Clinical and preclinical pulmonary disease in newly diagnosed rheumatoid arthritis: a two-year follow-up study.

OBJECTIVE: Pulmonary disease is a major cause of excess mortality among patients with rheumatoid arthritis (RA). Interstitial lung disease (ILD) is a feared complication, but the benefit of screening is unknown. The aim of this study was to assess the frequency of pulmonary disease, including ILD, in early RA. METHOD: Patients with newly diagnosed RA were recruited prospectively at a single centre and underwent systematic pulmonary function tests (PFTs) and computed tomography (CT) scans at inclusion and after two years. RESULTS: The study included 150 patients (mean age 57 years, 63% female; 59% current or former smokers). Of these, 136 underwent baseline PFTs and 137 CT. Mean forced expiratory volume in one second was 99% predicted and forced vital capacity 106%. Mean diffusing capacity of the lungs for carbon monoxide (DLCO) was 84% predicted. Frequently detected CT abnormalities were pulmonary nodules (42%), bronchiectasis (29%), and emphysema (20%). Two patients had clinically significant ILD and six had mild reticulation suggestive of preclinical ILD. No ILD progression was identified at two-year follow-up. Smoking was associated with DLCO<80% (p=0.004), combined hyperinflation and diffusion impairment (residual volume>120% and DLCO<80%) (p=0.004), and visual emphysema on CT (p<0.001). CONCLUSION: Emphysema and bronchiectasis were common, but most patients had mild disease with preserved lung function. Preclinical or clinical ILD was seen in a minority in this early phase of RA. These findings suggest symptom-based screening and primary intervention focusing on smoking cessation rather than screening for ILD at the time of RA diagnosis.

Prevalence of survey-based criteria for fibromyalgia and impact on hospital burden: a 7 year follow-up study from an outpatient clinic.

OBJECTIVES: To evaluate the prevalence of survey-based criteria for fibromyalgia (FM) among newly referred patients in a rheumatic outpatient clinic, and to compare the use of secondary healthcare services between survey-based FM and non-FM cases. METHOD: Newly referred patients to an outpatient clinic were screened for the fulfilment of the 2011 FM survey criteria during a 6 month period in 2013 in this observational cohort study. Demographic data were obtained at baseline. Patients' medical files were evaluated and comparisons between groups were made regarding the use of hospital healthcare facilities during the 7 year observation period. RESULTS: Out of 300 invited patients, 248 (83%) completed the questionnaire; 90 patients (36%) fulfilled survey-based criteria for FM at enrolment. FM cases were primarily women (80% vs 54% of non-FM cases), and received more medications (median 4 vs 3 drugs) and public economic support (62% vs 20%). At the 7 year follow-up, crude analyses showed that FM cases had a higher number of hospital courses (median 10 vs 8) and had undergone more invasive procedures (78% vs 60%). Neurologists (42% vs 28%), gastroenterologists (30% vs 13%), endocrinologists (40% vs 21%), pain specialists (13% vs 3%), psychiatrists (20% vs 7%), and abdominal surgeons (43% vs 30%) were consulted more often by FM than by non-FM cases. CONCLUSION: Fulfilment of FM survey criteria among newly referred patients to a rheumatic outpatient clinic is frequent. Our study findings show that FM continues to present a challenge for healthcare professionals as well as for patients. RESEARCH HIGHLIGHTS: ● Fulfilment of FM survey criteria among newly referred patients to a rheumatic outpatient clinic is frequent.● The burden on the secondary healthcare system for these patients is significant.● This study suggests the need for increased awareness about the diagnosis of FM among certain medical and surgical specialties.

Anti-cyclic citrullinated peptide antibodies, 28-joint Disease Activity Score, and magnetic resonance imaging bone oedema at baseline predict 11 years' functional and radiographic outcome in early rheumatoid arthritis.

