Morphology of the Spleen in Oreochromis niloticus: Splenic Subregions and the Blood-Spleen Barrier

The spleen is a separate organ of the teleost, playing an essential role in immune reactions. The morphology of the spleen is different from the fish species. Little knowledge about the spleen structure and the blood splenic barrier (BSB) in Nile tilapia has been reported. To address this issue, we studied the histology of the spleen and the BSB in healthy Nile tilapia. The morphology of the spleen was observed, then H&E staining, modified Jame’s staining, and ultrastructural techniques were performed to portion the spleen into three subregions and analyze the location of components and fibers. Thereafter, vital staining of Nile tilapia with Trypan blue was conducted to elucidate the composition and function of BSB. Histologically, the spleen could be divided into three subregions (inner, middle, and outer). The venules, clumps of lymphocytes, and vessels were separately characterized features of the outer, middle, and inner layers. Post injection, Trypan blue was intercepted in the endotheliocytes of ellipsoids in the middle layer (i.p.) or was deposited to the reticular fibers surrounding the ellipsoids (i.v.). Additionally, the amount of Trypan blue was shown to be positively correlated to that of the Acid phosphatase expressed. In conclusion, the spleen could be portioned into three subregions, and the BSB lay in the middle layer, composed of the cuboidal-shaped endotheliocytes and the surrounding reticular fibers of the ellipsoid capillaries. The present study enriched the research of immune tissues and system in tilapia and provided reference for the study of spleen in other fish species.

Detection of Serum Levels of IL-17 and CCL-5 in a Sample of Iraqi Pulmonary Tuberculosis Patients

Cytokines and chemokines are small-secreted proteins involved in many aspects of cell development, differentiation, and activation functions. A prominent characteristic of these molecules is their effect on the immune system in relation to the development of cell trafficking and immune tissues and organs. Furthermore, they play an important role in initiating and coordinating the organized and sequential recruitment and activation of cells into Mycobacterium tuberculosis-infected lungs. We aimed to evaluate the levels of interleukin -17 (IL-17) and the chemotactic chemokine (C-C motif) ligand 5 (CCL5) in the sera of pulmonary tuberculosis (PTB) patients. About 90 subjects were included, involving 50 patients with pulmonary TB and 40 apparently healthy individuals who were selected as a control group. Sera were obtained for measuring IL-17 and CCL-5 levels by enzyme linked immunosorbent assay (ELISA). The results revealed that serum levels of IL-17 showed no significant differences between each patient's group and control. In contrast, the serum level of CCL-5 was significantly increased in pulmonary tuberculosis patients compared to control (P ≤0.01). The mean ±SE values of IL-17 level in PTB patients and controls were 43.06 ±3.64 and 41.009 ± 0.009 pg/ml, respectively. While, the mean ±SE values of CCL-5 level in PTB patients and controls were 455.40 ±25.35 and 80.86 ± 5.96 ng/L, respectively. The results of the current study suggest that high levels of CCL-5 in the sera of PTB patients may indicate an important role in the immunopathogenesis of the disease. Therefore, this chemokine could be considered as a useful biomarker for the severity of PTB infections.

Beauveria bassiana Ribotoxin (BbRib) Induces Silkworm Cell Apoptosis via Activating Ros Stress Response

The BbRib gene participates in the infection process of Beauveria bassiana (B. bassiana). It also helps pathogenic fungi to escape and defeat the insect host immune defense system by regulating the innate immune response. However, model insects are rarely used to study the mechanism of fungal ribosomal toxin protein. In this study, BbRib protein was produced by prokaryotic expression and injected into silkworm (Bombyx mori) larvae. The physiological and biochemical indexes of silkworm were monitored, and the pathological effects of BbRib protein on immune tissues of silkworm were examined by Hematoxylin and Eosin (HE) staining. BbRib protein can significantly affect the growth and development of the silkworm, causing poisoning, destroying the midgut and fat body and producing physiological changes. The ROS stress response in the adipose tissue and cells of the silkworm was activated to induce apoptosis. These results indicated that the BbRib gene not only participates in the infection process of B. bassiana, it also helps the pathogenic fungi escape the immune system by regulating the innate immune system of the silkworm, allowing it to break through the silkworm’s immune defense. This study reveals the potential molecular mechanism of BbRib protein to insect toxicity, and provides a theoretical basis and material basis for the development and use of novel insecticidal toxins.

