Cutaneous biomarkers of therapeutic efficacy in early treatment of hereditary ATTR amyloid polyneuropathy with tafamidis.

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. OBJECTIVES: 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.

Persistent Organic Pollutants in Whale Shark (Rhincodon typus) Skin Biopsies from Bahía de Los Ángeles, Mexico.

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.

Quality parameters for RNA preparations from biopsies of ulcerated human skin.

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.

Antemortem diagnosis of rabies from skin: comparison of nested RT-PCR with TaqMan real time PCR

Molecular techniques were adopted to diagnose rabies viral RNA from skin biopsy samples collected from 20 animals. Nested RT-PCR and TaqMan real time PCR techniques have successfully diagnosed rabies viral RNA in 9 and 11 skin samples, respectively. The sensitivity of both techniques was calculated in comparison to FAT applied on brain samples. Sensitivity of 76.47% was obtained with nested RT-PCR on skin biopsy samples while Taqman real time PCR revealed sensitivity of 92.85%. It was concluded that TaqMan real time PCR is a useful, specific, sensitive and better molecular approach for antemortem rabies diagnosis from skin samples of rabies suspected animals.

Antemortem Diagnosis of Rabies From Skin: Comparison of nested RT-PCR with TaqMan real time PCR

Molecular techniques were adopted to diagnose rabies viral RNA from skin biopsy samples collected from 20 animals. Nested RT-PCR and TaqMan real time PCR techniques have successfully diagnosed rabies viral RNA in 9 and 11 skin samples, respectively. The sensitivity of both techniques was calculated in comparison to FAT applied on brain samples. Sensitivity of 76.47% was obtained with nested RT-PCR on skin biopsy samples while Taqman real time PCR revealed sensitivity of 92.85%. It was concluded that TaqMan real time PCR is a useful, specific, sensitive and better molecular approach for antemortem rabies diagnosis from skin samples of rabies suspected animals.(AU)

Histopathological and parasitological analysis of skin tissues biopsies from two distinct anatomical areas of the ears of dogs naturally infected with Leishmania (Leishmania) chagasi

Canine visceral leishmaniasis is an endemic disease in Latin America caused by Leishmania (Leishmania) chagasi and transmitted to man and animals by infected blood-sucking sandflies) of the genus Lutzomyia. Dogs are considered to be the primary domestic reservoir of disease because they present an intense cutaneous parasitism. The aim of this study was to evaluate the intensity of the inflammatory process and to compare it to the parasite load of tissue from two different sites of the ear skin of dogs naturally infected with Leishmania chagasi. We think that exist a specific anatomical region that exhibits a relatively higher rate of parasitism. For diagnostic analysis, serological tests were carried out using the indirect fluorescence antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA). Twelve animals naturally infected with Leishmania chagasi were euthanatized with a lethal dose of Sodium Thiopental and T61. During the necropsy, fragments of the extremity and middle anatomical regions of the ear were collected. All tissues were fixed in a 10% formalin solution and then paraffin-embedded for histopathological (HE) and immunohistochemical analysis. The streptoavidin-peroxidase immunohistochemistry method was used to detect tissue amastigotes using optical microscopy. Our results indicated a chronic inflammatory reaction, ranging from discrete to an intense

Histopathological and parasitological analysis of skin tissues biopsies from two distinct anatomical areas of the ears of dogs naturally infected with Leishmania (Leishmania) chagasi

Canine visceral leishmaniasis is an endemic disease in Latin America caused by Leishmania (Leishmania) chagasi and transmitted to man and animals by infected blood-sucking sandflies) of the genus Lutzomyia. Dogs are considered to be the primary domestic reservoir of disease because they present an intense cutaneous parasitism. The aim of this study was to evaluate the intensity of the inflammatory process and to compare it to the parasite load of tissue from two different sites of the ear skin of dogs naturally infected with Leishmania chagasi. We think that exist a specific anatomical region that exhibits a relatively higher rate of parasitism. For diagnostic analysis, serological tests were carried out using the indirect fluorescence antibody test (IFAT) and the enzyme-linked immunosorbent assay (ELISA). Twelve animals naturally infected with Leishmania chagasi were euthanatized with a lethal dose of Sodium Thiopental™ and T61™ During the necropsy, fragments of the extremity and middle anatomical regions of the ear were collected. All tissues were fixed in a 10% formalin solution and then paraffin-embedded for histopathological (HE) and immunohistochemical analysis. The streptavidin-peroxidase immunohistochemistry method was used to detect tissue amastigotes using optical microscopy. Our results indicated a chronic inflammatory reaction, ranging from discrete to an intense magnitude. The inflammatory process was more frequently observed in the extremity of the ear than in the middle portion of the ear (p<0.05). The presence of parasites in the ear extremity was higher than in other evaluated regions. A positive correlation between the tissue inflammation, parasitism, and serological data was confirmed at both ear positions (p<0.05). Skin biopsies are an important tool for CVL diagnosis and the ear extremity represents an appropriated area to perform the assays.