Dexamethasone impairs the expression of antimicrobial mediators in lipopolysaccharide-activated primary macrophages by inhibiting both expression and function of interferon ß.

Glucocorticoids potently inhibit expression of many inflammatory mediators, and have been widely used to treat both acute and chronic inflammatory diseases for more than seventy years. However, they can have several unwanted effects, amongst which immunosuppression is one of the most common. Here we used microarrays and proteomic approaches to characterise the effect of dexamethasone (a synthetic glucocorticoid) on the responses of primary mouse macrophages to a potent pro-inflammatory agonist, lipopolysaccharide (LPS). Gene ontology analysis revealed that dexamethasone strongly impaired the lipopolysaccharide-induced antimicrobial response, which is thought to be driven by an autocrine feedback loop involving the type I interferon IFNß. Indeed, dexamethasone strongly and dose-dependently inhibited the expression of IFNß by LPS-activated macrophages. Unbiased proteomic data also revealed an inhibitory effect of dexamethasone on the IFNß-dependent program of gene expression, with strong down-regulation of several interferon-induced antimicrobial factors. Surprisingly, dexamethasone also inhibited the expression of several antimicrobial genes in response to direct stimulation of macrophages with IFNß. We tested a number of hypotheses based on previous publications, but found that no single mechanism could account for more than a small fraction of the broad suppressive impact of dexamethasone on macrophage type I interferon signaling, underlining the complexity of this pathway. Preliminary experiments indicated that dexamethasone exerted similar inhibitory effects on primary human monocyte-derived or alveolar macrophages.

Defective T-cell response to COVID-19 vaccination in acute myeloid leukaemia and myelodysplastic syndromes.

Limited data exist on COVID-19 vaccination efficacy in patients with acute myeloid leukemia and myelodysplasia with excess blasts (AML/MDS-EB2). We report results from a prospective study, PACE (Patients with AML and COVID-19 Epidemiology). 93 patients provided samples post-vaccine 2 or 3 (PV2, PV3). Antibodies against SARS-COV-2 spike antigen were detectable in all samples. Neutralization of the omicron variant was poorer than ancestral variants but improved PV3. In contrast, adequate T-cell reactivity to SARS-COV-2 spike protein was seen in only 16/47 (34%) patients PV2 and 23/52 (44%) PV3. Using regression models, disease response (not in CR/Cri), and increasing age predicted poor T cell response.

An Inflammatory Reaction to Stored Fascia Lata 37 Years Postimplantation.

We report a rare case of a suspected inflammatory reaction to stored fascia lata 37 years post-placement. Clinical, imaging, histopathological, and immunohistochemical findings are presented, with a literature review on reactions to stored fascia lata. A 39-year-old woman had upper eyelid congenital ptosis repaired successfully at 2 years with bilateral frontalis suspension procedures using stored fascia lata. Thirty-seven years later, the patient presented with swelling of her eyelids and forehead, which was tender to the touch, in the same pattern as the fascia lata slings placed earlier. Histopathological examination disclosed a non-necrotizing granulomatous inflammatory infiltrate with numerous asteroid bodies. Initially, it was responsive to oral prednisone, but with recurrent inflammation, long-term methotrexate was required to control the inflammation. To our knowledge, this type of delayed inflammatory reaction has not been previously reported. It raises a concern about the use of allogeneic donor tissue and accepted sterilization techniques that may not be 100% effective in deactivating all components of the donor graft, including potential infectious pathogens, leading to a subsequent latent reaction.

Ex vivo modelling of PD-1/PD-L1 immune checkpoint blockade under acute, chronic, and exhaustion-like conditions of T-cell stimulation.

Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1-revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1-to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.

Design, synthesis and evaluation of tryptophan analogues as tool compounds to study IDO1 activity.

