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.

Application of knockout mice to the experimental analysis of infections with bacteria and protozoa.

Knockout mice with distinct gene deletions are valuable tools for in vivo analyses of the immune response against infectious agents. Studies of bacterial and protozoal infections have shown that antimicrobial immunity is generally defective in knockout mice. At one extreme, deletion of just one gene may completely compromise resistance, while at the other extreme, redundancy in the immune system allows partial compensation for the deleted part.

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.

Localisation of human peripheral blood leukocytes after transfer to C.B-17 scid/scid mice.

Severe combined immunodeficient (scid) mice of the inbred strain C.B-17 lack functional T and B cells and, because of this, they tolerate xenografts. We reconstituted scid mice with human peripheral blood leukocytes (PBL) by i.p. injection. In order to determine the human PBL in lymphoid organs of these reconstituted scid mice, we labelled the human PBL prior to transfer with the fluorescent dye PKH 26-GL. With this experimental approach it was possible to detect the human cells in lymphoid organs and peritoneal exudate of the reconstituted scid mice by cytofluorimetrical and histological methods. This method is thus helpful for the determination of xenografts transplanted upon scid mice as well as in other experimental settings including adoptive transfer.

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.

Role of T cell subsets in immunity against intracellular bacteria: experimental infections of knock-out mice with Listeria monocytogenes and Mycobacterium bovis BCG.

The generation of knock-out mice with targeted gene deletions has already proven its enormous value for our understanding of the antimicrobial immune response. Here, we describe studies with knock-out mice deficient in the TCR-beta gene, lacking alpha/beta T cells; in the TCR-delta gene, lacking gamma/delta T cells; in the beta 2m gene, lacking beta 2-microglobulin, and hence cell surface expressed MHC class I and functional CD8 T cells; and in the H-2I-A beta gene, lacking cell surface expressed MHC class II and hence functional CD4 T cells. These mice were infected with Listeria monocytogenes or Mycobacterium bovis BCG as representative microbes which primarily activate CD8 T cells or CD4 T cells, respectively. Data described in this treatise demonstrate that the different gene deletions had an impact of varying degree on antibacterial defense and on the formation of granulomatous lesions. At the same time, the data point to a compensatory potential of the incomplete immune system. We assume that deletions in the major immune effector cells promote the emergence of a second line of defenders which frequently remain silent in the normal immune system. Thus, our data illustrate an enormous redundancy of the immune system, which, however, is not abundant since it takes over essential functions in the immunodeficient situation.

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.

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.

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.

[Histological and cytofluorometric investigations of immunodeficient CB17 scid/scid mice]. / Histologische und zytofluorometrische Untersuchungen von immundefekten Mäusen des Stammes CB17 scid/scid.

Blood cell differentiation and development is under the control of a number of genes, and mutations in one of these genes could result in an immunodefective phenotype. Mice of the CB17 scid/scid ("severe combined imunodeficient") strain are characterized by a mutation which affects early lymphoid differentiation and lack functional T and B lymphocytes. We investigated the serum level of murine immunoglobulins of CB17 scid/scid mice in comparison with the wild type CB17. Furthermore we performed FACS-analysis of the peripheral blood cells as well as cells from the spleen and thymus of these animals. Histological methods were used to study the morphology of the lymphoid tissues. In the sera of CB17 scid/scid mice murine immunoglobuline levels below 50 ng/ml were found. Mice with a level between this and the normal level (5-10 mg/ml) were called "leakys". Cytofluorometric as well as histological investigations revealed no differences between scid and leaky mice, which both appeared quite different than immunocompetent CB17 (wildtype). On the other hand animals with lymphomas show mononuclear infiltrations in the lymphoid tissues. Inflammatory reactions in scid are characterized by leukocytic infiltrations in the draining lymphnodes.

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.

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.

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.

Studies with MHC-deficient knock-out mice reveal impact of both MHC I- and MHC II-dependent T cell responses on Listeria monocytogenes infection.

Mutant mice with a defined genetic defect in the beta 2-microglobulin (beta 2m) or the H2-I-A beta chain, which are virtually devoid of functional CD8 or CD4 alpha beta T cells, respectively, were employed for analyzing immune mechanisms involved in acquired resistance against Listeria monocytogenes. Although the lethal dose of L. monocytogenes was markedly lower for either mouse mutant as compared with their heterozygous control littermates, both beta m -/- and A beta -/- mutants were able to resolve low dose infection. However, in both mouse mutants, the course of disease was exacerbated and clearance was markedly delayed. Vaccine induced immunity against a secondary high dose infection lethal for naive animals was also impaired in beta 2m -/- and A beta -/- mice. However, both mutant mice were still capable of controlling secondary infection. Based on numbers of L. monocytogenes organisms in spleens, beta 2m -/- mutants suffered more dramatically from primary and secondary infection than A beta -/- mice. Ag-induced IFN-gamma secretion was impaired during the early phase of infection in beta 2m -/- mice and at later stages in A beta -/- mice. Modulation of gamma delta T cells by mAb treatment led to significant increase in bacterial load of spleens in both beta 2m -/- and A beta -/- mice. Finally, the development of granulomatous lesions was markedly affected in both mutants. In beta 2m -/- mutants, infiltrative lesions appeared and in A beta -/- mice few inflammatory islets with necrotic centers developed. These data demonstrate the importance of both MHC I- and MHC II-dependent immune mechanisms in acquired resistance to L. monocytogenes and point to the necessity of a coordinated interaction between CD8 and CD4 alpha beta T cells (and probably gamma delta T cells) in anti-L. monocytogenes resistance.

Antigen-specific T-cell responses during primary and secondary Listeria monocytogenes infection.

Although murine listeriosis is a widely used experimental model for the analysis of cell-mediated immunity, there is little information about individual T-cell antigens of Listeria monocytogenes which are recognized during primary and secondary infection. To study the antigen responses of L. monocytogenes-reactive T cells, somatic and secreted listerial proteins were separated by two-dimensional gel electrophoresis and subsequently divided into 480 liquid fractions. Antigen-specific T cells isolated from mice at different times of primary and secondary listeriosis were tested for their capacity to proliferate with distinct protein fractions. Supernatants of these cultures were screened for the production of gamma interferon, interleukin-4 (IL-4), and IL-10. Proliferation of antigen-specific T cells correlated with the production of high concentrations of gamma interferon, whereas IL-4 and IL-10 production in response to listerial protein fractions could not be detected. During both primary and secondary listeriosis, T cells recognized a multitude of somatic and secreted proteins rather than one or a few dominant antigens. Secreted proteins were recognized before somatic proteins, and T cells recognized different fractions in secreted and somatic proteins.

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.