Vismodegib, a sonic hedgehog signalling blockade, ameliorates ovalbumin and ovalbumin/lipopolysaccharide-induced airway inflammation and asthma phenotypical models.

AIMS: Asthmatics exhibit clinical fluctuations between manageable and treatment-resistant phenotypes as a worldwide socioeconomic health burden. Sonic Hedgehog (Shh) genes mediate regulatory pulmonary cell renewal in adults and contribute to the pathogenesis of high phenotypic asthma which depends mainly on T helper-2 (Th-2) cells and related cytokines. However, the exact pathophysiological roles of Shh molecular signalling in the Th-17-dependent low phenotypic allergic airway inflammation and asthma are not evidenced previously. MAIN METHODS: Ovalbumin (OVA) and OVA/lipopolysaccharide (LPS)-sensitized and challenged BALB/c mice were enrolled currently to assess the Shh signalling proteins. Furthermore, the effects of vismodegib, a Smo inhibitor, on the modulation of Shh signalling were compared to dexamethasone. The asthma phenotypes were confirmed by serum total immunoglobulin-E (IgE), bronchoalveolar lavage (BAL) fluid white blood cell counts, lung interleukins, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-ß1, and histopathological changes, and scoring. KEY FINDINGS: Mice challenged with OVA or OVA/LPS showed upregulated lung Shh, patched (Ptch1), smoothened (Smo), and Gli1 proteins. Vismodegib in the two experimental phenotypes of asthma showed reduced airway inflammation and remodelling. Additionally, vismodegib reduced the eosinophilia and neutrophilia reported in high and low asthma types, respectively. Moreover, vismodegib and dexamethasone exhibited negative feedback control throughout the enhanced Shh signalling cascades, including Shh, Ptch1, and Gli1 in several asthma models. SIGNIFICANCE: In conclusion, Shh signalling partially elucidates the OVA/LPS-challenged mice with severe asthma, which proposes a new promising molecular therapeutic target. Furthermore, Smo inhibition by vismodegib has therapeutic potential in both experimental eosinophilic and neutrophilic allergic airway diseases.

Fibroblast Growth Factor 2 Augments Transforming Growth Factor Beta 1 Induced Epithelial-mesenchymal Transition in Lung Cell Culture Model.

Impaired lung epithelial cell regeneration following injury may contribute to the development of pulmonary fibrosis. Epithelial-mesenchymal transition (EMT) is a critical event in embryonic development, wound healing following injury, and even cancer progression. Previous studies have shown that the combination of transforming growth factor beta-1 (TGFß1) and fibroblast growth factor 2 (FGF2) induces EMT during cancer metastasis. However, this synergy remains to be elucidated in inducing EMT associated with wound healing after injury. We set out this study to determine the effect of fibroblast growth factor 2 (FGF2) on TGFß1-induced EMT in the human lung epithelium. BEAS-2B and A549 cells were treated with TGFß1, FGF2, or both. EMT phenotype was investigated morphologically and by measuring mRNA expression levels; using quantitative real-time PCR. E-cadherin expression was assayed by western blot and immunofluorescence staining. Cell migration was confirmed using a wound-healing assay. TGFß1 induced a morphological change and a significant increase in cell migration of BEAS-2B cells. TGFß1 significantly reduced E-cadherin (CDH1) mRNA expression and markedly induced expression of N-cadherin (CDH2), tenascin C (TNC), fibronectin (FN), actin alpha 2 (ACTA2), and collagen I (COL1A1). While FGF2 alone did not significantly alter EMT gene expression, it enhanced TGFß1-induced suppression of CDH1 and upregulation of ACTA2, but not TNC, FN, and CDH2. FGF2 significantly inhibited TGFß1-induced COL1A1 expression. Furthermore, FGF2 maintained TGFß1-induced morphologic changes and increased the migration of TGFß1-treated cells. This study suggests a synergistic effect between TGFß1 and FGF2 in inducing EMT in lung epithelial cells, which may play an important role in wound healing and tissue repair after injury.

