Pegylated interferons Lambda-1a and alfa-2a display different gene induction and cytokine and chemokine release profiles in whole blood, human hepatocytes and peripheral blood mononuclear cells.

Pegylated interferon-lambda-1a (Lambda), a type III interferon (IFN) in clinical development for the treatment of chronic HCV infection, has shown comparable efficacy and an improved safety profile to a regimen based on pegylated IFN alfa-2a (alfa). To establish a mechanistic context for this improved profile, we investigated the ex vivo effects of Lambda and alfa on cytokine and chemokine release, and on expression of IFN-stimulated genes (ISGs) in primary human hepatocytes and peripheral blood mononuclear cells (PBMCs) from healthy subjects. Our findings were further compared with changes observed in blood analysed from HCV-infected patients treated with Lambda or alfa in clinical studies. mRNA transcript and protein expression of the IFN-λ-limiting receptor subunit was lower compared with IFN-α receptor subunits in all cell types. Upon stimulation, alfa and Lambda induced ISG expression in hepatocytes and PBMCs, although in PBMCs Lambda-induced ISG expression was modest. Furthermore, alfa and Lambda induced release of cytokines and chemokines from hepatocytes and PBMCs, although differences in their kinetics of induction were observed. In HCV-infected patients, alfa treatment induced ISG expression in whole blood after single and repeat dosing. Lambda treatment induced modest ISG expression after single dosing and showed no induction after repeat dosing. Alfa and Lambda treatment increased IP-10, iTAC, IL-6, MCP-1 and MIP-1ß levels in serum, with alfa inducing higher levels of all mediators compared with Lambda. Overall, ex vivo and in vivo induction profiles reported in this analysis strongly correlate with clinical observations of fewer related adverse events for Lambda vs those typically associated with alfa.

Results of a phase-I/II randomized, masked, placebo-controlled trial of recombinant human interleukin-11 (rhIL-11) in the treatment of subjects with active rheumatoid arthritis.

Interleukin-11 (IL-11) is a pleiotropic cytokine that regulates the growth and development of hematopoietic stem cells and decreases the proinflammatory mediators of cytokine and nitric oxide production. In animal models of arthritis, treatment with recombinant human IL-11 (rhIL-11) reduces both the level of synovitis and the histologic lesion scores in the joints. The goal of this phase-I/II study in adults with rheumatoid arthritis (RA) was to evaluate the safety and clinical activity of different doses and schedules of rhIL-11 in patients with active RA for whom treatment with at least one disease-modifying antirheumatic drug had failed. This was a multicenter, randomized, placebo-controlled trial that evaluated the safety and tolerability of rhIL-11 in 91 patients with active RA. rhIL-11 was administered subcutaneously; patients were randomized into one of five treatment groups (ratio of rhIL-11 to placebo, 4:1). Patients were treated for 12 weeks with either 2.5 or 7.5 microg/kg of rhIL-11 or placebo twice per week or 5 or 15 microg/kg of rhIL-11 or placebo once per week. The status of each subject's disease activity in accordance with the American College of Rheumatology (ACR) criteria was assessed before, during, and after completion of administration of the study drug. Administration of rhIL-11 was well tolerated at all doses and schedules. The most frequent adverse event was a reaction at the injection site. The data suggest a statistically significant reduction in the number of tender joints (P < 0.008) at the 15 microg/kg once-weekly dose schedule but showed no overall significant benefit at the ACR criterion of a 20% response. The trial showed rhIL-11 to be safe and well tolerated at a variety of doses and schedules over a 12-week treatment period in patients with active RA. The only adverse event clearly associated with rhIL-11 administration was reaction at the injection site.

Rofecoxib.

