Effect of the Intralesional Forms of Methylprednisolone Acetate and Methotrexate on Psoriatic Nails.

Individuals with psoriatic nails often have a lower quality of life relative to their counterparts with healthy nails. Methotrexate (MTX), an anti-neoplastic agent, is a longstanding treatment option for nail psoriasis. In the current study, we compared the effects of MTX to that of a corticosteroid, namely, methylprednisolone acetate (i.e., Depo-Medrol®) across individuals with nail psoriasis. We used a cohort study design, and both agents were administered intralesionally. Outcome variables were based on the Nail Psoriasis Severity Index (NAPSI). We quantified the effect in terms of change in NAPSI, complete cure at week 16, and cure between 32 and 36 weeks. Our regressions demonstrated that reduced NAPSI scores with Depo-Medrol were, on average, greater than that with MTX by 2.27 (n = 48, P = 0.000255) at week 16. Similarly, the odds of complete cure at week 16 was greater with Depo-Medrol® than with MTX (odds ratio = 18.6, P < 0.0001). In terms of both complete cure and change in NAPSI, Depo-Medrol® was significantly more effective than MTX at a follow-up period of 32-36 weeks. Our study established that intralesional Depo-Medrol® is more effective than intralesional methotrexate for treating nail psoriasis.

Chiral Monitoring Across Both Enantiomeric Excess and Concentration Space: Leveraging Quantum Cascade Lasers for Sensitive Vibrational Circular Dichroism Spectroscopy.

Recently, high-throughput quantum cascade laser-based vibrational circular dichroism (QCL-VCD) technology has reduced the measurement time for high-quality vibrational circular dichroism spectra from hours to a few minutes. This study evaluates QCL-VCD for chiral monitoring using flow-through measurement of a changing sample in a circulating loop. A balanced detection QCL-VCD system was applied to the enantiomeric pair R/S-1,1'-bi-2-naphthol in solution. Different mixtures of the two components were used to simulate a racemization process, collecting spectral data at a time resolution of 6 min, and over three concentration levels. The goal of this experimental setup was to evaluate QCL-VCD in terms of both molar and enantiomeric excess (EE) sensitivity at a time resolution relevant to chiral monitoring in chemical processes. Subsequent chemometric evaluation by partial least squares regression revealed a cross-validated prediction accuracy of 2.8% EE with a robust prediction also for the test data set (error = 3.5% EE). In addition, the data set was also treated with the least absolute shrinkage and selection operator (LASSO), which also achieved a robust prediction. Due to the operating principle of LASSO, the obtained coefficients constituted a few discrete spectral frequencies, which represent the most variance. This information can be used in the future for dedicated QCL-based instrument design, gaining a higher time resolution without sacrificing predictive capabilities.

Antibody Selection via Phage Display in Microtiter Plates.

The most common and robust in vitro technology to generate monoclonal human antibodies is phage display. This technology is a widely used and powerful key technology for recombinant antibody selection. Phage display-derived antibodies are used as research tools, in diagnostic assays, and by 2022, 14 phage display-derived therapeutic antibodies were approved. In this review, we describe a fast high-throughput antibody (scFv) selection procedure in 96-well microtiter plates. The given detailed protocol allows the antibody selection ("panning"), screening, and identification of monoclonal antibodies in less than 2 weeks. Furthermore, we describe an on-rate panning approach for the selection of monoclonal antibodies with fast on-rates.

Antibody Batch Cloning.

The antigen-binding ability of each antibody clone selected by phage display is usually initially ranked by a screening ELISA using monovalent scFv antibody fragments. Further characterization often requires bivalent antibody molecules such as IgG or scFv-Fc fusions. To produce these, the V region encoding genes of selected hits have to be cloned into a mammalian expression vector and analyzed as a bivalent molecule, requiring a laborious cloning procedure. We established a high-throughput procedure allowing rapid screening of candidates in bivalent formats. This protocol allows for the parallelized cloning of all selected antibody fragments into a mammalian expression vector in the 96-well plate format. The bivalent antibody molecules can then be produced and purified in 96-well plates for further analysis in microtiter plate assays.

Mapping Epitopes by Phage Display.