Fibroblast growth factor 2 decreases bleomycin-induced pulmonary fibrosis and inhibits fibroblast collagen production and myofibroblast differentiation.

Fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of pulmonary fibrosis. Mice lacking FGF2 have increased mortality and impaired epithelial recovery after bleomycin exposure, supporting a protective or reparative function following lung injury. To determine whether FGF2 overexpression reduces bleomycin-induced injury, we developed an inducible genetic system to express FGF2 in type II pneumocytes. Double-transgenic (DTG) mice with doxycycline-inducible overexpression of human FGF2 (SPC-rtTA;TRE-hFGF2) or single-transgenic controls were administered intratracheal bleomycin and fed doxycycline chow, starting at either day 0 or day 7. In addition, wild-type mice received intratracheal or intravenous recombinant FGF2, starting at the time of bleomycin treatment. Compared to controls, doxycycline-induced DTG mice had decreased pulmonary fibrosis 21 days after bleomycin, as assessed by gene expression and histology. This beneficial effect was seen when FGF2 overexpression was induced at day 0 or day 7 after bleomycin. FGF2 overexpression did not alter epithelial gene expression, bronchoalveolar lavage cellularity or total protein. In vitro studies using primary mouse and human lung fibroblasts showed that FGF2 strongly inhibited baseline and TGFß1-induced expression of alpha smooth muscle actin (αSMA), collagen, and connective tissue growth factor. While FGF2 did not suppress phosphorylation of Smad2 or Smad-dependent gene expression, FGF2 inhibited TGFß1-induced stress fiber formation and serum response factor-dependent gene expression. FGF2 inhibition of stress fiber formation and αSMA requires FGF receptor 1 (FGFR1) and downstream MEK/ERK, but not AKT signaling. In summary, overexpression of FGF2 protects against bleomycin-induced pulmonary fibrosis in vivo and reverses TGFß1-induced collagen and αSMA expression and stress fiber formation in lung fibroblasts in vitro, without affecting either inflammation or epithelial gene expression. Our results suggest that in the lung, FGF2 is antifibrotic in part through decreased collagen expression and fibroblast to myofibroblast differentiation. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In vitro and in vivo antitumor effects of the Egyptian scorpion Androctonus amoreuxi venom in an Ehrlich ascites tumor model.

Scorpion venom is a highly complex mixture of about 100-700 different components, where peptides are the major constituents with various biological and pharmacological properties including anticancer activities. In this study, anticancer efficacy of the venom of the Egyptian scorpion Androctonus amoreuxi has been evaluated. In vitro, the human breast cancer MCF-7 cell line was treated with the venom and the IC50 was estimated. In vivo studies, Ehrlich ascites carcinoma (EAC) cells were inoculated into CD-1 mice intraperitoneally to form liquid tumor or subcutaneously to form solid tumor and then treated with intraperitoneal injection with venom (0.22 mg/kg) every other day. The total tumor cells in the ascitic fluid and the size of the solid tumor were assessed after 14 and 30 days, respectively. In addition, the mean survival time (MST), body weight, tumor volume, PCV, viability of tumor cells, CBC, AST, ALP, creatinine, oxidative stress biomarkers (GSH, MDA, PCC), tumor marker Ki67, growth factor VEGF and caspase-3 were measured in normal control, EAC control and venom-treated groups (n = 6). Treatment with venom induced anti-tumor effects against liquid and in solid tumors as indicated by a significant (P < 0.05) reduction in tumor volume/size, count of viable EAC cells, expression of Ki67 and VEGF as well as by remarkable increases in MST and caspase-3 expression as compared to non-treated group. Interestingly, the venom restored the altered hematological and biochemical parameters of tumor-bearing animals and significantly increased their life span. These data indicate to (1) the cytotoxic potential effects of A. amoreuxi on tumor cells via anti-proliferative, apoptotic and anti-angiogenic activities; (2) opening a new avenue for further studies on the anti-cancer effects of this agent.

Immunomodulatory effects of IL-12 released from poly-N-acetyl glucosamine gel matrix during schistosomiasis infection.

