Test-retest precision and longitudinal cartilage thickness loss in the IMI-APPROACH cohort.

OBJECTIVE: To investigate the test-retest precision and to report the longitudinal change in cartilage thickness, the percentage of knees with progression and the predictive value of the machine-learning-estimated structural progression score (s-score) for cartilage thickness loss in the IMI-APPROACH cohort - an exploratory, 5-center, 2-year prospective follow-up cohort. DESIGN: Quantitative cartilage morphology at baseline and at least one follow-up visit was available for 270 of the 297 IMI-APPROACH participants (78% females, age: 66.4 ± 7.1 years, body mass index (BMI): 28.1 ± 5.3 kg/m2, 55% with radiographic knee osteoarthritis (OA)) from 1.5T or 3T MRI. Test-retest precision (root mean square coefficient of variation) was assessed from 34 participants. To define progressor knees, smallest detectable change (SDC) thresholds were computed from 11 participants with longitudinal test-retest scans. Binary logistic regression was used to evaluate the odds of progression in femorotibial cartilage thickness (threshold: -211 µm) for the quartile with the highest vs the quartile with the lowest s-scores. RESULTS: The test-retest precision was 69 µm for the entire femorotibial joint. Over 24 months, mean cartilage thickness loss in the entire femorotibial joint reached -174 µm (95% CI: [-207, -141] µm, 32.7% with progression). The s-score was not associated with 24-month progression rates by MRI (OR: 1.30, 95% CI: [0.52, 3.28]). CONCLUSION: IMI-APPROACH successfully enrolled participants with substantial cartilage thickness loss, although the machine-learning-estimated s-score was not observed to be predictive of cartilage thickness loss. IMI-APPROACH data will be used in subsequent analyses to evaluate the impact of clinical, imaging, biomechanical and biochemical biomarkers on cartilage thickness loss and to refine the machine-learning-based s-score. GOV IDENTIFICATION: NCT03883568.

Tissue distribution of sprifermin (recombinant human fibroblast growth factor 18) in the rat following intravenous and intra-articular injection.

Objective: Fibroblast growth factor 18 (FGF18) is involved in chondrogenesis and articular cartilage repair. We investigated tissue distribution and pharmacokinetics of radioactive [3H]sprifermin, a recombinant human FGF18, in rats after a single intravenous (i.v.) or intra-articular (i.a.) injection. Design: In two studies (48-96-h [n = 23] and 28-day [n = 12]), 35 male albino (Sprague Dawley) rats received single i.v. or i.a. dose [3H]sprifermin (0.24 mg/kg). Radioactivity was measured in blood, serum, and (in animals receiving i.a. administration) in the knee joint by liquid scintillation counting. Radioactivity in organs, tissues, and distribution in the whole body were measured with whole-body autoradiography. Results: After i.v. injection, radioactivity peaked in serum and whole blood after 4 and 24 h, respectively, with greater total radioactivity in serum. After i.a. injection, radioactivity peaked in serum and whole blood after 24 and 48 h, respectively; intact [3H]sprifermin was not detected in vena caval serum and systemic exposure was low, approximately 20% of that with i.v. injection. Following i.v. injection, radioactivity was mainly found in the liver, adrenal glands, kidney, and spleen; following i.a. injection, radioactivity was preferentially concentrated in articular cartilage after initial distribution in the joint capsule, and still evident in the joint after 28 days. Conclusions: After i.a. injection of [3H]sprifermin in rats, radioactivity was concentrated in the knee joint, particularly articular cartilage, with low levels in other investigated tissues. Systemic exposure to sprifermin was greater with i.v. than i.a. injection. Subsequent clinical investigation in patients with osteoarthritis has reported consistent results.

Participation of group 2 CD1 molecules in the control of murine tuberculosis.

Besides the classical major histocompatibility complex (MHC) class I and MHC class II molecules, human CD1 molecules have been shown to present mycobacterial antigens in vitro. In this study, in vivo treatment of mice with anti-CD1 monoclonal antibodies resulted in exacerbated tuberculosis at very early time points. In CD1-modulated mice, Mycobacterium tuberculosis-specific production of the type 1 cytokines, IL-12, TNF, and IFN-gamma as well as of TGF beta was reduced. These findings suggest an antigen-presenting role of CD1 molecules in tuberculosis.

