Pre-treatment with IL2 gene therapy alleviates Staphylococcus aureus arthritis in mice.

BACKGROUND: Staphylococcus aureus (S. aureus) arthritis is one of the most detrimental joint diseases known and leads to severe joint destruction within days. We hypothesized that the provision of auxiliary immunoregulation via an expanded compartment of T regulatory cells (Tregs) could dampen detrimental aspects of the host immune response whilst preserving its protective nature. Administration of low-dose interleukin 2 (IL2) preferentially expands Tregs, and is being studied as a treatment choice in several autoimmune conditions. We aimed to evaluate the role of IL2 and Tregs in septic arthritis using a well-established mouse model of haematogenously spred S. aureus arthritis. METHODS: C57BL/6 or NMRI mice we intravenously (iv) injected with a defined dose of S. aureus LS-1 or Newman and the role of IL2 and Tregs were assessed by the following approaches: IL2 was endogenously delivered by intraperitoneal injection of a recombinant adeno-associated virus vector (rAAV) before iv S. aureus inoculation; Tregs were depleted before and during S. aureus arthritis using antiCD25 antibodies; Tregs were adoptively transferred before induction of S. aureus arthritis and finally, recombinant IL2 was used as a treatment starting day 3 after S. aureus injection. Studied outcomes included survival, weight change, bacterial clearance, and joint damage. RESULTS: Expansion of Tregs induced by IL2 gene therapy prior to disease onset does not compromise host resistance to S. aureus infection, as the increased proportions of Tregs reduced the arthritis severity as well as the systemic inflammatory response, while simultaneously preserving the host's ability to clear the infection. CONCLUSIONS: Pre-treatment with IL2 gene therapy dampens detrimental immune responses but preserves appropriate host defense, which alleviates S. aureus septic arthritis in a mouse model.

Antibiotics with Interleukin-15 Inhibition Reduce Joint Inflammation and Bone Erosions but Not Cartilage Destruction in Staphylococcus aureus-Induced Arthritis.

Staphylococcus aureus-induced arthritis causes rapid joint destruction, often leading to disabling joint damage despite antibiotics. We have previously shown that interleukin-15 (IL-15) inhibition without antibiotics is beneficial in S. aureus-induced arthritis. We therefore hypothesized that the inhibition of IL-15, in combination with antibiotics, might represent a useful therapy that would reduce inflammation and joint destruction but preserve the host's ability to clear the infection. Female wild-type C57BL/6 mice were intravenously inoculated with the toxic shock syndrome toxin 1 (TSST-1)-producing LS-1 strain of S. aureus with 0.8 × 108 CFU S. aureus LS-1/mouse. Three days later, treatment consisting of cloxacillin, followed by flucloxacillin, together with either anti-IL-15 antibodies (aIL-15ab) or control antibodies, was started. Studied outcomes included survival, weight change, bacterial clearance, and joint damage. The addition of aIL-15ab to antibiotics in S. aureus-induced arthritis reduced synovitis and bone erosions compared to controls. The number of bone-resorbing osteoclasts in the joints was reduced, whereas cartilage destruction was not significantly altered. Importantly, the combination therapy did not adversely affect the clinical outcome of S. aureus-induced arthritis, such as survival or weight change, or compromise the host's ability to clear the infection. Since the clinical outcome of S. aureus-induced arthritis was not affected, the addition of aIL-15ab to antibiotics ought to be safe. Taken together, the combination of aIL-15ab and antibiotics is a beneficial, but not optimal, treatment of S. aureus-induced arthritis since it reduces synovitis and bone erosions but has a limited effect on cartilage destruction.

Collagen epitope expression on B cells is sufficient to confer tolerance to collagen-induced arthritis.

