The impact of single amino acid substitutions in CD3gamma on the CD3epsilongamma interaction and T-cell receptor-CD3 complex formation.

The human T-cell receptor-CD3 complex consists of at least eight polypeptide chains; CD3gamma- and delta-dimers associate with the disulfide linked alphabeta- and zetazeta-dimers to form a functional receptor complex. The exact structure of this complex is still unknown. We now have examined the interaction between CD3gamma and CD3 in human T-cells. For this purpose, we have generated site-directed mutants of CD3gamma that were introduced in human T-cells defective in CD3gamma expression. Cell-surface and intracellular expression of the introduced CD3gamma chains was determined, as was the association with CD3delta, CD3, and the T-cell receptor. Although the introduction of wild type CD3gamma and CD3gamma (78Y-F) fully restored T-cell receptor assembly and expression, the introduction of CD3gamma (82C-S), CD3gamma (85C-S), and CD3gamma (76Q-E) all resulted in an impaired association between CD3gamma and CD3 and a lack of cell-surface expressed CD3gamma. Finally, the introduction of CD3gamma (76Q-L) and CD3gamma (78Y-A) restored the expression of TCR-CD3deltagammazeta2 complexes, although the association between CD3gamma and CD3 was impaired. These results indicate that several amino acids in CD3gamma are essential for an optimal association between CD3gamma and CD3 and the assembly of a cell-surface expressed TCR-CD3deltagammazeta2 complex.

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Clinical utility of diagnostic tests for rheumatoid factor.

OBJECTIVE: To investigate and compare the accuracy and usefulness of diagnostic tests for rheumatoid factor (RF). METHODS: In a cross-sectional study sera derived from patients admitted to the Section of Rheumatology were tested for presence of RF using either nephelometry or the Waaler test. Diagnostic sensitivity and predictive values of the tests were calculated and compared. The accuracy of the tests was compared using receiver-operating characteristics (ROC) methodology. RESULTS: Good agreement was found between the tests (kappa approximately 0.7). At cut-off 19 IU/mL nephelometry showed the highest sensitivity (82.4%) and specificity (95.9%) for rheumatoid arthritis (RA). In comparison, the Waaler test had a sensitivity of 60.3% and specificity of 95.9% at cut-off titer 128. The tests showed nearly equal performance characteristics when predicting SS. CONCLUSION: Although both tests exhibit good performance characteristics, nephelometry has a higher accuracy when predicting RA and SS. The common practice of using both tests for detection of RF is not recommended.

Evaluation of diagnostic tests for antinuclear antibodies in rheumatological practice.

To investigate and compare the accuracy and usefulness of diagnostic tests for antinuclear antibodies (ANA) a cross-sectional study of sera derived from patients admitted to the Department of Rheumatology was tested for the presence of ANA using either indirect immunofluorescence on HEp-2 cells, indirect immunoperoxidase techniques on HEp-2 cells and mouse kidney, or two commercial enzyme-linked immunosorbent assays (ELISA). The diagnostic sensitivity and predictive values of the tests were calculated and compared. The accuracy of tests was compared using receiver-operating characteristics (ROC) methodology. All ANA-positive sera were further analysed for the presence of antibodies against extractable nuclear antigens (anti-ENA) and anti-DNA. A moderate to good agreement was found between tests, with kappa ranging from 0.469 to 0.659. Highest sensitivity for systemic lupus erythematosus (SLE; 93.3%) and primary Sjögren's syndrome (SS; 70%) was found using immunofluorescence on HEp-2 cells. Immunofluorescence on HEp-2 cells performed statistically better than the other tests in predicting SLE but not SS. All tests except mouse kidney showed good and comparable performance in detecting sera with anti-ENA and anti-DNA. At the given cut-off values indirect immunofluorescence on HEp-2 cells performed best. All assays except mouse kidney showed performance characteristics sufficient for use in routine analysis of ANA.

Neural compressive symptoms appearing during steroid treatment in a patient with intracranial lipoma.

Intracranial lipoma is a rare condition, and it is usually asymptomatic. We describe a 67 year old woman who developed blurred vision, diplopia, left sided oculomotor palsy, and ipsilateral ptosis during steroid treatment for giant cell arteritis. These symptoms were considered to be associated with aggressive giant cell arteritis, and the steroid dose was raised. Surprisingly, the symptoms increased, and further examination revealed an intracranial lipoma situated in the Meckel's cave. During tapering of the steroids her symptoms gradually improved. This is the first report demonstrating that steroids may induce hypertrophy of the fat tissue in the intracranial lipoma, causing compression of the cranial nerves passing through the cavernous sinus thereby mimicking the ocular symptoms sometimes associated with aggressive giant cell arteritis.

Identification and characterization of SIGIRR, a molecule representing a novel subtype of the IL-1R superfamily.

