Initial investigation of glucose metabolism in mouse brain using enriched 17O‐glucose and dynamic 17O‐MRS

In this initial work, the in vivo degradation of ¹⁷O‐labeled glucose was studied during cellular glycolysis. To monitor cellular glucose metabolism, direct ¹⁷O‐magnetic resonance spectroscopy (MRS) was used in the mouse brain at 9.4 T. Non‐localized spectra were acquired with a custom‐built transmit/receive (Tx/Rx) two‐turn surface coil and a free induction decay (FID) sequence with a short TR of 5.4 ms. The dynamics of labeled oxygen in the anomeric 1‐OH and 6‐CH₂OH groups was detected using a Hankel–Lanczos singular value decomposition (HLSVD) algorithm for water suppression. Time‐resolved ¹⁷O‐MRS (temporal resolution, 42/10.5 s) was performed in 10 anesthetized (1.25% isoflurane) mice after injection of a 2.2 M solution containing 2.5 mg/g body weight of differently labeled ¹⁷O‐glucose dissolved in 0.9% physiological saline. From a pharmacokinetic model fit of the H₂¹⁷O concentration–time course, a mean apparent cerebral metabolic rate of ¹⁷O‐labeled glucose in mouse brain of CMR_Glc = 0.07 ± 0.02 μmol/g/min was extracted, which is of the same order of magnitude as a literature value of 0.26 ± 0.06 μmol/g/min reported by ¹⁸F‐fluorodeoxyglucose (¹⁸F‐FDG) positron emission tomography (PET). In addition, we studied the chemical exchange kinetics of aqueous solutions of ¹⁷O‐labeled glucose at the C1 and C6 positions with dynamic ¹⁷O‐MRS. In conclusion, the results of the exchange and in vivo experiments demonstrate that the C6‐¹⁷OH label in the 6‐CH₂OH group is transformed only glycolytically by the enzyme enolase into the metabolic end‐product H₂¹⁷O, whereas C1‐¹⁷OH ends up in water via direct hydrolysis as well as glycolysis. Therefore, dynamic ¹⁷O‐MRS of highly labeled ¹⁷O‐glucose could provide a valuable non‐radioactive alternative to FDG PET in order to investigate glucose metabolism.     

Pyruvate‐lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1‐13C]pyruvate MRS and [18F]FDG‐PET

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1‐¹³C]pyruvate MRS and [¹⁸F]FDG‐PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1‐¹³C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1‐¹³C]pyruvate to lactate conversion was followed by ¹³C MRS. Subsequently [¹⁸F]FDG uptake was investigated by static and dynamic PET measurements. Standard uptake values (SUVs) for [¹⁸F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant k_pl of ¹³C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [¹⁸F]FDG PET measurements and k_pl values for the xenografts of both tumor types. The k_pl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [¹⁸F]FDG‐PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1‐¹³C]pyruvate.     

Treatment of collagen‐induced arthritis by Natura‐α via regulation of Th‐1/Th‐17 responses

Cytokines and CD4⁺ Th cells play a crucial role in the pathogenesis of rheumatoid arthritis. Among the Th populations, Th‐1 and Th‐17 have been described as pathogenic in collagen‐induced arthritis (CIA) whereas Th‐2 and Treg were found to have protective effects. The objective of this study was to examine the affect of Natura‐α, a newly developed cytokine regulator, on CIA and on Th cell development. Natura‐α treatment was administered before or during arthritis induction. Anti‐type II collagen antibodies and cytokine expression were evaluated by ELISA. Emergence of CD4⁺CD25⁺Foxp3⁺ T cells was assessed by flow cytometry. Th‐17 differentiation of naive CD4 T cells was assessed in cultures with anti‐CD3 and anti‐CD28. We showed that Natura‐α both prevented and treated CIA. We further demonstrated that in vivo treatment with Natura‐α inhibited IL‐17 production and anti‐type II collagen IgG development. We showed in vitro, using an APC‐free system, that Natura‐α acted directly on differentiating T cells and inhibiting the formation of Th‐1 and Th‐17 cells but did not affect Th‐2 cells. Since Natura‐α inhibits a large spectrum of important pathogenic factors in CIA, it may provide a new and powerful approach to the treatment of rheumatoid arthritis and other inflammatory diseases.     

