Significance of Intracellular Adhesion Molecule-1 Polymorphism and IP-10 among Breast Cancer Patients.

Breast cancer (BC) is the second leading cause of women's death worldwide. Intercellular adhesion molecule-1 (ICAM-1) is involved in cell-cell interaction, migration and recruitment of immune cells. Polymorphisms in ICAM-1 gene may be involved in BC progression. IFN-gamma inducible protein-10 (IP-10) has the ability to recruit T-cells to induce cellular immunity and may have protective effect against BC development. The current study aimed to shed light on the role of of ICAM-1 SNP and/or serum levels of IP10 in BC in Egyptian female patients and detect possible correlation between these two factors and pathological prognostic markers. 40 breast cancer patients and 40 healthy females were enrolled in the study. Genotyping of ICAM-1 rs281437 SNP was done using real time PCR and serum levels of IP-10 were measured using ELISA. Allelic distribution demonstrated high frequency of ICAM-1 rs281437 CC genotype among BC patients (60%) compared to CT and TT alleles (30% and 10%, respectively). ICAM-1 rs281437 CC genotype showed 9.8 folds more risk to develop BC than other genotypes (95% CI=5.8-21.8, P<0.05). Relation between the studied alleles and hormonal receptors (ER, PR) showed that both ICAM-1 rs281437 CC and CT genotype have 5 folds more to be ER+, PR+ BC compared to TT allele (95% CI=0.21-117.8 and 0.15-125.4, respectively). Serum IP-10 levels were markedly decreased among breast cancer patients when compared with healthy controls (P = 0.001). In conclusion, ICAM-1 rs281437 CC genotype is significantly associated with breast cancer; females carrying CC allele may be at higher risk to develop BC than those carrying CT or TT genotypes. On the other hand, IP-10 may have a protective effect against breast cancer.

Amino group of salicylic acid exhibits enhanced inhibitory potential against insulin amyloid fibrillation with protective aptitude toward amyloid induced cytotoxicity.

Protein misfolding and aggregation lead to amyloid generation that in turn may induce cell membrane disruption and leads to cell apoptosis. In an effort to prevent or treat amyloidogenesis, large number of studies has been paying attention on breakthrough of amyloid inhibitors. In the present work, we aim to access the effect of two drugs, that is, acetylsalicylic acid and 5-amino salicylic acid on insulin amyloids by using various biophysical, imaging, cell viability assay, and computational approaches. We established that both drugs reduce the turbidity, light scattering and fluorescence intensity of amyloid indicator dye thioflavin T. Premixing of drugs with insulin inhibited the nucleation phase and inhibitory potential was boosted by increasing the concentration of the drug. Moreover, addition of drugs at the studied concentrations attenuated the insulin fibril induced cytotoxicity in breast cancer cell line MDA-MB-231. Our results highlight the amino group of salicylic acid exhibited enhanced inhibitory effects on insulin fibrillation in comparison to acetyl group. It may be due to presence of amino group that helps it to prolong the nucleation phase with strong binding as well as disruption of aromatic and hydrophobic stacking that plays a key role in amyloid progression.

NY-BR-1 Antigen Expression and anti-NY-BR-1 IgG in Egyptian Breast Cancer Patients: Clinicopathological and Prognostic Significance.

Breast cancer is the most common gynecological malignancy in the world. In Egypt, it ranks the first among female malignancies with incidence of 37.7%. Over the last decades, the integration of prognostic and predictive markers in treatment decisions has led to more individualized and optimized therapy. NY-BR-1 antigen has been shown to be frequently expressed in breast cancers. The study aimed to assess the tissue expression of NY-BR-1 antigen and serum IgG antibody to this antigen in Egyptian breast cancer females. The study was conducted on 60 females (10 healthy, 10 having benign breast lesions, 40 with malignant breast cancer). NY-BR-1 Ag expression was evaluated by immunohistochemistry and anti-NY-BR-1 IgG was assessed by ELISA. Results revealed a significant difference in NY-BR-1 Ag expression between benign and malignant breast cancer patients. There was a significant correlation between NY-BR-1 antigen expression and estrogen receptor's status (P = 0.019), stage of the disease (P = 0.008), menopausal status (P = 0.008), lymph node involvement (P = 0.022) and anti-NY-BR-1 IgG (P = 0.032) among the studied individuals. In addition, there was a statistically significant increase in anti-NY-BR-1 IgG O.D. results among malignant breast cancer group. It is correlated with tumor type (P < 0.001) and progesterone receptor status (P = 0.038). In conclusion, our work may represent a step towards identification of a new prognostic marker specific for breast cancer.