OBJECTIVE: To investigate the clinical and radiographic status, and to identify baseline predictors of functional status and erosive progression at 11 years' follow-up of early rheumatoid arthritis (RA) patients. METHODS: Patients enrolled in the Danish investigator-initiated randomized controlled CIMESTRA trial, which investigated a 2 year treat-to-target intervention with methotrexate and intra-articular glucocorticoids with or without cyclosporine, were followed up. The 28-joint Disease Activity Score (DAS28), Health Assessment Questionnaire (HAQ) score, and total Sharp van der Heijde score (TSS) were assessed at baseline and 11 years. Baseline magnetic resonance imaging (MRI) of unilateral wrists was scored (OMERACT RAMRIS). Multivariable linear regression analyses of baseline variables [TSS, HAQ, DAS28, age, anti-cyclic citrullinated peptide (anti-CCP) status, gender, MRI erosion score, MRI synovitis score, MRI bone marrow oedema score] were performed in 96 patients with HAQ11yrs and ∆TSS0-11yrs as dependent variables. Since outcomes were similar in the two treatment arms, data were pooled. RESULTS: In total, 120 of 160 patients completed 11 years' follow-up. They were 63 (55-72) years old, 68% were in DAS28 remission (≤ 2.4), HAQ11yrs was 0.25 (0-0.75), mean ∆TSS0-11yrs was 0.96 ± 1.52 units/year; 53%, 20%, and 27% received conventional treatment, biologics, and no treatment, respectively; and 34% had not progressed radiographically since baseline. Increased DAS28 (p = 0.02) and anti-CCP (p = 0.03) predicted HAQ11yrs, whereas anti-CCP (p = 0.03) and MRI bone marrow oedema (p = 0.01) predicted ∆TSS0-11yrs in multivariable analyses. CONCLUSIONS: Early and strict synovitis suppression with methotrexate and intra-articular glucocorticoids led to persistently high remission rates and limited erosive progression at 11 years. In this well-treated cohort, baseline anti-CCP status, DAS28, and MRI bone marrow oedema predicted functional status and/or erosive progression.

Circulating serum interleukin-6, serum chitinase-3-like protein-1, and plasma vascular endothelial growth factor are not predictive for remission and radiographic progression in patients with early rheumatoid arthritis: post-hoc explorative and validation studies based on the CIMESTRA and OPERA trials.

OBJECTIVE: To investigate serum interleukin-6 (IL-6), serum chitinase-3-like protein-1 (YKL-40), and plasma vascular endothelial growth factor (VEGF) as measures of disease activity and predictors of clinical remission and radiographic progression in two early rheumatoid arthritis (RA) randomized controlled trials (RCTs). METHOD: Treatment-naïve patients with early RA (< 6 months' duration) and active disease, participating in two investigator-initiated RCTs, were treated according to a predefined treat-to-target algorithm aiming at inflammatory control, using methotrexate (MTX) + cyclosporine versus MTX + placebo (CIMESTRA study, n = 150, 5 year follow-up) or MTX + adalimumab versus MTX + placebo (OPERA study, n = 180, 2 year follow-up). The 28-joint Disease Activity Score (DAS28) and conventional radiography [bilateral hands and feet at baseline, 2 years and 5 years (only CIMESTRA)] were obtained at baseline and during follow-up. Serum IL-6, serum YKL-40, and plasma VEGF were measured in baseline blood samples and during follow-up. Hypotheses regarding the biomarkers' relation with DAS28 and ability to predict clinical remission (DAS28 < 2.6) and radiographic progression (change in total Sharp van der Heijde score ≥ 2) were generated in CIMESTRA and validated in OPERA, by Spearman's correlation and logistic regression analyses. RESULTS: Baseline IL-6, YKL-40, and VEGF correlated significantly with DAS28 in CIMESTRA (r = 0.50, r = 0.36, r = 0.36, respectively, all p < 0.01) and these results were confirmed in OPERA patients (r = 0.52, p < 0.01; r = 0.18, p = 0.01; r = 0.23, p = 0.002, respectively). None of the biomarkers (absolute values or change) was predictive of clinical remission or radiographic progression at 2 or 5 years in either study. CONCLUSION: Serum IL-6, serum YKL-40, and plasma VEGF were significantly correlated with DAS28 at baseline, but did not have consistent predictive value for clinical remission or radiographic progression in two early RA RCTs.

