Nail involvement in patients with epidermolysis bullosa: A systematic review.

Background: Nail changes in patients with congenital epidermolysis bullosa (EB) are caused by abnormalities of the nail matrix and bed secondary to pathogenic alterations of the dermoepidermal junction. Even though ungual alterations are extremely frequent in these patients, there are scarce studies about their frequency and/or association with subtypes or clinical course of EB. Objectives: To systematically review nail abnormalities in patients with EB reported in the literature. Methods: We searched all published articles in electronic databases until June 2020 reporting patients with EB with detailed descriptions of malformed/diseased nails using specific terms and inclusion/exclusion criteria. Clinical data were extracted by two independent authors. Descriptive statistics were used. Results: We included 36 articles reporting 74 individual patients with a mean age of 28.23 years: 29 (39.2%) had dominant dystrophic EB, 27 (36.4%) had junctional EB, 8 (10.8%) had EB simplex, 6 (8.1%) had Kindler syndrome and 4 (5.4%) had recessive dystrophic EB. The most common abnormalities were dystrophic nails (48.6%), anonychia (43.2%) and pachyonychia (40.5%). Anonychia was considered the most severe abnormality and was reported more frequently in patients with junctional (62.9%) and recessive dystrophic EB (50%). Multiple organ involvement was present in 52.7% of patients. Patients with severe junctional epidermolysis bullosa and recessive dominant epidermolysis bullosa presented anonychia since birth. Conclusions: In this summary of nail abnormalities in patients with EB, anonychia was more frequent in patients with severe EB subtypes and multiple organ involvement. Further prospective studies are required to understand the associations between nail abnormalities in specific EB subtypes and/or patient outcomes.

Characterization of immunoglobulin loci in the gigantic genome of Ambystoma mexicanum.

Background: The axolotl, Ambystoma mexicanum is a unique biological model for complete tissue regeneration. Is a neotenic endangered species and is highly susceptible to environmental stress, including infectious disease. In contrast to other amphibians, the axolotl is particularly vulnerable to certain viral infections. Like other salamanders, the axolotl genome is one of the largest (32 Gb) and the impact of genome size on Ig loci architecture is unknown. To better understand the immune response in axolotl, we aimed to characterize the immunoglobulin loci of A. mexicanum and compare it with other model vertebrates. Methods: The most recently published genome sequence of A. mexicanum (V6) was used for alignment-based annotation and manual curation using previously described axolotl Ig sequences or reference sequences from other vertebrates. Gene models were further curated using A. mexicanum spleen RNA-seq data. Human, Xenopus tropicalis, Danio rerio (zebrafish), and eight tetrapod reference genomes were used for comparison. Results: Canonical A. mexicanum heavy chain (IGH), lambda (IGL), sigma (IGS), and the putative surrogate light chain (SLC) loci were identified. No kappa locus was found. More than half of the IGHV genes and the IGHF gene are pseudogenes and there is no clan I IGHV genes. Although the IGH locus size is proportional to genome size, we found local size restriction in the IGHM gene and the V gene intergenic distances. In addition, there were V genes with abnormally large V-intron sizes, which correlated with loss of gene functionality. Conclusion: The A. mexicanum immunoglobulin loci share the same general genome architecture as most studied tetrapods. Consistent with its large genome, Ig loci are larger; however, local size restrictions indicate evolutionary constraints likely to be imposed by high transcriptional demand of certain Ig genes, as well as the V(D)J recombination over very long genomic distance ranges. The A. mexicanum has undergone an extensive process of Ig gene loss which partially explains a reduced potential repertoire diversity that may contribute to its impaired antibody response.

Bioinformatic Analysis of B- and T-cell Epitopes from SARS-CoV-2 Structural Proteins and their Potential Cross-reactivity with Emerging Variants and other Human Coronaviruses.

