Immunomodulatory Activity of Aqueous Extract of Polyherbal Formulation on Macrophage Function and Humoral Immune Response against SRBCs in Mice

Several herbal formulation were not properly documented due to poor scientific data as well as poor standard regulation in preparation and marketing. The study evaluate the effect of aqueous extract of Polyherbal formulation on Macrophages’ phagocytic function and Humoral immune response in Mice A total of Sixteen 16 Mice was used. Group I received normal saline, Group II-IV received 500 mg, 1000 mg, and 1500 mg of Polyherbal formulation respectively for 21 days each. The animals were sensitized and challenged with Sheep red blood cells at day 14th and 19th of the treatment respectively. On day 21st all the animals were injected with 0.1 ml Indian ink for carbon clearance assay and blood sample was collected at 1 minute and 15 minutes of the injection. The phagocytic function of Macrophages and humoral immune response were determined spectrophotometrically and Hemagglutination assay respectively. The results show that the highest carbonic particle clearances index (K) median score (Median=0.0228), Macrophage phagocytose index (α) median score (Median=3.249), organ weight index (g/100g) mean score (M=0.06633) and Hemagglutination antibody titre median score (Median=32.00) was recorded by Group IV when compared with other groups. The carbonic particle clearance (K) (p=0.02), phagocytic index of Macrophage (α) (p=0.03), and organ weight index (g/100g) (p<0.0001) significantly increase with increase in the Polyherbal formulation concentration. Hemagglutination antibody titre against Sheep red blood cells (p=0.02) also significantly increased. The extract has potential immunostimulatory activity on both Macrophages’ phagocytic function and humoral immune response in mice thus could be useful in improving immune responses.

IN VITRO EXPERIMENTAL REWIRING OF 4 NEUTROPHILIC GRANULOCYTE SUBSETS FROM THE PRO-INFLAMMATORY TO THE ANTI-INFLAMMATORY PHENOTYPE IN CHILDREN WITH SURGICAL PURULENT INFECTION OF SOFT TISSUE

Treatment of young children with atypical or recurrent purulent soft tissue infections (PSTD) that do not respond well to surgery and antibiotics is most challenging. PSTD occurs against the background of impaired functioning of the immune system and, first of all, the system of neutrophilic granulocytes (NG). The vector effect of immunotropic therapy on a specific NG subsets may allow the correction of NG dysfunctions without compromising host protection, including strategies to enhance, inhibit or restore their functions.The aim of study: to evaluate in vitro the modulating effects of arginyl-alpha-aspartyl-lysyl-valyl-tyrosyl-arginine (HP) on the transformed phenotype of 4 NG subsets, as well as on the functional activity of NG in children with purulent-inflammatory soft tissue diseases.We studied samples of peripheral blood (PB) from young children 2-4 years old: 17 children with atypical acute PSTD and 10 apparently healthy children. At stage I, a comparative assessment of the content and phenotype of 4 NG subsets CD16+CD62L+СD63- , CD16+CD62L+СD63+, СD64- CD16+CD32+CD11b+, СD64+CD16+CD32+CD11b+, phagocytic and microbicidal functions of NG was carried out. At stage II, the in vitro system determined the effects of HP on NG in children with PSTD according to the studied parameters. By the method of flow cytometry (FC500 “Beckman Coulter” (USA), conjugates of MkAT “Beckman Coulter International S.A.” (France)), the relative number of NGs of the studied subsets and the density of receptor expression (MFI) were determined. To assess the phagocytic function of NG a microbiological method was used to assess the completeness of phagocytosis with S. aureus (strain 209). The activity of NG NADPH oxidase was investigated in the NBT-spontaneous test (NBTsp.) and in the in vitro NBT-induced test (NBTind.). A comparative study of PB samples from conventionally healthy children and children with PSTD made it possible to identify various variants of transformation of the phenotype of the studied NG subsets, associated with defects in their functional activity. In the in vitro system the effects of HP were demonstrated, manifested by a decrease in the amount of CD16+CD62L+CD63+NG and an increase in CD16+CD62L+CD63- NG, modulation of the negatively altered phenotype of subsets CD64- CD32+CD16+CD11b+NG and CD64+CD32+CD16+CD11b+NG, aimed at restoring phagocytic function and maintaining the tension of NADPH oxidases.As a result of the study it was found the immunomodulatory effects of HP, which is manifested in the reorientation of NG from the pro-inflammatory phenotype to the anti-inflammatory one, which can be used in the future when creating personalized targeted immunotherapy aimed at correcting defective functioning NG in early children, suffering from PSTD.

