E. coli by expressing the StrepTagII antigen into outer membrane protein X (OmpX) and validated that these engineered bacteria were recognised by anti-StrepTagII antibodies. We then combined this antigen–antibody system with a purified complement assay to avoid interference of serum components and directly compare MAC-mediated bacterial killing via IgG1 and pentameric IgM. While both IgG1 and IgM could induce MAC-mediated killing, we show that IgM has an increased capacity to induce complement-mediated killing of E. coli compared to IgG1. While Fc mutations that enhance IgG clustering after target binding could not improve MAC formation, mutations that cause formation of pre-assembled IgG hexamers enhanced the complement activating capacity of IgG1. Altogether, we here present a system to study antibody-dependent complement activation on E. coli and show IgM’s enhanced capacity over IgG to induce complement-mediated lysis of E. coli.Antibodies play a key role in the immune defence against Gram-negative bacteria. After binding to bacterial surface antigens, IgG and IgM can activate the complement system and trigger formation of lytic membrane attack complex (MAC) pores. Molecular studies to compare functional activity of antibodies on bacteria are hampered by the limited availability of well-defined antibodies against bacterial surface antigens. Therefore, we genetically engineered  This cell envelope structure consists of an inner membrane, a peptidoglycan layer, and on top an outer membrane decorated with outer membrane proteins and lipopolysaccharides (LPS)2. The broad diversity of proteins and sugars displayed on Gram-negative bacteria are potential targets for antibodies. After antibodies bind their target, they can induce effector functions that range from neutralisation of the bacteria and activation of immune cells to the activation of the complement system3. Activation of the complement system on Gram-negative bacteria results in the attraction of phagocytes, opsonisation, and the direct killing of the bacteria via the formation of membrane attack complex (MAC) pores4. These MAC pores damage the bacterial outer membrane, leading to inner membrane damage and subsequent cell death. MAC pores also enable other immune components or antibiotics to pass the outer membrane and reach their inner membrane- or peptidoglycan-associated targets7. Complement activation is therefore an important effector function of antibodies directed against Gram-negative bacteria. However, our understanding of what renders antibodies efficacious in inducing effector functions on bacteria is limited.Antibodies play a key role in the immune defence against Gram-negative bacterial infections. Gram-negative bacteria are notoriously difficult to combat because of their complex cell envelope structure8. Antibodies are proteins that all share a Y-shaped structure with two functional domains. The Y-stem is the crystallisable fragment (Fc) and the two arms are the antigen-binding fragments (Fab), which are all linked together with a flexible hinge region. Antibody isotypes differ in their composition of the basic structure and Fc structures and therefore vary in molecular weight and function. IgG has a molecular weight of 150 kDa, is the most prevalent isotype in human serum, and occurs in four subclasses, IgG1-4. IgM is the first isotype to be produced during an infection, mainly occurs in pentameric form, and has a molecular weight of approximately 1000 kDa11. Of the five antibody isotypes, only IgG1-3 and IgM can activate the complement system via the classical pathway (CP). The CP initiates when the large C1q protein with six collagen arms binds to the Fc domains of an antibody-antigen complex. After C1q binds to antibodies, the C1r and C1s of the C1-complex activate and convert C4 and C2 into a C3-convertase (C4bC2b)12. This C3-convertase then converts C3 and deposits C3b on the bacterial surface. At high C3b density, C3 convertases switch substrate specificity to form C5 convertases that cleave C5 into C5a and C5b. C5b subsequently interacts with C6, C7, C8 and multiple copies of C9 to form MAC pores (C5b-9) that can directly kill Gram-negative bacteria14.In humans, antibodies occur in five different isotypes: IgG, IgM, IgA, IgE, and IgD19. Recent studies with monoclonal antibodies have shed light on the molecular dynamics of IgG hexamerisation and subsequent complement activation20. The importance of IgG hexamerisation for complement activation on bacteria is highlighted by engineering strategies improving hexamerisation that in turn enhance complement activation on bacteria26. In contrast to IgG, IgM has a multimeric structure and when it binds an antigen, it can directly bind C1q. Multiple lines of evidence have suggested that IgM is a more potent complement activator than IgG34. However, the comparison between the complement-activating capacity of IgM and IgG is mainly based on studies with polyclonal or chimeric IgM. Due to the size and multimeric structure of IgM, it has been challenging to produce recombinant monoclonal IgM. Recent advances now enable the production of recombinant monoclonal IgM which allows us to produce IgG and IgM molecules with the same target35. Previously we have studied the mechanisms of MAC-mediated killing on Escherichia coli as a model for Gram-negative bacteria37. However, the limited number of well-defined monoclonal antibody sequences that target E. coli structures hampers the study of how efficiently different antibody isotypes and subclasses activate the complement system on these bacterial membranes.In order for IgG to activate the complement system via the CP, six IgG molecules need to bind the surface and assemble in a hexameric structure to enable C1q to bind, as the affinity of C1q to a single IgG is relatively lowE. coli MG1655 using StrepTagII as an antigen incorporated into outer membrane protein X (OmpX)39. This enabled us to directly compare the binding and complement activation capabilities of monoclonal anti-StrepTagII IgG1 and pentameric IgM. We used this in combination with a purified CP complement assay to solely compare the effect of the antibody isotypes in the absence of other serum components. Using these tools, we show IgM’s enhanced capacity over IgG1 to induce complement-mediated lysis of E. coli. Altogether, this system can provide valuable insight into the molecular dynamics of how (engineered) IgG and IgM activate complement on bacterial membranes.Here, we set up an artificial antigen–antibody system in E. coli MG1655 via the classical pathway (CP), we first considered using serum as a complement source. However, since complement can also be activated via the lectin and alternative pathway in serum, this interferes with pinpointing the effect of antibodies alone. Additionally, serum contains proteins like lysozyme that destroy the bacterial particle after outer membrane permeation by MAC pores, which makes it difficult to solely study the effect of the complement system7. To circumvent these limitations, we reconstituted the CP with purified complement proteins  are added for 45 min at 37 °C . Therefore, we continued solely with IgM co-expressed with J-chain with subsequent purification by size exclusion chromatography to obtain pure pentameric IgM, hereafter referred to as IgM. To validate that the anti-StrepTagII mAbs specifically recognised the StrepTagII antigen on E. coli, we assessed the binding of these mAbs to both MG1655-OmpX-StrepTagII and MG1655 Wt. All three anti-StrepTagII forms, IgG1, IgG4, and IgM, did not bind MG1655 Wt, but bound efficiently to MG1655-OmpX-StrepTagII  described to target MG1655 surface structures. Therefore, we required a mAb in combination with an antigen that is expressed on the bacterial surface. For this, we used the anti-StrepTagII mAb, which is described to recognise the eight amino acid peptide StrepTagII with high affinity. To place the antigen on the bacterial surface, we genetically engineered StrepTagII into loop 2 of outer membrane protein X (OmpX) and overexpressed the protein in Having validated that IgG and IgM anti-StrepTagII bind their target comparably and can both induce inner membrane damage of MG1655-OmpX-StrepTagII bacteria, we aimed to compare the efficiency of IgM and IgG in activating the complement system on these bacteria. First, we compared C1q binding to IgM and IgG1 in the absence of downstream components by detecting C1q. C1q binding to surface-bound IgG1 was slightly more efficient than to surface-bound IgM Fig. a. In con50) of IgG1 and IgM, we fitted four-parameter logistic curves on the Sytox graphs and extracted the EC50 values  lower than the EC50 of IgG1. This difference increases to about 74-fold (61–83-fold) when comparing IgM and IgG1 in equimolar ratio  or E345K (Glu345 → Lys)) can enhance hexamerisation of target-bound antibodies48. On several bacterial species , these single amino acid mutations were shown to enhance C1q recruitment and downstream complement effector functions21. Therefore, we produced anti-StrepTagII IgG1s with the E430G single mutation. In addition, we generated a triple mutant (E345R (Glu345 → Arg), E430G (Glu430 → Gly), S440Y (Ser440 → Tyr), denoted as RGY), because of its ability to form IgG1 hexamers in solution without binding an antigen48. As counterpart for this pre-assembled hexamer, we produced IgM with the C575A (Cys575 → Ala) mutation that prevents IgM from forming pentamers and thus IgM exists as monomers in solution50. First, we analysed the antibodies with mass photometry to assess their conformation51. IgG1 E430G was present in solution as monomers of 150 kDa like wild-type IgG1  lower than the one of IgG1  antibodies in their capacity to trigger complement-mediated killing of E. coli.Our understanding of which antigen–antibody combinations most potently trigger complement-mediated killing of Gram-negative bacteria is limited by the lack of well-defined antibody-antigen combinations. Here we aimed to compare IgG and IgM in their ability to activate the complement system on Vibrio cholerae52. In contrast, another study with chimeric antibodies has shown that IgMs exhibited similar serum bactericidal activity as IgG1 when targeting porin A in the outer membrane of Neisseria meningitidis53. However, these chimeric IgG and IgM antibodies bound at EC50 values of about 2.5 µg/mL, indicating they have less affinity for their antigen than the mAbs used here. The differences of how IgG1 and IgM compare in activating the complement system might therefore be (partly) explained by the affinity of the Fab for the antigen. However, several other factors might also play a role, such as antigen location or antigen density. Our results are obtained with the StrepTagII antigen as part of overexpressed OmpX. Recent studies have demonstrated that on E. coli MG1655, there are patches rich in Omps and patches rich in lipopolysaccharides (LPS)54. These patches differ in membrane lipid composition which could interfere with MAC-insertion into the membrane. Therefore, the location of the antigen of the antibody could have implications for the capacity of MAC-insertion and thereby complement-mediated killing. Since different antibodies binding to the LPS are described to both activate and inhibit complement-mediated killing of Gram-negative bacteria55, we hypothesise that both the location, but also the distance of the antigen to the membrane might be an important determinant for whether an antibody can induce complement-mediated killing or not. On single membrane particles, an epitope further away from the membrane was demonstrated to induce less complement-dependent lysis than an epitope closer to the membrane56. We hypothesise that if complement activation by antibodies takes place too far from the membrane, the direct local anchoring of the MAC into the membrane might be impaired36. The importance of antigen density was previously highlighted by the fact that hexameric IgM has superior complement activating capabilities over pentameric IgM and IgG1 if the antigen density is low, whereas at high densities they all three matched57. Our results are obtained with an overexpressed antigen and the observed difference in complement activation between IgM and IgG1 should be investigated for this type of antigen at different expression levels and thus antigen densities. To study the influence of antigen density, the expression of OmpX-StrepTagII could be varied by using a variable inducible