Monoclonal antibodies (mAbs) are valuable biological molecules, serving for many applications. Therefore, it is advantageous to know the interaction pattern between antibodies and their antigens. Regions on the antigen which are recognized by the antibodies are called epitopes, and the respective molecular counterpart of the epitope on the mAbs is called paratope. These epitopes can have many different compositions and/or structures. Knowing the epitope is a valuable information for the development or improvement of biological products, e.g., diagnostic assays, therapeutic mAbs, and vaccines, as well as for the elucidation of immune responses. Most of the techniques for epitope mapping rely on the presentation of the target, or parts of it, in a way that it can interact with a certain mAb. Among the techniques used for epitope mapping, phage display is a versatile technology that allows the display of a library of oligopeptides or fragments from a single gene product on the phage surface, which then can interact with several antibodies to define epitopes. In this chapter, a protocol for the construction of a single-target oligopeptide phage library, as well as for the panning procedure for epitope mapping using phage display is given.

Generation of Chimeric Antigen Receptors against Tetraspanin 7.

Adoptive transfer of antigen-specific regulatory T cells (Tregs) has shown promising results in the treatment of autoimmune diseases; however, the use of polyspecific Tregs has limited effects. However, obtaining a sufficient number of antigen-specific Tregs from patients with autoimmune disorders remains challenging. Chimeric antigen receptors (CARs) provide an alternative source of T cells for novel immunotherapies that redirect T cells independently of the MHC. In this study, we aimed to generate antibody-like single-chain variable fragments (scFv) and subsequent CARs against tetraspanin 7 (TSPAN7), a membrane protein highly expressed on the surface of pancreatic beta cells, using phage display technology. We established two methods for generating scFvs against TSPAN7 and other target structures. Moreover, we established novel assays to analyze and quantify their binding abilities. The resulting CARs were functional and activated specifically by the target structure, but could not recognize TSPAN7 on the surface of beta cells. Despite this, this study demonstrates that CAR technology is a powerful tool for generating antigen-specific T cells and provides new approaches for generating functional CARs.

Diagnostic approach to tinea capitis with kerion: A retrospective study.

BACKGROUND: Kerion is an inflammatory type of tinea capitis manifesting as boggy crusted nodules. Diagnosis of kerion is often challenging due to high rates of false-negative mycological samples. METHODS: A retrospective study among children with kerion, prior to antifungal treatment, was conducted to assess rates of false-negative mycological samples. Specimens for direct microscopy and fungal culture were collected at baseline and after administration of an oral antibiotic course, with or without an oral steroid course. Kerion was categorized as highly inflammatory when a painful, moist scalp nodule with spontaneous purulent discharge or exuberant crust was present, or mildly inflammatory when an erythematous, dry scalp nodule was seen. RESULTS: Twenty-three children (mean age 7.9 ± 3.0 years) were included in the study. Trichophyton tonsurans was the most common species isolated (69.6%). Highly inflammatory kerions were significantly more likely to be culture negative before treatment than mildly inflammatory kerions (80% vs. 16.7%, p < .01). Non-inflammatory tinea capitis lesions (n = 13) were culture positive in all cases. Following a combined oral antibiotic and steroid course given to most highly inflammatory kerions (n = 11/13), higher rates of positive fungal cultures were found compared to baseline (90.9% vs. 18.2%, p < .01). CONCLUSION: High rates of negative fungal cultures were found only in highly inflammatory kerion. Sampling a highly inflammatory kerion after a combined oral antibiotic and steroid course improved rates of positive fungal cultures. In addition, sampling of non-inflammatory tinea capitis lesions (when present in addition to the kerion) had the highest culture sensitivity.

Efficacy of Fluocinonide\Bifonazole Cream for the Treatment of Trachyonychia: A Retrospective Case Series.

Background: Trachyonychia is a benign nail condition, most commonly seen in children. Trachyonychia manifests as excessive longitudinal ridging, nail roughness, and nail brittleness. Treatment is sought mainly for esthetic reasons as well as functional reasons. A number of therapeutic approaches exist, mainly based on case reports or small non-comparative case series. Aims: To report treatment outcome among patients with trachyonychia. Methods: A retrospective case-series study was conducted among patients who were treated trachyonychia between years 2017 and 2020. Patients were prescribed fluocinonide 0.05% with bifonazole 1% cream, applied with or without occlusion, and methylprednisolone 1-2 mg/nail, injected into the involved nail matrix, or oral cyclosporine 3 mg/kg. Complete response (over 90% improvement) and partial response (over 50% improvement) were assessed. Results: A total of 43 patients with trachyonychia were included [mean age 10.0 years (±5.7), 69.8% males, mean disease duration 4.7 years (±3.0)]. In 90.7% of the cases, topical fluocinonide\bifonazole cream was prescribed. Under-occlusion topical application was found to be highly effective, achieving complete response in 35.3% and partial response in an additional 52.9% of the patients. Occluded application was also found to be significantly more effective than non-occluded application. Treatment efficacy was not affected by the severity of nail roughness, trachyonychia morphology, or whether trachyonychia was idiopathic or coexisted with other dermatological conditions. Conclusion: Occluded application of fluocinonide plus bifonazole cream is efficient for the treatment of trachyonychia and should be considered a first line of treatment.

