α-Haemolysin production, as a single factor, causes fulminant sepsis in a model of Escherichia coli-induced bacteraemia.

α-Haemolysin (HlyA) from uropathogenic Escherichia coli has been demonstrated to be a significant virulence factor for ascending urinary tract infections. Once the E. coli reach the well-vascularised kidneys, there is a high risk of bacteraemia and a subsequent septic host response. Despite this, HlyA has the potential to accelerate the host response both directly and via its ability to facilitate adenosine triphosphate release from cells. It has not been settled whether HlyA aggravates bacteraemia into a septic state. To address this, we used an E. coli strain in a model of acute urosepsis that was either transfected with a plasmid containing the full HlyA operon or one with deletion in the HlyA gene. Here, we show that HlyA accelerates the host response to E. coli in the circulation. Mice exposed to HlyA-producing E. coli showed massively increased proinflammatory cytokines, a substantial fall in circulating thrombocytes, extensive haematuria, and intravascular haemolysis. This was not seen in mice exposed to either E. coli that do not secrete HlyA or vehicle controls. Consistent with the massive host response to the bacteria, the mice exposed to HlyA-producing E. coli died exceedingly early, whereas mice exposed to E. coli without HlyA production and vehicle controls survived the entire observation period. These data allow us to conclude that HlyA is a virulence factor that accelerates a state of bacteraemia into fulminant sepsis in a mouse model.

Comparison of ABO antibody titration, IgG subclasses and qualitative haemolysin test to reduce the risk of passive haemolysis associated with platelet transfusion.

BACKGROUND: One of the strategies used to reduce the risk of haemolysis due to ABO-minor incompatible platelet transfusions is to perform a screening test to identify group O donors with high titres of anti-A and anti-B. However, critical immunoglobulin M/ immunoglobulin G (IgM/IgG) titres remain unclear. OBJECTIVE: This study aimed to determine IgM titres of anti-A and anti-B in individual donor serum vs platelet products plasma and identify a possible association between IgM/IgG titres, haemolysin test and IgG subclasses in Brazilian blood donors from group O. METHODS: IgM anti-A and Anti-B titration tests were performed on single-donor serum and platelet product plasma by gel agglutination (GA) at room temperature. For IgG anti-A and anti-B titration, serum was first treated with 0.01 M dithiothreitol (DTT), and the test was performed by GA with incubation at 37°C. Dilution of 1:64 as the cut-off was considered for both IgM/IgG. The qualitative haemolysin test was performed in tube, adding AB fresh serum, with incubation at 37°C. IgG subclasses were determined by GA using specific monoclonal antibodies. RESULTS: An association between anti-A and anti-B IgM titres and haemolysin were demonstrated (P < .001). IgM titres in plasma samples from platelet components correlated to those in single-serum samples. IgG1/IgG3 subclasses were associated with total haemolysis and titres above 64, whereas IgG2/IgG4 subclasses were associated with the absence of haemolysis and titres below 64 (P < .001). CONCLUSION: Our data suggest that a value of 64 as a critical titre can be used as a screening test of anti-A and anti-B IgM to prevent transfusion reactions. This can be a safe and cost-effective approach for managing ABO-incompatible platelet transfusions.

Relieving Sore Throat Formula Exerts a Therapeutic Effect on Pharyngitis through Immunoregulation and NF-κB Pathway.

Relieving Sore Throat Formula (RSTF) is a formula approved by the China Food and Drug Administration and has been used for the treatment of pharyngitis in clinic for many years. However, the potential pharmacological mechanism still remains unknown. We combined multiple methods including bioinformatics data digging, network pharmacology analysis, and pathway analysis to predict the potential target of RSTF. We verified our in silico prediction results with an in vivo/vitro antibacterial effect test, mouse phagocytic index test, proliferation, transformation, and migration of mouse spleen lymphocytes. Alteration of NF-κB pathway was determined by Western blotting, immunofluorescence, and PCR. The in vivo experiments demonstrated that the RSTF could significantly relieve the symptoms of pharyngitis. A rat saliva secretion test showed that RSTF can effectively relieve the xerostomia symptom. A phenol red excretion test showed that RSTF has an eliminating phlegm effect. A hot plate method and granuloma experiment proved that RSTF also have analgesic and anti-inflammatory effects. In silico prediction demonstrates that 70 active compounds of RSTF were filtered out through ADME screening and 84 putative targets correlated with different diseases. Pathway enrichment analysis showed that the candidate targets were mostly related to the response to bacteria and immunity signalling pathways, which are known contributors to pharyngitis. Experimental results confirmed that RSTF exerted therapeutic effects on pharyngitis mainly by antibacterial effect and downregulation of NF-κB activities. It is demonstrated both in silico and in vivo/vitro that RSTF exerted therapeutic effects on pharyngitis mainly through an antibiotic effect and downregulation of NF-κB signalling pathway.

