Incomplete Freund's Adjuvant Research Articles

Effect of Salvia miltiorrhiza Bunge extracts on improving the efficacy and reducing the toxicity of Tripterygium wilfordii polyglycosides in the treatment of rheumatoid arthritis

Ethnopharmacological relevanceTripterygium wilfordii polyglycosides (TWP), extracted from the traditional Chinese herb Tripterygium wilfordii, has been widely used in the treatment of rheumatoid arthritis (RA). However, the toxicity of TWP to a variety of organs such as liver, kidney and testis greatly limits its clinical application. Salvia miltiorrhiza Bunge is often used in the treatment of RA due to its blood circulation promoting, stasis resolving, and anti-inflammatory effects. Salvia miltiorrhiza Bunge has also been reported to possess multiple organ protective effects. Aim of the studyTo investigate the influences of two main components of Salviorrhiza miltiorrhiza Bunge, hydrophilic salvianolic acids (SA) and lipophilic tanshinones (Tan), on the efficacy and toxicity of TWP in treating RA and to explore the underlying mechanisms. Materials and methodsSA and Tan were extracted from Salvia miltiorrhiza Bunge and the extracts were quantitated by HPLC and identified by UPLC-Q/TOF-MS. Then, a collagen-induced arthritis (CIA) rat model was established using bovine type II collagen (CII) and incomplete Freund's adjuvant (IFA). CIA rats were treated with TWP and/or SA/Tan. After 21 days of continuous treatment, arthritis symptoms and organs toxicity were evaluated. Meanwhile, serum metabolomics were investigated by the UPLC-Q/TOF-MS to understand the underlying mechanism. ResultsSA and Tan extracts could significantly alleviate arthritis symptoms in CIA rats and decrease the serum levels of inflammatory factors TNF-α, IL-1β and IL-6 when combined with TWP. Meanwhile, both extracts alleviated injury of liver, kidney and testis caused by TWP, and the hydrophilic extract SA was superior. Moreover, a total of 38 endogenous differential metabolites were identified between the CIA model group and the TWP group, among which 33 metabolites were significantly recovered after the combination of SA or Tan. Metabolic pathway analysis showed that SA and Tan can affect metabolic pathways including linoleic acid metabolism, glycerophospholipid metabolism, sphingolipid metabolism and steroid biosynthesis metabolism pathway. ConclusionsOur findings indicated for the first time that two Salviorrhiza miltiorrhiza Bunge extracts could improve the efficacy and reduce the toxicity of TWP in the treatment of RA by adjusting metabolic pathways, and the hydrophilic extract SA was superior.

Establishing feasibility for porcine zona pellucida immunocontraception in a large population of free-roaming horses inhabiting the Virginia Range of Nevada, USA

In North America, range constraints are increasingly encroaching on wild horse habitat, necessitating effective but humane reproductive management (Kirkpatrick and Turner. J. Zoo Wildl. Med. 1991; 23:445-452). The largest free-roaming wild horse fertility control program, both in terms of population (>4,000) and territory size (≈300 000-acres), inhabits Nevada's Virginia Range. This program, commenced in 2019, provided a unique opportunity for an extended temporal field study investigating feasibility of porcine zona pellucida (pZP) immunocontraception. Vaccine is administered via remote intra-muscular dart-delivery with an initial treatment of 0.5mL native pZP formulated with 0.5mL Freund's complete (primer) and incomplete (booster) adjuvant, respectively and thereafter at intervals ≈8-12 months. A database, continuously updated from field observations, provides individual animal records including date and type of intervention and of births, deaths and movements. This ongoing study aims to establish feasibility by evaluating vaccine efficacy measurable via annual birth rates and population demographics and concurrently analyze the effects of treatment frequency and season. For this reported initial phase, analyses included monthly data (January 2019 - December 2021; 36 months) for all females. This was characterized by:number of cumulative vaccinations subset into four further classifications considering vaccine having no efficacy loss or loss within a 6-, 12-, and 18- month period post vaccination; based on foaling data, likelihood of being in-foal; based on foaling data, likelihood of conceiving in that month; and based on age, considered as mature or immature (<1-year-old). Foaling rate and population size were descriptively presented over time at monthly intervals. Results described mean numbers of vaccinations per mature mare for any month, vaccination count in relation to conception rate (within mare) and population conception rates. A total of 1,501 individual mares were treated with administration of 4,795 vaccinations. Population data showed a decreased number of immature mares, declining pregnancy rate and a seasonal breeding peak that declined dramatically from 2020. The proportion of vaccinated mares increased steadily and by 2021 showed population coverage ≈40-80%. The most salient findings showed that the system reached stability, assuming a 12-month decay rate, at an average ≈1.0 vaccination/mare/year and can be recommended as a treatment frequency. As data collection and treatment is ongoing, future analyses will provide evidence-based and robust recommendations for best management practices.

