Whipple's Disease, An Overlooked Diagnosis

Whipple disease is a rare multisystem inflammatory disease. Because fewer than 1000 reported cases have been described, clinical experience with this disorder is sparse. We are reporting a case of a 46-year-old man who presented with fever, weight loss, and polyarthralgia for 2 months, and 1 month of diarrhea. The patient was thoroughly investigated for collagen diseases and COVID-19, with no definite diagnosis. A therapeutic trial by immunosuppressive drugs provided partial remission followed by a marked rebound of the symptoms. His occult blood in stool was positive and subsequent upper endoscopy with proximal small intestinal biopsies showed the pathological features of Whipple's disease. The patient showed a dramatic improvement following treatment with ceftriaxone and trimethoprim-sulfamethoxazole. Despite the rarity of Whipple's disease, its course mimics many rheumatological diseases, inflammatory bowel disease, and COVID-19 disease. It should always be a part of the differential diagnosis of obscure polyarthralgia and chronic diarrhea.Copyright © 2023 The Author(s).

Chilblain lupus erythematosus associated with systemic lupus erythematosus: A classic diagnosis in a current epidemiological context

Objectives: Chilblain lupus erythematosus (LE) is a rare chronic cutaneous lupus erythematosus (CCLE) characterized by the appearance of violaceous plaques in acral regions most exposed to cold. The isolated form affects middle-aged women, while the familial form manifests in early childhood and is associated with mutations in the TREX1 gene. Result(s): A 13-year-old adolescent, with no relevant family history, was referred in March 2021 for suspected chilblain-like lesions associated with COVID-19 infection. The patient presented with multiple violaceous papules on hands and feet. The lesions were slightly painful. Small hyperkeratotic papules were also observed on finger pads. Physical examination also revealed some aphthae affecting the lips. No other systemic symptoms were reported. A skin biopsy and blood tests were performed due to presumed chilblain LE with probable systemic involvement. Histology revealed basal vacuolar damage and intense perivascular and periadnexal lymphocytic inflammatory dermal infiltrate. Remarkably, mucin was noted among the collagen bundles. Leukopenia and positive ANA antibodies (titre 1:320) were detected. Complement levels were normal. SARS-CoV2 infection was ruled out. Skin lesions disappeared within 1 month under topical corticosteroids. Hydroxychloroquine was afterwards started by Rheumatology without recurrence of skin symptoms until last follow-up. Discussion(s): We present an uncommon case of an adolescent with systemic LE presenting as chilblain LE. Chilblain LE can be accompanied by other discoid CCLE. It can progress to systemic LE in up to 20% of patients, especially when concomitant CCLE is present. This rare presentation of CCLE should be differentiated from typical chilblain and other resembling lesions, such as SARS-CoV2-associated chilblain and acral purpuric lesions (COVID toes). The Mayo Clinic diagnostic criteria can be helpful, particularly in this last SARS-CoV2 outbreak scenario, when the reporting of similar skin lesions has been significant.

PANLAR 2023 Abstracts

The proceedings contain 238 papers. The topics discussed include: treatment with tofacitinib attenuates muscle loss through Myogenin activation in collagen-induced arthritis;plasma cytokine levels in a group of Colombian patients with moderate systemic lupus erythematosus and rheumatoid arthritis;prevalence of neoplastic disease in patients with systemic sclerosis in a south American cohort;characterization of rheumatic manifestations in patients with HIV infection from a south American hospital;anthropometric measures of central adiposity in the evaluation of metabolic syndrome in patients with idiopathic inflammatory myopathies;anti RO 52/60 antibodies and their clinical serological correlation. single center descriptive study;safety and immunogenicity of CoronaVac and CHADOX1 vaccines against SARS-COV-2 in patients with rheumatoid arthritis: Brazilian multicentric study;and effect of ABO and RH blood type on SARS-COV-2 infection severity in patients with rheumatic diseases: data from the national SAR-COVID registry.

Beyond Articular Cartilage: A Systematic Review of the Extracellular Matrix in Other Components of the Osteoarthritic Joint