Galectin-3 is Persistently Increased in Early Rheumatoid Arthritis (RA) and Associates with Anti-CCP Seropositivity and MRI Bone Lesions, While Early Fibrosis Markers Correlate with Disease Activity.

Galectin-3 has been suggested as a pro-inflammatory mediator in animal arthritis and rheumatoid arthritis (RA). We aimed to study the serum level of galectin-3 in patients with newly diagnosed RA and associations with disease profile, Magnetic resonance imaging (MRI) findings and seromarkers of synovial matrix inflammation. One hundred and sixty DMARD naïve patients newly diagnosed with RA were included (CIMESTRA study). Clinical, serological and imaging data were recorded before treatment and at 6 weeks, 3 and 12 months. Galectin-3 and hyaluronan (HYA) were measured by ELISA (R&D and Corgenix, USA), and the N-terminal propeptide of type III collagen (PIIINP) by radioimmunoassay (Orion Diagnostica, Finland). One hundred and nineteen, 87 and 60 blood donors served as controls for galectin-3, HYA and PIIINP, respectively. Baseline galectin-3 was significantly elevated in anti-CCP positive (4.2 µg/l IQR [3.6;6.1]) patients as compared with anti-CCP negatives (4.0 µg/l [2.6;4.9], P = 0.05) and controls (3.8 µg/l [3.0;4.8], P < 0.01). During treatment, galectin-3 remained elevated, but increased transiently with peak values at 6 weeks. Galectin-3 correlated with baseline smoking, anti-CCP, and with MRI erosion score after 1 year of follow-up. HYA and PIIINP were elevated (P < 0.001) irrespective of anti-CCP status and correlated positively with synovitis assessed clinically and by MRI. HYA and PIIINP did not correlate with galectin-3. These observations indicate that HYA and PIIINP mainly reflect expansive synovitis proliferation while galectin-3 is more closely linked to autoimmunity, smoking and joint destructive processes.

Prediction of treatment response to adalimumab: a double-blind placebo-controlled study of circulating microRNA in patients with early rheumatoid arthritis.

At least 30% of patients with rheumatoid arthritis (RA) do not respond to biologic agents, which emphasizes the need of predictive biomarkers. We aimed to identify microRNAs (miRNAs) predictive of response to adalimumab in 180 treatment-naïve RA patients enrolled in the OPtimized treatment algorithm for patients with early RA (OPERA) Study, an investigator-initiated, prospective, double-blind placebo-controlled study. Patients were randomized to adalimumab 40 mg (n=89) or placebo-adalimumab (n=91) subcutaneously in combination with methotrexate. Expressions of 377 miRNAs were determined using TaqMan Human MicroRNA LDA, A Card v2.0 (Applied Biosystems). Associations between miRNAs and treatment response were tested using interaction analyses. MiRNAs with a P-value <0.05 using three different normalizations were included in a multivariate model. After backwards elimination, the combination of low expression of miR-22 and high expression of miR-886.3p was associated with EULAR good response. Future studies to assess the utility of these miRNAs as predictive biomarkers are needed.

Interactions between smoking, increased serum levels of anti-CCP antibodies, rheumatoid factors, and erosive joint disease in patients with early, untreated rheumatoid arthritis.

