Efficient Generation of Skin Organoids from Pluripotent Cells via Defined Extracellular Matrix Cues and Morphogen Gradients in a Spindle-Shaped Microfluidic Device.

Pluripotent stem cell-derived skin organoids (PSOs) emerge as a developmental skin model that is self-organized into multiple components, such as hair follicles. Despite their impressive complexity, PSOs are currently generated in the absence of 3D extracellular matrix (ECM) signals and have several major limitations, including an inverted anatomy (e.g., epidermis inside/dermis outside). In this work, a method is established to generate PSOs effectively in a chemically-defined 3D ECM environment. After examining various dermal ECM molecules, it is found that PSOs generated in collagen -type I (COLI) supplemented with laminin 511 (LAM511) exhibit increased growth compared to conventional free-floating conditions, but fail to induce complete skin differentiation due in part to necrosis. This problem is addressed by generating the PSOs in a 3D bioprinted spindle-shaped hydrogel device, which constrains organoid growth longitudinally. This culture system significantly reduces organoid necrosis and leads to a twofold increase in keratinocyte differentiation and an eightfold increase in hair follicle formation. Finally, the system is adapted as a microfluidic device to create asymmetrical gradients of differentiation factors and improves the spatial organization of dermal and epidermal cells. This study highlights the pivotal role of ECM and morphogen gradients in promoting and spatially-controlling skin differentiation in the PSO framework.

A New Treatment Strategy that Reduces Growth/Differentiation Factor-15 in Type-2 Diabetes Mellitus: Major Autohemotherapy: A Pilot Study.

Context: Growth/differentiation factor-15 (GDF-15) is a diagnostic and prognostic marker associated with inflammation, renal damage, and cardiovascular risk in type-2 diabetes mellitus. Researchers have proposed treatment targets that reduce GDF-15 levels. Objective: Our aim in this study is to investigate the effect of major autohemotherapy on GDF-15 levels and to evaluate it as a complementary therapy. Design: The research team designed a laboratory study to examine the effect of major hemotherapy on GDF-15 levels in patients with type-2 diabetes mellitus and healthy volunteers. Blood was drawn from the participants in a closed system, infused with ozone gas, and reinfused into the patients. Setting: The study was carried out from 15 August-5 October at Kirsehir Training and Research Hospital Traditional and Complementary Medicine Center, Turkey. Participants: The study was conducted prospectively, and two groups were formed, consisting of those with type-2 diabetes mellitus (n = 21) and healthy volunteers (n = 14). Outcome Measures: All participants received 10 sessions of major autohemotherapy at a concentration of 25-35 micrograms/milliliter twice a week. Before and after the application, GDF-15, fasting glucose, glycosylated hemoglobin, and lipid panel levels were studied and compared. Results: Age, GDF-15, fasting glucose, glycosylated hemoglobin, and triglyceride levels were found to be higher in the type-2 diabetes mellitus group compared to the healthy group, and high-density lipoprotein cholesterol levels were found to be lower. After major autohemotherapy, GDF-15 and low-density lipoprotein cholesterol decreased significantly in the type-2 diabetes mellitus group. No change was observed in the healthy group. Conclusions: As a new treatment strategy, major autohemotherapy reduces GDF-15 levels in type-2 diabetes mellitus and contributes to the therapeutic effects of ozone therapy.

Human skin-on-a-chip for mpox pathogenesis studies and preclinical drug evaluation.

Timely intervention of preventative and therapeutic measures abated a 2022 mpox global outbreak. However, the high transmissibility and unique pathological characteristics of mpox demand further investigation. Here, we discuss the potentials of human skin-on-a-chip as a valuable model for mpox disease evaluation, to achieve in-depth physiological understanding and desirable therapeutic predictive capabilities.

Clinical, virological, imaging and pathological findings in a SARS CoV-2 antibody positive cat.