Background/Aims Traditionally viewed from the perspective of cartilage degeneration, osteoarthritis is increasingly seen as a disease of global joint dysfunction. Connective tissue extracellular matrix (ECM) is a crucial determinant of joint mechanobiology, providing cells with scaffolding, topographical cues, and a reservoir of soluble factors. While ECM dysregulation has been extensively studied in osteoarthritic cartilage, it remains poorly defined in other joint tissues. Here, we systematically review the composition, architecture, and remodelling of non-cartilage soft joint tissue ECM in human osteoarthritis and animal disease models. Methods A systematic search strategy was run through the MEDLINE, EMBASE and Scopus databases on 30 October 2020 and repeated on 1 October 2021. The search criteria included disease nomenclature, relevant tissues, as well as structural ECM components and architectural features. All papers were independently screened by two reviewers on the Covidence platform according to predefined eligibility criteria. Relevant clinical, demographic, and biological data were extracted from included studies, which were assessed for bias using the OHAT Risk of Bias Rating Tool for Human and Animal Studies. Results 148 of 8,156 identified studies met all eligibility criteria. 113 papers evaluated human osteoarthritis;of 35 animal studies, the most frequently used models involved surgical joint destabilisation in small mammals. ECM was best defined in menisci, ligaments, and synovium;fewer papers assessed skeletal muscles, tendons, and fat pads. Compared to the healthy joint, osteoarthritis is associated with qualitative and quantitative alterations in structural ECM components, most notably collagens and proteoglycans. In recent years, whole proteome sequencing has been employed to address these changes systematically. The mechanical properties of ECM change significantly in osteoarthritis in response to post-translational modifications, extensive calcification, and the marked loss of matrix organisation across the joint. Notably, some aspects of ECM remodelling in these tissues appear to precede discernible cartilage dysregulation. Similar ECM dysregulation is also observed in animal models, although intermodel variability in arthritogenic precipitant and the range of reported outcomes make comparisons difficult. Many studies are limited by significant bias, notably in the infrequent reporting of investigator blinding, and in the poor demographic matching of osteoarthritic and control patients. Encouragingly, the quality of methodology reporting and use of age-matched control populations have improved in recent years. Conclusion Current data provide compelling evidence of whole joint ECM changes in osteoarthritis and importantly suggest that these changes occur early in the disease process. How ECM dysfunction affects the behaviour of tissue-resident cells remains less well understood. Our work will support the design of disease-relevant biomaterials used to model osteoarthritis in vitro, helping to address this issue, by more accurately recreating the extracellular environment. Furthermore, the development of imaging modalities sensitive to connective tissue ECM changes warrants investigation from both diagnostic and prognostic perspectives.

Parietex Mini Male Sling for Stress Urinary Incontinence at the Time of Three Piece Penile Prosthesis: Technique and Outcomes

INTRODUCTION AND OBJECTIVE: Male stress urinary incontinence (SUI) and climacturia are often comorbid with erectile dysfunction (ED) post prostatectomy. While the inflatable penile prosthesis (IPP) is the gold standard surgical option for ED, there are many surgical options for male SUI as well as climacturia including bulking agents, Virtue male sling, AdVance>= male sling, mini Jupette with IPP, and artificial urinary sphincter (AUS). We present our technique and outcomes for mini male sling (MMS) with IPP insertion for mild SUI and climacturia using a hydrophilic monofilament polyester mesh. METHOD(S): After obtaining IRB approval, a retrospective review of all IPPs performed by a single high volume surgeon was performed. Those men who underwent IPP insertion with concomitant MMS were analyzed. The technique of MMS placement involves measurement of an approximately 5x3cm Parietex>= mesh (Covidien, MN) and overlying this ventrally over the bulbar urethra. The Parietex>= mesh includes a hydrophilic absorbable collagen film to minimize tissue reaction and risk of erosion. The lateral edges of the mesh are sutured to the 3- and 9 o'clock positions on the left and right corporal bodies, respectively, using a nonabsorbable, braided suture in an interrupted fashion. Mesh is sutured just proximal to the corporotomy sites. Tightness of mesh was measured using the proximal end of a Debakey forceps, with ease of passage when IPP deflated and inability to pass forceps when IPP inflated indicative of proper sizing. RESULT(S): A total of 63 men underwent IPP with MMS between January 2018 and October 2022. All patients had ED after radical prostatectomy. Average patient age was 67 years old, and average IPP size was 21cm with 1cm rear tip extender (RTE). A total of 59 men had SUI, with average pad per day (PPD) of 1.5. Twenty-five men with SUI had comorbid climacturia, and 4 men had preoperative climacturia without SUI. Average PPD post IPP with MMS was 0.1. Of the 29 men with preoperative climacturia, only one did not have resolution of his symptoms post MMS. There was only one reported complication of acute urinary retention requiring temporary foley catheter placement. Two patients required subsequent AUS insertion for persistent SUI. There were no reported cases of infection or mesh erosion. CONCLUSION(S): The hydrophilic Parietex>= mesh is a safe, easy to use, nonreactive, and effective material for mini male sling insertion at the time of IPP placement for men with ED and mild/moderate SUI or climacturia.