A novel member of the interleukin 1 receptor (IL-1R) superfamily, SIGIRR (single Ig IL-1R-related molecule) was identified in mouse and human. Although it shows the typical conserved motifs that characterize the IL-1R and Toll superfamily, it is structurally and functionally distinct from both. SIGIRR has only one Ig domain in its extracellular portion whereas the IL-1R family contains three Ig folds. An unusually long cytoplasmic domain is reminiscent of the structure of drosophila Toll, yet the SIGIRR peptide sequence is more closely related to IL-1RI. The human SIGIRR gene maps to 11p15. 5 and thus is not located in the same cluster on chromosome 2 that is known to contain four members of the IL-1R family. It failed to bind to the known IL-1-family members and, when co-expressed with the IL-1RI, had no effect on the binding of IL-1 and on subsequent nuclear factor kappaB (NFkappaB) activation. A chimera, in which the SIGIRR intracellular domain was fused to the IL-1R extracellular domain, did not activate NFkappaB unlike similar fusion proteins of other IL-1R related molecules. We conclude that the SIGIRR protein represents a novel subtype of the IL-1R superfamily.

Cloning of a novel receptor subunit, AcPL, required for interleukin-18 signaling.

We have identified a novel member of the interleukin-1 (IL-1) receptor family, which we have termed AcPL. In transient transfection assays, we were unable to demonstrate a role for AcPL in IL-1-induced activation of NFkappaB. Interleukin-18 (interferon-gamma-inducing factor) is another member of the IL-1 family of cytokines, and it has recently been shown that IL-18 has a weak affinity for IL-1R-rp1. We examined whether AcPL might function alone or in concert with IL-1R-rp1 to mediate IL-18 signaling. We found that both IL-1R-rp1 and AcPL expression were required for induction of NFkappaB activity and for activation of c-Jun N-terminal kinase in response to IL-18. Furthermore, a dominant negative version of AcPL specifically inhibited IL-18 signaling. In vitro immunoprecipitation assays demonstrated that AcPL alone was unable to bind IL-18 with any appreciable affinity. We propose that although IL-1R-rp1 binds the cytokine, IL-1R-rp1 and AcPL proteins are both required for IL-18 signaling, analogous to the requirement for both IL-1R and IL-1RAcP in IL-1-mediated responses.

Binding of interleukin-18 to the interleukin-1 receptor homologous receptor IL-1Rrp1 leads to activation of signaling pathways similar to those used by interleukin-1.

Interleukin-18 (IL-18) is an inflammatory cytokine that has been shown to enhance a variety of Th1 type T cell responses. Because IL-18 is homologous to IL-1, we tested binding of IL-18 to the known IL-1R family members. We could show binding of IL-18 to the orphan receptor IL-1Rrp1 but not to other IL-1R homologous proteins. IL-1Rrp1 and IL-1RI share highly conserved domains within their cytoplasmic regions. Comparison of the IL-1 and IL-18 signaling mechanisms showed that they activate identical cytoplasmic messengers. IL-18, like IL-1, induced association of its receptor with IRAK and subsequent recruitment of TRAF6. IL-18 activated p38 MAP kinase, jun kinase, and beta casein kinase (TIP kinase), an apparently novel kinase previously thought to be specifically activated by IL-1 and tumor necrosis factor (TNF). IL-18 activated NF-kappaB in EL4/6.1 thymoma cells but not in COS-7 cells, even though the latter presumably contain all components required for the IL-1 signaling pathway. From our binding and signaling studies, we conclude that the IL-18 receptor complex consists of IL-18, the IL-1Rrp1, and another thus far unidentified receptor molecule.

Secreted and membrane-bound isoforms of T1, an orphan receptor related to IL-1-binding proteins, are differently expressed in vivo.

The murine T1 gene encodes a membrane-bound glycoprotein (T1-M), highly similar to interleukin-1 (IL-1) receptor type I, and a soluble variant (T1-S) representing its isolated extracellular domain. In vivo, the expression pattern of both T1 isoforms differs drastically. The T1-M receptor is abundantly expressed in single cells of the major hemopoietic organs (embryonic liver, spleen, bone marrow). It is restricted to few hemopoietic cell types throughout ontogenesis. By contrast, the soluble T1-S protein is predominantly expressed in selected nonhemopoietic embryonic tissues (developing skin, bone, and retina) and deposited in extracellular matrix. Despite the similarity of the T1 ligand-binding domain to all IL-1-binding proteins, it does not exhibit affinity to either IL-1 alpha or -beta. Thus, T1-M likely represents a novel orphan receptor of selected hemopoietic cells. The matrix-associated T1-S variant might act to create a reservoir of the putative T1 ligand in some differentiating tissues.

Role of cell type-specific promoters in the developmental regulation of T1, an interleukin 1 receptor homologue.