Rituximab‐induced interleukin‐15 reduction associated with clinical improvement in rheumatoid arthritis

Rituximab therapy alters all aspects of B‐cell participation in the disturbed immune response of rheumatoid arthritis patients. To determine the impact of B‐cell depletion on other immune compartments, we analysed levels of soluble and surface interleukin‐15 (IL‐15) along with the frequency of IL‐15‐related subsets after rituximab treatment. We then studied the correlation of observed changes with clinical activity. Heparinized blood samples from 33 rheumatoid arthritis patients were collected on days 0, 30, 90 and 180 after each of three rituximab cycles. Serum cytokine levels were determined by ELISA. Interleukin‐15 trans‐presentation was analysed by cytometry. Flow cytometry with monoclonal antibodies was performed to analyse circulating cell subsets. Interleukin‐15 was detected in the serum of 25 patients before initiating the treatment. Rituximab then progressively reduced serum IL‐15 (138 ± 21 pg/ml at baseline, 48 ± 18 pg/ml after third cycle, P = 0·03) along with IL‐17 (1197 ± 203 pg/ml at baseline, 623 ± 213 pg/ml after third cycle, P = 0·03) and tended to increase the frequency of circulating regulatory T cells (3·1 ± 1 cells/μl at baseline, 7·7 ± 2 cells/μl after third cycle). Rituximab also significantly decreased IL‐15 trans‐presentation on surface monocytes of patients negative for IL‐15 serum (mean fluorescence intensity: 4·82 ± 1·30 at baseline, 1·42 ± 0·69 after third cycle P = 0·05). Reduction of serum IL‐15 was associated with decrease in CD8⁺ CD45RO⁺/RA⁺ ratio (1·17 ± 0·21 at baseline, 0·36 ± 0·06 at third cycle, P = 0·02). DAS28, erythrocyte sedimentation rate and C‐reactive protein correlated significantly with CD8⁺ CD45RO⁺/RA⁺ ratio (R = 0·323, R = 0·357, R = 0·369 respectively, P < 0·001). Our results suggest that sustained clinical improvement after rituximab treatment is associated with IL‐15/memory T‐cell‐related mechanisms beyond circulating B cells.     

Three‐dimensional ultrashort echo time cones T1ρ (3D UTE‐cones‐T1ρ) imaging

We report a novel three‐dimensional (3D) ultrashort echo time (UTE) sequence employing Cones trajectory and T_1ρ preparation (UTE‐Cones‐T_1ρ) for quantitative T_1ρ assessment of short T₂ tissues in the musculoskeletal system. A basic 3D UTE‐Cones sequence was combined with a spin‐locking preparation pulse for T_1ρ contrast. A relatively short TR was used to decrease the scan time, which required T₁ measurement and compensation using 3D UTE‐Cones data acquisitions with variable TRs. Another strategy to reduce the total scan time was to acquire multiple Cones spokes (N_sp) after each T_1ρ preparation and fat saturation. Four spin‐locking times (TSL = 0–20 ms) were acquired over 12 min, plus another 7 min for T₁ measurement. The 3D UTE‐Cones‐T_1ρ sequence was compared with a two‐dimensional (2D) spiral‐T_1ρ sequence for the imaging of a spherical CuSO₄ phantom and ex vivo meniscus and tendon specimens, as well as the knee and ankle joints of healthy volunteers, using a clinical 3‐T scanner. The CuSO₄ phantom showed a T_1ρ value of 76.5 ± 1.6 ms with the 2D spiral‐T_1ρ sequence, as well as 85.7 ± 3.6 and 89.2 ± 1.4 ms for the 3D UTE‐Cones‐T_1ρ sequences with N_sp of 1 and 5, respectively. The 3D UTE‐Cones‐T_1ρ sequence provided shorter T_1ρ values for the bovine meniscus sample relative to the 2D spiral‐T_1ρ sequence (10–12 ms versus 16 ms, respectively). The cadaveric human Achilles tendon sample could only be imaged with the 3D UTE‐Cones‐T_1ρ sequence (T_1ρ = 4.0 ± 0.9 ms), with the 2D spiral‐T_1ρ sequence demonstrating near‐zero signal intensity. Human studies yielded T_1ρ values of 36.1 ± 2.9, 18.3 ± 3.9 and 3.1 ± 0.4 ms for articular cartilage, meniscus and the Achilles tendon, respectively. The 3D UTE‐Cones‐T_1ρ sequence allows volumetric T_1ρ measurement of short T₂ tissues in vivo.     