BACKGROUND: The mechanisms underlying tolerance induction and maintenance in autoimmune arthritis remain elusive. In a mouse model of rheumatoid arthritis, collagen type II (CII)-induced arthritis, we explore the contribution of B cells to antigen-specific tolerance. METHODS: To generate expression of the CII-peptide specifically on B-cell major histocompatibility complex type II, lentiviral-based gene therapy including a B-cell-specific Igk promoter was used. RESULTS: Presentation of the CII-peptide on B cells significantly reduced the frequency and severity of arthritis as well as the serum levels of CII -specific IgG antibodies. Further, both frequency and suppressive function of regulatory T cells were increased in tolerized mice. Adoptive transfer of regulatory T cells from tolerized mice to naïve mice ameliorated the development of CII-induced arthritis. CONCLUSION: Our data suggest that endogenous presentation of the CII-peptide on B cells is one of the key contributors to arthritis tolerance induction and maintenance.

Gene Therapy Induces Antigen-Specific Tolerance in Experimental Collagen-Induced Arthritis.

Here, we investigate induction of immunological tolerance by lentiviral based gene therapy in a mouse model of rheumatoid arthritis, collagen II-induced arthritis (CIA). Targeting the expression of the collagen type II (CII) to antigen presenting cells (APCs) induced antigen-specific tolerance, where only 5% of the mice developed arthritis as compared with 95% of the control mice. In the CII-tolerized mice, the proportion of Tregs as well as mRNA expression of SOCS1 (suppressors of cytokine signaling 1) increased at day 3 after CII immunization. Transfer of B cells or non-B cell APC, as well as T cells, from tolerized to naïve mice all mediated a certain degree of tolerance. Thus, sustainable tolerance is established very early during the course of arthritis and is mediated by both B and non-B cells as APCs. This novel approach for inducing tolerance to disease specific antigens can be used for studying tolerance mechanisms, not only in CIA but also in other autoimmune diseases.

Systemic signs of neutrophil mobilization during clinically stable periods and during exacerbations in smokers with obstructive pulmonary disease.

BACKGROUND: It is still unclear whether signs of neutrophil mobilization in the blood of patients with chronic obstructive pulmonary disease represent true systemic events and how these relate to bacterial colonization in the airways. In this study, we evaluated these issues during clinically stable periods and during exacerbations in smokers with obstructive pulmonary disease and chronic bronchitis (OPD-CB). METHODS: Over a period of 60 weeks for each subject, blood samples were repeatedly collected from 60 smokers with OPD-CB during clinically stable periods, as well as during and after exacerbations. Myeloperoxidase (MPO) and neutrophil elastase (NE) protein and mRNA, growth of bacteria in sputum, and clinical parameters were analyzed. Ten asymptomatic smokers and ten never-smokers were included as controls. RESULTS: We found that, during clinically stable periods, neutrophil and NE protein concentrations were increased in smokers with OPD-CB and in the asymptomatic smokers when compared with never-smokers. During exacerbations, neutrophil and MPO protein concentrations were further increased in smokers with OPD-CB, without a detectable increase in the corresponding mRNA during exacerbations. However, MPO and NE protein and mRNA displayed positive correlations. During exacerbations, only increased neutrophil concentrations were associated with growth of bacteria in sputum. Among patients with low transcutaneous oxygen saturation during exacerbations, PaO2 (partial oxygen pressure) correlated with concentrations of MPO and NE protein and neutrophils in a negative manner. CONCLUSION: There are signs of systemic neutrophil mobilization during clinically stable periods and even more so during exacerbations in chronic obstructive pulmonary disease. In this condition, MPO and NE may share a cellular origin, but its location remains uncertain. Factors other than local bacteria, including hypoxemia, may be important for driving systemic signs of neutrophil mobilization.

Systemic cytokine signaling via IL-17 in smokers with obstructive pulmonary disease: a link to bacterial colonization?