This paper reports a presumptive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a cat. A cat with respiratory disease living with three individuals with coronavirus disease 2019 showed bilateral ground-glass opacities in the lung on X-ray and computed tomography. The clinical swabs were negative for SARS-CoV-2 RNA, but the serum was positive for SARS-CoV-2 antibodies. Interstitial pneumonia and prominent type 2 pneumocyte hyperplasia were noted on histopathology. Respiratory tissues were negative for SARS-CoV-2 RNA or antigen, but the cat was positive for feline parvovirus DNA. In conclusion, the respiratory disease and associated pathology in this cat could have been due to exposure to SARS-CoV-2.

An In Vitro Selection Platform to Identify Multiple Aptamers against Multiple Cell-Surface Markers Using Ligand-Guided Selection.

Aptamer ligand discovery against multiple molecules expressed on whole cells is an essential component in molecular tool development. However, owing to their intrinsic structural characteristics, cell-surface receptors have proven to be challenging targets in ligand discovery. Several variants to systematic evolution of ligands by exponential enrichment (SELEX) have been introduced to address the ″target problem″ for aptamer screening. To this end, we introduced a variant of SELEX, termed ligand-guided selection (LIGS), to identify highly specific aptamers against complex cell-surface markers in their native state. So far, the application of LIGS has been aimed at identifying aptamers against the most dominant receptors on the cell surface. Here, we report that LIGS can be expanded to identify two receptors on the same cell surface, paving the way to generate a multiplexed ligand discovery platform based on SELEX-targeting membrane receptors in their native functional state. Using CD19 and CD20 expressed on Toledo cells as a model system, multiple aptamer families were evolved against Toledo cells. We then utilized two monoclonal antibodies (mAbs) against CD20 and CD19 to selectively partition specific aptamers against CD19 and CD20. Following biochemical characterization, we introduce two specific aptamers against CD19 and two specific aptamers against CD20 with high affinity. Multi-target LIGS, as reported here, demonstrates a successful combinatorial approach for nucleic acid library screening to generate multiple artificial nucleic acid ligands against multiple receptors expressed on a single cell.

Engineering human skin model innervated with itch sensory neuron-like cells differentiated from induced pluripotent stem cells.

Atopic dermatitis (AD), driven by interleukins (IL-4/IL-13), is a chronic inflammatory skin disease characterized by intensive pruritus. However, it is unclear how immune signaling and sensory response pathways cross talk with each other. We differentiated itch sensory neuron-like cells (ISNLCs) from iPSC lines. These ISNLCs displayed neural markers and action potentials and responded specifically to itch-specific stimuli. These ISNLCs expressed receptors specific for IL-4/IL-13 and were activated directly by the two cytokines. We successfully innervated these ISNLCs into full thickness human skin constructs. These innervated skin grafts can be used in clinical applications such as wound healing. Moreover, the availability of such innervated skin models will be valuable to develop drugs to treat skin diseases such as AD.

Clinical, virological, imaging and pathological findings in a SARS CoV-2antibody positive cat

This paper reports a presumptive severe acute respiratory syndrome coronavirus 2 (SARSCoV-2) infection in a cat. A cat with respiratory disease living with three individuals with coronavirus disease 2019 showed bilateral ground-glass opacities in the lung on X-ray and computed tomography. The clinical swabs were negative for SARS-CoV-2 RNA, but the serum was positive for SARS-CoV-2 antibodies. Interstitial pneumonia and prominent type 2 pneumocyte hyperplasia were noted on histopathology. Respiratory tissues were negative for SARS-CoV-2 RNA or antigen, but the cat was positive for feline parvovirus DNA. In conclusion, the respiratory disease and associated pathology in this cat could have been due to exposure to SARS-CoV-2.

Emergence of West Nile Virus Lineage-2 in Resident Corvids in Istanbul, Turkey.