Simultaneous imaging of hyperpolarized [1,4‐13C2]fumarate, [1‐13C]pyruvate and 18F–FDG in a rat model of necrosis in a clinical PET/MR scanner

A co‐polarization scheme for [1,4‐¹³C₂]fumarate and [1‐¹³C]pyruvate is presented to simultaneously assess necrosis and metabolism in rats with hyperpolarized ¹³C magnetic resonance (MR). The co‐polarization was performed in a SPINlab polarizer. In addition, the feasibility of simultaneous positron emission tomography (PET) and MR of small animals with a clinical PET/MR scanner is demonstrated. The hyperpolarized metabolic MR and PET was demonstrated in a rat model of necrosis. The polarization and T₁ of the co‐polarized [1,4‐¹³C₂]fumarate and [1‐¹³C]pyruvate substrates were measured in vitro and compared with those obtained when the substrates were polarized individually. A polarization of 36 ± 4% for fumarate and 37 ± 6% for pyruvate was obtained. We found no significant difference in the polarization and T₁ values between the dual and single substrate polarization. Rats weighing about 400 g were injected intramuscularly in one of the hind legs with 200 μL of turpentine to induce necrosis. Two hours later, ¹³C metabolic maps were obtained with a chemical shift imaging sequence (16 × 16) with a resolution of 3.1 × 5.0 × 25.0 mm³. The ¹³C spectroscopic images were acquired in 12 s, followed by an 8‐min ¹⁸F‐2‐fluoro‐2‐deoxy‐d ‐glucose (¹⁸F–FDG) PET acquisition with a resolution of 3.5 mm. [1,4‐¹³C₂]Malate was observed from the tissue injected with turpentine indicating necrosis. Normal [1‐¹³C]pyruvate metabolism and ¹⁸F–FDG uptake were observed from the same tissue. The proposed co‐polarization scheme provides a means to utilize multiple imaging agents simultaneously, and thus to probe various metabolic pathways in a single examination. Moreover, it demonstrates the feasibility of small animal research on a clinical PET/MR scanner for combined PET and hyperpolarized metabolic MR.     

Evaluation of membrane‐bound and soluble forms of human leucocyte antigen‐G in systemic sclerosis

Systemic sclerosis (SSc) is a complex disease characterized by immune dysregulation, extensive vascular damage and widespread fibrosis. Human leucocyte antigen‐G (HLA‐G) is a non‐classic class I major histocompatibility complex (MHC) molecule characterized by complex immunomodulating properties. HLA‐G is expressed on the membrane of different cell lineages in both physiological and pathological conditions. HLA‐G is also detectable in soluble form (sHLA‐G) deriving from the shedding of surface isoforms (sHLA‐G1) or the secretion of soluble isoforms (HLA‐G5). Several immunosuppressive functions have been attributed to both membrane‐bound and soluble HLA‐G molecules. The plasma levels of sHLA‐G were higher in SSc patients (444·27 ± 304·84 U/ml) compared to controls (16·74 ± 20·58 U/ml) (P < 0·0001). The plasma levels of transforming growth factor (TGF)‐β were higher in SSc patients (18 937 ± 15 217 pg/ml) compared to controls (11 099 ± 6081 pg/ml; P = 0·003), and a significant correlation was found between TGF‐β and the plasma levels of total sHLA‐G (r = 0·65; P < 0·01), sHLA‐G1 (r = 0·60; P = 0·003) and HLA‐G5 (r = 0·47; P = 0·02). The percentage of HLA‐G‐positive monocytes (0·98 ± 1·72), CD4⁺ (0·37 ± 0·68), CD8⁺ (2·05 ± 3·74) and CD4⁺CD8⁺ double‐positive cells (14·53 ± 16·88) was higher in SSc patients than in controls (0·11 ± 0·08, 0·01 ± 0·01, 0·01 ± 0·01 and 0·39 ± 0·40, respectively) (P < 0·0001). These data indicate that in SSc the secretion and/or shedding of soluble HLA‐G molecules and the membrane expression of HLA‐G by peripheral blood mononuclear cells (PBMC) is clearly elevated, suggesting an involvement of HLA‐G molecules in the immune dysregulation of SSc.     