OBJECTIVES: To determine to what extent shared epitopes, smoking, and anti-cyclic citrullinated peptide (anti-CCP) antibodies are associated with disease activity and erosive disease in patients with rheumatoid arthritis (RA) at disease onset. METHOD: RA patients not previously treated with disease-modifying anti-rheumatic drugs (DMARDs) and with a disease duration of < 6 months (CIMESTRA study) were examined for shared epitopes, anti-CCP antibodies, immunoglobulin M rheumatoid factor (IgM-RF) and IgA-RF, radiographic erosive changes in hands and feet, and clinical disease activity. RESULTS: The study comprised 153 patients, of whom 104 (68%) were ever-smokers. The prevalence of patients with 0, 1, or 2 shared epitopes was 40 (48%), 71 (49%), and 33 (23%), respectively. Anti-CCP antibodies, IgM-RF, and IgA-RF were present in 89 (58%), 99 (65%), and 82 (54%) patients, respectively. Among smokers, erosive disease was associated with anti-CCP antibodies [odds ratio (OR) 3.9, 95% confidence interval (CI) 1.6-9.3], IgM-RF (OR 4.9, 95% CI 1.9-12), and IgA-RF (OR 2.8, 95% CI 1.2-6.4) but absent with regard to shared epitopes. Among never-smokers, erosive disease was not associated with either shared epitopes or antibodies. All antibody levels measured were associated with smoking and shared epitopes. CONCLUSIONS: Shared epitopes and smoking were associated with the production of anti-CCP antibodies and rheumatoid factors of IgM and IgA isotypes, which again were associated with erosive disease at presentation only in smokers. As shared epitopes and smoking were not directly associated with erosive disease, smoking may enhance the development of erosive disease in RA at different levels or through separate pathways.

Up-regulated dipeptidyl-peptidase IV (CD26) on monocytes was unaffected by effective DMARD treatment in early steroid and DMARD-naive rheumatoid arthritis.

OBJECTIVES: To study the CD26 density on monocytes and CD4+ T-lymphocytes in steroid and DMARD-naïve, early rheumatoid arthritis (RA) patients and to analyse for correlations with disease activity, including long-term radiographic progression. METHODS: Forty patients with active, early steroid and DMARD naïve RA (<6 months' duration) were randomised to treatment with methotrexate (MTX) versus MTX and cyclosporine A (CYA). Controls were 15 healthy age and gender matched subjects. Peripheral blood mononuclear cells were analysed for CD26 density by flow cytometry at baseline and after 52 weeks. Radiographic progression was scored by delta total Sharp-van der Heijde score (TSS) from 0 to 5 years. RESULTS: The density of CD26 on monocytes (CD3-CD14+) in RA was up-regulated compared to healthy controls (p<0.0001) and remained unaffected by clinically effective DMARD treatment after 52 weeks. In anti-CCP positive RA patients (n=18) baseline CD26 density on monocytes correlated to 5-year radiographic progression (p=0.008, r=0.60). The density of CD26 did not correlate to DAS28, the swollen or tender joint count or CRP-level at baseline or at year one. The CD26 density on CD4+ T-lymphocytes at week 0 was comparable to healthy controls (p=0.34). CONCLUSIONS: The up-regulated density of CD26 on monocytes in steroid and DMARD naïve active early RA was unaffected by 52 weeks of effective DMARD treatment and correlated to 5-year radiographic progression.

Presence and interaction of inflammatory cells in the spleen of Atlantic cod, Gadus morhua L., infected with Francisella noatunensis.

Serious infectious diseases, accompanied by macrophage-dominated chronic inflammation, are common in farmed Atlantic cod. To increase knowledge relating to morphological aspects of such inflammatory responses, cod were challenged with Francisella noatunensis, an important bacterial pathogen of this fish species. Tissue and cell dynamics in the spleen were examined sequentially over 60 days. Small clusters of mainly macrophage-like cells (MLCs) staining for non-specific esterase and acid phosphatase developed with time. These foci were transiently infiltrated by pleomorphic proliferating cells of unknown nature and by granulocyte-like cells (GCLCs) staining for peroxidase and lysozyme. The latter cell type, which appeared to be resident in the red pulp of control fish, migrated into the inflammatory foci of infected fish. Cells expressing genes encoding IFN-γ and IL-8 increased in number during the study period. Bacteria were detected only in the MLCs and their number increased despite the extensive inflammation. Our results demonstrate an intimate spatial relationship in inflammatory foci between at least three cell types. The presence of GCLCs, together with MLCs, suggests pyogranulomatous inflammation as a more appropriate descriptive term than granulomatous inflammation.

MRI bone oedema is the strongest predictor of subsequent radiographic progression in early rheumatoid arthritis. Results from a 2-year randomised controlled trial (CIMESTRA).

