Histopathological changes and oxidative damage in hepatic tissue of rats experimentally infected with Babesia bigemina.

The present study was aimed to investigate oxidative stress, DNA damage, and histopatholog- ical alterations in hepatic tissues of splenectomized Wistar rats experimentally infected with Ba- besia bigemina. Rats were challenged with 5x106 infected erythrocytes. Babesia infection was con- firmed both with Giemsa's staining blood smears and nested-PCR amplified region of apical membrane antigen-1 (AMA-1) gene. Parasitemia reached approximately 10 % at day 5 post-in- fection. Livers of infected rats were enlarged and darker in color, became extremely brittle with marked congestion. Microscopic evaluation showed cytoplasmic clearing of hepatocytes and se- vere hydropic changes with significantly dilated sinusoids containing macrophages and also intra- sinosoidal parasitized erythrocytes. Severe infiltration of lymphoplasma cells was also present throughout the liver parenchyma. Furthermore, Kupffer cells were enlarged and, occasionally, containing Babesia-parasitized erythrocytes. The activity of Glutathione (GSH) and catalase (CAT), and total antioxidant capacity (TAC) were also significantly decreased (p ⟨ 0.05) after infection of rats with B. bigemina. B. bigemina infection also induced a significant increase (p ⟨ 0.05) in hepatic malondialdehyde (MDA) and nitric oxide-derived products (NOx) concentra- tions as well as amount of endogenous hepatocytes DNA damage. Hepatic damage was also re- flected through the measurement of lactic acid dehydrogenase (LDH) and protein carbonyl con- tent (PCO) in liver cells. These two indices of liver injury were also significantly elevated (p ⟨ 0.5) during B. bigemina infection. Evaluation of correlation between assayed variables in infected rats revealed that MDA levels were positively correlated with PCO, NOx, LDH and DNA damage in the infected group and negatively correlated with GSH, CAT and TAC. There was also an inverse relationship between the antioxidant enzymes activities of GSH, CAT and TAC with PCO, NOx and DNA damage in infected rats. However, NOx showed positive correlation with PCO and DNA damage in infected rats. On the basis of the above results it can be concluded that the Ba- besia infection increases oxidative stress markers, protein carbonyl content and DNA damage and decreases antioxidant enzymes activities in the liver. These results suggest that B. bigemina infec- tion could alter the liver histopathology and causes DNA damage following oxidative stress in hepatic tissue. Further studies are needed to precisely define how hepatic tissue damage takes place in B. bigemina infection.

Differential response of head and neck cancer cell lines to TRAIL or Smac mimetics is associated with the cellular levels and activity of caspase-8 and caspase-10.

BACKGROUND: Current treatment strategies for head and neck cancer are associated with significant morbidity and up to 50% of patients relapse, highlighting the need for more specific and effective therapeutics. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and Smac mimetics (SMs) are promising anticancer agents, but their effect on head and neck squamous cell carcinoma (HNSCC) remains unknown. METHODS: We examined the response of a panel of nine HNSCC cell lines to TRAIL and SMs and investigated the mechanism of cell type-specific response by functional analysis. RESULTS: Head and neck cancer cell lines revealed a converse response pattern with three cell lines being highly sensitive to Smac-164 (SM) but resistant to TRAIL, whereas the other six were sensitive to TRAIL but resistant to SM. Distinct protein expression and activation patterns were found to be associated with susceptibility of HNSCC cell lines to TRAIL and SM. Tumour necrosis factor-related apoptosis-inducing ligand sensitivity was associated with high caspase-8 and Bid protein levels, and TRAIL-sensitive cell lines were killed via the type II extrinsic apoptotic pathway. Smac mimetic-sensitive cells expressed low levels of caspase-8 and Bid but had high TNF-α expression. Smac mimetic-induced cell death was associated with caspase-10 activation, suggesting that in the absence of caspase-8, caspase-10 mediates response to SM. Cotreatment with TNF-α sensitised the resistant cells to SM, demonstrating a decisive role for TNF-α-driven feedback loop in SM sensitivity. CONCLUSIONS: Tumour necrosis factor-related apoptosis-inducing ligand and SMs effectively kill HNSCC cell lines and therefore represent potential targeted therapeutics for head and neck cancer. Distinct molecular mechanisms determine the sensitivity to each agent, with levels of TNF-α, caspase-8, Bid and caspase-10 providing important predictive biomarkers of response to these agents.