Development of the Oral Health Literacy Profile (OHLP)-Psychometric properties of the oral health and dental health system knowledge scales.

OBJECTIVES: Current oral health literacy (OHL) instruments vary in focus, methodology, administration time and burden. The widely used word recognition tests fail to encompass important OHL domains, while others exceed time frames for use in practical settings, require an interviewer or mainly measure self-assessments. This paper describes the development of the Oral Health Literacy Profile (OHLP), introduces its components and evaluates the psychometric properties of its two core modules, the knowledge tests. METHODS: A preliminary version of the questionnaire was developed and assessed for content validity, relevance and redundancy by an expert panel. It was tested in a convenience sample (n = 95) leading to the creation of a 28-item questionnaire, which was afterwards tested in a second convenience sample (n = 193). Item difficulty, discrimination, internal reliability and construct validity were assessed for the oral health knowledge (OHK) and dental health system knowledge (DHSK) modules. RESULTS: The items showed acceptable range of difficulty (ideal: 7 items; easy: 5 items; and difficult: 3 items) and good to very good discriminatory power (the point-biserial index (PBI) > 0.30: all items). Construct validity was considered to be fulfilled when 75% of the hypotheses of expected group differences were met. Satisfactory internal reliability was observed. CONCLUSION: With all its components, the OHLP is a suitable short instrument to assess the most relevant dimensions of the multifaceted construct of OHL. PRACTICE IMPLICATION: The OHLP can be widely used in research, especially suitable in practical settings, and thereby identify patients who may benefit from oral health education.

Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy.

Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications.

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients binds to the ACE2-RBD interface and is tolerant to most known RBD mutations.

The novel betacoronavirus severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) causes a form of severe pneumonia disease called coronavirus disease 2019 (COVID-19). To develop human neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor-binding domain (RBD) of the spike protein were selected by phage display. The antibody STE90-C11 shows a subnanometer IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody is demonstrated in the Syrian hamster and in the human angiotensin-converting enzyme 2 (hACE2) mice model. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD is solved at 2.0 Å resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibition of STE90-C11 is not blocked by many known emerging RBD mutations. STE90-C11-derived human IgG1 with FcγR-silenced Fc (COR-101) is undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19.

Management of tinea capitis in Israel: A comparative study.

BACKGROUND: Tinea capitis is a common fungal infection in Israel, most commonly caused by the dermatophyte Trichophyton tonsurans. OBJECTIVES: To investigate the effectiveness of oral antifungal monotherapy in producing clinical or complete cure. We also evaluated the impact of topical therapy (bifonazole 1% shampoo and/or betamethasone valerate 0.1% solution), prior to oral treatment, on patients' likelihood of clinical or complete cure. METHODS: A retrospective chart review was conducted. Patients with mycologically confirmed tinea capitis were treated with one of four regimens: (1) terbinafine (greater than 40 kg: 250 mg/day, 20 to 40 kg: 125 mg/day, less than 20 kg: 62.5 mg/day), (2) itraconazole 5 mg/kg daily, (3) fluconazole 6 mg/kg daily, or (4) griseofulvin 20 mg/kg daily. We used generalized linear models (GLM) to determine whether there was a significant association between the odds of cure and choice of treatment. RESULTS: The causative species was Trichophyton tonsurans in all but 6 cases that grew T violaceum. For pediatric patients, the odds of having complete or clinical cure within 6 weeks was greater if they used terbinafine compared to itraconazole, fluconazole, or griseofulvin (odds ratio [OR] = 9.06, P = .047). The likelihood of complete or clinical cure within 8 weeks of oral therapy was lower if topical steroids were previously used compared to if topical antifungals were used prior to systemic treatment (OR = 0.29, P = .046). CONCLUSIONS: Our findings substantiate prior literature demonstrating that terbinafine is non-inferior to griseofulvin, itraconazole, and fluconazole in the therapy of pediatric tinea capitis caused by T tonsurans.