The metabolism of l-tryptophan to N-formyl-l-kynurenine by indoleamine-2,3-dioxygenase 1 (IDO1) is thought to play a critical role in tumour-mediated immune suppression. Whilst there has been significant progress in elucidating the overall enzymatic mechanism of IDO1 and related enzymes, key aspects of the catalytic cycle remain poorly understood. Here we report the design, synthesis and biological evaluation of a series of tryptophan analogues which have the potential to intercept putative intermediates in the metabolism of 1 by IDO1. Functionally-relevant binding to IDO1 was demonstrated through enzymatic inhibition, however no IDO1-mediated metabolism of these compounds was observed. Subsequent T m-shift analysis shows the most active compound, 17, exhibits a distinct profile from known competitive IDO1 inhibitors, with docking studies supporting the hypothesis that 17 may bind at the recently-discovered Si site. These findings provide a start-point for development of further mechanistic probes and more potent tryptophan-based IDO1 inhibitors.

Primary ductal adenocarcinoma of the lacrimal gland: A review and report of five cases.

Primary ductal adenocarcinoma (PDA) is a rare epithelial tumor of the lacrimal gland. Herein we report 5 cases and review 29 published cases of PDA of the lacrimal gland. Among these 5 cases, the most common clinical presentation was painless swelling and/or proptosis of their eye. The size of the lesions ranged from 1.6 to 2.5 cm. Histopathologic examination revealed proliferations of ductal or gland-like cells with vesiculated pleomorphic nuclei and prominent nucleoli. Tumor cells stained positive for epithelial and apocrine differentiation markers. Immunohistochemistry for human epidermal growth factor 2 was positive in 2 of the 4 cases. Four of the five patients were alive at the last follow-up visit. One died with bone metastases, which were diagnosed 25 months after exenteration and then survived an additional 51 months. On reviewing of twenty-nine previously published cases of PDA, the mean age of diagnosis was 58 years, with a male predominance (75%). Fifteen patients (54%) had distant metastases, 1 (4%) had local recurrence, and 10 (37%) suffered from a PDA-related death. PDA is a high-grade aggressive epithelial tumor of the lacrimal gland. Although rare, awareness and recognition of this malignancy are important to help determine prognosis and treatment options.

Immunohistochemical analysis of benign and malignant melanocytic lesions of the conjunctiva using double-staining.

OBJECTIVE: To implement a double-staining technique to identify the most sensitive and specific combinations of melanoma antigen recognized by T cells (Melan-A), microphthalmia-associated transcription factor (MITF), human melanoma black 45 (HMB45), and Ki67 aiming to assist in the diagnosis of atypical melanocytic conjunctival lesions that are more prone to malignant progression. METHODS: Eight specimens of conjunctival melanoma and of primary acquired melanosis with moderate to severe atypia were double-immunostained with a combination of a cytoplasmic marker (anti-Melan-A or anti-HMB45), and a nuclear marker (anti-MITF or anti-Ki67). Eight specimens of normal conjunctiva and of conjunctival nevi served as controls. The specimens were processed using 3,3-diaminobenzidine substrate for nuclear stains and the fast-red substrate for cytoplasmic stains. Each slide was analyzed by light microscopy and provided a percent scale and a 0 to 4+ score for each nuclear and cytoplasmic component. RESULTS: Melan-A and MITF were strongly positive markers for all melanocytic cells, whereas Ki67 and HMB45 provided a variable response for identifying potentially proliferative or aggressive cells. HMB45 and MITF proved to be the best combination for differentiating between atypical and benign lesions on a percent scale and a 0 to 4+ scale (p = 0.0004), with the 3 other combinations providing mainly confirmatory diagnostic information (p < 0.05). CONCLUSIONS: Our study used an immunohistochemical double-staining approach to differentiate between atypical and benign melanocytic lesions of the conjunctiva. Our findings should aid in a more complete immunohistopathological diagnosis of conjunctival melanocytic lesions, particularly in diagnostically difficult cases.

Regression of Sebaceous Carcinoma of the Eyelid after a Small Incisional Biopsy: Report of Two Cases.

