Improved virological response to highly active antiretroviral therapy in HIV-1-infected patients carrying the CCR5 Delta32 deletion

Background

Patients heterozygous for the C‐C chemokine receptor 5 (CCR5) Δ32 deletion spontaneously progress less rapidly to AIDS and death than do wild‐type patients. We investigated whether the CCR5 Δ32 deletion has an impact on immunological, virological and clinical responses to highly active antiretroviral therapy (HAART) in HIV‐1‐infected patients.

Patients and methods

We included in the study 565 HIV‐1‐infected patients from the French HIV‐1 infected cohort with documented date of serconversion (SEROCO)/haemophiliacs HIV‐1 infected (HEMOCO) cohorts, who started HAART after 1996. We investigated virological responses to HAART at 6 months (defined as a plasma HIV‐1 RNA measurement below the threshold of detection or a 2 log HIV‐1 RNA decrease) and at 12 months (defined as a plasma HIV‐1 RNA measurement below the threshold of detection) and clinical response to HAART by Kaplan–Meier survival curves, with AIDS and death as outcomes.

Results

The Δ32 heterozygous patients (n=83; 15%) had a better virological response to HAART than wild‐type patients (73 vs 53% at 6 months, P=0.01; and 60 vs 44% at 12 months, P=0.01). This better virological response was still observed after adjustment for antiretroviral status (whether or not patients were naïve to antiretroviral therapy), year of HAART initiation, number of new antiretroviral drugs and baseline viral load. There was no statistical difference between heterozygous patients and wild‐type patients in terms of survival and AIDS‐free survival.

Conclusions

CCR5 Δ32 heterozygous patients were more likely to have a virological response to HAART than wild‐type patients at 6 and 12 months. However, this virological response did not produce better immunological and clinical responses. The long‐term impact of the Δ32 deletion on survival in HIV‐1‐infected treated patients should be investigated in a meta‐analysis.

     

Is long‐term virological response related to CCR5 Δ32 deletion in HIV‐1‐infected patients started on highly active antiretroviral therapy?

Objective

The aim of the study was to determine whether the chemokine (C‐C motif) receptor 5 (CCR5) Δ32 deletion is associated with long‐term response to combination antiretroviral treatment (cART) in HIV‐1‐infected patients.

Methods

The genetic substudy of the Agence Nationale de Recherche sur le SIDA (ANRS) CO8 APROCO‐COPILOTE cohort included 609 patients who started protease inhibitor‐containing cART in 1997–1999. Patients were considered to have a sustained virological response if all plasma HIV RNA measurements in the period considered were <500 HIV‐1 RNA copies/ml, allowing for a single blip. Virological response was compared between patients heterozygous for CCR5 Δ32 (Δ32/wt) and wild‐type patients (wt/wt) from month 4 to year 3 and from month 4 to year 5. Logistic regression analysis was used to adjust for baseline demographical data, HIV RNA, CD4 cell count, antiretroviral exposure status, time spent on antiretroviral therapy at years 3 and 5 and adherence to treatment (month 4 to year 3 or 5).

Results

A sustained virological response was more frequent in Δ32/wt than in wt/wt patients from month 4 to year 3, with 66%vs. 52% of patients, respectively, showing a sustained response (P=0.02); after adjustment for potential confounders, the association of Δ32 with a sustained response was nearly significant (P=0.07). A sustained virological response was also more frequent in Δ32/wt patients up to year 5, with 48% showing a sustained response vs. 35% of wt/wt patients (P=0.01); after adjustment, Δ32 remained significantly associated with a sustained virological response up to year 5 (P=0.04). There was no association with CD4 response.

Conclusion

The Δ32 deletion in Δ32/wt patients is associated with a beneficial virological response to cART in the long term. Whether this association is a direct effect of the Δ32 deletion remains unclear and requires confirmation in further observational studies.

     

Persistence of CCR5 usage among primary human immunodeficiency virus isolates of individuals receiving intermittent interleukin‐2

Objective

To investigate the impact of intermittent interleukin‐2 (IL‐2) plus combination antiretroviral therapy (cART) on HIV‐1 entry co‐receptor use.

Methods

Primary HIV‐1 isolates were obtained from 54 HIV‐1‐positive individuals at baseline and after 12 months using co‐cultivation of peripheral blood mononuclear cells (PBMC) with activated PBMC of HIV‐negative healthy donors. HIV‐1 co‐receptor use was determined on U87‐CD4 cells.