BACKGROUND: The mutations in SARS-CoV-2 variants of concern (VOC) facilitate the virus' escape from the neutralizing antibodies induced by vaccines. However, the protection from hospitalization and death is not significantly diminished. Both vaccine boosters and infection improve immune responses and provide protection, suggesting that conserved and/or cross-reactive epitopes could be involved. While several important T- and B-cell epitopes have been identified, mainly in the S protein, the M and N proteins and their potential cross-reactive epitopes with other coronaviruses remain largely unexplored. AIMS: To identify and map new potential B- and T-cell epitopes within the SARS-CoV-2 S, M and N proteins, as well as cross-reactive epitopes with human coronaviruses. METHODS: Different bioinformatics tools were used to: i) Identify new and compile previously-reported B-and T-cell epitopes from SARS-CoV-2 S, M and N proteins; ii) Determine the mutations in S protein from VOC that affect B- and T-cell epitopes, and; iii) Identify cross-reactive epitopes with coronaviruses relevant to human health. RESULTS: New, potential B- and T-cell epitopes from S, M and N proteins as well as cross-reactive epitopes with other coronaviruses were found and mapped within the proteins' structures. CONCLUSION: Numerous potential B- and T-cell epitopes were found in S, M and N proteins, some of which are conserved between coronaviruses. VOCs present mutations within important epitopes in the S protein; however, a significant number of other epitopes remain unchanged. The epitopes identified here may contribute to augmenting the protective response to SARS-CoV-2 and its variants induced by infection and/or vaccination, and may also be used for the rational design of novel broad-spectrum coronavirus vaccines.

Advances in Our Understanding of the Interaction of Drugs with T-cells: Implications for the Discovery of Biomarkers in Severe Cutaneous Drug Reactions.

Drugs can activate different cells of the immune system and initiate an immune response that can lead to life-threatening diseases collectively known as severe cutaneous adverse reactions (SCARs). Antibiotics, anticonvulsants, and antiretrovirals are involved in the development of SCARs by the activation of αß naïve T-cells. However, other subsets of lymphocytes known as nonconventional T-cells with a limited T-cell receptor repertoire and innate and adaptative functions also recognize drugs and drug-like molecules, but their role in the pathogenesis of SCARs has only just begun to be explored. Despite 30 years of advances in our understanding of the mechanisms in which drugs interact with T-cells and the pathways for tissue injury seen during T-cell activation, at present, the development of useful clinical biomarkers for SCARs or predictive preclinical in vitro assays that could identify immunogenic moieties during drug discovery is an unmet goal. Therefore, the present review focuses on (i) advances in the understanding of the pathogenesis of SCARs reactions, (ii) a description of the interaction of drugs with conventional and nonconventional T-cells, and (iii) the current state of soluble blood circulating biomarker candidates for SCARs.

Nutritional status of pediatric patients with epidermolysisbullosa. A cross-sectional study / Estado nutricional de pacientes pediátricos con epidermólisis ampollosa. Un estudio transversal

Epidermolysis bullosa (EB) is a genetic disorder with con-tinuous formation of blisters and erosions in the skin and mu-cous membranes as well as multi-systemic involvement.Patients are at high-risk of malnutrition due to decreased foodintake and increased nutrient demand. This cross-sectionalretrospective study evaluated the daily caloric intake and nu-tritional status of pediatric patients with EB at a specializedclinic through anthropometric measurements and estimationof the daily intake by 24-hour dietary recall. We used the Waterlow and World Health Organization (WHO) malnutritionclassification schemes. Descriptive statistics were used. Weincluded 17 patients with a mean age of 8.4 years (SD 4.6),82.3% had malnutrition. Those with more severe subtypes,junctional and recessive dystrophic EB, had acute superim-posed on chronic malnutrition (100% and 63.4% respec-tively), wasting (100% and 72.6%), and stunting (0% and54.4%) more frequently. Most patients required supplemen-tation (caloric 76.4% and vitamin/mineral 100%). We concluded that there is a high frequency of malnutritionin our EB patients. Although their energy requirements is cal-culated to be increased in 100-150% of the estimate, our pa-tients only reach 73.1% of that, thus requiring supplementa-tion. Patients with more severe subtypes of EB had chronicmalnutrition more frequently. Even though malnutrition isclosely linked to wound healing and adequate growth and de-velopment of patients, there are few studies about nutritionin EB worldwide. We believe evaluating the nutritional status of these patients is the first step to identifying deficiencies, of-fering adequate comprehensive medical care and establishingnutritional interventions in a timely manner.(AU)

A bioinformatic prediction of antigen presentation from SARS-CoV-2 spike protein revealed a theoretical correlation of HLA-DRB1*01 with COVID-19 fatality in Mexican population: An ecological approach.

SARS-CoV-2 infection is causing a pandemic disease that is reflected in challenging public health problems worldwide. Human leukocyte antigen (HLA)-based epitope prediction and its association with disease outcomes provide an important base for treatment design. A bioinformatic prediction of T cell epitopes and their restricted HLA Class I and II alleles was performed to obtain immunogenic epitopes and HLA alleles from the spike protein of the severe acute respiratory syndrome coronavirus 2 virus. Also, a correlation with the predicted fatality rate of hospitalized patients in 28 states of Mexico was done. Here, we describe a set of 10 highly immunogenic epitopes, together with different HLA alleles that can efficiently present these epitopes to T cells. Most of these epitopes are located within the S1 subunit of the spike protein, suggesting that this area is highly immunogenic. A statistical negative correlation was found between the frequency of HLA-DRB1*01 and the fatality rate in hospitalized patients in Mexico.

