Elevated Fab glycosylation of anti-hinge antibodies.

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by systemic inflammation and the presence of anti-citrullinated protein antibodies (ACPAs), which contain remarkably high levels of Fab glycosylation. Anti-hinge antibodies (AHAs) recognize immunoglobulin G (IgG) hinge neoepitopes exposed following cleavage by inflammation-associated proteases, and are also frequently observed in RA, and at higher levels compared to healthy controls (HCs). Here, we investigated AHA specificity and levels of Fab glycosylation as potential immunological markers for RA. METHOD: AHA serum levels, specificity, and Fab glycosylation were determined for the IgG1/4-hinge cleaved by matrix metalloproteinase-3, cathepsin G, pepsin, or IdeS, using enzyme-linked immunosorbent assay and lectin affinity chromatography, in patients with early active RA (n = 69) and HCs (n = 97). RESULTS: AHA reactivity was detected for all hinge neoepitopes in both RA patients and HCs. Reactivity against CatG-IgG1-F(ab´)2s and pepsin-IgG4-F(ab´)2s was more prevalent in RA. Moreover, all AHA responses showed increased Fab glycosylation levels in both RA patients and HCs. CONCLUSIONS: AHA responses are characterized by elevated levels of Fab glycosylation and highly specific neoepitope recognition, not just in RA patients but also in HCs. These results suggest that extensive Fab glycosylation may develop in response to an inflammatory proteolytic microenvironment, but is not restricted to RA.

Effects of high-intensity exercise training on physical fitness, quality of life and treatment outcomes after oesophagectomy for cancer of the gastro-oesophageal junction: PRESET pilot study.

BACKGROUND: Treatment for cancer of the gastro-oesophageal junction (GOJ) can result in considerable and persistent impairment of physical fitness and health-related quality of life (HRQoL). This controlled follow-up study investigated the feasibility and safety of postoperative exercise training. METHODS: Patients with stage I-III GOJ cancer were allocated to 12 weeks of postoperative concurrent aerobic and resistance training (exercise group) or usual care (control group). Changes in cardiorespiratory fitness, muscle strength and HRQoL were evaluated. Adherence to adjuvant chemotherapy, hospitalizations and 1-year overall survival were recorded to assess safety. RESULTS: Some 49 patients were studied. The exercise group attended a mean of 69 per cent of all prescribed sessions. After exercise, muscle strength and cardiorespiratory fitness were increased and returned to pretreatment levels. At 1-year follow-up, the exercise group had improved HRQoL (+13·5 points, 95 per cent c.i. 2·2 to 24·9), with no change in the control group (+3·7 points, -5·9 to 13·4), but there was no difference between the groups at this time point (+9·8 points, -5·1 to 24·8). Exercise was safe, with no differences in patients receiving adjuvant chemotherapy (14 of 16 versus 16 of 19; relative risk (RR) 1·04, 95 per cent c.i. 0·74 to 1·44), relative dose intensity of adjuvant chemotherapy (mean 57 versus 63 per cent; P = 0·479), hospitalization (7 of 19 versus 6 of 23; RR 1·41, 0·57 to 3·49) or 1-year overall survival (80 versus 79 per cent; P = 0·839) for exercise and usual care respectively. CONCLUSION: Exercise in the postoperative period is safe and may have the potential to improve physical fitness in patients with GOJ cancer. No differences in prognostic endpoints or HRQoL were observed. Registration number: NCT02722785 ( https://www.clinicaltrials.gov).