Quantitative evidence of suppressed TMEM119 microglial immunohistochemistry in fatal morphine intoxications

The aim of this pilot study was to investigate the diagnostic potential of TMEM119 as a useful microglia-specific marker in combination with immunostainings for phagocytic function and infiltrating capacity of monocytes in cases of lethal monosubstance intoxications by morphine (MOR), methamphetamine (METH), and of ethanol-associated death (ETH) respectively. Human brain tissue samples were obtained from forensic autopsies of cases with single substance abuse (MOR, n = 8; ETH, n = 10; METH, n = 9) and then compared to a cohort of cardiovascular fatalities as controls (n = 9). Brain tissue samples of cortex, white matter, and hippocampus were collected and stained immunohistochemically with antibodies against TMEM119, CD68KiM1P, and CCR2. We could document the lowest density of TMEM119-positive cells in MOR deaths with highly significant differences to the control densities in all three regions investigated. In ETH and METH deaths, the expression of TMEM119 was comparable to cell densities in controls. The results indicate that the immunoreaction in brain tissue is different in these groups depending on the drug type used for abuse.

2-O, 3-O desulfated heparin (ODSH) increases bacterial clearance and attenuates lung injury in cystic fibrosis by restoring HMGB1-compromised macrophage function

Background High mobility group box 1 protein (HMGB1) is an alarmin following its release by immune cells upon cellular activation or stress. High levels of extracellular HMGB1 play a critical role in impairing the clearance of invading pulmonary pathogens and dying neutrophils in the injured lungs of cystic fibrosis (CF) and acute respiratory distress syndrome (ARDS). A heparin derivative, 2-O, 3-O desulfated heparin (ODSH), has been shown to inhibit HMGB1 release from a macrophage cell line and is efficacious in increasing bacterial clearance in a mouse model of pneumonia. Thus, we hypothesized that ODSH can attenuate the bacterial burden and inflammatory lung injury in CF and we conducted experiments to determine the underlying mechanisms. Methods We determined the effects of ODSH on lung injury produced by Pseudomonas aeruginosa (PA) infection in CF mice with the transmembrane conductance regulator gene knockout (CFTR−/−). Mice were given ODSH or normal saline intraperitoneally, followed by the determination of the bacterial load and lung injury in the airways and lung tissues. ODSH binding to HMGB1 was determined using surface plasmon resonance and in silico docking analysis of the interaction of the pentasaccharide form of ODSH with HMGB1. Results CF mice given 25 mg/kg i.p. of ODSH had significantly lower PA-induced lung injury compared to mice given vehicle alone. The CF mice infected with PA had decreased levels of nitric oxide (NO), increased levels of airway HMGB1 and HMGB1-impaired macrophage phagocytic function. ODSH partially attenuated the PA-induced alteration in the levels of NO and airway HMGB1 in CF mice. In addition, ODSH reversed HMGB1-impaired macrophage phagocytic function. These effects of ODSH subsequently decreased the bacterial burden in the CF lungs. In a surface plasmon resonance assay, ODSH interacted with HMGB1 with high affinity (KD = 3.89 × 10–8 M) and induced conformational changes that may decrease HMGB1’s binding to its membrane receptors, thus attenuating HMGB1-induced macrophage dysfunction. Conclusions The results suggest that ODSH can significantly decrease bacterial infection-induced lung injury in CF mice by decreasing both HMGB1-mediated impairment of macrophage function and the interaction of HMGB1 with membrane receptors. Thus, ODSH could represent a novel approach for treating CF and ARDS patients that have HMGB1-mediated lung injury. Graphic abstract

Neutrophil Elastase-Regulated Macrophage Sheddome/ Secretome and Phagocytic Failure

Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE) a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of E. coli bio-particles. We performed LC-MS/MS proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and down-regulated 211 proteins. NE released not only cell surface proteins into the media but also released cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors (including LRP1), 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/ secretome included proteins from other macrophage cellular domains suggesting that NE may globally regulate macrophage structure and function.

Causal relationships between the parameters of gas discharge visualization and phagocytosis

Background. Previously we have been shown that between parameters of GDV and principal neuroendocrine factors of adaptation exist strong canonical correlation. In the next study, we detected very strong (R=0,994) integral canonical correlation between the parameters of GDV and Immunity. This study, conducted in the same contingent, will analyze the relationships between GDV parameters, on the one hand, and Phagocytosis parameters, on the other. Material and Methods. We observed twice ten women and ten men aged 33-76 years without clinical diagnose. In the morning in basal conditions at first registered kirlianogram by the method of GDV by the device “GDV Chamber” (“Biotechprogress”, SPb, RF). Than we estimated the parameters of Phagocytic function of neutrophils. Results processed by method of canonical analysis, using the software package “Statistica 5.5”. Results. According to the value of the canonical correlation coefficient R with GDV parameters, the registered Phagocytosis parameters are arranged in the following order: activity (0,616), bactericidal capacity (0,493), completeness (0,489) and intensity (0,484) of Phagocytosis of E. coli; completeness (0,482), bactericidal capacity (0,448), activity (0,364) and intensity (0,338) of Phagocytosis of Staph. aureus. Coefficient of canonical correlation between parameters of GDV, on the one hand, and Phagocytosis, on the other hand, makes 0,847. Conclusion. The above data, taken together with the previous ones, state that between parameters of Neuroendocrine-Immune complex and GDV exist strong canonical correlation suggesting suitability of the latter method.