Murine T1, an orphan receptor related to interleukin 1 receptors, exhibits a bimodal expression in mouse development. The molecular analysis of cultured cell lines now reveals the contribution of alternate promoters of the T1 gene to its differential expression. In nonhemopoietic cell types, where T1 synthesis in vivo is restricted to organogenesis and neoplasia, a recently characterized AP-1-dependent promoter directs a proliferation-associated expression of the gene. In hemopoietic cells, which express the T1 receptor throughout ontogenesis in vivo, T1 gene activity is driven by a novel serum factor-independent, constitutive promoter. The tissue-specific use of constitutive versus growth factor-dependent alternate promoters thus directs the permanent activity of the T1 gene in hemopoietic tissue versus the developmentally restricted expression of the gene in nonhemopoietic tissues in vivo.

Kienböck's disease--late results by non-surgical treatment. A follow-up study.

Two groups of patients with Kienböck's disease were followed. Twenty-three wrists had been immobilised with plaster and twenty-six had no treatment. At follow up there was a marked improvement in both groups. Eighty-three percent of the wrists in the new treated group were pain free, or reported pain only on heavy work, and in the nontreated group this was valid for 77%. Examining X-rays at follow up we did not find a single wrist in which the lunate was normal or less deformed than at the time of diagnosis. In all forty-nine wrists the lunate was deformed and in 67% osteoarthrosis in the radiocarpal joint was evident. It is concluded, that Kienböck's disease has a naturally benign course, the remaining symptoms at follow-up might be caused by osteoarthrosis and nothing seems to be gained by rigorous immobilisation. If pain persists efficient treatment must be based on surgical methods.

Ulnar variance determination.

Surgical procedures concerning the distal articular surfaces of the radius and ulna, demand an accurate method of measurement of ulnar variance. A new method, which is a modification of the method described by Palmer (1982), is introduced. 100 randomly selected healthy persons were submitted to X-ray of the wrist and the ulnar variance was determined independently by three observers using both methods. By "weighted kappa" statistics the results, expressed in intra- and interobserver agreement, showed a significantly higher reliability in favour of the Modified method.

Ulnar variance in Kienböck's disease.

Forty four patients with forty seven wrists suffering from Kienböck's disease were re-examined. The mean observation time was 20.5 years. In all forty seven wrists the treatment had been immobilization. Using a standard X-ray projection, and a reliable method of ulnar variance measuring, the ulnar variance was determined by three observers independently. Comparing the result with the ulnar variance in normal wrists we found the so-called "ulnar minus variant" overrepresented in patients with Kienböck's disease. However, comparing X-rays taken at the time of diagnosis with X-rays at re-examination, we found in eight out of forty seven wrists that a subchondral bone formation in the distal radium opposite the lunate bone had taken place. This bone formation will tend to enhance the negative value of ulnar variance measurements, and suggests an explanation of the overrepresentation of "ulnar minus variants" in Kienböck's disease. Excluding these eight wrists from the material and comparing the mean ulnar variance value in the remaining thirty nine wrists with the mean value in normal wrists no statistical difference was shown. Based on these observations it seems unlikely that the "ulnar minus variant" has any bearing on the cause of Kienböck's disease.

Two thymidylate synthetases in Bacillus subtilis.

Bacillus subtilis grown at temperatures below 37 degrees contains two thymidylate synthetases, TSaseA and TSaseB. Their presence is dependent on functional thyA and thyB genes, respectively. When cells are grown at 46 degrees they only contain TSaseA activity. This allous an easy positive selection for thyA and thyA, THYB mutants. The two TSases have been physically separated, and they show similar overall requirements for activity. However, they differ significantly in both their kinetic and their physicochemical properties.

Thymidine-requiring mutants of Salmonella typhimurium that are defective in deoxyuridine 5'-phosphate synthesis.

In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the presence of thymidine were selected. One was shown to be defective in deoxyuridine 5'-phosphate (dUMP) synthesis; it requires deoxyuridine or thymidine for growth and is sensitive to trimethoprim in the presence of deoxyuridine. Genetic studies showed that the mutant is mutated in two genes, dcd and dum, located at 70 and 18 min, respectively, on the Salmonella linkage map. The dcd gene cotransduces 95% with udk, the structural gene for uridine kinase. Both mutations are necessary to create a deoxyuridine requirement, providing evidence for the existence of two independent pathways for dUMP synthesis. Pool studies showed that a dum mutation by itself causes a small decrease in the deoxythymidine 5'-triphosphate (dTTP) pool of the cells, whereas a dcd mutation results in a much more marked decrease. The double mutant dcd dum, when incubated in the absence of deoxyuridine, contains barely detectable levels of dTTP. Enzyme analysis revealed that dcd encodes deoxycytidine 5'-triphosphate deaminase. The gene product of the dum gene has not yet been identified; it does not encode either subunit of ribonucleoside diphosphate reductase or deoxyuridine 5'-triphosphate pyrophosphatase. Mutants deleted for the dcd-udk region of the S. typhimurium chromosome were isolated.