TlyA protein of Mycobacterium leprae: a probable bio-marker of active infection.

The extent of pathogenicity of the mycobacterial infections depends on virulence factors that mediate survival inside macrophages. Virulence factors are generally believed to be specific for pathogenic species and mutated/non-functional in nonpathogenic strains. Mycobacterial TlyA can modulate the phagolysosome maturation pathway, immediately after entry into macrophages. Over-expression of open reading frame (ORF) ML1358 (tlyA) in tissues of leprosy patients by partial DNA chip and real time PCR analysis during active infection attracted our interest to explore the properties of this gene at molecular and serological levels, to understand its role in the host. Molecular properties were studied by cloning and expression of the corresponding gene in pASK-iba 43(þ) expression vector in E. coli and bioinformatics tools while sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and ELISA were applied to investigate the serological significance of rTlyA protein in different clinical states of leprosy. We observed that TlyA has a close relation among mycobacteria with specific protein domains in slow growing intracellular adapted pathogenic species. The presence of trans-membrane domains indicates its association to the cell membrane. The study revealed its highly significant sero-reactivity (P value , 0·001) in borderline lepromatous (BL) patients, and those with reversal reaction (RR) and erythema nodosum leprosum (ENL). Its role in active infection, association with the cell membrane, presence in pathogenic species and high sero-reactivity, suggested the tlyA gene as a strong disease progression marker.

On the mechanism of erythrocyte hemolysis induced by photooxidized psoralen.

Contemporary concepts on a possible mechanism of erythrocyte hemolysis induced by photooxidized psoralen - the medicinal photosensitizing furocoumarin - are reviewed. The hypothesis on the mechanochemical mechanism of hemolysis is considered in view of recent data on photoinduced aggregation in photooxidized psoralen solutions. Appropriate chemical structures of photoproduct hemolysins and aggregating photoproducts are discussed.

Prevalence of anti-A and anti-B hemolysis among blood group O donors in Lagos.

BACKGROUND: Group O donor blood is more readily available and is frequently used as universal red cell donor in our environment. The presence of hemolysins in the donors may however lead to hemolysis in the recipients. Attempts have been made to study the prevalence of hemolysins in various populations with results from our environment showing wide variation (20-80%). AIMS: To determine the prevalence and titer of anti-A and anti B hemolysins among blood donors at the Lagos University Teaching Hospital and compare results with that obtained elsewhere. Determine if the practice of transfusion of group O blood to nongroup O recipients is permissible in this environment. MATERIALS AND METHODS: Test for hemolysis was done using the standard tube method. Samples positive for hemolysis were then scored and titrated with the titers read visually and photometrically at 540 nm. RESULTS: Three hundred and fifty blood group O donors with age range 18-58 years and median age of 28 ΁ 8.4 years were enrolled in the study. The overall prevalence of anti-A and/or anti-B hemolysins obtained was 30.3%. Prevalence of anti-A and anti-B hemolysins only was 15.4% and 5.1% respectively whereas both anti-A and anti-B hemolysins were present in 9.7% donor samples. Though anti-A hemolysins were more prevalent than anti-B hemolysins, anti-B hemolysins had higher mean visual (6:7) and spectrophotometric titers (81:101). A visual titer of 8 and above which is considered significant was seen in 18.6% of donor samples. CONCLUSION: Anti-A and anti-B hemolysins exist in significant frequencies and titers among blood group O donors in Lagos. It is recommended that the use of group O donor blood for recipients who are non-O be discouraged. Clinical studies to determine the frequency and severity of hemolysis in non-group O recipients of blood group O are required.

