Cutaneous leishmaniasis (CL) is a vector-borne parasitic disease, routinely diagnosed by direct light microscopy. The sensitivity of this method is dependent on the number of parasites present in the lesion. Immunoexpression of CD1a surface antigen by Leishmania amastigotes and its application as a diagnostic tool has been recently demonstrated in several species including Leishmania major, Leishmania tropica and Leishmania infantum. Leishmania donovani is the only reported species in Sri Lanka primarily causing CL and its CD1a status remains unexplored. We studied CD1a expression by amastigotes of L. donovani in skin biopsies from 116 patients with suspected CL. The biopsy sections were stained with CD1a clones O10 and MTB1 separately. Slit skin smear (SSS) results were considered the gold standard for diagnosis of CL. 103 cases were confirmed through SSS where 73 of them showed positive parasite staining for CD1a clone MTB1 with 70.9% sensitivity. Positivity was seen mostly in parasites closer to the epidermis. CD1a clone O10 failed to detect any amastigotes. Test sensitivity improved to 74.1% when the analysis was applied only to patients with low/no discernible Leishman-Donovan (LD) bodies in histology. Our findings show that CD1a clone MTB1 successfully stains amastigotes of L. donovani species and can be used as a supplementary diagnostic tool in detecting CL, especially when LD bodies are low in number. This method could be validated to detect other forms of leishmaniasis caused by L. donovani in Indian and sub-Saharan regions.

This research paper addresses the hypothesis that mast cells (MCs) contribute to the formation of mammary fibrosis. MCs are important immune regulatory and immune modulatory cells that play major roles in the inflammatory process. Since there is no detailed knowledge, this research study aimed to comparatively investigate the presence, localization, and immunophenotypes of MCs in healthy and mastitic mammary tissues. A total of 264 mammary samples were evaluated for the examination of mast cells and fibrosis. The mean mast cell number in both acute and chronic mastitis samples were very significantly higher than the control group P < 0.001). A 7.9-fold increase in the number of mast cells was found when the chronic mastitis group was compared with the control (healthy) group. Immunohistochemistry revealed presence of all three immune phenotypes in control and mastitic mammary samples (tryptase + (MCT), chymase + (MCC) and both chymase and tryptase + (MCTC). The mean MCT, MCC, and MCTC numbers in the chronic mastitis group were found to be significantly higher than the control (P < 0.001 for all three phenotypes) but did not differ significantly between control and acute mastitis samples. When the mean numbers of MCT, MCC, and MCTC in the control group and chronic mastitis group were compared, a 10.5, 7.8, and a 4.1-fold increase was observed, respectively. The amount of connective tissue was strongly increased in tissues with chronic mastitis and a 3.01-fold increase was detected compared to the control group. A statistically significant relation was also found between the amount of fibrosis and the increased number of total MCs (P < 0.001).

A survey on Anisakis simplex (sensu stricto (s.s.)) from blue whiting, Micromesistius poutassou, in the north-eastern Atlantic Ocean revealed the occurrence of high infection levels of third larval stages in visceral organs and flesh. Larvae were genetically identified with a multilocus approach as A. simplex (s.s.). Histochemical, immunohistochemical and ultrastructural observations were conducted on 30 M. poutassou specimens. Gonads, pyloric caeca and flesh harboured encapsulated larvae of A. simplex (s.s.) but no intense host reaction was encountered around the parasite in the above organs. In the liver, the most infected organ, the larvae co-occurred with the coccidian Goussia sp. Within the granuloma around the A. simplex (s.s.) larvae, two concentric layers were recognized, an inner mostly comprising electron-dense epithelioid cells and an outer layer made of less electron-dense epithelioid cells. Macrophages and macrophage aggregates (MAs) were abundant out of the granulomas, scattered in parenchyma, and inside the MAs, the presence of engulfed Goussia sp. was frequent. In liver tissue co-infected with Goussia sp. and A. simplex (s.s.), hepatocytes showed cytoplasmic rarefaction and acute cell swelling. Results suggest that the host-induced encapsulation of A. simplex (s.s.) larvae is a strategic compromise to minimize collateral tissue damage around the larval infection sites, to facilitate the survival of both parasite and host.