Results

Fourteen out of the 21 (67%) IL‐2‐treated individuals harbouring a primary CCR5‐dependent (R5) HIV‐1 isolate at baseline confirmed an R5 virus isolation after 12 months in contrast to 3 out of 7 (43%) of those receiving cART only. After 12 months, only 1 R5X4 HIV‐1 isolate was obtained from 21 cART+IL‐2‐treated individuals infected with an R5 virus at entry (5%) vs. 2/7 (29%) patients receiving cART alone, as confirmed by a 5‐year follow‐up on some individuals.

Conclusions

Intermittent IL‐2 administration plus cART may prevent evolution towards CXCR4 usage in individuals infected with R5 HIV‐1.

     

Coexpression of chemokine receptors CCR5, CXCR3, and CCR4 and ligands for P‐ and E‐selectin on T lymphocytes of patients with juvenile idiopathic arthritis

Objective

To investigate P‐ and E‐selectin ligand coexpression with chemokine receptors (CKRs) on T cells in the synovial fluid (SF) and blood of children with juvenile idiopathic arthritis (JIA).

Methods

Sixteen patients with polyarticular or persistent oligoarticular JIA (ages 5.3–15.1 years) were studied. SF and venous blood were collected, and immunostaining for the expression of CCR4, CCR5, CXCR3, and P‐ or E‐selectin ligands was performed.

Results

Compared to blood, SF was greatly enriched for CD4+ T cells bearing CCR5, CCR4, CXCR3, and both P‐ and E‐selectin ligand. Twenty‐five percent of the CD4+ T cells in SF expressed both CCR5 and CCR4, some also coexpressing CXCR3. Such cells were rare in blood. Half of the few CCR5+ T cells in blood coexpressed P‐ or E‐selectin ligand, a phenotype that was enriched up to 50‐fold in SF. A minority of CCR4+ and CXCR3+ cells in blood (∼25%) coexpressed selectin ligand; these were enriched 4–8‐fold in SF. Most CCR4‐expressing CD4+ T cells expressed both E‐selectin ligand and cutaneous lymphocyte antigen.

Conclusion

CCR4‐, CCR5‐, CXCR3‐, and selectin ligand–expressing CD4+ T cells preferentially accumulate in the joints of children with JIA. The marked enrichment of CCR5+ T cells coexpressing P‐selectin and/or E‐selectin ligand in CD4+ SF T cells suggests that the few such cells in blood selectively migrate to inflamed joints via endothelial P‐ and E‐selectin– and CCR5‐activating chemokines. The predominance of CCR4‐expressing CD4+ T cells coexpressing E‐selectin ligand suggests that such cells migrate not only to areas of cutaneous inflammation, as previously reported, but also to the joints in JIA. Combined targeting of CCR5‐ and E‐selectin–dependent mechanisms may be a relevant treatment strategy.

     

CCR5 is involved in resolution of inflammation in proteoglycan‐induced arthritis

Objective

CCR5 and its ligands (CCL3, CCL4, and CCL5) may play a role in inflammatory cell recruitment into the joint. However, it was recently reported that CCR5 on T cells and neutrophils acts as a decoy receptor for CCL3 and CCL5 to assist in the resolution of inflammation. The aim of this study was to determine whether CCR5 functions as a proinflammatory or antiinflammatory mediator in arthritis, by examining the role of CCR5 in proteoglycan (PG)–induced arthritis (PGIA).

Methods

Arthritis was induced by immunizing wild‐type (WT) and CCR5‐deficient (CCR5^−/−) BALB/c mice with human PG in adjuvant. The onset and severity of PGIA were monitored over time. Met‐RANTES was used to block CCR5 in vivo. Arthritis was transferred to SCID mice, using spleen cells from arthritic WT and CCR5^−/− mice. The expression of cytokines and chemokines was measured by enzyme‐linked immunosorbent assay.

