How to Detect Antibodies Against Babesia divergens in Human Blood Samples.

Background: Today only indirect fluorescent antibody assays (IFAs) are commercially available to detect antibodies against Babesia divergens in humans. IFA is subjective and requires highly experienced staff. We have therefore developed an enzyme-linked immunosorbent assay (ELISA)-based method for measuring anti-B. divergens immunoglobulin G antibodies in human blood samples. Methods: Crude merozoite extract from in vitro cultures of a new B. divergens isolate was used in ELISA to detect antibodies in different sets of samples: Borrelia burgdorferi-positive samples, healthy individuals, tick-bitten individuals including follow-up samples 3 months later, positive control samples from patients with an active Babesia infection, and samples from malaria-endemic regions. As a reference, IFA was used to detect antibodies in the tick-bitten samples. Western blot was used to evaluate reactions against specific bands in extracts with/without parasites. Results: Using IFA as the reference method, the sensitivity and specificity of the ELISA were 86% (12/14) and 100% (52/52). There was a very high correlation (r = -0.84; P = .0004) between IFA dilution factors and ELISA absorbances among the samples classified as positive. Five percent of the B. burgdorferi-positive samples were judged as weakly positive and 5% as strongly positive in our ELISA. Western blot showed that the immunodominant antigens (∼120 kDa) were from merozoites and not from erythrocytes. Conclusions: This ELISA can detect antibodies directed against B. divergens, and it can be a useful and easy assay to handle compared with IFA. The ELISA can also measure high and low levels of antibodies, which could give insight into the recency of a B. divergens infection.

Acquisition of complement fixing antibodies targeting Plasmodium falciparum merozoites in infants and their mothers in Uganda.

Background: Antibody-mediated complement fixation has previously been associated with protection against malaria in naturally acquired immunity. However, the process of early-life development of complement-fixing antibodies in infants, both in comparison to their respective mothers and to other immune parameters, remains less clear. Results: We measured complement-fixing antibodies in newborns and their mothers in a malaria endemic area over 5 years follow-up and found that infants' complement-fixing antibody levels were highest at birth, decreased until six months, then increased progressively until they were similar to birth at five years. Infants with high levels at birth experienced a faster decay of complement-fixing antibodies but showed similar levels to the low response group of newborns thereafter. No difference was observed in antibody levels between infant cord blood and mothers at delivery. The same result was found when categorized into high and low response groups, indicating placental transfer of antibodies. Complement-fixing antibodies were positively correlated with total schizont-specific IgG and IgM levels in mothers and infants at several time points. At nine months, complement-fixing antibodies were negatively correlated with total B cell frequency and osteopontin concentrations in the infants, while positively correlated with atypical memory B cells and P. falciparum-positive atypical memory B cells. Conclusion: This study indicates that complement-fixing antibodies against P. falciparum merozoites are produced in the mothers and placentally-transferred, and they are acquired in infants over time during the first years of life. Understanding early life immune responses is crucial for developing a functional, long lasting malaria vaccine.

Babesia divergens Shows Equal Predilection for Human ABO Blood Types in an In Vitro Erythrocyte Preference Assay.

Babesia is spread to humans via ticks or blood transfusions. Severity of Plasmodium falciparum malaria is strongly correlated to the ABO blood group of the patient. Babesia divergens is an intraerythrocytic parasite with many similarities to malaria, but the impact of ABO on the susceptibility to and progression of the infection in humans is unknown. We have now cultured B. divergens in human group A, B and O erythrocytes in vitro and measured rates of multiplication. The predilection for the different erythrocyte types was also determined using an in vitro erythrocyte preference assay when the parasites were grown in group A, B or O erythrocytes over time and then offered to invade differently stained erythrocytes of all the blood types at the same time. The results showed no difference in multiplication rates for the different blood types, and the parasite exhibited no obvious morphological differences in the different blood types. When cultured first in one blood type and then offered to grow in the others, the preference assay showed that there was no difference between the A, B or O blood groups. In conclusion, this indicates that individuals of the different ABO blood types are likely to be equally susceptible to B. divergens infections.