Comparison between MRI, microbiology and histology in evaluation of antibiotics in a murine model of thigh infection.

Although in vivo magnetic resonance imaging (MRI) is rapidly becoming a recognised tool in experimental pharmacological research, at the best of our knowledge, scarce application in the field of antibacterial drug research has been reported so far. In this last field, animal models of bacterial infections are used to test the efficacy of novel compounds. In this paper we have explored the potential usefulness of MRI in monitoring the chronological evolution of experimental bacterial infections and the effect of different therapeutic treatments. A murine model of thigh infection induced by Staphylococcus aureus has been used and the efficacy of vancomycin and imipenem/cilastatin has been tested. Three groups of infected animals were studied by microbiology, histology and MRI methods. The results obtained show that in vivo MRI data are highly consistent with microbiological and histological data, allowing, similarly to these commonly used techniques, the efficacy of different antibacterial treatments to be quantified. Our findings suggest that MRI could be used to assess the efficacy of new chemical entities in antibacterial pharmacological research. The advantages of MRI, as a non invasive technique, in comparison with commonly used microbiological and histological methods are discussed.

Cutting edge: anti-CD1 monoclonal antibody treatment reverses the production patterns of TGF-beta 2 and Th1 cytokines and ameliorates listeriosis in mice.

Protection against intracellular bacteria by T cells is regulated by Ag-presenting molecules, which comprise classical MHC class I molecules, MHC class II molecules, and nonclassical MHC class Ib molecules. The role of CD1 molecules, which are structurally similar to classical MHC class I gene products, but less polymorphic, is not understood so far. We show that CD1 surface expression increased on APC in Listeria-infected mice. The in vivo treatment with anti-CD1 mAb reduced TGF-beta 2 levels and concomitantly increased secretion of the proinflammatory cytokine TNF, the Th1 cell promoting cytokine IL-12, and the Th1 cell cytokine IFN-gamma at the onset of listerial infection. These findings point to a regulatory role of CD1-reactive cells in the immune response against listeriosis.

Lethal tuberculosis in interleukin-6-deficient mutant mice.

Tuberculosis is a chronic infectious disease which causes major health problems globally. Acquired resistance is mediated by T lymphocytes and executed by activated macrophages. In vitro studies have emphasized the importance of macrophage activation for mycobacterial growth inhibition. In vivo, the protective host response is focused on granulomatous lesions in which Mycobacterium tuberculosis is contained. A cellular immune response of the T helper 1 (Th1) type is considered central for control of tuberculosis. Using interleukin-6 (IL-6)-deficient mice, we here demonstrate a crucial role of this pluripotent cytokine in protection against M. tuberculosis but not against Mycobacterium bovis BCG. Infection with M. tuberculosis was lethal for the IL-6-deficient mice at inocula that were still controlled by IL-6-competent mice. Spleen cells from M. tuberculosis-infected IL-6-/- mouse mutants produced elevated levels of IL-4 and reduced levels of gamma interferon compared to the control levels. Cytofluorometric analyses of spleen cells from M. tuberculosis-infected mice revealed more-profound alterations in T-cell ratios in IL-6-/- mice than in control mice. We assume that IL-6 contributes to host resistance by its proinflammatory activity and by its influence on cytokine secretion.

Interleukin-12 secretion by Mycobacterium tuberculosis-infected macrophages.

Infection with Mycobacterium tuberculosis or phagocytosis of large latex beads induced interleukin-12 (IL-12) production in macrophages. In contrast, tumor necrosis factor (TNF) was produced only in response to M. tuberculosis infection, not after phagocytosis of latex beads. Comparable results were obtained with cells from immunocompetent C57BL/6 and gamma interferon receptor-deficient mutant mice. Thus, phagocytosis by mechanisms not specific for M. tuberculosis was a sufficient trigger for IL-12 secretion, emphasizing the central role of this cytokine in the initiation of anti-infective immunity.

Abscess formation in Listeria monocytogenes-infected gamma delta T cell deficient mouse mutants involves alpha beta T cells.

Although mutant mice lacking gamma delta T cells resolve Listeria monocytogenes infection, extensive abscesses are formed. Manifestation of these inflammatory lesions is prevented by in vivo depletion with monoclonal antibodies of CD4, CD8 or both T cell subsets. We conclude that these inflammatory tissue reactions develop when alpha beta T cells of either CD4 or CD8 phenotype are released from control by gamma delta T cells.

