BACKGROUND: ATTR (ATTRv) amyloidosis neuropathy is characterized by progressive sensorimotor and autonomic nerve degeneration secondary to amyloid deposition caused by a misfolded transthyretin protein (TTR). Small nerve fiber neuropathy is an early clinical manifestation of this disease resulting from the dysfunction of the Aδ and C small nerve fibers. Tafamidis, a selective TTR stabilizer, has proven its efficacy in the earlier stages of hATTR.
OBJECTIVE: To evaluate the clinical course and utility of cutaneous pathological biomarkers in patients with ATTR amyloidosis treated with tafamidis compared to control patients.
METHODS: Forty patients diagnosed with early stages of ATTRv amyloidosis (polyneuropathy disability [PND] scores 0-II) underwent small and large nerve fiber neurological evaluations, and annual skin biopsies for intraepidermal nerve fiber density (IENFD) and amyloid deposition index (ADI) estimation. Thirty patients were allocated to receive tafamidis, and 10 patients served as controls. Tafamidis pharmacokinetics analysis was performed in patients who received the treatment.
RESULTS: At baseline, 12% of patients in stage PND 0 and 28% in PND I displayed small nerve fiber denervation in the distal thigh, whereas 23% and 38%, respectively, in the distal leg. Similarly, 72% and 84% had amyloid deposition in the distal thigh and 56% and 69% in the distal leg. Following 1 year of treatment, the tafamidis group showed significant clinical improvement compared to the control group, revealed by the following mean differences (1) -9.3 versus -4 points (p = <.00) in the patient\'s neuropathy total symptom score 6 (NTSS-6) questionnaire, (2) -2.5 versus +2.8 points (p = <.00) in the Utah Early Neuropathy Score (UENS), and (3) +1.2°C versus -0.6 (p = .01) in cold detection thresholds. Among the patients who received tafamidis, 65% had stable or increased IENFD in their distal thigh and 27% in the distal leg. In contrast, all patients in the control group underwent denervation. The ADI either decreased or remained constant in 31% of the biopsies in the distal thigh and in 24% of the biopsies in the distal leg of the tafamidis-treated patients, whereas it rose across all the biopsies in the control group. At the 4-year follow-up, the tafamidis group continued to display less denervation in the distal thigh (mean difference [MD] of -3.0 vs. -9.3 fibers/mm) and the distal leg (mean difference [MD] -4.9 vs. -8.6 fibers/mm). ADI in tafamidis-treated patients was also lower in the distal thigh (10 vs. 30 amyloid/mm2) and the distal leg (23 vs. 40 amyloid/mm2) compared to control patients. Plasma tafamidis concentrations were higher in patients with IENFD improvement and in patients with reduced amyloid deposition. Patients without amyloid deposition in the distal leg at baseline displayed delayed disease progression at 4 years.
CONCLUSIONS: Cutaneous IENFD and amyloid deposition assessments in the skin of the distal thigh and distal leg are valuable biomarkers for early diagnosis of ATTR amyloidosis and for measuring the progression of small nerve fiber neuropathy. Early treatment with tafamidis slows the clinical progression of the disease, skin denervation, and amyloid deposition in the skin. Higher plasma concentrations of tafamidis are associated with better disease outcomes, suggesting that increasing the drug dose could achieve better plasma concentrations and response rates. This study describes the longest small nerve fiber neuropathy therapeutic trial with tafamidis and is the first to report small fiber symptoms, function, and structural assessments as outcomes.

BACKGROUND: Bovine besnoitiosis (elephant skin disease) caused by Besnoitia besnoiti is a costly endemic disease in the Middle East, Asia, and tropical and subtropical Africa and is also emerging as a significant problem in Europe. This study is aimed at determining the prevalence of B. besnoiti in blood and skin biopsies of cattle as well as evaluating the risk factors associated with the infection among cattle in Mosul, Iraq.
RESULTS: To achieve this aim, four hundred and sixty apparently healthy cattle of different breeds, ages, and sexes were sampled from seven different locations in Mosul, Iraq. Blood and skin biopsies were carefully collected from each cattle, and these samples were subjected to molecular analysis. The detection of B. besnoiti was molecularly confirmed by the presence of 231 bp of ITS-1 in the rDNA gene of the protozoan. Besnoitia besnoiti DNA was present in 74 (16.09%; 95% CI = 13.01-19.72) and 49 (10.65%; 95% CI = 8.15-13.80) of the blood and skin biopsies, respectively, that were analyzed. Age, breed, and sex were significantly (p < 0.05) associated with the occurrence of B. besnoiti among cattle in the study area.
CONCLUSIONS: Findings from this study will serve as baseline data in the epidemiology, prevention, and control of the protozoan among cattle in Iraq.