Metabolic changes induced by complete Freund’s adjuvant resemble the features of trained immunity

Abstract Using the mouse model of viral myocarditis, we recently reported that animals immunized with complete Freund’s adjuvant (CFA) conferred complete protection against Coxsackievirus B3 infection. Because that Bacillus Calmette-Guérin (BCG) is a well-known inducer of trained immunity and both CFA and BCG possess similar mycobacterial components, we sought to test the hypothesis that the trained immunity might be the underlying mechanism of CFA-mediated protection against CVB3. By using incomplete Freund’s adjuvant (IFA) as a negative control, we investigated the metabolic profiles by mass spectrometry in bone marrow-derived monocytes and CD11b+ cells sorted from splenocytes using LPS as a secondary stimulus. The analysis revealed elevated levels of lactate, α-ketoglutarate, fumarate, malate, and oxaloacetate in the CFA group as compared with the control or IFA groups. In contrast, S-adenosyl methionine and homoserine levels were high in the IFA group. Overall, our data suggest that the metabolites produced in response to CFA were uniquely different from those of IFA. Whether these profiles correlate with the epigenetic changes are currently being investigated. Supported by grants from NIH (R21, A116673)

Interleukin-6 Blockers Improve Inflammation-Induced Lipid Metabolism Impairments but Induce Liver Fibrosis in Collagen-Induced Arthritis.

Interleukin-6 (IL-6) receptor blockers improve systemic inflammation, however, their inconsistent effects on lipid metabolism and drug-induced liver injuries warrant further investigation. This study aimed to determine the effects of IL-6 receptor blocker therapy on lipid metabolism and liver morphology in collagen-induced arthritis. Sixty three Sprague Dawley rats were divided into control (n = 24), inflammation (n = 24), and IL-6 blocker (n = 15) groups. Inflammation was induced in the inflammation and IL-6- blocker groups using Bovine type-II collagen and incomplete Freund's adjuvant. At first signs of arthritis, the IL-6 blocker group received an IL-6 blocker, tocilizumab for six weeks. Serum concentrations of low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C) and ATP-binding cassette transporter-A1 (ABCA1) were measured. Liver fibrosis was determined by histological stains and liver enzymes were measured using the colorimetric-chemistry analyzer. In the inflammation group, HDL-C and ABCA1 were reduced compared to control (p < 0.0001 and p = 0.04, respectively) and IL-6 blocker (p = 0.0003 and p < 0.0001, respectively) groups. LDL-C was increased in the inflammation compared to control (p = 0.02). Markers of liver fibrosis were increased in the IL-6 blocker group compared to control and inflammation groups (picrosirius red collagen area fraction: p < 0.0001 and p = 0.0008, respectively; Masson's trichrome collagen area fraction: p = 0.0002 and p = 0.01, respectively). Alkaline phosphatase concentrations were increased in the IL-6 blocker group compared to the control (p < 0.0001) and inflammation (p = 0.002) groups. IL-6 blockers ameliorated inflammation-induced lipid metabolism impairments, however they induced liver fibrosis. Although IL-6 blockers may reduce inflammation-induced metabolic impairments in chronic inflammatory disorders, routine monitoring of liver function is warranted while on treatment.

Inoculation of Freund's adjuvant in European eel (Anguilla anguilla) revealed key KEGG pathways and DEGs of host anti-Edwardsiella anguillarum infection

Freund's complete (FCA) and incomplete adjuvants (FIA), generally applied in subunit fishery vaccine, have not been explored on the molecular mechanism of the nonspecific immune enhancement. In this study, we examined the RNA-seq in the spleen of European eel (Anguilla anguilla) inoculated with FCA and FIA (FCIA group) to elucidate the key KEGG pathways and differential expressed genes (DEGs) in the process of Edwardsiella anguillarum infection and A. anguilla anti-E. anguillarum infection using genome-wide transcriptome. After eels were challenged by E. anguillarum at 28 d post the first inoculation (dpi), compared to the control uninfected eels (Con group), the control infected eels (Con_inf group) showed severe pathological changes in the liver, kidney and spleen, although infected eels post the inoculation of FCIA (FCIA_inf group) also formed slight bleeding. Compared to the FCIA_inf group, there was more than 10 times colony forming unit (cfu) in the Con_inf group per 100 μg spleen, kidney or blood, and the relative percent survival (RPS) of eels was 44.4% in FCIA_inf vs Con_inf. Compared to the Con group, the SOD activity in the FCIA group increased significantly in the liver and spleen. Using high-throughput transcriptomics, DEGs were identified and 29 genes were verified using fluorescence real-time polymerase chain reaction (qRT-PCR). The result of DEGs clustering showed 9 samples in 3 groups of Con, FCIA and FCIA_inf were similar, contrast to distinct differences of 3 samples in the Con_inf group. We found 3795 up and 3548 down regulated DEGs in the compare of FCIA_inf vs Con_inf, of which 5 enriched KEGG pathways of “Lysosome”, “Autophagy”, “Apoptosis”, “C-type lectin receptor signaling” and “Insulin signaling” were ascertained, and 26 of 30 top GO terms in the compare were significantly enriched. Finally, protein-protein interactions between the DEGs of the 5 KEGG pathways and other DEGs were explored using Cytoscape 3.9.1. The compare of FCIA_inf vs Con_inf showed 110 DEGs from the 5 pathways and 718 DEGs from other pathways formed total of 9747° in a network, of which 9 hub DEGs play vital roles in anti-infection or apoptosis. Together, the interaction networks revealed that 9 DEGs involved in the 5 pathways underlies the key process of A. anguilla anti-E. anguillarum infection or host cell apoptosis.

