Epidermal innervation morphometry by immunofluorescence and bright‐field microscopy

We investigated the agreement between simple indirect immunofluorescence (IF) and bright‐field immunohistochemistry (BFI) on free‐floating sections for intraepidermal nerve fiber density (IENFD) quantification. Fifty‐five healthy subjects and 63 patients with probable small fiber neuropathy (SFN) underwent two adjacent skin biopsies at the distal leg processed by IF and BFI technique. Agreement between IENFD pairs obtained by each method was assessed by Bland–Altman testing. The area under the curve of the receiving operating characteristics (ROC) curves was used to compare the discrimination ability. The diagnostic judgment was based on sex and age‐adjusted normative values. IF and BFI showed good correlation (r = 0.81), with a ratio of about 2:1 and a mean difference of 5.5 ± 3.0 IENF per millimeter between paired measures, as demonstrated by linear regression and Bland–Altman test analyses. The square root transformation confirmed a Poisson distribution of the data and a fixed bias between IF and BFI measurements. The ROC curves analysis demonstrated a striking overlap between IF and BFI (0.83 and 0.82; p = 0.72). The diagnosis of SFN disagreed in only 6.7% of cases when the judgment was based on a difference of >1 IENF from 5% cut‐off value. IF and BFI showed comparable diagnostic efficiency when referred to appropriate normative reference values.     

A unifying working hypothesis for juvenile polyposis syndrome and Ménétrier's disease: Specific localization or concomitant occurrence of a separate entity?

Background

Juvenile polyposis syndrome with gastric involvement may mimic Ménétrier's disease, which is correlated to transforming growth factor (TGF)α overproduction and PDX1 upregulation in the gastric fundus.

Aim

We report a family with juvenile polyposis syndrome where one member showed typical features of Ménétrier's disease and concomitant Helicobacter pylori infection.

Methods

We studied a 31-year-old woman belonging to a family with juvenile polyposis syndrome, who exhibited a particular form of hyperplastic gastropathy diagnosed as Ménétrier's disease with Helicobacter pylori infection.

Results

TGFα overexpression and undetectable PDX1 expression were demonstrated in the fundic gastric biopsy specimens. In all affected members of the family we identified a 4-bp deletion in exon 9 of SMAD4 gene, a mutation usually associated with a more virulent form of juvenile polyposis syndrome with a higher incidence of gastric and colonic polyposis.

Conclusion

To explain the association of juvenile polyposis syndrome with Ménétrier's disease we hypothesized a new mechanism that involves TGFβ-SMAD4 pathway inactivation and TGFα overexpression related to Helicobacter pylori infection.     

Synthesis,biological evaluation,and in silico studies of novel chalcone- and pyrazoline-based 1,3,5-triazines as potential anticancer agents

A novel series of triazin-chalcones (7,8)a–g and triazin-N-(3,5-dichlorophenyl)pyrazolines (9,10)a–g were synthesized and evaluated for their anticancer activity against nine different cancer strains. Triazine ketones 5 and 6 were synthesized from the cyanuric chloride 1 by using stepwise nucleophilic substitution of the chlorine atom. These ketones were subsequently subjected to a Claisen–Schmidt condensation reaction with aromatic aldehydes affording chalcones (7,8)a–g. Then, N-(3,5-dichlorophenyl)pyrazolines (9,10)a–g were obtained by cyclocondensation reactions of the respective chalcones (7,8)a–g with 3,5-dichlorophenylhydrazine. Among all the evaluated compounds, chalcones 7d,g and 8g exhibited more potent in vitro anticancer activity, with outstanding GI₅₀ values ranging from 0.422 to 14.9 μM and LC₅₀ values ranging from 5.08 μM to >100 μM. In silico studies, for both ligand- and structure-based, were executed to explore the inhibitory nature of chalcones and triazine derivatives. The results suggested that the evaluated compounds could act as modulators of the human thymidylate synthase enzyme.

A novel series of triazin-chalcones (7,8)a–g and triazin-N-(3,5-dichlorophenyl)pyrazolines (9,10)a–g were synthesized and evaluated for their anticancer activity against nine different cancer strains.     