The whale shark (Rhincodon typus) is a filter-feeding organism that can be considered a sentinel species, and Bahía de los Ángeles (BLA) in the Gulf of California is an important sighting site for these elasmobranchs. This filter-feeding organism can be considered a pollutant sampler from the marine environment. Persistent organic pollutants are toxic compounds with high mobility and environmental persistence, bioaccumulation and trophic transfer. Among these are polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs). The present work aimed to determine concentrations of PAHs and OCPs in whale shark skin biopsies, collected in 2021 at BLA. Mean detected levels of PAHs and OCPs were 279.4 ng/g dw (dry weight) and 1478.1 ng/g dw, respectively. Analysis of similarities between the ordered sizes (4.2-7.6 m) and the concentrations of PAHs and OCPs indicated no significant differences. Individual PAHs detected indicate pyrogenic and petrogenic sources; the presence of pesticides at levels higher than those of hydrocarbons may be related to agricultural activity in the areas surrounding the Baja California peninsula. This study is the first report of PAH levels in R. typus for the Gulf of California and Mexico.

Epigenetic approaches for estimating the age of living organisms are revolutionizing studies of long-lived species. Molecular biomarkers that allow age estimates from small tissue biopsies promise to enhance studies of long-lived whales, addressing a fundamental and challenging parameter in wildlife management. DNA methylation (DNAm) can affect gene expression, and strong correlations between DNAm patterns and age have been documented in humans and nonhuman vertebrates and used to construct \"epigenetic clocks\". We present several epigenetic clocks for skin samples from two of the longest-lived cetaceans, killer whales and bowhead whales. Applying the mammalian methylation array to genomic DNA from skin samples we validate four different clocks with median errors of 2.3-3.7 years. These epigenetic clocks demonstrate the validity of using cytosine methylation data to estimate the age of long-lived cetaceans and have broad applications supporting the conservation and management of long-lived cetaceans using genomic DNA from remote tissue biopsies.

UNASSIGNED: It is important to evaluate the agreement between clinical diagnosis and histopathological diagnosis. The aim of this study was to review studies and to calculate the degree of agreement between clinical and histopathological diagnosis.
UNASSIGNED: The aim of this review was to find out the concordance level between clinical and histopathological diagnosis; to find out in which type of pathology concordance was the highest; to identify the types of pathologies for which biopsy was mostly performed and the anatomical region selected for biopsy.
UNASSIGNED: Review was carried out in accordance with the PRISMA 2020 flow diagram for the selection of articles that met the criteria (years 2005-2021). Articles were found in Google scholar, PubMed, Scopus databases using different keywords. The main criterion was to involve studies that reported data about clinical and histopathological diagnosis. A mean concordance value was calculated and resulted of 72.8 % (95% CI).
UNASSIGNED: To our knowledge, our study was the first review done regarding concordance between clinical and histopathological diagnosis in skin diseases with the aim to provide researchers with enriched literature and encourage them to bring about an appropriate systematic review study.

Background: Obtaining high quality RNA from skin biopsies is complex due the physical composition and high content of nucleases of this tissue. This becomes particularly challenging when using compromised skin samples with necrotic, inflammed or damaged areas, such as those from patients suffering skin conditions, which affect more than 900 million people annually. We evaluated the impact of the biopsy size and tissue preservation method on the quality and quantity of RNA extracts. Methods: Skin lesion biopsies were obtained from patients with cutaneous leishmaniasis (CL). Biopsy specimens of 2 mm (n = 10) and 3 mm (n = 59) were preserved in Allprotect® reagent, and 4 mm biopsies in OCT (n = 54). Quality parameters were evaluated using Nanodrop and Bioanalyzer. The informativeness of the extracted samples for downstream analyses was evaluated using RT-qPCR and RNA-Seq. Results: The success rate, based on quality parameters of RNA extraction from tissue biopsies stored in OCT and 2 mm biopsies stored in Allprotect®, was 56% (30/54) and 30% (3/10), respectively. For 3 mm skin biopsies stored in Allprotect® was 93% (55/59). RNA preparations from 3 mm-Allprotect® biopsies had an average RIN of 7.2 ± 0.7, and their integrity was not impacted by sample storage time (up to 200 days at -20°C). RNA products were appropriate for qRT-PCR and RNA-seq. Based on these results, we propose a standardized method for RNA extraction from disrupted skin samples. This protocol was validated with lesion biopsies from CL patients (n = 30), having a success rate of 100%. Conclusions: Our results indicate that a biopsy size of 3 mm in diameter and preservation in Allprotect® for up to 200 days at -20°C, are best to obtain high quality RNA preparations from ulcerated skin lesion biopsy samples.

