfer of 500 Mexican pesos (approximately US$40) every month, which can be collected every two months. At the start of the program in 2007, the established eligibility criteria included being 70 years and over and residing in a locality with 2,500 or less inhabitants . It should be noted that our evaluation is based on the two eligibility criteria and does not account for the fact that the program expanded the eligibility criteria to those residing in a locality up to 30,000 inhabitants after 2011 The second program objective related to health-oriented social participation and social protection actions was late to start, and during the period of this evaluation, it only had enrolled a very small percentage of beneficiaries. For that reason, this work will focus on evaluating the impact of the program's cash transfer feature only.The Research and Ethics Committees of Mexico's National Institute of Public Health approved the original study. Participants received a detailed explanation of the procedures and signed an informed consent declaration before data collection occurred.70 y más was derived from both quantitative and qualitative analyses of data. Both components  of the study are briefly presented in the next paragraphs.The estimated impact of 70 y más established two eligibility criteria: (1) beneficiaries had to be 70 years old and over and (2) had to reside in localities of 2,500 or less inhabitants. We used these eligibility criteria to design our impact evaluation study and to identify the group exposed to the program  and to establish three control groups: Group 1 ; Group 2 , and Group 3 . Details about intervention and control group selection can be found in Appendix S1 in In 2007, at the start of the program, Sample size was determined according to 35 indicators related to household characteristics, household expenditures, health conditions of the OA, and use of healthcare services. Sample size was calculated using unilateral statistical tests with a 95% confidence level and a power of 80%. Additionally, the sample size calculation accounted for several scenarios based on the effect of the program, including expected effects of 1, 2, 3, 4, 5, 10, 15, and 20 percentage points for each of the 35 indicators proposed. The results of the analysis revealed that a sample size of 1,500 elderly per evaluation group would suffice to detect program effects of up to 4 or 5 percentage points in all of the variables.The baseline survey was conducted in October-December 2007 among a total of 516 localities in seven states of Mexico. Out of the targeted 6,000 interviews of OA, 5,465 OA (a 91% response rate) were interviewed during baseline. The follow-up survey was conducted in November-December 2008, and it was possible to locate and interview 5,270 OA, or 96% of the OA interviewed at baseline.70 y más program. As can be seen from the size of the originally estimated sample , 5,465 OA were interviewed at baseline, and 5,270 of these were interviewed during follow-up. The final analytical sample was defined based on the common rule from studies on OA that only mentally apt individuals or their caregivers (defined as the people who provide assistance to the OA in their basic and daily activities) can respond to survey questions. By this definition, caregivers can actually answer all questions related to the OA  except for the ones related to perception. For instance, a caregiver should not answer a question which requests information on the OA's emotions. Thus, any survey questions where the OA have to express their feelings or emotions are not answered by caregivers. Thus, of the 5,270 observations existing in both baseline and follow-up  recommendation on active aging One of the most immediate and likely effects of the program 7Assuming that our evaluation design succeeded in replicating the environmental conditions of the program and its beneficiaries, it was only necessary to carry out a simple comparison between the average of any indicator of interest for the intervention group and the average of that same indicator for the control group to estimate the program effect. However, this assumption can bias the results greatly, since it is possible that not all observable and unobservable differences between the intervention and control groups will have been removed by our design.For this reason, we took advantage of the differences-in-differences (DD) model to estimate the program effect. Instead of analyzing the differences between the variables across treatment and control, this model allows us to analyze the differences in change between treatment and control groups by accounting for two types of potential differences between the groups: (1) the differences that existed prior to the intervention (i.e. at baseline or pre-intervention) between treatment and control groups, and (2) the differences arising from unobserved factors at the local level that do not change between baseline and follow-up data collection, which in this case is 2007 and 2008. The DD model is then based on the assumption that in the absence of the program, the change observed in the intervention group would have been the same as the change observed in the control group, or more succinctly, the trends of both groups would be equal. If there were differences between the groups for unobserved characteristics that vary over time and these were associated with program exposure, the DD model would generate biased estimates of the program effect. However, it is expected that the DD model removes a large proportion of the possible causes of bias in its estimates.ijt is an outcome variable for individual i who lives in locality j at time t. Tijt is an indicator variable that takes a value of 1 if the measurement of individual i is in the post-intervention survey (2008) or 0 if it is in the baseline survey (2007). Pij represents an indicator variable that takes the value of 1 if individual