Most commonly described as sporadic, pulmonary adenocarcinoma with enteric differentiation (PAED) is a rare variant of invasive lung cancer recently established and recognised by the World Health Organization. This tumour is highly heterogeneous and shares several morphological features with pulmonary and colorectal adenocarcinomas. Our objective is to summarise current research on PAED, focusing on its immunohistochemical and molecular features as potential tools for differential diagnosis from colorectal cancer, as well as prognosis definition and therapeutic choice. PAED exhibits an ‘entero-like’ pathological morphology in more than half cases, expressing at least one of the typical immunohistochemical markers of enteric differentiation, namely CDX2, CK20 or MUC2. For this reason, this malignancy appears often indistinguishable from a colorectal cancer metastasis, making the differential diagnosis laborious. Although standard diagnostic criteria have not been established yet, in the past few years, a number of approaches have been addressed, aimed at defining specific immunohistochemical and molecular signatures. Based on previously published literature, we have collected and analysed molecular and immunohistochemical data on this rare neoplasm, and have described the state of the art on diagnostic criteria as well as major clinical and therapeutic implications.

The analysis of data from 295 patients from 58 published articles allowed us to identify the most represented immunohistochemical and molecular markers, as well as major differences between Asian PAEDs and those diagnosed in European/North American countries. The innovative molecular approaches, exploring driver mutations or new gene alterations, could help to identify rare prognostic factors and guide future tailored therapeutic approaches to this rare neoplasm.

Significant experimental evidence supports fat as a taste modality; however, the associated peripheral mechanisms are not well established. Several candidate taste receptors have been identified, but their expression pattern and potential functions in human fungiform papillae remain unknown. The aim of this study is to identify the fat taste candidate receptors and ion channels that were expressed in human fungiform taste buds and their association with oral sensory of fatty acids. For the expression analysis, quantitative RT-PCR (qRT-PCR) from RNA extracted from human fungiform papillae samples was used to determine the expression of candidate fatty acid receptors and ion channels. Western blotting analysis was used to confirm the presence of the proteins in fungiform papillae. Immunohistochemistry analysis was used to localise the expressed receptors or ion channels in the taste buds of fungiform papillae. The correlation study was analysed between the expression level of the expressed fat taste receptors or ion channels indicated by qRT-PCR and fat taste threshold, liking of fatty food and fat intake. As a result, qRT-PCR and western blotting indicated that mRNA and protein of CD36, FFAR4, FFAR2, GPR84 and delayed rectifying K+ channels are expressed in human fungiform taste buds. The expression level of CD36 was associated with the liking difference score (R −0·567, β=−0·04, P=0·04) between high-fat and low-fat food and FFAR2 was associated with total fat intake (ρ=−0·535, β=−0·01, P=0·003) and saturated fat intake (ρ=−0·641, β=−0·02, P=0·008).

Tularemia caused by the bacterium Francisella tularensis is a zoonotic disease. Tularemia is a common disease in the hare, and as a game species can be an important source of infection for humans. In this study, hares diagnosed with tularemia were examined with the aim to investigate whether the muscle (meat) had any pathological changes and/or contained F. tularensis. Real-time PCR and/or immunohistochemistry (IHC) detected the bacteria in muscle samples from 40 out of 43 investigated hares. IHC showed that bacteria were few and most commonly located in the peri- and endomysium. Histopathology showed occasional perimysial necroses and mild inflammation in association to the bacteria. Attempts to culture from 14 muscle samples were successful in two cases, both stored in the freezer <1 year. The result of this study shows that since F. tularensis is present in the muscle of infected hares, there is a risk for human infection when consuming undercooked hare meat. The risk is enhanced by the fact that some hares do not have easily detected gross lesions. The study contributes to a better understanding of sources of infection and risk factors for humans to contract tularemia.

The growing request for healthier fatty acid composition of animal products is raising the necessity of a deeper knowledge of the main factors controlling fatty acids storage in muscle and backfat. Perilipin (PLIN) 5, and the whole Perilipin family, seem to play a crucial role in the regulation of lipids deposition as code for proteins coating intracellular lipid droplets surface. Nevertheless, the knowledge of these genes in pig is still incomplete. The present research was aimed at investigating in different pig breeds the PLIN5 gene, analysing its expression level and the associations of the variability in its downstream gene region with pork meat and carcass quality traits. Moreover, the PLIN5 protein localisation in porcine skeletal muscle was investigated through immunofluorescence, resulting to be widespread in Semimembranosus muscle (SM) myofibers. The identified single nucleotide polymorphism (SNP) rs327694326 (NC_010444.4:g.74314701T>C) located in PLIN5 downstream region was analysed in different pig populations, represented by 512 Italian Large White (ILW) pigs, 300 Italian Duroc (IDU) samples, 100 Italian Landrace, 100 Pietrain and 60 pigs belonging to three Italian native breeds (20 samples of Cinta Senese, 20 Calabrese and 20 Casertana pigs). The C allele was found in ILW, IDU and Pietrain pigs. In ILW pigs this SNP showed results indicating a possible association with oleic and cis-vaccenic fatty acid contents in backfat tissue. Furthermore, as PLINs are known to regulate lipases activity, we tested if the rs327694326 SNP was associated with differences in Hormone-sensitive lipase (LIPE) gene expression levels. In SM of ILW pigs, PLIN5 C allele was associated with significantly lower LIPE transcription levels than T allele (P=0.02 for Student’s t test of TT v. CT samples, P<0.0001 for TT v. CC pigs), whereas in IDU breed no significant difference was noticed in LIPE transcription levels between TT and TC animals. The results may suggest that variations in the PLIN5 sequence may be linked to LIPE expression through a still poorly known regulative molecular process.