ANTECEDENTES: El tratamiento del cáncer de la unión gastroesofágica (gastroesophageal junction, GEJ) puede determinar un deterioro considerable y persistente de la condición física y de la calidad relacionada con la salud (health-related quality of life, HRQoL). El objetivo de este estudio controlado de seguimiento fue investigar la factibilidad y seguridad del entrenamiento físico postoperatorio. MÉTODOS: Pacientes con cáncer de GEJ en estadio I-III fueron asignados a 12 semanas de entrenamiento postoperatorio simultáneo aeróbico y de resistencia o a cuidados médicos habituales. Se evaluaron los cambios en el estado cardiorrespiratoria, fuerza muscular y HRQoL. Se recogieron datos de la adherencia a la quimioterapia adyuvante, hospitalizaciones y supervivencia global a 1 año para evaluar la seguridad. RESULTADOS: Se estudiaron un total de 49 pacientes. El grupo con ejercicio asistió al 69% de todas las sesiones planificadas. Después del ejercicio, la fuerza muscular y el estado cardiorrespiratorio aumentaron y volvieron a los niveles previos al tratamiento. Si bien al año de seguimiento, el grupo con ejercicio presentó una mejoría de la HRQoL (+13,5 puntos (i.c. del 95% 2,2 a 24,9)), sin cambios en el grupo con atención médica habitual (+3,7 puntos (i.c. del 95% −5,9 a 13,4)), no hubo diferencias entre los grupos en ese momento (+9,8 puntos (i.c. del 95% −5,1 a 24,8)). El ejercicio fue seguro, sin diferencias entre el ejercicio o la atención médica habitual en pacientes que recibían quimioterapia adyuvante 87,5% versus 84,2% (RR 1,04 (i.c. del 95% 0,74 a 1,44)), intensidad relativa de la dosis de quimioterapia adyuvante 56,8% versus 63,3% (P = 0,479), hospitalizaciones 36,8% versus 26,1% (RR 1,41 (i.c. del 95% 0,57 a 3,49)) o supervivencia global a 1 año 80,0% versus 79,3% (P = 0,839). CONCLUSIÓN: El ejercicio en el periodo postoperatorio es seguro y puede tener potencial para mejorar la condición física en pacientes con cáncer de GEJ. No se observaron diferencias en los resultados pronósticos o en la HRQoL.

Safety and feasibility of preoperative exercise training during neoadjuvant treatment before surgery for adenocarcinoma of the gastro-oesophageal junction.

Background: Neoadjuvant chemotherapy or chemoradiotherapy is used widely before tumour resection in cancer of the gastro-oesophageal junction (GOJ). Strategies to improve treatment tolerability are warranted. This study examined the safety and feasibility of preoperative exercise training during neoadjuvant treatment in these patients. Methods: Patients were allocated to a standard-care control group or an exercise group, who were prescribed standard care plus twice-weekly high-intensity aerobic exercise and resistance training sessions. The primary endpoint was the incidence of serious adverse events (SAEs) that prevented surgery, including death, disease progression or physical deterioration. Preoperative hospital admission, postoperative complications, changes in patient-reported quality of life and pathological treatment response were also recorded. In the exercise group, adherence to exercise and changes in aerobic fitness, muscle strength and body composition were measured. Results: The incidence of SAEs was not increased in the exercise group. The risk of failure to reach surgery was 5 versus 21 per cent in the control group (risk ratio (RR) 0·23, 95 per cent c.i. 0·04 to 1·29), the risk of preoperative hospital admission was 15 versus 38 per cent respectively (RR 0·39, 0·12 to 1·23) and the risk of postoperative complications was 58 versus 57 per cent (RR 1·06, 0·61 to 1·73). The exercise group attended a mean of 17·5 sessions, and improved fitness, muscle strength and Functional Assessment of Cancer Therapy - Esophageal (FACT-E) total score compared with the baseline level. Conclusion: Preoperative exercise training during neoadjuvant treatment in patients with GOJ cancer is safe and feasible, with improvements in fitness, strength and quality of life. Preoperative exercise training may be associated with a lower risk of critical SAEs that preclude surgery or result in hospitalization.

Increasing signal homogeneity and image quality in abdominal imaging at 3 T with very high permittivity materials.