Rofecoxib (Vioxx, Merck & Co., Inc.) is a new orally-effective non-steroidal anti-inflammatory drug (NSAID) approved for treatment of acute pain, fever, primary dysmenorrhea and pain and inflammation in osteoarthritis (OA). It is also being evaluated for treatment of rheumatoid arthritis and adenomatous polyps of the colon. Rofecoxib is a specific inhibitor of cyclooxygenase-2 (COX-2), thereby inhibiting prostanoid synthesis in cells that express COX-2, including inflammatory cells. As cells in the gastrointestinal (GI) tract principally express COX-1, a different isoform of cyclooxygenase, it is predicted that rofecoxib will have less GI toxicity than other less selective NSAIDs. In clinical trials, rofecoxib was found to be as effective as other NSAIDs for management of pain and inflammation. In trials that compare rofecoxib with ibuprofen, diclofenac and indomethacin, less GI toxicity has been observed, as assayed by a decrease in lesions visible on endoscopy, by GI blood loss and, in a meta-analysis, by frequency of serious adverse GI events. The presence of COX-2 in cells other than inflammatory cells results in side effects common among NSAIDs, including peripheral oedema and hypertension. These side effects are dose-dependent. Rofecoxib, together with other branded NSAIDs, are relatively expensive, which has led to concern regarding costs versus benefits. There is also concern regarding potential risks associated with the use of rofecoxib by populations that would otherwise not tolerate NSAIDs.

Pharmacology and pharmacokinetics of methotrexate in rheumatic disease. Practical issues in treatment and design.

Methotrexate (MTX) is among the most effective drugs for treatment of rheumatoid arthritis and has been proven valuable in the treatment of multiple other disorders of immune regulation. MTX has been administered at a wide range of doses and dose intervals, in conjunction with multiple other drugs, and in patients with a broad range of concomitant disorders. To design a safe and effective MTX treatment plan for an individual patient, the provider must have knowledge of the pharmacology and drug interactions of this effective but potentially dangerous medication. The first section of the article reviews MTX structure, pharmacology pharmacokinetics, and mechanisms of action in rheumatic disease. The second section examines factors that can be used to increase MTX efficacy and decrease toxicity.

An unbiased analysis of V(H)-D-J(H) sequences from B-1a, B-1b, and conventional B cells.

Previous studies conclude that the repertoire of B-1a (CD5+ B) cells is highly restricted. Studies here, which use FACS sorting and single-cell PCR methodology to develop an unbiased representation of the IgH repertoires of B-1a, B-1b, and conventional B cells from the peritoneal cavity, demonstrate that the B-1a cell repertoire is more diverse than previously thought. Furthermore, adult B-1a cells have significantly fewer noncoded nucleotide (N) insertions than conventional B cells. However, B-1a cells are not defined by the absence of these regions, since such insertions are present in two-thirds of B-1a cell transcripts. All three B cell populations use a wide spectrum of V(H), D, and J(H) elements and display considerable diversity in complementarity-determining region 3 (CDR3). However, characteristic differences in the repertoires of all three B cell populations also exist, suggesting different selective and/or developmental forces act to shape each repertoire.

Analysis of three new idiotypes on human monoclonal autoantibodies.

We have identified and characterised three new idiotypes on human IgM McAbs generated from the splenocytes of a SLE patient with active disease. RT-6, which binds H1 and Sm/RNP, expresses essentially a private Id. Its expression is limited to a small number of human McAbs and the sera from patients with infectious diseases. In contrast RT-72Id and RT-84Id, expressed on McAbs which are polyreactive for two or more antigens, have a public distribution. RT-72Id and RT-84Id are found on McAbs from murine and human adult, and foetal tissues. In sera, significant numbers of SLE, RA and patients with other autoimmune diseases are positive for both Ids. RT-84Id is also elevated in SLE relatives and spouses, and in patients with Klebsiella infection. No correlation with disease activity, IgM or IgG levels was observed with either Id. However, RT-72Id was significantly associated with anti-ssDNA antibodies and RhF. RT-6Id and RT-72Id are located on the framework regions of the mu heavy chain, whereas RT-84Id is present on the kappa light chain, within the binding site. The McAbs are encoded by mainly germline genes: heavy chains of RT-6, RT-72 and RT-84 are encoded by the genes VH26, VH4.22 and VH4.21, respectively, and the light chain sequences of RT-6 and RT-72 are derived from DPL11 and HK102. Immunofluorescent staining revealed the presence of RT-72Id and RT-84Id positive immunoglobulin deposits in 18% and 45%, respectively, of the lupus renal sections compared with none in the disease control group, suggesting that these Ids may contribute to the pathology of the disease.