We have reported recently that Interleukin-12 (IL-12) released from poly-N-acetyl glucosamine gel matrix (F2 gel/IL-12) is more effective than free IL-12 to enhance vaccination of mice with Schistosoma soluble worm antigen preparation. The aim of this study is to evaluate the effect of F2 gel/IL-12 on the inflammatory responses in mice undergoing schistosomiasis infection in absence of vaccination. To achieve this, mice undergoing Schistosoma mansoni infection or cured from this infection, after treatment with praziquantil (PZQ), were treated with subcutaneous injection of IL-12 for 3 consecutive days or once with F2 gel loaded with IL-12 (F2 gel/IL-12). The treatment was started on day 35 days after infection. For infection, mice were infected with 100 cercariae of S. mansoni using tail immersion method. We found that treatment with F2 gel/IL-12 induced significant decreases in the egg burden with a moderate reduction in the size of granuloma and decrease in the cellular granulomatous reaction in the lung as compared to infected mice treated with IL-12. These effects of F2 gel/IL-12 were more pronounced in infected mice previously treated with the anti-schistosomal drug PZQ. The total numbers of white blood cells in all treated mice showed similar profile. Treatment with IL-12 or F2 gel/IL-12, however, showed significant reduction in the number of mononuclear cells when compared with non-treated infected mice. In conclusion, this study showed the ability of IL-12 released from F2 gel to lower the inflammatory response to Schistosoma infection even in absence of vaccination.

Histopathological changes in liver of mice after experimental envenomation with Androctonus amoreuxi scorpion venom.

A total of 78 adult male Albino mice were divided into thirteen groups (6 mice in each). One served as a control group and the other twelve groups were venom treated groups. The mice of treated groups were injected with 0.1 ml saline solution in which a particular amount of scorpion venom. The first 6 groups were subcutaneously injected with 1/2 LD50 (0.05 microg/g body weight), while the other 6 groups were injected with 1/4 LD 50 (0.025 microg/g body weight) by the same route. The animals from each group were anesthetized with ethyl ether and sacrificed at different time intervals (3, 6, 9, 12 hrs, 4 & 7days post toxin administration). The microscopic examination of liver tissue obtained from envenomed animals showed variable histopathological changes being severely increased with the time interval of envenoming. The most obvious changes in the liver were acute cellular swelling, hydropic degeneration, congestion of central veins and portal blood vessels. Besides, extramedullary hematopoiesis and invaginations in nuclei of hepatic cells, with formation of intranuclear cytoplasmic inclusions were observed.

Aedes aegypti (Linnaeus) re-emerging in southern Egypt.

Aedes aegypti is one of the demonstrated vector-borne diseases worldwide particularly in the Sub-Sahara of Africa. Its re-emergence in the Egyptian southern border (Aswan) and now in Toshka is an integration mark. Ae. aegypti medical importance, epidemiological implications, ecological behaviors and control measures were discussed.

Clinicopathological investigations on mice envenomed with scorpion venom (Androctonus amoreuxi).

The present study assessed the toxicity of Androctonus amoreuxi crude venom on blood and biochemical serum parameters of mice. Adult male Albino mice were divided into three groups, in the control group mice were injected S.C. with saline solution. The second group and the third were injected with the venom S.C. in mice in the following doses 1/4 and 1/2 dose of LD50 respectively. Blood and serum samples were taken after 3 hours, 6 hours, 9 hours, 12 hours, 4 days and 7 days. Hematocrit (Ht), red blood cells (RBC) count, hemoglobin, MCV, MCH & MCHC were performed. Serum biochemical parameters, the levels of total proteins, albumin, globulin, glucose, cholesterol, triglycerides, ALT, AST, ALP, creatinine, uric acid and urea were measured. RBCs, Hob, PCV, MCV, MCH & MCHC showed significant increase, and increase in total protein, albumin and globulin within the experiment. Glucose and cholesterol levels were significantly increase from the beginning. Triglycerides showed significant decrease after 6 hours. Liver enzymes and kidney functions revealed significant changes post-injection.