i belongs to the intervention group or 0 if he or she belongs to the control group 1, while the term (Tijt*Pij) represents the estimate of the program impact, X it's a time-varying covariates vector, μi represents a fixed effect at individual level, and εijt is the error term.The general DD linear probability model for estimating the impact of the program is specified as follows:p<0.05, and considered marginally significant if 0.05<p<0.10. All analyses were performed using STATA 13.1.The DD model permits the identification of the treatment effect under the assumption that the change in the treated group in the absence of the program would have been the same as the observed change in the control group. When applying this model, the DD model already controls for fixed characteristics over time, thus, only time-varying covariates will be used, both individual/household and locality levels. Differences were considered statistically significant if We also conducted additional analyses to complement the DD models (with fixed effects) in combination with propensity score matching as a strategy to verify the robustness of our results, and we verified the assumption of parallelism regarding the DD models. The results of these analyses and their rationale are presented in the Appendix S2 in The qualitative study was conducted in January-February 2009 and was based on the data collected during the baseline measurement of the quantitative component. The objectives and research questions were established in accordance with the quantitative component in order to utilize the qualitative exploration to generate a complementary and expanded triangulation of the results.The qualitative sample was purposively selected with maximum variation criteria to achieve maximum representation of the different subgroups observed We included OA that have been captured in the quantitative baseline survey. Four localities were selected in two of the seven participating states at baseline. To select the participating states, certain criteria were used on structural characteristics that could determine the experience of the beneficiaries with respect to the program. Selected states shared the same levels of deprivation, migratory rates, and indigenous population proportion. For the selection of localities, the difficulty of accessing health services and the ethnicity composition of its inhabitants were also considered so that the perceived experience of the program could be determined by those characteristics. Sampling scheme for qualitative component can be found in Appendix S1 in Total sample consisted of: 129 semi-structured face to face interviews: 99 program beneficiaries; 16 potential beneficiaries; and 6 suspended OA beneficiaries; 8 interviews with local key actors , and 4 observations from support delivery, one in each selected locality. No refusals were faced during fieldwork.Interview guide had four sections, self-perception of mental and physical health, use of program's transfer and decision taking over the money, self-perception of social network  and evaluation of perceived impact of the program. Interview guide was piloted with 5 OA in a non-selected locality.The fieldwork was conducted in February-April 2009 by a team of five female cultural anthropologists (two of them with M.A.) who lived in the study sites during data collection. In those localities where OA only spoke indigenous language three female indigenous translators helped in order to translate questions and answers for the interviews. All team members were previously trained and have extensively experience doing fieldwork. All interviews and observations were audio-recorded, previous signed of the informed consent, and totally transcribed along with the ethnographic notes, and recorded in field diaries. Notes from the field diaries underwent content analysis The information generated by the qualitative component of the study consists of ethnographic data composed of selected excerpts from the transcripts collected from semi-structured interviews The interviews were coded by all team doing fieldwork using pre-defined analytical codes, and live or empirical codes using the program NVivo 2. A content analysis Additionally, by using triangulation, the internal validity of the data was achieved. By stating the limitations of the qualitative data implicit in the variation of the characteristics of the participants involved in the qualitative exploration (e.g. men and women belonging to indigenous and mixed communities in two states of the country), we sought to create reflexivity to reduce selection bias.The quantitative baseline findings can be found in The findings for the depressive symptoms indicator show the program had a significant overall effect on them. The negative value of the associated coefficient , and now yes, now we have this. I do feel better.Q. And about how you feel, do you feel any change?R. No, I feel happy (laughter). I do, maybe there is a person who is not happy, but I do feel happy.Q. And do you think that how you feel is related somehow to the program, or is it because you are indeed a happy kind of person anyway?R. Well, indeed I did not feel sad, I was ashamed because the money was not enough to buy needed things, and then, when I got sick, I used to have to beg for money. But now, at least we have some money. If I get sick at least I have to buy medicine that [the health services] don't have. On the other hand, the indigenous man expresses his joy at receiving the program:Q. Please ask him how he feels now that he is receiving the program? How does he feel about getting [the support], does he feel comfortable, quiet, or does he feel bad or stressed, how does he feel?Translator R. (Laughter) He says that when he receives the support he feels happy and brings some meat and more things. When he is home, he meets my godmother [elder's wife], close together, happy because they receive