BTK inhibitors synergise with 5-FU to treat drug-resistant TP53-null colon cancers

Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK – a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

The role of VLA-4 binding for experimental melanoma metastasis and its inhibition by heparin

Introduction

Heparin is known to efficiently attenuate metastasis in various tumour models by different mechanisms including inhibition of tumour cell contacts with soluble and cellular components such as inhibition of heparanase or P- and L-selectin. We recently showed that heparin efficiently binds to VLA-4 integrin in melanoma cells in vitro. Here we describe VLA-4 integrin as a mediator of melanoma metastasis that is inhibited by the low molecular weight heparin (LMWH) Tinzaparin.

Materials and Methods

sh-RNA-mediated knock-down of VLA-4 integrin in B16F10 murine melanoma cells (B16F10-VLA-4kd) was performed and cell binding characteristics were investigated in vitro. Experimental metastasis of B16F10-VLA-4kd and B16F10 cells and interference by Tinzaparin were analysed in mice.

Results

VLA-4 knock-down of B16F10 cells resulted in loss of VCAM-1 binding, but preserved the capacity to bind platelets through P-selectin. The observed reduced metastasis of B16F10-VLA-4kd cells confirmed the role of VLA-4 in this process. However, loss of melanoma VLA-4 function hardly further affected reduction of metastasis in P-selectin deficient mice. Tinzaparin treatment of mice injected with B16F10 and B16F10-VLA-4kd cells significantly reduced metastasis suggesting its potential to block both P- and L-selectin and VLA-4 in vivo. The use of N-acetylated heparin, which has no VLA-4 binding activity but blocks P- and L-selectin was less efficient than Tinzaparin in mice injected with B16F10 cells and B16F10-VLA-4kd cells.

Conclusion

These findings provide evidence that heparin inhibits experimental melanoma metastasis primarily by blocking VLA-4 and P-selectin.     

Update on laser-evoked potential findings in fibromyalgia patients in light of clinical and skin biopsy features

In fibromyalgia (FM), reduced habituation of laser-evoked potentials (LEPs) suggests a dysfunction of pain processing at a central level. In this study, we aimed to further examine the nociceptive pathways at the peripheral to the central level in a large group of FM patients by means of LEPs and skin biopsy, in light of healthy controls findings and main clinical features. One hundred and ninety-nine FM patients and 109 age- and sex-matched controls were submitted to LEPs by the dorsum of the right hand and the skin over the right chest and knee tender point stimulation. Skin biopsy was performed in 21 randomly selected FM patients and 60 age- and sex-matched controls. The mean N2–P2 amplitude was reduced in the whole FM group, with normal or even increased values in patients with migraine as comorbidity and reduced values in other patients including those presenting with distal sensory deficits. All patients had reduced N2–P2 habituation in respect to controls. In the FM group, LEPs habituation was correlated with pain at tender points and bad quality of life. Epidermal fiber density was significantly reduced in FM patients versus controls, and correlated with N2–P2 amplitude by the hand and chest tender-point stimulation. Dysfunction in the nociceptive system at both the central and peripheral levels may concur to explain phenotypical eterogeneity and clinical symptom complexity in fibromyalgia.     

Long-term efficacy and safety of subcutaneous pasireotide in acromegaly: results from an open-ended,multicenter, Phase II extension study

Pasireotide has a broader somatostatin receptor binding profile than other somatostatin analogues. A 16-week, Phase II trial showed that pasireotide may be an effective treatment for acromegaly. An extension to this trial assessed the long-term efficacy and safety of pasireotide. This study was an open-label, single-arm, open-ended extension study (primary efficacy and safety evaluated at month 6). Patients could enter the extension if they achieved biochemical control (GH ≤ 2.5 μg/L and normal IGF-1) or showed clinically relevant improvements during the core study. Thirty of the 60 patients who received pasireotide (200–900 μg bid) in the core study entered the extension. At extension month 6, of the 26 evaluable patients, six were biochemically controlled, of whom five had achieved control during the core study. Normal IGF-1 was achieved by 13/26 patients and GH ≤ 2.5 μg/L by 12/26 at month 6. Nine patients received pasireotide for ≥24 months in the extension; three who were biochemically controlled at month 24 had achieved control during the core study. Of 29 patients with MRI data, nine had significant (≥20 %) tumor volume reduction during the core study; an additional eight had significant reduction during the extension. The most common adverse events were transient gastrointestinal disturbances; hyperglycemia-related events occurred in 14 patients. Twenty patients had fasting plasma glucose shifted to a higher category during the extension. However, last available glucose measurements were normal for 17 patients. Pasireotide has the potential to be an effective, long-term medical treatment for acromegaly, providing sustained biochemical control and significant reductions in tumor volume.