Global series: Complex regional pain syndrome: abstracts from the International Association for the Study of Pain complex regional pain syndrome SIG virtual symposia 2021.

The aim of this IASP complex regional pain syndrome (CRPS) SIG Global Series 2021 was to bring together clinicians including those from developing countries to better understand the clinical presentation of complex regional pain syndrome in countries with less well-published patient populations. The purpose was to learn from each other about the range of treatments, successful outcomes, and challenges experienced. These meeting proceedings comprise abstracts from nine countries that span 4 continents and are summaries of online presentations delivered by speakers representing these countries over the course of 2 symposia. The symposia were attended by a global audience of approximately 360 people. Patients with CRPS were described and treated by clinicians from countries across Asia (Pakistan, Jordan, South Korea, Taiwan, and Singapore), South America (Brazil and Peru), Africa (South Africa), and Europe (Norway). This reflects that CRPS exists across borders, ethnicities, and cultures. These proceedings provide a broader perspective within the international pain community about how we can better understand and treat CRPS across the globe.

Mediators of Pain and Physical Function in Female and Male Patients with Chronic Pain.

PURPOSE: Chronic pain is often multifactorial and accompanied by psychological distress, catastrophizing thoughts, reduced physical function, and socio-economic worries. In this explorative study, we investigated potential mediators in the relationships of psychological and demographic variables with chronic pain and physical function in women and men. PATIENTS AND METHODS: The study included 301 patients admitted to a multidisciplinary pain clinic. Prior to their first consultation, patients completed a questionnaire including items on demographics (age, education, occupational and financial situation), catastrophizing thoughts, psychological distress, pain intensity, and physical function. Hierarchical multiple regression analyses examined demographic and psychological factors associated with pain intensity and physical function. Mediation and reversed mediation models were tested and developed based on calculated relations in the regression analyses between demographic, psychological, pain intensity and physical function variables. RESULTS: Fifty-eight percent were females and mean age 43.8 and 46.0 years for women and men, respectively. In the regression analyses, psychological factors accounted better for pain intensity than demographic variables, while physical function was best accounted for by demographic variables. Among women, catastrophizing thoughts mediated significantly the relationships between education and pain intensity, and between education and physical function. Psychological distress mediated significantly the relationships between financial situation and pain intensity, and between financial situation and physical function in women. In men, the only significant mediation model was psychological distress mediating the relationship between financial situation and pain intensity. Some of the reversed models revealed indirect effects, indicating bidirectionality. CONCLUSION: The results indicate that there might be gender-specific mediators in how demographic variables are associated with pain intensity and physical function. This suggests an awareness among clinicians of potential gender-specific factors mediating pain problems, and the need for a gender-specific, multidisciplinary approach in the treatment of chronic pain.

Mirror therapy for phantom limb and stump pain: a randomized controlled clinical trial in landmine amputees in Cambodia.

Background and aims The aim of the study was to examine the effect of mirror and tactile therapy on phantom and stump pain in patients with traumatic amputations, with particular reference to amputees in low-income communities. Methods The study was conducted with an open, randomized, semi-crossover case-control design in rural Cambodia. A study sample of 45 landmine victims with trans-tibial amputations was allocated to three treatment arms; mirror therapy, tactile therapy, and combined mirror-and-tactile therapy. Non-responders from the mono-therapy interventions were crossed over to the alternative intervention. The intervention consisted of 5 min of treatment every morning and evening for 4 weeks. Endpoint estimates of phantom limb pain (PLP), stump pain, and physical function were registered 3 months after the treatment. Results All three interventions were associated with more that 50% reduction in visual analogue scale (VAS)-rated PLP and stump pain. Combined mirror-tactile treatment had a significantly better effect on PLP and stump pain than mirror or tactile therapy alone. The difference between the three treatment arms were however slight, and hardly of clinical relevance. After treatment, the reduction of pain remained unchanged for an observation period of 3 months. Conclusions The study documents that a 4-week treatment period with mirror and/or tactile therapy significantly reduces PLP and stump pain after trans-tibial amputations. Implications The article reports for the first time a randomized controlled trial of mirror therapy in a homogenous sample of persons with traumatic amputations. The findings are of special relevance to amputees in low-resource communities.

