SYNERGISTIC IMMUNOMODULATORY ACTIVITY OF AQUEOUS ROOT EXTRACT OF ASPARAGUS RACEMOSUS WILLD AND ETHANOL WHOLE PLANT EXTRACT OF BOERHAVIA DIFFUSA LINN

Objective: On the basis of traditional use and Ethno pharmacological evidences Boerhavia diffusa whole plant and root part of Asparagus racemosus (Shatavari) both are widely used to enhance the immunity. But in combination there is no scientific evidence so current study was designed. Methods: Laboratory based study, namely, carbon clearance, cyclophosphamide induced immune suppression and neutrophil adhesion was designed using mice as an experimental animals in different combination of aqueous whole plant extract of B. diffusa and methanol extract of root part of A. racemosus were used as a test drug in the ratio 1:1, 1:2, and 2:1 (100 mg/kg) against the established standard drug Ashwagandha. Results: The results revealed that animals treated with combined extract (1:1, 1:2, and 2:1) at a dose of 100 mg/kg increase rate of carbon clearance from blood, there is significance alternation in blood parameter in cyclophosphamide group and also improve the Neutrophil adhesion when treated with different combination of polyherbal formulation treated groups. Conclusion: The polyherbal formulation in different ratio showing good significant immunomodulatory activity as compare to standard.

Neutrophil DREAM Promotes Neutrophil Recruitment in Vascular Inflammation Via Nuclear Factor Kappa B-Dependent and Independent Mechanisms

The interaction between neutrophils and endothelial cells (ECs) is critical for the pathogenesis of vascular inflammation. Neutrophil recruitment to inflamed tissues is initiated by rolling on activated ECs through the interactions between P-/E-selectins and their ligands. Subsequently, activated integrins (mainly αLβ2 and αMβ2) and chemokine receptors bind to their ligands on ECs and mediate slow-rolling, adhesion, crawling, and transmigration of neutrophils. Although many neutrophil adhesion receptors have been identified, the regulation of their ligand-binding function remains not fully understood. Using real-time intravital microscopy with mice lacking downstream regulatory element antagonist modulator (DREAM) and their bone marrow chimeric mice, we demonstrated that hematopoietic cell DREAM contributes to neutrophil recruitment to sites of vascular inflammation induced by TNF-α- but not a G protein-coupled receptor ligand, MIP-2 or fMLP. Our studies using adoptive neutrophil transfers and flow chamber assays revealed that neutrophil DREAM positively regulates the neutrophil recruitment processes under TNF-α-induced inflammatory conditions. Using RNA-seq and biochemical and cell biological studies, we found that neutrophil DREAM upregulates numerous pro-inflammatory molecules and down-regulates anti-inflammatory molecules after TNF-α treatment. In particular, neutrophil DREAM repressed expression of A20, a negative regulator of NF-κB signaling, and enhanced phosphorylation of IκB kinase (IKK) in response to TNF-α, suggesting the role of neutrophil DREAM in NF-κB activity. Furthermore, we observed that DREAM deletion and IKK inhibition significantly diminishes the ligand-binding activity of β2 integrins in neutrophils after short-term treatment with TNF-α and that deletion of neutrophil DREAM does not affect the expression of other neutrophil adhesion receptors, such as PSGL-1, L-selectin, CD44, CXCR2, and CXCR4. As assessed by flow cytometry using conformation-specific reporter antibodies, knockdown of DREAM in neutrophil-like HL-60 cells decreased TNF-α-induced activation of β2 integrins. Neutrophil DREAM promoted degranulation through IKK-mediated SNAP-23 phosphorylation after short-term treatment with TNF-α, implying the role of neutrophil DREAM-IKK signaling in NF-κB-independent signaling. Using intravital microscopy with Berkeley mice (a mouse model of sickle cell disease) deficient in hematopoietic or nonhematopoietic DREAM, we demonstrated that hematopoietic cell DREAM is crucial for inducing intravascular cell-cell aggregation and vaso-occlusive events in microvessels following the TNF-α challenge. Furthermore, infusion of DREAM KO neutrophils, compared with WT neutrophils, significantly reduced neutrophil recruitment and vaso-occlusive events in TNF-α-challenged SCD mice. These results demonstrate that neutrophil DREAM positively regulates β2 integrin function and promotes neutrophil recruitment during sterile inflammation via NF-κB-dependent and independent mechanisms. Our study provides evidence that targeting DREAM might be a novel therapeutic strategy to reduce excessive neutrophil recruitment in inflammatory diseases. Disclosures No relevant conflicts of interest to declare.