West Nile fever is a vector-borne viral disease affecting animals and humans causing significant health and economic problems globally. This study was aimed at investigating circulating West Nile virus (WNV) strains in free-ranging corvids in Istanbul, Turkey. Brain, liver, and kidney were collected from corvids (n = 34) between June 2019 and April 2020 and analyzed for the presence of WNV-specific RNA by quantitative RT-PCR. In addition, histopathologic and immunohistochemical examinations were also performed. Samples found to be positive by qRT-PCR were partially sequenced. WNV-specific RNA was detected in 8 of 34 corvids analyzed, which included 7 hooded crows (Corvus cornix) and 1 Eurasian magpie (Pica pica). Phylogenetic analysis based on partial WNV sequences from the 8 WNV-positive corvids identified in this study revealed that all sequences clustered within the WNV lineage-2; they were at least 97% homologues to WNV lineage-2 sequences from Slovakia, Italy, Czechia, Hungary, Senegal, Austria, Serbia, Greece, Bulgaria, and Germany. WNV sequences showed a divergence (87.94-94.46%) from sequences reported from Romania, Central African Republic, South Africa, Madagascar, Israel, and Cyprus, which clustered into a different clade of WNV lineage-2. Common histopathologic findings of WNV-positive corvids included lymphoplasmacytic hepatitis, myocarditis, and splenitis. The liver and heart were found to be the tissues most consistently positive for WNV-specific antigen by immunohistochemistry, followed by the kidney and brain. This study demonstrates for the first time the existence of WNV virus belonging to the genetic lineage-2 in resident corvids in Istanbul, Turkey. We hypothesize that the WNV strains circulating in Istanbul are possibly the result of a spillover event from Europe. Since WNV is a zoonotic pathogen transmitted by mosquito vectors, the emergence of WNV in Istanbul also poses a risk to humans and other susceptible animals in this densely populated city and needs to be addressed by animal and public health authorities.

Causes of Visual Impairment Among the Registered Visually Disabled: A Retrospective Study.

Purpose To study the causes of visual impairment among Bahraini patients registered as visually disabled. Materials and methods A retrospective descriptive study of all patients referred to the Ministry of Social Development for visual disability from January 2014 to December 2019 was performed. Information recorded were age, gender, the cause of the visual impairment, and visual acuity in the better eye. If a patient had multiple ophthalmic diseases, the untreatable disease causing visual impairment was recorded. Patients were considered to have visual impairment according to World Health Organization criteria. Results A total of 484 Bahraini patients were included in the study. The mean age was 57.3 years of age ranging from 3 to 100 years; 63% of the total cases were males. The most common cause of visual impairment was diabetic retinopathy (DR) 201 (41.53%), followed by glaucoma 161 (33.26%). This is followed by hereditary and congenital disorders 34 (7.02%), glaucoma combined with DR 21 (4.34%), other retinal diseases 17 (3.51), retinitis pigmentosa 14 (2.89), optic atrophy 9 (1.86), corneal disorders 8 (1.65%), age-related macular degeneration 8 (1.65%), and others 11 (0.83%). Conclusion DR and glaucoma are the major causes of visual impairment among adults. Complications leading to visual impairment of both disorders are avoidable. Prevention measures to be taken control these diseases and prevent their morbidity. Congenital and hereditary disorders are the most common causes of visual impairment among children.

Wet Cupping Therapy Improves the Parameters of Ventricular Repolarization.

BACKGROUND: Cupping therapy (CT) is an ancient medical treatment since antiquity and is used for the treatment of such various disease states as contagious diseases, chronic or acute inflammatory disease, and autoimmune disorders. Ventricular repolarization is represented by QT and corrected-QT (QTc) intervals from surface electrocardiography. OBJECTIVES: As novel repolarization parameters, Tpeak-toTend (Tp-Te) interval, and Tp-Te/QT and Tp-Te/QTc ratios are suggested to correlate better with ventricular arrhythmia risk in various clinical conditions than sole QT and QTc intervals. In this study, we aimed to determine whether these parameters changed significantly after CT in healthy individuals. METHODS: One hundred and twenty participants (57 women and 63 men; mean age: 49.0 ± 13.0 years) participated in this study. ECGs strips were recorded 1 hour before and 1 hour after CT from each participant, and relevant ECG parameters were compared. RESULTS: Tp-Te interval [69.51 ± 11.54 msec vs 63.15 ± 10.89 msec, p = 0.001], Tp-Te/QT ratio [0.191 ± 0.030 vs 0.174 ± 0.031, p = 0.002] and Tp-Te/QTc ratio [0.175 ± 0.030 vs 0.159 ± 0.026, p = 0.001] were found to be significantly decreased 1 hour after the procedure compared with the pre-procedure values. However, no statistically significant change was observed in mean heart rate, QT and QTc intervals, QT/QRS and cQT/QRS, and frontal QRS/T angle after the procedure compared with the same parameters before the procedure (p > 0.05). CONCLUSIONS: In accordance with the results of our study, it is plausible to conclude that CT may exert cardioprotective effect. However, larger scale prospective studies are needed to support our findings.