expression vector. Furthermore, the antigen location could be varied by attaching StrepTagII to the LPS of E. coli via click chemistry, by clicking DBCO-StrepTagII to KDO-azide which can be incorporated into the LPS58. Lastly, to alter the distance of StrepTagII to the membrane, we could make use of flexible linkers to attach the antigen to the LPS.Using the experimental setups described above, we showed that pentameric IgM is an about 70-fold more efficient complement activator and inducer of MAC-dependent bacterial killing than IgG1. Anti-StrepTagII antibody has been described to have a high affinity Fab for StrepTagII, and the affinity might be an important determinant for this effector function of antibodies. In line with our results, high affinity O-antigen targeting IgMs have previously been described to be 20–140-fold more vibriocidal than IgG on E. coli. RGY mutations in IgG1, that trigger the formation of pre-assembled hexamers in solution18, enhance the activity of IgG1 to induce killing of MG1655 as efficiently as pentameric IgM. In contrast, the monomeric variant of IgM could not induce killing of MG1655, most likely due to its inability to bind C1q. In addition, we showed that mutations in IgG1 that enhance on-target hexamerisation48 could not overcome the difference between IgG1 and IgM. This contradicts our previous findings where these enhanced on-target hexamerisation forming IgGs significantly improved complement activation on Streptococcus pneumoniae22. The here-presented results were obtained with the Gram-negative bacterium E. coli in which the antigen type, density, and location differs from the Gram-positive S. pneumoniae which could explain the observed differences.In this study, we show that pentameric IgM is more efficient than IgG1 in driving complement-mediated killing of 60, studying the relationship between antigens and antibodies is a key prerequisite to guide the design of new therapeutic antibodies.In conclusion, we here present an antigen–antibody system which allows us to directly compare the capacity of (engineered) antibodies to activate complement on live bacteria. This system could also be used to study the effect of antibody isotype on other antibody effector functions like phagocytosis. We also employed a purified complement assay to compare the effect of antibodies alone in their ability to induce complement-mediated killing. In the future, this assay could be used to study patient derived antibodies to understand their immune response during or after bacterial infections. Since the broad range of effector functions of antibodies render them an attractive alternative to antibiotics to treat or prevent bacterial infections61. Complement components C1-complex, C1-inhibitor, C2, C4, and C8 were obtained from Complement Technologies. His-tagged C5, C6, and C7 were expressed in HEK293E cells at U-Protein Express as described previously36. C9-3xGGGGS-LPETG-6xHis was recombinantly expressed in Expi293F cells13. C3 was isolated from plasma as previously described61.Normal human serum (NHS) was obtained from a pool of healthy donors as previously describedPolyclonal IgM was isolated from NHS over a POROS™ CaptureSelect™ IgM Affinity Matrix (ThermoScientific) using an ÄKTA Pure . The IgM was dialysed overnight against PBS with 0.5 M NaCl (PBS500). The dialysed IgM was run over a Superose 6 Increase 10/300 GL column (Cytiva) and fractions pooled to increase and assess purity to ≥ 95%. Polyclonal IgG was isolated from NHS using a HiTrap Protein G High Performance column . After capture, IgG was eluted according to the manufacturer’s instructions and dialysed overnight against PBS. For both polyclonal IgM and IgG a working stock was stored at 4 °C and a long-term storage stock at −80 °C.62. For monoclonal IgM production, gBlocks containing codon optimised VH and VL sequences with an upstream KOZAK and HAVT20 signal peptide were cloned into adapted pcDNA34 vectors (ThermoFisher Scientific), upstream the IgM heavy and kappa light chain constant regions, respectively, using Gibson assembly (New England Biolabs), as described previously22. BamHI and BsiWI were used as the 3’ cloning sites for the VH and VL respectively, in order to preserve the immunoglobulin heavy and kappa light chain amino acid sequence. A plasmid coding for J-chain expression with the following sequence: QEDERIVLVD NKCKCARITS RIIRSSEDPN EDIVERNIRI IVPLNNRENI SDPTSPLRTR FVYHLSDLCK KCDPTEVELD NQIVTATQSN ICDEDSATET CYTYDRNKCY TAVVPLVYGG ETKMVETALT PDACYPD was kindly provided by Theo Rispens. For the production of monomeric IgM, a C575A mutation was introduced in the IgM heavy chain by PCR and cloned in an adapted pcDNA34 vector. Subsequent cloning of the VH region was performed as described above. After verification of the correct sequence, the plasmids were used to transfect EXPI293F cells (ThermoFisher Scientific). EXPI293F cells were grown in EXPI293 medium in culture filter cap Erlenmeyer bottles (Corning) on a rotation platform (125 rotations/min) at 37 °C, 8% CO2. One day before transfection, cells were diluted to 2 × 106 cells/mL. The next day, cells were diluted to 2 × 106 cells/mL with SFM4Transfx-293 medium containing UltraGlutamine I  prior to transfection using PEI . For transfection, 1 µg DNA/mL cells (ratio of heavy and light chain plasmids is 2:3 for IgM and 1:3 for monomeric IgM) was added to OPTIMEM  and gently mixed. For expressions of IgM containing the J-chain, the J-chain plasmid was used as 50% of total plasmid. After adding PEI , the mixture was incubated at room temperature for 20 min and then added dropwise to the cells while manually rotating the Erlenmeyer culture bottle. After 4 to 5 h, 1 mM Valproic Acid (Sigma) was added to the transfected cells. After 5 days of expression, the cell supernatant was collected by centrifugation and filtration (0.45 µm) and subsequently dialysed against PBS. After dialysis, extra NaCl was added to the IgM and monomeric IgM preparation to a final of 0.5 M, before application to a POROS™ CaptureSelect™ IgM Affinity matrix column. IgM and monomeric IgM were eluted with 0.1 M Glycine–HCL pH 3.0. on the ÄKTA Pure. 0.5 M NaCl was added to the pooled fractions, which were neutralised with 1 M Tris pH 7.5. Both IgM isolates were dialysed against PBS500. Finally, IgM with or without J-chain and monomeric IgM were isolated on a Superose 6 Increase 10/300 GL to ≥ 95% purity. For all mAbs, a working stock was stored at 4 °C and a long-term storage stock at −80 °C.The heavy and light chain variable region (VH and VL) amino acid sequences of monoclonal anti-StrepTagII were from patent . IgG1, IgG4, IgG1 E430G, and IgG1 RGY were produced as described previouslyE. coli MG1655 wildtype or MG1655-OmpX-StrepTagII as specified with the results. To obtain MG1655-OmpX-StrepTagII, MG1655 was transformed with a modified pULTRA vector containing a kanamycin resistance cassette and the following sequence for OmpX-StrepTagII with StrepTagII underlined: MKKIACLSAL AAVLAFTAGT SVAATSTVTG GYAQSDAQGQ MNKMGGFNLK YRYEEDNSPL GVIGSFTYTE KSRTASGQSG QWSHPQFEKG GSSGDYNKNQ YYGITAGPAY RINDWASIYG VVGVGYGKFQ TTEYPTYKHD TSDYGFSYGA GLQFNPMENV ALDFSYEQSR IRSVDVGTWI AGVGYRF.All experiments were performed with 600 0.4–0.6) shaking at 37 °C. Once at mid-log phase, bacteria were pelleted by centrifugation and resuspended in RPMI 1640 medium (Thermofisher/Gibco) containing 0.05% human serum albumin (RPMI-HSA) to OD600 = 1 (~ 1 * 109 bacteria/mL).For all experiments, a single bacterial colony was picked from Lysogeny Broth (LB) agar plate (MG1655-OmpX-StrepTagII with 50 µg/mL kanamycin) and grown overnight in LB medium (for MG1655-OmpX-StrepTagII with 50 µg/mL kanamycin) whilst shaking at 37 °C. Bacteria were diluted 1/30 and 1/100 in fresh LB medium and grown until mid-log phase  detection antibody for 30 min at 4 °C whilst shaking. In case of polyclonal antibodies, the detection antibodies were 3 µg/mL of anti-IgM-PE/Dazzle594  or anti-IgG-FITC . After incubation, the bacteria were washed in RPMI-HSA, pelleted, and resuspended in RPMI-HSA. The antibody binding to bacteria was measured by flow cytometry on a MACSQuant VYB (Miltenyi Biotec).Bacteria were grown as described above and incubated with an antibody concentration as denoted per experiment at a bacterial concentration of OD63. Per component in 100% serum: C1-complex 135 µg/mL; C1-inhibitor 180 µg/mL; C2 20 µg/mL; C3 1250 µg/mL; C4 400 µg/mL; C5 70 µg/mL; C6 64 µg/mL; C7 56 µg/mL; C8 55 µg/mL; and C9 60 µg/mL. As first step of the purified classical pathway assay, bacteria (OD600 = 0.025) were incubated with an indicated concentration of antibody and C1-complex for 15 min, shaking at 4 °C. Subsequently, the remaining components C1-inhibitor, C2, C3, C4, C5, C6, C7, C8, and C9 were added at twice the concentration of the desired final concentration in a volume that is equivalent of that of the first step, and incubated for 45 min shaking at 37 °C. This renders the final concentration of C1-complex half that of the other complement components and the final bacteria concentration OD600 = 0.0125.Purified complement components were used in concentrations in physiological ratio as they occur in (diluted) serum, denoted as percentage serum equivalent64), randomly labelled with NHS-Alexa Fluor 488  as done previously13, or monoclonal anti-C5b-9-AF488 (clone aE11) and incubated for 30 min at 4 °C whilst shaking.Bacterial viability was measured in either an indicated percentage of normal human serum (NHS) for 45 min at 37 °C or the purified CP assay as described above. To measure Sytox influx, Sytox Blue (Thermofisher) was added during the incubation at a final concentration of 1 µM. After the incubation, the samples were diluted in cold RPMI-HSA and immediately measured by flow cytometry on a MACSQuant VYB. To analyse C3b and MAC deposition, the samples were treated similarly as for Sytox measurements and after a total incubation of 1 h, the bacteria were washed with RPMI-HSA, resuspended in 1 µg/mL monoclonal mouse anti-C3b  and incubated for 30 min at 4 °C whilst shaking.To analyse C4b deposition, bacteria were incubated with the specified antibodies in the first step of the purified CP assay as described above. After 15 min, C1-inhibitor and C4 were added twice concentrated of the desired concentration in a volume that is equivalent of that of the first step, and incubated for 15 min shaking at 37 °C. Afterwards, the bacteria were washed with RPMI-HSA, resuspended in 3 µg/mL murine monoclonal anti-human C4d  and incubated for 30 min at 4 °C whilst shaking. Again, the bacteria were washed with RPMI-HSA, resuspended in 3 µg/mL Goat F(ab’)2-anti-mouse-IgG-FITC  and incubated for 30 min at 4 °C whilst shaking. After incubation with a detection antibody, the bacteria were washed in RPMI-HSA, pelleted, and resuspended in cold RPMI-HSA. The detection antibody binding to bacteria was measured by flow cytometry on a MACSQuant VYB.51. Microscope coverslips  were prepared by sequential cleaning in sonication baths of isopropanol and then MilliQ water (2x), followed by placement of a CultureWell gasket (Grace Biolabs). About 15 µL of PBS was placed in a well for focusing, after which about 3 µL of diluted sample was mixed in. Measurement concentrations were typically around 5–20 nM. Measurements were recorded using medium field-of-view settings for 120 s. An in-house calibration mix consisting of IgG4Δhinge-L368A (73 kDa), IgG1-Campath (149 kDa), apoferritin (483 kDa), and GroEL (800 kDa) was used. Recordings were processed in DiscoverMP (Refeyn), from which also the relative abundances of protein complexes were obtained. Further data analysis and plotting were performed in Jupyter Notebook using an in-house Python library.Mass photometry experiments were performed on a Refeyn OneMP mass photometer (Refeyn) similar to described beforeFlow cytometry data was analysed using FlowJo V10 and graphs including standard deviation were constructed using Graphpad Prism 9. Mass photometry data was processed in DiscoverMP (Refeyn) and analysed using an in-house python library. To obtain the fitted curves and half-maximum values for these, the nonlinear analysis function ‘Sigmoidal, 4PL, X is concentration’ function of Graphpad Prism 9 was used.Human blood was isolated after informed consent was obtained from all subjects in accordance with the Declaration of Helsinki. Approval was obtained from the medical ethics committee of the UMC Utrecht, The Netherlands.Supplementary Figures.