Increased sHLA-G Is Associated with Improved COVID-19 Outcome and Reduced Neutrophil Adhesion

Human leukocyte antigen (HLA) is a group of molecules involved in inflammatory and infective responses. We evaluated blood sHLA-E and sHLA-G levels in hospitalized COVID-19 patients with respiratory failure and their relationship with clinical evolution, changes in endothelial activation biomarker profile, and neutrophil adhesion. sHLA-E, sHLA-G, and endothelial activation biomarkers were quantified by ELISA assay in plasma samples. Neutrophil adhesion to endothelium was assessed in the presence/absence of patients’ plasma samples. At admission, plasma levels of sHLA-G and sHLA-E were significantly higher in COVID-19 patients with respiratory failure compared to controls. COVID-19 clinical improvement was associated with increased sHLA-G plasma levels. In COVID-19, but not in control patients, an inverse correlation was found between serum sICAM-1 and E-selectin levels and plasma sHLA-G values. The in vitro analysis of activated endothelial cells confirmed the ability of HLA-G molecules to control sICAM-1 and sE-selectin expression via CD160 interaction and FGF2 induction and consequently neutrophil adhesion. We suggest a potential role for sHLA-G in improving COVID-19 patients’ clinical condition related to the control of neutrophil adhesion to activated endothelium.

Probiotic Potential and Immunomodulatory Properties in Enterococcus Faecium GMB24 and Enterococcus Hirae SMB16 Isolated from Goat and Sheep Milk

Probiotic attributes of lactic acid bacteria isolated from goat and sheep milk samples were analysed by culturing them on MRS (de Man, Rogosa and Sharpe) agar media. The most potential isolates, GMB24 and SMB16, were identified by biochemical tests, which were further identified as Enterococcus faecium GMB24 and Enterococcus hirae SMB16 by 16S rRNA gene sequencing approach. The probiotic potential of the GMB24 and SMB16 were possessed for probiotics attributes including antimicrobial activity against five pathogenic bacteria viz., Escherichia coli (MTCC118), Staphylococcus aureus (MTCC7443), Pseudomonas aeruginosa (MTCC424), Listeria monocytogens (MTCC657) and Salmonella typhimurium (MTCC733), and antibiotic susceptibility test. These bacterial isolates had ability to tolerate different concentrations of acid, bile and phenol resistance. Further, immunomodulating activities of potential probiotic bacteria were tested on neutrophil adhesion test, haemagglutinating antibody titer and delayed type hypersensitivity. The data from these experiments were used for the principal component analysis (PCA) for assessing survivability of isolates at different factors. The probiotic bacterial isolates showed good response for the enhanced antibody production and delayed type hypersensitivity (DTH) activity. Probiotic isolates E. faecium GMB24 and E. hirae SMB16, at 109 cells/ml doses per day, increased the neutrophil adhesion, haemagglutinating antibody titer and DTH in comparison to the untreated control group. These differences were statistically significant (P < 0.05). The isolates showed negative test for hemolytic and gelatinase activities and hence were considered safe. E. faecium GMB24 and E. hirae SMB16 possessed an excellent probiotic potential and had a strong potential immune-stimulant activity.