Corvus R. ruficollis (desert or brown necked raven): a reservoir host for zoonotic parasites in Egypt.

Egypt includes many desert and rural areas. The small uptown fertile areas are placed under illegal enormous pressure of existing resources, where intensive agricultural practices are performed in combination with high population densities. The brown necked ravens (Corvus ruficollis) are attracted in huge numbers to such areas. The birds are omnivorous, very aggressive pest and seriously affect human welfare. The study focused on zoonotic role of ravens.

Efficacy of Ixodes ricinus as a vector of zoonotic babesiosis in Sinai Peninsula, Egypt.

Ticks and blood samples were collected every month from March 2009 through April 2010 from different sites in Sinai to detect babesial parasites using PCR assay based on nuclear small subunit rRNA gene. Ticks were found to contain babesial DNA. Sequence determination and analysis of amplified portions of nss-rDNA revealed their identity with B. bovis and a high degree of homology with B. bigemina and B. divergens. The results represent the first genetic evidence of different species of Babesia and identified the role of Ixodes ricinus as a vector of zoonotic B. microti infection. Rodent isolate (HK) and American isolate (GI) were studied in transmission experiments. The present study used in vitro culture of zoonotic Babesia sp. EU1 from blood samples of rodent in Sinai. This study provides an evidence of transovarian and transstadial transmissions of the parasite within I. ricinus, which emphasizes that this tick could be a vector and reservoir of EU1

Identification of Egyptian Fasciola species by PCR and restriction endonucleases digestion of the nuclear small subunit ribosomal RNA gene.

Fascioliasis is one of the familiar zoonotic health problems of worldwide distribution including Egypt. In this study, a simple and rapid polymerase chain reaction/restriction fragment length polymorphisms (PCR/RFLPs) assay, using the common restriction endonucleases Aval, EcoRI, Eael, Sac11 and Avail was applied to differentiate between both Fasciola gigantica and F. hepatica. The five restriction endonucleases were used to differentiate between the two species of Fasciola based on -1950 bp long sequence of the 18S nuclear small subunit ribosomal RNA gene. Aval and EcoRI restriction endonucleases failed to differentiate between the two Fasciola species when each restriction enzyme gave the same restriction patterns in both of them. However, F. gigantica and F. hepatica were well-differentiated when their small subunit ribosomal DNA were digested with Eael and Sac 11 restriction endonucleases.

Studies on zoonotic cryptosporidiosis parvum in Ismailia Governorate, Egypt.

A total of 390 stool samples from children less than 8 years old attending the MOHP central hospital in Ismailia District were examined for cryptosporidiosis. Stools were subjected to direct wet smear method and Sheather's sugar flotation and stained with Modified Z.N. Among the 390 children 204 were diarrheic of whom C. parvum was positive in 68 (33.3%). The highest infection rate was 26/46 among children less than 2 months, 40/150 among children less than 2 years and 2/8 among children less than 7 years. Of these children the clinical pictures ranged from diarrhea (20.7%), to dehydration (20%), abdominal pain and mild fever (19.2%), and the lowest was tenesmus (6.25%). The infection rate was 88.2% among cryptosporidiosis children compared to 11.8% that not in contact with animals. Water samples examined showed was 0.0% in bottled water up to 9.33% in water tank. C. parvum in farm animals was 20.9% in sheep, 22.5% in buffaloes, 23.7% in cows and 25.9% in goats.

ELISA-seroprevalence of Toxoplasma gondii in draught horses in Greater Cairo, Egypt.

Toxoplasma gondii is one of the important zoonotic parasites of worldwide. In this paper the seroprevalence of T. gondii in draught horses (3-15 years) including 90 males and 10 females in the first half of the year 2009 was studied. The result showed that the overall ELISA-T. gondii antibodies were 25% of the horses in Greater Cairo, 50% (females) and 22.2% (males).

Laboratory studies on the effect of magnetic technologies on some biological parameters of Biomphalaria alexandrina in Egypt.

