Gene expression profiles of antigenic proteins of third stage larvae of the zoonotic nematode Anisakis pegreffii in response to temperature conditions

Anisakis pegreffii, a recognised etiological agent of human anisakiasis, is a parasite of homeothermic hosts at the adult stage and of ectothermic hosts at the third larval stage. Among distinct factors, temperature appears to be crucial in affecting parasite hatching, moulting and to modulate parasite-host interaction. In the present study, we investigated the gene transcripts of proteins having an antigenic role among excretory secretory products (ESPs) (i.e., a Kunitz-type trypsin inhibitor, A.peg-1; a glycoprotein, A.peg-7; and the myoglobin, A.peg-13) after 24 h, in A. pegreffii larvae maintained in vitro, under controlled temperature conditions. Temperatures were 37 °C and 20 °C, resembling respectively homeothermic and ectothermic hosts conditions, and 7 °C, the cold stress condition post mortem of the fish host. Primers of genes coding for these ESPs to be used in quantitative real-time PCR were newly designed, and qRT-PCR conditions developed. Expression profiles of the genes A.peg-1 and A.peg-13 were significantly up-regulated at 20 °C and 37 °C, with respect to the control (larvae kept at 2 °C for 24 h). Conversely, transcript profiles of A.peg-7 did not significantly change among the chosen temperature conditions. In accordance with the observed transcript profiles, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of the three target ESPs at 37 °C, while only A.peg-13 was observed at 7 °C. The results suggest that temperature conditions do regulate the gene expression profiles of A.peg-1 and A.peg-13 in A. pegreffii larvae. However, regulation of the glycoprotein A.peg-7 is likely to be related to other factors such as the host's immune response

Species-specific Real Time-PCR primers/probe systems to identify fish parasites of the genera Anisakis, Pseudoterranova and Hysterothylacium (Nematoda: Ascaridoidea)

Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.), P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum, a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency (E) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers

Cantú syndrome: A new case and evolution of clinical conditions during first 2-year follow-up

: Cantú syndrome, or hypertrichotic osteochondrodysplasia, is a rare autosomal dominant disease characterized by congenital hypertrichosis, characteristic dysmorphisms, skeletal abnormalities and cardiomegaly. We report on a 7-year-old girl with congenital generalized hypertrichosis, coarse facial appearance and cardiac involvement, with a de novo heterozygous mutation (c.3461G > A) in the ABCC9 gene. During the annual cardiac follow-up at the age of nine the echocardiogram showed mild left ventricular dilatation in consideration of which she started ramipril treatment. The progression of the clinical manifestations of Cantú syndrome highlights the relevance of an early diagnosis, including genetic analysis, and a multidisciplinary approach with long-term follow-up

First molecular identification of the zoonotic parasite Anisakis pegreffii (Nematoda: Anisakidae) in a paraffin-embedded granuloma taken from a case of human intestinal anisakiasis in Italy

<p>Abstract</p> <p>Background</p> <p>Anisakiasis is an important fish-borne zoonosis provoked by larval stages of nematodes belonging to the genus <it>Anisakis</it>. The detection and identification of human infections is difficult. This is due to: a) the low specificity of the clinical features and symptomatology related to human infections; b) the paucity of diagnostic features of larvae found in granulomatous lesions characteristic of "invasive anisakiasis"; and c) the lack morphological characters diagnostic at the specific level when larvae of <it>Anisakis </it>are detected. Thus, molecular-based diagnostic approaches are warranted.</p> <p>Method</p> <p>We have developed a PCR method that amplifies the DNA of <it>Anisakis </it>spp. in fixed paraffin-embedded tissues. This method was applied to a granuloma removed from a human case of intestinal anisakiasis in Italy. Specific primers of the mtDNA <it>cox2 </it>gene were used and sequence analysis was performed according to the procedures already established for species of <it>Anisakis</it>.</p> <p>Results</p> <p>The sequence obtained (629 bp) was compared with those of the other species of <it>Anisakis </it>which have so far been genetically characterized and with sequences obtained from larval stages of <it>Anisakis </it>collected from the Mediterranean fish <it>Engraulis encrasicolus</it>. This enabled the genetic identification of the larva in the human tissue as <it>A. pegreffii</it>. This is the first instance of human intestinal anisakiasis diagnosed using PCR of DNA purified from a fixed eosinophilic granuloma embedded in paraffin.</p> <p>Conclusion</p> <p>The case of human anisakiasis presented reinforces the pathological significance of the species <it>A. pegreffii </it>to humans. The molecular/genetic methodological approach based on mtDNA <it>cox2 </it>sequence analysis, described here, can allow easy and rapid identification of <it>Anisakis </it>spp. in formalin-fixed and paraffin embedded tissues removed from cases of either gastric or intestinal human anisakiasis.</p