We examined whether systemic cytokine signaling via interleukin (IL)-17 and growth-related oncogene-α (GRO-α) is impaired in smokers with obstructive pulmonary disease including chronic bronchitis (OPD-CB). We also examined how this systemic cytokine signaling relates to bacterial colonization in the airways of the smokers with OPD-CB. Currently smoking OPD-CB patients (n=60, corresponding to Global initiative for chronic Obstructive Lung Disease [GOLD] stage I-IV) underwent recurrent blood and sputum sampling over 60 weeks, during stable conditions and at exacerbations. We characterized cytokine protein concentrations in blood and bacterial growth in sputum. Asymptomatic smokers (n=10) and never-smokers (n=10) were included as control groups. During stable clinical conditions, the protein concentrations of IL-17 and GRO-α were markedly lower among OPD-CB patients compared with never-smoker controls, whereas the asymptomatic smoker controls displayed intermediate concentrations. Notably, among OPD-CB patients, colonization by opportunistic pathogens was associated with markedly lower IL-17 and GRO-α, compared with colonization by common respiratory pathogens or oropharyngeal flora. During exacerbations in the OPD-CB patients, GRO-α and neutrophil concentrations were increased, whereas protein concentrations and messenger RNA for IL-17 were not detectable in a reproducible manner. In smokers with OPD-CB, systemic cytokine signaling via IL-17 and GRO-α is impaired and this alteration may be linked to colonization by opportunistic pathogens in the airways. Given the potential pathogenic and therapeutic implications, these findings deserve to be validated in new and larger patient cohorts.

Cytokine expression in patients with bladder pain syndrome/interstitial cystitis ESSIC type 3C.

PURPOSE: Bladder wall nitric oxide production in patients with bladder pain syndrome type 3C is increased compared to undetectable nitric oxide in patients with nonHunner bladder pain syndrome and healthy controls. However, the underlying mechanism/s of the increased nitric oxide production is largely unknown. We compared mRNA expression of a select group of cytokines in patients with bladder pain syndrome/interstitial cystitis type 3C and in pain-free controls. MATERIALS AND METHODS: Cold cup biopsies from 7 patients with bladder pain syndrome type 3C and 6 healthy subjects were analyzed. mRNA expression of IL-4, 6, 10 and 17A, iNOS, TNF-α, TGF-ß and IFN-γ was estimated by real-time polymerase chain reaction. IL-17 protein expression was determined by immunohistochemistry. Mast cells were labeled with tryptase to evaluate cell appearance and count. RESULTS: IL-6, 10 and 17A, and iNOS mRNA levels as well as the number of mast cells infiltrating the bladder mucosa were significantly increased in patients with bladder pain syndrome type 3C compared to healthy controls. TNF-α, TGF-ß and IFN-γ mRNA levels were similar in patients and controls. IL-17A expression at the protein level was up-regulated and localized to inflammatory cells and urothelium in patients with bladder pain syndrome type 3C. CONCLUSIONS: Patients with bladder pain syndrome/interstitial cystitis had increased mRNA levels of IL-17A, 10 and 6, and iNOS. IL-17A might be important in the inflammatory process. To our knowledge the increase in IL-17A is a novel finding that may have new treatment implications.

Glucocorticoid receptor function is decreased in neutrophils during endotoxic shock.

OBJECTIVES: It remains unclear whether glucocorticoid treatment can improve the outcome of sepsis. The aim of the present study was to investigate if glucocorticoid receptor (GR) expression and function is impaired in lipopolysaccharide (LPS) induced shock, and whether the time point for start of glucocorticoid treatment affects the outcome. METHODS: Male C57BL/6J mice were administered LPS i.p. and GR expression and binding ability in blood and spleen leukocytes were analysed by flow cytometry. GR translocation was analysed using Image Stream technique. The effect of dexamethasone treatment started 2 h before or 2, 12 or 36 h after LPS administration on survival was studied. RESULTS: Despite increased GR expression in neutrophils after LPS administration, the GR binding capacity was reduced. In addition, GR translocation was decreased in neutrophils and T lymphocytes from endotoxic mice at 12 h compared to control animals. Dexamethasone treatment improved survival only when started early (2 h) after LPS administration. CONCLUSION: The decreased glucocorticoid responsiveness displayed by neutrophils, in combination with their increased numbers, may explain why survival is increased only when dexamethasone treatment is given early during LPS induced shock.

Antigen-specific gene therapy after immunisation reduces the severity of collagen-induced arthritis.