Prevalence of Neospora caninum as an etiologic agent of animal abortion in Kurdistan Region of Iraq.

Neospora caninum ( N. caninum) is the etiologic agent of neosporosis, a potential cause of severe reproductive disorders in cattle, small ruminants, equines, wild animals and canids across the world. The current study is performed to estimate molecular prevalence of N. caninum in small ruminants and equines that had abortion in Kurdistan region of Iraq. A total of 64 tissue samples (brain, placenta, heart, lung and liver) were taken from aborted foetuses, with a total of 122 dam blood samples taken from 63 sheep, 39 goats, 12 mares and 8 jennies in local breed fields. Besides, a risk factor analysis for N. caninum positive animals was performed. The observed prevalence of N. caninum DNA in the blood of sheep, goats, horses and donkeys were 20.6%, 17.9%, 21.4% and 25.0%, respectively, and 19.3%, 17.6%, 18.1 and 20.0% in the aborted foetuses of the animals, respectively. Moreover, occurrence of N. caninum was 20.3% in the blood of aborted dams, while it was 18.7% in their aborted foetuses. Confirmatory analysis was also done through constructing a phylogenetic tree to compare the partial sequences of the Nc-5 gene in our isolates (OP771519, OP771520, OP771521 and OP771522) with the GenBank sequences. This showed 98-100% sequence identity with other N. caninum strains in the GenBank database. Older small ruminants and equines had a higher risk of being positive for N. caninum and exposure to dogs were considered as significant risk factors for N. caninum infection in the studied animals (p<0.05). Thus, the results of this study suggest that N. caninum is one of the microbial abortive agents in small ruminants and equines in Kurdistan region of Iraq. It is hoped that the results of this study will help to control animal abortion in livestock and reduce the economic losses.

Apoptin induces apoptosis by changing the equilibrium between the stability of TAp73 and ΔNp73 isoforms through ubiquitin ligase PIR2.

Apoptin, a protein derived from the chicken anaemia virus, induces cell death in various cancer cells but shows little or no cytotoxicity in normal cells. The mechanism of apoptin-induced cell death is currently unknown but it appears to induce apoptosis independent of p53 status. Here we show that p73, a p53 family member, is important in apoptin-induced apoptosis. In p53 deficient and/or mutated cells, apoptin induced the expression of TAp73 leading to the induction of apoptosis. Knockdown of p73 using siRNA resulted in a significant reduction in apoptin-induced cytotoxicity. The p53 and p73 pro-apoptotic target PUMA plays an important role in apoptin-induced cell death as knockdown of PUMA significantly reduced cell sensitivity to apoptin. Importantly, apoptin expression resulted in a marked increase in TAp73 protein stability. Investigation into the mechanisms of TAp73 stability showed that apoptin induced the expression of the ring finger domain ubiquitin ligase PIR2 which is involved in the degradation of the anti-apoptotic ∆Np73 isoform. Collectively, our results suggest a novel mechanism of apoptin-induced apoptosis through increased TAp73 stability and induction of PIR2 resulting in the degradation of ∆Np73 and activation of pro-apoptotic targets such as PUMA causing cancer cell death.

Pediatric patients with common variable immunodeficiency: long-term follow-up.

BACKGROUND: Common variable immunodeficiency (CVID) is the most common form of symptomatic primary immunodeficiency disease. It is characterized by hypogammaglobulinemia, increased predisposition to infections, autoimmunity, and cancer. OBJECTIVES: This study was performed to evaluate the clinical and immunological features of a group of pediatric patients with CVID. METHODS: The study population comprised 69 individuals with CVID diagnosed during childhood. RESULTS: The patients were followed up for a mean (SD) period of 5.2 (4.3) years. The mean diagnostic delay was 4.4 (3.6) years, which was significantly lower in patients who were diagnosed recently. Children were classified according to 5 clinical phenotypes: infections only (n=39), polyclonal lymphocytic infiltration (n=17), autoimmunity (n=12), malignancy (n=7), and enteropathy (n=3). Postdiagnosis survival (10-year) was 71%. CONCLUSIONS: The high percentages of pediatric patients with CVID in Iran may be due to the considerable prevalence of parental consanguinity in the region and an underlying genetic background.