Anaesthetizing children-From a nurse anaesthetist's perspective-A qualitative study.

AIM: The aim of this study was to describe nurse anaesthetists' experiences of encountering and caring for children in connection to anaesthesia. DESIGN: Qualitative design. METHODS: Sixteen written narratives based on eight nurse anaesthetists' experiences of meeting children was analysed using qualitative content analysis. RESULTS: The overarching theme was: "anaesthetizing children is a complex caring situation, including interactions with the child and parents as well as ensuring patient safety, affected by the perioperative team and organizational prerequisites". The nurses stated that in their interaction with the family, their goal was to ensure that children and parents felt secure and calm. "Striving to work in confidence" underlined the team and organizational influences. Encountering children involves more than knowledge about technical equipment, procedures and drugs. Knowledge about children's development and fears and parents' needs are essential for an optimal caring situation. Organizations need to realize that extra time, skills and resources are needed to safely anaesthetize children.

A novel monoclonal antibody targeting carboxymethyllysine, an advanced glycation end product in atherosclerosis and pancreatic cancer.

Advanced glycation end products are formed by non-enzymatic reactions between proteins and carbohydrates, causing irreversible lysine and arginine alterations that severely affect protein structure and function. The resulting modifications induce inflammation by binding to scavenger receptors. An increase in advanced glycation end products is observed in a number of diseases e.g. atherosclerosis and cancer. Since advanced glycation end products also are present in healthy individuals, their detection and quantification are of great importance for usage as potential biomarkers. Current methods for advanced glycation end product detection are though limited and solely measure total glycation. This study describes a new epitope-mapped single chain variable fragment, D1-B2, against carboxymethyllysine, produced from a phage library that was constructed from mouse immunizations. The phage library was selected against advanced glycation end product targets using a phage display platform. Characterization of its binding pattern was performed using large synthetic glycated peptide and protein libraries displayed on microarray slides. D1-B2 showed a preference for an aspartic acid, three positions N-terminally from a carboxymethyllysine residue and also bound to a broad collection of glycated proteins. Positive immunohistochemical staining of mouse atherosclerotic plaques and of a tissue microarray of human pancreatic tumors confirmed the usability of the new scFv for advanced glycation end product detection in tissues. This study demonstrates a promising methodology for high-throughput generation of epitope-mapped monoclonal antibodies against AGE.

Epitope mapping of a new anti-Tn antibody detecting gastric cancer cells.

Here, we introduce a novel scFv antibody, G2-D11, specific for two adjacent Tn-antigens (GalNAc-Ser/Thr) binding equally to three dimeric forms of the epitope, Ser-Thr, Thr-Thr and Thr-Ser. Compared to other anti-Tn reagents, the binding of G2-D11 is minimally influenced by the peptide structure, which indicates a high degree of carbohydrate epitope dominance and a low influence from the protein backbone. With a high affinity (KDapp = 1.3 × 10-8 M) and no cross-reactivity to either sialyl-Tn epitope or blood group A antigens, scFv G2-D11 is an excellent candidate for a well-defined anti-Tn-antigen reagent. Detailed immunohistochemical evaluation of tissue sections from a cohort of 80 patients with gastric carcinoma showed in all cases positive tumor cells. The observed staining was localized to the cytoplasm and in some cases to the membrane, whereas the surrounding tissue was completely negative demonstrating the usefulness of the novel Tn-antigen binding antibody.