The appearance of severe signal drop-outs in abdominal imaging at 3 T arises primarily from areas of very low B1+ transmit field in the body, and is problematic in both obese as well as very thin subjects. In this study, we show how thin patient-friendly pads containing new high permittivity materials can be designed and optimized, and when placed around the subject increase substantially the B1+ uniformity and the image quality. Results from nine healthy volunteers show that inclusion of these dielectric pads results in statistically significant decreases in the coefficient of variance of the B1+ field, with stronger and more uniform fields being produced. In addition there are statistically significant decreases in time-averaged power required for scanning. These differences are present in both quadrature-mode operation (coefficient of variance decrease, P<0.0001, mean 25.4±10%: power decrease, P=0.005, mean 14±14%) and also for the RF-shimmed case (coefficient of variance decrease, P=0.01, mean 16±13%: power decrease, P=0.005, mean 22±11%) of a dual-transmit system.

Closed reduction intermetacarpal Kirschner wire fixation in the treatment of unstable fractures of the base of the first metacarpal.

The purpose of this study was to describe the results of extra-articular and intra-articular fractures, at the base of the first metacarpal, treated with closed reduction and percutaneous fixation with intermetacarpal Kirschner wires. Outcome was evaluated by experienced pain, functional outcome and radiographic indications for arthritis. In total, 25 patients with unstable fractures at the base of the first metacarpal underwent closed reduction and percutaneous fixation of the fracture. Prospectively collected data of 25 consecutive patients were evaluated retrospectively, assessing stability of fixation, operation time and the occurrence of fracture dislocation during and after treatment. All patients were assessed at 1, 3, 6 and 24 months. Follow-up included questionnaires: functional tests including grip and pinch measurement and radiographic analysis for post-traumatic arthritis, using the modified Eaton-Littler classification. In total, 15 patients with extra-articular fractures and 10 patients with intra-articular fractures were treated with this technique. In the group of extra-articular fractures of 15 patients, only one patient had loss of grip strength greater than 20% in comparison with the contra-lateral side (corrected for hand dominance). No clinically important difference was found for pinch strength. One patient experienced functional limitations and was unable to return to a previous hobby. In the patients group with intra-articular fractures, seven patients had a Bennett fracture and three a Rolando fracture. One patient with a Bennett fracture had a loss of pinch strength greater than 20% corrected for hand dominance. One of the three patients with a Rolando fracture had grip loss greater than 20%. None of the patients with intra-articular fractures experienced any functional limitations. The described fixation procedure results in a stable fixation of the fracture fragments, and no secondary dislocation of the fracture occurred. Fractures consolidated within 32 (26-50) days and no new fractures were observed. These results suggest that this technique can be safely used in the treatment of extra-articular fractures as well as intra-articular fractures at the base of the first metacarpal.

Evaluation by double-blind placebo-controlled oral challenge of the clinical relevance of IgE antibodies against plant glycans.

BACKGROUND: The clinical relevance of immunoglobulin E (IgE) to plant glycans is a longstanding debate. We sought to evaluate their clinical reactivity using the human glycoprotein lactoferrin expressed in rice. METHODS: Allergic patients with IgE antibodies against plant glycans were analyzed for the presence of IgE against rice-produced lactoferrin. The potency of IgE to induce mediator release was assessed by basophil histamine release and skin prick tests (SPTs). Clinical relevance was evaluated by double-blind placebo-controlled oral challenge (DBPCOC). RESULTS: Twenty-four of 29 sera (82.7%) with IgE antibodies against plant glycans demonstrated IgE binding to transgenic lactoferrin. In three of five cases transgenic lactoferrin induced histamine release. Compared to a control major grass pollen allergen lactoferrin concentrations needed for biological activity of IgE were 5-6 orders of magnitude higher. Skin prick test and DBPCOC were negative in five patients with potential clinical reactivity that volunteered to undergo these in vivo challenges. CONCLUSIONS: Poor or no biological activity and lack of clinical relevance of IgE-binding plant glycans (five out of five) was demonstrated using human lactoferrin expressed in rice as a model.

Combined expression of the non-receptor protein tyrosine kinases FAK and Src in primary colorectal cancer is associated with tumor recurrence and metastasis formation.