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface.

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.

Baculovirus-free insect cell expression system for high yield antibody and antigen production.

Antibodies are essential tools for therapy and diagnostics. Yet, production remains expensive as it is mostly done in mammalian expression systems. As most therapeutic IgG require mammalian glycosylation to interact with the human immune system, other expression systems are rarely used for production. However, for neutralizing antibodies that are not required to activate the human immune system as well as antibodies used in diagnostics, a cheaper production system would be advantageous. In our study, we show cost-efficient, easy and high yield production of antibodies as well as various secreted antigens including Interleukins and SARS-CoV-2 related proteins in a baculovirus-free insect cell expression system. To improve yields, we optimized the expression vector, media and feeding strategies. In addition, we showed the feasibility of lyophilization of the insect cell produced antibodies. Furthermore, stability and activity of the antibodies was compared to antibodies produced by Expi293F cells revealing a lower aggregation of antibodies originating from High Five cell production. Finally, the newly established High Five expression system was compared to the Expi293F mammalian expression system in regard of yield and costs. Most interestingly, all tested proteins were producible in our High Five cell expression system what was not the case in the Expi293F system, hinting that the High Five cell system is especially suited to produce difficult-to-express target proteins.

Cold sub-atmospheric and atmospheric pressure plasma for the treatment of Trichophyton rubrum onychomycosis: An in-vitro study.

Previous studies have suggested the applicability of cold atmospheric pressure plasma for the treatment of onychomycosis. Whether delivering cold plasma in sub-atmospheric pressure would be beneficial for this purpose is yet to be established. The current study aimed to evaluate efficacy of cold sub-atmospheric and atmospheric pressure plasma in Trichophyton rubrum growth inhibition. Bovine nails infected with T. rubrum were treated by a cold air plasma device, which enables utilizing plasma in sub-atmospheric pressures (Low = 100 millibar; High = 300 millibar) or atmospheric pressure. The infected foci were exposed to the plasma source directly or indirectly. Treatment with high sub-atmospheric pressure setting achieved T. rubrum growth reduction of 94.0% and 73.0%, for direct and indirect exposure to the plasma source, respectively (P < .001). Low sub-atmospheric pressure setting achieved similar T. rubrum growth reduction of 86.2% for direct exposure to the plasma source (P < .001), but only marginally significant 58.8% reduction rate for indirect exposure to the plasma source (P = .056). None statistically significant fungal growth reduction was attained with the use of atmospheric pressure setting. Cold plasma was shown to effectively inhibit T. rubrum nail growth, with sub-atmospheric pressure setting achieving better outcome than atmospheric pressure.

In situ based surface-enhanced Raman spectroscopy (SERS) for the fast and reproducible identification of PHB producers in cyanobacterial cultures.

The production of polyhydroxybutyrate (PHB) by autotrophic fermentation of cyanobacteria has received increasing interest in the light of carbon emission reducing process strategies. Biotechnological approaches are in development to optimize the yield of PHB, including adapted cultivation media, characterized by a limitation of key nutrients: cyanobacteria accumulate PHB as energy storage molecules under limited growth conditions. Since there is an increasing demand for fast, simple and reliable analytics, we report the establishment of surface enhanced Raman spectroscopy (SERS) as a suitable monitoring tool for up scaled PHB production processes. Both, pure Ag-colloids mixed with bacterial culture, and in situ prepared colloids (Ag-Synechocystis), generated on the cell surface directly, were successfully applied and evaluated for this purpose. SERS measurements with in situ prepared Ag-colloids improved the reproducibility of Raman signals from 54.8% to 93.9%. The measurement time could be reduced significantly, completing our secondary goal. The quality of classically and in situ prepared Ag-colloids was monitored by zeta potential measurements and scanning electron microscopy (SEM) respectively. For data interpretation and statistical model-building an in house written code in the open source software RStudio was implemented. It was applied for the differentiation of PHB producers at the cellular level, revealing heterogeneities within sample groups regarding the PHB amount accumulated. The results obtained using the statistical model were validated as well and were complementary to the reference HPLC analysis. Therefore, a fast and reliable identification in situ SERS tool for the selection of the most promising cyanobacterial PHB production was established.

Clinical significance of Candida isolation from dystrophic fingernails.

