Thabo Lapp

TLR-mediated inflammatory responses to Streptococcus pneumoniae are highly dependent on surface expression of bacterial lipoproteins.

Streptococcus pneumoniae infections induce inflammatory responses that contribute toward both disease pathogenesis and immunity, but the host–pathogen interactions that mediate these effects are poorly defined. We used the surface lipoprotein-deficient ∆lgt pneumococcal mutant strain to test the hypothesis that lipoproteins are key determinants of TLR-mediated immune responses to S. pneumoniae. We show using reporter assays that TLR2 signaling is dependent on pneumococcal lipoproteins, and that macrophage NF-κB activation and TNF-α release were reduced in response to the ∆lgt strain. Differences in TNF-α responses between Δlgt and wild-type bacteria were abrogated for macrophages from TLR2- but not TLR4-deficient mice. Transcriptional profiling of human macrophages revealed attenuated TLR2-associated responses to ∆lgt S. pneumoniae, comprising many NF-κB–regulated proinflammatory cytokine and chemokine genes. Importantly, non-TLR2–associated responses were preserved. Experiments using leukocytes from IL-1R–associated kinase-4–deficient patients and a mouse pneumonia model confirmed that proinflammatory responses were lipoprotein dependent. Our data suggest that leukocyte responses to bacterial lipoproteins are required for TLR2- and IL-1R–associated kinase-4–mediated inflammatory responses to S. pneumoniae.

Identification of Therapeutic Targets of Inflammatory Monocyte Recruitment to Modulate the Allogeneic Injury to Donor Cornea

PURPOSE We sought to test the hypothesis that monocytes contribute to the immunopathogenesis of corneal allograft rejection and identify therapeutic targets to inhibit monocyte recruitment. METHODS Monocytes and proinflammatory mediators within anterior chamber samples during corneal graft rejection were quantified by flow cytometry and multiplex protein assays. Lipopolysaccharide or IFN-γ stimulation of monocyte-derived macrophages (MDMs) was used to generate inflammatory conditioned media (CoM). Corneal endothelial viability was tested by nuclear counting, connexin 43, and propidium iodide staining. Chemokine and chemokine receptor expression in monocytes and MDMs was assessed in microarray transcriptomic data. The role of chemokine pathways in monocyte migration across microvascular endothelium was tested in vitro by chemokine depletion or chemokine receptor inhibitors. RESULTS Inflammatory monocytes were significantly enriched in anterior chamber samples within 1 week of the onset of symptoms of corneal graft rejection. The MDM inflammatory CoM was cytopathic to transformed human corneal endothelia. This effect was also evident in endothelium of excised human cornea and increased in the presence of monocytes. Gene expression microarrays identified monocyte chemokine receptors and cognate chemokines in MDM inflammatory responses, which were also enriched in anterior chamber samples. Depletion of selected chemokines in MDM inflammatory CoM had no effect on monocyte transmigration across an endothelial blood-eye barrier, but selective chemokine receptor inhibition reduced monocyte recruitment significantly. CONCLUSIONS We propose a role for inflammatory monocytes in endothelial cytotoxicity in corneal graft rejection. Therefore, targeting monocyte recruitment offers a putative novel strategy to reduce donor endothelial cell injury in survival of human corneal allografts.

Analysis of Filaggrin Mutations and Expression in Corneal Specimens from Patients with or without Atopic Dermatitis

Background: Filaggrin is expressed in the epidermis and is essential for the maintenance of the epidermal barrier. Null mutations within the filaggrin gene (FLG) lead to a disturbed epidermal barrier and are associated with a significantly increased risk of atopic dermatitis (AD). The association of AD with ocular surface disorders prompted us to speculate that common FLG mutations may be particularly prevalent in AD patients with ocular comorbidities. Methods: Corneal buttons and biopsies from AD patients with ocular involvement (n = 11) and from non-atopic patients (n = 9) with a histological diagnosis of keratitis were included in the study. DNA samples obtained from paraffin-embedded corneal specimens were genotyped for the two most common FLG mutations (R501X and 2282del4). Filaggrin protein expression was analysed by immunohistochemistry. Results: Normal skin and corneal specimens (n = 6) were positive for filaggrin, which could be detected in the stratum corneum of the skin and in the basal epithelial layer of the cornea. Interestingly, all AD corneal specimens as well as the specimens from keratitis patients without AD were negative for filaggrin expression. Genotyping of the FLG mutations R501X and 2282del4 revealed wild-type alleles in all analysed samples. Conclusions: The lack of filaggrin expression observed in the analysed corneal specimens from AD patients is not due to the two most common FLG mutations (R501X, 2282del4) but is most likely secondary to inflammation, as all keratitis specimens of non-AD patients showed lack of filaggrin expression as well.