Recapitulating T cell infiltration in 3D psoriatic skin models for patient-specific drug testing.

Drug screening studies for inflammatory skin diseases are currently performed using model systems that only partially recapitulate human diseased skin. Here, we developed a new strategy to incorporate T cells into human 3D skin constructs (HSCs), which enabled us to closely monitor and quantitate T cell responses. We found that the epidermis promotes the activation and infiltration of T cells into the skin, and provides a directional cue for their selective migration towards the epidermis. We established a psoriatic HSC (pHSC) by incorporating polarized Th1/Th17 cells or CCR6+CLA+ T cells derived from psoriasis patients into the constructs. These pHSCs showed a psoriatic epidermal phenotype and characteristic cytokine profiles, and responded to various classes of psoriasis drugs, highlighting the potential utility of our model as a drug screening platform. Taken together, we developed an advanced immunocompetent 3D skin model to investigate epidermal-T cell interactions and to understand the pathophysiology of inflammatory skin diseases in a human-relevant and patient-specific context.

Ligand Guided Selection (LIGS) of Artificial Nucleic Acid Ligands against Cell Surface Targets.

With the success of RNA-based therapeutic drugs, the demand has increased for sophisticated nucleic-acid-based targeting agents. Nucleic acid aptamers (NAAs), in this regard, represent a suitable class of molecules with synthetic versatility. Aptamers are composed of single-stranded RNA/DNA/XNA molecules, which can be identified using a method called systematic evolution of ligands by exponential enrichment (SELEX) against any molecule. This Spotlight summarizes the recent introduction of ligand guided selection (LIGS), which will permit the identification of a wide range of functional aptamers against complex targets such as cell surface receptors while maintaining their native functional state. Aptamers identified from LIGS will allow researchers to develop aptamers in biomedicine as low-cost, stable therapeutic agents and diagnostic molecules or biochemical devices.

Endovascular treatment of intracranial aneurysms using the Woven EndoBridge (WEB) device: retrospective analysis of a single center experience.

INTRODUCTION: Woven EndoBridge (WEB) is an innovative device for the treatment of intracranial aneurysms especially wide-necked bifurcation aneurysms. Here we present our experience with the WEB device. MATERIAL AND METHODS: Patients treated using only the WEB device between September 2014 and November 2018 were included in the study. Follow up imaging studies and medical records of the patients were retrospectively reviewed. RESULTS: Forty-two aneurysm of 42 patients (27 female, 15 male; median age: 56, range: 32-76) were treated using the WEB device. The mean diameter of the aneurysms was 6.6 mm (range: 3-12 mm). The neck diameter was ≥4 mm in 57% of the aneurysms. The locations of the aneurysms were the middle cerebral artery bifurcation in 29 (69%), basilar tip in 5 (12%), anterior communicating artery in 5 (12%), internal carotid artery tip in 2 (5%), and M1 segment of the middle cerebral artery in 1 (2%) of the patients. Five patients had subarachnoid hemorrhage due to aneurysm rupture. The device could be successfully deployed in all of the cases. There were control imaging studies available for 36 patients who were followed up for a median of 7 months (range: 1-33 months). Adequate occlusion was observed in 35 of these 36 patients (97%). There was no treatment related morbidity or mortality. CONCLUSION: Although long term follow-up data are not available, WEB intrasaccular flow disruptor seems to be effective and safe for intracranial bifurcation aneurysm treatment in the mid-term follow up.

CRISPR/Cas9-based targeted genome editing for correction of recessive dystrophic epidermolysis bullosa using iPS cells.