IL-4 neutralization or TNF-alpha treatment ameliorate disease by an intracellular pathogen in IFN-gamma receptor-deficient mice.

Successful control of infectious disease depends on aquisition of an appropriate protective immune response. IFN-gamma, first produced by NK cells and then by Th1 cells, is central to acquired resistance against intracellular bacteria, including Listeria monocytogenes. In contrast, IL-4 is not generated to a measurable degree. Here we show that IL-4 is produced during listeriosis by IFN-gamma receptor gene disruption (IFN-gammaR(-/-)) mutant mice. Production of TNF was diminished, whereas IL-12 production was virtually unchanged in these mutants. Neutralization of IL-4 with anti-IL-4 mAb, as well as TNF-alpha reconstitution with rTNF-alpha, ameliorated listeriosis. These findings demonstrate the detrimental effects of IL-4 in listeriosis independent of IFN-gamma down-regulation and document the far-reaching consequences of a single cytokine deficiency on other cytokines. In cases where the primary gene defect cannot be restored, precise identification of secondary effects will promote rational immunotherapy based on neutralization or reconstitution of secondary immune deviations.

Control of natural killer cell-mediated innate resistance against the intracellular pathogen Listeria monocytogenes by gamma/delta T lymphocytes.

Listeria monocytogenes is an intracellular bacterium which causes an acute infectious disease in mice. Initial host resistance depends on innate immunity mediated primarily by natural killer (NK) cells followed by specific alpha/beta T cells, which are central to acquired specific immunity. Gamma/delta T lymphocytes seem to provide a link between the innate and the specific immune response. All these lymphocyte populations produce gamma interferon (IFN-gamma), which, because of its macrophage-activating potential, is central to antibacterial protection. IFN-gamma from NK cells not only contributes to early host resistance but also promotes development of protective T-cell responses of helper T type 1 (Th1) type. Here, we show that innate resistance and early IFN-gamma production in listeriosis are markedly impaired in T-cell receptor (TCR)-delta-/- but not TCR-beta-/- gene disruption mutant mice. By two-color cytofluorimetry, we demonstrate that NK cells rather than gamma/delta T lymphocytes are the major cellular source of IFN-gamma in immunocompetent mice and that IFN-gamma production by NK cells is impaired in the TCR-delta-/- mutants. Probably, reduced tumor necrosis factor production in listeria-infected TCR-delta-/- mutants contributed to impaired NK cell activation. Our data reveal a novel function of gamma/delta T cells as regulators of innate resistance against sublethal infection with an intracellular pathogen.

Activation of natural killer cells by heat-killed Listeria monocytogenes requires additional signals from lymphoid cells.

Regulatory and protective functions have been attributed to murine natural killer (NK) cells in a number of infectious diseases including listeriosis. We have developed an in vitro model to study parameters underlying the activation of naive NK cells using heat-killed Listeria monocytogenes (HKL) as stimulator. Independent from expression of the cell surface marker NK1.1, NK cells lysed YAC-1 cells after in vitro stimulation with HKL or HKL + Interleukin (IL)-2, but not medium or IL-2 alone. In contrast, NK cells from severely immunocompromised SCID or RAG-1-/-mutant mice failed to respond to HKL alone, but required exogenous IL-2. Using single-gene-disruption mutant mice, we show that NK-cell activation can be supported by either T-cell receptor (TCR) alpha beta cells, TCR- gamma delta cells. MHC class I or MHC class II gene products. We conclude from these data that recognition of listerial components alone is insufficient for activation of naive NK cells, and that additional costimulatory signals are necessary. These can be provided by various lymphoid cells and appear to be cytokines.

Stimulation of protective CD8+ T lymphocytes by vaccination with nonliving bacteria.