Introduction: Outpatient exercise training has been shown to be beneficial for solid organ transplant recipients. Little is known about the effects of inpatient rehabilitation programs for recipients with a more complicated postoperative course. Research Question: This study was designed to (1) describe the changes in functional outcomes after an inpatient rehabilitation program, and (2) determine whether the changes in lower body strength and quadriceps strength are associated with changes in functional exercise capacity. Design: This was a single-arm prospective longitudinal study. The recipients participated in an inpatient rehabilitation program twice a day, 7 days a week for 3 to 4 weeks. Outcome Measures Included: 2-Minute Walking Test, Timed Up and Go, Berg Balance Scale, 30-Second Sit to Stand, biceps and quadriceps strength, Functional Independence Measure, SF-36, and Canadian Occupational Performance Measure. Results: Twenty-eight patients  completed the study. Participants were mostly liver (42%) and lung recipients (35%). There were statistically significant improvements in all outcomes after the intervention. There was no relationship between changes in functional exercise capacity and quadriceps strength or lower body strength. Conclusion: An inpatient rehabilitation program may improve several functional outcomes and health-related quality of life in transplant recipients with a complicated postoperative course. Pretransplant factors such as deconditioning, anemia, nutritional depletion, and depression as well as posttransplant factors such as effects of immunosuppressant medications, respiratory failure, prolonged hospitalization, myopathies, neuropathies, and episodes of organ rejections can negatively impact recipients’ health and physical function. Poor physical function in recipients can be associated with rehospitalizations, poor health-related quality of life (HRQoL), and increased mortality.Solid-organ transplantation is one of the major medical advances of the 20th century. It is proven to decrease mortality and improve the quality of life of people living with end-stage liver, heart, kidney, pancreas, or lung conditions.5 It improves HRQoL, physical function, maximal exercise capacity, quadriceps muscle strength, and diastolic blood pressure. However, most of the published studies have focused on recipients who were independent and able to participate in an outpatient program. There is limited research on recipients with a more complicated course who need additional care and may be referred to a specialized inpatient rehabilitation program. The few existing studies examining the effects of inpatient rehabilitation for recipients have shown that this type of program is feasible, safe, and well-tolerated by patients.68 These studies have also shown that an inpatient rehabilitation program for recipients may improve physical function, muscle strength, functional exercise capacity, activities of daily living, and independence.8 In addition to the clinical benefits, an inpatient rehabilitation program may impact readmission to hospitals and reduce healthcare utilization.The benefits of exercise training in transplantation are well established. Despite the preliminary evidence for the effectiveness of an inpatient rehabilitation program for recipients, most of the studies on inpatient rehabilitation focused on a single organ group and were either case studies or retrospective studies that consequently had limited control over the choice of outcome measures and how they were measured.A retrospective study with over 3000 patients examined the association between different discharge locations post-transplant and 30-day readmissions to the hospital and observed that recipients who were discharged to an inpatient rehabilitation program had less risk for readmission within 30 days posttransplant. Considering that the main goal of inpatient rehabilitation is to improve patients’ independence, understanding the relationship between lower body strength and functional exercise capacity is critical to planning effective interventions to improve walking and consequently independence.Lower limb muscle strength has been shown to be associated with decreased exercise and functional capacity of individuals with chronic conditions.The aims of this study were (1) to describe the changes in functional outcomes after an inpatient rehabilitation program for solid organ transplant recipients, and (2) to determine whether the changes in lower body strength and quadriceps strength are associated with changes in functional exercise capacity. After obtaining informed consent, patients were required to attend 2 pre and 2 postintervention assessment sessions. The preintervention assessment sessions occurred within the first 2 days of admission. The postintervention assessment sessions occurred within 1 or 2 days of hospital discharge. The first preintervention assessment session was conducted by the physiotherapist of the inpatient rehabilitation program. Patients were asked to perform the Two-Minute Walk Test (2MWT), the Berg Balance Scale (BBS), and the Timed Up and Go (TUG). A research coordinator was responsible for the second pre-intervention assessment that included a second 2MWT, muscle strength testing, the 30-second Sit-to-Stand test (30-STS), the Canadian Occupational Performance Measure (COPM), and the SF-36 Health Survey. During the second assessment session, measurements were collected of anthropometry, demographics, type of transplant, length of acute hospital stay, and functional independence measure (FIM). Once the outcome measures were collected at the preintervention assessment sessions, patients commenced the multidisciplinary inpatient rehabilitation program. The outcome measures were again collected after the rehabilitation program over 2 different days. The same assessors administered the same tests or questionnaires postintervention.This was a single-arm prospective longitudinal study conducted at St John's Rehab (SJR) Program of the Sunnybrook Health Sciences Centre in Toronto, Canada. The study was approved by the Research Ethics Board. The inpatient rehabilitation program is a dedicated program for transplant recipients.The Multi-Organ Transplant Program of the Toronto General Hospital performs approximately 621 solid organ transplants per year. In 2022, 37% of the recipients were female and the mean age of all recipients was 55.4. The most common problems that patients experienced were extreme fatigue, severe deconditioning, nutritional depletion, metabolic toxicity, myopathy, neuropathy, cognitive loss secondary to the disease or its treatment, and depression. Medical complications could include rejection of the transplanted organ, anemia, cardiac failure, respiratory failure, and infection.Recipients of liver, kidney, heart, lung, and pancreas transplants who were referred from the Multi-Organ Transplant Program of the Toronto General Hospital to a multidisciplinary inpatient rehabilitation program from August 2012 to March 2014. The Toronto General Hospital referred patients to the inpatient rehabilitation program if they had problems with body functions, structures, activities, and participation.The exclusion criteria for the study were (a) inability to understand English; (b) known cognitive impairment; (c) evidence of a neurological or musculoskeletal condition that could severely limit mobility and (d) transplantation not being performed within 6 months of inpatient rehabilitation admission. Discharge from the program occurs when the initial established goals for rehabilitation are reached and if discharge home is deemed safe by the healthcare team. Necessary equipment and follow-up services and appointments need to be arranged before discharge. The physical therapy program was offered twice a day, 7 days a week for 3 to 4 weeks. The physical therapy exercises were individualized, tailored to each patient's functional abilities, and consisted of (a) supervised mobility/transfer exercises; (b) range of motion and resistance exercises ; (c) endurance exercises  and (d) balance training. All exercises were individualized based on the patient's baseline assessment and each session lasted about 45 min.The inpatient rehabilitation program is a multidisciplinary program that comprises Physical Therapy, Occupational Therapy, Nursing, Psychology, Physiatry, Hospitalist Physician, and nutritional support. The test was administered in a corridor of approximately 30 m and supervised by a clinician and research assistant. Patients were instructed to walk as far as they could in 2 min and standardized instructions were given every 30 s. Two 2MWT were performed on 2 different days to account for learning effects. Individuals were allowed to use their gait aid if required. The highest walked distance in meters was used for analysis. The 2MWT was chosen over the 6MWT as the 2MWT was routinely used in the program and because the 6MWT can be burdensome for patients with higher disability levels.The 2MWT is a valid and reliable test that measures functional exercise capacity in different clinical populations. It includes items such as single-legged stance, transfers, reaching, and turning around. Each item consists of a 5-point ordinal scale ranging from 0 to 4, with 0 indicating the lowest level of function and 4 the highest level of function. The maximum score is 56. Scores below 45 points indicate a higher risk of falling. The BBS has been shown to be responsive to an inpatient rehabilitation program in a sample of heart and lung recipients.The BBS is a 14-item performance-based instrument that measures functional balance and that can be used to assess response to treatments. The test requires the patient to rise from a standard armchair, walk 3 m at a comfortable and safe pace, walk back to the chair and sit down. The time to complete the test is presented in seconds. After a practice test, the second trial was recorded on the same day and the best results would be used for analysis. Individuals were allowed to use their gait aid if required.The TUG is a psychometrically robust test that was designed to evaluate the risk of falls by observing the patient's postural stability, gait, stride