Phylogenetic Diversity of Dermanyssus gallinae (Dermanyssidae) based on Mitochondrial Cytochrome Oxidase-1 Gene Sequence Collected from Different Bird Species in Iran.

A wide range of hosts, especially birds, can be infested with Dermanyssus gallinae (D. gallinae), as an obligate hematophagous mite. In this study, cytochrome oxidase 1 (CO1) gene sequences were employed to perform molecular and phylogenetic analyses of D. gallinae collected from different bird species in Iran. Adult mites were collected from the body surface and cage material of ornamental and wild birds in industrial farms located in the Western and Northwestern regions of Iran. The infestation was identified in layer poultry farming by inspecting the eggs and the whole surfaces of the birds' bodies. The holding area and body surface of the ornamental and wild birds were also thoroughly examined. The D. gallinae samples were assigned to two subgroups of haplogroup A (i.e., A1 and A2). The phylogenetic tree suggested that the D. gallinae samples collected from wild birds in the A1 sub-haplogroup should be placed beside Japanese, Norwegian, Italian, and French samples isolated from wild birds in the A2 sub-haplogroup. Additionally, the highest phylogenetic similarity in the A2 sub-group was observed between mites isolated from ornamental and industrial birds in Australia. The findings of the present study suggest that crows and sparrows may play an important role in the transmission of D. gallinae infestation to other species of wild birds due to their high population, as well as their presence in most areas.

Impact of WWOX alterations on p73, ΔNp73, p53, cell proliferation and DNA ploidy in salivary gland neoplasms.

OBJECTIVE: WWOX gene is altered in a variety of neoplasms. Wwox is pro-apoptotic through interaction with p73 and may be involved in chromosomal stability by interaction with p73 and p53. The aims of this study were to characterize WWOX transcription, methylation status and immunoexpression in salivary neoplasms and to determine whether these were associated with p73, p53, cell proliferation and DNA ploidy. MATERIALS AND METHODS: Seven malignant and 21 benign fresh salivary neoplasms were included. WWOX expression was determined by RT-PCR and sequencing of transcripts, quantitative PCR and immunohistochemistry. Methylation-specific PCR was used to assess the methylation of its first exon. For p73, ΔNp73, p53 and ki67 immunohistochemistry and ploidy analysis, 29 malignant samples from archives were included. RESULTS: No consistent pattern of WWOX exon 1 methylation was found, but aberrant and novel transcripts were observed in 17/28 neoplasms; 55% of tumours showed reduced WWOX RNA. WWOX RNA levels were associated with p53 immunopositivity. Immunohistochemical Wwox expression did not correlate with methylation status, p53 or p73 expression or proliferation. p73, proliferation and DNA ploidy were associated with malignant phenotype. CONCLUSION: Aberrant WWOX transcription and decreased expression are frequent in salivary neoplasms and WWOX transcription is associated with p53 staining.

Molecular Characterization and Phylogenetic Analysis of Pathogenic Theileria spp. Isolated from Cattle and Sheep based on Cytochrome b Gene in Iran.

The present study investigated the phylogenetic relationship based on cytochrome b gene sequences among pathogenic Theileria species (spp.) in Iran, including Theileria annulata and Theileria lestoquardi, along with other data available in GenBank. A total of 136 (cattle) and 80 (sheep) blood samples suspected of piroplasm infection were obtained from six different provinces of Iran. Both microscopic and molecular methods using species-specific primers were used for screening T. annulata and T. lestoquardi positive samples. Finally, the partial cytochrome b gene of 30 T. annulata and 5 T .lestoquardi were amplified, sequenced, and deposited in GenBank. The results indicated that there were 12 different genotypes among T. annulata isolates, while only one genotype was observed among T. lestoquardi isolates. T. lestoquardi infection in cattle was detected in one sample, and no T. annulata and T. lestoquardi coinfection were detected in sheep and cattle. In the phylogenetic tree, different Theileria spp. were placed in separate clades, and the reliability of depicted tree and monophyly of T. annulata and T. lestoquardi ingroups were supported by the bootstrap value of 94% which significantly indicated that these two species evolved from a common ancestor. The tree also showed that these two pathogenic spp. shared a more recent common ancestor, compared to another species of Theileria parasites. To the best of our knowledge, this study is the first phylogenic analysis of pathogenic Theileria spp. in Iran based on the cytochrome b gene sequences. In addition, the first T. lestoquardi cytochrome b gene was sequenced and deposited in GenBank.