Effects of oral Bt-maize (MON810) exposure on growth and health parameters in normal and sensitised Atlantic salmon, Salmo salar L.

Responses to GM maize Bt-maize, MON810) expressing Cry1Ab protein from the soil bacterium Bacillus thuringiensis (Bt) in diets for both normal and immune-sensitised (with soyabean meal (SBM)-induced enteropathy) post-smolt Atlantic salmon were investigated following 33 and 97 d of exposure. Triplicate tanks of salmon were fed one of four diets, all containing 20% whole-kernel meal maize, either Bt-maize or its near-isogenic maternal line, without or with 15% extracted SBM inclusion. The fish fed Bt-maize utilised the feed less efficiently, as revealed by lower protein and mineral digestibilities and lower lipid and energy retention efficiencies. Higher intestinal weight, as well as increased interferon-γ and decreased sodium-glucose co-transporter mRNA expression, and a transient increase in T-helper cell presence, as measured by cluster of differentiation 4 (CD4) protein in the distal intestine (DI), may partly explain the lower nutrient digestibilities and retentions. The Bt-maize seemed to potentiate oxidative cellular stress in the DI of immune-sensitised fish, as indicated by increases in superoxide dismutase and heat shock protein 70 mRNA expression. The data suggest that Cry1Ab protein or other antigens in Bt-maize have local immunogenic effects in salmon DI. No systemic immune responses could be detected, as indicated by haematology, differential leucocyte counts, plasma clinical chemistry, as well as absence of Cry1Ab-specific antibodies and Cry1Ab protein in plasma. The responses to Bt-maize observed in the present study differed from results from earlier studies in salmon and other animals fed the same event Bt-maize. Longer-term experiments and more in-depth studies on intestinal physiology and immune responses are needed to evaluate health implications.

Flavone reduces the production of virulence factors, staphyloxanthin and α-hemolysin, in Staphylococcus aureus.

Staphylococcus aureus is a leading cause of nosocomial infections due to its resistance to diverse antibiotics. This bacterium produces a large number of extracellular virulence factors that are closely associated with specific diseases. In this study, diverse plant flavonoids were investigated to identify a novel anti-virulence compound against two S. aureus strains. Flavone, a backbone compound of flavonoids, at subinhibitory concentration (50 µg/mL), markedly reduced the production of staphyloxanthin and α-hemolysin. This staphyloxanthin reduction rendered the S. aureus cells 100 times more vulnerable to hydrogen peroxide in the presence of flavone. In addition, flavone significantly decreased the hemolysis of human red blood by S. aureus, and the transcriptional level of α-hemolysin gene hla and a global regulator gene sae in S. aureus cells. This finding supported the usefulness of flavone as a potential antivirulence agent against antibiotic-resistant S. aureus.

Experimental investigation of the immunoregulatory and anti-inflammatory effects of the traditional Chinese medicine "Li-Yan Zhi-Ke Granule" for relieving chronic pharyngitis in rats.

Chronic pharyngitis, a chronic inflammation of the pharyngeal mucous membrane and submucous lymphoid tissues, is often caused by unsatisfactory treatment of acute pharyngitis or repeated occurrences of upper respiratory tract infection and is related to a high-dust environment. Traditional herbal pharmacotherapy is well known for combining plant species to create complex phytochemical mixtures in the attempt to ameliorate pathophysiological processes. The aim of current study is to investigate the effect of immunoregulation and anti-inflammation with the traditional Chinese medicine (TCM) "Li-Yan Zhi-Ke Granule" in rats. Determination of serum hemolysin and the carbon particle clearance test were performed. The results demonstrate that administration of the TCM "Li-Yan Zhi-Ke Granule" may improve the effect of phagocytosis by mononuclear macrophages and immune function in rats, and may also increase the immunoregulatory and anti-inflammatory responses of rats with chronic pharyngitis. This traditional drug could relieve the symptoms of sore throat and cough in rats with chronic pharyngitis.