Flk‐1+ mesenchymal stem cells aggravate collagen‐induced arthritis by up‐regulating interleukin‐6

The immunomodulatory ability of mesenchymal stem cells (MSCs) may be used to develop therapies for autoimmune diseases. Flk‐1⁺ MSCs are a population of MSCs with defined phenotype and their safety has been evaluated in Phase 1 clinical trials. We designed this study to evaluate whether Flk‐1⁺ MSCs conferred a therapeutic effect on collagen‐induced arthritis (CIA), an animal model of rheumatic arthritis, and to explore the underlying mechanisms. Flk‐1⁺ MSCs, 1–2 × 10⁶, were injected into CIA mice on either day 0 or day 21. The clinical course of arthritis was monitored. Serum cytokine profile was determined by cytometric bead array kit or enzyme‐linked immunosorbent assay. Flk‐1⁺ MSCs and splenocytes co‐culture was conducted to explore the underlying mechanisms. Flk‐1⁺ MSCs did not confer therapeutic benefits. Clinical symptom scores and histological evaluation suggested aggravation of arthritis in mice treated with MSCs at day 21. Serum cytokine profile analysis showed marked interleukin (IL)‐6 secretion immediately after MSC administration. Results of in vitro culture of splenocytes confirmed that the addition of Flk‐1⁺ MSCs promoted splenocyte proliferation and increased IL‐6 and IL‐17 secretion. Moreover, splenocyte proliferation was also enhanced in mice treated with MSCs at day 21. Accordingly, MSCs at low concentrations were found to promote lipopolysaccharide‐primed splenocytes proliferation in an in vitro co‐culture system. We propose that Flk‐1⁺ MSCs aggravate arthritis in CIA model by at least up‐regulating secretion of IL‐6, which favours Th17 differentiation. When Flk‐1⁺ MSCs are used for patients, we should be cautious about subjects with rheumatoid arthritis.     

Interleukin‐27 suppresses osteoclastogenesis via induction of interferon‐γ

Interleukin (IL)‐27 is a heterodimeric cytokine that is known to have both stimulatory and inhibitory functions during immune responses. We investigated the effects of IL‐27 on arthritis and bone erosion in the murine collagen‐induced arthritis (CIA) model. We demonstrate that the inhibitory effect of IL‐27 on osteoclastogenesis is associated with interferon‐γ (IFN‐γ) production by using an IFN‐γ knockout mouse model. The IL‐27‐Fc was injected into both CIA and IFN‐γ‐deficient mice. The effects of IL‐27‐Fc on osteoclast differentiation were evaluated both in vitro and in vivo. The IL‐27‐Fc‐injected mice showed significantly lower arthritis indices and fewer tartrate‐resistant acid‐phosphatase‐positive osteoclasts in their joint tissues than untreated mice. Interleukin‐27 inhibited osteoclastogenesis from bone marrow‐derived mononuclear cells in vitro, which was counteracted by the addition of anti‐IFN‐γ antibody. The IL‐27‐Fc did not affect arthritis in IFN‐γ knockout mice. Interleukin‐27 also suppressed osteoclast differentiation in human and intriguingly, it could promote the expression of IFN‐γ on priming osteoclasts. These results imply that IL‐27 suppressed the generation of CIA and osteoclastogenesis, which were mediated by the induction of IFN‐γ.     

Decreased lactate concentration and glycolytic enzyme expression reflect inhibition of mTOR signal transduction pathway in B‐cell lymphoma