PURPOSE: To report 2 cases of regression of sebaceous carcinoma of the eyelid after a small incisional biopsy. METHODS: Clinical, imaging, and histopathological findings are presented, with a literature review on regressing ocular tumors. RESULTS: Our first patient was a 79-year-old man who presented with a 10-month history of progressive left upper eyelid ptosis caused by an eyelid tumor with orbital involvement and confirmed on magnetic resonance imaging. Our second patient was a 70-year-old woman who presented with ptosis with a left upper eyelid mass. Both patients underwent a small incisional biopsy of their lesion. The histopathological diagnoses in both cases were consistent with sebaceous carcinoma. Both patients refused exenteration. Follow-up clinical examination and imaging disclosed total regression of the ptosis and of the neoplasm with no sign of recurrence in both patients over a 4-year period for Case 1 and a 7-year period for Case 2. CONCLUSION: Regression following incisional biopsy of basal cell, squamous cell, and Merkel cell carcinoma, including of the eyelid, is well documented. To the best of our knowledge, our 2 cases of sebaceous carcinoma are the first to be reported with total involution clinically and on imaging of the tumor following partial incisional biopsy.

Pilomatrixoma of the ocular adnexa: report of 3 cases with variations in the histopathological findings.

OBJECTIVE: To report the clinical and variations in the histopathological features of pilomatrixoma of the ocular adnexa in 3 young individuals. DESIGN: A retrospective case series was performed with clinical, histological, and immunohistochemical analysis. PARTICIPANTS: Case 1 is an 18-year-old male who presented with a reddish-blue swelling under the left eyebrow. The lesion measured 2 × 1 cm. Case 2 is a 2-year-old female who presented with a reddish-blue nodule inferior to the right eyebrow with telangiectatic vessels. The lesion measured 6 × 4 × 4 mm. Case 3 is a 14-year-old female who presented with a subcutaneous lesion under the right upper eyebrow with fluctuating inflammation. The lesion measured 12 × 3 × 2 mm. Histopathological examination of case 1 disclosed peripheral basaloid cells and central shadow cells containing calcific foci, separated by a transition zone. In case 2, histopathological analysis revealed central calcific foci in islands of shadow cells with more peripheral basaloid cells. In case 3, we observed numerous clusters of shadow cells with focal calcifications, as well as basaloid cells in a disorganized configuration. CONCLUSION: Pilomatrixoma is an uncommon benign skin neoplasm originating from the matrix of the hair root. We describe a spectrum of histopathological findings in pilomatrixoma of the ocular adnexal in 3 young individuals.

Histopathological Study on the Proposed Pathogenesis of Intratarsal Keratinous Cysts.

PURPOSE: Intratarsal keratinous cysts (IKCs) are a recently described entity that is frequently misdiagnosed clinically as chalazia and mislabeled in the literature as "intratarsal epidermal inclusion cysts" or "epidermoid cysts." It is important to accurately diagnose IKCs and distinguish them from chalazia because IKCs require a complete surgical excision and can exhibit multiple recurrences following curettage. The authors performed a retrospective case series to further elucidate the pathogenesis of IKCs and to determine the diagnostically optimal panel of stains for diagnosis. METHODS: A study group of 8 specimens of IKCs and control specimens of epidermal inclusion cysts were obtained from their pathology laboratories. The authors compared the histological and immunohistochemical profile of IKCs and epidermal inclusion cysts by staining sections from each specimen with hematoxylin and eosin, periodic acid-Schiff, Masson trichrome, cytokeratin 5, cytokeratin 17, carcinoembryonic antigen, and epithelial membrane antigen. The immunoreactivity data were then analyzed using a 2-tailed Mann-Whitney test, assuming a nonparametric population (p < 0.05 is significant). RESULTS: Histopathologically, IKCs are embedded in the tarsus lined by stratified squamous epithelium with an inner undulating cuticle filled with a compact keratinous-appearing material. The authors demonstrate that IKCs develop progressively from dilated meibomian ducts to the formation of complete cysts with their markers. The most valuable immunochemical stains to diagnose IKC were cytokeratin 17, carcinoembryonic antigen, and epithelial membrane antigen (p < 0.05 with each). CONCLUSIONS: These findings provide a better understanding of the pathogenesis and the immunohistochemical findings of this relatively new entity allowing for more appropriate diagnosis of IKCs aiming to reduce future complications from their management.

Benign Solitary Fibrous Histiocytoma of Xanthomatous Subtype of the Perilimbal Conjunctiva and Adjacent Sclera in a Youth.

