A longitudinal study of the leukocyte protein L1 as an indicator of disease activity in patients with rheumatoid arthritis

L1 is a major granulocyte and monocyte protein. It is released during leukocyte activation, and the plasma level is thought to reflect the inflammatory activity. Fifteen patients with classical or definite rheumatoid arthritis were examined monthly during one year. The laboratory tests included L1, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), orosomucoid, haptoglobin, ceruloplasmin, alpha 1-antitrypsin, immunoglobulins and blood cell counts. The clinical tests included articular index, grip strength, morning stiffness and pain. The L1 protein was found to have highly significant correlations (p less than 0.0001) with orosomucoid (r = 0.86), CRP (r = 0.79), ESR (r = 0.78), haptoglobin (r = 0.75), alpha 1-antitrypsin (r = 0.63) and ceruloplasmin (r = 0.44). Significant correlation was also found between L1 and IgA. None of the laboratory variables showed significant correlation with pain, but when they were correlated with articular index, grip strength and morning stiffness, L1 was found to have the highest average correlation coefficient (p less than 0.0001).     

L1, a major granulocyte protein; isolation of high quantities of its subunits

L1 is a major granulocyte and monocyte protein with a Mr of 36.5 kDa. It is found mainly in the cytosol of these cells. Purified L1 is shown, on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), to contain three subunits. In this study, 6 mol/l concentration of urea was found to be sufficient for disassembly of the polypeptides, and urea-containing preparative isoelectric focusing gel was used for separation of high quantities of the subunits. The pI of the eluted subunits were 5.8, 6.1 and 7.1. When tested on 2D-PAGE, the isolated subunits were found at their typical locations. Polyclonal rabbit antibodies were produced against the subunits, and the antisera were, on dot-blot, found to react with the different subunits as well as the purified L1.     

Prolonged and increased expression of soluble Fc receptors, IgG and a TCR-Ig fusion protein by transiently transfected adherent 293E cells

In studies of the relation between structure and function of proteins of the immune system, there is a continuous need for screening of a large number of protein variants. To optimise the yield following transient gene expression in small or medium culture volumes, several parameters were investigated. First, secretion levels of a soluble form of human Fcgamma receptor IIA (FcgammaRIIA) were measured after transfection of 293, 293E, 293T as well as COS-7 cell lines. The transgene was under cytomegalovirus (CMV) promoter control on the expression vector pcDNA3, which also contains an SV40 origin of replication (SV40 ori). All 293 cell lines secreted more protein than COS-7 cells. Introduction of the Epstein Barr virus (EBV) origin of replication (oriP) greatly increased the protein expression from the 293E cells, both the amount of protein produced per day and the duration of production. At best, 293E cells secreted fully functional protein for 3-4 weeks provided supernatant was harvested every 2-3 days followed by medium replacement. This method was then used for expression of soluble forms of human FcgammaRI, FcgammaRIIB, the human neonatal Fc receptor (FcRn), a T cell receptor (TCR)-immunoglobulin (Ig) fusion protein, and human IgG3. With an initial culture volume of 5 ml, the yield was approximately 200 microg for FcgammaRIIA, 1.5 microg for FcgammaRI, 5 microg for FcRn, 20 microg for FcgammaRIIB, 40 microg for the TCR-Ig fusion protein and 850 microg for IgG3. Culture expansion during the 3 weeks of culture further increased the yield. Protein yield was also improved by scaling up the initial volume. This approach can provide sufficient amounts of protein for screening experiments, and in the case of antibody, milligrams of recombinant protein for extensive structural analysis can be obtained from one single transient transfection. The approach should be of interest to laboratories that do not have access to a bioreactor but still have a requirement for reasonable amounts of protein to be produced in an easy and cost-effective manner.     

The Medi-Jector II: efficacy and acceptability in insulin-dependent diabetic patients with and without needle phobia