The structural basis of germline-encoded VH3 immunoglobulin binding to staphylococcal protein A.

The ability of human VH3 immunoglobulins (Ig) to bind to staphylococcal protein A (SPA) via their Fab region is analogous to the binding of bacterial superantigens to T cell receptors. The present report establishes the structural basis for the interaction of SPA and VH3 Ig. We have studied a panel of 27 human monoclonal IgM that were derived from fetal B lymphocytes. As such, these IgM were expected to be encoded by unmutated germline genes. Binding to SPA in ELISA occurred with 15 of 15 VH3 IgM, but none of 12 IgM from the VH1, VH4, VH5, or VH6 families. The VH sequences of the 27 IgM were derived from 20 distinct VH elements, including 11 from the VH3 family. Use of D, JH, and CL genes was similar among VH3 and non-VH3 IgM. A comparison of the corresponding VH protein sequences, and those of previously studied IgM, identified a probable site for SPA binding that includes VH3 residues in framework region 3 (FR3), and perhaps FR1 and 3' complementary determining region 2. The results thus demonstrate that among human IgM, specificity for SPA is encoded by at least 11 different VH3 germline genes. Furthermore, like the T cell superantigens, SPA likely binds to residues in the VH framework region, outside the classical antigen-binding site of the hypervariable loops.

Emerging human B cell repertoire. Influence of developmental stage and interindividual variation.

In B cell precursors developing in fetal lymphopoietic tissue, the selection of VH, DH, and JH gene segments for initial H chain gene assembly is biased. The present study was designed to determine whether these biases persist in fully developed human fetal B cells and to examine specificities encoded by the favored elements. B cells were prepared from two sites representing different stages of development, i.e., fetal liver as a source of cells newly generated in that lymphopoietic environment and fetal spleen as a source of more mature cells, potentially subject to selective environmental factors. The expressed repertoires were sampled by two methods. EBV transformation so binding and structure could be examined simultaneously and generation of cDNA from individual, sorted, unstimulated B cells. We found that mature B cells in liver and secondary lymphoid tissue exhibit the same degree of bias in VH use we previously reported in lymphopoietic tissue of the same gestational age. However, the pattern of DH and JH use more nearly resembled that of the adult, suggesting that some constraints imposed by the rearrangement process are normalized rapidly. Sequences recovered from EBV-transformed clones were not distinguishable from transcripts recovered from single cells by direct amplification. Among antibodies expressed by the EBV clones, binding to self-Ag was common, binding profiles varied, and, in contrast to mice, there was little relationship between specificity and VH element. Interestingly, the two individuals studied differed in the VH element most commonly used. One resembled previously studied fetal repertoires in that VH56p1 encoded about 20% of expressed antibodies, whereas the other did not express VH56p1 and used VH26 in 25% of expressed antibodies. This was found to reflect a lack of the genomic VH56p1 allele, suggesting that genetic variation at the VH locus may significantly influence the emerging human antibody repertoire.

Sequence analysis and idiotypic relationships of BEG-2, a human fetal antibody reactive with DNA.

Monoclonal antibody (mAb) BEG-2 is a dsDNA binding IgM lambda derived from a 12-week human fetus. Two binding site idiotypes (BEG-2 Id alpha and BEG-2 Id beta) have been defined with the use of polyclonal rabbit anti-idiotypic anti-serum. BEG-2 Id alpha is located on the lambda light chain and has been described previously. The BEG-2 Id beta is present on the mu heavy chain. By means of a direct binding ELISA, BEG-2 Id beta has been identified on EBV-derived mAbs from human fetal liver or spleen (5%), human cord blood (2.7%) and adult peripheral blood (1%). In addition, the Id is present on 8.5% of adult spleen-derived hybridoma antibodies and 6% of RA synovium-derived hybridoma antibodies. In all populations the presence of the Id is strongly associated with binding to DNA and other polyanions. Competition assays indicated that the Id was located at or near the antigen-binding site on these molecules. To explore the structural basis of this binding, a major part of the BEG-2 heavy chain was sequenced and found to be encoded by a member of the VH4 family joined to a variant of JH5 by a very short Diversity or N region. Of the BEG-2 Id beta positive mAbs for which the VH family has been determined, five are encoded by VH4 and two are encoded by VH6, but none is encoded by other families. Thus, the BEG-2 Id beta identifies a set of polyreactive antibodies that are common in fetal life, persist into adulthood and are encoded by VH6 and, a subset of VH4 genes.