Reestablishment of tolerance induction in rheumatoid arthritis (RA) would be an optimal treatment with few, if any, side effects. However, to develop such a treatment further insights in the immunological mechanisms governing tolerance are needed. We have developed a model of antigen-specific tolerance in collagen type II (CII) induced arthritis (CIA) using lentivirus-based gene therapy. The immunodominant epitope of CII was inserted into a lentivirus vector to achieve expression on the MHC class II molecule and the lentiviral particles were subsequently intravenously injected at different time points during CIA. Injection of lentiviral particles in early phases of CIA, that is, at day 7 or day 26 after CII immunisation, partially prevented development of arthritis, decreased the serum levels of CII-specific IgG antibodies, and enhanced the suppressive function of CII-specific T regulatory cells. When lentiviral particles were injected during manifest arthritis, that is, at day 31 after CII immunisation, the severity of arthritis progression was ameliorated, the levels of CII-specific IgG antibodies decreased and the proportion of T regulatory cells increased. Thus, antigen-specific gene therapy is effective when administered throughout the inflammatory course of arthritis and offers a good model for investigation of the basic mechanisms during tolerance in CIA.

Negative feedback on IL-23 exerted by IL-17A during pulmonary inflammation.

It is now established that IL-17 has a broad pro-inflammatory potential in mammalian host defense, in inflammatory disease and in autoimmunity, whereas little is known about its anti-inflammatory potential and inhibitory feedback mechanisms. Here, we examined whether IL-17A can inhibit the extracellular release of IL-23 protein, the upstream regulator of IL-17A producing lymphocyte subsets, that is released from macrophages during pulmonary inflammation. We characterized the effect of IL-17A on IL-23 release in several models of pulmonary inflammation, evaluated the presence of IL-17 receptor A (RA) and C (RC) on human alveolar macrophages and assessed the role of the Rho family GTPase Rac1 as a mediator of the effect of IL-17A on the release of IL-23 protein. In a model of sepsis-induced pneumonia, intravenous exposure to Staphylococcus aureus caused higher IL-23 protein concentrations in cell-free bronchoalveolar lavage (BAL) samples from IL-17A knockout (KO) mice, compared with wild type (WT) control mice. In a model of Gram-negative airway infection, pre-treatment with a neutralizing anti-IL-17A Ab and subsequent intranasal (i.n.) exposure to LPS caused higher IL-23 and IL-17A protein concentrations in BAL samples compared with mice exposed to LPS, but pre-treated with an isotype control Ab. Moreover, i.n. exposure with IL-17A protein per se decreased IL- 23 protein concentrations in BAL samples. We detected IL-17RA and IL-17RC on human alveolar macrophages, and found that in vitro stimulation of these cells with IL-17A protein, after exposure to LPS, decreased IL-23 protein in conditioned medium, but not IL-23 p19 or p40 mRNA. This study indicates that IL-17A can partially inhibit the release of IL-23 protein during pulmonary inflammation, presumably by stimulating the here demonstrated receptor units IL-17RA and IL-17RC on alveolar macrophages. Hypothetically, the demonstrated mechanism may serve as negative feedback to protect from excessive IL-17A signaling and to control antibacterial host defense once it is activated.

Disease-dependent local IL-10 production ameliorates collagen induced arthritis in mice.

Rheumatoid arthritis (RA) is a chronic destructive autoimmune disease characterised by periods of flare and remission. Today's treatment is based on continuous immunosuppression irrespective of the patient's inflammatory status. When the disease is in remission the therapy is withdrawn but withdrawal attempts often results in inflammatory flares, and re-start of the therapy is commenced when the inflammation again is prominent which leads both to suffering and increased risk of tissue destruction. An attractive alternative treatment would provide a disease-regulated therapy that offers increased anti-inflammatory effect during flares and is inactive during periods of remission. To explore this concept we expressed the immunoregulatory cytokine interleukin (IL)-10 gene under the control of an inflammation dependent promoter in a mouse model of RA - collagen type II (CII) induced arthritis (CIA). Haematopoetic stem cells (HSCs) were transduced with lentiviral particles encoding the IL-10 gene (LNT-IL-10), or a green fluorescence protein (GFP) as control gene (LNT-GFP), driven by the inflammation-dependent IL-1/IL-6 promoter. Twelve weeks after transplantation of transduced HSCs into DBA/1 mice, CIA was induced. We found that LNT-IL-10 mice developed a reduced severity of arthritis compared to controls. The LNT-IL-10 mice exhibited both increased mRNA expression levels of IL-10 as well as increased amount of IL-10 produced by B cells and non-B APCs locally in the lymph nodes compared to controls. These findings were accompanied by increased mRNA expression of the IL-10 induced suppressor of cytokine signalling 1 (SOCS1) in lymph nodes and a decrease in the serum protein levels of IL-6. We also found a decrease in both frequency and number of B cells and serum levels of anti-CII antibodies. Thus, inflammation-dependent IL-10 therapy suppresses experimental autoimmune arthritis and is a promising candidate in the development of novel treatments for RA.

