Disruption of the CREBBP gene and decreased expression of CREB,NFκB p65, c-JUN,c-FOS,BCL2 and c-MYC suggest immune dysregulation

Genomic aberrations in theCREBBP (CREB-binding protein – CREBBP or CBP) gene such as point mutations, small insertions or exonic copy number changes are usually associated with Rubinstein-Taybi syndrome (RTs). In this study, the disruption of the CREBBP gene on chromosome 16p13.3, as revealed by CGH-array and FISH, suggests immune dysregulation in a patient with the Rubinstein Taybi syndrome (RTs) phenotype. Further investigation with Western blot techniques demonstrated decreased expression of CREB, NFκB, c-Jun, c-Fos, BCL2 and cMyc in peripheral blood mononuclear cells, thus indicating that the CREBBP gene is essential for the normal expression of these proteins and the regulation of immune responses.     

Thyroid fine‐needle aspiration cytology: Is there a place to virtual cytology?

Telecytology has been used for education, training, and consultation. Cytological studies from gynecological, nongynecological and fine‐needle aspiration cytology (FNAC) specimens (including studies of thyroid FNAC) analyzed the diagnostic accuracy and reproducibility of telecytology‐based predominantly on static digital images. The aim of this study was to evaluate the diagnostic reproducibility of virtual cytology by measuring intraobserver and interobserver agreements among two cytopathologists, using the Bethesda System for Reporting Thyroid Cytopathology (BSRTC) nomenclature. 502 glass slides from 222 cases of thyroid FNAC were retrieved and scanned by a high‐resolution scanner generating whole slides images (virtual cytology). Conventional and virtual cytology were analyzed by a skilled cytopathologist and the intraobserver agreement rate was 77.5% with the corresponding κ value of 0.54, suggesting a moderate agreement between both methods. A second cytopathologist analyzed the same slides only by virtual cytology and the interobserver agreement rate was 80.2% with the corresponding κ value of 0.57, suggesting a moderate agreement between both cytopathologists. The virtual cytology resulted in a higher proportion of aspirates classified as nondiagnostic (20.3 and 14.9% for the first and second cytopathologist, respectively) as compared to conventional cytology (8.1%). Regarding specific diagnostic categories as defined by the BSRTC nomenclature, the follicular lesion of undetermined significance category presented the lowest concordance rates, corresponding to 5.9% intraobserver agreement and no (0.0%) interobserver agreement. We suggest that virtual cytology can be an alternative to conventional cytology in assessment of thyroid FNAC specimens, but nondiagnostic aspirates obtained by virtual cytology should be reassessed by conventional cytology. Diagn. Cytopathol. 2013;41:793–798. © 2013 Wiley Periodicals, Inc.     

Presentation of a viral peptide assembled on the carbohydrate moieties of immunoglobulin does not require processing

We have previously demonstrated that an immunodominant CD4 T cell epitope, HA110-120 of the hemagglutinin (HA) of the A/PR/8/34 influenza virus, enzymatically assembled on the carbohydrate moieties of self immunoglobulins (Ig) primed the precursors of peptide-specific T cells and induced efficient proliferation in vivo of naive lymphocytes from transgenic mice expressing the peptide-specific T cell receptor. Here, we show that an immuno-galacto-peptide construct, IgG-gal-HA, does not require intracellular or extracellular processing to present the peptide to the specific T cells. The presentation occurs following the binding of the IgG-gal-HA construct to Fcγ receptor on the surface of antigen-presenting cells (APC), with concurrent interaction of the peptides to their neighboring major histocompatibility complex class II molecules. This mechanism of peptide presentation may harness the immune response in vivo by the engagement of APC with a low capacity of antigen processing, such as neonatal B cells. In addition, the enzymatic method of assembling various aminated compounds on the sugar moieties of Ig may offer novel perspectives on immunotargeting of antagonist peptides, cytostatic drugs, and biologically active ligands of therapeutic use.     

Biallelic variants in CENPF causing a phenotype distinct from Strømme syndrome

Biallelic loss-of-function (LoF) variants in CENPF gene are responsible for Strømme syndrome, a condition presenting with intestinal atresia, anterior ocular chamber anomalies, and microcephaly. Through an international collaboration, four individuals (three males and one female) carrying CENPF biallelic variants, including two missense variants in homozygous state and four LoF variants, were identified by exome sequencing. All individuals had variable degree of developmental delay/intellectual disability and microcephaly (ranging from −2.9 SDS to −5.6 SDS) and a recognizable pattern of dysmorphic facial features including inverted-V shaped interrupted eyebrows, epicanthal fold, depressed nasal bridge, and pointed chin. Although one of the cases had duodenal atresia, all four individuals did not have the combination of internal organ malformations of Strømme syndrome (intestinal atresia and anterior eye segment abnormalities). Immunofluorescence analysis on skin fibroblasts on one of the four cases with the antibody for ARL13B that decorates primary cilia revealed shorter primary cilia that are consistent with a ciliary defect. This case-series of individuals with biallelic CENPF variants suggests the spectrum of clinical manifestations of the disorder that may be related to CENPF variants is broad and can include phenotypes lacking the cardinal features of Strømme syndrome.     