Results

In CCR5^−/− mice and WT mice treated with the CCR5 inhibitor Met‐RANTES, exacerbated arthritis developed late in the disease course. The increase in arthritis severity in CCR5^−/− mice correlated with elevated serum levels of CCL5. However, exacerbated arthritis was not intrinsic to the CCR5^−/− lymphoid cells, because the arthritis that developed in SCID mouse recipients was similar to that in WT and CCR5^−/− mice. CCR5 expression in the SCID mouse was sufficient to clear CCL5, because serum levels of CCL5 were the same in SCID mouse recipients receiving cells from either WT or CCR5^−/− mice.

Conclusion

These data demonstrate that CCR5 is a key player in controlling the resolution of inflammation in experimental arthritis.

     

The CCL5/CCR5 axis promotes interleukin‐6 production in human synovial fibroblasts

Objective

CCL5 (RANTES) was originally identified as a product of activated T cells and plays a crucial role in the inflammatory response. This study was undertaken to investigate the intracellular signaling pathways involved in CCL5‐induced interleukin‐6 (IL‐6) production in human synovial fibroblasts.

Methods

CCL5‐mediated IL‐6 expression was assessed by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. The mechanisms of action of CCL5 in different signaling pathways were studied using Western blotting. Knockdown of CCR5 and protein kinase Cδ (PKCδ) protein was achieved by transfection of small interfering RNA (siRNA). Chromatin immunoprecipitation assays were used to study in vivo binding of c‐Jun to the IL‐6 promoter. Transient transfection was used to examine IL‐6 and activator protein 1 (AP‐1) activity.

Results

Osteoarthritis synovial fibroblasts (OASFs) showed significant expression of CCL5 and CCR5, and expression was higher than that in normal synovial fibroblasts. Stimulation of OASFs with CCL5 induced concentration‐ and time‐dependent increases in IL‐6 production. CCL5‐mediated IL‐6 production was attenuated by CCR5 monoclonal antibody, CCR5 inhibitor (Met‐RANTES), and CCR5 siRNA. Pretreatment with a PKCδ inhibitor (rottlerin), a c‐Src inhibitor (PP2), or an AP‐1 inhibitor (tanshinone IIA) also blocked the potentiating action of CCL5. Treatment of OASFs with CCL5 increased the accumulation of phosphorylated c‐Jun in the nucleus, AP‐1 luciferase activity, and c‐Jun binding to the AP‐1 element on the IL‐6 promoter. CCL5‐mediated AP‐1 luciferase activity and c‐Jun binding to the AP‐1 element were inhibited by Met‐RANTES, rottlerin, and PP2.

Conclusion

The present results suggest that the interaction between CCL5 and CCR5 increases IL‐6 production in human synovial fibroblasts via the PKCδ/c‐Src/c‐Jun and AP‐1 signaling pathways.

     

HFE mutations in patients with hereditary haemochromatosis in Sweden

Cardoso EMP, Stål P, Hagen K, Cabeda JM, Esin S, De Sousa M, Hultcrantz R (Karolinska Hospital, Stockholm; Huddinge University Hospital, Huddinge; and Karolinska Institute, Stockholm, Sweden; Santo António Hospital; Abel Salazar Institute for Biomedical Sciences, Porto, Portugal). HFE mutations in patients with hereditary haemochromatosis in Sweden. J Intern Med 1998; 243 : 203–8.

Objective

To determine the frequency of mutations (C282Y and H63D) in a newly identified gene HFE in patients with hereditary haemochromatosis (HH) in Sweden.

Design

Molecular genetic analyses of the HFE gene (polymerase chain reaction (PCR) followed by enzyme restriction) were performed in genomic DNA from unrelated patients with a clinical diagnosis of HH and in healthy subjects.

Settings

Patients with HH treated with phlebotomies at Karolinska Hospital and Huddinge Hospital were analyzed.

Subjects

Eighty-seven unrelated patients with HH and 117 healthy controls.

Results

It was found that the HFE C282Y mutation occurs in 94.2% of chromosomes from patients with HH. Eighty patients (92.0%) were homo- zygous for the C282Y mutation and one was heterozygous. Three patients were heterozygous for both C282Y and H63D mutations. One patient was homozygous and one was heterozygous for the H63D mutation. One patient carried normal alleles. In healthy controls, the C282Y mutation occurred in nine subjects (7.7%), all of which were heterozygous. The H63D mutation was found in 28 control subjects, one of which was homozygous.

Conclusions

We found that the majority of patients with HH have the C282Y mutation in the HFE gene. The frequency of the H63D mu- tation was higher in controls than in patients with HH, although in chromosomes at risk the frequency of the H63D mutation was higher in patients.