The present study characterized the biological function of the asparaginyl peptidase legumain-1 (LEG-1) of the bovine lungworm Dictyocaulus viviparus and its suitability as a recombinant vaccine against dictyocaulosis. Quantitative real-time PCR and immunoblot analysis revealed LEG-1 to be almost exclusively transcribed and expressed in parasitic lungworm stages. Immunohistochemistry localized the enzyme in the parasite's gut, which was confirmed by immunoblots detecting LEG-1 in the gut as well as male testes. LEG-1 was recombinantly (rLEG-1) expressed in the yeast Pichia pastoris and subsequently analysed in activity assays for its enzyme functions and substrate specificity. For sufficient functionality, rLEG-1 needed trans-activation through D. viviparus cathepsin L-2, indicating a novel mechanism of legumain activation. After trans-activation, rLEG-1 worked best at pH 5·5 and 35–39 °C and cleaved a legumain-specific artificial substrate as well as the natural substrates bovine collagen types I and II. In a clinical vaccination trial, rLEG-1 did not protect against challenge infection. Results of in vitro characterization, transcription pattern and localization enhance the presumption that LEG-1 participates in digestion processes of D. viviparus. Since rLEG-1 needs trans-activation through a cathepsin, it is probably involved in an enzyme cascade and therefore remains interesting as a candidate in a multi-component vaccine.

Objectives: The aim of this report was to assess the clinical effectiveness of two Gene expression profiling (GEP) and two expanded immunohistochemistry (IHC) tests compared with current prognostic tools in guiding the use of adjuvant chemotherapy in patients with early breast cancer.

Methods: A systematic review of the evidence on clinical effectiveness of OncotypeDX, IHC4, MammaPrint, and Mammostrat, compared with current clinical practice using clinicopathological parameters, in women with early breast cancer was conducted. Ten databases were searched to include citations to May 2016.

Results: Searches identified 7,064 citations, of which forty-one citations satisfied the criteria for the review. A narrative synthesis was performed. Evidence for OncotypeDX demonstrated the impact of the test on decision making and there was some support for OncotypeDX predicting chemotherapy benefit. There were relatively lower levels of evidence for the other three tests included in the analysis. MammaPrint, Mammostrat, and IHC4 tests were limited to a small number of studies. Limitations in relation to study design were identified for all tests.

Conclusions: The evidence base for OncotypeDX is considered to be the most robust. Methodological weaknesses relating to heterogeneity of patient cohorts and issues arising from the retrospective nature of the evidence were identified. Further evidence is required for all of the tests using prospective randomized controlled trial data.

The cholinergic system is involved in cortical plasticity, attention, and learning. Within the visual cortex the cholinergic system seems to play a role in visual perception. The cholinergic neurons which project into the visual cortex are located in the basal forebrain. It has been shown that mice deficient for the low-affinity neurotrophin receptor p75NTR display increased numbers of cholinergic neurons in the basal forebrain and a denser cholinergic innervation of the hippocampus. This prompted us to analyze whether the cholinergic system is altered in adult p75NTR deficient mice. By analyzing the densities of cholinergic fibers within layer IV as well as within layer V of the visual cortex, we found that adult p75NTR deficient mice display increased cholinergic fiber densities. However, this increase was not accompanied by an increase in the density of local cholinergic neurons within the visual cortex. This indicates that the enhanced cholinergic innervation of the visual cortex is due to alteration of the cholinergic neurons located in the basal forebrain, projecting to the visual cortex. The increased cholinergic innervation of the visual cortex makes the p75NTR deficient mice an attractive model to study the necessity of the cholinergic system for the visual cortex.