MicroRNA-26a systemic administration attenuates tumor formation in hepatocellular carcinoma mouse model.

MicroRNA (miRNA)-26a is one of the tumor suppressor genes that has been down regulated during the development of hepatocellular carcinoma (HCC). This work was conducted to evaluate the possible preventive effect of exogenous miRNA-26a administration on diethylnitrosamine (DEN)-mediated HCC. Balb/C mice were intraperitoneally injected with saline (Normal group), DEN (HCC group) or miRNA-26a (HCC+miRNA-26a group). On week 8, 12, 16 and 20, the concentrations of alpha-fetoprotein (AFP), des-gamma carboxyprothrombin (DCP), the levels of helper T cells-associated cytokines, and the vascular endothelial growth factor (VEGF), were measured. Flow cytometry determined the frequencies of regulatory T (Treg) cells. The concentrations of AFP, DCP and VEGF, as well as the frequency of Treg cells showed significantly lower values following miRNA-26a administration than in HCC group. miRNA-26a administration has reduced the levels of IL (interleukin)-2 and TNF (tumor necrosis factor)-α, in contrast, IL-10 level was markedly elevated in comparison to HCC model at all experimental time points. The restore of miRNA-26a function significantly (P<0.001) down regulated the expression levels of survivin & caspase-3 compared to HCC group. The obtained data introduce an evidence for the suppressive impact of miRNA-26a on liver tumor formation and its possible manipulation as a therapeutic design for HCC.

Tumor microenvironmental plasmacytoid dendritic cells contribute to breast cancer lymph node metastasis via CXCR4/SDF-1 axis.

PURPOSE: Plasmacytoid dendritic cells (PDCs) infiltration into breast cancer tissues is associated with poor prognosis. Also, CXCR4 shows compelling evidences to be exploited by cancer cells to migrate to distant sites. The present study investigated lymph node metastasis in the light of PDCs infiltration and the potential cross talk with CXCR4/SDF-1 chemokine axis. METHODS: We assessed circulating PDCs proportions drained from the axillary tributaries, and the in situ expression of both CD303 and CXCR4 in breast cancer patients with positive lymph nodes (pLN) and negative lymph nodes (nLN) using immunohistochemistry and flow cytometry. We also analyzed the expression of SDF-1 in lymph nodes of pLN and nLN patients. We studied the effect of the secretome of PDCs of pLN and nLN patients on the expression of CXCR4 and activation of NF-κB in human breast cancer cell lines SKBR3 and MCF-7. TNF-α mRNA expression level in PDCs from both groups was determined by qPCR. RESULTS: Our findings indicate increased infiltration of PDCs in breast cancer tissues of pLN patients than nLN patients, which correlates with CXCR4+ cells percentage. Interestingly, SDF-1 is highly immunostained in lymph nodes of pLN patients compared to nLN patients. Our in vitro experiments demonstrate an upregulation of NF-κB expression and CXCR4 cells upon stimulation with PDCs secretome of pLN patients than those of nLN patients. Also, PDCs isolated from pLN patients exhibited a higher TNF-α mRNA expression than nLN patients. Treatment of MCF-7 cell lines with TNF-α significantly upregulates CXCR4 expression. CONCLUSIONS: Our findings suggest a potential role for microenvironmental PDCs in breast cancer lymph node metastasis via CXCR4/SDF-1 axis.

Pathogenic Potential of Fresh, Frozen, and Thermally Treated Anisakis spp. Type II (L3) (Nematoda: Anisakidae) after Oral Inoculation into Wistar Rats: A Histopathological Study.