Interleukin 15 mediates joint destruction in Staphylococcus aureus arthritis.

BACKGROUND: Staphylococcus aureus arthritis causes severe and rapid joint damage despite antibiotics. Thus, there is a need to identify new treatment targets in addition to antibiotics. Lately, interleukin 15 (IL-15) has been implicated both in osteoclastogenesis and in bacterial clearance-2 important issues in S. aureus-induced joint destruction. This has prompted us to investigate the importance of IL-15 in S. aureus-induced arthritis. METHODS: Toxic shock syndrome toxin-1 producing S. aureus was intravenously inoculated in IL-15 knockout and wildtype mice and in wildtype mice treated with anti-IL-15 antibodies (aIL-15ab) or isotype control antibody. RESULTS: Absence of IL-15, either in knockout mice or after treatment with aIL-15ab, significantly reduced weight loss compared with controls during the infection. The severity of synovitis and joint destruction was significantly decreased in IL-15 knockout and aIL-15ab treated mice compared with controls. In IL-15 knockout mice there was a reduced number of osteoclasts in the joints. The host's ability to clear bacteria was not influenced in the IL-15 knockout mice, but significantly increased after treatment with aIL-15ab. CONCLUSIONS: IL-15 is a mediator of joint destruction in S. aureus-induced arthritis and contributes to general morbidity, which makes this cytokine an interesting treatment target in addition to conventional antibiotics.

Rip2 deficiency leads to increased atherosclerosis despite decreased inflammation.

RATIONALE: The innate immune system and in particular the pattern-recognition receptors Toll-like receptors have recently been linked to atherosclerosis. Consequently, inhibition of various signaling molecules downstream of the Toll-like receptors has been tested as a strategy to prevent progression of atherosclerosis. Receptor-interacting protein 2 (Rip2) is a serine/threonine kinase that is involved in multiple nuclear factor-κB (NFκB) activation pathways, including Toll-like receptors, and is therefore an interesting potential target for pharmaceutical intervention. OBJECTIVE: We hypothesized that inhibition of Rip2 would protect against development of atherosclerosis. METHODS AND RESULTS: Surprisingly, and contrary to our hypothesis, we found that mice transplanted with Rip2(-/-) bone marrow displayed markedly increased atherosclerotic lesions despite impaired local and systemic inflammation. Moreover, lipid uptake was increased whereas immune signaling was reduced in Rip2(-/-) macrophages. Further analysis in Rip2(-/-) macrophages showed that the lipid accumulation was scavenger-receptor independent and mediated by Toll-like receptor 4 (TLR4)-dependent lipid uptake. CONCLUSIONS: Our data show that lipid accumulation and inflammation are dissociated in the vessel wall in mice with Rip2(-/-) macrophages. These results for the first time identify Rip2 as a key regulator of cellular lipid metabolism and cardiovascular disease.

Interleukin-17A during local and systemic Staphylococcus aureus-induced arthritis in mice.