     

Clinical disease among patients heterozygous for familial mediterranean fever

Objective

To define the molecular basis of familial Mediterranean fever (FMF) in patients with only 1 mutation in the MEFV gene.

Methods

Genetic analysis was performed in 20 FMF patients, including full sequencing of complementary DNA (cDNA) samples and multiplex ligation‐dependent probe amplification analysis. In patients with first‐degree relatives with FMF, haplotype analysis was also performed.

Results

A second mutation was found in 2 patients. In the other 18 patients, we could not identify additional mutations, large genomic deletions, or duplications. Analysis of single‐nucleotide polymorphisms along the cDNA ruled out a lack of expression of 1 of the alleles. In 2 of the 3 families in which more than 1 sibling had FMF, we showed that the affected siblings inherited a different MEFV allele from the parent who did not have the MEFV mutation.

Conclusion

These findings are highly consistent with the existence of a clinical phenotype among some patients who are heterozygous for FMF and could explain the vertical transmission in some families. A single mutation in the MEFV gene may be much more common than was previously thought and may include up to 25% of patients who are diagnosed as having FMF.

     

CXCR5‐ and CCR7‐dependent lymphoid neogenesis in a murine model of chronic antigen‐induced arthritis

Objective

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease with unknown etiology and only partially defined pathogenesis. The aim of this study was to establish a murine model of chronic arthritis in which the development of tertiary lymphoid tissue, a hallmark of human RA, is locally induced, and to characterize the roles of the homeostatic chemokine receptors CXCR5 and CCR7 in this process.

Methods

We developed a modified model of chronic antigen‐induced arthritis (AIA) in mice with a strong bias toward inflammation. Disease pathology was assessed up to 9 months in wild‐type, CXCR5‐deficient, and CCR7‐deficient mice by determination of knee joint swelling and cellular and humoral immune responses, as well as by histologic analysis of arthritic knee joints.

Results

In this novel model of AIA, mice developed organized ectopic lymphoid follicles with topologically segregated B cell and T cell areas, high endothelial venules, and germinal center formation within the chronically inflamed synovial tissue. Analysis of the initiation and progression of AIA in wild‐type, CXCR5^−/−, and CCR7^−/− mice revealed a reduction of acute inflammatory parameters in both knockout strains as well as significantly reduced joint destruction in CXCR5^−/− mice. Most importantly, the development and organization of tertiary lymphoid tissue were significantly impaired in CXCR5‐deficient and CCR7‐deficient mice.

Conclusion

Our results suggest that an inflammatory microenvironment efficiently triggers lymphoid neogenesis in autoimmune diseases such as RA. Moreover, the generation of autoreactive tertiary lymphoid tissues, which is entirely dependent on homeostatic chemokines, may in turn maintain local aberrant chronic immune responses.

     

Targeting of Gr‐1+,CCR2+ monocytes in collagen‐induced arthritis

Objective

The chemokine receptor CCR2 is highly expressed on monocytes and considered a promising target for treatment of rheumatoid arthritis. However, blockade of CCR2 with a monoclonal antibody (mAb) during progression of collagen‐induced arthritis results in a massive aggravation of the disease. In this study we investigated why CCR2 antibodies have proinflammatory effects, how these effects can be avoided, and whether CCR2+ monocytes are useful targets in the treatment of arthritis.

Methods

Arthritis was induced in DBA/1 mice by immunization with type II collagen. Mice were treated with mAb against CCR2 (MC‐21), IgE, or isotype control antibodies at various time points. Activation of basophils and depletion of monocyte subsets were determined by fluorescence‐activated cell sorter analysis and enzyme‐linked immunosorbent assay.

Results

Crosslinkage of CCR2 activated basophils to release interleukin‐6 (IL‐6) and IL‐4. In vivo, IL‐6 release occurred only after exposure to high doses of MC‐21, whereas application of low doses of the mAb circumvented the release of IL‐6. Regardless of the dose level used, the antibody MC‐21 efficiently depleted Gr‐1+,CCR2+ monocytes from the synovial tissue, peripheral blood, and spleen of DBA/1 mice. Activation of basophils with high doses of MC‐21 or with antibodies against IgE resulted in a marked aggravation of collagen‐induced arthritis and an increased release of IL‐6. In contrast, low‐dose treatment with MC‐21 in this therapeutic setting had no effect on IL‐6 and led to marked improvement of arthritis.