The third-stage (L3) larvae of Anisakis are the etiological agents of human anisakiasis caused by consumption of raw or undercooked seafood infected with anisakid nematodes. Infection with these worms is associated with abdominal pain, nausea, and diarrhea and can lead to massive infiltration of eosinophils and the formation of granulomas in the gastrointestinal tract if the larvae are not removed. Food allergy affects populations worldwide, and despite several reports on the presence of the potentially zoonotic nematodes among edible fishes in Egypt, there are few immunological and molecular studies investigating the epidemiology of these parasites. Anisakidosis, a human infection with nematodes of the family Anisakidae, is caused most commonly by Anisakis spp. In the present study, seventy specimens of the European seabass Dicentrarchus labrax commercialized in Alexandria city along the Mediterranean Sea were acquired during the period from July to December, 2015. Fish were necropsied and dissected to investigate the presence of nematode larvae. Thirty fish (42.9%) of the total were parasitized by nematode larvae which were morphologically identified as Anisakis spp. Type II (L3) according to light and scanning electron microscopy. The pathogenic potential of oral inoculation of fresh, frozen, and thermally treated larvae into Wistar rats was elucidated by histological examination of their thymus and spleen. Results obtained indicated that neither cooling nor freezing of the parasite could destroy their allergenic capacity. So, it is important to create a wider awareness of this potential risk to human health. It is becoming increasingly likely that the impact of Anisakis spp. on human health has been underestimated, and it is perhaps time to consider more sweeping measures than those currently enforced to protect the public health.

New host records of three juvenile nematodes in Egypt: Anisakis sp. (Type II), Hysterothylacium patagonense (Anisakidae), and Echinocephalus overstreeti (Gnathostomatidae) from the greater lizard fish Saurida undosquamis of the Red Sea.

Three juvenile nematode parasites were collected naturally from 90 (75 %) out of 120 specimens of the marine greater lizard fish Saurida undosquamis captured from water coasts at Hurghada City along the Red Sea in Egypt during the period from September 2013 to April 2014. Worms were identified on the basis of light and scanning electron microscopy. Two of the recovered worms were isolated from the peritoneal cavity of the infected fish around the wall of the stomach as encapsulated larvae. The anisakid juvenile Anisakis sp. (Type II) was characterized by an anteroventrally triangular mouth, with a boring tooth; its postanal tail was rounded, without a terminal mucron or spine. The gnathostomatid Echinocephalus overstreeti was characterized by the presence of a cephalic bulb armed with six transverse rows of spines which were slightly more compact near the anterior end of bulb with maximal separation near the midbulb; the cephalic bulb terminated at a pseudolabia which situated dorsoventrally and reached its greatest width at the posterior one third of the body, The postanal tail terminated at a pointed mucron. The third juvenile species, Hysterothylacium patagonense (Anisakidae), was isolated from the intestine of the infected fish; they are characterized by a small-sized body with a conical tail provided by a nodulose apex, and the anterior end was equipped with three lips. A dorsal lip slightly smaller than the two subventrals left a deep postlabial groove and prominent lateral flanges in between, and the proximal part of each lip was smooth. The three described species were compared morphologically and morphometrically with some of the previously recorded species of the same genus. From this comparison, the similarity and variations between these species were described and concluded that the present study should be considered as a new host record in Egypt.

Cytokine signature and antibody-mediated response against fresh and attenuated Anisakis simplex (L3) administration into Wistar rats: implication for anti-allergic reaction.

The third larval stage (L3) of Anisakis simplex (Anisakidae) is one of the zoonotic parasitic nematodes in the musculature and visceral organs of marine fishes belonging to family Moronidae. The consumption of these high-commercial-value fish is widespread in many countries around the Mediterranean Sea including Egypt. The presence of these larvae in fish muscles poses a potential consumer hazard due to the parasite's ability to cause anisakidosis. Forty-two out of 60 (70%) of the European seabass Dicentrarchus labrax were found to be naturally infected by L3 of A. simplex in the form of encapsulated juveniles in the fish musculature. Morphological examination of recovered parasites by light and scanning electron microscopy showed that, in general, all specimens examined closely resembled A. simplex (L3). To evaluate the allergenicity of this nematode, white blood cell count; levels of T helper 1 (Th1) [interferon (IFN)-γ and tumor necrosis factor (TNF)-α)], Th2 [IL-4, IL-5, and IL-6], and Th17 [IL-17] related cytokines; total IgE and IgG antibodies; and nitric oxide (NO) were measured in the plasma of Wistar rats sensitized by oral inoculation with fresh, frozen, and heat-treated A. simplex L3 or rats intraperitoneally injected with L3 crude extract. Rats sensitized with fresh and frozen L3 larvae produced significantly higher levels of IFN-γ, IL-5, IL-17, and total IgE as compared to control rats. Heat-treated larvae administration resulted in a significant rise of IFN-γ, TNF-α, IL-5, and total IgE in comparison to control rats. Intraperitoneal sensitizations enhanced release of IFN-γ, TNF-α, and total IgE. Oral sensitization led to a significant production of NO. Thereby, frozen or cooked larval L3 cannot inhibit the release of Th-related cytokines and IgE, which might impact on the overall anti-parasitic immunity.