length, and sway. The test requires the patient to cross their arm across their chest and stand up and sit down as many times as possible in 30 s. Results were recorded in the number of repetitions completed.The 30-STS test is a valid and reliable test to assess lower body strength in older adults. In addition, the research assistant practiced the assessment with 10 healthy individuals before initiating the study to ensure good intrarater reliability. Elbow flexion and knee extension force are associated with performance in upper and lower limb functional tests and independence in activities of daily living.Peripheral muscle force was assessed using an isometric hand-held dynamometer . Measurements of elbow flexion and knee extension were performed on the dominant side and results were reported in Newtons. The test was performed with patients in a sitting position. The average of the highest 3 measures within 5% of each other were used for analysis. Good reproducibility of these tests has been previously shown. The scale contains 18 items each scored from 1  to 7 (fully independent). For this study, the FIM motor subscales were used, which included the areas of self-care , sphincter control, mobility, and locomotion.The FIM is a disability scale widely used in rehabilitation settings. It has been shown to be reliable and valid as a measure of assessment of functional independence. It includes questions regarding self-care, productivity, and leisure. Scores were calculated by summing individual problem scores and dividing them by the number of problems.Problematic activities of daily living (ADL) were identified using the COPM. It is an individualized, patient-centered measure designed to identify participants’ self-perception of occupational performance and can be used with individuals with a variety of disabilities. Each domain is transformed to a 0-100 scale on the assumption that each question carries equal weight. Lower scores indicate a lower HRQoL.The SF-36 is a generic measure and short-form health survey with 36 questions. It assesses functional health and well-being from the patient's perspective and reports on eight domains. It is a practical, reliable, and valid measure of physical and mental health that has been tested among kidney transplant recipients previously.t-test and SD of 16.36. However, the aim was to include 30 patients for several reasons: to account for 20% of dropouts, be within a range of sample size suggested for rehabilitation studies for power above 80% and to be within the study budget.The within-subject difference in the 2MWT following rehabilitation represents the primary outcome. To calculate the sample size, preliminary data were collected from 10 recipients that had participated in the inpatient rehabilitation program. The data showed that the mean change in 2MWT after rehabilitation was 23.46 (16.36) meters. Using an online calculator, it was determined that 6 patients would be required to yield 80% power (α = .05) to detect a mean difference of at least 23 m in the 2MWT using a two-tailed paired t-test was used for normally distributed variables and Wilcoxon for nonnormally distributed. To address the second objective, we used the Pearson correlation coefficient to estimate the associations between the scores of the 30-STS and quadriceps strength with the 2MWT. A P-value ≤ .05 was considered significant. Data from all patients recruited to participate in the study were analyzed irrespective of their attendance or compliance with the rehabilitation program.Statistical analyses were performed using SPSS v 26 . All variables were tested for normality. To address the first objective, a student two-tailed paired During the study period of 1 year and 4 months, 64 patients were approached, 17 patients declined to participate in the study, and 11 did not meet the eligibility criteria. Thirty-six recipients agreed to participate in the study, 8 dropped out and 28 completed the study. A detailed flowchart of the study and reasons for dropouts can be found in P < .001, CI 95%). The mean changes of each outcome can be found in All functional outcomes improved significantly post-intervention (r = .102). A similar result was found between changes in 2MWT and quadriceps strength (r = .127). There were no statistically significant differences in mean changes of any outcome when participants of each organ type were compared, except in the performance subscale of the COPM.Pearson coefficients of correlation between the changes in 2MWT and 30-STS demonstrated no association , BBS, and TUG scores after an inpatient program among heart and lung recipients. Patcai et al have shown improvements in FIM after an inpatient rehabilitation program in solid-organ transplant recipients.The findings were consistent with other inpatient rehabilitation studies of specific organ groups. Shiner et al in a 2011 study that evaluated the effects of inpatient rehabilitation in lung transplant recipients, found no difference in HRQoL between intervention and control groups. Considering the lack of a comparison group in this study, no confirmation of changes in the HRQoL and physical function were a result of the inpatient program or natural recovery after transplant. Longitudinal studies (where rehabilitation was not offered) have shown natural recovery in functional outcomes and HRQoL in recipients at 6- and 12-months posttransplant. The postintervention assessment in this study occurred earlier (within 2-3 months posttransplant); therefore, due to the magnitude of change found, the improvements seen were likely to be a result of the rehabilitation program rather than natural recovery. For example, the mean change in the primary outcome (2MWT), which was assessed early posttransplant (within 2-3 months), was 3 times higher (31 [19.2] m) than the minimal detectable change in older adults (9.1 m). In a previous study where an exercise program was offered to lung transplant candidates, the mean change between the six-minute walk distance at a time for assessment for transplant and 1-month posttransplant was 37 m, which was slightly above the minimally important difference for adults with chronic lung disease (30 m).In contrast to these findings, Ihle et al used the 60-second sit-to-stand test in a sample of lung recipients in the acute care setting and found a mean of 13 repetitions. This confirmed how severely impaired the sample in this study was (mean 30-STS at baseline = 0.35 repetitions). This severe impairment in lower limb muscle strength may be partly explained by the long length of stay in the hospital posttransplant (mean 56 days). The median length of stay in the inpatient ward in the study by Tarrant et al was 7 days for medical readmission patients and 19 days for immediate postoperative lung transplant recipients.The 30-STS or dynamometry has not been used to assess lower limb strength or biceps or quadriceps muscle strength, respectively, in transplant recipients participating in an inpatient rehabilitation program. However, Tarrant et al The improvements in lower body strength and biceps and quadriceps strength found were expected as the exercise training included resistance exercises. After the intervention, the number of patients who were able to perform the 30-STS test was 4 times higher. This increase is relevant as this was an important functional ability to prevent falls.The mean change in the 30-STS in this study was only 1 repetition. In a recent study conducted among older adults undergoing rehabilitation, the minimal detectable change for the 30-STS was found to be 0.70, which suggests that an additional repetition was likely not a measurement error and may represent a real functional improvement.r = .127, P < .02) or 30-STS . A study from 2015 by Kjølhede et al found a moderate positive correlation between 2MWT scores and maximal muscle strength measured by an isokinetic dynamometer in people with multiple sclerosis. In 2018, Bokaeian et al found a weak positive correlation between quadriceps strength and the 2MWT among patients with knee osteoarthritis. In this study, an isokinetic dynamometer to measure quadriceps strength was not used, which makes it difficult to compare the findings with these previous studies. Studies with a larger sample size are needed to confirm whether impairments in lower body muscle strength impact functional exercise capacity in transplant recipients.No correlations were found between the 2MWT and quadriceps strength  may have had an impact on the improvements of physical function in the participants; however, no determination of the contribution of each component was assessed. Forty-four percent of the approached patients did not want to participate or were excluded from the study. There was no data on these patients; however, the reasons for declining participation and for exclusion were recorded. Most of the excluded patients had had their transplant between 2 and 4 years prior to their admission to inpatient rehabilitation. These patients were not referred by the Toronto General Hospital like other patients with recent transplants. They were admitted to the program for other reasons than issues related to their transplant  and because they had already been a patient in the program early posttransplant.Although the data were collected between 2012 and 2013, the results of the study remain relevant as the program and criteria for referral remain the same. There is limited availability of such programs for transplant recipients worldwide and these findings may influence the development of new programs. Finally, as a single-centered study, generalization bias is expected, but with the lack of inpatient rehabilitation programs in Canada, multi-center sampling was impossible.A multidisciplinary inpatient rehabilitation program may improve several functional outcomes and health-related quality of life among transplant recipients who have experienced a more complicated course of transplantation. The findings of this study may encourage patients and healthcare professionals working in transplantation to advocate for more inpatient rehabilitation programs for recipients who are not able to be discharged home following transplantation.