PURPOSE: The protein tyrosine kinase focal adhesion kinase (FAK) and Src in association with phosphorylation of the adapter protein paxillin are essential in tumor metastasis formation. Elevated levels of FAK, Src and paxillin may increase the metastatic potential of colorectal tumor cells. The aim of the current study was to examine the expression of FAK, Src, and paxillin using immunohistochemistry in the context of disease progression and to evaluate its clinical significance as a prognostic factor. EXPERIMENTAL DESIGN: The relationship between FAK, Src and paxillin levels and colorectal cancer progression was evaluated by immunohistochemistry in 104 colorectal cancer specimens with clinical follow up. In addition, FAK, Src and paxillin expression levels were quantified in 68 colorectal tumors and corresponding liver metastases. RESULTS: FAK and paxillin expression individually did not significantly impact time to recurrence (p=0.09, and p=0.89 respectively). Src expression was associated with tumor recurrence p=0.03. However, tumors that expressed both high FAK and Src levels had a significant shorter time to recurrence (p=0.004, hazard ratio: 2.98, 95% CI 1.14-6.31). FAK, Src and paxillin showed equivalent levels in corresponding liver metastases compared to the primary tumors (p=0.67, p=0.28 and p=0.34 respectively). CONCLUSIONS: These findings show that high levels of FAK and Src combined were predictive for recurrence of colorectal cancer. In addition, expression of FAK, Src and paxillin in colorectal cancer were maintained in corresponding distant metastases.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Quality of life in adults following bone marrow transplantation during childhood.

SUMMARY: Quality of life (QOL) was assessed in 22 young adults, 14 years - on average- after having received bone marrow transplantation (BMT) during childhood at the Leiden University Medical Center. All were disease-free and >16 years when interviewed. The sickness impact profile and the Medical Outcome Study 36-item Short Form Health Survey were used as generic questionnaires in the assessment of QOL. The Functional Assessment of Cancer Therapy-Bone Marrow Transplant Scale (FACT-BMT) was used as a disease-specific measure of QOL. Coping was assessed by means of the Utrecht coping list. BMT-related variables were obtained from medical files. Of the generic QOL measures, most results fell within the normal range of functioning, although some illness-related impairment was reported on subscales for general and work-related functioning. Compared to a reference sample of patients who had received BMT as adults, patients involved in this study scored significantly higher on the 'emotional well-being' subscale of the FACT-BMT, indicating significantly better emotional functioning. The age at BMT and total body irradiation (TBI) were not related to patients' QOL. We can conclude that at long term, having received BMT during childhood does not negatively affect the QOL of patients. Bone Marrow Transplantation (2004) 33, 329-336. doi:10.1038/sj.bmt.1704345 Published online 1 December 2003

Immunochemical characterization of two Pichia pastoris-derived recombinant group 5 Dactylis glomerata isoallergens.

BACKGROUND: Grass pollen of the Poaceae grasses are known to be highly allergenic. Major allergens from the species Lolium, Phleum, Poa and Holcus have been cloned and expressed as recombinant proteins, but of the important species Dactylis glomerata no recombinants are available. METHODS: Dac g 5 was cloned by PCR on the basis of homology with Lol p 5 and expressed in Pichia pastoris. Recombinant Dac g 5 (rDac g 5) was affinity purified and compared to natural Dac g 5 (nDac g 5) by immunoblot, radioallergosorbent test (RAST), RAST inhibition, basophil histamine release assay (HRA), competitive radioimmunoassay (RIA) and sandwich enzyme-linked immunosorbent assay (ELISA). In addition, N-terminal sequencing, concanavalin A (Con A) binding, circular dichroism spectrum measurements and matrix-assisted laser desorption ionization-time of flight mass-spectrometric analysis were performed. RESULTS: Clones were obtained that coded for pro-Dac g 5 and two mature isoforms of Dac g 5; the deduced amino acid sequences of both isoforms differed by 4 amino acids. Both mature isoforms were expressed in Pichia at a concentration of approximately 15 mg/l. SDS-PAGE analysis showed that rDac g 5 had an apparent M(r) approximately 10 kD above nDac g 5. By mass spectrometry this difference was shown to be around 2.5 kD. Positive Con A staining suggested (O-linked) glycosylation as an explanation for this increase in M(r). Whereas both purified recombinants showed a tendency to dimerize, purified nDac g 5 contained a 12-kD peptide not observed for rDac g 5. RAST, RAST inhibition and HRA showed that the IgE reactivity of rDac g 5 was similar to that of nDac g 5. A small subgroup, however, clearly demonstrated decreased IgE reactivity to rDac g 5.02. Differences in immune reactivity of both isoforms were confirmed by monoclonal antibody (mAb)-based sandwich ELISA. CONCLUSIONS: Dac g 5 was successfully cloned and expressed in P. pastoris. Minor differences in primary structure between isoforms influence their immune reactivity.