Immunomodulatory activity of polysaccharide-protein complex of longan (Dimocarpus longan Lour.) pulp.

The immunomodulatory function of longan pulp polysaccharide-protein complex (LP3) was investigated in immunosuppressed mice models. Compared with the model control, peroral administration of 100 mgkg⁻¹d⁻¹ LP3 could significantly increase/enhance antibody production against chicken red blood cell (CRBC), concanavalin A (ConA)-induced splenocyte proliferation, macrophage phagocytosis, NK cell cytotoxicity against YAC-1 lymphoma cell, and interferon-gamma (INF-γ) and interleukin-2 (IL-2) secretion in serum (P < 0.05). The immunomodulatory effects, except for those on splenocytes and macrophages (P > 0.05), were also observed in mice administered with 50 or 200 mgkg⁻¹d⁻¹ LP3 (P < 0.05). The beneficial effects of 50-200 mgkg⁻¹d⁻¹ LP3 were comparable to those of 50 mgkg⁻¹d⁻¹ ganoderan. The strong immunomodulatory activity of LP3 confirmed its good potential as an immunotherapeutic adjuvant.

Increased Risk of Thrombocytopenia and Death in Patients with Bacteremia Caused by High Alpha Toxin-Producing Methicillin-Resistant Staphylococcus aureus.

Alpha toxin (Hla) is a major virulence factor of Staphylococcus aureus that targets platelets but clinical data on Hla pathogenesis in bacteremia (SAB) is limited. We examined the link between in vitro Hla activity and outcome. Study isolates obtained from 100 patients with SAB (50 survivors; 50 non-survivors) were assessed for in vitro Hla production by Western immunoblotting in a subset of isolates and Hla activity by hemolysis assay in all isolates. Relevant demographics, laboratory and clinical data were extracted from patients' medical records to correlate Hla activity of the infecting isolates with outcome. Hla production strongly correlated with hemolytic activity (rs = 0.93) in vitro. A trend towards higher hemolytic activity was observed for MRSA compared to MSSA and with high-risk source infection. Significantly higher hemolytic activity was noted for MRSA strains isolated from patients who developed thrombocytopenia (median 52.48 vs. 16.55 HU/mL in normal platelet count, p = 0.012) and from non survivors (median 30.96 vs. 14.87 HU/mL in survivors, p = 0.014) but hemolytic activity of MSSA strains did not differ between patient groups. In vitro Hla activity of MRSA strains obtained from patients with bacteremia is significantly associated with increased risk for thrombocytopenia and death which supports future studies to evaluate feasibility of bedside phenotyping and therapeutic targeting.

Initiation of antibody responses by different classes of lymphocytes. I. Types of thoracic duct lymphocytes involved in primary antibody responses of rats.

Thoracic duct cells and spleen cells were tested for their ability to restore the primary antibody response of X-irradiated rats to bovine serum albumin (BSA), sheep red blood cells (SRBC), horse spleen femtin (HSF), and Salmonella typhi flagella. Spleen cells were at least as efficient as thoracic duct cells in restoring the response to BSA, HSF, and Salmonella typhi flagella. In further experiments thoracic duct cells lacking large dividing lymphocytes were tested for their ability to restore the primary response. Large lymphocytes were eliminated by the in vitro incubation of thoracic duct cells for 24 hr at 37 degrees C or by treatment of thoracic duct cell donors with the mitotic inhibitor vinblastine sulfate 24 hr prior to cannulation of the thoracic duct. Experiments with SRBC show that incubated cells and cells from vinblastine-treated donors are as efficient as normal cells in restoring the primary antibody response. On the other hand, experiments with HSF and Salmonella typhi flagella show that incubated cells and cells from vinblastine-treated donors are about five times less efficient than normal cells in restoring the response. Normal thoracic duct cells were more efficient than incubated cells but less efficient than cells from vinblastine-treated donors in restoring the early response to BSA. The experimental findings indicate that the classes of thoracic duct lymphocytes which initiate the primary antibody response to SRBC differ from the classes which initiate the response to HSF and Salmonella typhi flagella, or BSA.