Although normothermic machine perfusion (NMP) provides superior preservation of liver grafts compared to static cold storage and allows for viability testing of high-risk grafts, its effect on the liver immune compartment remains unclear. We investigated the innate immune response during 6 h of continuous NMP (cNMP) of livers that were directly procured  or procured after 60 min warm ischemia , followed by 12 h of whole blood (WB) reperfusion. WI livers showed elevated transaminase levels during cNMP but not after WB reperfusion. Perfusate concentrations of TNF-α were lower in WI livers during cNMP and WB reperfusion, whereas IL-8 concentrations did not differ significantly. TGF-β concentrations were higher in WI livers during NMP but not after WB reperfusion, whereas IL-10 concentrations were similar. Endoplasmic stress and apoptotic signaling were increased in WI livers during cNMP but not after WB reperfusion. Additionally, neutrophil mobilization increased to a significantly lesser extent in WI livers at the end of NMP. In conclusion, WI livers exhibit a distinct innate immune response during cNMP compared to DP livers. The cytokine profile shifted towards an anti-inflammatory phenotype during cNMP and WB reperfusion, and pro-apoptotic signaling was stronger during cNMP. During WB reperfusion, livers exhibited a blunted cytokine release, regardless of ischemic damage, supporting the potential reconditioning effect of cNMP. The success of liver transplantation (LT) in the treatment of end-stage liver failure has resulted in an important imbalance between the numbers of patients on the waiting list and donor organ availability . To overStatic cold storage (SCS) is the gold standard preservation method, but it falls short in adequately protecting these DCD livers from the inevitable IRI. Normothermic machine perfusion (NMP) has been proposed as an alternative preservation strategy , additioDuring NMP, the liver is connected ex situ to a perfusion device that recirculates an oxygenated and nutrient-enriched packed red blood cell perfusate at physiological body temperature. When used to replace preservation with SCS, NMP is applied throughout the entire preservation phase and is also referred to as continuous NMP (cNMP). Alternatively, NMP can be used as a means to assess graft quality after a period of SCS or hypothermic machine perfusion, in which case it is referred to as end-ischemic NMP (eNMP) . A recenTo date, most mechanistic studies on NMP have focused on hepatocellular and cholangiocellular function and injury. In contrast, little is known about the effects of NMP on the immune compartment of the liver. Lee et al. used eNMP to investigate the immune profile of six discarded human livers and showed that dynamic changes in leukocyte frequencies occurred, with the mobilization of neutrophils and T cells, together with significant secretion of pro-inflammatory cytokines . EquallyThe aim of the current study was to provide insights into the innate immune response during cNMP and subsequent whole blood (WB) reperfusion as a surrogate for transplantation, as well as how this is affected by prior exposure to WI. To this end, porcine livers were either procured directly  or after 60 min of WI (WI group). All livers underwent 6 h of NMP followed by 12 h of allogenic WB reperfusion, and a comparative assessment of hepatocellular injury and function, cytokine secretion, endoplasmic reticulum (ER) stress, and leukocyte mobilization was performed.p = 0.008). This difference was no longer significant during WB reperfusion  (p = 0.03) (p = 0.3) and during WB reperfusion   A,C. Tiss = 0.03) E,F. In cp = 0.7) B,D. Equap = 0.7) G,H.p = 0.004) p = 0.008) and WB reperfusion . The same was observed for Interleukin-8 (IL-8) . Notably, IL-8 concentrations were only increased after three hours of NMP and peaked at six hours in both groups, with again significantly lower levels during NMP of WI (49.1 (26.9–108.7) pg/mL/g) than DP (205.6 (106.3–349.4) pg/mL/g, p = 0.047) at three hours. Moreover, we observed a distinct IL-8 dynamic after WB reperfusion compared to NMP in both groups . Perfusate concentrations of Interleukin (IL)-10 increased at three hours and peaked at six hours NMP, but overall, they were very low in both groups, and no significant difference could be observed during NMP  or WB reperfusion  (p = 0.06) and throughout WB reperfusion (WI AUC 149 (67.9–272) vs. DP 6.5 (3.1–8.4), p = 0.008). Equally, the TGF-β/IL-8 ratio was higher in the WI group during NMP (WI AUC 15.5 (11.8–22.6) vs. DP 3 (2.4–4.3), p = 0.016) and WB reperfusion (WI AUC 4.6 (2.8–12.7) vs. DP 0.8 (0.3–1), p = 0.057), whereas the IL-10/TNF-α was higher only during WB reperfusion (WI AUC 0.4 (0.3–0.8) vs. DP 0.03 (0.02–0.1), p = 0.008). No difference was observed between IL-10/IL-8 ratios.The overall cytokine release during NMP and subsequent WB reperfusion was assessed by calculating the AUC of the cytokine perfusate concentration corrected for liver weight A,B. In bh groups D. Perfush groups E. In con > 0.99) F. To exp > 0.99) . The ovep = 0.008) and WB reperfusion  (p = 0.008) and WB reperfusion  (p = 0.008) but not during WB reperfusion  (To investigate the effect of WI on unfolded protein response (UPR), which is activated in IRI to restore protein homeostasis and prevent cell death caused by the accumulation of misfolded proteins, we assessed the expression of two mRNAs known to be specifically induced upon endoplasmic reticulum (ER) stress: glucose-regulating protein-78 (GRP78), which is an ER chaperone, and C/EBP homologous protein (CHOP), a transcription factor that plays an important role in the ER stress-induced apoptosis pathway. Additionally, expression of the apoptosis-related downstream effectors B-cell lymphoma 2 (Bcl2) and Bcl2-associated X protein (Bax) were also measured. The expression of GRP78 was significantly increased in WI compared to DP livers, both during NMP  B. CHOP e = 0.15) C. There  = 0.84) D,E. To v = 0.84) F,G.p = 0.015). The proportion of lymphocytes decreased in both groups throughout NMP, but this decrease was significantly greater in DP livers compared to WI livers at three hours . The proportion of mononuclear cells remained similar throughout the NMP period in both groups.White blood cell frequencies were quantified using perfusate smears taken at different timepoints throughout NMP. Cell counts during WB reperfusion could not be performed, because total cell recovery was too low to determine accurate frequencies. No differences were observed during the first hour of NMP; however, at three and six hours, there were notable differences . NeutropThis study shows that livers exposed to severe WI damage display a distinct innate immune response during cNMP compared to livers that were directly procured, with a shift towards an anti-inflammatory cytokine profile and altered leukocyte mobilization. Despite suffering significantly more hepatocellular damage, as reflected by increased AST release, perfusate concentrations of pro-inflammatory cytokines TNF-α and IL-8 were significantly lower in WI livers during NMP. In contrast, IL-10 release was similar and TGF-β release was higher in WI livers, resulting in significantly greater TGF-β/TNF-α and TGF-β/IL-8 ratios. Moreover, proportionally fewer neutrophils were mobilized into the perfusate in WI livers. These findings imply that ischemic injury alters the liver’s biological response during NMP, suggesting different preservation modalities might suit livers of different quality.DCD livers inevitably suffer a period of WI prior to the in situ cold flush, which aggravates IRI and the associated release of pro-inflammatory cytokines . HoweverAlong with a decreased pro-inflammatory response, WI livers displayed a higher release of TGF-β and, consequently, a shift in the balance towards anti-inflammatory signals. Jassem et al. showed that NMP of human livers upregulated activation of regenerative pathways . TGF-β iAdditionally, we investigated immune cell mobilization during NMP and observed a relative increase in neutrophils in the perfusate at 3 and 6 h. Interestingly, this increase was significantly lower in WI ischemic livers at the end of NMP. In a recent study, Hautz et al. described immune cell dynamics during NMP using single-cell RNA sequencing and flow cytometry . They foDuring the WB reperfusion that followed the 6 h cNMP, the cytokine release pattern did not show the typical profile seen after hepatic IRI, regardless of exposure to WI. Whereas TNF-α typically reaches an early peak within 6 h after reperfusion, in our study, TNF-α concentrations remained relatively stable throughout the entire 12 h WB reperfusion phase. As TNF-α plays a critical role in propagating the pro-inflammatory responses, a blunted TNF-α response may protect the graft from IRI by creating a “less hostile” microenvironment. This is consistent with our observation that, at the end of WB reperfusion, there were no differences in AST release and histological injury in WI livers compared to controls despite the more severe injury at baseline. Our group has previously shown that porcine transplantation of livers that were exposed to 60 min WI and preserved with SCS inevitably led to primary non-function and recipient death . In contOur porcine experimental model inevitably presents with some limitations. First, one may argue that WB reperfusion with allogenic WB recruits alloreactive immune activity that should also be factored into the equation. However, early IRI mainly results from inflammatory responses generated by innate immune cells, whereas the adaptive immune response involved in alloreactive reactivity comes into play only at later stages, and hyperacute rejection in non-sensitized animals is very rare ,30. MoreIn conclusion, this study shows that livers exposed to WI exhibit a distinct innate immune response during cNMP compared to livers that are procured without WI damage. The cytokine profile of WI-damaged livers shifted towards a more anti-inflammatory phenotype during NMP, which persisted after WB reperfusion, whereas neutrophil mobilization during NMP was decreased in WI livers. Moreover, following six hours of NMP, a blunted cytokine release was observed during WB reperfusion, potentially indicating a protective effect against IRI. Whether this also improves long-term graft outcome should be the subject of future investigations. Overall, these findings imply that liver grafts respond differently to cNMP when suffering WI injury, potentially suggesting a greater protective effect on DCD livers, and therefore support the evolution towards more organ-tailored preservation, whereby different preservation modalities might suit organs of different quality.Porcine livers were either exposed to 60 min of WI  before procurement or were directly procured , followed by NMP for six hours. After six hours, the perfusate was changed to allogenic WB from a donor pig, and the liver was reperfused for 12 h to mimic transplantation . The allMale TOPIGS TN70 pigs  with a body weight of approximately 30 kg and liver weight of 600–700 g were used for this study protocol. Animals were kept in the animal facility of the University Hospitals of Leuven under a 12 h day/night rhythm in single pens with free access to food  and tap water, with visual, olfactory, and auditory contact between them. The animals were kept for at least two days prior to the surgery to get accustomed to their surroundings. Animals were euthanized by exsanguination under general anaesthesia during liver procurement or WB collection. The KU Leuven Animal Care Committee approved the study, and the experiments were conducted in line with European guidelines . ResultsThe surgical model has been reported in detail elsewhere ,34. Brie2) of 70–100 mmHg and warmed up to 38 °C. The circuit was primed with 500 mL of Gelufosine .Porcine NMP experiments were performed as previously described . BrieflyDuring NMP, the volume of washed red blood cells to be added in order to reach a hematocrit of 30% was calculated with the formula: [(weight of the liver + priming volume) × desired hematocrit]/hematocrit after washing. The same formula was used for the allogenic WB reperfusion. The perfusate was supplemented with heparin, antibiotics, calcium gluconate, and bicarbonate as previously reported. In line with current clinical practice of liver NMP preservation, perfusion was started after a period of SCS of circa 90 min, which corresponds to