Molecular basis of an autoantibody-associated restriction fragment length polymorphism that confers susceptibility to autoimmune diseases.

Recently, combined serological and molecular studies of autoantibodies have revealed that these antibodies play an important role in the normal function of the immune system and in the development of the B cell repertoire. Accordingly, we hypothesized that a homozygous deletion of a critical autoantibody-associated Ig variable (V) gene may alter the immune system and thus predispose the host to autoimmune disorders. Initial experiments revealed several restriction fragment length polymorphisms (RFLP) of the Humhv3005 gene, that is likely to encode heavy chains of rheumatoid factors, and the closely related 1.9III gene. By probing EcoR1-digested DNA with the Humhv3005/P1 probe, we found that one of the four major hybridizing bands was missing in approximately 20% of patients with either rheumatoid arthritis or systemic lupus erythematosus, but only 2% of normal subjects. To delineate the genetic basis of this polymorphism, we have now employed the PCR to amplify and analyze hv3005, 1.9III, and homologous genes in individuals with characteristic RFLP genotypes. Our results indicate that the human Vh gene repertoire contains several hv3005- and 1.9III-like genes, and that a complete deletion of the hv3005-like genes is relatively restricted to a subset of autoimmune patients. These findings provide initial evidence for deletion of developmentally regulated autoreactive V genes in autoimmune diseases.

Autoantibodies and the fetal antibody repertoire.

Developing fetal B cells preferentially rearrange a restricted subset of the encoded antibody gene segments. There are striking structural similarities between elements expressed early in man and in mouse, most evident on comparison of murine VH elements from the VH7183 family to human VH elements of the VH3 family. The similarity is pronounced in two framework regions which together encode a possible binding site that is distinct from the classical antigen-combining site. By comparing all known human and murine VH gene sequences, we have demonstrated that these regions have been conserved in a family-specific manner throughout the mammalian radiation. The "non-conserved" spacer of the recombinase recognition signal is also highly conserved in a family-specific manner, suggesting a mechanism by which the expression of family-dependent features may be regulated. The evidence that such features contribute to the high incidence of self- and poly-specificity in the fetal antibody repertoire is reviewed.

Structure and evolution of mammalian VH families.

Antibodies are encoded by a limited number of germline gene segments that undergo somatic diversification through rearrangement and mutation. Because these mutation processes are efficient, it is widely believed that there is little environmental selection pressure for the maintenance of specific antibody gene sequences. We have performed pairwise comparisons of known germline (as opposed to somatically generated) antibody VH elements with the hope of identifying conserved structural features common to sets of VH gene segments. These studies reveal that VH families arose prior to the mammalian radiation and have since been conserved, that this conservation appears to reflect selection at the level of protein sequence, and that the conserved regions are discretely localized on a solvent-exposed face of the heavy chain, at some distance from the antibody combining site. A family-specific region was also identified within the recombinase recognition sequences. Our results provide a context for theories that address the physiological significance of variations in VH family utilization during the development of the immune repertoire.

Early restriction of the human antibody repertoire.

Diversification of the antibody repertoire in mammals results from a series of apparently random somatically propagated gene rearrangement and mutational events. Nevertheless, it is well known that the adult repertoire of antibody specificities is acquired in a developmentally programmed fashion. As previously shown, rearrangement of the gene segments encoding the heavy-chain variable regions (VH) of mouse antibodies is also developmentally ordered: the number of VH gene segments rearranged in B lymphocytes of fetal mice is small but increased progressively after birth. In this report, human fetal B-lineage cells were also shown to rearrange a highly restricted set of VH gene segments. In a sample of heavy-chain transcripts from a 130-day human fetus the most frequently expressed human VH element proved to be closely related to the VH element most frequently expressed in murine fetal B-lineage cells. These observations are important in understanding the development of immunocompetence.