A standard of care for individuals with PIK3CA-related disorders: An international expert consensus statement

Growth promoting variants in PIK3CA cause a spectrum of developmental disorders, depending on the developmental timing of the mutation and tissues involved. These phenotypically heterogeneous entities have been grouped as PIK3CA-Related Overgrowth Spectrum disorders (PROS). Deep sequencing technologies have facilitated detection of low-level mosaic, often necessitating testing of tissues other than blood. Since clinical management practices vary considerably among healthcare professionals and services across different countries, a consensus on management guidelines is needed. Clinical heterogeneity within this spectrum leads to challenges in establishing management recommendations, which must be based on patient-specific considerations. Moreover, as most of these conditions are rare, affected families may lack access to the medical expertise that is needed to help address the multi-system and often complex medical issues seen with PROS. In March 2019, macrocephaly-capillary malformation (M-CM) patient organizations hosted an expert meeting in Manchester, United Kingdom, to help address these challenges with regards to M-CM syndrome. We have expanded the scope of this project to cover PROS and developed this consensus statement on the preferred approach for managing affected individuals based on our current knowledge.     

Bariatric Surgery Outcomes in Patients with Latent Autoimmune Diabetes of the Adult

Bariatric surgery is a very effective treatment for obesity-associated type 2 diabetes. However, the benefits of bariatric surgery in patients with obesity and autoimmune diabetes, such as type 1 diabetes and latent autoimmune diabetes in adults (LADA), are controversial. We report 3 female patients with obesity and LADA who underwent laparoscopic Roux-en-Y gastric bypass >10 years ago. The patients were diagnosed with LADA both 1 and 9 years before (n = 2) or 11 years after the surgery (n = 1). Patients preoperative body mass index ranged from 36 to 47 kg/m² and improved to 23–37 kg/m² in the last follow-up visit, 10–15 years after surgery. Daily insulin dose also decreased from an average of 0.68 to 0.45 IU/kg in those patients treated with insulin before bariatric surgery. Only one patient developed diabetes-related target organ damage. This study shows that patients with LADA depict remarkable reduction of body weight and insulin requirements over long-term after bariatric surgery. So, LADA should not be considered a contraindication for bariatric surgery yet should only be recommended for patients with concomitant obesity with the primary aim of achieving sustained weight loss.     

Colorectal cancer cells activate adjacent fibroblasts resulting in FGF1/FGFR3 signaling and increased invasion

Cancer-associated fibroblasts expressing fibroblast activation protein (FAP) have been implicated in the invasive behavior of colorectal cancer. In this study, we use FAP expression as a marker of fibroblast activation and analyze the effect of activated fibroblasts on colorectal cancer migration and invasion in experimental cell studies. We also investigated the expression pattern of FAP in cancer-associated fibroblasts during transformation from benign to malignant colorectal tumors. In immunohistochemical analyses, FAP was expressed in fibroblasts in all colorectal cancer samples examined, whereas all normal colon, hyperplastic polyps, or adenoma samples were negative. In in vitro studies, conditioned medium from colon cancer cells, but not adenoma cells, activated fibroblasts by inducing FAP expression. These activated fibroblasts increased the migration and invasion of colon cancer cells in Boyden chamber experiments and in a three-dimensional cell culture model. We identify fibroblast growth factor 1/fibroblast growth factor receptor 3 (FGF1/FGFR-3) signaling as mediators leading to the increased migration and invasion. Activated fibroblasts increase their expression of FGF1, and by adding a fibroblast growth factor receptor inhibitor, as well as an FGF1-neutralizing antibody, we reduced the migration of colon cancer cells. Our findings provide evidence of a possible molecular mechanism involved in the cross talk between cancer cells and fibroblasts leading to cancer cell invasion.     