AIMS: To report the clinical and pathological features of a benign fibrous histiocytoma of the xanthomatous subtype in the perilimbal conjunctiva and adjacent sclera in a youth. METHODS: An 11-year-old Caucasian boy presented with a yellowish dome-shaped conjunctival mass abutting the inferotemporal limbus of the left eye. The tumor measured 4 mm in its maximum diameter. The lesion was excised and was noted to extend into the sclera. Three years postoperatively, there was no evidence of recurrence. RESULTS: Histopathological examination disclosed a highly cellular lesion composed predominantly of benign-appearing foamy histiocytes without multinucleated giant cells. The tumor contained sparse fibrous septa without a storiform configuration. Immunohistochemical analysis showed diffuse cytoplasmic positive staining with adipophilin, CD34, and CD163. CONCLUSION: A lesion comprised of numerous homogeneous foamy histiocytes without multinucleated giant cells in the perilimbal conjunctiva and adjacent sclera of a youth is presented as a unique case of a rare variant of fibrous histiocytoma of the xanthomatous subtype.

Langerhans cell histiocytosis in an 18-month-old child presenting as periorbital cellulitis.

Langerhans cell histiocytosis (LCH) is a rare multi-system disease. It presents infrequently as a childhood orbital tumor, and can mimic more common inflammatory orbital disease processes. We report the clinical, histopathological, and electron microscopic findings of orbital LCH in an 18-month-old child, along with a review of the recent literature regarding molecular pathogenetic analysis of LCH. The child presented with a two-week history of progressive left periorbital edema and redness. He was initially diagnosed and treated empirically for bacterial periorbital cellulitis, but subsequently underwent ophthalmological consultation after he failed to improve. Histopathological examination of an orbital biopsy specimen revealed numerous Langerhans-type cells, which stain positive for CD1A and CD207 (langerin). Electron microscopic examination demonstrated characteristic Birbeck granules within the Langerhans-type cells. Three year follow-up did not demonstrate recurrence or disease progression.

Gestational diabetes and the long-term risk of cataract surgery: A longitudinal cohort study.

AIMS: We assessed the long-term risk of cataract following a pregnancy complicated by gestational diabetes. METHODS: We carried out a longitudinal cohort study of 1,108,541 women who delivered infants between 1989-2013 in Quebec, Canada, with follow-up extending up to 25years later. The cohort included 71,862 women with gestational diabetes and 5247 with cataracts. We used Cox regression models to estimate hazard ratios (HR) and 95% confidence intervals (CI) for the association of gestational diabetes with subsequent risk of cataract, adjusted for age, parity, socioeconomic status, time period, comorbidity, and type 2 diabetes. RESULTS: Women with gestational diabetes had an elevated incidence of cataract (22.6 per 1000) compared with no gestational diabetes (15.1 per 1000), with 1.15 times the risk (95% CI 1.04-1.28). Women with gestational diabetes who subsequently developed type 2 diabetes had a higher risk of cataract compared with no gestational and type 2 diabetes (HR 3.62, 95% CI 3.01-4.35), but women with gestational diabetes who did not develop type 2 diabetes continued to be at risk (HR 1.12, 95% CI 1.00-1.25). CONCLUSIONS: Gestational diabetes may be an independent risk factor for cataract later in life, although risks are greatest for women who subsequently develop type 2 diabetes.

Macrophage responses to lipopolysaccharide are modulated by a feedback loop involving prostaglandin E2, dual specificity phosphatase 1 and tristetraprolin.

In many different cell types, pro-inflammatory agonists induce the expression of cyclooxygenase 2 (COX-2), an enzyme that catalyzes rate-limiting steps in the conversion of arachidonic acid to a variety of lipid signaling molecules, including prostaglandin E2 (PGE2). PGE2 has key roles in many early inflammatory events, such as the changes of vascular function that promote or facilitate leukocyte recruitment to sites of inflammation. Depending on context, it also exerts many important anti-inflammatory effects, for example increasing the expression of the anti-inflammatory cytokine interleukin 10 (IL-10), and decreasing that of the pro-inflammatory cytokine tumor necrosis factor (TNF). The tight control of both biosynthesis of, and cellular responses to, PGE2 are critical for the precise orchestration of the initiation and resolution of inflammatory responses. Here we describe evidence of a negative feedback loop, in which PGE2 augments the expression of dual specificity phosphatase 1, impairs the activity of mitogen-activated protein kinase p38, increases the activity of the mRNA-destabilizing factor tristetraprolin, and thereby inhibits the expression of COX-2. The same feedback mechanism contributes to PGE2-mediated suppression of TNF release. Engagement of the DUSP1-TTP regulatory axis by PGE2 is likely to contribute to the switch between initiation and resolution phases of inflammation.