Development of a Lymphoid Organ-Chip to Evaluate Covid Vaccine Boosting Strategies

Background: Secondary lymphoid organs provide the adequate microenvironment for the development of antigen (Ag)-specific immune responses. The tight collaboration between CD4+ T cells and B cells in germinal centers is crucial to shape B cell fate and optimize antibody maturation. Dissecting these immune interactions remains challenging in humans, and animal models do not always recapitulate human physiology. To address this issue, we developed an in vitro 3D model of a human lymphoid organ. The model relies on a microfluidic device, enabling primary human cells to self-organize in an extracellular matrix (ECM) under continuous fluid perfusion. We applied this Lymphoid Organ-Chip (LO chip) system to the analysis of B cell recall responses to SARS-CoV-2 antigens. Method(s): We used a two-channel microfluidic Chip S1 from Emulate, where the top channel is perfused with antigen (spike protein or SARS-CoV-2 mRNA vaccine), while the bottom channel contains PBMC (n = 14 independent donors) seeded at high-density in a collagen-based ECM. Immune cell division and cluster formation were monitored by confocal imaging, plasmablast differentiation and spike-specific B cell amplification by flow cytometry, antibody secretion by a cell-based binding assay (S-flow). Result(s): Chip perfusion with the SARS-CoV-2 spike protein for 6 days resulted in the induction CD38hiCD27hi plasmablast maturation compared to an irrelevant BSA protein (P< 0.0001). Using fluorescent spike as a probe, we observed a strong amplification of spike-specific B cell (from 3.7 to 140-fold increase). In line with this rapid memory B cell response, spike-specific antibodies production could be detected as early as day 6 of culture. Spike perfusion also induced CD4+ T cell activation (CD38+ ICOS+), which correlated with the level of B cell maturation. The magnitude of specific B cell amplification in the LO chip was higher than in 2D and 3D static cultures at day 6, showing the added value of 3D perfused culture for the induction of recall responses. Interestingly, the perfusion of mRNA-based SARS-CoV-2 vaccines also led to strong B cell maturation and specific B cell amplification, indicating that mRNA-derived spike could be expressed and efficiently presented in the LO chip. Conclusion(s): We developed a versatile Lymphoid Organ-Chip model suitable for the rapid evaluation of B cell recall responses. The model is responsive to protein and mRNA-encoded antigens, highlighting its potential in the evaluation of SARS-CoV-2 vaccine boosting strategies.

Polymerized type I collagen as a potential treatment against COVID-19.

In this document we make a brief compilation of current treatments, focusing on a Mexican drug, from the Mexican industry, which has attracted attention as a potential treatment of COVID-19 in its moderate to severe forms, due to its anti-inflammatory efficacy that is it has been shown to contribute to the treatment of the forms that occur with cytokine storm, so we make a recapitulation of the main research in this regard.Copyright © 2022 Comunicaciones Cientificas Mexicanas S.A. de C.V.. All rights reserved.

Antibacterial, antiviral, and biodegradable collagen network mask for effective particulate removal and wireless breath monitoring.

Functional face masks that can effectively remove particulate matter and pathogens are critical to addressing the urgent health needs arising from industrial air pollution and the COVID-19 pandemic. However, most commercial masks are manufactured by tedious and complicated network-forming procedures (e.g., meltblowing and electrospinning). In addition, the materials used (e.g., polypropylene) have significant limitations such as a lack of pathogen inactivation and degradability, which can cause secondary infection and serious environmental concerns if discarded. Here, we present a facile and straightforward method for creating biodegradable and self-disinfecting masks based on collagen fiber networks. These masks not only provide superior protection against a wide range of hazardous substances in polluted air, but also address environmental concerns associated with waste disposal. Importantly, collagen fiber networks with naturally existing hierarchical microporous structures can be easily modified by tannic acid to improve its mechanical characteristics and enable the in situ production of silver nanoparticles. The resulting masks exhibit excellent antibacterial (>99.99%, 15 min) and antiviral (>99.999%, 15 min) capabilities, as well as high PM2.5 removal efficiency (>99.9%, 30 s). We further demonstrate the integration of the mask into a wireless platform for respiratory monitoring. Therefore, the smart mask has enormous promise for combating air pollution and contagious viruses, managing personal health, and alleviating waste issues caused by commercial masks.

Vitamin C Regulates the Profibrotic Activity of Fibroblasts in In Vitro Replica Settings of Myocardial Infarction.