Lipoxin A₄ inhibits porphyromonas gingivalis-induced aggregation and reactive oxygen species production by modulating neutrophil-platelet interaction and CD11b expression

Porphyromonas gingivalis is an etiological agent that is strongly associated with periodontal disease, and it correlates with numerous inflammatory disorders, such as cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. Lipoxin A₄ (LXA₄) is an endogenous anti-inflammatory and proresolving mediator that is protective of inflammatory disorders. The aim of this study was to investigate the effect of LXA₄ on the P. gingivalis-induced activation of neutrophils and platelets and the possible involvement of Rho GTPases and CD11b/CD18 integrins. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry and fluorescence microscopy. Integrin activity was studied by flow cytometry, detecting the surface expression of CD11b/CD18 as well as the exposure of the high-affinity integrin epitope, whereas the activation of Rac2/Cdc42 was examined using a glutathione S-transferase pulldown assay. The study shows that P. gingivalis activates Rac2 and Cdc42 and upregulates CD11b/CD18 and its high-affinity epitope on neutrophils, and that these effects are diminished by LXA₄. Furthermore, we found that LXA₄ significantly inhibits P. gingivalis-induced aggregation and ROS generation in whole blood. However, in platelet-depleted blood and in isolated neutrophils and platelets, LXA₄ was unable to inhibit either aggregation or ROS production, respectively. In conclusion, this study suggests that LXA₄ antagonizes P. gingivalis-induced cell activation in a manner that is dependent on leukocyte-platelet interaction, likely via the inhibition of Rho GTPase signaling and the downregulation of CD11b/CD18. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.Periodontitis is one of the most prevalent inflammatory diseases in humans, the key etiologic agent being the Gram-negative anaerobic rod Porphyromonas gingivalis (54). This bacterium not only is involved in tooth loss but also may cause recurrent bacteremias and contribute to systemic disorders, such as cardiovascular disease (10, 22, 23, 39, 46, 65). P. gingivalis expresses a broad range of virulence factors, such as cysteine proteinases (gingipains), fimbriae, lipopolysaccharide (LPS), and capsular polysaccharide. Infection with the bacterium may lead to chronic inflammation in which hyperresponsive neutrophils contribute to host-mediated tissue destruction. P. gingivalis has been found in human atherosclerotic plaques (15, 27) and has been shown to promote the phenotypic switch of murine monocytes into foam cells, e.g., by inducing reactive oxygen species (ROS) generation and the oxidation of low-density lipoprotein (LDL) (31, 38, 57).We have recently reported that the exposure of human blood to P. gingivalis causes the formation of atherogenic LDL through a gingipain-mediated cleavage of apoB-100 (5). Furthermore, P. gingivalis, unlike other periodontopathic bacteria, has been shown to trigger platelet aggregation in vitro (55, 66), mainly through the interaction between bacterial gingipains and protease-activating receptors (PARs) on the platelets (49). Since platelet aggregation precedes thromboembolic events, this is an important pathogenic feature of the bacterium (1, 32).CD11b/CD18 (complement receptor 3 or Mac-1), the main β₂ integrin expressed on leukocytes, plays an important role in inflammation by promoting leukocyte adhesion and transmigration to sites of infection and by stimulating iC3b-mediated phagocytosis and cytokine production (21). In neutrophils, CD11b/CD18 binds to the platelet GPIIb/IIIa receptor via fibrinogen, thereby mediating neutrophil-platelet interaction and ROS production (11). In accordance with this, we have shown previously that platelet-leukocyte aggregation and ROS production in whole blood are mediated through selectin- and integrin-dependent interactions involving P-selectin and CD11b/CD18 (4).CD11b/CD18 requires inside-out signaling to expose and activate its high-affinity epitope and to enable ligand binding (6). P. gingivalis has been shown to induce inside-out activation of CD11b/CD18 in monocytes/macrophages (25, 29) and to upregulate the CD11b/CD18 receptors on human neutrophils via LPS (68). The P. gingivalis-induced activation of CD11b/CD18 has been most extensively studied in monocytes/macrophages, where two main signaling pathways have been implicated. First, the CD14-mediated binding of fimbriae and LPS to toll-like receptor 2 (TLR-2) stimulates CD11b/CD18 activation through a Rac1- and phosphatidylinositol 3-kinase (PI3K)-mediated pathway (26, 28, 29). Second, the bacteria can bind and activate PAR2 via gingipains, which induces CD11b/CD18 activation (34), possibly via a Rho-dependent pathway (69). Interestingly, these two pathways have been suggested to work synergistically (67). P. gingivalis also mediates platelet and neutrophil activation by acting on platelet TLR-2 and the P13K/Akt pathway (7, 35). Harokopakis and Hajishengallis (29) have shown previously that fimbriae of P. gingivalis induce CD11b/CD18 activation in human neutrophils; however, the mechanisms by which the whole bacterium interacts with CD11b/CD18 and the associated intracellular signaling in neutrophils need to be clarified.In neutrophils, Rac2 accounts for >96% of the Rac protein expressed (33, 58) and is involved in oxidase activity (13). Upon the binding of GTP, Rac and the closely related Rho GTPase Cdc42 interact with the downstream effector p21-activated kinase (PAK) (43). In human neutrophils, CD11b/CD18-mediated adhesion and phagocytosis activates Rac2 as well as Cdc42, which correlates with ROS production (9). The involvement of Rac2 in ROS generation has been demonstrated repeatedly (13), whereas Cdc42 is suggested to have an antagonistic role in oxidative activation (14).Lipoxins (LXs) are endogenously produced eicosanoids with potent anti-inflammatory and proresolving effects (41, 63). Merched et al. (44) proposed that a failure in the endogenous synthesis of LXA₄ underlies the unremitting inflammation that fuels atherosclerosis. LXA₄ functions mainly through the G protein-coupled receptor ALXR (18) and has been shown repeatedly to be protective in periodontal disease (36, 37, 61). Mouse models demonstrate that the administration of stable LXA₄ analogues significantly inhibits P. gingivalis-induced neutrophil influx, cyclooxygenase-2 expression, and prostaglandin E₂ secretion, which is done without promoting any further spreading of the infection (56). Moreover, 15-lipoxygenase transgenic rabbits, overexpressing LXA₄, show significantly diminished bone loss upon infection with P. gingivalis compared to the bone loss of control animals (64). Lipoxins also have been shown to downregulate CD11b/CD18 expression on whole-blood leukocytes (17). We have shown previously that LXA₄ modulates the inside-out activation of CD11b/CD18 in neutrophils (51), and Godson and coworkers showed that LXA₄ may influence the activation of integrins in monocytes/macrophages by modulating Rho GTPases (40, 59).In this study, we demonstrate that LXA₄ inhibits P. gingivalis-induced platelet/leukocyte aggregation and ROS production in whole blood. Furthermore, LXA₄ blocks the bacterium-induced expression and function of CD11b/CD18 on neutrophils, possibly by inhibiting Rac2 and Cdc42 signaling pathways. Interestingly, the effects of LXA₄ in P. gingivalis-induced cellular responses appear to be dependent on platelet-neutrophil interactions.     