Effect of murine exposure to gamma rays on the interplay between Th1 and Th2 lymphocytes.

Gamma radiation radiotherapy is one of the widely used treatments for cancer. There is an accumulating evidence that adaptive immunity is significantly contributes to the efficacy of radiotherapy. This study is carried out to investigate the effect of gamma rays on the interplay between Th1/Th2 response, splenocyte lymphoproliferative response to polyclonal mitogenic activators and lymphocytic capacity to produce IL-12 and IL-10 in mice. Results showed that exposure of intact spleens to different doses of γ-rays (5, 10, 20 Gy) caused spontaneous and dose-dependent immune stimulation manifested by enhanced cell proliferation and elevated IL-12 production with decreased IL-10 release (i.e., Th1 bias). While exposure of splenocytes suspension to different doses of γ-rays (5, 10, 20 Gy) showed activation in splenocytes stimulated by PWM at 5 Gy then a state of conventional immune suppression that is characterized by being dose-dependent and is manifested by decreased cell proliferation and IL-12 release accompanied by increase in IL-10 production (i.e., Th2 bias). In addition, we investigated the exposure of whole murine bodies to different doses of γ-rays and found that the exposure to low dose γ-rays (0.2 Gy) caused a state of immune stimulation terminated by a remarkable tendency for immune suppression. Exposure to 5 or 10 Gy of γ-rays resulted in a state of immune stimulation (Th1 bias), but exposure to 20 Gy showed a standard state of immune suppression (Th2 bias). The results indicated that apparently we can control the immune response by controlling the dose of γ-rays.

Effect of ultra violet irradiation on the interplay between Th1 and Th2 lymphocytes.

UNLABELLED: Although ultraviolet (UV) radiation is used to treat several types of diseases, including rickets, psoriasis, eczema, and jaundice, the prolonged exposure to its radiation may result in acute and chronic health effects particularly on the skin, eyes, and the immune system. AIM: This study was carried out to show the effect of UV on both of the lymphoproliferative response and their capacity to produce IL-12 and IL-10 in mice. METHODS: Mice were exposed to whole body UVB and tested for the effect of recovery times on lymphocyte proliferation and cytokine production. In addition, direct irradiation of spleens and lymphocyte suspension was carried out. Basal and mitogens-stimulated lymphocyte proliferation was assessed by MTT assay while IL-10 and IL-12 were measured using ELISA. RESULTS: There was a significant suppression in lymphocyte proliferation in comparison with control. IL-12 level was significantly reduced while the level of IL-10 was increased. Con A and PWM mitogens had no significant changes in IL-10 while Con A caused a highly significant increase in IL-12 at day 6 of recovery in UVB body irradiation. CONCLUSION: Exposure to UVB radiation could cause a state of immune suppression and shifts Th1/Th2 cell response. This effect is closely associated with the reduction of Th1 cytokines' expression and increase in Th2 cytokines' levels.

Inhibition of the recombinant cattle tick Rhipicephalus (Boophilus) annulatus glutathione S-transferase.

Rhipicephalus (Boophilus) annulatus is a bloodsucking ectoparasite that causes severe production losses in the cattle industry. This study aims to evaluate the in vitro effects of tannic acid, hematin (GST inhibitors) and different plant extracts (rich in tannic acid) on the activity of the recombinant glutathione S-transferase enzyme of the Egyptian cattle tick R. annulatus (rRaGST), in order to confirm their ability to inhibit the parasitic essential detoxification enzyme glutathione S-transferase. Extraction with 70% ethanol of Hibiscus cannabinus (kenaf flowers), Punica granatum (red and white pomegranate peel), Musa acuminata (banana peel) (Musaceae), Medicago sativa (alfalfa seeds), Tamarindus indicus (seed) and Cuminum cyminum (cumin seed) were used to assess: (i) inhibitory capacities of rRaGST and (ii) their phenolic and flavonoid contents. Ethanol extraction of red pomegranate peel contained the highest content of phenolic compounds (29.95mg gallic acid/g dry tissue) compared to the other studied plant extracts. The highest inhibition activities of rRaGST were obtained with kenaf and red pomegranate peel (P. granatum) extracts with IC50 values of 0.123 and 0.136mg dry tissue/ml, respectively. Tannic acid was the more effective inhibitor of rRaGST with an IC50 value equal to 4.57µM compared to delphinidine-HCl (IC50=14.9±3.1µM). Gossypol had a weak inhibitory effect (IC50=43.7µM), and caffeic acid had almost no effect on tick GST activity. The IC50 values qualify ethacrynic acid as a potent inhibitor of rRaGST activity (IC50=0.034µM). Cibacron blue and hematin showed a considerable inhibition effect on rRaGST activity, and their IC50 values were 0.13µM and 7.5µM, respectively. The activity of rRaGST was highest for CDNB (30.2µmol/min/mg protein). The enzyme had also a peroxidatic activity (the specific activity equals 26.5µmol/min/mg protein). Both tannic acid and hematin inhibited rRaGST activity non-competitively with respect to GSH and competitively with respect to CDNB. While red pomegranate extracts inhibited rRaGST activity competitively with respect to GSH, uncompetitive inhibition was observed with respect to CDNB.