Icos gene disruption in NOD mice elicits myositis associated with anti-troponin T3 autoantibodies.

Idiopathic inflammatory myopathies (IIM) are autoimmune inflammatory disorders leading to skeletal muscle weakness and disability. The pathophysiology of IIM is poorly understood due to the scarcity of animal disease models. Genetic deletion of Icos or Icosl (Inducible T-cell co-stimulator deficient/ligand) in non-obese diabetic (NOD) mice leads to muscle disease. Our aim was to characterise Icos-/- NOD myopathy and to search for novel autoantibodies (aAbs) in this model. Diabetes, weight, myopathy incidence/clinical score and grip strength were assessed over time. Locomotor activity was analysed with the Catwalk XT gait analysis system. Muscle histology was evaluated in haematoxylin/eosin and Sirius red-stained sections, and immune infiltrates were characterised by immunofluorescence and flow cytometry. 2D gel electrophoresis of muscle protein extracts and mass spectrometry were used to identify novel aAbs. NOD mice were immunised with troponin T3 (TNNT3) in incomplete Freund's adjuvant (IFA) and R848. An addressable laser-bead immunoassay (ALBIA) was developed to measure aAb IgG serum levels. Icos-/- NOD mice did not exhibit diabetes but developed spontaneous progressive myositis with decreased muscle strength and altered locomotor activity. Muscle from these mice exhibited myofibre necrosis, myophagocytosis, central nuclei, fibrosis and perimysial and endomysial cell infiltrates with macrophages and T cells. We identified anti-TNNT3 aAbs in diseased mice. Immunisation of NOD mice with murine TNNT3 protein led to myositis development, supporting its pathophysiological role. These data show that Icos-/- NOD mice represent a spontaneous model of myositis and the discovery of anti-TNNT3 aAb suggests a new autoantigen in this model.

Temporomandibular joint arthritis increases canonical Wnt pathway expression in the articular cartilage and trigeminal ganglion in rats

The canonical Wnt pathway participates in inflammatory diseases and it is involved in neuropathic pain. This study evaluated the immunoexpression of the canonical Wnt signaling pathway in the articular cartilage of the temporomandibular joint (TMJ) and along the nociceptive trigeminal pathway in arthritic rats. For this, male Wistar rats were divided into Control (C) and Arthritic (RA) groups. Arthritis induction was performed through subcutaneous injection of methylated bovine serum albumin (mBSA) and complete Freund Adjuvant (CFA)/ Incomplete Freund Adjuvant (IFA) on the first 14 days (once a week), followed by 3 weekly intra-articular injections of mBSA (10 μl/joint; left TMJ). The following parameters were evaluated: nociceptive threshold, inflammatory infiltrate, type I and III collagen birefringence, immunohistochemistry for IL-1β, TNF-α, IL-6, Wnt10b, β-catenin, cyclin-D1 in articular cartilage, c-Myc in synovial membrane, and immunofluorescence analysis for c-Fos, Wnt-10b and β-catenin in the trigeminal ganglion and the trigeminal subnucleus caudalis. The RA group showed intense articular cartilage damage with proliferation of type III collagen, increased immunoexpression of proinflammatory cytokines and Wnt-10b, β-catenin and cyclin-D1 in the articular cartilage and c-Myc in the synovial membrane. In the RA group, a reduction in the nociceptive threshold was observed, followed by a significant increase in the expression of Wnt-10b in neurons and β-catenin in satellite cells of the trigeminal ganglion. c-Fos immunoexpression was observed in neurons, peripherally and centrally, in arthritic rats. Our data demonstrated that TMJ arthritis in rats causes articular cartilage damage and nociceptive behavior, with increased immunoexpression of canonical Wnt pathway in the articular cartilage and trigeminal ganglion.

Determination of the antigenic properties of quinoidr adiotoxin using the indirect hemagglutination test (IHA)