Extracellular collagen remodeling is one of the central mechanisms responsible for the structural and compositional coherence of myocardium in patients undergoing myocardial infarction (MI). Activated primary cardiac fibroblasts following myocardial infarction are extensively investigated to establish anti-fibrotic therapies to improve left ventricular remodeling. To systematically assess vitamin C functions as a potential modulator involved in collagen fibrillogenesis in an in vitro model mimicking heart tissue healing after MI. Mouse primary cardiac fibroblasts were isolated from wild-type C57BL/6 mice and cultured under normal and profibrotic (hypoxic + transforming growth factor beta 1) conditions on freshly prepared coatings mimicking extracellular matrix (ECM) remodeling during healing after an MI. At 10 µg/mL, vitamin C reprogramed the respiratory mitochondrial metabolism, which is effectively associated with a more increased accumulation of intracellular reactive oxygen species (iROS) than the number of those generated by mitochondrial reactive oxygen species (mROS). The mRNA/protein expression of subtypes I, III collagen, and fibroblasts differentiations markers were upregulated over time, particularly in the presence of vitamin C. The collagen substrate potentiated the modulator role of vitamin C in reinforcing the structure of types I and III collagen synthesis by reducing collagen V expression in a timely manner, which is important in the initiation of fibrillogenesis. Altogether, our study evidenced the synergistic function of vitamin C at an optimum dose on maintaining the equilibrium functionality of radical scavenger and gene transcription, which are important in the initial phases after healing after an MI, while modulating the synthesis of de novo collagen fibrils, which is important in the final stage of tissue healing.

Valorization of Salmo salar Skin Waste for the Synthesis of Angiotensin Converting Enzyme-1 (ACE1) Inhibitory Peptides.

One of potential inhibitors which is widely used for the clinical treatment of COVID-19 in comorbid patients is Angiostensin Converting Enzyme-1 (ACE1) inhibitor. A safer peptide-based ACE1 inhibitor derived from salmon skin collagen, that is considered as the by-product of the fish processing industry have been investigated in this study. The inhibitory activity against ACE1 was examined using in vitro and in silico methods. In vitro analysis includes the extraction of acid-soluble collagen, characterization using FTIR, Raman, UV-Vis, XRD, cytotoxicity assay, and determination of inhibition against ACE1. In silico method visualizes binding affinity, molecular interaction, and inhibition type of intact collagen and active peptides derived from collagen against ACE1 using molecular docking. The results of FTIR spectra detected amide functional groups (A, B, I, II, III) and imine proline/hydroxyproline, while the results of Raman displayed peak absorption of amide I, amide III, proline/hydroxyproline ring, phenylalanine, and protein backbone. Furthermore, UV-Vis spectra showed typical collagen absorption at 230 nm and based on XRD data, the chain types in the samples were α-helix. ACE1 inhibition activity was obtained in a concentration-dependent manner where the highest was 82.83% and 85.84% at concentrations of 1000, and 2000 µg/mL, respectively, and showed very low cytotoxicity at the concentration less than 1000 µg/mL. In silico study showed an interaction between ACE1 and collagen outside the active site with the affinity of - 213.89 kcal/mol. Furthermore, the active peptides of collagen displayed greater affinity compared to lisinopril, namely HF (His-Phe), WYT (Trp-Tyr-Thr), and WF (Trp-Phe) of - 11.52; - 10.22; - 9.58 kcal/mol, respectively. The salmon skin-derived collagen demonstrated ACE1 inhibition activity with a non-competitive inhibition mechanism. In contrast, the active peptides were predicted as potent competitive inhibitors against ACE1. This study indicated that valorization of fish by-product can lead to the production of a promising bioactive compound to treat COVID-19 patient with diabetic comorbid.

Moderate COVID-19 Disease Is Associated With Reduced Bone Turnover.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection has been associated with musculoskeletal manifestations, including a negative effect on bone health. Bone formation was found to be reduced in coronavirus disease 2019 (COVID-19) patients. The aim of this case-control study was to determine whether bone metabolism is coupled or uncoupled in COVID-19 patients with moderately severe disease, the latter expressed by the requirement of hospitalization but not intensive care treatment, no need for mechanical ventilation, and a C-reactive protein level of (median [quartiles], 16.0 [4.0; 52.8]) mg/L in serum. Besides standard biochemical markers, serum levels of C-terminal telopeptide of type 1 collagen, tartrate-resistant acid phosphatase, osteocalcin, bone-specific alkaline phosphatase, sclerostin, dickkopf-1, and osteoprotegerin were evaluated in COVID-19-infected patients at the time of hospital admission, along with those of age- and sex-matched noninfected controls. The median age of the 14 female and 11 male infected patients included in the matched-pair analysis was (67 [53; 81]) years. C-terminal telopeptide of type 1 collagen was significantly lower in COVID-19 patients (0.172 [0.097; 0.375] ng/mL) than in controls (0.462 [0.300; 0.649] ng/mL; p = 0.011). The patients' osteocalcin levels (10.50 [6.49; 16.26] ng/mL) were also lower than those of controls (15.33 [11.85, 19.63] ng/mL, p = 0.025). Serum levels of sclerostin and dickkopf-1 were significantly higher in infected patients relative to controls. The remaining parameters did not differ between cases and controls. A limitation of the study was that patients and controls were recruited from different hospitals. Nevertheless, due to the geographical proximity of the two centers, we assume that this fact did not influence the results of the study. Given this limitation, the investigation showed that bone metabolism is altered but remains coupled in patients with moderately severe COVID-19. Therefore, it is important to evaluate bone turnover markers and fracture risk in these patients during the postinfection period. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Severe epidermolysis bullosa acquisita in a patient with graft-versus-host disease: causal or coincidental?