The application of kinase inhibitors in cancer treatment is growing rapidly. However, methods for monitoring the effectiveness of the inhibitors are still poorly developed and currently rely mainly on the tracking of changes in the tumor volume, a rather late and relatively insensitive marker of the therapeutic response. In contrast, MRS can detect changes in cell metabolism and has the potential to provide early and patient‐specific markers of drug activity. Using human B‐cell lymphoma models and MRS, we have demonstrated that the inhibition of the mTOR signaling pathway can be detected in malignant cells in vitro and noninvasively in vivo by the measurement of lactate levels. An mTOR inhibitor, rapamycin, suppressed lactic acid production in lymphoma cell line cultures and also diminished steady‐state lactate levels in xenotransplants. The inhibition was time dependent and was first detectable 8 h after drug administration in cell cultures. In xenotransplants, 2 days of rapamycin treatment produced significant changes in lactic acid concentration in the tumor measured in vivo, which were followed by tumor growth arrest and tumor volume regression. The rapamycin‐induced changes in lactate production were strongly correlated with the inhibition of expression of hexokinase II, the key enzyme in the glycolytic pathway. These studies suggest that MRS or ¹⁸F‐fluorodeoxyglucose positron emission tomography (FDG PET) detection of changes in glucose metabolism may represent effective noninvasive methods for the monitoring of mTOR targeting therapy in lymphomas and other malignancies. Furthermore, the measurement of glucose metabolic inhibition by MRS or FDG PET imaging may also prove to be effective in monitoring the efficacy of other kinase inhibitors given that the rapamycin‐sensitive mTOR lies downstream of many oncogenic signaling pathways. Copyright © 2012 John Wiley & Sons, Ltd.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

Novel therapeutic compound tuftsin–phosphorylcholine attenuates collagen‐induced arthritis

Treatment with helminthes and helminthes ova improved the clinical symptoms of several autoimmune diseases in patients and in animal models. Phosphorylcholine (PC) proved to be the immunomodulatory molecule. We aimed to decipher the tolerogenic potential of tuftsin–PC (TPC), a novel helminth‐based compound in collagen‐induced arthritis (CIA) a mouse model of rheumatoid arthritis (RA). CIA DBA/1 mice were treated with TPC subcutaneously (5 µg/0.1 ml) or orally (250 µg/0.1 ml), starting prior to disease induction. The control groups were treated with PBS. Collagen antibodies were tested by enzyme‐linked immunosorbent assay (ELISA), cytokine protein levels by ELISA kits and regulatory T (T_reg) and regulatory B (B_reg) cell phenotypes by fluorescence‐activated cell sorter (FACS). TPC‐treated mice had a significantly lower arthritis score of 1.5 in comparison with control mice 11.8 (P < 0.0001) in both subcutaneous and orally treated groups at day 31. Moreover, histology analysis demonstrated highly inflamed joints in control mice, whereas TPC‐treated mice maintained normal joint structure. Furthermore, TPC decreased the titres of circulating collagen II antibodies in mice sera (P < 0.0001), enhanced expression of IL‐10 (P < 0.0001) and inhibited production of tumour necrosis factor (TNF)‐α, interleukin (IL)?17 and IL‐1β (P < 0.0001). TPC significantly expanded the CD4⁺CD25⁺ forkhead box protein 3 (FoxP3⁺) T_reg cells and CD19⁺IL‐10⁺CD5^highCD1d^highT cell immunoglobulin mucin‐1 (TIM‐1⁺) B_reg cell phenotypes (P < 0.0001) in treated mice. Our data indicate that treatment with TPC attenuates CIA in mice demonstrated by low arthritic score and normal joints histology. TPC treatment reduced proinflammatory cytokines and increased anti‐inflammatory cytokine expression, as well as expansion of T_reg and B_reg cells. Our results may lead to a new approach for a natural therapy for early rheumatoid arthritis onset.     

<Emphasis Type="Italic">Lactobacillus casei</Emphasis> reduces the Inflammatory Joint Damage Associated with Collagen-Induced Arthritis (CIA) by Reducing the Pro-Inflammatory Cytokines

This study evaluated the therapeutic efficacy of Lactobacillus casei in treating rheumatoid arthritis using collagen-induced arthritis (CIA) animal model. Healthy female Wistar rats (weight—180–200 g) were included in this study. Oral administration of L. casei was started on the same day. Indomethacin was used as standard reference drug. Serum level of IL-6, α-TNF, and IL-10 were observed. Four-point arthritis indexes were also assessed at the end of week for 28th day. L. casei-treated rats had shown normal histopathology without any synovial infiltration, pannus formation, cartilage, and bone destruction. Arthritis score was also lower for the group treated with L. casei. Oral administration of L. casei significantly decreased the pro-inflammatory cytokines. Present study suggests that L. casei has potent antiarthritic effect in CIA model. Inhibition of COX-2 via inhibiting the pro-inflammatory cytokines is an understanding of the complex interactions involved in these pathways.     

