STEP-CD study: ustekinumab use in paediatric Crohn's disease-a multicentre retrospective study from paediatric IBD Porto group of ESPGHAN.

Ustekinumab is an effective therapy for adult Crohn's disease (CD), but data in paediatric CD patients are scarce. The aim of the study was to describe the real-life effectiveness and safety of ustekinumab in paediatric CD. This is a multicentre review of children with Crohn's disease treated with ustekinumab. The aim of our study was to describe the effectiveness and safety of ustekinumab in paediatric real-life practice. This is a study of the Paediatric IBD (inflammatory bowel disease) Porto group of ESPGHAN. Corticosteroid (CS)- and exclusive enteral nutrition (EEN)-free remission, defined as weighted Paediatric Crohn's Disease Activity Index (wPCDAI) < 12.5, and physician global assessment (PGA) were determined at weeks 12 and 52. A total of 101 children were included at a median age of 15.4 years (IQR 12.7-17.2) with a median follow-up of 7.4 months (IQR 5.6-11.8). Ninety-nine percent had received prior anti-TNF, 63% ≥ 2 anti-TNFα therapies and 22% vedolizumab. Baseline median wPCDAI was 39 (IQR 25-57.5) (71 (70%) patients with moderate-severe activity). Weeks 12 and 52 CS- and EEN-free remission were both 40.5%. Clinical response at week 6, iv induction route and older age at onset of ustekinumab treatment were predictive factors associated with clinical remission at week 12. Seven minor adverse events probably related to ustekinumab were reported. One patient died from an unrelated cause.  Conclusion: Our results suggest that ustekinumab is effective and safe in children with chronically active or refractory CD. What is Known: • Ustekinumab is an effective therapy for adult moderate to severe Crohn's disease (CD). • Off-label use of ustekinumab in children is increasing especially in anti-TNF refractory CD. What is New: • Is the largest cohort of real-world use of ustekinumab in paediatric CD to date. • Clinical response at week 6, iv induction and older age at onset of ustekinumab were predictive factors associated with clinical response at week 12.

Blood gene expression biomarkers of response to anti-TNF drugs in pediatric inflammatory bowel diseases before initiation of treatment.

BACKGROUND/AIMS: Changes in gene expression profiles among individuals with inflammatory bowel diseases (IBDs) could potentially influence the responsiveness to anti-TNF treatment. The aim of this study was to identify genes that could serve as predictors of early response to anti-TNF therapies in pediatric IBD patients prior to the initiation of treatment. METHODS: We conducted a prospective, longitudinal, and multicenter study, enrolling 24 pediatric IBD patients aged less than 18 years who were initiating treatment with either infliximab or adalimumab. RNA-seq from blood samples was analyzed using the DESeq2 library by comparing responders and non-responders to anti-TNF drugs. RESULTS: Bioinformatic analyses unveiled 102 differentially expressed genes, with 99 genes exhibiting higher expression in responders compared to non-responders prior to the initiation of anti-TNF therapy. Functional enrichment analyses highlighted defense response to Gram-negative bacteria (FDR = 2.3 ×10-7) as the most significant biological processes, and hemoglobin binding (FDR = 0.002), as the most significant molecular function. Gene Set Enrichment Analysis (GSEA) revealed notable enrichment in transcriptional misregulation in cancer (FDR = 0.016). Notably, 13 genes (CEACAM8, CEACAM6, CILP2, COL17A1, OLFM4, INHBA, LCN2, LTF, MMP8, DEFA4, PRTN3, AZU1, and ELANE) were selected for validation, and a consistent trend of increased expression in responders prior to drug administration was observed for most of these genes, with findings for 4 of them being statistically significant (CEACAM8, LCN2, LTF2, and PRTN3). CONCLUSIONS: We identified 102 differentially expressed genes involved in the response to anti-TNF drugs in children with IBDs and validated CEACAM8, LCN2, LTF2, and PRTN3. Genes participating in defense response to Gram-negative bacterium, serine-type endopeptidase activity, and transcriptional misregulation in cancer are good candidates for anticipating the response to anti-TNF drugs in children with IBDs.

Neuroinflammatory Findings of Corneal Confocal Microscopy in Long COVID-19 Patients, 2 Years after Acute SARS-CoV-2 Infection.