Graft-versus-host disease (GvHD) is common after haematopoietic cell transplantation (HCT). Mucocutaneous manifestations are variable and may simulate autoimmune bullous dermatoses. However, the association of GvHD with autoimmune disorders, including bullous dermatoses, is also well recognized. We describe a patient with GvHD in whom severe and relapsing epidermolysis bullosa acquisita (EBA) was diagnosed 3 years after transplant and propose a causal association with GvHD. A 66-year-old woman developed GvHD following allogeneic HCT for acute myeloid leukaemia in 2016. This affected her gastrointestinal tract and skin but improved with oral corticosteroids and ciclosporin. In 2019 she presented with a widespread rash consisting of large, tense, haemorrhagic blisters. Histological features were in keeping with EBA. Direct immunofluorescence was also consistent with EBA, demonstrating linear positivity for IgG and C3 confined to the blister base, as was detection of collagen VII antibodies on indirect immunofluorescence. She was admitted and treated with high-dose oral steroids, ciclosporin and intravenous immunoglobulin (IVIg) with eventual resolution of blistering. Although further IVIg administration was planned as an outpatient, this coincided with the start of the COVID-19 pandemic and she elected not to attend and also stopped all medication. Despite this, her EBA remained quiescent until September 2021 when she was readmitted with a severe deterioration in blistering and significant dysphagia due to an oesophageal stricture, with a weight of 31.7 kg. Once again, she responded rapidly to oral prednisolone and IVIg. Dapsone was considered but precluded by G6PD deficiency and there were clinical and adherence concerns about using mycophenolate mofetil. Upon discharge she was again nonadherent to medication and failed to attend for planned IVIg. She flared and was admitted for a third time in December 2021, requiring gastrostomy for nutritional support;her weight at this time was 26.4 kg. Her EBA is currently well controlled on prednisolone and IVIg. EBA is a rare, acquired blistering disorder secondary to autoantibodies targeting type VII collagen. Previous studies have found circulating basement membrane zone (BMZ) antibodies in 24% of chronic GvHD patients, possibly generated in response to chronic BMZ damage (Hofmann SC, Kopp G, Gall C et al. Basement membrane antibodies in sera of haematopoietic cell recipients are associated with graft-versushost disease. J Eur Acad Dermatol Venereol 2010;24: 587-94). Corresponding clinical manifestations are rare, with bullous pemphigoid the most frequently reported. EBA is much less common with four previously reported cases [Brassat S, Fleury J, Camus M, et al. (Epidermolysa bullosa acquisita and graftversus- host disease). Ann Dermatol Venereol 2014;141: 369-73 (in French)]. As a fifth case of EBA, our patient provides further evidence of a likely pathophysiological relationship between GvHD and autoimmune subepidermal bullous dermatoses, and highlights the significant challenges of managing these vulnerable patient groups during the COVID-19 pandemic.

A review of fibroblast-like synoviocytes in the pathogenesis of Rheumatoid arthritis: Their activation and the inhibition of their apoptosis

Rheumatoid Arthritis (RA) is a systemic, autoimmune, inflammatory disease characterized by synovial hyperplasia, inflammatory cell infiltration in the synovial tissues, and progressive destruction of cartilage and bones. This disease often leads to chronic disability. More recently, activation of synovial fibroblasts (SFs) has been linked to innate immune responses and several cellular signalingpathways that ultimately result in the aggressive and invasive stages of RA. SFs are the major sources of pro-inflammatory cytokines in RA synovium. They participate in maintaining the inflammatory state that leads to synovial hyperplasia and angiogenesis in the inflamed synovium. The altered apoptotic response of synovial and inflammatory cells has been connected to these alterations of inflamed synovium. RA synovial fibroblasts (RASFs) have the ability to inhibit several apoptotic proteins that cause their abnormal proliferation. This proliferation leads to synovial hyperplasia. Apoptotic pathway proteins have thus been identified as possible targets for modifying the pathophysiology of RA. This review summarizes current knowledge of SF activation and its roles in the inhibition of apoptosis in the synovium, which is involved in joint damage during the effector phase of RA development.Copyright © 2022 Biomedpress.