OBJECTIVE: To identify predictors of radiographic progression in a 2-year randomised, double-blind, clinical study (CIMESTRA) of patients with early rheumatoid arthritis (RA). METHODS: Patients with early RA (n = 130) were treated with methotrexate, intra-articular betamethasone and ciclosporin/placebo-ciclosporin. Baseline magnetic resonance imaging (MRI) of the wrist (wrist-only group, n = 130) or MRI of wrist and metacarpophalangeal (MCP) joints (wrist+MCP group, n = 89) (OMERACT RAMRIS), x-ray examination of hands, wrists and forefeet (Sharp/van der Heijde Score (TSS)), Disease Activity Score (DAS28), anti-cyclic citrullinated peptide antibodies (anti-CCP), HLA-DRB1-shared epitope (SE) and smoking status were assessed. Multiple regression analysis was performed with delta-TSS (0-2 years) as dependent variable and baseline DAS28, TSS, MRI bone oedema score, MRI synovitis score, MRI erosion score, anti-CCP, smoking, SE, age and gender as explanatory variables. RESULTS: Baseline values: median DAS28 5.6 (range 2.4-8.0); anti-CCP positive 61%; radiographic erosions 56%. At 2 years: DAS28 2.0 (0.5-5.7), in DAS remission: 56%, radiographic progression 26% (wrist+MCP group, similar for wrist-only group). MRI bone oedema score was the only independent predictor of delta-TSS (wrist+MCP group: coefficient = 0.75 (95% CI 0.55 to 0.94), p<0.001; wrist-only group: coefficient = 0.59 (95% CI 0.40 to 0.77), p<0.001). Bone oedema score explained 41% of the variation in the progression of TSS (wrist+MCP group), 25% in wrist-only group (Pearson's r = 0.64 and r = 0.50, respectively). Results were confirmed by sensitivity analyses. CONCLUSION: In a randomised controlled trial aiming at remission in patients with early RA, baseline RAMRIS MRI bone oedema score of MCP and wrist joints (and of wrist only) was the strongest independent predictor of radiographic progression in hands, wrists and forefeet after 2 years. MRI synovitis score, MRI erosion score, DAS28, anti-CCP, SE, smoking, age and gender were not independent risk factors. TRIAL REGISTRATION NUMBER: NCT00209859.

In active chronic rheumatoid arthritis, dipeptidyl peptidase IV density is increased on monocytes and CD4(+) T lymphocytes.

The effect of low-dose methotrexate (MTX) treatment on the CD26 density on circulating monocytes and CD4(+) T lymphocytes or levels of soluble CD26 (sCD26) has not yet been described in rheumatoid arthritis (RA). While CD26 in T lymphocytes is involved in the activation and proliferation of T lymphocytes, little is known of the role of CD26 in monocytes as it has only recently been localized to monocytes. We analysed the CD26 density by flow cytometry and levels of sCD26 in plasma before initiation of MTX treatment and 12 weeks later. This was done on 34 RA patients fulfilling the 1987 American College of Rheumatology (ACR) criteria followed for 16 weeks after starting MTX treatment. CD26 density on monocytes was increased in RA patients compared with healthy controls before MTX treatment (P < 0.01). After 12 weeks of MTX treatment, the CD26 density on monocytes decreased significantly in the ACR-50% group (P = 0.03), but not in the ACR-20% and the non-responder group (P = 0.15 and 0.87). The increased CD26 density on CD4(+) T lymphocytes (P < 0.01) was unaffected by the reduction in disease activity in relation to MTX treatment. The percentage of monocytes and CD4(+) T lymphocytes among peripheral blood circulating mononuclear cells did not change during MTX treatment. No effect of MTX treatment was observed on the plasma levels of sCD26. Active chronic RA is characterized by enhanced CD26 density on circulating monocytes and CD4(+) T lymphocytes. MTX treatment decreased CD26 density on monocytes in the ACR-50% responder group and was associated with decreased disease activity. The enhanced CD26 density on CD4(+) T lymphocytes was uninfluenced by MTX treatment.