Prevalence of gastrointestinal parasites in working horses.

Fecal samples for detection of gastrointestinal parasites were collected from 221 working horses from September 2002 to May 2003 from 14 villages in Urmia, North West of Iran. Fecal samples of 46 horses (20.8%) were negative for parasite eggs or oocysts. One hundred and seventy five positive horses (48.9%) were infected with a single parasite type and 49 (22.2%) and 18 (8.1%) of horses had multiple infections with two and three parasites, respectively. The highest prevalence and intensity rate belonged to small strongyles. The overall prevalence of intestinal parasites eggs and oocyst in the positive horses were: strongyles 72.9%, Oxyuris equi 22.6%, Parascaris equorum 12.2%, Anoplocephalidae 6.3%, Fasciola spp. 3.2% and Eimeria leuckarti 0.5%. Larval identification showed that small strongyle larvae were most frequent (97.6%) followed by Strongylus edentatus (22.6%), S. equinus (18.5%) and S. vulgaris (6.5%). This study suggests that the high rate of infection with gastrointestinal parasites could contribute to low performance and life expectancy of working horses in the region.

Liver endothelium mediates the hepatocyte's uptake of ceruloplasmin.

The mode of transport of ceruloplasmin (CP) into the liver was investigated in fractionated liver cell suspensions. Incubation of 125I-CP at 4 degrees C with these different fractions led to its binding only to endothelial cells but not Kupffer cells and hepatocytes. Incubation at 37 degrees C led to rapid uptake of 125I-CP by endothelium, but cell-associated radioactivity declined after 15 min, which suggests the release of the labeled substance. Internalization was confirmed by fractionation of surface-bound and internalized ligand. The released label now acquired binding potential for fresh target hepatocytes, and the binding was inhibitable with asialoceruloplasmin but not native CP. This suggested that the released molecule was modified in the endothelium by desialation. Desialation was confirmed by incubation of endothelium with double-labeled CP (3H label on sialic acid and 125I on the protein part). We conclude that in the liver, CP is first recognized and taken up by endothelial cells that are endowed with appropriate surface receptors for the protein. Endothelium then modifies the molecule by desialation to expose the penultimate galactosyl residues. The modified molecule is then released, recognized, and taken up by hepatocytes through their membrane galactosyl-recognition system. These findings are consistent with the role of endothelium as an active mediator of molecular transport between blood and tissue, and further assign a biological role for the galactosyl-recognition system in hepatocytes.

Fate of the nucleus of the marrow erythroblast.

Nucleated red cells lose their nuclei during passage through the endothelium of marrow sinuses. The passage occurs through cytoplasmic pores which are not gaps at the junction of two endothelial cells but perforations within the endothelium. Enucleation occurs because the pores are of relatively fixed size. Whereas the cytoplasm is flexible and squeezes through the pore, the nucleus is rigid and cannot conform to the pore size. It is, thus, caught, and the red cell becomes enucleated.

Bone marrow histogenesis: a comparison of fatty and red marrow.

Histogenesis of fatty marrow in extramedullary sites was compared to histogenesis of the red marrow. Both tissues undergo a similar sequence of events leading to reconstruction of hemopoietic marrow nodules. Histogenesis of yellow marrow then continues; such implants become nodules of fatty marrow, whereas implants of red marrow remain hemopoietically active. This observation supports the concept that histogenesis of the bone marrow recapitulates the ontogeny of this organ and indicates an intrinsic difference between the stroma of red and yellow marrow.

Transplantation of marrow to extramedullary sites.