BACKGROUND: Histopathology protocols for processing dermatopathology specimens vary among laboratories.
OBJECTIVE: To determine an optimal histopathology protocol to minimize cost and turnaround time (TAT) for biopsy specimens in a dermatopathology laboratory.
METHODS: A prospective, 4-month study compared the mean cost and TAT of producing one versus two initial H&E slides, and zero versus three unstained slides that could be used for frequently used special or immunohistochemical (IHC) stains.
RESULTS: For all cases, cost was lower for one versus two initial H&E slides, with an insignificant increase in TAT. Producing three vs zero unstained slides incurred higher cost, with no reduction in TAT. In a subset of cases in which frequently used special or IHC stains were performed, cost and TAT were optimized by producing one initial H&E and three unstained slides.
CONCLUSIONS: A protocol of one initial H&E slide and zero unstained slides optimizes cost and TAT in our dermatopathology laboratory. Pigmented lesions and inflammatory dermatoses may benefit from the addition of unstained slides. Further study is needed to quantify this benefit and evaluate for other cases for which an alternative protocol is advantageous.

Leishmania infantum is a protozoan causing human zoonotic visceral leishmaniasis (ZVL) and visceral-cutaneous canine leishmaniosis (CanL) in the Mediterranean Basin. L. infantum is able to infect a large number of wild and domestic species, including cats, dogs, and horses. Since the 1990s, clinical cases of equine leishmaniasis (EL), typically characterized by cutaneous forms, have been increasingly diagnosed worldwide. The aim of the present study was to evaluate the presence of clinical forms of EL in CanL-endemic areas in Italy, where exposure of equine populations was ascertained from recent serological surveys. For this purpose, formalin-fixed and paraffin-embedded skin biopsies of 47 horses presenting chronic dermatitis compatible with EL were retrospectively selected for the study and subjected to conventional and q-PCR. A singular positivity for L. infantum was found; BLAST analysis of sequence amplicons revealed a 99-100% homology with L. infantum sequences. The histological examination revealed a nodular lymphoplasmacytic and histiocytic infiltrate; immunohistochemistry showed rare macrophages containing numerous positive amastigotes. The present retrospective study reports, for the first time, a case of a cutaneous lesion by L. infantum occurring in an Italian horse. Pathological and healthy skin samples should be investigated on a larger scale to provide information on the potential clinical impact of EL in the practice, and to define the role of horses in epidemiological ZVL and CanL scenarios.

Non-inflammatory alopecia is a frequent skin problem in dogs, causing damaged coat integrity and compromised appearance of affected individuals. In this study, we examined the Cesky Fousek breed, which displays atypical recurrent flank alopecia (aRFA) at a high frequency. This type of alopecia can be quite severe and is characterized by seasonal episodes of well demarcated alopecic areas without hyperpigmentation. The genetic component responsible for aRFA remains unknown. Thus, here we aimed to identify variants involved in aRFA using a combination of histological, genomic, and transcriptomic data. We showed that aRFA is histologically similar to recurrent flank alopecia, characterized by a lack of anagen hair follicles and the presence of severely shortened telogen or kenogen hair follicles. We performed a genome-wide association study (GWAS) using 216 dogs phenotyped for aRFA and identified associations on chromosomes 19, 8, 30, 36, and 21, highlighting 144 candidate genes, which suggests a polygenic basis for aRFA. By comparing the skin cell transcription pattern of six aRFA and five control dogs, we identified 236 strongly differentially expressed genes (DEGs). We showed that the GWAS genes associated with aRFA are often predicted to interact with DEGs, suggesting their joint contribution to the development of the disease. Together, these genes affect four major metabolic pathways connected to aRFA: collagen formation, muscle structure/contraction, lipid metabolism, and the immune system.

Quantitative grading of testing has research and clinical relevance. QASAT (quantitative scale for grading of cardiovascular reflex tests, transcranial Doppler, sudomotor testing, and small fiber densities from skin biopsies) is an objective instrument for grading dysautonomia, related small fiber neuropathy and cerebral blood flow. QASAT uses established autonomic tests (deep breathing, Valsalva maneuver, tilt test, sudomotor test) and skin biopsies for assessment of small fibers. Calculations of scores are complex. This paper presents a qpack-an open source software package that implements QASAT in a Python programming language. The qpack automatically generates reproducible scores of each test and reduces calculation errors. Datasets for verifying the correct qpack implementation are provided. The goal of qpack is to facilitate availability, reproducibility, and quality of autonomic studies and skin biopsies for assessment of small fibers. Qpack is easy to use with standard Python distributions, can be incorporated into routine clinical or research autonomic testing and it is freely available.