Machine Learning analysis of human pulmonary extracellular matrix architecture identifies disease-specific remodeling patterns

Background: Normal organ function is critically dependent on an intact three-dimensional architecture. Structural abnormalities induced by pathological situations instruct cells to behave abnormally and promoting disease progression oftentimes leading to organ failure. Current approaches do not allow for high-resolution (HR) threedimensional (3D) visualisation and analysis of human organ structure. Method(s): Here, we develop a method to perfuse human tissue segments to remove cells and study the 3D structural scaffold, which could be applied to any organ. Our approach enables HR-3D imaging of organ architecture, which we apply to study healthy and diseased human lung, specifically emphysema, usual interstitial pneumonia, pulmonary sarcoidosis, and COVID-19. Result(s): Our imaging reveals major structural abnormalities previously unseen by existing methodologies. Furthermore, we identify disease-specific patterns of structural remodelling using machine learning, including the altered spatial relationship between extracellular matrix (ECM) proteins collagen type IV, elastin and fibrillar collagen present across all diseases. Conclusion(s): Given the importance of organ structure on function, our approach opens the possibility to understand human physiology in a new way, which may assist in future disease diagnosis and explain the detrimental pulmonary effects of the diseases studied here.

Developing advanced cell culture models to study COVID19-associated pulmonary fibrosis

Patients with severe COVID-19-associated pneumonia are at risk to develop pulmonary fibrosis. To study the underlying mechanisms, we aim to develop advanced cell culture models that reliably reflect COVID-19-related profibrotic microenvironment. To identify key cellular players, we performed pilot immunohistochemistry analysis on lung tissue from COVID-19 patients with fibrosis collected during autopsy. Results revealed diffuse alveolar damage with macrophage infiltration, and myofibroblast accumulation with enriched collagen deposition surrounding the damaged alveoli. To mimic SARS-CoV-2 infection in alveoli, we infected human primary type II alveolar epithelial cells (AEC2) and found enhanced signaling of profibrotic cytokine transforming growth factor beta (TGFbeta) in some donors. To recreate the early fibrotic niche, an alveolar-macrophage-fibroblast (AMF) tri-culture model was established. After infecting AEC2 with SARS-CoV-2 in this AMF model, gene expression analysis provided evidence for fibroblast-to-myofibroblast transition. Furthermore, we found that overexpression of SARS-CoV-2 papain-like protease (PLpro) can promote TGFbeta signaling in HEK293T and A549 cells. After infecting AEC2 with SARS-CoV-2 PLpro lentivirus in the AMF model, we found signs of epithelial-to-mesenchymal transition and fibroblast-to myofibroblast transition. In future studies, we will use a detailed analysis of COVID-19-associated lung fibrosis with other types of lung fibrosis, to further refine COVID-19-related fibrosis models, including lung-on-chip models.

Characterization of Mesenchymal Cell from BAL of post COVID-19 patient whit fibrosis

Increasing number of severe COVID 19 patients develop pulmonary Fibrosis, but the management of this complication is still unclear due to a lack of clinical trials. Aim of this study was to characterize mesenchymal cells (MC) isolated from 10 broncho-alveolar lavage (BAL, at 2 months after discharge) from patients with COVID19 fibrosis (COVID19-f) and to compare them with those isolated from 8 patients with collagen tissue diseaseassociated interstitial fibrosis(CTD-ILD). BAL fluid (BALf) levels of TGFbeta, VEGF, TIMP2, RANTES, IL6, IL8, and PAI1 were assessed by ELISA. Primary MC foci were cultured and expanded in D-MEM +10% FBS, characterized by flow cytometry and osteogenic and adipogenic differentiation. Collagen 1 production (+/-TGF-beta) was tested by WB and mRNA expression. BALf cytokine and GF levels were comparable in the two groups. Efficiency of MC isolation from BAL was 100% in COVID-f compared to 65% in CTD-ILD. MC antigen surface expression of CD105, CD73, CD90 (>90%, respectively), CD45, CD34, CD19 and HLA-DR (<5%, respectively) was comparable. None of MC samples differentiated in adipocytes, while COVID19-f were positive for calcium deposition. COVID19-f MC showed at WB, higher Collagen 1 production with respect to CTD-ILD with TGF-beta stimulation. Our preliminary data suggest MC from COVID19-f share several features with CTD-ILD but might have a higher response to fibrogenic and differentiation signals.