Immunomodulatory activity of Alcoholic extracts of Tinospora cordifolia Stem

Tinospora cordifolia is a plant well known for its medicinal value in Indian ayurveda and Indian traditional medicine system. However, to prove its efficiency for the clinical utilization, more experimental data will be beneficial. In the present investigation, evaluated the immunomodulatory activity of the alcoholic extracts of Tinospora cordifolia stem on various in-vivo experimental models such as neutrophil adhesion test, phagocytic index by carbon clearance test, Hemagglutinating antibody (HA) titre and delayed type hypersensitivity (DTH) responses. The evaluation of immunomodulatory potential by oral administration of alcoholic stem extracts (50, 100, 200 and 300mg/kg b.w, p.o) evoked a significant increase in percent neutrophil adhesion to nylon fibers as well as a dose dependent increased in antibody titre values, and potentiated delayed type hypersensitivity reaction induced by sheep red blood cells and significant response towards phagocytosis in carbon clearance assay (*p<0.05, *p<0.01, ***p<0.001). This may be due to concentrations of active phytochemicals present in particular plant extract. Hence, it was concluded that the plant extracts increased humoral as well as cell mediated immunity.

Immunomodulatory Activity on Specific Immune Function in Albino Mice Exposed to Extract and Fractions of Zapoteca portoricensis (Jacq) HM. Hernández Roots

Zapoteca portoricensis (Jacq) HM. Hernández popularly called “Elugelu” belongs to the Fabaceae family. It is reportedly used in traditional medicine as anti-diarrhoea, anticonvulsant, antispasmodic and in management of autoimmune disorders. The immunomodulatory activity of methanol root extract and fractions of Zapoteca portoricensis was evaluated using three experimental models: neutrophil adhesion, haemagglutination antibody (HA) titre and delayed hypersensitivity (DTH) of an immune response in mice. Five (5) groups of mice (n=5) were used; group I was the normal control; group II was the standard control (levamisole, 2.5 mg/kg) while groups IIIA–C, IVA–C and VA–C received graded doses (25, 50, 100 mg/kg) of methanol extract (ME), ethyl acetate fraction (EF) and methanol fraction (MF), respectively. Sheep red blood cells (SRBCs; 0.1 ml) were injected subcutaneously to sensitize the animals. The study results showed that the methanol fraction (MF) exhibited the highest percentage (46.12%) in neutrophil adhesion followed by the ethyl acetate fraction (EF) (37.06%) at 100 mg/kg dose, respectively, compared with the normal control. The highest percentage increase in both primary and secondary antibody titre was found to be ME (85.19%, 43.20%), EF (91.53%, 102.67%) and MF (128.31%, 111.89%) at 50 mg/kg dose, respectively compared to the normal control. The EF at the doses of 25 and 50 mg/kg produced the highest percentage inhibition (56.57%, 58.33%) in DTH response, respectively, followed by MF (42.46%) at a dose of 25 mg/kg. Oral administration of Z. portoricensis exhibited immunomodulatory effects on specific components of the immune system in mice.

Neutrophil Adhesion and the Release of the Free Amino Acid Hydroxylysine

During infection or certain metabolic disorders, neutrophils can escape from blood vessels, invade and attach to other tissues. The invasion and adhesion of neutrophils is accompanied and maintained by their own secretion. We have previously found that adhesion of neutrophils to fibronectin dramatically and selectively stimulates the release of the free amino acid hydroxylysine. The role of hydroxylysine and lysyl hydroxylase in neutrophil adhesion has not been studied, nor have the processes that control them. Using amino acid analysis, mass spectrometry and electron microscopy, we found that the lysyl hydroxylase inhibitor minoxidil, the matrix metalloproteinase inhibitor doxycycline, the PI3K/Akt pathway inhibitors wortmannin and the Akt1/2 inhibitor and drugs that affect the actin cytoskeleton significantly and selectively block the release of hydroxylysine and partially or completely suppress spreading of neutrophils. The actin cytoskeleton effectors and the Akt 1/2 inhibitor also increase the phenylalanine release. We hypothesize that hydroxylysine release upon adhesion is the result of the activation of lysyl hydroxylase in interaction with matrix metalloproteinase, the PI3K/Akt pathway and intact actin cytoskeleton, which play important roles in the recruitment of neutrophils into tissue through extracellular matrix remodeling.