RON2, a novel gene in Babesia bigemina, contains conserved, immunodominant B-cell epitopes that induce antibodies that block merozoite invasion.

Bovine babesiosis is the most important protozoan disease transmitted by ticks. In Plasmodium falciparum, another Apicomplexa protozoan, the interaction of rhoptry neck protein 2 (RON2) with apical membrane antigen-1 (AMA-1) has been described to have a key role in the invasion process. To date, RON2 has not been described in Babesia bigemina, the causal agent of bovine babesiosis in the Americas. In this work, we found a ron2 gene in the B. bigemina genome. RON2 encodes a protein that is 1351 amino acids long, has an identity of 64% (98% coverage) with RON2 of B. bovis and contains the CLAG domain, a conserved domain in Apicomplexa. B. bigemina ron2 is a single copy gene and it is transcribed and expressed in blood stages as determined by RT-PCR, Western blot, and confocal microscopy. Serum samples from B. bigemina-infected bovines were screened for the presence of RON2-specific antibodies, showing the recognition of conserved B-cell epitopes. Importantly, in vitro neutralization assays showed an inhibitory effect of RON2-specific antibodies on the red blood cell invasion by B. bigemina. Therefore, RON2 is a novel antigen in B. bigemina and contains conserved B-cell epitopes, which induce antibodies that inhibit merozoite invasion.

Pamidronate Administration During Pregnancy and Lactation Induces Temporal Preservation of Maternal Bone Mass in a Mouse Model of Osteogenesis Imperfecta.

During pregnancy and lactation, the maternal skeleton undergoes significant bone loss through increased resorption to provide the necessary calcium supply to the developing fetus and suckling neonate. This period of skeletal vulnerability has not been clearly associated with increased maternal fracture risk, but these physiological conditions can exacerbate an underlying metabolic bone condition like osteogenesis imperfecta. Although bisphosphonates (BPs) are commonly used in postmenopausal women, there are cases where premenopausal women taking BPs become pregnant. Given BPs' long half-life, there is a need to establish how BPs affect the maternal skeleton during periods of demanding metabolic bone changes that are critical for the skeletal development of their offspring. In the present study, pamidronate- (PAM-) amplified pregnancy-induced bone mass gains and lactation-induced bone loss were prevented. This preservation of bone mass was less robust when PAM was administered at late stages of lactation compared with early pregnancy and first day of lactation. Pregnancy-induced osteocyte osteolysis was also observed and was unaffected with PAM treatment. No negative skeletal effects were observed in offspring from PAM-treated dams despite lactation-induced bone loss prevention. These findings provide important insight into (1) a treatment window for when PAM is most effective in preserving maternal bone mass, and (2) the maternal changes in bone metabolism that maintain calcium homeostasis crucial for fetal and neonatal bone development. © 2019 American Society for Bone and Mineral Research.

Low Dose of Bisphosphonate Enhances Sclerostin Antibody-Induced Trabecular Bone Mass Gains in Brtl/+ Osteogenesis Imperfecta Mouse Model.

Osteogenesis imperfecta (OI) is a genetic disorder characterized by altered bone quality and imbalanced bone remodeling, leading to skeletal fractures that are most prominent during childhood. Treatments for OI have focused on restoring pediatric bone density and architecture to recover functional strength and consequently reduce fragility. Though antiresorptive agents like bisphosphonates (BPs) are currently the most common intervention for the treatment of OI, a number of studies have shown efficacy of sclerostin antibody (SclAb) in inducing gains in bone mass and reducing fragility in OI mouse models. In this study, the effects of the concurrent use of BP and SclAb were evaluated during bone growth in a mouse harboring an OI-causing Gly→Cys mutation on col1a1. A single dose of antiresorptive BP facilitated the anabolic action of SclAb by increasing availability of surfaces for new bone formation via retention of primary trabeculae that would otherwise be remodeled. Chronic effects of concurrent administration of BP and SclAb revealed that accumulating cycles conferred synergistic gains in trabecular mass and vertebral stiffness, suggesting a distinct advantage of both therapies combined. Cortical gains in mass and strength occurred through SclAb alone, independent of presence of BP. In conclusion, these preclinical results support the scientific hypothesis that minimal antiresorptive treatment can amplify the effects of SclAb during early stages of skeletal growth to further improve bone structure and rigidity, a beneficial outcome for children with OI. © 2018 American Society for Bone and Mineral Research.