Dietary source of polyunsaturated fatty acids influences cell cytotoxicity in broiler chickens

The current study aims to investigate the effects of dietary source of n-3 polyunsaturated fatty acids (PUFA) on immune response in broiler chickens, represented by cytotoxic cell activity. A total of 255 one-day-old male Cobb 500 broiler chickens were fed on fish oil (FO)-, flaxseed oil-enriched diets at 50 and 19 g/kg, respectively, in addition to the soybean-based control diet. At slaughter, samples of blood and spleen were harvested from 20 birds/treatment (n = 20). The immune tissues' fatty acid profile was analyzed by gas chromatography, and the cytotoxic cell activity was investigated. The results showed that supplementing broiler chickens with diets rich in n-3 PUFA had a substantial effect on the broiler immune tissues' fatty acid profile. Cytotoxic cell activity was significantly higher in splenocytes and peripheral blood mononuclear cells (PBMCs) from broilers fed flaxseed oil than those provided FO and the soybean control diet. These results suggest that flaxseed oil may be used to enrich chickens with n-3 PUFA and improve the immune status of chicken flocks to resist diseases.

Molecular characterization of a new IgZ3 subclass in common carp (Cyprinus carpio) and comparative expression analysis of IgH transcripts during larvae development

Background Immunoglobulins (Igs) distributed among systemic immune tissues and mucosal immune tissues play important roles in protecting teleosts from infections in the pathogen-rich aquatic environment. Teleost IgZ/IgT subclasses with different tissue expression patterns may have different immune functions. Results In the present study, a novel secreted IgZ heavy chain gene was cloned and characterized in common carp (Cyprinus carpio). This gene exhibited a different tissue-specific expression profile than the reported genes IgZ1 and IgZ2. The obtained IgZ-like subclass gene designated CcIgZ3, had a complete open reading frame contained 1650 bp encoding a protein of 549 amino acid residues. Phylogenetic analysis revealed that CcIgZ3 was grouped with carp IgZ2 and was in the same branch as IgZ/IgT genes of other teleosts. Basal expression detection of the immunoglobulin heavy chain (IgH) in healthy adult common carp showed that CcIgZ3 transcripts were widely expressed in systemic immune tissues and mucosal-associated lymphoid tissues. CcIgZ3 was expressed at the highest levels in the head kidneys, gills, and gonads, followed by the spleen, hindgut, oral epithelium, liver, brain, muscle, foregut, and blood; it was expressed at a very low level in the skin. The transcript expression of CcIgZ3 in leukocytes isolated from peripheral blood cells was significantly higher than that in leukocytes isolated from the spleen. Different groups of common carp were infected with Aeromonas hydrophila via intraperitoneal injection or immersion. RT-qPCR analysis demonstrated that significant differences in CcIgZ3 mRNA levels existed between the immersion and injection groups in all the examined tissues, including the head kidney, spleen, liver, and hindgut; in particular, the CcIgZ3 mRNA level in the hindgut was higher in the immersion group than in the injection group. The different routes of A. hydrophila exposure in common carp had milder effects on the IgM response than on the CcIgZ3 response. Further study of the relative expression of the IgH gene during the development of common carp showed that the tissue-specific expression profile of CcIgZ3 was very different from those of other genes. RT-qPCR analysis demonstrated that the CcIgZ3 mRNA level increased gradually in common carp during the early larval development stage from 1 day post fertilization (dpf) to 31 dpf with a dynamic tendency similar to those of IgZ1 and IgZ2, and IgM was the dominant Ig with obviously elevated abundance. Analyses of the tissue-specific expression of IgHs in common carp at 65 dpf showed that CcIgZ3 was expressed at mucosal sites, including both the hindgut and gill; in contrast, IgZ1 was preferentially expressed in the hindgut, and IgZ2 was preferentially expressed in the gill. In addition to RT-qPCR analysis, in situ hybridization was performed to detect CcIgZ3-expressing cells and IgM-expressing cells. The results showed that CcIgZ3 and IgM transcripts were detectable in the spleens, gills, and hindguts of common carp at 65 dpf. Conclusions These results reveal that CcIgZ3 gene transcripts are expressed in common carp during developmental stage not only in systemic tissues but also in mucosal tissues. CcIgZ3 expression can be induced in immune tissues by A. hydrophila challenge via immersion and intraperitoneal injection with significantly different expression profiles, which indicates that CcIgZ3 is involved in the antimicrobial immune response and might play an important role in gut mucosal immunity.