Capnocytophaga canimorsus: a rare case of conservatively treated prosthetic valve endocarditis

We describe a rare case of prosthetic valve endocarditis caused by the canine bacterium Capnocytophaga canimorsus in a male aged 73 years. The diagnosis of infective endocarditis was unequivocal, as it blood cultures were positive for C. canimorsus and vegetations were detected on transesophageal echocardiography; the modified Duke criteria were fulfilled. PET‐CT showed intense ¹⁸F‐FDG uptake of the prosthetic valve area. The patient was treated with antibiotics alone (no surgery), and is now on life‐long suppressive antibiotic therapy. To our knowledge, this is the third reported case of prosthetic valve endocarditis caused by C. canimorsus and the first one to have been treated conservatively.     

Relationship between natural and infection‐induced antibodies in systemic autoimmune diseases (SAD): SLE,SSc and RA

Infection or vaccine‐induced T cell‐dependent immune response and the subsequent high‐affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease‐specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine‐induced and disease‐related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme‐linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti‐CS) and F4 fragment (anti‐F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n = 92), rheumatoid arthritis (n = 73) and systemic sclerosis (n = 157) disease groups. Anti‐measles IgG and anti‐dsDNA IgG/IgM autoantibodies were measured using commercial and in‐house indirect ELISA tests. In all SAD groups the anti‐measles IgG‐seropositive cases showed significantly higher anti‐CS IgG titers (P = 0·011). In anti‐dsDNA IgG‐positive SLE patients, we detected significantly higher levels of anti‐CS and anti‐F4 IgG nAAbs (P = 0·001 and < 0·001, respectively). Additionally, we found increased levels of IgM isotypes of anti‐CS and anti‐F4 nAAbs in anti‐dsDNA IgM‐positive SLE patients (P = 0·002 and 0·016, respectively). The association between IgG isotypes of pathogen‐ or autoimmune disease‐related antibodies and the IgG nAAbs may underscore the immune response‐inducible nature of the diseases investigated. The relationship between protective anti‐dsDNA IgM and the IgM isotype of anti‐F4 and anti‐CS may provide immunoserological evidence for the beneficial roles of nAAbs in SLE patients.     

Co‐expression of TLR‐9 and MMP‐13 is associated with the degree of tumour differentiation in prostate cancer

In vitro experiments demonstrated that stimulation of Toll‐like receptor 9 (TLR‐9) by synthetic TLR‐9 ligands induces the invasion of TLR‐9‐expressing prostate cancer cells through matrix metalloproteinase 13 (MMP‐13). However, the clinical value of TLR‐9 and MMP‐13 co‐expression in the pathophysiology of the prostate is unknown. In the study, we evaluated the expression levels and clinical significance of the TLR‐9 and MMP‐13 in a series of prostate tissues. One hundred and eighty prostate tissues including prostate cancer (PCa) (n = 137), high‐grade prostatic intraepithelial neoplasia (HPIN) (n = 18) and benign prostatic hyperplasia (BPH) (n = 25) were immunostained for the TLR‐9 and MMP‐13 markers. Subsequently, the correlation between the TLR‐9 and MMP‐13 staining scores and clinicopathological parameters was obtained. Higher expressions of TLR‐9 and MMP‐13 were found in PCa and high‐grade prostatic intraepithelial neoplasia compared to benign prostatic hyperplasia tissues. Among PCa samples, a positive relationship was revealed between the MMP‐13 expression and Gleason score (P < 0.001). There was a significant correlation between TLR‐9 expression and regional lymph node involvement (P = 0.04). The expression patterns of TLR‐9 and MMP‐13 markers demonstrated a reciprocal significant correlation between the two markers in the same series of prostate samples (P < 0.001). Furthermore, the Gleason score of TLR‐9^high/MMP‐13^high and TLR‐9^low/MMP‐13^low phenotypes showed a significant difference (P = 0.002). Higher expressions of TLR‐9 and MMP‐13 can confer aggressive behaviour to PCa. Therefore, these markers may be used as a valuable target for tailored therapy of PCa.     

