PCR-restriction fragment length polymorphism assay for detection of gyrA mutations associated with fluoroquinolone resistance in Campylobacter coli

A fragment of the gyrA gene was sequenced from 34 isolates of Campylobacter coli, including 23 isolates resistant to ciprofloxacin. All ciprofloxacin-resistant isolates examined by DNA sequencing carried a point mutation at position Thr-86 on the gyrA gene product, involving the replacement of Thr-86 by Ile. A combined PCR-restriction fragment length polymorphism technique using RsaI was developed to detect this mutation.     

New syngeneic inflammatory‐related lung cancer metastatic model harboring double KRAS/WWOX alterations

New mouse models with specific drivers of genetic alterations are needed for preclinical studies. Herein, we created and characterized at the genetic level a new syngeneic model for lung cancer and metastasis in Balb‐c mice. Tumor cell lines were obtained from a silica‐mediated airway chronic inflammation that promotes tumorigenesis when combined with low doses of N‐nitrosodimethylamine, a tobacco smoke carcinogen. Orthotopic transplantation of these cells induced lung adenocarcinomas, and their intracardiac injection led to prominent colonization of various organs (bone, lung, liver and brain). Driver gene alterations included a mutation in the codon 12 of KRAS (G–A transition), accompanied by a homozygous deletion of the WW domain‐containing oxidoreductase (WWOX) gene. The mutant form of WWOX lacked exons 5–8 and displayed reduced protein expression level and activity. WWOX gene restoration decreased the in vitro and in vivo tumorigenicity, confirming the tumor suppressor function of this gene in this particular model. Interestingly, we found that cells displayed remarkable sphere formation ability with expression of specific lung cancer stem cell markers. Study of non‐small‐cell lung cancer patient cohorts demonstrated a deletion of WWOX in 30% of cases, with significant reduction in protein levels as compared to normal tissues. Overall, our new syngeneic mouse model provides a most valuable tool to study lung cancer metastasis in balb‐c mice background and highlights the importance of WWOX deletion in lung carcinogenesis.     

Two patients with rituximab associated low gammaglobulin levels and relapsed covid-19 infections treated with convalescent plasma

Patients with haematological malignancies are considered to be a risk group for developing severe Coronavirus disease (Covid-19). Because of the limitations of therapeutic options, the development of new treatment strategies is mandatory, such as convalescent plasma (CP). Herein we report the use of CP therapy as an off-label indication in two lymphoma patients with relapsed COVID-19 in the setting of low gammaglobulin levels because of previous rituximab chemo-immunotherapy. Both were PCR positive for SARS-CoV-2 but had an absence of antibodies to the virus more than one month later of symptoms initiation. They developed important respiratory and neurological complications. After CP infusion, neutralising antibodies were detected and viral load dissapeared in both patients leading to clinical improvement with no more Covid-19 relapse.     

Brush border myosin Ia inactivation in gastric but not endometrial tumors

Brush border Myosin Ia (MYO1A) has been shown to be frequently mutated in colorectal tumors with microsatellite instability (MSI) and to have tumor suppressor activity in intestinal tumors. Here, we investigated the frequency of frameshift mutations in the A8 microsatellite in exon 28 of MYO1A in MSI gastric and endometrial tumors and found a high mutation rate in gastric (22/47; 46.8%) but not endometrial (3/48; 6.2%) tumors. Using a regression model, we show that MYO1A mutations are likely to confer a growth advantage to gastric, but not endometrial tumors. The mutant MYO1A^7A protein was shown to lose its membrane localization in gastric cancer cells and a cycloheximide‐chase assay demonstrated that the mutant MYO1A^7A protein has reduced stability compared to the wild type MYO1A. Frequent MYO1A promoter hypermethylation was also found in gastric tumors. Promoter methylation negatively correlates with MYO1A mRNA expression in a series of 58 non‐MSI gastric primary tumors (Pearson's r = ?0.46; p = 0.0003) but not in a cohort of 54 non‐MSI endometrial tumors and treatment of gastric cancer cells showing high MYO1A promoter methylation with the demethylating agent 5‐aza‐2′‐deoxycytidine, resulted in a significant increase of MYO1A mRNA levels. We found that normal gastric epithelial cells, but not normal endometrial cells, express high levels of MYO1A. Therefore, when considered together, our findings suggest that MYO1A has tumor suppressor activity in the normal gastric epithelium but not in the normal endometrium and inactivation of MYO1A either genetically or epigenetically may confer gastric epithelial cells a growth advantage.     

Detection of cdtA, cdtB, and cdtC genes in Campylobacter jejuni by multiplex PCR

A multiplex PCR was developed for simultaneous detection of the cytolethal distending toxin (cdt) genes of Campylobacter jejuni. Three primer pairs targeting each one of the cdtA, cdtB and cdtC genes were designed and combined in the same PCR reaction. The assay was evaluated with 100 C. jejuni strains recovered from humans and animals and it was found to be rapid and specific. Two isolates presented several deletions affecting both cdtA and cdtB genes. High prevalence (98%) of the three cdt genes was found among isolates of different geographic origins.