Density distribution analysis of antigen-sensitive cells in the rat.

Analysis of the cell populations capable of initiating a response to sheep and horse erythrocyte antigens has been carried out by means of equilibrium density gradient centrifugation. The results indicate that there are at least six distinguishable AS cell populations for sheep erythrocytes, but only three for horse erythrocytes in the spleen of the Lewis rat. Evidence is presented for the existence of metabolic, physiological, and immunological differences among these populations. It is suggested that at least one population of AS cells responds only to the specific antigen and at least one other population is sensitive to stimulation by a broad range of antigens. It is assumed that the difference between these two AS cells results from a process of differentiation of AS cells primed into DNA synthesis by antigen stimulation.

[Studies on chemistry component and the biological activity of petroleum ether extraction from pre-and post-processed of Cornus officinalis].

OBJECTIVE: To investigate substance basis for improving pharmacodynamics by comparing the chemical constituents of petroleum ether extraction from crude and processed Cornus officinalis and the effects on the immunologic function of mice with immunosuppression induced. METHODS: The volatile components in petroleum ether extraction were analyses by GC-MS. Non-specific immune function was determined by cleaning carbon particle index K, Swallow index a, spleen index and thymus index. The specific humoral immune function was evaluated by detecting the content of serum hemolysin. RESULTS: The chemical constituents of petroleum ether extraction of Cornus officinalis before and after being processed had significant changes. After being processed, Vitamin E increased by 46.6%, linoleic acid increased by 18.3% and methyl linen increased by 30.9%. The extraction increased clearance rate of charcoal carbon particles index K, swallow index alpha, spleen index, thymus index and the level of serum hemolysin. CONCLUSION: The extraction can markedly improve non-specific immune function and the specific humoral immune function which are active sites of improving immunologic function and post-processed is better. Substance basis could be Vitamin E, Linoleic acid, methyl linen and so on.

Study on the effect of regulation of Cordyceps militaris polypeptide on the immune function of mice based on a transcription factor regulatory network.

BACKGROUND: The pathogenesis of the abnormality of the immune system is still not clear at present. Chemosynthetic drugs, human or animal immune products and microbiological drugs are used as the main drugs in clinics currently, but these drugs have different side effects. So researchers turned to safer natural products in order to find immunomodulatory active substances from natural products and their extracts. METHODS: Immunosuppressed mice were induced by cyclophosphamide and administered with Cordyceps militaris polypeptide (CMP) for the study on the effect of CMP on the immune function of mice and its mechanism. Based on the 1748 differential gene sets selected in our previous work, the transcription factors and their corresponding target genes were screened by integrating the TRED (Transcriptional Regulatory Element Database), a transcriptional factor-target gene regulatory network was constructed, then the role of transcription factors in the regulatory network was elucidated by statistically analyzing the key nodes, and finally, the correlation of network genes with diseases was analyzed by using the DAVID database. RESULTS: The results of animal experiments showed that CMP could increase the immune organ indexes, the number of white blood cells, the degree of delayed allergy and the content of hemolysin in the serum of mice. CMP was found to be involved in the regulation of immune function in mice through genes Kdr, Spp1, Ptgs2, Rel, and Smad3, and transcription factors Ets1, E2f2 and E2f1. E2F2 and E2F1 are members of the E2F family, so we speculated that the E2F family might play an important role, and its main regulatory pathways were the PI3K-Akt signaling pathway and TNF signaling pathway. CONCLUSION: CMP can improve the immunity of mice. CMP can regulate the immune function of mice through multiple genes and transcription factors, and may also play a role in immune-related diseases, such as cancer.

Antioxidant and immunity activity of water extract and crude polysaccharide from Ficus carica L. fruit.