Staphylococcus aureus is one of the dominant pathogens that induce septic arthritis in immunocompromised hosts, e.g., patients suffering from rheumatoid arthritis treated with immunosuppressive drugs. S. aureus-induced arthritis leads to severe joint destruction and high mortality despite antibiotic treatment. Recently, interleukin-17A (IL-17A) has been discovered to be an important mediator of aseptic arthritis both in mice and humans, but its function in S. aureus-induced arthritis is largely unknown. Here, we investigated the role of IL-17A in host defense against arthritis following systemic and local S. aureus infection in vivo. IL-17A knockout mice and wild-type mice were inoculated systemically (intravenously) or locally (intra-articularly) with S. aureus. During systemic infection, IL-17A knockout mice lost significantly more weight than the wild-type mice did, but no differences were found in the mortality rate. The absence of IL-17A had no impact on clinical arthritis development but led to increased histopathological erosivity late during systemic S. aureus infection. Bacterial clearance in kidneys was increased in IL-17A knockout mice compared to the level in wild-type mice only 1 day after bacterial inoculation. During systemic S. aureus infection, serum IL-17F protein levels and mRNA levels in the lymph nodes were elevated in the IL-17A knockout mice compared to the level in wild-type mice. In contrast to systemic infection, the IL-17A knockout mice had increased synovitis and erosions and locally decreased clearance of bacteria 3 days after local bacterial inoculation. On the basis of these findings, we suggest that IL-17A is more important in local host defense than in systemic host defense against S. aureus-induced arthritis.

15-Lipoxygenase-2 expression in human macrophages induces chemokine secretion and T cell migration.

BACKGROUND: We determined previously that hypoxia results in increased 15-lipoxygenase type 2 (15-LOX-2) expression and CXCL8 secretion in macrophages. This study sought to determine whether 15-LOX-2 expression links directly with the secretion of inflammatory molecules in macrophages and also investigated its subsequent effects on T cell migration. METHODS: Adenovirus-mediated gene delivery caused overexpression of 15-LOX-2 in human macrophages. We used cytometric bead array to measure chemokine secretion, and assessed T cell migration by counting cells in chemotaxis chambers. Expression of chemokine receptors was determined by FACS analysis. Using siRNA, we reduced 15-LOX-2 expression in human macrophages. We used scrambled siRNA as control. RESULTS: Macrophages that overexpress 15-LOX-2 showed increased secretion of chemokine CXCL10 after 24h incubation. In addition, preconditioned medium from 15-LOX-2-overexpressing cells increased T cell migration and surface expression of CXCR3, the CXCL10 receptor. Knockdown of 15-LOX-2 expression decreased CXCL10 secretion from hypoxic macrophages and also reduced T cell migration. CONCLUSION: In macrophages, overexpression of 15-LOX-2 results in increased secretion of CXCL10 and CCL2. Products released in response to increased 15-LOX-2 activation lead to increased expression of CD69, the T cell activation marker as well as increased T cell migration. Therefore, increased expression of 15-LOX-2 induced by hypoxia may participate in T cell recruitment in diseases such as atherosclerosis.

A non-conservative polymorphism in the IL-6 signal transducer (IL6ST)/gp130 is associated with myocardial infarction in a hypertensive population.

Inflammation is a key component in the development of atherosclerosis, and myocardial infarction (MI); therefore we investigated the association between an interleukin-6 signal transducer (IL6ST)/gp130 polymorphism, gp130 function and risk of MI. Structural modeling suggested that a non-conservative single nucleotide polymorphism in the gp130, Gly148Arg, can change the stability and functional properties of the molecule. In vitro studies were done with BAF/3 cells lacking endogenous gp130. Cells stably transfected with the gp130 148Arg variant proliferated less and showed slightly lower STAT-3 phosphorylation in response to gp130 stimulation as compared to cells transfected with gp130 148Gly. In a prospectively followed hypertensive cohort we identified 167 patients who suffered a MI during the study and compared them to matched controls (mean age 57 years, 73% males, n=482). Carriers of the 148Arg variant (f(Arg)=0.12) of the gp130 receptor had decreased odds ratio for MI in univariate analysis (0.56, 95% CI 0.34-0.91, p=0.02). In conclusion, a genetically determined structural variant of the IL-6 receptor subunit gp130 is, independently of other known risk factors, associated with decreased risk of MI. The variant is also associated with decreased IL-6 responsiveness and could lead to a configuration change in the gp130 receptor.

Retention of low-density lipoprotein in atherosclerotic lesions of the mouse: evidence for a role of lipoprotein lipase.