Severe bleeding eyelid after trivial trauma: conjunctival metastasis of a renal cell carcinoma

Dear Editor, A 70-year-old woman was referred to our clinic with a persistent bleeding of the eye following a minor injury, having slipped and bumped her head against a bedside table that morning. The right upper eyelid contained a subtarsal lesion and a hyposphagma in the temporal superior quadrant of her right eye. There was no evidence of a penetrating eye injury. Due to a permanent conjunctival hemorrhage, inspection of the wound was difficult. As the source of bleeding could not be identified using the slit-lamp, surgical exploration and wound care were planned. The wound exploration and haemostasis were performed as an emergency procedure. Next to the wound, a whitish gelatinous nodule could be identified as the source of the bleeding (Fig. 1a). An excisional biopsy of this nodule was taken and the bleeding was stopped with an electrosurgical cauter. The patient’s medical history revealed a renal cell carcinoma (initial pT3, pNx cM0 G2 R0 L0 V1). A nephrectomy on the left side had been performed in July 2006 as well as consecutive resections of metastases of the diaphragm, the right lung, and the right breast in November 2007. In September 2008, partial resection of the 6th rib was performed, as well as a resection of a second metastasis of the right lung. Since January 2009, a further 20 smaller metastases of the lungs had been removed, and in February 2009 the patient presented with cerebral metastases. These were treated with a radiosurgical procedure in April 2009. The patient is still alive and in moderate condition. Histology of the conjunctival specimen showed a low differentiated malignant epithelial tumor (Fig. 1b). There were only a few small vessels supplying the tumor. Immunohistochemistry was positive for pancytokeratine AE1/AE3 (Fig. 1c) and CD10 (Fig. 1d). Examination of the primary tumor showed identical histological and immunohistochemical findings. In summary, the histopathological findings confirmed the nodule as a conjunctival metastasis of the known renal cell carcinoma. In 2004, Shields and co-worker analyzed a series of 1,643 conjunctival tumors [1]. In this large cohort they found only 13 metastases, i.e., metastases accounted for less than 1% of all conjunctival tumors. Five of these 13 metastases were associated with breast cancer, three with pulmonary cancer, two with a malignant melanoma of the skin, one with a carcinoid, one with a laryngeal carcinoma, and one metastasis of unknown origin. These data illustrate the rarity of conjunctival metastases in comparison to primary conjunctival tumors. However, renal cell carcinomas (RCC) represent approximately only 1–3% of all adult visceral cancers [2]. One of the common characteristics of RCCs is their tendency to metastasize without showing early clinical symptoms. The most common locations of metastases are the lungs (>50%) and the bones (up to 30%), followed by regional lymph nodes, the liver, and the brain [3]. Only in very rare cases do RCCs affect the eye. In a T. Lapp (*) Division of Infection and Immunity, University College London, The Cruciform Building, Gower Street, London WC1E 6BT, UK e-mail: T.Lapp@ucl.ac.uk

Collagen Characteristics and Refractive Outcomes

High satisfaction after refractive surgery fundamentally depends on the long-term refractive stability. Several factors have an influence on postoperative refractive results, i.e. long-term stability of preoperative refraction, preoperative difference between subjective and objective refractive measurements. Individual tissue characteristics might have an influence on postoperative results as well. However, there are no options up to now to evaluate tissue parameters preoperatively.

Humanes Leukozytenantigensystem in der Augenheilkunde

Various inflammatory and non-inflammatory eye diseases are associated with specific HLA isotypes. Therefore, HLA isotyping can be a useful diagnostic tool for these diseases and has already been shown to reduce the rejection rate of corneal allografts. Unfortunately, the volume of published data and the varying quality of these publications complicate obtaining good overview in this field. This review briefly summarizes the genetic structure of the HLA system and elucidates differences between HLA classes I and II in the context of antigen presentation. Possible mechanisms of HLA associations in the field of ophthalmology are discussed, and finally different tools (e.g. genome wide association studies) for assessing associations of HLA isotypes with different ocular diseases are examined.

Altes Immunsystem – Neue Erkenntnisse?