Relevance. The article presents the results of experiments to determine the antigenic properties (the ability of quinoid radiotoxin to synthesize antiradiotoxic antibodies) of plant radiotoxin using IHA.Methods. An antigenic variant of the erythrocyte diagnosticum (AGED), which is a ram erythrocytes sensitized by radiation antigens, was used to conduct the IHA-test. As control (positive) antiradiation antibodies in the reaction, we used antiradiation antibodies obtained by hyperimmunization of rabbits with a radiation antigen — radiotoxin isolated from the liver tissue of lethally irradiated sheep, tested antitoxic antibodies — hyperimmune sera from sheep hyperimmunized with quinoid (plant) radiotoxin, negative serum obtained from intact (non-irradiated, not vaccinated with any vaccines) sheep, heterologous sera (antiburn, anticolibacillary). The reaction was placed on microplates in a volume of 50 ml and was taken into account according to a four-point system in crosses.Results. The study of the antigenic activity of the test radiation antigen — plant quinoidr adiotoxin (QRT) — using the IHA-test showed that the test antigen has a high antigenic activity, inducing in the body of immunized animals (sheep) the synthesis of antiradiotoxic antibodies, the titers of which significantly exceed those immunized with liver (animal) antigen — animal radiotoxin. Quinoidr adiotoxin, obtained from plant tissue (potato tubers) and conjugated with a protein-lipoid group (incomplete Freund's adjuvant — IFA), has a high antigenicity, inducing the synthesis of specific antiradiotoxic antibodies, which can be used as an essential component of the immunochemical test system — sensitin used for sensitization of micro- and nanoparticles of bentonite in the design of antiradiation antibody variant of bentonited iagnosticum (ABBD).

Olfactory Bulbs in Arthritis Model Mouse Persistently Express Interleukin-6 before the Onset of Arthritis: Relationship to Food Intake

Introduction: Rheumatoid arthritis (RA) can be comorbid with psychiatric symptoms. Brain abnormalities in RA patients and in arthritis models have been reported. However, it remains unclear when these abnormalities occur and where they are distributed. In this study, we analyzed spatiotemporal changes in gene expression in the brains of mice with collagen-induced arthritis (CIA). Methods: Mice were divided into three groups: (i) CIA (all mice developed arthritis on day 35): complete Freund’s adjuvant (CFA) and type II collagen at initial immunization, and incomplete Freund’s adjuvant (IFA) and type II collagen at booster immunization; (ii) C(+/−) (50% mice developed arthritis on day 35): only IFA at booster immunization; and (iii) C(−/−) (no arthritis): only CFA at initial immunization and only IFA at booster immunization. Whole brains were collected at ten stages of arthritis and divided into six sections. Real-time polymerase chain reaction was performed using RNA extracted from the brain, and the expression of proinflammatory cytokines and glial markers was semi-quantified. Arthritis score, body weight, and food and water intakes were recorded and analyzed for correlations with brain gene expression. We also investigated the effect of interleukin-6 (IL-6) injection in the olfactory bulbs (OBs) on the food intake. Results: After booster immunization, a transient increase in Integrin subunit α-M and IL-1β was observed in multiple areas in CIA. IL-6 is persistently expressed in the OB before the onset of arthritis, which is correlated with body weight loss and decreased food intake. This change in the OB was observed in the C(+/−) but not in the C(−/−) groups. In the C(+/−) group, non-arthritic mice showed the same changes in the OB as the arthritic mice. This elevation in IL-6 levels persisted throughout the chronic phase until day 84. In addition, IL-6 injection into the OB reduced food intake. Conclusion: Persistent elevation of IL-6 in the OB from the early stage of arthritis may be an important finding that might explain the neuropsychiatric pathophysiology of RA, including appetite loss, which is present in the early stages of the disease and manifests as a variety of symptoms over time.

The antimicrobial peptide MetchnikowinII enhances Ptfa antigen immune responses against avian Pasteurella multocida in chickens.

Immunostimulants and vaccines are the main means for controlling infectious diseases and searching highly effective and low toxic immunestimulants has always been the focus of researchers. The MetchnikowinII (MetII) had been expressed by us and exhibited both antibacterial and antifungal activities, in this study, we evaluated its potential for an adjuvant effect. In chickens, antigen-specific immunoglobulin Gs (IgGs) were increased after MetII adjuvanted vaccination using the Ptfa protein. Compared to group Ptfa + iFA, which was only adjuvanted with incomplete Freund's adjuvant (iFA), the antibody titers of the group Ptfa + iFA + Met20 μg·mL-1 (PFM20) and Ptfa + iFA + Propolis (PFP) significantly increased (P<0.05). Likewise, Interleukin-2 (IL-2) and Interferon-γ (IFN-γ) cytokines in group Ptfa + iFA + Met20 μg·mL-1 (PFM20) and Ptfa + iFA + Propolis (PFP) were significantly higher than those of the other three experimental groups (P<0.05). The stimulation index (SI) value in chickens of group PFM20 was significantly higher than that of the other four experimental groups (P<0.05). Chickens that received MetII adjuvanted vaccinations benefitted from higher protection rate (88%) when challenged with Pasteurella multocida (P. multocida), which was significantly higher than those of group PF and PFP (P<0.05). These results suggested that the antimicrobial peptide MetII may play an adjuvant role in the immune response in chickens but need a proper usage, because the higher usage of 40 μg·mL-1 and 60 μg·mL-1 resulted poor effect. Whether MetII could be a potential adjuvant or a biomolecule as part of a complex adjuvant for vaccines needs more experimental evidence, the study still provides an examples for understanding vaccine adjuvants.

Epicutaneous Application of Mannan Induces Psoriasis-like Inflammation in an Inbred Mouse Strain.