OBJECTIVE: To describe corneal confocal microscopy findings in patients with long COVID-19 with persistent symptoms over 20 months after SARS-CoV-2 infection. DESIGN: A descriptive cross-sectional study that included a total of 88 patients; 60 patients with Long COVID-19 and 28 controls. Long COVID-19 diagnosis was established according to the World Health Organization criteria. Corneal confocal microscopy using a Heidelberg Retina Tomograph II (Heidelberg Engineering, Heidelberg, Germany) was performed to evaluate sub-basal nerve plexus morphology (corneal nerve fiber density, nerve fiber length, nerve branch density, nerve fiber total branch density, nerve fiber area, and nerve fiber width). Dendritic cell density and area, along with microneuromas and other morphological changes of the nerve fibers were recorded. RESULTS: Long COVID-19 patients presented with reduced corneal nerve density and branch density as well as shorter corneal nerves compared to the control group. Additionally, Long COVID-19 patients showed an increased density of dendritic cells also with a greater area than that found in the control group of patients without systemic diseases. Microneuromas were detected in 15% of Long COVID-19 patients. CONCLUSIONS: Long COVID-19 patients exhibited altered corneal nerve parameters and increased DC density over 20 months after acute SARS-CoV-2 infection. These findings are consistent with a neuroinflammatory condition hypothesized to be present in patients with Long COVID-19, highlighting the potential role of corneal confocal microscopy as a promising noninvasive technique for the study of patients with Long COVID-19.

Polymorphisms indicating risk of inflammatory bowel disease or antigenicity to anti-TNF drugs as biomarkers of response in children.

Few genetic polymorphisms predict early response to anti-TNF drugs in inflammatory bowel disease (IBD), and even fewer have been identified in the pediatric population. However, it would be of considerable clinical interest to identify and validate genetic biomarkers of long-term response. Therefore, the aim of the study was to analyze the usefulness of biomarkers of response to anti-TNFs in pediatric IBD (pIBD) as long-term biomarkers and to find differences by type of IBD and type of anti-TNF drug. The study population comprised 340 children diagnosed with IBD who were treated with infliximab or adalimumab. Genotyping of 9 selected SNPs for their association with early response and/or immunogenicity to anti-TNFs was performed using real-time PCR. Variants C rs10508884 (CXCL12), A rs2241880 (ATG16L1), and T rs6100556 (PHACTR3) (p value 0.049; p value 0.03; p value 0.031) were associated with worse long-term response to anti-TNFs in pIBD. DNA variants specific to disease type and anti-TNF type were identified in the pediatric population. Genotyping of these genetic variants before initiation of anti-TNFs would enable, if validated in a prospective cohort, the identification of pediatric patients who are long-term responders to this therapy.

Association between HLA DNA Variants and Long-Term Response to Anti-TNF Drugs in a Spanish Pediatric Inflammatory Bowel Disease Cohort.

The genetic polymorphisms rs2395185 and rs2097432 in HLA genes have been associated with the response to anti-TNF treatment in inflammatory bowel disease (IBD). The aim was to analyze the association between these variants and the long-term response to anti-TNF drugs in pediatric IBD. We performed an observational, multicenter, ambispective study in which we selected 340 IBD patients under 18 years of age diagnosed with IBD and treated with anti-TNF drugs from a network of Spanish hospitals. Genotypes and failure of anti-TNF drugs were analyzed using Kaplan-Meier curves and Cox logistic regression. The homozygous G allele of rs2395185 and the C allele of rs2097432 were associated with impaired long-term response to anti-TNF drugs in children with IBD after 3 and 9 years of follow-up. Being a carrier of both polymorphisms increased the risk of anti-TNF failure. The SNP rs2395185 but not rs2097432 was associated with response to infliximab in adults with CD treated with infliximab but not in children after 3 or 9 years of follow-up. Conclusions: SNPs rs2395185 and rs2097432 were associated with a long-term response to anti-TNFs in IBD in Spanish children. Differences between adults and children were observed in patients diagnosed with CD and treated with infliximab.

Ammonia Concentration in the Eluent Influences Fragmentation Pattern of Triacylglycerols in Mass Spectrometry Analysis.