The antioxidative activities of water extract (WE) and crude hot-water soluble polysaccharide (PS) from Ficus carica L. fruit were investigated using various assays in vitro, including scavenging abilities on DPPH, superoxide and hydroxyl radicals and reducing power. The immunity activities of PS were evaluated using the carbon clearance test and serum hemolysin analysis in mice. In addition, total phenolics and flavonoids contents were also determined. Both WE and PS have notable scavenging activities on DPPH with the EC(50) values of 0.72 and 0.61 mg/ml, respectively. The PS showed higher scavenging activity than WE on superoxide radical (EC(50), 0.95 mg/ml) and hydroxyl anion radical (scavenging rate 43.4% at concentration of 4 mg/ml). The PS (500 mg/kg) also has a significant increase in the clearance rate of carbon particles and serum hemolysin level of normal mice. The results indicate that both WE and PS might be applicable in healthy medicine and food industry.

[Protecting effect of Chaenomeles speciosa broth on immunosuppressive mice induced by cyclophosphamide].

OBJECTIVE: To investigate the effects of Chaenomeles speciosa broth on immunoregulation for anti-tumor chemotherapy. METHODS: Immunosuppressive model was induced by cyclophosphamide (CTX) in mice. The mice were treated with the broth for 15 days. The serum hemolysin was observed in mouse sera. Spleen lymphocyte transformation and gene transcription related to the immunoregulation in spleen lymphocytes were detected. RESULTS: After administrated the broth, the serum hemolysin and lymphocyte transformation rates significantly increased and the mRNA expression of foxp3, TGF-beta, PD1, Fas, Bax were downregulated compared with CTX-group. CONCLUSION: Chaenomeles speciosa broth has protective effects on the immunosuppressive mouse induce by CTX.

Glycosaminoglycan from Apostichopus japonicus induces immunomodulatory activity in cyclophosphamide-treated mice and in macrophages.

This study was designed to systematically elucidate the immunomodulation effect of glycosaminoglycan from Apostichopus japonicus (AHG) in cyclophosphamide (CY)-induced immunosuppression model and potential mechanism responsible for the activation of macrophages. The results showed that the treatment with AHG could increase natural killer (NK) cell cytotoxicity, carbon clearance and marker enzymes activities in CY-induced immunosuppression mice, indicating that the innate immunity experienced recovery to some extent. Moreover, CY-induced reductions in thymus and spleen indices, serum levels of cytokines, immunoglobulins and hemolysin, as well as the ratio of spleen lymphocyte subsets were recovered by AHG, suggesting that AHG could improve the adaptive immunity through cellular immunity and humoral immunity. Delightedly, it was found that AHG at 10 mg/kg body weight could restore the CY-induced immunosuppression in mice to normal level on both innate and adaptive immunity. Furthermore, AHG also promoted both the expression of NO, TNF-α, IL-6, IL-1ß, IL-18 and MCP-1 protein and related mRNA in macrophages. It was revealed that AHG activated macrophages through the phosphorylation of mitogen-activated protein kinase (MAPK) and nuclear factor-B (NF-κB). In conclusion, AHG exerts remarkable immunomodulatory activities in both innate and adaptive immune system. These findings should have great value for further study on the immunopotentiating mechanisms of this biomacromolecule.

Protective effects of Ulva pertusa polysaccharide and polysaccharide­iron (III) complex on cyclophosphamide induced immunosuppression in mice.

A water-soluble polysaccharide was isolated from marine green algae Ulva pertusa and then chelated with iron to prepare the polysaccharide­iron (III) complex. The immunomodulatory activities of sulfated polysaccharide and polysaccharide­iron (III) complex were investigated through a mice immune-deficiency model. Cyclophosphamide (Cy) was utilized to establish mice immunodeficiency model. Both polysaccharide and polysaccharide­iron (III) complex were proved to promote the proliferation of lymphocyte and enhance the activities of mice macrophages. In mice serum, the levels of cytokines including TNF-α, IFN-γ and IL-10 restored and the contents of hemolysin were also found elevated after treatment with polysaccharide and its iron complex. Besides, it has been shown that both polysaccharide and polysaccharide­iron (III) complex increased the contents of Hb, RBC and HCT in mice blood, and the effect of iron complex was better. All these results suggested that Ulva pertusa polysaccharide could be developed as a healthy function food. It was also noteworthy that the polysaccharide­iron (III) complex showed no negative effect upon the immunomodulatory activity of polysaccharide. Instead, the polysaccharide­iron (III) complex showed excellent hematopoietic capacity perhaps due to the supplement of iron.