Mannan from yeast induces psoriasis-like inflammation in the skin of inbred mouse strains. Limitations of available models led us to develop a new psoriasis model with a rapid disease onset, severe disease course, short duration, and a simple and easy-to-induce protocol with much more practically convenient features and cost-benefits. Mannan-induced skin inflammation (MISI) is more severe than the classical imiquimod (IMQ)-induced skin inflammation (IISI), with characteristic features resembling human plaque psoriasis but with relatively fewer toxicity issues. Epicutaneous application of mannan (5 mg) in incomplete Freund's adjuvant or Vaseline induces severe psoriasis in BALB/c female mice. Psoriasis area and severity index (PASI) and histological evaluation of the skin could help assess the disease development. MISI mimics natural environmental factors affecting the skin relatively more closely than IISI. This disease model can be used to dissect inflammatory pathways in the skin, identify genetic and environmental factors affecting psoriasis, and test potential pharmacological agents or new combinations of available drugs for treatment before designing clinical trials. Key features • S. cerevisiae mannan induces psoriasis-like skin inflammation (MISI) when applied on the skin of inbred mice. • The MISI model has a rapid onset, severe disease, short duration, and simple and easy-to-induce protocol. • MISI is more severe than imiquimod-induced skin inflammation (IISI). • Female mice had a more severe disease than males in the MISI model, thereby allowing the study of sex-dependent disease mechanisms. • The MISI model identifies skin inflammatory pathways and genetic/environmental factors affecting psoriasis. • The MISI model can be used as a drug testing platform for potential pharmaceuticals to develop new therapeutics for psoriasis patients. • The MISI model can be used to explore the relative contribution of different pattern recognition receptors in the development and severity of psoriasis.

Rat resistance to rheumatoid arthritis induction as a function of the early-phase adrenal-pineal crosstalk.

Chronic inflammatory diseases are triggered by causal stimuli that might occur long before the appearance of the symptoms. Increasing evidence suggests that these stimuli are necessary but not always sufficient to induce the diseases. The murine model of type II collagen emulsified in Freund's incomplete adjuvant (collagen-induced arthritis) to induce rheumatoid arthritis (RA) follows this pattern as some animals do not develop the chronically inflamed phenotype. Considering that in the immune-pineal axis (IPA) theory adrenal-pineal cross-talk adjusts early phases of inflammatory processes, we investigated whether differences in IPA activation could explain why some animals are resistant (RES) while others develop RA. We observed a similar increase in 6-sulfatoxymelatonin (aMT6s) excretion from day 3 to 13 in both RES and RA animals, followed by a significant decrease in RA animals. This pattern of aMT6s excretion positively correlated with plasma corticosterone (CORT) in RES animals. Additionally, RA animals presented a lower aMT6s/CORT ratio than saline-injected or RES animals. Plasmatic levels of tumour necrosis factor were similar in both groups, but interleukin (IL)-1β and monocyte chemotactic protein 1 (MCP-1) levels were lower in RES compared to RA animals. IL-2 and IL-4 were decreased in RES animals compared to saline-injected animals. The aMT6s/CORT ratio inversely correlated with the paw thickness and the inflammatory score (levels of IL-1β, MCP-1, IL-2 and IL-4 combined). Thus, adrenocortical-pineal positive interaction is an early defence mechanism for avoiding inflammatory chronification. KEY POINTS: Immune-pineal axis imbalance is observed in early-phase rheumatoid arthritis development. Only resistant animals present a positive association between adrenal and pineal hormones. The 6-sulfatoxymelatonin/corticosterone ratio is decreased in animals that develop rheumatoid arthritis. The inflammatory score combining the levels of nocturnal interleukin (IL)-1β, monocyte chemotactic protein 1, IL-2 and IL-4 presents a very strong positive correlation with the size of inflammatory lesion. The 6-sulfatoxymelatonin/corticosterone ratio presents a strong negative correlation with the inflammatory score and paw oedema size.

Dextran-enhanced CEST MRI reveals the size effect of BBB dysfunction associated with neuroinflammation