Genetic heterogeneity in rheumatoid arthritis mouse models induced by extrinsic and intrinsic factors

A cumulative effect of the susceptibility genes with polymorphic alleles may be responsible for rheumatoid arthritis (RA). The objective of this study was to clarify whether susceptibility to RA is under the control of common allelic loci between two different RA models induced by extrinsic and intrinsic factors, collagen‐induced arthritis (CIA) in DBA/1 mice and arthritis in MRL/Mp (MRL) mice associated with the Fas deficient mutant gene, Fas^lpr, respectively. CIA was examined in mice of parental DBA/1 and MRL, (MRL × DBA/1) F1 and (MRL × DBA/1) F2 progenies. In genome‐wide screening of the severity in the F2 using microsatellite markers, significant linkage was observed on chromosomes 5 and 17 at map position of D5Mit259 and H‐2, respectively, associated with DBA/1 alleles, while there was no loci associated with arthritis of MRL‐Fas^lpr mice previously identified. In a quantitative trait locus (QTL) analysis, the locus on chromosome 5 showed the highest peak at map position 35 cM (LOD score 6.0). This study may indicate that the arthritis induced by extrinsic and intrinsic factors is under the control of a different combination of susceptibility genes with common and different alleles, possibly simulating the genetic heterogeneity of RA.     

KIR‐HLA‐A and B alleles of the Bw4 epitope against HIV infection in discordant heterosexual couples in Chaco Argentina

Activating and inhibitory killer immunoglobulin‐like receptors (KIR) and their ligands HLA‐Bw4 (loci A and B) were studied by way of establishing whether they can contribute to protection against HIV‐1 infection in highly exposed and persistently seronegative (HESN) patients. Twenty‐three HIV‐1 serodiscordant heterosexual couples, 100 HIV‐1⁺ patients and 200 healthy individuals were included in this retrospective case–control study. HLA typing was performed by means of PCR followed by sequence‐specific oligonucleotide probe reverse hybridization. KIR3DL1 and KIR3DS1 were studied by PCR sequence‐specific primers. The frequency of KIR3DS1(3DS1/3DL1)‐Bw4 combination was significantly higher in HESN patients versus the discordant couples (P = 0·0003) and HIV‐1⁺ patients (P = 0·0001). Conversely, the KIR3DL1/KIR3DL1 homozygosity was significantly decreased in HESN patients versus the discordant couples (P = 0·00003), and HIV‐1⁺ patients (P = 0·00066). The frequency of HLA‐A*32 and HLA‐B*44 was higher in HESN versus their discordant couples (P = 0·009; P = 0·049), and HIV‐1⁺ patients (P = 0·00002; P = 0·0001). This had greater significance in combination with KIR3DS1 (3DS1/3DL1). KIR3DS1(3DS1/3DL1) could have a greater effect on protection against HIV‐1 infection in HESN patients when bound to a specific HLA allele, in this case HLA‐A*32 and HLA‐B*44, both Bw4 alleles. The differences probably arise both in the HLA alleles and in the subtypes of KIR receptors depending on the ethnic group studied.     

Human monoclonal Fab and human plasma antibodies to carbamyl‐epitopes cross‐react with malondialdehyde‐adducts

Oxidized low‐density lipoprotein (OxLDL) plays a crucial role in the development of atherosclerosis. Carbamylated LDL has been suggested to promote atherogenesis in patients with chronic kidney disease. Here we observed that plasma IgG and IgM antibodies to carbamylated epitopes were associated with IgG and IgM antibodies to oxidation‐specific epitopes (ρ = 0·65–0·86, P < 0·001) in healthy adults, suggesting a cross‐reaction between antibodies recognizing carbamyl‐epitopes and malondialdehyde (MDA)/malondialdehyde acetaldehyde (MAA) ‐adducts. We used a phage display technique to clone a human Fab antibody that bound to carbamylated LDL and other carbamylated proteins. Anti‐carbamyl‐Fab (Fab106) cross‐reacted with oxidation‐specific epitopes, especially with MDA‐LDL and MAA‐LDL. We showed that Fab106 bound to apoptotic Jurkat cells known to contain these oxidation‐specific epitopes, and the binding was competed with soluble carbamylated and MDA‐/MAA‐modified LDL and BSA. In addition, Fab106 was able to block the uptake of carbamyl‐LDL and MDA‐LDL by macrophages and stained mouse atherosclerotic lesions. The observed cross‐reaction between carbamylated and MDA‐/MAA‐modified LDL and its contribution to enhanced atherogenesis in uraemic patients require further investigation.