Correct assessment of the fatty acyl at the glycerol sn-2 position in triacylglycerol (TAG) analysis by liquid chromatography and mass spectrometry (LC-MS) is challenging. Ammonium hydroxide (NH4OH) is the preferred choice for the solvent additive for the formation of the ammonium adduct ([M + NH4]+). In this study, the influence of different NH4OH concentrations in the eluents on TAG adduct formation and fragmentation under LC-MS analysis was assessed. Increasing NH4OH concentrations delayed the chromatographic elution time according to a power function. The [M + NH4]+ and [M + ACN + NH4]+ adducts (where ACN means acetonitrile) were formed at all ammonium concentrations assayed. [M + ACN + NH4]+ predominated above 18.26 mM [NH4OH], and the intensity of [M + NH4]+ dropped. TAG fragmentation for fatty acyl release in the MSE was reduced with increasing [M + ACN + NH4]+ adduct, which suggests that ACN stabilizes the adduct in a way that inhibits the rupture of the ester bonds in TAGs. A linear equation (Hsn-I = a × H[M+NH4]+, where sn-I refers to the sn position of the glycerol (I = 1, 2, or 3) and H is the peak height) was deduced to quantify the dehydroxydiacylglycerol fragment intensity in relation to [M + NH4]+ intensity in the full scan. This equation had a slope mean value of 0.369 ± 0.058 for the sn-1 and sn-3 positions, and of 0.188 ± 0.007 for the sn-2 position.

Update on Corneal Confocal Microscopy Imaging.

In vivo corneal confocal microscopy (IVCM) is a non-invasive ophthalmic imaging technique that provides images of the cornea at the cellular level. Despite the uses in ocular surface pathologies, in the last decades IVCM has been used to provide more knowledge in refractive surgery wound healing, in neuropathies diagnosis, etc. The observation of the corneal cells, both normal and inflammatory, and the possibility of quantification of the corneal nerve density with manual or automated tools, makes IVCM have a significant potential to improve the diagnosis and prognosis in several systemic and corneal conditions.

Differential Membrane Lipid Profiles and Vibrational Spectra of Three Edaphic Algae and One Cyanobacterium.

The membrane glycerolipids of four phototrophs that were isolated from an edaphic assemblage were determined by UPLC-MS after cultivation in a laboratory growth chamber. Identification was carried out by 18S and 16S rDNA sequencing. The algal species were Klebsormidium flaccidum (Charophyta), Oocystis sp. (Chlorophyta), and Haslea spicula (Bacillariophyta), and the cyanobacterium was Microcoleus vaginatus (Cyanobacteria). The glycerolipid profile of Oocystis sp. was dominated by monogalactosyldiacylglycerol (MGDG) species, with MGDG(18:3/16:4) accounting for 68.6%, whereas MGDG(18:3/16:3) was the most abundant glycerolipid in K. flaccidum (50.1%). A ratio of digalactosyldiacylglycerol (DGDG) species to MGDG species (DGDG/MGDG) was shown to be higher in K. flaccidum (0.26) than in Oocystis sp. (0.14). This ratio increased under high light (HL) as compared to low light (LL) in all the organisms, with its highest value being shown in cyanobacterium (0.38-0.58, LL-HL). High contents of eicosapentaenoic acid (EPA, C20:5) and hexadecenoic acid were observed in the glycerolipids of H. spicula. Similar Fourier transform infrared (FTIR) and Raman spectra were found for K. flaccidum and Oocystis sp. Specific bands at 1629.06 and 1582.78 cm-1 were shown by M. vaginatus in the Raman spectra. Conversely, specific bands in the FTIR spectrum were observed for H. spicula at 1143 and 1744 cm-1. The results of this study point out differences in the membrane lipid composition between species, which likely reflects their different morphology and evolutionary patterns.

Whole Transcription Profile of Responders to Anti-TNF Drugs in Pediatric Inflammatory Bowel Disease.