INTRODUCTION: The blood-brain barrier (BBB) is compromised in multiple central nervous system (CNS) disorders associated with neuroinflammation, including multiple sclerosis (MS). Currently available magnetic resonance imaging (MRI) methods, however, are only able to measure BBB leakage in the lower molecular size range with the use of small molecular tracers, i.e., gadolinium (Gd) agents (&lt;1 kDa)1,2 and water (18 Da).3,4 The goal of this study is to adopt a dextran-based chemical exchange saturation transfer (CEST) MRI approach for assessing BBB leakage in the larger size range and studying the size characteristics of BBB dysfunction.&#x0D; METHODS: All animal experiments will be approved by the Animal Care and Use Committee of Johns Hopkins University. EAE MS mouse model: C57Bl/6 mice (F/6-10w), were injected s.c. with myelin peptide (MOG35-55, 200 μL, 0.5 mg/mL) emulsified in incomplete Freund's adjuvant supplemented with M. tuberculosis H37Ra (5 mg/mL) and i.p. with 300 ng of pertussis toxin on days 0 and 2. Mice were observed daily for signs of paralysis using a 0-5 rating system. Fluorescent imaging. EAE mice (n=3) were injected with the combination of fixable Dex40-TRITC and Dex3-FITC (i.v.) at the dose of 80 mg/kg, and sacrificed at 30 min after injection (without perfusion) to collect brains. Fluorescence microscopy was then performed on tissue sections. MRI: all in vivo MRI was acquired using a Biospec 11.7 T horizontal MRI scanner (Bruker, Ettlingen, Germany). According to our previously reported protocol,5 CEST MRI was performed before and after the i.v. injection of 200 µL dex40 saline solution (750 mg/kg b.w), using parameters: B1=1.8 µT, Tsat=3 s, Δω=-3 to +3 ppm with a step size of 0.2 ppm. MTRasym=(S-Δω–S+Δω)/S0 was computed after the B0 correction using the WASSR method. ΔMTRasym (1 ppm) at each time point was calculated by MTRasym (t)- MTRasym (pre).&#x0D; RESULTS: 1. The size-dependent BBB disruption in MS can be detected by fluorescent dextran-tracers of different sizes: Immunofluorescent results show dextrans of smaller sizes (e.g., 3 kDa) penetrated the brain parenchyma deeper than larger sizes (e.g., 40 kDa). Our study proves the feasibility to use dextrans as a group of tracers with different sizes for probing the size effect of BBB dysfunction. 2. Dex-enhanced CEST MRI: As shown in Figure 1, mice with high clinical disability scores have BBB impairment in the mouse brain, confirmed with Gd-enhanced MRI (Figure 1B). Dex-enhanced MRI results (Figure 1C) showed substantial contrast enhancement in the corresponding brain regions. Interestingly, while the size of Dex (40 kDa) is larger than the size of Gd-DOTA (559 Da), the area showing enhanced Dex-CEST signal is slightly larger than that of Gd-enhancement, suggesting that, besides size, other particle properties such as shape and surface properties of a given agent/particle may also contribute to the permeation across BBB.&#x0D; CONCLUSIONS: We have established a dextran-based imaging protocol for assessing the biodistribution of dextrans in the brains of EAE mice. We will continue studying the size effect of dextrans and determining the optimal dextran size for accurately mentoring the disease progression.

Production of Anti-Camel IgY for diagnosis of infectious diseases affecting camels located in Kingdom of Saudi Arabia

Chicken can be used for the production of hyper-immune serum for many type of antigens specially, the antigens of mammals and other species. Therefore, the current study aimed to use the chickens for the productions of anti-camel IgY- antibodies using ammonium sulphate and ammonium sulphate - caprylic acid methods for detection of any infectious disease affecting camels.One ml of purified camel IgG of mixed with complete freund's adjuvant was injected intramuscularly and S/C to four groups of laying hens (Hisex strain, 140 days old). The first booster dose was administrated two week after first immunization with one ml of purified camel IgG mixed with incomplete freund's adjuvant and the hens were repeatedly boostered (three times) at two weeks intervals. During the immunization periods we collect the eggs every day and the anti-camel immunoglobulins Y was separated by using two extraction methods; the ammonium sulphate precipitation method and the ammonium sulphate caprylic acid method, the sensitivity and the specificity of the recovered antibody against the camel IgG were evaluated using SDS-PAGE and ELISA.Two weeks after the initial dose of immunizing the hens the anti-camels IgY-antibodies were detected then the mean antibody titer increased significantly (P < 0.05) till became the highest level at 10 weeks post immunization. The antibody titer remain at high level without significant increase (P > 0.05) up to 12 weeks after the first dose of immunization then started to decrease.Therefore, there are several benefits of using immunized chicken to produce IgY antibodies rather than using other species such as mammals in order to prepare anti-camels antibodies conjugates for diagnosis of infectious disease affecting camels.

Phytosomal curcumin alleviates collagen-induced arthritis by downregulating Th17 and upregulating Treg cell responses in rats

Objective: To explore the effects of a nano-formulation of curcumin (phytosomal curcumin) on the clinical and pathological symptoms of collagen-induced arthritis (CIA) in rats. Methods: Forty male Wistar rats were immunized with an emulsion containing bovine type II collagen and incomplete Freund's adjuvant and then administered phytosomal curcumin post-immunization. Clinical symptoms and histological analysis of the synovial tissues were performed. The effect of phytosomal curcumin on Th17 and Treg parameters was also evaluated. Results: Phytosomal curcumin reduced the clinical severity and paw swelling in CIA-induced rats, which was accompanied by a reduction in the number of inflammatory cell infiltration in the synovial tissue. Additionally, treatment with phytosomal curcumin significantly inhibited CIA-associated mediators as well as increased the anti-inflammatory mediators in comparison to the control groups. Conclusions: Phytosomal curcumin could improve CIA autoimmune responses and can be considered a potential candidate for the treatment of rheumatoid arthritis.

Involvement of TLR2-TLR4, NLRP3, and IL-17 in pain induced by a novel Sprague-Dawley rat model of experimental autoimmune encephalomyelitis.