the time needed for cold flush in the donor, hepatectomy, and back-table preparation. Vasodilators, insulin, heparin, and sodium taurocholate were continuously infused during NMP, as previously described.During perfusion, portal vein and hepatic artery flows  and pressures  were continuously monitored, and pointwise data were recorded at 15 min, 30 min, one hour, three hours, and six hours during NMP . Intrahepatic vascular resistance was calculated as the pressure (mmHg)/flow (mL/min) ratio.g at 4 °C, aliquoted in 1 mL microtubes, snap frozen, and stored at −80 °C. AST was measured according to the International Federation of Clinical Chemistry method . Lactate perfusate concentration was measured with a point-of-care instrument .Perfusate samples were collected in EDTA tubes, centrifuged at 2876× Tissue samples were stored in 4% formalin, paraffin-embedded, and stained with hematoxylin-eosin (H&E). Histological injury was scored semi-quantitively with the SUZUKI score  by an exEnzyme-linked immunosorbent assays (ELISA) were used to determine perfusate concentrations of TNF-α, IL-8, IL-10, and TGF-β  following manufacturer instructions.Total RNA from frozen liver sections stored in Trizol  was isolated using miRNeasy Mini kit . The resultant RNA was quantified using a NanoDrop one . Subsequently, equal amounts of RNA were converted to complementary DNA using MMLV . Gene expression assays were performed using the following primers: CHOP (Ss03821509_s1), GRP78 (Ss03374255_m1), Bcl-2 (Ss03375167_s1), and Bax (Ss03375842_u1) . Amounts of specific RNA were normalized to established housekeeping genes (β-actin) (Ss03376563_uH) , which remained constant in the experimental groups.Apoptosis was quantified by terminal deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL assay) using the In Situ Cell Death Detection Kit, TMR red ) on formalin-fixed, paraffin-embedded liver tissue samples. Five µm thin sections were prepared on Superfrost Plus Gold adhesion microscope slides, deparaffinized (2× 5 min in xylene), rehydrated , and rinsed in phosphate-buffered saline (PBS) (3× 5 min) at room temperature. After a Proteinase K treatment  (1.24568.0100) (20 µg/mL in PBS for 15 min at 37 °C), slides were rinsed (4× 5 min in PBS at room temperature) and incubated with the TUNEL reaction mixture for 1 h at 37 °C in a humidified chamber. Afterwards, the slides were rinsed with PBS (4× 5 min at room temperature), and nuclei were stained with DAPI (1/2000 diluted in PBS) , Waltham, MA, USA). After a final wash, a drop of Slow Fade Diamond antifade , Waltham, MA, USA) was applied. Staining was visualized using a fluorescence microscope (TUNEL (Ex 540/Det 580); DAPI (Ex372/Det456)).Positive and negative deoxynucleotidyl transferase dUTP nick-end labeling cells were counted by an assessor (TW) who was blinded for the group allocation on 4 to 8 complete sections per sample at 20× magnification, then the apoptosis index was calculated as the ratio between the number of positive cells and the number of total cells.p-value of <0.05 in a two-sided test was considered significant. Statistical analyses were performed with GraphPad Prism .As this was a preliminary explorative study, a sample size calculation was not possible. The sample size in each group was chosen to account for biological variation between animals. Data are summarized as median (interquartile range). Data were plotted in relation to perfusion time and single timepoints. To assess the overall inflammatory response, the AUCs for cytokine, AST, and lactate perfusate concentrations were calculated and compared between groups with a Mann–Whitney U test. A similar approach was used for ER stress-related markers in tissue samples. Complete case analysis was used for dealing with missing data in AUC calculation. Concentrations below the inferior detection limit of the used assays were rounded to said value for the calculation of the AUCs. To explore the balance between anti- and pro-inflammatory signals, ratios of perfusate concentrations of IL-10 and TGF-β to that of TNF-α and IL-8 were calculated at every time point and compared between groups. Correction for multiple testing was performed with the Sidak method when appropriate. A


The consequences of increasing opioid misuse in the U.S. over the last two decades have been severe, contributing to hundreds of thousands of lives lost and heavy tolls on individuals, families, and society. The Appalachian Region has been hit particularly hard, with its predominantly rural landscape seeing disproportionate increases in opioid misuse and overdoses. These cases have been difficult to address due to poor treatment access and capacity constraints in many areas of Appalachia.The current study focuses on evaluating The Kentucky Access to Recovery Program (KATR), which provides services to individuals recovering from opioid addiction residing in several counties in Eastern Kentucky. Its purpose is to understand the impact of KATR on service recipients’ access to recovery services and supports.Semi-structured interviews were conducted with 12 service recipients, three service providers, and four vendors of support services related to housing, transportation, medical/dental care, employment, and childcare. Qualitative data were analyzed using thematic analysis.Themes related to individual-level impacts were identified and discussed, including behavioral changes related to recovery, physical and mental health improvements, relationship repair, regaining custody of children, provision of needed supports, and ability to gain employment and improve finances. Study findings showed that KATR had meaningful impacts on the lives of service recipients by helping meet needs and reducing barriers to their ongoing recovery.Through its use of vouchers for support services and basic-needs provision, KATR demonstrates a potentially effective strategy for increasing access to health-related social services for persons in recovery in predominantly rural areas. Since 1999, opioid-related overdose deaths have more than quadrupled.Consequences of opioid misuse can be life-threatening, as this behavior coincides with a significant rise in both morbidity and mortality, exacting a heavy toll on patients, physicians, and societal efficiency. Other societal costs that exist due to the opioid epidemic include declines in work productivity and attendance, as well as increases in use of emergency services, correctional facilities, and public insurance.The Appalachian Region is highly rural, with 42% of its area classified as rural compared to 20% of the U.S. as a whole.10The present study considers how this crisis unfolds in Eastern Kentucky, the 31-county Appalachian portion of one U.S. state. It examines an agency which provides services to one Eastern Kentucky county and the four nearest surrounding counties. These five counties have experienced substantial effects from the opioid crisis across the past decade. In County 1, the age-adjusted rate of drug overdose deaths in 2018 was approximately 60% higher than the national average.13The Kentucky Access to Recovery (KATR) program helps men and women recovering from opioid addiction. All services offered are free to low-income adult residents of Kentucky who are in treatment or early recovery (2 years or less). KATR has three offices throughout Eastern Kentucky. The offices offer funds for various services: the provision of clothing, support locating housing, medical/dental care, transportation assistance, vocational/employment services, and childcare assistance. These services are offered to eligible participants who are resident in counties in and adjacent to KATR offices. The resources and material support must be connected to the eligible participant’s treatment and/or recovery management plan. KATR has been established as a supplement and not a replacement plan, meaning other resources—such as insurance, Medicaid, Medicare, and state block grant dollars—must be used first.The authors conducted a program evaluation of KATR. The evaluation considered program effectiveness in its entirety, the effects on the program related to the social determinants of health, and how the program impacts individuals. The purpose of this study is to understand the impact of KATR on service recipients and the barriers they encounter in their recovery journey while part of the program.To achieve the study goals, semi-structured interviews were conducted with three primary groups of individuals involved with the KATR program: (1) 12 service recipients—persons who received services from KATR; (2) three service providers—the program coordinators of KATR; and (3) four vendors of support services related to mechanical needs, housing assistance, dental work, and childcare. Service recipients were randomly selected to participate in the study. Using a random number generator, fifty participants were identified to receive the initial recruitment postcard and a follow-up phone call. Interviews were scheduled or conducted on the phone during the first initial phone contact which took place approximately three to five days after postcards were received. For program coordinators and vendors, investigators were provided contact information, and all potential respondents were emailed about the current study.Semi-structured interviews were completed with all three groups from August to October 2020 via telephone or Zoom and recorded via Zoom. When recording the telephone calls, the interviewer opened Zoom and used Zoom to record while conducting the interview on speaker phone in a secure and private office space. Participants were given the option to decline the recording of their interview. All participants provided consent to have their interview recorded. Interviews with program coordinators lasted about an hour, and vendor interviews lasted approximately 15 minutes. The service participant interviews averaged about 15 minutes in length. The Zoom recordings were saved as MP3 files without video. Recordings were transcribed primarily using Rev, a third-party transcribing service, and reviewed by the research team for accuracy. Each participant group addressed topics related to the impact of the opioid support services program on the service users, community, and organization .Interviews with service recipients lasted, on average, 15 to 20 minutes. Service recipients received a $15 gift card to Walmart for participating in the study. Three of the authors conducted the semi-structured interviews with service recipients until data saturation was reached. The three authors discussed weekly the context of the interviews until all authors agreed that data saturation had been reached for this subset of respondents.All interviews with the program coordinators were conducted by one of the investigators and lasted approximately 50 minutes each. No incentives were provided to program coordinators.One of the authors conducted semi-structured interviews with four of the KATR participating vendors. Each interview lasted, on average, 15 minutes. Incentives were not provided for vendors.Data analysis was guided by the social determinants of health (SDOH) framework which includes five domains: economic stability; education access and quality; healthcare access and quality; neighborhood and built environment; and social and community context.16The average age of participants was 42 years, with an even mix of self-identified males and females. Regarding race, the sample was mostly homogenous with only one participant identifying as black and the remainder as white. The referral sources for these participants included counselors involved in their recovery, the court system, other social services, and friends. The participants, on average, traveled 28 miles to access KATR services; however, during the COVID-19 pandemic, most services were conducted via phone and internet. Ten out of the 12 participants noted that they had access to internet.Three program coordinators of KATR also provided their insights. Each of the coordinators had lived in the local community their entire lives and had previously worked in public service positions prior to employment with the agency delivering KATR.Four vendors were interviewed as part of the study. All four businesses had been in existence for several years . Two vendors were automotive repair garages and two were clothing stores. The business representatives noted that they were well known in the community.Participants were