The effect of magnetic water on some biological parameters of B. alexandrina was investigated. The growth rate, egg laying capacity, hatchability of deposited eggs, development of the newly hatched snails and the mortality rate of the treated snails (maintained in magnetic water for 12 weeks) were determined and compared with control ones maintained in normal water. The mean length of the diameter of treated snails after 12 weeks exposure to magnetic water (6.8 +/- 0.93) was slightly greater than the control group (6.4 +/- 0.8). Also, a highly significant increase in number of eggs and egg-masses of treated snails (8.06 +/- 6.55 & 1.14 +/- 0.8, respectively) as compared with controls (2.5 +/- 1.2 & 0.33 +/- 0.14, respectively). Hatchability rate of treated eggs in magnetic water for a period of 5 days was higher (63.4%) than the control group which recorded 28.2%. However, a highly significant reduction in the survival rate of newly hatched snails (22.5%) maintained in magnetic water for another 7 days after hatching as compared with the control (61.5%). Also a highly significant reduction in the survival rate of adults in magnetic water for 12 weeks which recorded 40% in contrast with the control ones, recorded 88%, (P < 0.001). There was slight decrease in the calcium content of the shells of treated snails (25.44% of wt.) as compared to controls (29.58% of wt.). The haermaphrodite gland acini of snails in magnetic water for 12 weeks were most frequently in final stages of oogensis and spermatogenesis. There were a significant increase in the mean number of 2ry oocytes and mature ova in acini of treated snails comparing with the control group (P < 0.05).

Influence of certain fertilizers on the activity of some molluscicides against Biomphalaria alexandrina and Lymnaea natalensis snails.

Effect of the fertilizers (ammonium nitrate, potassium sulphate and urea) on molluscicidal activity of the molluscicides (copper sulphate, niclosamide & mollutox) against B. alexandrina and L. natalensis was investigated. The molluscicides were more potant than fertilizers. Snails were exposed for 24 hr to a fertilizers using LC0 (1/10 & LC50) then, to molluscicides. Pre-exposure to potassium sulphate caused a synergistic action with copper sulphate, niclosamide and mollutox on L. natalen-sis. Pre-exposure to urea caused an additive effect with niclo-samide and mollutox against L. natalensis and B. alexandrina respectively. Pre-exposure to ammonium nitrate caused an additive action to niclosamide on L. natalensis. Snails were exposed for 24hr to one molluscicide, then exposed to fertilizers, showed that pre-exposure to niclosamide or mollutox caused an additive effect with ammonium nitrate and potassium sulphate. Pre-exposure to mollutox caused an additive effect with urea on the two snails' sp. juvenile or adult B. alexandrina were ex-posed to LC0 of molluscicide-fertilizer mixture, showed that urea when mixed with each molluscicides showed greatly reduced on the growth rate percent (0.00), survival rate and snail fecundity. Molluscicides and fertilizers mixed at different ratios of LC (40:10, 30:20, 25:25, 20:30 & 10:40), the toxicity of the mixtures caused antagonistic effect on adult B. alexandrina, but a mixture of niclosamide-ammonium nitrate caused a potent effect (synergism or additive) except at ratio 20:30 which showed an antagonism on L. natalensis. Mixtures of copper sulphatepotassium sulphate (10:40), niclosamide-potassium sulphate (20:30), mollutox-ammonium nitrate (25:25) revealed an additive effect on L. natalensis.

Effect of Commiphora molmol on Bithynia connollyi with special reference to their morphology and medical importance.

Morphological studies on B. connollyi dealt with the shell description, concerning colour, number of whorls, measurements, the correlation coefficient between shell shape and thickness, weight, length and width. Electrophoretic studies were carried out on snail's foot. Examination of B. connollyi showed a lot of gymnocephalus cercariae of liver fluke, Opisthorchis sp. Commiphora mohmol (Myrrh) has molluscicidal effect on B. connollyi at concentration (80 ppm) after 72 hr exposure. The mortality rate increased with the increasing the exposure time (death 100% at 72 hr. with 80 ppm & death 100% at 96 hr. with 40 ppm). Based on safety to man, animals and environment, C. molmol is highly recommended as a cheap herbal molluscicide.