Up to 92% of patients suffering from multiple sclerosis (MS) experience pain, most without adequate treatment, and many report pain long before motor symptoms associated with MS diagnosis. In the most commonly studied rodent model of MS, experimental autoimmune encephalomyelitis (EAE), motor impairments/disabilities caused by EAE can interfere with pain testing. In this study, we characterize a novel low-dose myelin-oligodendrocyte-glycoprotein (MOG)-induced Sprague-Dawley (SD) model of EAE-related pain in male rats, optimized to minimize motor impairments/disabilities. Adult male SD rats were treated with increasing doses of intradermal myelin-oligodendrocyte-glycoprotein (MOG1−125) (0, 4, 8, and 16 μg) in incomplete Freund's adjuvant (IFA) vehicle to induce mild EAE. Von Frey testing and motor assessments were conducted prior to EAE induction and then weekly thereafter to assess EAE-induced pain and motor impairment. Results from these studies demonstrated that doses of 8 and 16 μg MOG1−125 were sufficient to produce stable mechanical allodynia for up to 1 month in the absence of hindpaw motor impairments/disabilities. In the follow-up studies, these doses of MOG1−125, were administered to create allodynia in the absence of confounded motor impairments. Then, 2 weeks later, rats began daily subcutaneous injections of the Toll-like receptor 2 and 4 (TLR2–TLR4) antagonist (+)-naltrexone [(+)-NTX] or saline for an additional 13 days. We found that (+)-NTX also reverses EAE-induced mechanical allodynia in the MOG-induced SD rat model of EAE, supporting parallels between models, but now allowing a protracted timecourse to be examined completely free of motor confounds. Exploring further mechanisms, we demonstrated that both spinal NOD-like receptor protein 3 (NLRP3) and interleukin-17 (IL-17) are necessary for EAE-induced pain, as intrathecal injections of NLRP3 antagonist MCC950 and IL-17 neutralizing antibody both acutely reversed EAE-induced pain. Finally, we show that spinal glial immunoreactivity induced by EAE is reversed by (+)-NTX, and that spinal demyelination correlates with the severity of motor impairments/disabilities. These findings characterize an optimized MOG-induced SD rat model of EAE for the study of pain with minimal motor impairments/disabilities. Finally, these studies support the role of TLR2–TLR4 antagonists as a potential treatment for MS-related pain and other pain and inflammatory-related disorders.

Comprehensive Relationship Analysis of the Long Noncoding RNAs (lncRNAs) and the Target mRNAs in Response to the Infection of Edwardsiella anguillarum in European eel (Anguilla anguilla) Inoculated with Freund's Adjuvant.

Freund's complete adjuvant (FCA) and incomplete adjuvant (FIA), generally applied in subunit fishery vaccine, have not been explored on the molecular mechanism of the non-specific immune enhancement. As long noncoding RNAs (lncRNAs) play vital regulating roles in various biological activities, in this study, we examined the genome-wide expression of transcripts in the liver of European eel (Anguilla anguilla, Aa) inoculated with FCA and FIA (FCIA) to elucidate the regulators of lncRNAs in the process of Edwardsiella anguillarum (Ea) infection and Aa anti-Ea infection using strand-specific RNA-seq. After eels were challenged by Ea at 28days post the first inoculation (dpi), compared to the control uninfected eels (Li group), the control infected eels (Con_Li group) showed severe bleeding, hepatocyte atrophy, and thrombi formed in the hepatic vessels of the liver, although eels inoculated with FCIA (FCIA_Li group) also formed slight thrombi in the hepatic vessels. Compared to the FCIA_Li group, there was about 10 times colony-forming unit (cfu) in the Con_Li group per 100μg liver tissue, and the relative percent survival (RPS) of eels was 50% in FCIA_Li vs Con_Li. Using high-throughput transcriptomics, differential expressed genes (DEGs) and transcripts were identified and the results were verified using fluorescence real-time polymerase chain reaction (qRT-PCR). Interactions between the differential expressed lncRNAs (DE-lncRNAs) and the target DEGs were explored using Cytoscape according to their co-expression and co-location relationship. We found 13,499 lncRNAs (10,176 annotated and 3423 novel lncRNAs) between 3 comparisons of Con_Li vs Li, FCIA_Li vs Li, and FCIA_Li vs Con_Li, of which 111, 110, and 129 DE-lncRNAs were ascertained. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DEGs targeted by DE-lncRNAs revealed these DEGs mainly involved in single-organism cellular process in BP, membrane in CC and binding in MF, and KEGG pathways showed that the target DEGs in co-expression and co-location enriched in cell adhesion molecules. Finally, 118 DE-lncRNAs target 1161 DEGs were involved in an interaction network of 8474 co-expression and 333 co-location-related links, of which 16 DE-lncRNAs play vital roles in anti-Ea infection. Taken together, the interaction networks revealed that DE-lncRNAs underlies the process of Ea infection and Aa anti-Ea infection.