the support.Q. And now, finally I only wanted to ask if he feels that after receiving this money has something changed in his health and mood?Translator R. He feels better because before that support, he had no support, but since he already has support he feels better, more comfortable. But before, he says he did not, because sometimes he had no money to buy a little something for the kitchen. Well now he feels better because he already has the support. Many of OA declared that pension was their only source of income at the moment. Moreover, because of the conditions in rural areas, which can also be characteristically peasantry, where most of the beneficiaries had lived their entire lives, receiving a stable and fixed monetary income was declared by OA, especially women, to be an event which had never before been experienced. This suggests that there was a significant perceived impact for these beneficiaries.participates in household decision making 70 y más is the first economic transaction of their entire lives, which has very revealing impact implications on autonomy, and empowerment. These women expressed in the semi structured interviews how the money gave both parts, themselves and their children, a kind of relief, feeling better for not only reduce the economic burden in their children, but gaining independence in terms of how to spend their own money.Having one's own resources also gives a sense of economic independence for OA who in many cases are financially dependent on their children, whose own families demand the majority of their resources. For many women, the money they receive through the program In these interviews a man and a woman express their economic independence from their networks and children.R. I, what I have left, I keep it [for savings] and… when I'm sick, well, not telling anyone to borrow me money. Q. and how do you feel now that you have the Program?R. Oh! It is a great help, because I don't have to ask my children anymore for help.Q. And about them, how do you feel they feel about this?R. I believe that they are ok with this. I say it because they do not worry anymore. They are not thinking that they have to help me. reduce suffering', 'give themselves value', 'increase happiness', as well as to improve the emotional health.So then, by being able to offer to their household financial support to purchase food, OA creates reciprocity among they and their families obtaining acknowledgement from the household members and are empowering themselves to decide on issues that concern their lives, as well as the collective life of the household. In general, the diverse elements described around the noncontributory pensions contribute in different ways through complex social mechanisms to 'In the narrative of this indigenous woman reciprocity and empowerment are feelings expressed in relation to the program:T. Mmm… She says that she feels good with the 70 y más program. She feels that it has helped her. She says she feels more important with this money, mainly in relation to her family because with this money she can help her daughter a little bit. She also says that she helps her daughter in embroideries; when her daughter cannot finish them, she helps her.Q. And could you ask her what the most important thing is that she feels she gives her family?T. She says that she cannot give them more than sharing her food. She says she always cooks for her grandchildren because they help her, too. When [her grandchildren] come, they give her some 1000 pesos, or they bring some [soda beverage] or some clothes. She says that it is because that she always prepares some food for them. That is all of what she gives them. 70 y más, given its eligibility criteria, is that OA a little younger than 70 years old and residing in localities with less than 2,500 inhabitants could modify some of their habits and behaviors related to income, and at the same time, they may be able to modify some dietary habits or some of their social, family-related, or personal expectations, particularly in terms of their emotional health. Although it has been referred to as different names in the literature, this process can be designated as the anticipation effect.A possible effect of the program An advantage of our evaluation design is that we can identify the presence, or absence, of a potential anticipation effect and its magnitude. These reflect not only the extent the program would have on impacting future beneficiaries, but also a generalized idea of the real magnitude of the program effect. In order to estimate the anticipation effect, the same specification of our models described earlier was used with the inclusion of OA aged 65 to 69 years old from control groups 2 and 3. For depressive symptoms  we obserResults of the analysis that included OA aged 69 (and who were in the control group at baseline) in the intervention group are in the sensitivity analysis panel of For the mental well-being outcomes using DD model estimates combined with the propensity score matching algorithms the results can be found in 70 y más could have an impact, related to the economic transfer, on the analyzed mental well-being indicators, because the transfer can be seen as a component of the SES among OA beneficiaries. The most conclusive result found in our analyses was the effect of the program on the presence of depressive symptoms, which can be interpreted from the OA transcripts. This is a very important finding, since of all mental health problems experienced by OA, the most important based on prevalence is depression. This issue is even more pressing because depression is associated with increased mortality and suicide, as well as morbidity, in terms of functional dependence In our analysis context, we hypothesized that the program This same argument could be generalized as follows. If non-contributory pensions have a positive effect on economic and well-being indicators