The presence of N-terminal secretion signal sequences leads to strong stimulation of the total expression levels of three tested medically important proteins during high-cell-density cultivations of Escherichia coli.

Genetic optimizations to achieve high-level production of three different proteins of medical importance for humans, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon alpha 2b (IFN-alpha2b), and single-chain antibody variable fragment (scFv-phOx), were investigated during high-cell-density cultivations of Escherichia coli. All three proteins were poorly expressed when put under control of the strong Pm/xylS promoter/regulator system, but high volumetric yields of GM-CSF and scFv-phOx (up to 1.7 and 2.3 g/liter, respectively) were achieved when the respective genes were fused to a translocation signal sequence. The choice of signal sequence, pelB, ompA, or synthetic signal sequence CSP, displayed a high and specific impact on the total expression levels for these two proteins. Data obtained by quantitative PCR confirmed relatively high in vivo transcript levels without using a fused signal sequence, suggesting that the signal sequences mainly stimulate translation. IFN-alpha2b expression remained poor even when fused to a signal sequence, and an alternative IFN-alpha2b coding sequence that was optimized for effective expression in Escherichia coli was therefore synthesized. The total expression level of this optimized gene remained low, while high-level production (0.6 g/liter) was achieved when the gene was fused to a signal sequence. Together, our results demonstrate a critical role of signal sequences for achieving industrial level expression of three human proteins in E. coli under the conditions tested, and this effect has to our knowledge not previously been systematically investigated.

Monocyte chemoattractant protein 1 (MCP-1) in temporal arteritis and polymyalgia rheumatica.

OBJECTIVE: To examine the localisation of monocyte chemoattractant protein 1 (MCP-1) in the inflamed vessel wall in temporal arteritis (TA) and to measure MCP-1 in plasma both in patients with TA and patients with polymyalgia rheumatica (PMR). METHODS: By immunohistochemical techniques MCP-1 was localised to the vessel wall in patients with TA. In TA, PMR, and healthy controls MCP-1 was quantified by enzyme linked immunosorbent assay (ELISA) in plasma. RESULTS: MCP-1 was localised to the majority of mononuclear cells, some smooth muscle cells, and giant cells in the arterial biopsy specimens from 12 patients with histologically verified TA. In all sections, including the vasa vasorum, the endothelium stained positive. In the intima 73% (range 57-91%), in the media 49% (range 32-67%), and in the adventitia 74% (range of 62-91%) of all cells stained positive. In plasma MCP-1 was significantly raised in untreated TA (n=33) and untreated PMR (n=27) compared with healthy controls (n=12). Untreated TA plasma levels of MCP-1 (mean 391 pg/ml (range 82-778 pg/ml)) were similar to untreated PMR plasma levels (mean 402 pg/ml (range 29-1153 pg/ml)), and no significant difference was found between the two groups of patients. In both patients with TA and patients with PMR no correlation was found between the plasma level of MCP-1 and the erythrocyte sedimentation rate, haemoglobin concentration, and CD4/CD8 ratio. CONCLUSIONS: These results show that MCP-1 plays a part in the disease processes of TA and PMR.

Distribution of intraperitoneally injected microspheres labeled with the alpha-emitter astatine (211At) compared with phosphorus (32P) and yttrium (90Y) colloids in mice.

The alpha-emitter 211At was bound to polymer microspheres with a diameter of 1.8 microns. The distributions in mice of intraperitoneally injected 211At microspheres, 90Y silicate colloid, and 32P chromic phosphate colloid were compared. The microspheres with 211At spread rapidly in the peritoneal cavity and remained mainly on the intraperitoneal surfaces. Intraperitoneal injection of 90Y colloid resulted in high levels in intraperitoneal fat and the diaphragm, but 1 day after injection 8.5% of the injected dose per gram was found in blood and after 6 days 2.5% was observed in bone. The highest accumulation of 32P was found in liver and spleen. The injection of additional nonradioactive chromic phosphate colloid resulted in an even higher accumulation of 32P in spleen and liver. The same phenomenon was not observed with 211At microspheres. It is suggested that it is not only the particle size which is important in the distribution of intraperitoneally injected colloid, but the amount of colloid, the type of colloid, the addition or presence of other substances such as ascites, and the animal species might also influence the distribution. In conclusion, the intraperitoneal distribution of 211At-labeled microspheres in mice was favorable compared with 90Y and 32P colloid. These data must be viewed cautiously since the distribution might be different in other animal species or humans.