Differentially expressed genes in Diffuse Alveolar Damage (DAD) patterns of COVID-19

Severe COVID-19 induces DAD, a condition with temporal-spatial heterogeneity. We determined the differentially expressed genes (DEGs) in the histological patterns of DAD. Twelve fatal COVID-19 cases were classified in acute DAD (n=5) and intermediate/advanced (IA) DAD (n=7). Autopsy lung RNA was extracted from COVID-19 and 4 control cases. RNA sequencing was performed on the Illumina NovaSeq 6000. Enrichment analysis was performed with clusterProfiler using Genome-wide annotation for Human R package. GO terms and KEGG pathways were considered enriched if adjusted p<=0.05. Principal component analysis showed that IA-DAD samples were grouped, while acute DAD samples were scattered. The differential expression analysis between these two groups and the control cases revealed: 261 DEGs in the acute DAD (143 Up- and 53 Down-regulated), 244 DEGs in the IA- DAD tissues (67 Up- and 116 Down-regulated), and 61 DEGs were shared between them (45 Up- and 16 Downregulated). Patients with acute DAD had up-regulated genes related to oxidative phosphorylation, blood coagulation, megakaryocytes differentiation/regulation, and platelet degranulation/activation. Patients with IA-DAD had DEGs related to immunoglobulins and extracellular matrix. The shared up-regulated DEGs between both patterns are involved in innate and adaptive immune responses. We selected 3 DEGs in each DAD pattern for validation by realtime PCR. There were no differences in acute DAD DEGs, but DEGs overexpressed in intermediate DAD (COL3A1, IGLV3-19, IGHV1-58) were significantly higher. Genes related to thrombotic events occur at the acute stage of DAD, whereas immunoglobulin production and remodeling occur at later stages of DAD.

Serological markers of tissue remodeling are elevated in patients with COVID-19 who develop interstitial lung disease

Background: Coronavirus disease-19 (COVID-19) is an infectious disease that can result in serious respiratory illness. It is associated with extensive systemic inflammation and changes to the lung extracellular matrix (ECM), which may result in diffuse alveolar damage and pulmonary fibrosis. In this study, the aim was to investigate whether ECM remodeling, wound healing, and neutrophil activity is altered in patients with COVID-19, with or without interstitial lung disease (ILD). Method(s): Serum was collected from 72 patients with COVID-19 infection 3 months after diagnosis, 10 of whom developed ILD, and from 16 healthy controls. A panel of neo-epitope specific ELISAs reflecting type III collagen crosslinking (PC3X), type III and VI collagen formation (PRO-C3 and PRO-C6), type I, III, and VI collagen degradation (C1M, C3M, and C6M), fibrin crosslinking (X-FIB), fibrin clot formation (PRO-FIB), elastin degradation (EL-NE and ELP-3), and calprotectin degradation (CPa9-HNE) were assessed in serum of patients. Result(s): Mean serum neo-epitopes PC3X (p<0.0001), PRO-C3 (p=0.0024), C3M (p=0.0085), PRO-FIB (p<0.0001), ELP-3 (p<0.0001), and CPa9-HNE (p<0.0001) were significantly elevated in patients with COVID-19 compared to healthy controls. Additionally, PC3X (p=0.023) and PRO-C3 (p=0.0317) were significantly elevated in COVID-19 patients who developed ILD when compared to those who did not. Conclusion(s): Non-invasive serological biomarkers reflecting tissue remodeling were significantly elevated in patients infected with COVID-19. Additionally, type III collagen crosslinking and formation may differentiate patients who develop ILD consequent to COVID-19 infection.

Establishment of an in vitro assay to detect antibody-mediated procoagulant platelet formation on a single cell level in real time