Inadvertent BSA-induced elution of IgE in the BSA-RAST.

BACKGROUND: Bovine serum albumin (BSA) is widely used to block nonspecific binding in immunochemical assays. Whereas a previous study had indicated that soluble allergen present during the incubation with anti-IgE in the RAST did not affect bound IgE, we reinvestigated this in the current study, using IgE elution from BSA by soluble BSA as a test system. METHODS: Sepharose-coupled BSA (0.08, 0.4, 2, or 10 microg BSA/test) was incubated overnight with serum and washed. The Sepharose was then incubated with different concentrations of soluble BSA (0, 12, 60, 300, or 1500 microg/test), washed again, and incubated with radioactive anti-IgE. The effect on IgE binding was investigated for various incubation periods (t=0, 1, 2, 4, and 20 h). RESULTS: Incubation in buffer without BSA did not change IgE binding. Soluble BSA eluted IgE antibodies from immobilized BSA by up to 85%. If the BSA density on the solid phase was > or =2 microg/test, the elution efficiency was dependent on the levels of both immobilized BSA and soluble BSA. At lower densities, the dissociation was dependent only on the concentration of soluble BSA. The time needed to obtain 50% IgE elution (t(1/2)) was less if the density of immobilized BSA decreased. Below the critical density (0.8 microg BSA/mg solid phase), t(1/2) was independent of the coating density (45 min). Probably all IgE antibodies are monovalently bound below this density. CONCLUSIONS: Dissociation of IgE from immobilized protein in the presence of soluble protein should be taken into account, particularly when IgE to mammalian serum albumin is involved (milk, meat, or animal dander).

The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum.

BACKGROUND: For the detection of allergen-specific IgE in serum, IgE-binding assays such as the radioallergosorbent assay (RAST) are commonly used. In this study, the applicability and sensitivity of the stripped basophil histamine release bioassay was investigated and compared to the RAST. METHODS: Basophils were stripped of their IgE by an acidic buffer, sensitized by human serum and stimulated by allergen with or without interleukin (IL)-3. The histamine release was determined by fluorometric analysis. RESULTS: We showed that for enhancement of the maximal histamine release and the sensitivity of the stripped basophil assay, the priming cytokine IL-3 can be added to the basophils simultaneously to the stimulus. Preincubation of the cells with IL-3, as described in other studies, was not necessary. The bioassay can be used to study the specificity of IgE-mediated reactions. Basophils sensitized by serum absorbed to a particular allergen did not respond to this allergen anymore. This method is very suitable to study cross-reactivity between allergens. The results obtained in the bioassay were comparable to those obtained in the RAST. Using the RAST, lower concentrations of allergen-specific IgE were detected than in the bioassay. However, sera containing IgE against minor allergenic components were negative in the RAST, but strongly positive in the basophil assay. CONCLUSIONS: The stripped basophil histamine release bioassay is useful to complement and extend serological detection of allergen-specific IgE. Especially with sera containing IgE against minor components, this assay is more suitable than the RAST. Furthermore, in this assay, the dependency of IgE and of allergen-specific IgE in reactions can be studied in more detail.