3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration.

Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1-2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing.

Reversing bone loss by directing mesenchymal stem cells to bone.

Bone regeneration by systemic transplantation of mesenchymal stem cells (MSCs) is problematic due to the inability to control the MSCs' commitment, growth, and differentiation into functional osteoblasts on the bone surface. Our research group has developed a method to direct the MSCs to the bone surface by conjugating a synthetic peptidomimetic ligand (LLP2A) that has high affinity for activated α4ß1 integrin on the MSC surface, with a bisphosphonates (alendronate) that has high affinity for bone (LLP2A-Ale), to direct the transplanted MSCs to bone. Our in vitro experiments demonstrated that mobilization of LLP2A-Ale to hydroxyapatite accelerated MSC migration that was associated with an increase in the phosphorylation of Akt kinase and osteoblastogenesis. LLP2A-Ale increased the homing of the transplanted MSCs to bone as well as the osteoblast surface, significantly increased the rate of bone formation and restored both trabecular and cortical bone loss induced by estrogen deficiency or advanced age in mice. These results support LLP2A-Ale as a novel therapeutic option to direct the transplanted MSCs to bone for the treatment of established bone loss related to hormone deficiency and aging.

Prolonged alendronate treatment prevents the decline in serum TGF-ß1 levels and reduces cortical bone strength in long-term estrogen deficiency rat model.

INTRODUCTION: While the anti-resorptive effects of the bisphosphonates (BPs) are well documented, many questions remain about their mechanisms of action, particularly following long-term use. This study evaluated the effects of alendronate (Ale) treatment on TGF-ß1 signaling in mesenchymal stem cells (MSCs) and osteocytes, and the relationship between prolonged alendronate treatment on systemic TGF-ß1 levels and bone strength. METHODS: TGF-ß1 expression and signaling were evaluated in MSCs and osteocytic MLO-Y4 cells following Ale treatment. Serum total TGF-ß1 levels, a bone resorption marker (DPD/Cr), three-dimensional microCT scans and biomechanical tests from both the trabecular and cortical bone were measured in ovariectomized rats that either received continuous Ale treatment for 360 days or Ale treatment for 120 days followed by 240 days of vehicle. Linear regression tests were performed to determine the association of serum total TGF-ß1 levels and both the trabecular (vertebrae) and cortical (tibiae) bone strength. RESULTS: Ale increased TGF-ß1 signaling in the MSCs but not in the MLO-Y4 cells. Ale treatment increased serum TGF-ß1 levels and the numbers of TGF-ß1-positive osteocytes and periosteal cells in cortical bone. Serum TGF-ß1 levels were not associated with vertebral maximum load and strength but was negatively associated with cortical bone maximum load and ultimate strength. CONCLUSIONS: The increase of serum TGF-ß1 levels during acute phase of estrogen deficiency is likely due to increased osteoclast-mediated release of matrix-derived latent TGF-ß1. Long-term estrogen-deficiency generally results in a decline in serum TGF-ß1 levels that are maintained by Ale treatment. Measuring serum total TGF-ß1 levels may help to determine cortical bone quality following alendronate treatment.

Mixed-mode toughness of human cortical bone containing a longitudinal crack in far-field compression.

Bone is generally loaded under multiaxial conditions in vivo; as it invariably contains microcracks, this leads to complex mixed-mode stress-states involving combinations of tension, compression and shear. In previous work on the mixed-mode loading of human cortical bone (using an asymmetric bend test geometry), we found that the bone toughness was lower when loaded in far-field shear than in tension (opposite to the trend in most brittle materials), although only for the transverse orientation. This is a consequence of the competition between preferred mechanical vs. microstructural crack-path directions, the former dictated by the direction of the maximum mechanical "driving force" (which changes with the mode-mixity), and the latter by the "weakest" microstructural path (which in human bone is along the osteonal interfaces or cement lines). As most microcracks are oriented longitudinally, we investigate here the corresponding mixed-mode toughness of human cortical bone in the longitudinal (proximal-distal) orientation using a "double cleavage drilled compression" test geometry, which provides a physiologically-relevant loading condition for bone in that it characterizes the toughness of a longitudinal crack loaded in far-field compression. In contrast to the transverse toughness, results show that the longitudinal toughness, measured using the strain-energy release rate, is significantly higher in shear (mode II) than in tension (mode I). This is consistent, however, with the individual criteria of preferred mechanical vs. microstructural crack paths being commensurate in this orientation.