Microsatellite instability profiling in Egyptian bladder cancer patients: A pilot study.

Microsatellite alterations have been implicated in the pathogenesis of many cancers; however, they are still not well addressed in the bladder cancer (BC) of Egyptian population. We assessed microsatellite instability (MSI) profile and loss of heterozygosity (LOH) using 13 microsatellite markers in tumor tissue samples and urine sediments obtained from 30 Egyptian patients with BC. The concordance between MSI in tumor tissue and urine samples was determined, and correlated to relevant clinicopathologic features. We found that MSI was more frequent than LOH (100% and 46.7%, respectively). D16S310, MBP, and IFN-α showed the highest MSI frequency in urine samples (70%, 70%, and 66.67%, respectively), while MBP, ACTBP2, and D9S171 (66.67%, 63.33%, and 60%, respectively) were the most frequently detected in tumor tissues. All assessed MSI markers correlated significantly with pathologic subtype (being more frequent in TCC) and with hematuria. The concordance between tissue and urine samples was statistically significant for D16S476, D9S171, FGA, and ACTBP2 (P = 0.04, 0.015, 0.02, and 0.007, respectively). When we combined D16S476 and D9S171, the sensitivity, specificity, positive predictive value, and negative predictive value for the diagnosis of BC were 80.0%, 75.0%, 82.8%, and 71.4%, respectively. Accordingly, we concluded that MSI in urine sediments could be a potential tool for the diagnosis of BC.

Role of relevant immune-modulators and cytokines in hepatocellular carcinoma and premalignant hepatic lesions.

AIM: To assess the levels of different immune modulators in patients with hepatocellular carcinoma (HCC), in relation to other hepatic diseases. METHODS: Eighty-eight patients were included in the current study and represented patients with HCC (20), liver cirrhosis (28) and chronic hepatitis (CH; 25), and normal controls (NC; 15). Peripheral blood was isolated for immunophenotyping of active myeloid dendritic cells (mDCs; CD1c and CD40), mature inactive myeloid cells (CD1c and HLA), active plasmacytoid cells (pDCs; CD303 and CD40), mature inactive pDCs (CD30 and HLA), active natural killer (NK) cells (CD56 and CD161), active NK cells (CD56 and CD314) and inactive NK cells (CD56 and CD158) was done by flow cytometry. Serum levels of interleukin (IL)-2, IL-10, IL-12, IL-1ß, interferon (IFN)-α, IFN-γ and tumor necrosis factor (TNF)-αR2 were assessed by ELISA. RESULTS: Active mDCs (CD1C+/CD40+) and inactive mDCs (CD1c+/HLA+) were significantly decreased in HCC patients in relation to NC (P < 0.001). CD40+ expression on active pDCs was decreased in HCC patients (P < 0.001), and its level was not significantly changed among other groups. Inactive pDCs (CD303+/HLA+), inactive NKs (CD56+/CD158+) and active NKs (CD56+/CD161+) were not statistically changed among the four groups studied; however, the latter was increased in CH (P < 0.05). NKG2D was statistically decreased in HCC, CH and cirrhosis (P < 0.001), and it was not expressed in 63% (12/20) of HCC patients. There was significant decrease of IL-2, IFN-α and IFN-γ (P < 0.001), and a significant increase in IL-10, IL-1ß, and TNF-αR2 (P <0.01, P < 0.001 and P < 0.001; respectively) in HCC patients. There was inverted correlation between IL-12 and IL-1ß in HCC (r = -0.565, P < 0.01), with a strong correlation between pDCs (CD303+/CD40+) and NKs (CD56+/CD161+; r = 0.512, P < 0.05) as well as inactive mDCs (CD1c+/HLA+) and inactive NK cells (CD56+/CD158+; r = 0.945, P < 0.001). CONCLUSION: NKG2D, CD40, IL-2 and IL-10 are important modulators in the development and progression of HCC.

Effect of artemether on cytokine profile and egg induced pathology in murine schistosomiasis mansoni.