Patients with gastrointestinal complains due to enteric parasites, with reference to Entamoeba histolytica/dispar as dected by ELISA E. histolytica adhesion in stool.

A total of 210 patients with gastrointestinal troubles, of both sex and a mean age of 32 +/- 6.1 years, selected from the outpatient's clinics of Al-Azhar University Hospitals. 115 (54.76%) had dysentery, 95 (45.23%) did not have dysentery, 15 (14%) suffered flatulence, 20 (9.52%) had epi-gastric pain, 19 (9.05%) had vague abdominal pain, 5 vomiting (5.2%) and 10 (4.9%) had fever. Two symptoms were in 29 (13.81%) patients and three symptoms in 12 (5.71%). Of the 210 patients, 20 (9.9%) had helminthes infection, 121 (57.6%) had intestinal protozoa and 69 (32.9%) had no parasitic infection. Of these parasite-free patients, 16 had Shigella sp. and nine had Campylobacter sp. Of the patients with intestinal protozoa, 34 (16.2%) had E. histolytica/dispar by stool examination of stained smears. By using ELISA for detection of E. histolytica adhesion in stool samples of 115 with diarrhea only 18 had true E. histolytica infection and of 3 without diarrhea only one had E. histolytica infection. Mean-while, ELISA did not cross-reacted E. coli, Giardia lamblia, Cryptosporidium parvum, Endolimax nana or Blastocystis hominis. So, ELISA for detection of E. histolytica adhesion in stool samples was more specific than microscopy and safe direction to the E. histolytica treatment. Apart from intestinal protozoan and bacteria, helminthes were seen in stool analysis. These were Schistosoma mansoni (0.95%), Capillaria sp. (0.95%), Enterobius vermicularis (1.90%) macroscopically, Hymenolepis nana (4.3%) and Ascaris lumbricoides (1.43%).

Studies on rodents role as reservoir hosts of leishmaniasis with specical reference to their ectoparasites in Suez Governorate.

The distribution of rodents was studied in three different habitats. Seven rodent species were identified: Rattus norvegicus, R. alexandrinus, R. frugivorous, Mus musculus, Acomys russatus, Meriones sacramenti and Gerbillus pyramidum. The species distribution varied with the habitat type. The highest density of rodents was in July and August and the lowest one was in January. However, some species were collected all the year round. The rodents were investigated for the endo- and ecto-parasites. No Leishmania parasites were found. The ectoparasites were: Xenopsylla cheopis, Leptopsylla segnis and Ctenocephalides felis, Polyplax spinulos, Hyalomma dromedarii (nymph) and Echinolaelaps echidninus and Hemolaelaps glassgowi. Ecto-parasites were on rodents all year-round in domestic habitat and peridomestic habitats. In wild one, ecto-parasites activity was from March to December. The rodents' role as reservoir for L. major was experimentally studied. Rodents inoculated with L. major together with hamster and BALB-c mice developed cutaneous lesions. The active lesions, the rodents' ecological habitats and the presence of insect-vector may pave the way to an epidemic zoonotic leishmaniasis role.

Ecology, biology and susceptibility of Phlebotomus papatasi to Leishmania experimental infection in Suez Governorate.

Zoonotic cutaneous leishmaniasis (ZCL) is endemic in Sinai Peninsula. The sand fly and reservoirs were investigated in Suez G., since new settlements and land reclamation programs are ongoing. The results showed that Phlebotomus papatasi reached its highest density in September. The successfully colonized P. papatasi facilitated its biology and competence study. An autogenous trait was proven within P. papatasi population indicating its ability to survive and breed during adverse conditions. The vector competence was carried out under laboratory condition through feeding on lesion of a L. major experimentally infected hamster and by membrane feeding technique. Both hamsters and BALB-c mice inoculated with L. major developed ZCL lesions.