A significant effect was also observed for the OA empowerment, measured through the decision making at home and decision making on household expenditure, which can be found in the literature related to empowerment Following a constructivist interpretative framework The interviews with OA help us to understand the mechanisms behind income appropriation and decision making within the household. Furthermore, the results presented here indicate that social mechanisms are at play under which the beneficiaries experience an effect from the pension that redefines the meaning of money in their lives. Thus, besides meaning a perceived reduction of poverty, the pension the OA receive has a positive effect on self-determination and decision making at both the individual and household levels. This decision-making power is translated into economic autonomy, self-sense of value, feeling of reciprocity and worth and the reduction of economic dependence on their children.no longer suffering', 'feeling valuable', and 'being happy'.In turn, OA share or redistribute the income they receive across their household as found in the study by Marquez-Serrano Despite the beneficial aspect that is associated with increasing levels of decision making, it is also possible that there is a downside. For example, it is possible that older adults perceive the decision as a burden, as they lack strong social networks. While this is something that could not be analyzed in this study, it is important that future research will address this issue.A central finding of the study was related to the use of the pension. The fact that OA make their own decisions on how and on what to spend their money is a key element linked to the feelings of empowerment described earlier. Decision making and the effects of decisions make up the key mechanisms that largely explain the feelings of reduced sadness, which can be associated with depressive symptoms.70 y más program pension, the family dynamics can begin to change towards greater respect and consideration for the potential OA beneficiary, because they anticipate an economic transfer and along with it, potential economic benefits for all household members derived from the OA's decision to share their new income. However, it is important to emphasize that family dynamics may also be modified in an undesired manner. For example, it is possible that household members starting to treat the OA better knowing that in the short term could take the pension money. This is an issue that also must be analyzed in future research.Regarding anticipation effects it can be argued that since empowerment is associated with a higher income among OA, it is possible that in households where an OA is about to receive the 70 y más beneficiary would not have had a modification to empowerment. In fact, and in our estimates, the difference observed between beneficiaries and non-beneficiaries would be larger.Beyond this discussion, presence of anticipation effects implies that the program's impact would be even greater, since the absence of expectations of becoming a 70 y más program has on various indicators of mental well-being in OA residing in rural areas of Mexico.Using matching techniques in our analysis confirms the robustness of our results regarding the significant effect that the We have mentioned that a great body of evidence on non-contributory pension's impact has mainly focused on socio economic indicators. This is an important flaw because research indicates that in old age health mental problems are highly prevalent among OA. Due to its devastating consequences, mental health represents an important public health issue Even so, some studies have analyzed the effect of non-contributory pension on OA's health status. A first set of studies demonstrated that the South African pension improved the self-report health status of its beneficiaries and the co-residing members of the household 70 y más impact is a local estimator; this means that the observed effects are valid only for OA aged 70–74. So it is still pending analyze the impact of the program in older ages. Second, since 70 y más started in rural localities, our analytical sample is also restricted to rural areas of Mexico, so nothing is known about the program's effect in urban areas where the program also operates nowadays. Third, we just had a short time of the exposure to pension (11 or 12 months), so our impact estimate reflects only an immediate effect, and it is important to determine the medium and long terms effects of the program. And fourth, the qualitative component of the study does not have a baseline measure and only took into account OA beneficiaries of 70 y más. Even and when there is a debate about the relevance of using a control group from a constructivist point of view, the study could be have more robust results if qualitative component had been included a control group and/or a baseline measurement.Several limitations can be noted in our study. First, given we used a discontinuity regression approach to form our comparison groups, our estimate of the Finally, some of our results showed that if an intervention were implemented to increase the income and the SES of OA, some mental health outcomes may change, just as proposed in the literature File S1This file contains Appendix S1, Appendix S2, Table S1, Table S2, Table S3, Table S4 and Table S5. Appendix S1, Intervention and control group selection and Sampling scheme for qualitative component. Appendix S2, Propensity score matching and Parallelism assumption of DD model. Table S1, Probit regression to predict enrollment in 70 y más program. Table S2, Results of matching using caliper algorithm. Table S3, Results of matching using kernel algorithm. Table S4, Testing the parallelism assumption: alternative control groups. Table S5, Testing the parallelism assumption: alternative outcomes.(DOCX)Click here for additional data file.