Artemether (ART), the methylated derivative of artemisinin, is an efficacious antimalarial drug that also displays antischistosomal properties. This study was designed to evaluate the immunomodulatory action of a single intramuscular dose (50 mg/kg body weight) of ART in comparison with PZQ treatment (42 days PI). ART administration was 7, 14, 21 and 45 days PI. ART effect was studied parasitologically, histopathologically and immunologically. It was found that maximum effect was reached when ART treatment interfered with 14 or 21 days old schistosomula. ART treatment 14 or 21 days PI was associated with shift from Th2 to Th1 predominancy (decrease in IL-4 and upgrading of serum IFN-γ levels). In conclusion, ART is a promising drug in control of schistosomiasis mansoni due to its reductive effect on worm burden and its role in improvement of hepatic granulomatous lesions.

Immunodiagnosis of fascioliasis using sandwich enzyme-linked immunosorbent assay for detection of Fasciola gigantica paramyosin antigen.

BACKGROUND: Many immunological techniques have been developed over years using the different Fasciola antigens for diagnosis of parasitic infection and to replace the parasitological techniques, which are time consuming and usually lack sensitivity and reproducibility. MATERIALS AND METHODS: In this study, Fasciola gigantica paramyosin (Pmy) antigen was early detected in cattle sera using sandwich enzyme-linked immunosorbent assay (ELISA), to evaluate the Pmy antigen performance in diagnosis. This work was conducted on 135 cattle blood samples, which were classified according to parasitological investigation into, healthy control (30), fascioliasis (75), and other parasites (30) groups. RESULTS: The sensitivity of Sandwich ELISA was 97.33%, and the specificity was 95%, in comparison with parasitological examination, which recorded 66.66% sensitivity and 100% specificity, respectively. CONCLUSIONS: It was clear that the native F. gigantica Pmy is considered as a powerful antigen in early immunodiagnosis of fascioliasis, using a highly sensitive and specific sandwich ELISA technique.

Evaluation of the diagnostic potential of different immunological techniques using polyclonal antibodies against Fasciola gigantica excretory/secretory antigens in sheep.

The early detection of Fasciola antigens (Ags) in serum or stool could be more valuable in diagnosis as early treatment would be applied before irreparable damage occurs. In this study, fresh adult Fasciola gigantica (F. gigantica) worms were cultivated for 16 hrs. Excretory/secretory (E/S) Ags were extracted from the culturemedium and used to raise rabbit antibodies (Abs) to Fasciola. The purified Abs were then used in sandwich ELISA (S-ELISA) to detect Fasciola Ags in serum and stool samples from a total of 152 sheep, and sandwich-Dot-ELISA (S-D-ELISA) for the serum samples. Gross inspection of liver for flukes or other parasites was performed and results of parasitological stool examination were recorded. Accordingly, sheep were divided into healthy control group (25 sheep), Fasciola positive group (97 sheep) and other helminthic infection groups (30 sheep). S-ELISA for serum samples showed 91.9% sensitivity and 89% specificity. Fasciola Ags, detected in serum of sheep by S-D-ELISA, showed 97.2% sensitivity and 95% specificity and coproantigens detected by S-ELISA, showed 95.8% sensitivity and 92.7% specificity. Although, the specificity of stool examination was higher than that recorded for serum, the sensitivity of ELISA techniques to diagnose Fasciola Ags was higher than that recorded for parasitological examination. It is concluded that, S-D-ELISA has better sensitivity and specificity than S-ELISA for both stool and serum, and may prove useful for field applications.

Ontogeny of hemopoietic and lymphopoietic tissues in the lizard Chalcides ocellatus (Reptilia, Sauna, Scincidae).

The first and major blood-forming organ to develop in the viviparous lizard Chalcides ocellatus is the yolk sac, which exhibits prominent erythropoietic activity from as early as stage 21 through birth (stage 41). Myeloid cells and megakaryocytes are produced in the yolk sac from stage 23 onward. During lizard embryogenesis hemopoietic activity is also observed in spleen and bone marrow but in neither kidney nor liver. Cells capable of giving rise to lymphocytes both in vivo and in vitro are first found in the thymus at stage 35. Active lymphopolesis in thymus and spleen begins at stages 36 and 39, respectively. In contrast, the gut-associated lymphoid aggregates are not evident before birth.