Molecular cloning of Ra-sHSPI, a novel member of the HSP20 family from Rhipicephalus annulatus salivary glands.

Infestation of cattle by ticks of Rhipicephalus spp. results in severe veterinary and economical losses. Identification of novel proteins from tick salivary glands will enhance our understanding of several aspects of tick physiology and will aid in the development of anti-tick vaccines. Small heat shock proteins (HSPs) have important roles in infection and immunity, especially between invertebrate vectors and mammalian hosts while initially performing their molecular chaperone activity. Here, we report the identification of a small HSP gene from the salivary glands of Rhipicephalus annulatus ticks through immunoscreening of the corresponding cDNA expression library. The identified cDNA contained a 742bp sequence with 543bp open reading frame. It was subsequently cloned, expressed and successfully purified under both native and denaturing conditions. Sequence analysis and functional investigations showed that the protein belongs to the HSP20 family, hence the annotated name Ra-sHSPI. Indeed, recombinant Ra-sHSPI showed two typical in vitro activities of holdase chaperones, including thermal protection of bacterial cellular extracts and the recombinant HindIII at elevated temperatures. Moreover, the recombinant Ra-sHSPI showed strong immunogenic effect in animal model. These results pave the way toward further investigation of Ra-sHSPI role in ticks feeding and its potential use as protective antigen.

Curcumin protection activities against γ-rays-induced molecular and biochemical lesions.

BACKGROUND: Curcumin is a yellow-pigment phenolic compound used as a food spice and has a broad spectrum of antioxidant, anti-carcinogenic, anti-mutagenic and anti-inflammatory properties. METHODS: Radio-protective efficacy of curcumin; diferuloylmethane (C21H20O6) was evaluated using molecular and biochemical assays in male mice after exposure to 3 Gy γ-rays. Curcumin was given at a dose of 400 µmol/ kg body weight via gastric tubes for 5 following days either pre-, post- or both pre- and post-exposure. RESULTS: The incidence of aberrant cells and aberration types (mostly chromatids, breaks and fragments) was reduced with curcumin dosage as compared to irradiated group. Thiobarbituric acid reactive substances (TBARS), hydroperoxide (HP), xanthine oxidase (XO) and apoptotic markers (DNA- fragmentation and caspase-3 activation) were increased significantly, whereas levels of glutathione (GSH) and the enzymatic antioxidants [Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] were significantly depleted in γ-irradiated mice. Curcumin treatments of mice groups including the 5 days pre-irradiation treated group (protected), the 5 days post-irradiation treated group (treated), and the curcumin treated group 5 days pre- and post-irradiation (protracted), have attenuated the liver toxic effects of γ-rays as manifested by reducing the levels of TBARS, HP, XO and DNA fragmentation. Curcumin has also rescued the depletion of GSH and the enzymatic-antioxidant status. CONCLUSIONS: Curcumin has significant radio-protective and radio-recovery activities in γ-irradiated mice. It has antioxidant potential against γ-rays-induced cytogenetic, molecular and biochemical lesions in mice.

Identification of four novel Rhipicephalus annulatus upregulated salivary gland proteins as candidate vaccines.

The control of Rhipicephalus annulatus ticks in Egypt and other countries relies principally on the application of acaricides which have many drawbacks. Recently, cattle vaccination against ticks showed a potential unconventional approach to control ticks. As a target, salivary glands contain various proteins that may play specific roles during attachment, feeding and may modulate the immune system of the host. We have performed immunoscreening on expression normalized cDNA library to identify unique R. annulatus proteins from salivary gland (RaSal) that are particularly expressed during engorgement. We also present the cloning and sequencing of four novel cDNAs (RaSal1-4) from salivary glands that are expressed during feeding. RaSal4 shows 13 cysteine amino acid residues forming 6 potential disulfide bonds. We detected the expression level of the four genes during embryogenesis in eggs collected at 6, 12 and 18 days after oviposition. RT-PCR analysis detected these proteins at days 12 and 18 while slight amplification was detected at day 6 for only RaSal2. The expression of these salivary genes may put forward new vaccines to control tick infestations and tick-borne diseases.