Conclusion

These results show that depletion of CCR2+ monocytes may prove to be a therapeutic option in inflammatory arthritis, as long as the dose‐dependent proinflammatory effects of CCR2 mAb are taken into account.

     

Preclinical and clinical investigation of a CCR5 antagonist,AZD5672, in patients with rheumatoid arthritis receiving methotrexate

Objective

To investigate both the preclinical effects of blocking the chemokine receptor CCR5 and the clinical effects of this approach on the signs and symptoms of rheumatoid arthritis (RA) in patients with active disease.

Methods

Preclinical evaluations of AZD5672, a small‐molecule antagonist of CCR5, were performed, including studies of ligand binding and chemotaxis. The pharmacokinetics of AZD5672 were assessed in both single‐ and multiple‐dose studies in healthy volunteers. A randomized, placebo‐controlled, phase IIb study was conducted in patients with active RA receiving methotrexate. Treatment arms were AZD5672 (20, 50, 100, or 150 mg orally, once daily), matched placebo, or open‐label etanercept (50 mg subcutaneously, once weekly). The primary end point was the proportion of patients achieving a 20% improvement response on the American College of Rheumatology improvement criteria (ACR20) at week 12. Secondary end points included the ACR20 over time, as well as 50% (ACR50) and 70% (ACR70) improvement responses, changes in individual components of the ACR improvement criteria, and disease activity measured with the Disease Activity Score based on the 28‐joint count.

Results

AZD5672 was a highly potent and selective antagonist of CCR5, displaying nonproportional steady‐state pharmacokinetics while inhibiting internalization of CCR5 in an ex vivo macrophage inflammatory protein 1β stimulation assay in which AZD5672 was evaluated over the 20–150‐mg dose range. In the phase IIb study testing this dose range in patients with RA (n = 371 patients randomized to received treatment), AZD5672 was generally well tolerated, with no unexpected adverse events. There was no statistically significant difference in the proportion of patients achieving an ACR20 response at week 12 between those receiving any dose of AZD5672 and those receiving placebo; etanercept was significantly more efficacious than AZD5672 and placebo.

Conclusion

Despite a clear rationale for targeting CCR5, this clinical study showed that AZD5672, administered orally, did not have any clinical benefit, suggesting that CCR5 antagonism alone is unlikely to be a viable therapeutic strategy in RA.

     

Genetic loci contributing to hemophagocytic lymphohistiocytosis do not confer susceptibility to systemic‐onset juvenile idiopathic arthritis

Objective

To investigate whether single‐nucleotide polymorphisms (SNPs) within the genes PRF1, GZMB, UNC13D, and Rab27a, which are involved in natural killer cell dysfunction and known to contribute to the risk of hemophagocytic lymphohistiocytosis (HLH), confer an increased risk of susceptibility to systemic‐onset juvenile idiopathic arthritis (JIA).

Methods

Four SNPs across the PRF1 gene locus, 5 for GZMB, 7 for UNC13D, and 11 for Rab27a were investigated using MassArray genotyping in 133 UK Caucasian patients with systemic‐onset JIA and 384 ethnically matched unrelated control subjects. Additional control genotypes were accessed from the data generated by the Wellcome Trust Case Control Consortium.

Results

No significant association was found between any SNP within the 4 selected loci and systemic‐onset JIA, by either single‐point or haplotype analysis.

Conclusion

The results of this study demonstrate that genes involved in HLH do not confer a significant risk of association with systemic‐onset JIA.

     

Inhibition of the development of collagen‐induced arthritis in rhesus monkeys by a small molecular weight antagonist of CCR5

Objective

Collagen‐induced arthritis (CIA) in the rhesus monkey is a nonhuman primate model of rheumatoid arthritis (RA). The close phylogenetic relationship between humans and the rhesus monkey makes this model useful for the preclinical safety and efficacy testing of new therapies that are inactive in animals more distinctly related to humans. In this study, we tested the therapeutic potential of a novel, small molecular weight antagonist of CCR5, SCH‐X, in this model.