Monosized, magnetic polymer particles: their use in separation of cells and subcellular components, and in the study of lymphocyte function in vitro.

By employing the principles of "activated swelling", monosized, superparamagnetic polymer particles have been prepared ranging in size from 1-100 microns. Both during and after the swelling process, the particles can be modified to meet a series of specific demands making them potentially very interesting for many separation and assay purposes. Using monoclonal antibodies to direct the magnetic beads to their targets, immunomagnetic separation has turned out to be one of the most specific, reliable and, above all, the fastest technique available today to isolate particulate material for further studies. So far, most efforts have been concentrated on methodology for fractionation of cells in suspension, such as removal of tumour cells from bone marrow or isolation of lymphoid cells from peripheral blood. These studies have both established the parameters necessary for optimal performance and at the same time laid the groundwork for future developments making immunomagnetic separation an exciting new tool in many research areas. High speed and specificity are the most conspicuous features of immunomagnetic cell separation. These properties have been exploited in the successful development of a new technique for tissue typing of cells directly from peripheral blood specimens. Both higher sensitivity and specificity have been obtained. The same principles can be used for fast and safe quantification of cell populations and subpopulations in blood and cell suspensions. The functions of, and interactions between, peripheral blood cell populations or subpopulations in the immune response have also been studied with high precision. The significance of direct cell contact on the one hand, and soluble factors on the other, can now be established in detail. Immunomagnetic beads have also been used to study the interaction between various T lymphocyte membrane molecules in the early phases of the activation process. Finally, the usefulness of specially developed particles for the fractionation of subcellular components is described.

Biochemistry of the autolytic processes in Antarctic krill post mortem. Autoproteolysis.

1. Autoproteolysis post mortem was examined at 0 degree C by following the changes in the major classes of krill (Euphausia superba and Euphausia crystallorophias) proteins and by liberation of peptides and free amino acids, and was based on experiments conducted on board expedition vessels in the Antarctic. 2. Primarily salt-soluble proteins were broken down during the first week of incubation, whereas water-soluble and insoluble proteins were degraded to a much smaller extent. The enzymes responsible for the hydrolysis presumably originate primarily from the digestive apparatus of the krill. 3. In general, the individual amino acids were released at rates corresponding to their relative occurrence in the bulk protein of the krill. Alanine was liberated in larger amounts than would be expected from the composition of the krill protein, and was evidently formed also by reactions other than proteolysis. Glutamic acid, and certain amino acids which presumably occur with high frequency adjacent to glumatic acid residues in the krill protein, were liberated only to a limited extent, and accumulated in smaller peptides. 4. During proteolysis, arginine seemed to be converted to some degree into ornithine, and on prolonged incubation conversion of arginine and lysine into their corresponding decarboxylation products, agmatine and cadaverine, appeared to take place.

Hydrophilic monodisperse particles as solid-phase material in immunoassays: comparison of shell-and-core particles with compact particles.

Hydrophilic monodisperse shell-and-core particles with a density of 1.07 were superior to heavier compact particles as a solid-phase material for immunoassays. The shell-and-core particles formed a semistable suspension for 24 h and were easily collected by centrifugation. The hydroxyl groups of the particles were activated with two sulfonyl chlorides. The most reactive one, tresyl chloride, gave rapid chemical coupling of antibodies, whereas tosyl chloride favored a rapid hydrophobic adsorption which was followed by slow chemical coupling. The solid-phase sheep antirabbit IgG made was used as a separation agent in several immunoassays and gave solid-phase primary antibodies by immunoadsorption of rabbit antibodies.