Autologous fragments of transplanted marrow have survived in various extramedullary sites in the rat, rabbit, and dog. Survival of the fragments occurs with a complete reconstitution of the hemopoietic and adventitial structures. The process originates from a network of surviving reticular cells which proliferate and differentiate into osteoblasts and give rise to trabecular bone. Later, the reticular cells reconstruct the marrow's microcirculation. Hemopoietic repopulation of the marrow implant takes place only after its sinusoidal microcirculation has been established.

Clinical immunotherapeutic approaches for the treatment of head and neck cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide, accounting for more than 550,000 cases and 380,000 deaths annually. The primary risk factors associated with HNSCC are tobacco use and alcohol consumption; nevertheless genetic predisposition and oncogenic viruses also play important roles in the development of these malignancies. The current treatments for HNSCC patients include surgery, chemotherapy, radiotherapy, and cetuximab, and combinations of these. However, these treatments are associated with significant toxicity, and many patients are either refractory to the treatment or relapse after a short period. Despite improvements in the treatment of patients with HNSCC, the clinical outcomes of those who have been treated with standard therapies have remained unchanged for over three decades and the 5-year overall survival rate in these patients remains around 40-50%. Therefore, more specific and less toxic therapies are needed in order to improve patient outcomes. The tumour microenvironment of HNSCC is immunosuppressive; therefore immunotherapy strategies that can overcome the immunosuppressive environment and produce long-term tumour immunosurveillance will have a significant therapeutic impact in these patients. This review focuses on the current immunological treatment options under investigation or available for clinical use in patients with HNSCC.

p400 function is required for the adenovirus E1A-mediated suppression of EGFR and tumour cell killing.

We have recently shown that E1A protein of human adenovirus downregulates epidermal growth factor receptor (EGFR) expression and induces apoptosis in head and neck (HNSCC) and lung cancer cells independently of their p53 status. E1A has five isoforms of which the major ones E1A12S and E1A13S regulate transcription of cellular genes by binding to transcriptional modulators such as pRB, CtBP, p300 and p400. In this study, we have identified E1A12S isoform to have the highest effect on EGFR suppression and induction of apoptosis in HNSCC cells. Similar to Ad5, E1A12S from human adenovirus types 2, 3, 9 and 12 suppressed EGFR, whereas E1A12S of adenovirus types 4 and 40 had no effect on EGFR expression. Using deletion mutants of E1A12S we have shown that interaction of E1A with p400, but not p300 or pRB, is required for EGFR suppression and apoptosis. Inhibition of p400 by short hairpin RNA confirmed that HNSCC cells with reduced p400 expression were less sensitive to E1A-induced suppression of EGFR and apoptosis. p300 function was shown to be dispensable, as cells expressing E1A mutants that are unable to bind p300, or p300 knockout cells, remained sensitive to E1A-induced apoptosis. In summary, this study identifies p400 as an important mediator of E1A-induced downregulation of EGFR and apoptosis.

Laboratory evaluation of three strains of the entomopathogenic fungus Metarhizium anisopliae for controlling Dermanyssus gallinae.

The pathogenicity of three strains of the entomopathogenic fungus Metarhizium anisopliae on different life stages of Dermanyssus gallinae was evaluated in the laboratory. All the strains tested were virulent to D. gallinae but pathogenicity varied among the strains. Strain V245 induced a higher mortality rate using different concentrations than other two strains. The estimated median lethal concentration of different strains of M. anisopliae against D. gallinae varied depending on the exposure time of D. gallinae to M. anisopliae. It was concluded that the pathogenicity of the entomopathogenic fungus M. anisopliae on different life stages of D. gallinae was concentration and time dependent.

Effects of Essential Oils Combination on Sporulation of Turkey (Meleagris gallopavo) Eimeria Oocysts.

Avian coccidiosis is the most important parasitic disease in poultry production, which inflicts numerous losses to the industry. The extensive use of anticoccidial drugs leads to parasite resistance and drug residue in poultry products. In the present study, we aimed to investigate the effects of three famous essential oils (EOs) and their combination on inactivation of mixed oocysts of Eimeria adenoides, Eimeria dispersa, Eimeria meleagrimitis, and Eimeria meleagridis. The EOs of Thymus vulgaris, Artemisia sieberi, and Mentha pulegium were prepared. After inoculation of each turkey with 7&times;105 sporulated oocysts, fresh unsporulated oocysts were harvested from their feces. To evaluate the sporulation inhibition effect, 5&times;104 oocysts were used in each treatment. Each EO was used in increasing concentrations. Half maximal inhibitory concentration (IC50) was determined for each EO and they were blended in pairs based on IC50 line. Our results showed that the IC50 values for mentha, artemisia, and thyme were 22.92, 40.5, and 53.42 mg/ml, respectively. According to our results, artemisia and thyme combination has a synergistic effect, whereas the combination of a high concentration of mentha with a low concentration of thyme had an antagonistic effect. During this study, no interactions were observed between mentha and artemisia.