Two CD9 tetraspanin family members of Japanese flounder (Paralichthys olivaceus): characterization and comparative analysis of the anti-infectious immune function

CD9 is a glycoprotein of the transmembrane 4 superfamily that is involved in various cellular processes. Studies related to the immune functions and activities of CD9 in teleost fish are limited. In this study, we characterized two CD9 homologs, PoCD9.1 and PoCD9.3, from Japanese flounder (Paralichthys olivaceus). Sequence analysis showed that PoCD9.1 and PoCD9.3 possess characteristic transmembrane 4 superfamily (TM4SF) structures. PoCD9.1 shares 70.61% sequence identity with PoCD9.3. The expression of PoCD9.1 and PoCD9.3 in the three main immune tissues was significantly induced in a time-dependent manner by extracellular and intracellular pathogen infection, which indicates that the two CD9 homologs play an important role in the response to pathogenic infection. Following infection with the extracellular pathogen Vibrio anguillarum, the expression profiles of both PoCD9.1 and PoCD9.3 were similar. After infection with the intracellular pathogen Edwardsiella piscicida, the expression levels of PoCD9.1 and PoCD9.3 were different at different stages of infection, especially in the spleen. The spleen was the most important tissue for the PoCD9.1 and PoCD9.3 responses to pathogen infection among the three examined immune tissues. Knockdown of PoCD9.1 and PoCD9.3 attenuated the ability of host cells to eliminate pathogenic bacteria, and PoCD9.1 knockdown was more lethal than PoCD9.3 knockdown for host cells with E. piscicida infection. Overexpression of PoCD9.1 and PoCD9.3 promoted host or host cell defence against E. piscicida infection. These findings suggest that PoCD9.1 and PoCD9.3 serve as immune-related factors, play an important role in the immune defence system of Japanese flounder, and display different functions in response to different pathogens at different stages of infection.

The Radio-mitigative Effect of CpG-Oligodeoxynucleotides on Mice After Exposure to Carbon Ions Radiation.

Background: Heavy ion radiation constitutes a major health risk for astronaut in space flight, potential damage to healthy tissues surrounding the tumor target along its penetrating path should still be considered in hydrotherapy. Therefore, there is a demand for reliable countermeasure against heavy ions radiation. In this study, we will estimate the radiomitigative effect of CpG-ODN on immune tissues after carbon ions radiation (CIR). Methods: Firstly, the 30 days’ survival of mice was observed, peripheral blood cell was counted, the injury of three principal immune tissues (including bone marrow, thymus and spleen) was evaluated by histological examination, apoptosis and double strand breaks (DSB) were detected by TUNEL staining and γ-H2AX immunohistochemistry respectively, and cytokine (G-CSF, IL-6 and TNF-α) was measured by ELISA assay. Results: the 30 days’ survival improved, the injury of three principal immune tissues were obviously ameliorated, the number of γ-H2AX foci and TUNEL-positive nuclei decreased, and G-CSF, IL-6 and TNF-α expression increased by CpG-ODN treatment after CIR. Conclusion: CpG-ODN could enhanced mice survival, and ameliorate immune tissues injury, the mechanism may be that CpG-ODN induced cytokines production and inhibited the double strand breaks (DSB) and apoptosis in order to stimulate the generation and mobilization of the immune cells and reestablish immune system to combat bacterial infections.

Molecular Response in Intestinal and Immune Tissues to in Ovo Administration of Inulin and the Combination of Inulin and Lactobacillus lactis Subsp. cremoris