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe inherited skin disorder caused by mutations in the COL7A1 gene encoding type VII collagen (C7). The spectrum of severity depends on the type of mutation in the COL7A1 gene. C7 is the major constituent of anchoring fibrils (AFs) at the basement membrane zone (BMZ). Patients with RDEB lack functional C7 and have severely impaired dermal-epidermal stability, resulting in extensive blistering and open wounds on the skin that greatly affect the patient's quality of life. There are currently no therapies approved for the treatment of RDEB. Here, we demonstrated the correction of mutations in exon 19 (c.2470insG) and exon 32 (c.3948insT) in the COL7A1 gene through homology-directed repair (HDR). We used the clustered regulatory interspaced short palindromic repeats (CRISPR) Cas9-gRNAs system to modify induced pluripotent stem cells (iPSCs) derived from patients with RDEB in both the heterozygous and homozygous states. Three-dimensional human skin equivalents (HSEs) were generated from gene-corrected iPSCs, differentiated into keratinocytes (KCs) and fibroblasts (FBs), and grafted onto immunodeficient mice, which showed normal expression of C7 at the BMZ as well as restored AFs 2 mo postgrafting. Safety assessment for potential off-target Cas9 cleavage activity did not reveal any unintended nuclease activity. Our findings represent a crucial advance for clinical applications of innovative autologous stem cell-based therapies for RDEB.

Engineering tissue-specific blood vessels.

Vascular diversity among organs has recently become widely recognized. Several studies using mouse and human fetal tissues revealed distinct characteristics of organ-specific vasculature in molecular and functional levels. Thorough understanding of vascular heterogeneities in human adult tissues is significant for developing novel strategies for targeted drug delivery and tissue regeneration. Recent advancements in microfabrication techniques, biomaterials, and differentiation protocols allowed for incorporation of microvasculature into engineered organs. Such vascularized organ models represent physiologically relevant platforms that may offer innovative tools for dissecting the effects of the organ microenvironment on vascular development and expand our present knowledge on organ-specific human vasculature. In this article, we provide an overview of the current structural and molecular evidence on microvascular diversity, bioengineering methods used to recapitulate the microenvironmental cues, and recent vascularized three-dimensional organ models from the perspective of tissue-specific vasculature.

Case 269: Sacroiliac Joint Hydatid Disease.

HistoryA 20-year old woman living in Turkey presented with a 3-month history of lower back pain. She had no medical history of note and was taking no medications. Complete blood count, C-reactive protein level, sedimentation rate, and creatinine, alanine aminotransferase, and aspartate aminotransferase levels were within normal limits. Anteroposterior pelvic radiography and unenhanced pelvic CT were performed to rule out sacroiliitis. The imaging findings were abnormal, and the patient underwent contrast-enhanced sacroiliac MRI. A few days later, she underwent contrast-enhanced (100 mL iohexol, Omnipaque; GE Healthcare, Cork, Ireland) abdominal CT because of right upper quadrant pain.

Comparison of Topical, Systemic, and Combined Therapy with Steroids on Idiopathic Granulomatous Mastitis: A Prospective Randomized Study.

BACKGROUND: Idiopathic granulomatous mastitis (IGM) is a benign disorder of the breast, for which the optimal treatment modality remains missing. METHODS: A total of 124 patients with a histopathologically proven diagnosis of IGM were enrolled in a prospective, randomized parallel arm study. Patients were treated with topical steroids in Group T (n: 42), systemic steroids (0.8 mg/kg/day peroral) in Group S (n: 42), and combined steroids (0.4 mg/kg/day peroral + topical) in Group C (n: 40). Compliance with the therapy, response to the therapy, the duration of therapy, side effects and the recurrence rates were compared. RESULTS: Sixteen patients did not comply with the treatment, and the highest ratio of compliance with therapy was seen in Group T (p < 0.05). Complete clinical regression (CCR) was observed in 90 (83.3%) patients. Response to the treatment (RT) was evaluated radiologically and observed in 89.8% of the patients. There was no statistically significant difference between groups regarding CCR, RT and the recurrence rate. The longest duration of therapy was observed in Group T (22 ± 9.1-week), whereas the shortest was observed in Group S (11.7 ± 5.5-week) (p < 0.001). The systemic side effects were significantly lower in Group T in comparison with Groups S and C (2.4% vs. 38.2% and 30.3%, respectively) (p < 0.001). CONCLUSIONS: The efficiency of the treatment was similar for all groups, both clinically and radiologically. Although the duration of therapy was longer in Group T, the lack of systemic side effects increased the compliance of the patients with the therapy. Therefore, topical steroids would be among first-line treatment options of IGM.