Haem oxygenase-1 dictates intrauterine fetal survival in mice via carbon monoxide

Pregnancy establishment implies the existence of a highly vascularized and transient organ, the placenta, which ensures oxygen supply to the fetus via haemoproteins. Haem metabolism, including its catabolism by haem oxygenase-1 (HO-1), should be of importance in maintaining the homeostasis of haemoproteins and controlling the deleterious effects associated with haem release from maternal or fetal haemoglobins, thus ensuring placental function and fetal development. We demonstrate that HO-1 expression is essential to promote placental function and fetal development, thus determining the success of pregnancy. Hmox1 deletion in mice has pathological consequences for pregnancy, namely suboptimal placentation followed by intrauterine fetal growth restriction (IUGR) and fetal lethality. These pathological effects can be mimicked by administration of exogenous haem in wild-type mice. Fetal and maternal HO-1 is required to prevent post-implantation fetal loss through a mechanism that acts independently of maternal adaptive immunity and hormones. The protective HO-1 effects on placentation and fetal growth can be mimicked by the exogenous administration of carbon monoxide (CO), a product of haem catabolism by HO-1 that restores placentation and fetal growth. In a clinical relevant model of IUGR, CO reduces the levels of free haem in circulation and prevents fetal death. We unravel a novel physiological role for HO-1/CO in sustaining pregnancy which aids in understanding the biology of pregnancy and reveals a promising therapeutic application in the treatment of pregnancy pathologies.     

IL-1β promotes immunoregulatory responses in human blood basophils

Human basophils are essential effector cells of chronic allergic inflammation. IL-1 family cytokines such as interleukin (IL)-33 and IL-1β are elevated in serum and bronchoalveolar lavage fluid of allergic asthmatics. IL-33 is known to be a critical regulator of basophil's T2 immune responses. However, the effect of IL-1β on the function of basophils has not been well investigated. Here, we elucidate whether IL-1β regulates the function of human basophils and compared the effects of IL-1β and IL-33 on basophils of healthy and allergic subjects. We found that IL-1β activates the p38 MAPK signaling pathway and promotes IL-8 release in basophils of healthy donors, while FcεRI-mediated LCT₄and histamine secretion is not affected. Strikingly, in the presence of IL-3, IL-1β shows more potency than IL-33, as evidenced by the enhanced p38 phosphorylation and NF-κB activation, as well as the release of both IL-13 and IL-8. We found that the enhanced basophil responsiveness is achieved through IL-3-induced IL-1RI surface expression. Importantly, basophils of allergic donors release significantly higher amounts of IL-8 compared to those from healthy donors upon IL-33 and IL-1β stimulation. Consistently, we detected increased IL-1RI and decreased IL-3 receptor alpha-chain (CD123) and CCR3 expression on basophils of allergic subjects compared to healthy controls, suggesting an in vivo IL-3 priming in allergic donors. In summary, our results suggest enhanced sensitivity of basophils toward IL-33 and IL-1β in allergic subjects compared to those from healthy controls.