Purification and characterization of a cytotoxic neurotoxin-like protein from Naja haje haje venom that induces mitochondrial apoptosis pathway.

This study reported the purification and characterization of a cytotoxic, neurotoxin-like protein derived from the venom of the Egyptian cobra Naja haje haje, Elapidae family, and explored their mechanistic role in the cell death. The protein purification was performed through ion-exchange chromatography and gel-filtration chromatography and was characterized by SDS-PAGE, amino acid sequencing, and mass spectrum analysis. The antitumor activity of Naja haje venom (NHV) and its fractions (NHVI, NHV-Ia, NHV-Ib, NHV-Ic, NHV-II, NHV-III, and NHV-IV) were tested against different human cancer cell lines. The molecular cascade of cell death was explored through evaluation of apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase (HDAC) activity, mitochondrial transmembrane potential (Δψ(m)), cytochrome c release, total caspases, caspase-3, caspase-9, and cell cycle analysis by flow cytometry. Most of the separated fractions possessed variable cytotoxic effect against different cancer cells. The most potent antitumor fraction was NHV-Ic due to its ability to induce DNA damaging and fragmentation that was associated with a significant induction of apoptosis via mitochondrial pathway and disturbed cell cycle phases as well as an inhibition of HDAC activity. NHV-Ic induced the mitochondrial pathway initially by the impairment of Δψ(m) besides the DNA damage and in response to that the mitochondria-released cytochrome c that may in turn activated total caspases, caspase-3 and caspase-9 in lymphoblastic leukemia 1301 cells. The partial amino acid sequencing of NHV-Ic revealed 100, 95.65, and 91.3% homology with the Long neurotoxin 1 from Naja haje anchietae (Angolan cobra), Naja haje haje (Egyptian cobra), and Boulengerina annulata annulata (banded water cobra), respectively.

Phytotherapeutic effects of Echinacea purpurea in gamma-irradiated mice.

Echinacea (E.) purpurea herb is commonly known as the purple coneflower, red sunflower and rudbeckia. In this paper, we report the curative efficacy of an Echinacea extract in gamma-irradiated mice. E. purpurea was given to male mice that were divided into five groups (control, treated, irradiated, treated before irradiation & treated after irradiation) at a dose of 30 mg/kg body weight for 2 weeks before and after irradiation with 3 Gy of gamma-rays. The results reflected the detrimental reduction effects of gamma-rays on peripheral blood hemoglobin and the levels of red blood cells, differential white blood cells, and bone marrow cells. The thiobarbituric acid-reactive substances (TBARs) level, Superoxide dismutase (SOD) and glutathione peroxidase (GSPx) activities and DNA fragmentation were also investigated. FT-Raman spectroscopy was used to explore the structural changes in liver tissues. Significant changes were observed in the microenvironment of the major constituents, including tyrosine and protein secondary structures. E. purpurea administration significantly ameliorated all estimated parameters. The radio-protection effectiveness was similar to the radio-recovery curativeness in comparison to the control group in most of the tested parameters. The radio-protection efficiency was greater than the radio-recovery in hemoglobin level during the first two weeks, in lymphoid cell count and TBARs level at the fourth week and in SOD activity during the first two weeks, as compared to the levels of these parameters in the control group.

Purification and characterization of two larval glycoproteins from the cattle tick, Boophilus annulatus.

The present study was conducted to identify new target immunogenic molecules from the larval stage of the cattle tick, Boophilus annulatus (Acari: Ixodidae). Two specific larval glycoproteins (GLPs) were isolated by two-step affinity chromatography. The larval immunogens were first purified with CNBr-Sepharose coupled to rabbit anti-larval immunoglobulins, and the glycoproteins were then purified with Con-A Sepharose. These glycoproteins have molecular weights of approximately 32 and 15 kDa with isoelectric points between 6.8 and 7.2. Antibodies against the two GLPs, labeled I and II, were detected in the anti-whole tick, -whole larval, and -gut antigens through immunoblot analysis. These results suggest that these GLPs are good immunogens and can be useful in the vaccination of cattle against tick infestation.