Gain-of-Function Mutation of Tristetraprolin Impairs Negative Feedback Control of Macrophages In Vitro yet Has Overwhelmingly Anti-Inflammatory Consequences In Vivo.

The mRNA-destabilizing factor tristetraprolin (TTP) binds in a sequence-specific manner to the 3' untranslated regions of many proinflammatory mRNAs and recruits complexes of nucleases to promote rapid mRNA turnover. Mice lacking TTP develop a severe, spontaneous inflammatory syndrome characterized by the overexpression of tumor necrosis factor and other inflammatory mediators. However, TTP also employs the same mechanism to inhibit the expression of the potent anti-inflammatory cytokine interleukin 10 (IL-10). Perturbation of TTP function may therefore have mixed effects on inflammatory responses, either increasing or decreasing the expression of proinflammatory factors via direct or indirect mechanisms. We recently described a knock-in mouse strain in which the substitution of 2 amino acids of the endogenous TTP protein renders it constitutively active as an mRNA-destabilizing factor. Here we investigate the impact on the IL-10-mediated anti-inflammatory response. It is shown that the gain-of-function mutation of TTP impairs IL-10-mediated negative feedback control of macrophage function in vitro However, the in vivo effects of TTP mutation are uniformly anti-inflammatory despite the decreased expression of IL-10.

Dominant Suppression of Inflammation via Targeted Mutation of the mRNA Destabilizing Protein Tristetraprolin.

In myeloid cells, the mRNA-destabilizing protein tristetraprolin (TTP) is induced and extensively phosphorylated in response to LPS. To investigate the role of two specific phosphorylations, at serines 52 and 178, we created a mouse strain in which those residues were replaced by nonphosphorylatable alanine residues. The mutant form of TTP was constitutively degraded by the proteasome and therefore expressed at low levels, yet it functioned as a potent mRNA destabilizing factor and inhibitor of the expression of many inflammatory mediators. Mice expressing only the mutant form of TTP were healthy and fertile, and their systemic inflammatory responses to LPS were strongly attenuated. Adaptive immune responses and protection against infection by Salmonella typhimurium were spared. A single allele encoding the mutant form of TTP was sufficient for enhanced mRNA degradation and underexpression of inflammatory mediators. Therefore, the equilibrium between unphosphorylated and phosphorylated TTP is a critical determinant of the inflammatory response, and manipulation of this equilibrium may be a means of treating inflammatory pathologies.

Dual-Specificity Phosphatase 1 and Tristetraprolin Cooperate To Regulate Macrophage Responses to Lipopolysaccharide.

Dual-specificity phosphatase (DUSP) 1 dephosphorylates and inactivates members of the MAPK superfamily, in particular, JNKs, p38α, and p38ß MAPKs. It functions as an essential negative regulator of innate immune responses, hence disruption of the Dusp1 gene renders mice extremely sensitive to a wide variety of experimental inflammatory challenges. The principal mechanisms behind the overexpression of inflammatory mediators by Dusp1(-/-) cells are not known. In this study, we use a genetic approach to identify an important mechanism of action of DUSP1, involving the modulation of the activity of the mRNA-destabilizing protein tristetraprolin. This mechanism is key to the control of essential early mediators of inflammation, TNF, CXCL1, and CXCL2, as well as the anti-inflammatory cytokine IL-10. The same mechanism also contributes to the regulation of a large number of transcripts induced by treatment of macrophages with LPS. These findings demonstrate that modulation of the phosphorylation status of tristetraprolin is an important physiological mechanism by which innate immune responses can be controlled.