BACKGROUND: Candida onychomycosis mostly involves fingernails. Yet, in contrast to dermatophytes, Candida isolation from dystrophic fingernails does not prove casualty, as sample contamination and non-pathogenic Candida growth occur. Characterising treatment outcome of Candida-positive dystrophic nails is crucial to avoid unnecessary treatment. OBJECTIVE: To investigate predicators associated with treatment outcome among Candida-positive dystrophic fingernails. PATIENTS AND METHODS: A retrospective cohort study was carried out among 108 adults with Candida-positive dystrophic fingernails not cured with adequate systemic anti-fungal course. Diagnosis was based on a single mycological culture. Patients with treatment failure (n = 85; 78.7% of the cases) were compared to patients with partial response (mild to almost cure; n = 23; 21.3% of the cases) at 9 to 12 months following treatment initiation. RESULTS: Treatment failure was significantly associated with primary onycholysis (odds ratio [OR] 2.8; 95% confidence interval [CI] 1.1-7.4) and prolonged dystrophy (12.8 vs. 3.7 years in average), compared to partial treatment response. Non-responders had lower odds to present with distal lateral subungual onychomycosis, compared to partial responders (OR 0.3; 95% CI 0.1-0.7). Demographic and mycological characteristics, as well as number of nails affected, co-presence of paronychia, and treatment regime were not found to be associated with treatment response. CONCLUSION: Candida-positive primary onycholysis was shown to be non-responsive to systemic anti-fungal treatment, suggesting that anti-fungal treatment is not indicated. For other clinical scenarios, high proportions of treatment non-response suggest that determining causality of Candida should not be based on a single positive mycological culture.

Correction to: Pathogenesis, Clinical Signs and Treatment Recommendations in Brittle Nails: A Review.

Unfortunately, the co-author name was incorrectly published as "Jose L. López-Esterbaranz" instead of 'Jose L. López-Estebaranz" in the original article. The correct version of author name is updated here.The original article has been corrected.

Pathogenesis, Clinical Signs and Treatment Recommendations in Brittle Nails: A Review.

Nail plate brittleness (or fragility) is a common complaint affecting up to 20% of the population, especially women over 50 years of age, with fingernail fragility being more prevalent than toenail fragility. Nail brittleness is characterized by nails that split, flake and crumble, become soft and lose elasticity. The main clinical presentations are: onychoschizia, onychorrhexis, superficial granulation of keratin and worn-down nails. According to causative factors, we can distinguish 2 forms of nail fragility (NF): a primary "idiopathic or brittle nail syndrome" form and NF secondary to different causes such as inflammatory nail disorders, infections, systemic diseases and general conditions, traumas and alteration of the nail hydration. Optimal management requires treatment of the primary cause of brittle nails, when possible. In idiopathic NF oral supplementation, vitamins (especially biotin, also known as vitamin B7), trace elements and amino acids (especially cysteine) have been reported to be useful. In addition, several products, such as topical moisturizers and lacquers could be considered to restructure the affected nail plate and to reduce psychological impacts of this common problem.

Epitope Mapping via Phage Display from Single-Gene Libraries.

Antibodies are widely used in a large variety of research applications, for diagnostics and therapy of numerous diseases, primarily cancer and autoimmune diseases. Antibodies are binding specifically to target structures (antigens). The antigen-binding properties are not only dependent on the antibody sequence, but also on the discrete antigen region recognized by the antibody (epitope). Knowing the epitope is valuable information for the improvement of diagnostic assays or therapeutic antibodies, as well as to understand the immune response of a vaccine. While huge progress has been made in the pipelines for the generation and functional characterization of antibodies, the available technologies for epitope mapping are still lacking effectiveness in terms of time and effort. Also, no technique available offers the absolute guarantee of succeeding. Thus, research to develop and improve epitope mapping techniques is still an active field. Phage display from random peptide libraries or single-gene libraries are currently among the most exploited methods for epitope mapping. The first is based on the generation of mimotopes and it is fastened to the need of high-throughput sequencing and complex bioinformatic analysis. The second provides original epitope sequences without requiring complex analysis or expensive techniques, but depends on further investigation to define the functional amino acids within the epitope. In this book chapter, we describe how to perform epitope mapping by antigen fragment phage display from single-gene antigen libraries and how to construct these types of libraries. Thus, we also provide figures and analysis to demonstrate the actual potential of this technique and to prove the necessity of certain procedural steps.