Methods

CIA was induced in 10 rhesus monkeys. The animals were allocated to receive SCH‐X or saline as the control (n = 5 in each group). Treatment was initiated on the day of CIA induction and continued for 45 days. Monkeys were monitored before and 63 days after CIA induction for macroscopic signs of clinical arthritis, such as soft‐tissue swelling and body weight. Furthermore, markers of inflammation and joint degradation were monitored to follow the disease course.

Results

Only 2 of 5 animals in the SCH‐X–treated group displayed prominent soft‐tissue swelling, compared with all 5 saline‐treated monkeys. In addition to the suppression of joint inflammation, treatment with SCH‐X resulted in a reduction in joint destruction, as demonstrated by lower rates of urinary excretion of collagen crosslinks, with confirmation by histology. Whereas in all saline‐treated monkeys, marked erosion of joint cartilage was observed, this was absent in 4 of the 5 SCH‐X–treated monkeys.

Conclusion

The systemic effects of treatment with SCH‐X were a suppressed acute‐phase reaction (reduction in C‐reactive protein level) in the 3 treated monkeys with CIA that remained asymptomatic, and an altered antibody response toward type II collagen. The results suggest that the CCR5 antagonist SCH‐X might have a strong clinical potential for treatment during periods of active inflammation, as seen in RA.

     

PENTA guidelines for the use of antiretroviral therapy in paediatric HIV infection. Pediatric European Network for Treatment of AIDS

Objective

To produce European Guidelines for the use of antiretroviral therapy (ART) in HIV‐infected children.

Design

Systematic literature review using Medline, the major antiretroviral conference reports, and IDSA recommendations on guideline production.

Setting

Pediatric European Network for Treatment of AIDS (PENTA) Steering Committee.

Outcome measure

Guidelines have been produced for the use of antiretroviral therapy in HIV‐infected children in Europe. Recommendations on when to start ART and which ART to start, with dosages and a summary of the relevant literature, have been produced.

Conclusions

These guidelines are aimed at assisting paediatricians in Europe with ART prescribing, and provide a more cautious approach to starting therapy than current paediatric USA guidelines.

     

CARD15 mutations in familial granulomatosis syndromes: A study of the original Blau syndrome kindred and other families with large‐vessel arteritis and cranial neuropathy

Objective

To analyze the CARD15 gene in families with heritable multi‐organ granulomatoses, including the original Blau syndrome kindred as well as other families with related granulomatous conditions.

Methods

Linkage mapping was performed in 10 families. Observed recombination events were used to exclude regions centromeric or telomeric to 16q12.1, and the Blau gene critical region was refined to <3 cM, corresponding to a physical distance of 3.5 megabasepairs. Based on its known biochemical function, CARD15 was analyzed as a positional candidate for the Blau syndrome susceptibility gene, by direct DNA sequencing.

Results

These studies resulted in the identification, in 5 of the families, of 2 sequence variants at position 334 of the gene product (R334W and R334Q). Affected family members from the original Blau syndrome kindred were heterozygous for the R334W missense mutation; mutations at the same position were also observed in several unrelated Blau syndrome families, some of whose phenotypes included large‐vessel arteritis and cranial neuropathy. The missense mutations segregated with the disease phenotype in the families, and were not seen in 208 control alleles.

Conclusion

These findings demonstrate that CARD15 is an important susceptibility gene for Blau syndrome and for other familial granulomatoses that display phenotypic traits beyond those of classic Blau syndrome.

     

Osteoarthritis‐like changes and decreased mechanical function of articular cartilage in the joints of mice with the chondrodysplasia gene (cho)

Objective

To investigate whether heterozygosity for a loss‐of‐function mutation in the gene encoding the α1 chain of type XI collagen (Col11a1) in mice (chondrodysplasia, cho) causes osteoarthritis (OA), and to understand the biochemical and biomechanical effects of this mutation on articular cartilage in knee and temporomandibular (TM) joints.

Methods

Articular cartilage from the knee and TM joints of mice heterozygous for cho (cho/+) and their wild‐type littermates (+/+) was examined. The morphologic properties of cartilage were evaluated, and collagen fibrils were examined by transmission electron microscopy. Immunohistochemical staining was performed to examine the protein expression levels of matrix metalloproteinase 3 (MMP‐3) and MMP‐13 in knee joints. In 6‐month‐old animals, fixed‐charge density was determined using a semiquantitative histochemical method, and tensile stiffness was determined using an osmotic loading technique.