overwhelmingly positive about the effect that KATR has had on the lives of service recipients. Several themes emerged related to individual-level impacts of the recovery support services, including behavior change, physical and mental health improvements, relationship repair, regaining custody of children, provision of clothing, transportation, childcare, recovery support, and ability to gain employment and improve finances. These themes are detailed below.sic] hope, and they gave us something to work towards…they push us to do better.” Similarly, Service User 10 shared, “I think they show a lot of people that I did run with, look at me now and they know, and they realize that it's possible for a person to change. It's possible to beat an addiction. You know, although it's a lifelong disease, this is going to help you.” The optimistic attitude is not only beneficial for the service user, but as noted by Coordinator 3, for the community at large: “They’re on the climb up, and they are determined, and they're excited, and they feel hopeful. . . . They’re working towards a better future for them [sic] and their families…in turn, if they do well, they actually benefit our community.” Some service recipients also disclosed very grave personal situations prior to beginning their recovery journey and encountering KATR: “I couldn’t stay out of jail. I was in trouble all the time with pills. . . . I’d be dead. Or in jail. . . . It saved my life and got my family back” (Service User 12). Each of the coordinators noted perseverance as a shared strength of their service users.More than half of the service recipients shared that being part of KATR led to dramatic behavior changes and has helped them sustain the new habits learned as part of recovery from opioid addiction. For these individuals, being part of the program has provided a “fresh start,” hope for a new life ahead, and motivation to keep pushing through hardships. Service User 1 stated that “they give addicts like myself  gives them the option as well because they’re working. They’re able to pay their rent. We’re providing childcare. (Coordinator 3)The recovery journey is difficult and requires an immense amount of support. Service recipients noted that they face stressors of all kinds—triggers and cravings, self-guilt, and past traumas—as they strive to stay sober. While some KATR clients noted that their family and friends provide emotional and physical care, others are not able to utilize those relationships. Some service recipients have lost loved ones to overdoses, while others must distance themselves from people who continue to use substances, as removing negative influences is critical to recovery. For some families in rural Eastern Kentucky, substance use is generational, and leaving behind one’s family is distressing. KATR coordinators fill that void of a support system––and give extra support to others who still have family and friends: “It has helped folks and kind of sent them on a new path. Gave them encouragement to go forward into a beautiful place” (Vendor 3).The rural KATR site is in a persistently poor county. As such, financial vulnerability is a major challenge. Stresses of a financial nature can wreak havoc in the lives of vulnerable populations, especially those with a substance use disorder. Many KATR clients are starting over in most aspects of their lives, as opioid addiction cost them in many ways—including the loss of employment and assets. As discussed, KATR provides vouchers for clothing, childcare, healthcare costs, and transportation, among other needs. Several participants  shared that the cost of buying a new tire, required work shoes, or dentures could be detrimental to individuals on the road to recovery amidst persistent poverty. Many people have options to deal with life’s inevitable problems, but people in poverty often do not: “I’ve had them sit in front of us and cry . . . because we’ve helped them. . . . I guess we take a lot of things for granted. . . . I’ve got a credit card . . . but they don’t. . . . They don’t have the cash, or they gotta pay for something else and do without” (Coordinator 1). The support through vouchers has created some financial stability for the service recipients.One noted that the vouchers “gave me a little extra start, gave me that extra income…that I would have had to either take a loan or I would have had to borrow...kinda gave me that foot forward” (Service User 9). Further, the support from KATR—financial and emotional—provided numerous service recipients the ability and confidence to obtain employment:I think it works great. I think it’s helped me get back on track. It really has . . . I landed this job. I’ve been with it for the last year and a half ... in the past I never did care. I never did really worry about to get promoted and things. . . . I probably I shouldn’t be here, but, you know, it’s just really taught me to take care of business and how to manage my life better. (Service User 10)Residents in Appalachian areas are face economic decline, persistent and extreme poverty, and lack of access to health care and prevention, all of which can contribute to hardship for those living with an opioid use disorder and seeking recovery. Kentucky has the ninth-highest rate of opioid-involved overdoses in the U.S., and the county where KATR was implemented has seen a rate of age-adjusted overdose-related deaths that is 120% higher than the national average as recently as 2018.Coordinators indicated that this challenge could be due to lack of motivation, homelessness, no means to connect , or other family challenges. KATR meets the basic needs of their participants and the surrounding community, but it ultimately goes beyond this to fill gaps in the community. These gaps are felt not only by those receiving services, but also by those providing services. Many participants remarked that without KATR, they would not have received care for physical or mental health needs, such as dental services. They would also be without additional support, such as clothing needed for activities like job interviewing. With these services provided to them, service users experienced improved health, self-esteem and self-worth, as indicated in the interviews. The changes to self-esteem are felt not only by participants, but also by the coordinators and vendors who work with them.Many participants reported that the services provided by KATR helped them regain custody and improve their parenting skills. Alongside this, KATR provides childcare so parents can seek regular employment and pay their rent, without having to worry about the added exponential cost of childcare. Participants enrolled in KATR reported seeing their own strength, and ultimately having that strength is noticed by both KATR staff and vendors.In turn, the larger community feels the impact of KATR. This program is available in an area that has been ravaged by the opioid epidemic; it is therefore essential that it addresses stigma and helps change community attitudes toward those living with a substance use disorder. Vendors and service providers alike noted that the community has become more compassionate toward those living with opioid use disorder. The vouchers have been beneficial for the community’s small businesses and ultimately help overcome systemic problems in this Appalachian area . During the COVID-19 pandemic, many vendors noted that they would have had less income without the use of KATR vouchers.Considering systemic problems, KATR works to overcome community barriers to help individuals who are changing their way of life continue to meet their recovery goals, all while addressing community needs and fostering social change. In taking a holistic approach and seeing participants as both impacting and being impacted by their communities, KATR works to bridge gaps that exist when a person desires recovery and wants to meet essential recovery goals. Funding for the program comes through the Substance Abuse and Mental Health Services Administration (SAMHSA) and is administered through the state.It is clear that the KATR program has made substantial efforts to reduce longstanding health problems in rural counties across the state. The concentrated health disparities in this region elucidate the necessity for critical health, economic, and social intervention. In sum, KATR strives to eliminate health disparities surrounding the opioid epidemic and to provide supplemental tools that assist participants on their lifelong journey to recovery. The opioid epidemic is widespread and complex; thus, multifaceted programs and interventions such as KATR are deserving of implementation to assist in the treatment of opioid misuse and addiction. As programs are needed to assist with opioid misuse and addiction, evaluation of these programs is equally important to assess for the effectiveness and benefit to the community and individuals in which they serve.While the KATR program has overall shown to be helpful and impactful, it is not without challenges, such as programmatic limitations, community limitations and a need for increased marketing, and providers should work to address these as the program continues.Given that this study utilized qualitative research methods and evaluated a program in a specific region of Eastern Kentucky, results should not be assumed to be generalizable to other geographical areas and with other populations. While random selection was utilized to select service recipients to be sampled for the study, the research was conducted with a small sample of individuals, and participants may not be entirely representative of the service recipients, coordinators, and vendors. It is worth noting that the small sample size could be comprised of individuals who have more favorable views of the program. However, the authors were not affiliated with the KATR program and were identified as university researchers. By identifying as third-party evaluators, the research team is hopeful that participants felt autonomous enough to provide critical feedback of the program and their experiences.The purpose of this study was to understand the impact of KATR on service recipients and the barriers they encounter in their recovery journey while part of the program. Key findings from the study show that the KATR program assisted service users with provision of basic needs . Through the provision of these basic needs, service users indicated indirect impacts on their overall wellbeing, such as increased self-esteem and confidence, which enhanced their performance in job interviews. While there were many positive impacts, barriers still existed, such as individuals reporting guilt, stress, trauma experiences, and trying to overcome generational drug use and personal cravings. Programs are needed to help meet these needs and to reduce barriers to accessing recovery in rural areas like the area of Eastern Kentucky studied here. KATR demonstrates a useful strategy of increasing access to health-related services in predominantly rural areas through the use of social support schemes and vouchers.What is already known about this topic?The misuse of illicit and prescription opioids has become a nationwide epidemic, with particularly devastating effects in Appalachia. Many programs have arisen to address the complex determinants of addition and aid in recovery.What is added by this report?This study provides a comprehensive program evaluation of Kentucky Access To Recovery (KATR), an Eastern Kentucky-based program to provide supplemental supports to individuals in recovery from opioid use disorder. It details the experiences of service users, programmatic staff, and vendors contracted to provide services to program participants. Their interview responses—and the themes emerging from them—show that the KATR program has made substantial efforts to reduce longstanding health problems in rural counties across Kentucky.What are the implications for future research?KATR demonstrates a useful strategy of increasing access to health-related services in predominantly rural areas through the use of support schemes and vouchers. Because of the complex nature of the opioid epidemic, it is important that policymakers and health officials pursue multifaceted programs, like KATR, that combine health, economic, and social intervention. Future research should assess these programs not just in terms of individual impact, but also in respect to their impact on communities at large.