Annexin A1 regulates EGFR activity and alters EGFR-containing tumour-derived exosomes in head and neck cancers.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is the 6th most common cancer with approximately half a million cases diagnosed each year worldwide. HNSCC has a poor survival rate which has not improved for over 30 years. The molecular pathogenesis of HNSCCs remains largely unresolved; there is high prevalence of p53 mutations and EGFR overexpression; however, the contribution of these molecular changes to disease development and/or progression remains unknown. We have recently identified microRNA miR-196a to be highly overexpressed in HNSCC with poor prognosis. Oncogenic miR-196a directly targets Annexin A1 (ANXA1). Although increased ANXA1 expression levels have been associated with breast cancer development, its role in HNSCC is debatable and its functional contribution to HNSCC development remains unclear. METHODS: ANXA1 mRNA and protein expression levels were determined by RNA Seq analysis and immunohistochemistry, respectively. Gain- and loss-of-function studies were performed to analyse the effects of ANXA1 modulation on cell proliferation, mechanism of activation of EGFR signalling as well as on exosome production and exosomal phospho-EGFR. RESULTS: ANXA1 was found to be downregulated in head and neck cancer tissues, both at mRNA and protein level. Its anti-proliferative effects were mediated through the intracellular form of the protein. Importantly, ANXA1 downregulation resulted in increased phosphorylation and activity of EGFR and its downstream PI3K-AKT signalling. Additionally, ANXA1 modulation affected exosome production and influenced the release of exosomal phospho-EGFR. CONCLUSIONS: ANXA1 acts as a tumour suppressor in HNSCC. It is involved in the regulation of EGFR activity and exosomal phospho-EGFR release and could be an important prognostic biomarker.

Interaction of murine granulocyte-macrophage progenitors and supporting stroma involves a recognition mechanism with galactosyl and mannosyl specificities.

To study the molecular basis of "homing" of granulocyte-macrophage progenitors (CFU-C), we synthesized probes by covalent linking of sugars to bovine serum albumin. Long-term marrow cultures were established in the presence and absence of these probes. In the presence of galactosyl and mannosyl probes, total cell and CFU-C production in the supernate as well as the adherent layer were halted, and cobblestones (representing CFU-C bound to stroma) disappeared. Fucosyl probe and diffusible sugars had no effect on these parameters. These studies suggested membrane lectins with specificity for galactosyl and mannosyl residues may be responsible for the binding of CFU-C to supporting stroma. To determine if CFU-C possesses homing receptors with these specificities, we induced agglutination in marrow cell suspensions with these neoglycoprotein probes. Selective agglutination was observed only by galactosyl and mannosyl probes. The results suggest that CFU-C homing receptors are membrane lectins with specificity for galactosyl and mannosyl residues.

Desialation of transferrin by rat liver endothelium.

To examine the role of liver endothelium in desialation of transferrin (TF), pulse-chase studies were done by incubation of either 3H (sialic acid labeled)-, or 125I, or 59Fe (protein core labeled)-TF with fractionated liver endothelium. While 125I or 59Fe labels were externalized after initial binding and internalization, a large proportion of 3H label was internalized and remained within the cell. When the supernatant of these experiments was studied by isoelectricfocusing and Ricinus communis agglutinin (RCA120) affinity chromatography, generation of asialotransferrin was noted by both techniques. Incubation of liver endothelium with double-labeled TF (sialic acids with 3H and protein core with 125I or 59Fe) led initially to a concordant uptake of the two labels, which were then dissociated and 3H was retained by the cell. These findings indicate desialation of TF by liver endothelium. The significance of these findings in the pathogenesis of hepatic siderosis is discussed.