Infectious diseases caused by intracellular microbes are responsible for major health problems, and satisfactory control will ultimately depend on efficient vaccination strategies. The general assumption is that activation of protective immune responses against intracellular microbes dominated by CD8+ T cells are achieved only by live vaccines. In contrast, we here demonstrate stimulation of protective immunity in mice against the intracellular pathogen Listeria monocytogenes by vaccination with heat-killed listeriae. Vaccine-induced immunity comprised cytolytic and interferon gamma-producing CD8+ T lymphocytes. CD8+ T cells from vaccinated donor mice transferred protection against listeriosis. Moreover, vaccination with heat-killed listeriae induced production in CD4+ T-cell-deficient, H2-A beta gene-disrupted mutant mice. We conclude that antigens from killed listeriae are introduced into the major histocompatibility complex class I pathway and thus are recognized by CD8+ T cells. The practicability of killed vaccines against human infectious diseases therefore should be reevaluated.

Protective role of gamma/delta T cells and alpha/beta T cells in tuberculosis.

Tuberculosis is a chronic infectious disease which causes major health problems globally. Although acquired resistance crucially depends on alpha/beta lymphocytes, circumstantial evidence suggests that, in addition, gamma/delta T lymphocytes contribute to protection against tuberculosis. We have studied Mycobacterium tuberculosis infection in TcR-delta-/- or TcR-beta-/- gene deletion mutants which completely lack gamma/delta T cells or alpha/beta T cells, respectively. Low inocula of M. tuberculosis led to death of TcR-beta-/- mice and transient disease exacerbation in TcR-delta-/- mutants. Infection with higher inocula caused rapid death of TcR-delta-/- mice. The development of and bacterial containment in granulomatous lesions was markedly impaired in TcR-beta-/-, and less severely affected in TcR-delta-/- mutants. Mycobacteria-induced IFN-gamma production by spleen cells in vitro was almost abolished in TcR-beta-/- and virtually unaffected in TcR-delta-/- mice. Our data confirm the crucial role of alpha/beta T cells in protection against established tuberculosis and formally prove a protective role of gamma/delta T cells in early tuberculosis.

Influence of mouse strain and vaccine viability on T-cell responses induced by Mycobacterium bovis bacillus Calmette-Guérin.

C57BL/6 and BALB/c mice were vaccinated with either live or heat-killed Mycobacterium bovis bacillus Calmette-Guérin (BCG) organisms, and splenic T cells were used to screen the stimulatory potential of fractionated somatic and secreted mycobacterial proteins by production of gamma interferon (IFN-gamma). Maximum responses were obtained with fractionated secreted proteins of Mycobacterium tuberculosis. There was no single dominant antigen, but five regions of mycobacterial proteins induced high concentrations of IFN-gamma. However, only two of the five regions stimulated T cells from both mouse strains: two were exclusively recognized by T cells from BALB/c mice, and one was exclusively recognized by T cells from C57BL/6 mice. T cells from mice vaccinated with heat-killed M. bovis BCG organisms failed to respond to fractionated secreted proteins but recognized several somatic antigen fractions. As late as 1 year after primary vaccination, memory T cells responded to similar protein regions, and IFN-gamma production was intensified by secondary infection. Our data confirm a central role for secreted proteins in immunity to mycobacteria. Moreover, we demonstrate that a major set of mycobacterium-reactive T cells is stimulated only by vaccination with live but not with heat-killed M. bovis BCG organisms. Because a major impact of genetic host factors on antigen recognition was observed, we favor the use of live carrier organisms which secrete mycobacterial proteins over subunit vaccines as an improved antituberculosis vaccine.

Contribution of alpha/beta and gamma/delta T lymphocytes to immunity against Mycobacterium bovis bacillus Calmette Guérin: studies with T cell receptor-deficient mutant mice.

Mutant mice with defined T cell deficiencies were infected with Mycobacterium bovis bacillus Calmette Guérin (BCG) and the relative contribution of alpha/beta T cells and gamma/delta T cells to the host immune response was assessed. Recombinase activating gene (RAG-1)-/- mutants as well as T cell receptor (TcR) beta-/-, but not TcR-delta-/-, mutants succumbed to M. bovis BCG infection and failed to develop granulomatous lesions. Antigen-induced IFN-gamma production by spleen cells in vitro was abrogated in RAG-1-/- mutants and markedly diminished in TcR-beta-/- and TcR-delta-/- mice. Reconstitution experiments suggest that both alpha/beta and gamma/delta T cells are essential for antigen-specific IFN-gamma secretion. Our data formally prove the crucial role of alpha/beta T cells and reveal accessory functions of gamma/delta T cells in optimum immunity against M. bovis BCG.