Intestinal microbiota are a key factor in maintaining good health and production results in chickens. They play an important role in the stimulation of immune responses, as well as in metabolic processes and nutrient digestion. Bioactive substances such as prebiotics, probiotics, or a combination of the two (synbiotic) can effectively stimulate intestinal microbiota and therefore replace antibiotic growth promoters. Intestinal microbiota might be stimulated at the early stage of embryo development in ovo. The aim of the study was to analyze the expression of genes related to energy metabolism and immune response after the administration of inulin and a synbiotic, in which lactic acid bacteria were combined with inulin in the intestines and immune tissues of chicken broilers. The experiment was performed on male broiler chickens. Eggs were incubated for 21 days in a commercial hatchery. On day 12 of egg incubation, inulin as a prebiotic and inulin with Lactobacillus lactis subsp. cremoris as a synbiotic were delivered to the egg chamber. The control group was injected with physiological saline. On day 35 post-hatching, birds from each group were randomly selected and sacrificed. Tissues (spleen, cecal tonsils, and large intestine) were collected and intended for RNA isolation. The gene panel (ABCG8, HNF4A, ACOX2, APBB1IP, BRSK2, APOA1, and IRS2) was selected based on the microarray dataset and biological functions of genes related to the energy metabolism and immune responses. Isolated RNA was analyzed using the RT-qPCR method, and the relative gene expression was calculated. In our experiment, distinct effects of prebiotics and synbiotics following in ovo delivery were manifested in all analyzed tissues, with the lowest number of genes with altered expression shown in the large intestines of broilers. The results demonstrated that prebiotics or synbiotics provide a potent stimulation of gene expression in the spleen and cecal tonsils of broiler chickens. The overall number of gene expression levels and the magnitude of their changes in the spleen and cecal tonsils were higher in the group of synbiotic chickens compared to the prebiotic group.

Molecular Characterization of A New IgZ3 Subclass from Common Carp (Cyprinus Carpio) and Comparative Expression Analysis of IgH Transcripts During Ontogeny of Larvae

Background Immunoglobulins distributing among systemic immune tissues and mucosal immune tissues play an important role in teleost to protect them from infections in a pathogen-rich aquatic environment. Teleost IgZ/IgT subclasses with different tissue expression patterns may have varied immune functions.Results In the present study, a novel secreted IgZ heavy chain gene was cloned and characterized in common carp (Cyprinus carpio) which was different from the reported IgZ1 and IgZ2 in tissue-specific expression profile. The obtained IgZ-like subclass was designated as CcIgZ3 which complete open reading frame contained 1650 bp encoding a protein of 549 amino acid residues. The phylogenetic analysis revealed that CcIgZ3 was grouped with carp IgZ2 and was in the same branch as other teleosts IgZ/IgT. Basal expression detection of IgH in healthy adult common carp showed that CcIgZ3 transcripts were widely expressed in systemic immune tissues and Mucosal-associated lymphoid tissues. It was expressed at a higher level in the head kidney, gill, and gonad, followed by spleen, hindgut, oral epithelium, liver, brain, muscle, foregut, and blood, but at a very low level in the skin. The transcript level of CcIgZ3 mRNA in isolated leukocytes from peripheral blood cells was significantly higher than that of isolated leukocytes from the spleen. Different groups of common carp were infected with Aeromonas hydrophila via intraperitoneal injection and immersion respectively. The qRT-PCR analysis demonstrated that a significant difference in CcIgZ3 mRNA level between immersion and injection groups existed in all the detected tissues including head kidney, spleen, liver, and hindgut, especially in hindgut CcIgZ3 mRNA level was higher in immersion than in the injection group. Different routes for Aeromonas hydrophila challenge to common carp had comparatively less effect on IgM response. Further study on the relative expression of the IgH gene during the ontogeny of common carp presented that the tissue-specific expression profile of CcIgZ3 was very different from the others. In the early larval development stage of common carp from 1 dpf to 31 dpf, the qRT-PCR analysis demonstrated that the CcIgZ3 mRNA level increased gradually with a similar dynamic tendency of IgZ1 and IgZ2 and IgM was the dominant Ig with obviously higher abundance. The results of tissue-specific expression of IgH at 65 dpf of common carp showed that CcIgZ3 was expressed at mucosal sites including both hindgut and gill but IgZ1 was preferentially expressed in hindgut and IgZ2 in gill. Except for qRT-PCR analysis, the detection of the CcIgZ3-expressing cells and IgM-expressing cells by in situ hybridization was performed. The results showed that CcIgZ3 and IgM transcripts could be detected in the spleen, gill, and hindgut of the common carp at 65dpf. Conclusions These results revealed that CcIgZ3 gene transcript occurred in the early development stage of common carp not only in systemic tissues but also in mucosal tissues. CcIgZ3 could be induced with significantly different expression profiles at immune tissues after the challenge by Aeromonas hydrophila immersion and intraperitoneal injection, which indicated that CcIgZ3 might play a more important role in mucosal immunity than in systemic immunity.