A Combinatory Antibody-Antigen Microarray Assay for High-Content Screening of Single-Chain Fragment Variable Clones from Recombinant Libraries.

We have developed a combinatory antibody-antigen microarray for direct screening of multiple single-chain fragment variable (scFv) clones with no need for pre-purification or enrichment before screening. The straightforward workflow allows for early selection of binders to predefined peptide and glycopeptide targets. A capture antibody is contact printed on microarray slides, side by side with the antigens of interest. A large number of scFv clones, in supernatants, are printed on top of the capture antibody and the antigen in a "spot-on-spot" print. The printed scFv clones, which bind to the capture antibody, are detected using biotinylated antigen, while the binding of scFv clones to the printed antigen is detected through a mouse anti-tag antibody. Two different analyses are thus performed on the same slide, generating two kinds of information: one on the ability of an individual scFv clone to bind to the soluble form of the antigen, which may favour selection for higher affinity rather than avidity, while the other allows the identification of large numbers of clones, simultaneously, due to the binding of scFv clones to densely presented antigens, thus providing an overall increased hit rate. The functionality of the new screening approach was illustrated through the generation of antibodies against peptides from the chaperone complex Ku70/Ku80 and the GalNAcα-serine/threonine epitope on the IgA1 alpha chain hinge region. In total, 659 scFv clones were screened with a hit rate of approximately 20%. This approach allowed the identification of functional antibodies in both cases, illustrating the usefulness and capacity of this combinatory microarray screening technique for efficient analysis and validation of antibodies at an early stage of antibody generation.

Intra-tumour IgA1 is common in cancer and is correlated with poor prognosis in bladder cancer.

A high frequency of IgA1-positive tumour cells was found in tissue micro-arrays of oesophagus, colon, testis, lung, breast, bladder and ovarian cancer. IgA1 was observed in the cytoplasm and the plasma membrane. A correlation was found between intra-tumour IgA1 and poor overall survival in a large cohort of bladder cancer patients (n = 99, p = 0.011, log-rank test). The number of IgA1-positive tumour cells was also found to be higher in female than male bladder cancer patients. The presence of IgA1 was confirmed in formalin-fixed paraffin-embedded ovarian carcinoma samples using LC-MS/MS analysis. Uptake of IgA1 was also observed in breast cancer and melanoma cell lines when cultivated in the presence of serum from healthy individuals, indicating a possible origin of the IgA1 antibodies in cancer cells.

A human ICAM-1 antibody isolated by a function-first approach has potent macrophage-dependent antimyeloma activity in vivo.

We isolated a tumor B-cell-targeting antibody, BI-505, from a highly diversified human phage-antibody library, using a pioneering "function-first" approach involving screening for (1) specificity for a tumor B cell surface receptor, (2) induction of tumor programmed cell death, and (3) enhanced in vivo antitumor activity compared to currently used treatments. BI-505 bound to intercellular adhesion molecule-1, identifying a previously unrecognized role for this receptor as a therapeutic target in cancer. The BI-505 epitope was strongly expressed on the surface of multiple myeloma cells from both newly diagnosed and relapsed patients. BI-505 had potent macrophage-dependent antimyeloma activity and conferred enhanced survival compared to currently used treatments in advanced experimental models of multiple myeloma.

Charges drive selection of specific antibodies by phage display.

Phage display technology has emerged as a leading approach to select proteins with improved properties for many different types of applications. The selection typically selects not only for improved binding properties but also for other factors such as efficiency of protein production and folding in Escherichia coli, the host in which the proteins and the phage are produced. Furthermore, the selection methodology is likely to influence the character of retrieved variants. We have now defined the extent whereby the charge of the displayed proteins influence the selection process, resulting in an increased average positive charge among selected proteins in comparison to the proteins that are harbored in the library before selection. Implications of and possible routes to minimize this effect are discussed.