BACKGROUND: Up to 30% of patients with pediatric inflammatory bowel disease (IBD) do not respond to anti-Tumor Necrosis Factor (anti-TNF) therapy. The aim of this study was to identify pharmacogenomic markers that predict early response to anti-TNF drugs in pediatric patients with IBD. METHODS: An observational, longitudinal, prospective cohort study was conducted. The study population comprised 38 patients with IBD aged < 18 years who started treatment with infliximab or adalimumab (29 responders and nine non-responders). Whole gene expression profiles from total RNA isolated from whole blood samples of six responders and six non-responders taken before administration of the biologic and after two weeks of therapy were analyzed using next-generation RNA sequencing. The expression of six selected genes was measured for purposes of validation in all of the 38 patients recruited using qPCR. RESULTS: Genes were differentially expressed in non-responders and responders (32 before initiation of treatment and 44 after two weeks, Log2FC (Fold change) >0.6 or <-0.6 and p value < 0.05). After validation, FCGR1A, FCGR1B, and GBP1 were overexpressed in non-responders two weeks after initiation of anti-TNF treatment (Log2FC 1.05, 1.21, and 1.08, respectively, p value < 0.05). CONCLUSION: Expression of the FCGR1A, FCGR1B, and GBP1 genes is a pharmacogenomic biomarker of early response to anti-TNF agents in pediatric IBD.

Thoracic Cardiovascular Complications of Tuberculosis.

ABSTRACT: Mycobacterium infection remains a leading cause of morbidity and mortality worldwide. Although rare, thoracic cardiovascular complications are associated with devastating consequences if not promptly diagnosed using computed tomography. Intrapulmonary complications include tuberculous aortitis, Rasmussen aneurysms, involvement of bronchial and nonbronchial systemic arteries, and thromboembolic events. Extrapulmonary complications include pericarditis, myocarditis, endocarditis, involvement of coronary arteries, annular-subvalvular left ventricle aneurysms and mediastinal fibrosis. This article will review these complications and their computed tomography features.

Secondary syphilis with liver involvement in a liver transplant recipient.

Syphilis is capable of compromising almost any organ; however, syphilitic hepatitis is a rare manifestation that has been described most often in HIV-infected patients. Herein, we present a 33-year-old male liver transplant recipient who presented with progressive liver dysfunction characterized by mild ALT elevation and rising cholestasis, malaise, skin rash, and alopecia. Skin biopsy was characteristic of secondary syphilis, confirmed by both skin and liver biopsy-positive immunohistochemical staining for Treponema pallidum. The patient was treated with benzathine penicillin G 2.4 million units IM q week × 3 weeks. Three months later, the patient was asymptomatic and recovered from his general malaise. He showed no skin lesions and demonstrated complete regrowth of the hair on his scalp, beard, and eyebrows. The presence of liver dysfunction with cholestasis in a transplant recipient should alert transplant providers to the possibility of syphilitic hepatitis, particularly in men who have sex with men. Though not an early manifestation, cutaneous signs of secondary syphilis may be a helpful diagnostic indicator in most cases.

Actin reorganization at the centrosomal area and the immune synapse regulates polarized secretory traffic of multivesicular bodies in T lymphocytes.