Immune response to Mycobacterium bovis bacille Calmette Guérin infection in major histocompatibility complex class I- and II-deficient knock-out mice: contribution of CD4 and CD8 T cells to acquired resistance.

Knock-out mice with defined major histocompatibility complex (MHC) deficiencies were infected intravenously with Mycobacterium bovis bacille Calmette Guérin (M. bovis BCG) to assess the relative impact of MHC class I- and II-dependent immune responses. Heterozygous control mice were capable of controlling growth of M. bovis BCG, although infection progressed chronically, as assessed by determination of colony-forming units. Furthermore, infected controls developed granulomatous lesions at the site of mycobacterial growth and delayed-type hypersensitivity (DTH) reactions after challenge with purified protein derivative of tuberculin. In vitro, spleen cells from heterozygous control mice produced high concentrations of interferon-gamma (IFN-gamma) after restimulation with mycobacterial antigens. In contrast, the MHC class II-deficient A beta-/- mice, which are virtually devoid of functional CD4 T cells, succumbed to M. bovis BCG infection. Furthermore, A beta-/- mice lacked DTH reactions to tuberculin and only few minute picnotic lesions were formed in livers of infected mice. Finally, spleen cells from infected A beta-/- mice failed to produce measurable IFN-gamma concentrations after restimulation in vitro with various mycobacterial antigen preparations. The capacity of beta 2-microglobulin (beta 2m)-deficient mice, which are devoid of CD8 alpha/beta T cells, to inhibit growth of M. bovis BCG was only slightly affected at low inocula, although significantly higher colony-forming units were detected in spleens. These knock-out mice developed strong DTH responses to tuberculin and their spleen cells produced high levels of IFN-gamma once reactivated by mycobacterial antigens. Furthermore, in livers of infected beta 2m-deficient mice, extravascular infiltrates developed which were more diffuse than those in infected control littermates. Remarkably, the beta 2m-deficient mice were substantially more susceptible to higher inocula of M. bovis BCG than their control littermates. Our data formally prove the essential role of MHC class II-dependent immune mechanisms in all relevant aspects of immunity to M. bovis BCG. In addition, our findings emphasize an important contribution of MHC class I-dependent immunity to effective anti-mycobacterial protection. We assume that CD4 T cells are highly effective in containing M. bovis BCG within distinct granulomatous lesions, but fail to eradicate their intracellular pathogens. It appears most likely that CD8 T cells are also required to achieve this goal.

T cells and cytokines in intracellular bacterial infections: experiences with Mycobacterium bovis BCG.

Intracellular bacteria reside in mononuclear phagocytes, and protective immunity is dominated by T lymphocytes. Mycobacterium bovis bacillus Calmette-Guéin (BCG) infection of mice represents an excellent model for studying immune mechanisms involved in defence against persistent intracellular bacteria that cause chronic disease. Gene disruption mutant mice include: A beta-/-, which lack conventional CD4+ T cell receptor alpha/beta (TCR alpha/beta) T lymphocytes; beta 2 microglobulin -/-, which lack conventional CD8+ TCR alpha/beta lymphocytes; TCR beta-/-, which lack all TCR alpha/beta lymphocytes; TCR delta-/-, which lack all TCR gamma/delta lymphocytes; and RAG-1-/- mutants, which lack mature T and B lymphocytes. Studies of these mutants suggest that CD4+ TCR alpha/beta, CD8+ TCR alpha/beta and TCR gamma/delta T lymphocytes all contribute to immunity against M. bovis BCG. Activation of antibacterial effector functions in macrophages by T helper 1 (Th1) cell-derived gamma-interferon (IFN-gamma) is central to protection. In contrast, Th2 cells are only marginally involved. Activation of Th1 and Th2 cells is regulated by interleukin 10 (IL-10) and IL-12, which are induced early in infection with M. bovis BCG. Although IL-12 is stimulated by M. bovis BCG in immunocompetent mice, studies with IFN-gamma receptor-deficient and tumour necrosis factor alpha (TNF-alpha) receptor-deficient mutant mice suggest that M. bovis BCG-induced IL-12 secretion depends on IFN-gamma and TNF-alpha. Hence, IL-12 cannot be the first cytokine produced during M. bovis BCG infection.