Results

The diameter of collagen fibrils in articular cartilage of knee joints from heterozygous cho/+ mice was increased relative to that in control cartilage, and histologic analysis showed OA‐like degenerative changes in knee and TM joints, starting at age 3 months. The changes became more severe with aging. At 3 months, protein expression for MMP‐3 was increased in knee joints from cho/+ mice. At 6 months, protein expression for MMP‐13 was higher in knee joints from cho/+ mice than in joints from their wild‐type littermates, and negative fixed‐charge density was significantly decreased. Moreover, tensile stiffness in articular cartilage of knee joints from cho/+ mice was moderately reduced and was inversely correlated with the increase in articular cartilage degeneration.

Conclusion

Heterozygosity for a loss‐of‐function mutation in Col11a1 results in the development of OA in the knee and TM joints of cho/+ mice. Morphologic and biochemical evidence of OA appears to precede significant mechanical changes, suggesting that the cho mutation leads to OA through a mechanism that does not initially involve mechanical factors.

     

Influence of a monocyte chemoattractant protein 1 mutated allele on the response to protease inhibitor-based antiretroviral therapy

Background

Antiretroviral drug efficacy has been widely studied in relation to viral factors. Mutations in the HIV co‐receptors and their natural chemokines, however, may be critical in HIV infection and treatment response. We compared the efficacy of protease inhibitor (PI) treatment among PI‐naïve patients grouped according to whether they carried the chemokine CC motif receptor 2 (CCR‐2) 64I and monocyte chemoattractant protein 1 (MCP‐1)–2518G alleles.

Methods and results

HIV‐infected patients who were PI‐naive were selected for the study (n=164) but there was no restriction on lymphocyte CD4 count or plasma HIV viral load. Follow‐up was for the first 24 months of treatment. Clinical and laboratory data were obtained every 3 months. All the participants were genotyped for the MCP‐1–2518G, CCR‐2 64I, CCR‐5Δ32 and stromal derived factor 1 (SDF1) 3′A mutated alleles. The results indicated that patients carrying the mutated allele of MCP‐1 had a higher mean CD4 cell count throughout the follow‐up period than those with the common allele (P=0.01). Also, patients with the MCP‐1 and CCR‐2 mutated alleles were more likely to continue to have an undetectable viral load following treatment (P=0.05).

Conclusion

A better response to PI treatment appears to be conferred by mutations in the host MCP‐1 and CCR‐2 genes, and may be related to the cellular axis‐of‐entry used by the retrovirus.

     

Functional consequences of a germline mutation in the leucine‐rich repeat domain of NLRP3 identified in an atypical autoinflammatory disorder

Objective

To gain insight into the pathophysiology of an atypical familial form of an autoinflammatory disorder, characterized by autosomal‐dominant sensorineural hearing loss, systemic inflammation, increased secretion of interleukin‐1β (IL‐1β), and the absence of any cutaneous manifestations, and to assess the functional consequences of a missense mutation identified in the leucine‐rich repeat (LRR) domain of NLRP3.

Methods

Microsatellite markers were used to test the familial segregation of the NLRP3 locus with the disease phenotype. All NLRP3 exons were screened for mutations by sequencing. Functional assays were performed in HEK 293T cells to determine the effects of mutated (versus normal) NLRP3 proteins on NF‐κB activation, caspase 1 signaling, and speck formation.

Results

A heterozygous NLRP3 missense mutation (p.Tyr859Cys) was identified in exon 6, which encodes the LRR domain of the protein. This mutation was found to segregate with the disease phenotype within the family, and had a moderate activating effect on speck formation and procaspase 1 processing and did not alter the inhibitory properties of NLRP3 on NF‐κB signaling.

Conclusion

This report is the first to describe a familial form of a cryopyrinopathy associated with a mutation outside of exon 3 of NLRP3. This finding, together with the known efficacy of anti–IL‐1 treatments in these disorders, underlines the importance of screening all exons of NLRP3 in patients who present with atypical manifestations. In addition, the gain of function associated with this mutation in terms of activation of caspase 1 signaling was consistent with the observed inflammatory phenotype. Therefore, this study of the functional consequences of an LRR mutation sheds new light on the clinical relevance of in vitro assays.