T-cell receptor stimulation induces the convergence of multivesicular bodies towards the microtubule-organizing centre (MTOC) and the polarization of the MTOC to the immune synapse (IS). These events lead to exosome secretion at the IS. We describe here that upon IS formation centrosomal area F-actin decreased concomitantly with MTOC polarization to the IS. PKCδ-interfered T cell clones showed a sustained level of centrosomal area F-actin associated with defective MTOC polarization. We analysed the contribution of two actin cytoskeleton-regulatory proteins, FMNL1 and paxillin, to the regulation of cortical and centrosomal F-actin networks. FMNL1 ß phosphorylation and F-actin reorganization at the IS were inhibited in PKCδ-interfered clones. F-actin depletion at the central region of the IS, a requirement for MTOC polarization, was associated with FMNL1 ß phosphorylation at its C-terminal, autoregulatory region. Interfering all FMNL1 isoforms prevented MTOC polarization; nonetheless, FMNL1 ß re-expression restored MTOC polarization in a centrosomal area F-actin reorganization-independent manner. Moreover, PKCδ-interfered clones exhibited decreased paxillin phosphorylation at the MTOC, which suggests an alternative actin cytoskeleton regulatory pathway. Our results infer that PKCδ regulates MTOC polarization and secretory traffic leading to exosome secretion in a coordinated manner by means of two distinct pathways, one involving FMNL1 ß regulation and controlling F-actin reorganization at the IS, and the other, comprising paxillin phosphorylation potentially controlling centrosomal area F-actin reorganization. ABBREVIATIONS: Ab, antibody; AICD, activation-induced cell death; AIP, average intensity projection; APC, antigen-presenting cell; BCR, B-cell receptor for antigen; C, centre of mass; cent2, centrin 2; cIS, central region of the immune synapse; CMAC, CellTracker™ Blue (7-amino-4-chloromethylcoumarin); cSMAC, central supramolecular activation cluster; CTL, cytotoxic T lymphocytes; DAG, diacylglycerol; DGKα, diacylglycerol kinase α; Dia1, Diaphanous-1; dSMAC, distal supramolecular activation cluster; ECL, enhanced chemiluminescence; ESCRT, endosomal sorting complex required for traffic; F-actin, filamentous actin; Fact-low cIS, F-actin-low region at the centre of the immune synapse; FasL, Fas ligand; FMNL1, formin-like 1; fps, frames per second; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; HRP, horseradish peroxidase; ILV, intraluminal vesicles; IS, immune synapse; MFI, mean fluorescence intensity; MHC, major histocompatibility complex; MIP, maximal intensity projection; MVB, multivesicular bodies; MTOC, microtubule-organizing centre; NS, not significant; PBL, peripheral blood lymphocytes; PKC, protein kinase C; PKCδ, protein kinase C δ isoform; PLC, phospholipase C; PMA, phorbol myristate acetate; Pol. Index, polarization index; pSMAC, peripheral supramolecular activation cluster; PSF, point spread function; ROI, region of interest; SD, standard deviation; shRNA, short hairpin RNA; SEE, Staphylococcus enterotoxin E; SMAC, supramolecular activation cluster; TCR, T-cell receptor for antigen; T-helper (Th); TRANS, transmittance; WB, Western blot.

Imaging the Human Immunological Synapse.

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90° angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

Effect of Different Broad Waveband Lights on Membrane Lipids of a Cyanobacterium, Synechococcus sp., as Determined by UPLC-QToF-MS and Vibrational Spectroscopy.

Differential profile of membrane lipids and pigments of a Synechococcus sp. cyanobacterial strain cells exposed to blue, green, red and white light are determined by means of liquid chromatography and mass spectrometry or diode array detection. Raman and ATR-IR spectra of intact cells under the diverse light wavebands are also reported. Blue light cells exhibited an increased content of photosynthetic pigments as well as specific species of membrane glycerolipids as compared to cells exposed to other wavebands. The A630/A680 ratio indicated an increased content of phycobilisomes (PBS) in the blue light-exposed cells. Some differences in the protein conformation between the four light waveband-exposed cells were deduced from the variable absorbance at specific wavenumbers in the FT-Raman and ATR-FTIR spectra, in particular bands assigned to amide I and amide II. Bands from 1180 to 950 cm(-1) in the ATR-FTIR spectrum suggest degraded outer membrane polysaccharide in the blue light-exposed cells.

Recurrent hematuria caused by nutcracker syndrome.

A 26-year-old woman presented to the emergency department complaining of left flank pain, and proteinuria and hematuria were detected during urinalysis. A renal ultrasound did not reveal any disorder, and after performing a computed tomography angiography scan, compression of the left renal vein between the superior mesenteric artery and the aorta was seen. This compression is known as Nutcracker syndrome. From among the different treatment options available, it was decided, with patient consensus, to use open surgical management, performing a transposition of the left renal vein to a more distal level in the inferior vena cava. The immediate postoperative care progressed without complications and the symptoms resolved; after 1 year of surveillance, the patient continues to be asymptomatic. Nutcracker syndrome is a rare phenomenon, with few cases described. There are different therapeutic options for the treatment of Nutcracker syndrome, such as open surgery, endovascular treatment, or conservative treatment; because of the low prevalence of this syndrome, there are no sufficiently large series at present or with the necessary long-term surveillance to decide on the most suitable treatment. Distal transposition of the left renal vein in the inferior cava vein has proved to offer good long-term results, and this option offers a higher chance of resolution without the need for as many postsurgery controls as would be required with endovascular treatment.