Mononuclear phagocytes are derived from bone marrow precursor cells and are part of the innate immune system. These cells circulate in the blood as monocytes but differentiate in the peripheral circulation into tissue macrophages and dendritic cells under the influence of various cytokines. In addition to antimicrobial properties, macrophages also participate in wound healing; however, they also support degenerative and inflammatory processes. In cases of acute corneal allograft rejection, mononuclear cells initially form the main component of the cellular anterior chamber infiltrate. How monocytes are recruited into the anterior chamber is currently uncertain. Furthermore, no information is available about the possible cytotoxic effects on corneal endothelial cells. Gaining insight into these mechanisms may lead to potential pharmacological interventions.ZusammenfassungZellen des mononukleären phagozytären Systems entstammen Vorläuferzellen des Knochenmarks und sind ein Teil des angeborenen Immunsystems. Während sie im Blut noch als Monozyten zirkulieren, differenzieren sie sich in der Peripherie unter dem Einfluss verschiedener Zytokine in gewebeständige Makrophagen und dendritische Zellen. Neben antimikrobiellen Fähigkeiten sind Makrophagen auch an Wundheilungsprozessen beteiligt. Sie unterhalten allerdings auch degenerative und inflammatorische Prozesse. Im Fall einer akuten allogenen Hornhauttransplantatabstoßung bilden mononukleäre Zellen den Hauptteil des zellulären Vorderkammerinfiltrates. Hinsichtlich ihrer Funktion bei kornealen Abstoßungsreaktionen, der verschiedenen Wege ihrer Rekrutierung in die Vorderkammer, evtl. zytotoxischer Effekte auf korneale Endothelzellen sowie pharmakologischer Interventionsmöglichkeiten ist der derzeitige Kenntnisstand unzureichend.AbstractMononuclear phagocytes are derived from bone marrow precursor cells and are part of the innate immune system. These cells circulate in the blood as monocytes but differentiate in the peripheral circulation into tissue macrophages and dendritic cells under the influence of various cytokines. In addition to antimicrobial properties, macrophages also participate in wound healing; however, they also support degenerative and inflammatory processes. In cases of acute corneal allograft rejection, mononuclear cells initially form the main component of the cellular anterior chamber infiltrate. How monocytes are recruited into the anterior chamber is currently uncertain. Furthermore, no information is available about the possible cytotoxic effects on corneal endothelial cells. Gaining insight into these mechanisms may lead to potential pharmacological interventions.

Activation of human macrophages by human corneal allogen in vitro

Purpose To study distinct aspects of human monocyte-derived macrophage (MDM) activation by human corneal tissue as a possible initial stage in human corneal allograft rejection. Methods Human monocytes were isolated from peripheral blood mononuclear cells (PBMC) and differentiated into MDM. Human corneas with or without endothelium were fragmented using a standardized protocol. MDM were stimulated with human corneal fragments, corneal fragment supernatant, lipopolysaccharide (LPS) or interferon-gamma (IFNγ), and expression profiles for 34 cytokines were determined in MDM-conditioned media using a Luminex bead-based multiplex assay. Data from clinical aqueous humour samples served for comparison and validation. To assess cell recruitment, immunogenicity of corneal endothelial cells (CEC), monocyte survival and differentiation, we applied transwell migration assays, cell viability assays and fluorescence-activated cell sorting, respectively. Results Corneal fragments induced MDM to release distinct cytokines into the medium. Media thus conditioned in vitro by stimulated MDM shared cytokine patterns, namely MCP-1, MIP-1α and MIP-1β, with human aqueous humor samples obtained in human corneal allograft rejection. The presence of CEC in tissue fragments used for MDM stimulation attenuated the upregulation of distinct pro-inflammatory chemokines, like MCP-3 and IL-8, reduced the monocyte survival time, and diminished monocyte-to-macrophage differentiation induced by conditioned media. Distinct anti-inflammatory cytokines, like IL-4 and IL-13, were upregulated in the presence of corneal endothelium. Cornea fragment-stimulated MDMs induced recruitment of monocytes from a PBMC pool in a transwell migration model, modulated immune cell viability and promoted further immune cell recruitment and differentiation. Conclusions Human macrophages respond to allogenic corneal tissue and generate an inflammatory milieu. This can drive further recruitment of immunocompetent cells and modulate cell survival and differentiation of the cells recruited. These observations are consistent with the hypothesis that macrophages play a significant role in the initiation of corneal transplant rejection. Our data also indicate that distinct aspects of early human corneal transplant rejection can be modelled in vitro.