Atrazine induced In vivo immunotoxic studies in Bivalves

Atrazine is ubiquitously used broad-spectrum herbicide to control the weeds in agriculture. The present study aimed to evaluate the acute toxicity and immunotoxicity of Atrazine in two ecologically and economically important bivalves. Acute toxicity of atrazine evaluated in triplicates by taking control and six experimental groups each comprising of 30 animals and treated with a range of atrazine from 2 PPM to 12 PPM for 96 hours. Mortalities were recorded for every 24 hours until 96 hours and data analyzed by one-way ANOVA and Dunnett T-test. The results indicated a significant increase in mortalities with increase in dose and time of exposure in both species. The values of LC50 were determined as 6.10 PPM and 4.90 PPM respectively for Perna viridis and Paphia malabarica. Furthermore, the immunotoxic potential of atrazine assessed by treating mussels and clams with the five sub-lethal doses of atrazine for 14 days and quantifying the viability of hemocytes by using simple yet reliable Tryphan blue exclusion assay. The results of the present study suggest atrazine-induced immunotoxicity by decreasing the number of viable hemocytes in bivalves. Hemocytes with phagocytic function are indispensable to confer innate immunity in bivalves, decreased viability of these cells leads to compromised immunity. This study is first of its kind to implicate atrazine with the immunotoxicity in bivalves.

ВЛИЯНИЕ ВОЗРАСТА ПЕРВОГО ОТЕЛА КОРОВ ГОЛШТИНСКОЙ ПОРОДЫ НА УРОВЕНЬ ПРОДУКТИВНОСТИ И ФАКТОРЫ ЕСТЕСТВЕННОЙ РЕЗИСТЕНТНОТИ

Исследования проведены в Московском регионе на 2 группах первотелок голштинской породы. В I группу вошли животные, срок отела которых составил 26 мес, во II — первотелки, отел которых прошел в возрасте 31 мес. Изучены показатели молочной продуктивности, воспроизводительные качества и иммунологическая активность клеток крови животных в период лактации. В результате проведенных исследований установлено некоторое превосходство по удою у первотелок с более ранним периодом отела (на 333 кг) и снижение сервис-периода на 41,2 дня. У животных более позднего срока отела на фоне достаточно устойчивого показателя корреляции сегментоядерные нейтрофилы/лимфоциты установлено снижение стабильности работы иммунной системы, что выразилось в увеличении значимых корреляций (до 6) между клеточными факторами иммунитета по сравнению с животными более раннего срока отела, у которых таких корреляций было лишь 4. Усиление корреляции IgG/IgМ до -0,388 (Р≤0,05) свидетельствует о напряженности работы иммунной системы в период лактации у первотелок с более поздним сроком отела. Установлены значимые отрицательные корреляции сегментоядерные нейтрофилы /эозинофилы (r=-0,447 и r=-0,487 (Р≤0,05) соответственно по группам, что может быть связано с усилением фагоцитирующей функции организма животных. Полученные данные могут быть полезны при оценке состояния здоровья коров голштинской породы и оптимизации сроков их первого осеменения. INFLUENCE OF THE AGE OF THE FIRST CALVING OF HOLSTEIN COWS ON THE LEVEL OF PRODUCTIVITY AND FACTORS OF NATURAL RESISTANCE The study was conducted in the Moscow region on 2 groups of Holstein cows. The first group included animals whose calving period was 26 months, and the second group included first-calves, whose calving occurred at the age of 31 months. The indicators of milk productivity and reproductive qualities of animals were studied. Immunological activity of animal blood cells during lactation. As a result of the conducted studies, some superiority in milk yield was established in cows with an earlier calving period (by 333 kg) and a reduction in the period from calving to insemination by 41.2 days. In cows of the later calving period, against the background of a fairly stable index of segmented neutrophils/lymphocytes, a decrease in the stability of the immune system was found, which was expressed in an increase in the number of reliable correlations (up to 6) between cellular immunity factors compared to animals of the earlier calving period, in which there were only 4 such correlations. An increase in the IgG/IdM correlation to (r=-0.388) (P < 0.05) indicates an increase in the intensity of the work of the immune system during lactation in animals with a later calving period. Significant negative correlations of segmental neutrophils /eosinophils were found in the groups (r=-0.447 and r=-0.487 (P < 0.05), respectively), which may be associated with an increase in the phagocytic function of the animal body. The data obtained can be useful in assessing the health status of Holstein cows and optimizing the timing of the first insemination of animals.