Zoonotic transmission is a challenge for the control and elimination of malaria. It has been recorded in the Atlantic Forest, outside the Amazon which is the endemic region in Brazil. However, only very few studies have assessed the antibody response, especially of IgM antibodies, in Neotropical primates (NP). Therefore, in order to contribute to a better understanding of the immune response in different hosts and facilitate the identification of potential reservoirs, in this study, naturally acquired IgM antibody responses against Plasmodium antigens were evaluated, for the first time, in NP from the Atlantic Forest.Plasmodium species and different erythrocytic stage antigens were detected by ELISA.The study was carried out using 154 NP samples from three different areas of the Atlantic Forest. IgM antibodies against peptides of the circumsporozoite protein (CSP) from different Plasmodium vivax erythrocytic stage antigen. Levels of antibodies against PvAMA-1 were the highest compared to the other antigens. All families of NP showed IgM antibodies against CSP peptides, and, most strikingly, against erythrocytic stage antigens. Generalized linear models demonstrated that IgM positivity against PvCSP and PvAMA-1 was associated with PCR-detectable blood-stage malaria infection and the host being free-living. Interestingly, animals with IgM against both PvCSP and PvAMA-1 were 4.7 times more likely to be PCR positive than animals that did not have IgM for these two antigens simultaneously.Fifty-nine percent of NP had IgM antibodies against at least one CSP peptide and 87% against at least one Plasmodium spp. antigens are present in NP from the Atlantic Forest. High seroprevalence and antibody levels against blood-stage antigens were observed, which had a significant association with molecular evidence of infection. IgM antibodies against CSP and AMA-1 may be used as a potential marker for the identification of NP infected with Plasmodium, which are reservoirs of malaria in the Brazilian Atlantic Forest.IgM antibodies against different  P. vivax, 17% by Plasmodium falciparum, and <1% by Plasmodium malariae   . Plasmodn Brazil . Plasmodectively . Zoonotichniques . In Braz Janeiro .Plasmodium sp. and dominate the early memory response to recurrent malaria infections, providing evidence of additional mechanisms by which the immune system may control infection , Brazil, outside the Atlantic Forest in a non-transmission malaria area were used as negative controls.The NP samples that we studied came from three different locations in the Atlantic Forest: Indaial and Joinville, both in the state of Santa Catarina (SC) in south Brazil, and Guapimirim in the state of Rio de Janeiro (RJ) in southeast Brazil Table 1.Alouatta guariba clamitans) caught between 2008 and 2019: 63 from captive monkeys maintained at the Centre for Biological Research of Indaial (CEPESBI), and a further 12 plasma samples taken from free-living NP. In this group, 81% of samples were from adult NP  in Guapimirim, where serum samples were collected between January 2011 and October 2019 from 40 captive adult monkeys belonging to all NP families Table 1.s by PCR . Ninety s by PCR   and by the Brazilian Ministry of Environment .P. vivax CSP (PvCSP) were used: the classic VK210 (Pvc) DGQPAGDRAAGQPAG-(DRADGQPAG)2, VK247 (Pvk) (ANGAGNQPG)3-ANGAGN, and P. vivax-like (Pvl) (APGANQEGGAA)3. Additionally, CSP peptides from P. falciparum (NANP)8 (PfCSP) and P. malariae/P. brasilianum (GNAA)2-GNDA(GNAA)4 were utilized. The CSP repeat sequences of P. brasilianum and P. malariae are identical, and because of that, they were referred to as Pb/PmCSP , were used for the detection of immunoglobulin M (IgM) antibodies. Three variants of Pb/PmCSP .Escherichia coli , including aminoacids 43 to 487, was produced as a recombinant protein with 6xHis-tag in the eukaryotic expression system , which represents amino acids 1616–1704, was expressed as a 6xHis tag fusion protein in Pichia pastoris  from the Sal-1 reference strain, and recombinant EBP2 included amino acids 159–485 of the Erythrocyte Binding Protein 2 from C127 Cambodian isolate. These two recombinant proteins were expressed as 39 kDa 6xHis-tag fusion protein in hia coli . The ectn system . The Mern system . The 19-pastoris .Plasmodium antigens was performed, as previously described (19), 1.5 µg/mL (PvEBP-2 and PvAMA-1), 3 µg/mL (PvDBPII), and 5 µg/mL (PmMSP119 and PfMSP119). NP serum or plasma samples were diluted at 1:50. Anti-human IgM-peroxidase-conjugated antibody (Jackson Immunoresearch) was used as a secondary antibody at 1:2500 dilution for CSP peptides or 1:5000 for recombinant proteins. In each well was added 100 μL of the 3,3′,5,5′-Tetramethylbenzidine (TMB) single solution (Life Technologies). Reaction development was interrupted by adding 25 μL of 2N HCl in each well. Antibodies were detected using a microplate reader  at an optical density (OD) of 450 nm. Results were expressed as seroprevalence, i.e., the percentage of positives, and antibody levels were expressed as the reactivity index (RI), which was calculated by dividing the mean OD values of tested samples by the mean plus three standard deviations (SDs) of the negative control samples. Samples used as negatives were taken from three Alouatta g. clamitans from Belo Horizonte in the state of Minas Gerais, Brazil, a non-transmission malaria area. This species was selected as it corresponded to 77% of the non-control animals studied here. These samples were negative in the Plasmodium PCR protocols tested (P. vivax-like).The enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against escribed  with mods tested . SamplesX2), according to the data distribution. The differences between either the medians or means of the reactivity index (RI) of two groups were verified using either the t-test or the Mann-Whitney U test, respectively, according to the data distribution. The RI comparisons between more than two groups were performed using either Analysis of Variance (ANOVA) or the Kruskal-Wallis test, followed by Tukey’s or Dunn’s post-hoc tests, respectively, according to the data distribution. For analysis of the overall data set, i.e., the data from all three groups combined, the association between categorical variables  and seroprevalence and IgM titers was assessed using either the Chi-square test (X2) or Fisher’s exact test, and the t-test or Mann-Whitney U test, respectively. Heatmaps for antibody levels against Plasmodium antigens were created using the R package ComplexHeatmap, which showed the hierarchical clustering with families fixed (P. vivax erythrocytic stage antigens were plotted using the “UpSetR” R package (v1.4.0) , and the Gamma-binomial mixture model were used. Variables with P-values < = 0.20 in the likelihood ratio test went on to step 2, where all were jointly adjusted. From that moment on, all the variables that passed from the previous step were adjusted together. The significance of each was analyzed separately using the likelihood ratio test. Here, we compared whether there was a significant difference between the model without and with the variable in question. A model was fitted with all significant variables from the previous step. The process was repeated until only significant variables remained. In all analyses, a significance level of 5% was considered, i.e., values of P< 0.05.Analyses were done using GraphPad Prism version 8.0  and R version 4.0.2  . For eaces fixed . The int(v1.4.0) . AnalyseP. vivax “classic” VK210 (Pvc) in all three areas (P< 0.05)  of the NP showed an IgM response against at least one of the five CSP peptides assayed (P< 0.05). In Joinville/SC, antibody levels were significantly higher against PfCSP than against PvkCSP, PvlCSP, and Pb/PmCSP peptides (P< 0.05). The levels of antibodies were relatively low and without any statistically significant differences among all CSP peptides in Guapimirim/RJ  (P< 0.05) (P< 0.05)  of the NP studied against at least one of the three P. vivax), while 45% (61/135) of the NP showed IgM against all three variants of PvCSP and at least one erythrocytic stage antigen , and 53% (71/135) of the individuals showed IgM against at least one erythrocytic stage antigen but not against CSP (P< 0.001) between antibody levels against PvCSP and PvAMA-1 (ρ = 0.55), PvCSP and PvEBP-2 (ρ = 0.48), PvCSP and PvDBPII (ρ = 0.55), PvAMA-1 and PvEBP-2 (ρ = 0.72), PvAMA-1 and PvDBPII (ρ = 0.68), and PvEBP-2 and PvDBPII (ρ = 0.74) (Thirty-seven percent (50/135) of the NP showed IgM antibodies against all of the studied antigens  (P<0.05) (P>0.05) , while IgM titers were similar  compared to NP non-infected with P. simium (PCR- negative for P. simium)    were used. With respect to the response against the pre-erythrocytic stage protein, CSP, individuals that were PCR-positive for malaria infection showed a higher chance of having IgM antibodies against PfCSP, Pb/PmCSP, and PvCSP compared to PCR-negative NP  (P<0.008) (In order to identify which variables could explain the occurrence of IgM antibodies against ression) Figure 5P = 0.016), adult , and free-living  individuals were significantly more likely to respond against PvAMA-1 compared to PCR-negative, non-adult, and captive individuals, respectively (P<0.05). Interestingly, we observed that animals that had IgM against PvCSP and PvAMA-1 were 4.7 times more likely to be PCR-positive, compared to those individuals that did not respond to these proteins (P<0.05)  was identified that determined the chance of NP having IgM against erythrocytic stage proteins. PCR positive . Overall, 77% (33/43) of the selected NP samples showed IgM responses against PvMSP19, with 58% (25/43) having IgM antibodies to all P. vivax antigens tested. Twenty-three percent (10/43) showed IgM antibodies against one or up to three erythrocytic stage antigens, and 2% (1/43) did not respond against any of the studied antigens. Seroprevalence and antibody titers against PvMSP119 and the other erythrocytic stage P. vivax antigens studied were similar. However, there was a statistically significant difference only between PvMSP119 and PvCSP (P< 0.05). A significantly weak to moderate positive correlation was also identified between antibody levels against PvMSP119 and other antigens (P< 0.003)  showed IgM against Pb/PmCSP and PmMSP119, and 5% (2/43) did not respond against any Pb/Pm antigen. The frequency of responders and their level of antibody against PmMSP119 were higher than against Pb/PmCSP (P < 0.001). No correlation was observed between IgM levels against both Pb/Pm antigens  showed IgM against PmMSP119, 50% (22/43) showed IgM against the PfCSP and PfMSP119, and 16% (7/43) did not respond against any one of these antigens. Seroprevalences and antibody titers were similar against both of these antigens (P>0.05). A significant weak positive correlation was identified between antibody levels against PfCSP and PfMSP119 (ρ = 0.33 and P= 0.027) (Sixty percent (26/43) of NP showed IgM against PfMSP1The analyses of IgM responses among different NP families were performed using samples only from Guapimirim/RJ because the other areas had species only from the Atelidae family Table 1.Plasmodium was evaluated in NP from different Atlantic Forest areas of Brazil for the first time. The presence of IgM against the major pre-erythrocytic stage antigen (CSP) was used here as a potential marker of exposure to infected mosquito bites.In this study, the profile of acquired IgM antibody immune responses against both pre-erythrocytic and erythrocytic stage antigens of Plasmodium species, and the geographic areas were tested (ranging from 10% to 97%). Conversely, the IgM responses were very high for every erythrocytic stage antigen in all studied areas. IgM antibody levels were higher against the PvCSP variant VK210, which is the most common P. vivax CSP variant in humans, and NP, which is one of the two variants described for P. simium   . The lowe Amazon . The lowe Amazon . IgG ante Amazon .19 of P. vivax, P. falciparum, and P. malariae  compared to MSP119 specific for other Plasmodium species, while antibody levels were similar to other Pf antigens (PfCSP). A similar frequency of individuals showing IgG against PfMSP119 (approximately 50%) was reported in human populations in the Amazon region  against PvAMA-1 and malaria positivity  responsible for the IgM response identified here were the presence of PCR-detectable mic area . Free-lind India . OverallP. vivax transmission  and by the Brazilian Ministry of Environment .Plasmodium proteins and peptides. GA, DA, and LS performed the experiments. ES performed the statistic model. GA, DA, JS-A, TS, FK, LC, LP-R, CD-R, and CB analyzed and interpreted data. GA, DA, and CB wrote the first draft. All authors contributed to the article and approved the submitted version.CB, LC, and GA designed and conceived the study. GA, DA, AP-C, JJ, GG, AN, AP, SM, HT, VP, and ZH were responsible for sample collection. LT, HC, FK, IS, FN, JA, JF, and LP-R were responsible for the production of