Integrating Ligand-Receptor Interactions and In Vitro Evolution for Streamlined Discovery of Artificial Nucleic Acid Ligands.

To discover DNA ligands against a predetermined receptor protein complex, we introduce a comprehensive version of ligand-guided selection (LIGS). LIGS is, itself, a variant of systematic evolution of ligands by exponential enrichment (SELEX). Herein, we have optimized LIGS to identify higher affinity aptamers with high specificity. In addition, we demonstrate the expandability of LIGS by performing specific aptamer elution at 25°C, utilizing multiple monoclonal antibodies (mAbs) against cultured cells and primary cells obtained from human donors expressing the same receptor. Eluted LIGS libraries obtained through Illumina high-throughput (HT) DNA sequencing were analyzed by bioinformatics tools to discover five DNA aptamers with apparent affinities ranging from 3.06 ± 0.485 nM to 325 ± 62.7 nM against the target, T cell receptor-cluster of differentiation epsilon (TCR-CD3ε) expressed on human T cells. The specificity of the aptamers was validated utilizing multiple strategies, including competitive binding analysis and a double-knockout Jurkat cell line generated by CRISPR technology. The cross-competition experiments using labeled and unlabeled aptamers revealed that all five aptamers compete for the same binding site. Collectively, the data in this report introduce a modified LIGS strategy as a universal platform to identify highly specific multiple aptamers toward multi-component receptor proteins in their native state without changing the cell-surface landscape.

Case 269.

History A 20-year old woman living in Turkey presented with a 3-month history of lower back pain. She had no medical history of note and was taking no medications. Complete blood count, C-reactive protein level, sedimentation rate, and creatinine, alanine aminotransferase, and aspartate aminotransferase levels were within normal limits. Anteroposterior pelvic radiography and unenhanced pelvic CT were performed to rule out sacroiliitis. The imaging findings were abnormal, and the patient underwent contrast-enhanced sacroiliac MRI. A few days later, she underwent contrast-enhanced (100 mL iohexol, Omnipaque; GE Healthcare, Cork, Ireland) abdominal CT because of right upper quadrant pain. Figure 1: Anteroposterior pelvic radiograph. Figure 2: Axial unenhanced pelvic CT image. Figure 3a: (a) Coronal T2-weighted fat-saturated fast spin-echo (repetition time msec/echo time msec, 2220/57; section thickness, 4 mm), (b) axial unenhanced T1-weighted fat-saturated, and (c) axial contrast-enhanced (20 mL gadoteric acid, Dotarem; Guerbet, Roissy, France) T1-weighted fat-saturated (400/20; section thickness, 4 mm) sacroiliac images from MRI. Figure 3b: (a) Coronal T2-weighted fat-saturated fast spin-echo (repetition time msec/echo time msec, 2220/57; section thickness, 4 mm), (b) axial unenhanced T1-weighted fat-saturated, and (c) axial contrast-enhanced (20 mL gadoteric acid, Dotarem; Guerbet, Roissy, France) T1-weighted fat-saturated (400/20; section thickness, 4 mm) sacroiliac images from MRI. Figure 3c: (a) Coronal T2-weighted fat-saturated fast spin-echo (repetition time msec/echo time msec, 2220/57; section thickness, 4 mm), (b) axial unenhanced T1-weighted fat-saturated, and (c) axial contrast-enhanced (20 mL gadoteric acid, Dotarem; Guerbet, Roissy, France) T1-weighted fat-saturated (400/20; section thickness, 4 mm) sacroiliac images from MRI. Figure 4: Axial contrast-enhanced CT image of the abdomen.