Stereotactic body radiotherapy vs conventionally fractionated chemoradiation in locally advanced pancreatic cancer: A multicenter case‐control study (PAULA‐1)

The aim of this study was to compare two cohorts of LAPC patients treated with SBRT ± CHT vs CRT ± CHT in terms of local control (LC), distant metastases- free survival (DMFS), progression-free survival (PFS), overall survival (OS), and toxicity. Eighty patients were included. Patients in the two cohorts were matched ac- cording to: age ≤/&gt;65 years, tumor diameter (two cut-offs

Multidisciplinary studies on a sick-leader syndrome-associated mass stranding of sperm whales (Physeter macrocephalus) along the Adriatic coast of Italy

Mass strandings of sperm whales (Physeter macrocephalus) are rare in the Mediterranean Sea. Nevertheless, in 2014 a pod of 7 specimens stranded alive along the Italian coast of the Central Adriatic Sea: 3 individuals died on the beach after a few hours due to internal damages induced by prolonged recumbency; the remaining 4 whales were refloated after great efforts. All the dead animals were genetically related females; one was pregnant. All the animals were infected by dolphin morbillivirus (DMV) and the pregnant whale was also affected by a severe nephropathy due to a large kidney stone. Other analyses ruled out other possible relevant factors related to weather conditions or human activities. The results of multidisciplinary post-mortem analyses revealed that the 7 sperm whales entered the Adriatic Sea encountering adverse weather conditions and then kept heading northward following the pregnant but sick leader of the pod, thereby reaching the stranding site. DMV infection most likely played a crucial role in impairing the health condition and orientation abilities of the whales. They did not steer back towards deeper waters, but eventually stranded along the Central Adriatic Sea coastline, a real trap for sperm whales

Incidence of Adverse Cutaneous Reactions to Epidermal Growth Factor Receptor Inhibitors in Patients with Non-Small-Cell Lung Cancer

Background: Epidermal growth factor receptor (EGFR) inhibitors are routinely used in advanced non-small-cell lung cancer (NSCLC) harboring EGFR mutations. However, their use is associated with gastrointestinal and cutaneous toxicities, including acneiform eruptions, pruritus, xerosis, nail and hair changes. Aside from reducing patients' quality of life, such cutaneous reactions have a considerable impact on the oncologic treatment given that dose reduction or even drug discontinuation may be necessary, especially for the severe forms. Objectives: To assess the incidence, impact on treatment and management of EGFR inhibitor-related cutaneous reactions in patients with NSCLC. Methods: We conducted a prospective observational study on 87 consecutive patients with advanced NSCLC treated with EGFR-tyrosine kinase inhibitors from January to December 2019. Patients who developed mucocutaneous reactions were evaluated and treated by both oncologists and dermatologists, and underwent dermatologic follow-up until resolution of the cutaneous reaction. Demographic and clinical data were collected for each patient, and the severity of the cutaneous reaction was graded using the Common Terminology Criteria for Adverse Events. Results: Seventy-one patients (81.6%) developed cutaneous reactions. The number of cutaneous reactions per patient was 1 in 37%, 2 in 41% and 3 or more in 22%. The most common cutaneous reactions included acneiform eruptions (56.3%), xerosis +/- asteatotic eczema (48.3%), nail changes (39.1%), mucositis (29.9%), pruritus (24.1%) and hair changes (12.6%). Afatinib was associated with a higher rate of nail changes and mucositis (p &lt; 0.01 and p &lt; 0.005, respectively) compared to other agents, while no patient-related predictive factors were identified. Dose reduction was performed in 18% of patients. Multidisciplinary management involving dermatologists allowed to resume the drug in all patients who had discontinued it due to the cutaneous reactions. Conclusions: A multidisciplinary approach to EGFR inhibitor-related cutaneous reactions is advantageous and can reduce the need to discontinue oncologic treatment