Lack of immunogenicity of xenogeneic DNA from porcine biomaterials

BackgroundClinically useful biomaterials are derived from xenogeneic extracellular matrices, but extensive processes often used to remove all residual DNA are detrimental to their proper biological function. We hypothesized that deliberate and repeated injection of DNA extracted from clinically implantable, xenogeneic extracellular matrices might elicit an immune response in a well-established murine model that could ultimately lead to altered extracellular matrix remodeling. MethodsDNA was purified from unprocessed porcine extracellular matrices and processed extracellular matrices before sterilization (aseptic) and after sterilization. Groups of 10 mice were injected with these 3 purified DNAs and 3 controls: (1) DNA from E. coli; (2) DNA from unprocessed porcine extracellular matrices combined with interleukin-12 and methylated bovine serum albumin and emulsified in incomplete Freund's adjuvant; and (3) buffered saline. Immunizations occurred every 2 weeks for a total of 3 injections. Local cytokines and systemic anti-DNA antibodies were quantified 3 and 7 days after final injection. ResultsThe DNA extracted from unprocessed, aseptic, or sterilized porcine extracellular matrices failed to elicit a rejection response, and only with significant, proinflammatory adjuvant activation could such a response be seen. Without the adjuvants, biomaterial-derived DNA resulted in a mild accommodation cytokine response locally and no systemic anti-DNA antibody expression even at doses approximately 100-fold larger than would be clinically likely via extracellular matrix implantation. ConclusionThe immunological safety of porcine extracellular matrix biomaterials appears not to be related to DNA residues present. Such biomaterials need not be extensively processed, likely leading to detrimental changes in their bioactivity, solely in an effort to remove the mammalian DNA.

Antibodies targeting Candida albicans Als3 and Hyr1 antigens protect neonatal mice from candidiasis.

Pre-term infants in neonatal intensive care units are vulnerable to fungal sepsis. In this patient population, Candida albicans remains the predominant fungal pathogen causing high morbidity and mortality, despite antifungal therapy. Thus, new preventative/therapeutic strategies against neonatal candidiasis are needed. Previously, we have reported that vaccination with recombinant forms of the C. albicans N-termini of the cell wall proteins Als3 (rAls3p-N) and Hyr1 (rHyr1p-N) protected adult mice from disseminated candidiasis. Further, in a Phase 1b/2a NDV-3A (an rAls3p-N formulated with alum) protected women from recurrent vulvovaginal candidiasis, with anti-Als3p IgG2 isotype being a biomarker for efficacy. Here, we performed a proof of concept study to evaluate if anti-Als3p or anti-Hyr1p antibodies are important for prevention of disseminated candidiasis in neonates. Als3 and Hyr1 antigens when adjuvanted with complete Freund’s adjuvant (CFA)/incomplete Freund’s adjuvant (IFA) induced a robust antibody response with a ten-fold higher titer of IgG2, than attained by either antigen formulated with alum. Transplacental transfer of these antibodies significantly reduced fungal burden in the kidneys of mice pups, and adoptive transfer of vaccinated mothers’ sera into pups displayed similar levels of protection. Neutrophils were found important for this efficacy. Finally, anti-Hyr1 antisera potentiated the activity of fluconazole in protecting from C. albicans infection. Our current studies are the first in the field to emphasize the importance of anti-Als3 and anti-Hyr1 antibodies in preventing neonatal candidiasis. Considering that Candida infections in low birthweight infants is a lethal infection, active and passive vaccination strategies using these antigens could have profound clinical relevance.

Spray-drying-microencapsulated Minthostachys verticillata essential oil and limonene as innovative adjuvant strategy to bovine mastitis vaccines

Design of innovative adjuvant strategies with an appropriate safety profile is relevant to developed subunit or inactivated microorganism vaccines for bovine mastitis. Minthostachys verticillata essential oil (EO) has demonstrated ability to stimulate the innate immune response and adjuvant effect similar to Al(OH)3. Here we evaluated the adjuvant effect of EO and its metabolite, limonene (L) alone and microencapsulated by spray-drying, using an inactivated Enterococcus faecium strain bovine-mastitis inducer. The gas chromatography–mass spectrometry analysis showed that microencapsulation process did not alter the EO or L chemistry. Microencapsulated EO (McEO) or L (McL) (2.0, 2.5 and 5.0 mg/ml) decreased the viability of bovine mammary gland epithelial cells in a dose-dependent way. Balb/c mice (n = 32) were subcutaneously inoculated (day 0) and revaccinated (day 14 and 28) with saline solution, inactivated bacteria alone or combined with Incomplete Freund's Adjuvant; EO or L (2.5 mg/ml); McEO or McL (5.0 mg/ml); or microcapsule wall material (Mc) alone (2.5 mg/ml). EO, L, McEO and McL stimulated E. faecium-specific IgG (IgG1 or IgG2a) with opsonizing capacity and increased the proportion of CD4+ and CD8+ T cells producers of IFN-γ. Microencapsulation was an effective strategy to increase the adjuvant potential of EO or L. These new adjuvants deserve further study to evaluate their incorporation into vaccines for bovine mastitis.