Direct binding of apolipoprotein (apo)B-containing lipoproteins to proteoglycans is the initiating event in atherosclerosis, but the processes involved at later stages of development are unclear. Here, we investigated the importance of the apoB-proteoglycan interaction in the development of atherosclerosis over time and investigated the role of lipoprotein lipase (LPL) to facilitate low-density lipoprotein (LDL) retention at later stages of development. Atherosclerosis was analyzed in apoB transgenic mice expressing LDL with normal (control LDL) or reduced proteoglycan-binding (RK3359-3369SA LDL) activity after an atherogenic diet for 0 to 40 weeks. The initiation of atherosclerosis was delayed in mice expressing RK3359-3369SA LDL, but they eventually developed the same level of atherosclerosis as mice expressing control LDL. Retention studies in vivo showed that although higher levels of 131I-tyramine cellobiose-labeled control LDL (131I-TC-LDL) were retained in nonatherosclerotic aortae compared with RK3359-3369SA 131I-TC-LDL, the retention was significantly higher and there was no difference between the groups in atherosclerotic aortae. Lower levels of control 125I-TC-LDL and RK3359-3369SA 125I-TC-LDL were retained in atherosclerotic aortae from ldlr-/- mice transplanted with lpl-/- compared with lpl+/+ bone marrow. Uptake of control LDL or RK3359-3369SA LDL into macrophages with specific expression of human catalytically active or inactive LPL was increased compared with control macrophages. Furthermore, transgenic mice expressing catalytically active or inactive LPL developed the same extent of atherosclerosis. Thus, retention of LDL in the artery wall is initiated by direct LDL-proteoglycan binding but shifts to indirect binding with bridging molecules such as LPL.

Length of the antibody heavy chain complementarity determining region 3 as a specificity-determining factor.

The antigen binding site of an antibody is made up of residues residing in six hypervariable loops of the heavy and light chains. In most cases several or all of these loops are required for the establishment of the antigen-binding surface. Five of these loops display a limited diversity in length and sequence while the third complementarity determining region (CDR) of the heavy chain is highly different between antibodies not only with respect to sequence but also with respect to length. Its extensive diversity is a key component in the establishment of binding sites allowing for the recognition of essentially any antigen by humoral immunity. The relative importance of its sequence vs its length diversity in this context is however, not very well established. To investigate this matter further we have used an approach employing combinatorial antibody libraries and antigen-specific selection in the search for CDRH3 length and sequence diversity compatible with a given antigen specificity, the major antigenic determinant on the tumour-associated antigen mucin-1. In this way we have now defined heavy chain CDR3 length as a critical parameter in the creation of an antigen-specific binding site. We also propose that this may reflect a dependence of a particular structure of this hypervariable loop, the major carrier of diversity in the binding site, for establishment of a given specificity.

Antibody evolution from the centre to the periphery: applied to a human antibody fragment recognising the tumour-associated antigen mucin-1.

Mucin-1 has proven to be a suitable target for antibody-based diagnosis and therapy of certain tumours, but no appropriate human antibody or antibody fragment displaying slow dissociation rate kinetics against this target is available. Since a rapid dissociation character prevents an antibody fragment from remaining at the site of the antigen, this fact may prevent the successful application of a human mucin-1 specific antibody in diagnosis and therapy. We have now used iterative antibody libraries to evolve a human antibody fragment originally obtained from a naïve antibody library. A strategy was devised whereby molecular variants displaying slow dissociation kinetics against the repetitive mucin-1 tumour-associated antigen can be selected in vitro. The evolved clones, that allowed for a reduced dissociation from the tumour antigen, carried substitutions in the outer parts of the binding site. This demonstrated the ability of this in vitro evolution technique to mimic the process whereby antibodies evolve in vivo. We have thus devised a strategy through which molecular variants displaying slow dissociation from a repetitive target like the mucin-1 tumour-associated antigen can be obtained in vitro. These or related molecules obtained by this approach will serve as a starting point for the development of fully human antibodies for use in mucin-1 specific tumour therapy of diagnosis.