Introduction Procoagulant platelets (PLTs), a subpopulation of PLTs that is characterized by increased externalization of phosphatidylserine (PS), are increasingly identified to promote a prothrombotic environment in different diseases. Recently we observed that procoagulant PLT formation can be induced via engagement of immune receptor Fc-gamma-RIIA by COVID-19, VITT and HIT patient immunoglobulin subclass G (IgG) antibodies (Abs). Here, Fc-gamma- RIIA engagement by patient Abs resulted in significant formation of procoagulant PLTs and loss of mitochondrial potential that was associated with high thrombin formation as well as increased thrombus formation. In the cur- rent study, we aim to establish a PLT adhesion assay that allows investigation of PLT mitochondria during procoagulant PLT formation. Method PLTs were spread on human serum albumin, fibrinogen or collagen precoated glass slides. Adhesion and subsequent shape change of PLTs as well as procoagulant PLT formation were investigated in real time using immune fluorescence microscopy. For the detection of PLT shape change, mitochondrial dynamics and PS externalization, PLTs were double stained with MitoTracker green, a mitochondrial dye that binds to free thiol groups of cysteine residues in the mitochondrial membrane, and Annexin-V, respectively. For the visualization of mitochondrial release from PLTs intracellular compartment, a monoclonal Ab that binds to a subunit of the translocase of the outer membrane (TOM) complex on the mitochondrial membrane, namely TOM22, was used. Results During the observation period, a subgroup of PLTs that was spread on collagen became procoagulant as determined by an increased binding of Annexin- V on the PLT surface. Contrary, these changes were nearly absent in PLTs that adhered to fibrinogen (percentage [ %] of Annexin-V positive cells: 19.80 +/- 3.42 % vs. 1.92 +/- 0.62 %, p value 0.0357). Interestingly, procoagulant PLT formation was associated with a significant loss of MitoTracker green signal in PLTs while it remained constant in non-procoagulant PLTs attached on both extracellular matrix coatings. Loss of MitoTracker green signal was associated with translocation of mitochondrial proteins from intracellular to extracellular, as a higher count of TOM22 Ab-positive labelled structures, most likely extracellular mitochondria were detected on collagen but not on fibrinogen coated glass slides. Conclusion Our findings indicate, that the formation of procoagulant PLTs is associated with dramatic changes of the mitochondrial integrity in PLTs. Further attempts, that investigate the potential pathophysiological role of PLT mitochondrial release in Ab-mediated prothrombotic disorders may contribute to a further understanding of the role of PLT mitochondria in these complex diseases.

Family History of Alport Syndrome with Spectrum of Chronic Kidney Disease, Dialysis and Transplantation

Introduction: Alport syndrome should be considered in the differential diagnosis of patients with persistent microhematuria. Electron microscopic examination of renal tissue remains the most widely available and applied means for diagnosing AS. The presence of diffuse thickening and multilamellation of the GBM predicts a progressive nephropathy, regardless of family history. Unfortunately, ultrastructural information alone does not establish the mode of transmission in a particular family. Method(s): 18 years-old male patient was followed in the clinic due to persistent microscopic haematuria and proteinuria. Family history is significant for one brother in his early 20s, who started to have the presentation early in life and his initial biopsy showed thin basement membrane disease. The brother subsequently progressed to renal failure and a repeat biopsy confirmed the presence of Alport syndrome. Another brother had end-stage renal disease and underwent renal transplantation. The patient status was revised, and genetic studies confirmed the presence of an autosomal recessive type of Alport syndrome involving collagen for A3 chain COL4A3. His kidney function remained stable initially with an estimated GFR of approximately 90 mL/min/1.73 m2. The most recent eGFR is around 70 ml/min/1.73 m2. His proteinuria disappeared once Losartan 25 mg was added to Ramipril 5 mg. His blood pressure has been on target. Creatinine increased to 147 micromol/L and he was diagnosed as having acute kidney injury on chronic renal disease which was obvious post covid infection, then back to baseline. Current proteinuria 3 g/g Cr on Angiotensin receptor blockers. BP 110/70, all other systemic examination is unremarkable. No hearing or visual abnormalities. Result(s): The initial renal manifestations in early childhood include asymptomatic-persistent microscopic hematuria and rarely gross hematuria. At the onset, the serum creatinine and blood pressure are normal. Over time, proteinuria, hypertension, and progressive renal insufficiency develop. ESRD usually occurs between the ages of 16 and 35 years and rarely can occur between 45 and 60 years. Renal biopsy findings of thinning and multilaminar splitting of the glomerular capillary basement membrane seen on electron microscopic examination are pathognomonic. In 2013, an expert panel issued guidelines recommending genetic testing as the gold standard for the diagnosis of Alport syndrome. Currently, a skin biopsy using commercially available monoclonal antibody against the type IV collagen alpha-5 chain (COL4A5). If the protein is clearly absent in a suspected male, a diagnosis of Alport syndrome can be made without further testing. Conclusion(s): Males with X-linked AS due to a deletion mutation of the alpha 5 chain of type IV collagen usually progress to ESRD by the second or third decade of life. Likewise, patients with autosomal recessive AS due to mutations affecting alpha 3 or 4 chains of type IV collagen tend to progress to ESRD by age 30. Autosomal-dominant AS with heterozygous mutations of COL4A3 or COL4A4 usually has a slower progression of CKD. Treatment is blood pressure control with RAAS inhibitors where clinically appropriate. Cyclosporine may be helpful in some patients with stage I and II CKD with significant proteinuria. Caution using calcineurin inhibitors is indicated in all patients with more advanced CKD stages due to potential nephrotoxicity. No conflict of interestCopyright © 2023