Efficacy and acceptability of the Medi-Jector II, compared with conventional syringes, was evaluated in a cross-over study (two 4-week periods) in 14 adult insulin-dependent diabetic patients. In 6 out of these 14 patients the clinical diagnosis of needle phobia was confirmed by anxiety tests for trait (45.8 +/- 12.8 (SD) versus 34.0 +/- 6.2; p less than 0.05) and state (50.3 +/- 10.9 versus 28.9 +/- 5.2, p less than 0.05). Five patients (one needle-phobic) dropped out, all for Medi-Jector-related problems. No significant differences were found in body weight, insulin dosage, and number of hypoglycaemic reactions. HbA1 after the Medi-Jector period was significantly higher than after the conventional period (9.8 +/- 1.2 (SE) versus 9.1 +/- 1.1 (SE)%; p less than 0.05). The questionnaire, evaluating the Medi-Jector, revealed poor acceptance of the device (7/9 patients' general impression being moderate or bad). The use of the Medi-Jector in comparison with conventional injections caused the patients to complain about: more immediate pain (4/9 patients), delayed pain (often: 4/9, sometimes: 2/9), more insulin leakage (6/9), more bleeding (6/9), and more haematomas (5/9). Except for 'often occurring delayed pain' (4/5 needle-phobics versus 0/4 non-needle-phobics; p less than 0.05), needle-phobic patients scored the questionnaire similarly to the other patients. We conclude that the use of the Medi-Jector II did not cause a major short-term loss of metabolic control, but that the device was not well accepted by our patients, independent of the existence of needle phobia.     

Cytokine-Stimulating (In Vitro) Effect on Human Monocytes of Conjugates of Soluble Curdlan and Albumin Microbeads

Four kinds of conjugates (BSA-M-SC-Ra, BSA-M-SC-Re, AP-M-SC-Ra, and AP-M-SC-Re) composed of soluble fragments of [β](1 ± 3)-D-glucan (SC) and bovine serum albumin (BSA) or amine-modifled polystyrene (AP) microbeads were prepared: the contents of SC on the microbeads were found to be 13.6, 8.0, 3.6, and 2.6 μg per 1 X 10⁸ microbeads, respectively. Unconjugated BSA and AP microbeads and SC did not show cytokine-stimulating activity. BSA-M-SC-Re, BSA-M-SC-Ra, and AP-M-SC-Re showed strong cytokine-stimulating activity in vitro, indicating that conjugates composed of inactive fragments of polysaccharides and microbeads are able to induce cytokine release from mononuclear phagocytes. Conjugates with the SC linked to BSA microbeads by the reducing terminal (BSA-M-SC-Re) showed a stronger effect than conjugates in which SC was linked with BSA microbeads at random positions (BSA-M-SC-Ra).     

L1, a major granulocyte protein; isolation of high quantities of its subunits

L1 is a major granulocyte and monocyte protein with a M, of 36.5 kDa. It is found mainly in the cytosol of these cells. Purified LI is shown, on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), to contain three subunits. In this study, 6 mol/1 concentration of urea was found to be sufficient for disassembly of the polypeptides, and urea-containing preparative isoelectric focusing gel was used for separation of high quantities of the subunits. The pi of the eluted subunits were 5.8, 6.1 and 7.1. When tested on 2D-PAGE, the isolated subunits were found at their typical locations. Polyclonal rabbit antibodies were produced against the subunits, and the antisera were, on dot-blot, found to react with the different subunits as well as the purified LI.     

Levels of calprotectin (leukocyte L1 protein) during apheresis.

The plasma concentration of calprotectin was measured before, during and after apheresis in patients with Guillain-Barré Syndrome (GBS), Waldenstr?m's syndrome or hypercholesterolaemia and in healthy donors of platelets. Increased calprotectin levels were found after plasma exchange in the Waldenstr?m's syndrome patients, probably caused by release of the protein from activated leukocytes. The decreased calprotectin values observed in the other patients, may be due to plasma dilution. Unexpectedly, the GBS patients were found to have high initial calprotectin levels in plasma but not in cerebrospinal fluid. In donors, normal and unchanged calprotectin concentrations were found throughout.     

L1, a major granulocyte protein: Antigenic properties of its subunits

L1, a major granulocyte protein, was purified and analysed by use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Three subunits were visualized, and they were found to have molecular weights of 12.5 kDa, 13.3 kDa and 8.3 kDa. They were extracted from 2D-gels, and different combinations of subunits and two L1 antisera were analysed by immunodiffusion in agarose gel. The 8.3 kDa polypeptide in combination with one or both of the other polypeptides, gave immunoprecipitation with one of the L1 antisera, while no precipitation occurred when the three polypeptides were tested separately. Neither was there any precipitation when the two heavier polypeptides were tested in combination. By use of another L1 antiserum, all the L1 polypeptides were found to be antigenic and give immunoprecipitations. The L1 protein has a great affinity for calcium, and calcium was necessary for immunoprecipitation with the L1 subunits to occur. Autoradiographs of 2D-PAGE gels with labelled leucocytes visualized the L1 subunits in the same position as the subunits from purified, cold L1, indicating no significant alteration of the L1 protein during the purification procedure.