Neutrophil Necrosis Research Articles

Anti-Inflammatory Effects of Calcium Hydroxide Combined with Ellagic Acid as Pulp Capping Material: In Vivo Study.

Pulp capping is a pivotal treatment in dentistry aimed at preserving pulp vitality. While calcium hydroxide has long been considered the gold standard in pulp capping materials, its long-term use can induce chronic inflammation, ultimately leading to pulp necrosis and affecting human health. In this context, ellagic acid, a natural compound with potent anti-inflammatory properties, emerged as a promising adjunct to mitigate inflammation associated with calcium hydroxide application, thereby sustaining pulp vitality. This study aimed to investigate the inflammatory response by alterations in neutrophil, macrophage, lymphocyte, and tumor necrosis factor-α (TNF-α) expression following the treatment with a combination of calcium hydroxide and ellagic acid. Dental pulp perforation was made on 27 male Wistar rats on the upper first molar and then pulp capped with calcium hydroxide and ellagic acid. The pulp of the control positive group was capped with calcium hydroxide, and the control group was not capped. The teeth were then extracted after 1, 3, and 7 days posttreatment. The differences in the number of neutrophils, macrophages, lymphocytes, and TNF-α expression were analyzed using one-way analysis of variance (ANOVA) and Tukey's honestly significant difference (HSD) test. The treatment combination of calcium hydroxide and ellagic acid showed the lowest neutrophil number and TNF-α expression compared with the other groups (p < 0.05), while the macrophage and lymphocyte numbers were the highest compared with the other groups (p < 0.05). The combination of calcium hydroxide and ellagic acid as a pulp capping material exhibited a dual effect on the inflammatory response in dental pulp. These findings suggest that calcium hydroxide and ellagic acid modulate the inflammatory response in a complex manner, promoting a more controlled and potentially beneficial healing process.

Management of anti-melanoma differentiation-associated gene 5 antibody-induced refractory dermatomyositis complicated by interstitial pneumonia using tofacitinib and its outcomes: a case report

BackgroundClinical amyopathic dermatomyositis is characterized by cutaneous symptoms but lacks muscle symptoms. Anti-melanoma differentiation-associated gene 5 antibodies are frequently found in Japanese patients with clinical amyopathic dermatomyositis. Patients with rapidly progressive interstitial lung disease with positive anti-melanoma differentiation-associated gene 5 antibodies have poor prognoses, and majority of them are treated with combination immunosuppressive therapy; however, the best treatment is yet to be determined.Case presentationA 52-year-old Asian male patient presented with a chief complaint of dyspnea on exertion. He had a typical skin rash and rapidly progressive interstitial pneumonia. Additionally, anti-melanoma differentiation-associated gene 5 antibodies were detected; therefore, he was diagnosed with dermatomyositis-associated interstitial pneumonia. Respiratory failure worsened despite administering steroid pulse therapy, tacrolimus, and cyclophosphamide. Consequently, plasma exchange was performed on day 13 of admission. After a slight improvement, the patient’s respiratory failure worsened. Thus, cyclophosphamide was replaced by tofacitinib on day 28. Although respiratory failure improved and the progression of interstitial pneumonia seemed under control, βD-glucan level increased and Aspergillus antigen was detected on day 49. Micafungin and voriconazole were administered, but the patient succumbed to worsening respiratory failure on day 61. The pathological autopsy revealed multiple nodular lesions with cavity formation in both lungs and the presence of Aspergillus with severe neutrophilic infiltration and necrosis, which supported the diagnosis of invasive pulmonary aspergillosis.ConclusionThe patient with anti-melanoma differentiation-associated gene 5 antibody-related rapidly progressive interstitial lung disease, whose disease was difficult to control after the administration of triple immunosuppressive therapy (steroids, tacrolimus, and cyclophosphamide), showed good response with tofacitinib. Unfortunately, the patient died of invasive pulmonary aspergillosis owing to severe immunosuppression; thus, the signs of complications should be promptly detected.

Bear Bile Powder Ameliorates LPS-Induced Acute Lung Injury by Inhibiting CD14 Pathway and Improving Intestinal Flora: Exploration of "Fei (Lung)-Dachang (Large Intestine) Interaction".

To explore the effect of bear bile powder (BBP) on acute lung injury (ALI) and the underlying mechanism. The chemical constituents of BBP were analyzed by ultra-high-pressure liquid chromatography-mass spectrometry (UPLC-MS). After 7 days of adaptive feeding, 50 mice were randomly divided into 5 groups by a random number table (n=10): normal control (NC), lipopolysaccharide (LPS), dexamethasone (Dex), low-, and high-dose BBP groups. The dosing cycle was 9 days. On the 12th and 14th days, 20 µL of Staphylococcus aureus solution (bacterial concentration of 1 × 10-7 CFU/mL) was given by nasal drip after 1 h of intragastric administration, and the mice in the NC group was given the same dose of phosphated buffered saline (PBS) solution. On the 16th day, after 1 h intragastric administration, 100 µL of LPS solution (1 mg/mL) was given by tracheal intubation, and the same dose of PBS solution was given to the NC group. Lung tissue was obtained to measure the myeloperoxidase (MPO) activity, the lung wet/dry weight ratio and expressions of CD14 and other related proteins. The lower lobe of the right lung was obtained for pathological examination. The concentrations of inflammatory cytokines including interleukin (IL)-6, tumour necrosis factor α (TNF-α ) and IL-1β in the bronchoalveolar lavage fluid (BALF) were detected by enzyme linked immunosorbent assay, and the number of neutrophils was counted. The colonic contents of the mice were analyzed by 16 sRNA technique and the contents of short-chain fatty acids (SCFAs) were measured by gas chromatograph-mass spectrometer (GC-MS). UPLC-MS revealed that the chemical components of BBP samples were mainly tauroursodeoxycholic acid and taurochenodeoxycholic acid sodium salt. BBP reduced the activity of MPO, concentrations of inflammatory cytokines, and inhibited the expression of CD14 protein, thus suppressing the activation of NF-κB pathway (P<0.05). The lung histopathological results indicated that BBP significantly reduced the degree of neutrophil infiltration, cell shedding, necrosis, and alveolar cavity depression. Moreover, BBP effectively regulated the composition of the intestinal microflora and increased the production of SCFAs, which contributed to its treatment effect (P<0.05). BBP alleviates lung injury in ALI mouse through inhibiting activation of NF-κB pathway and decreasing expression of CD14 protein. BBP may promote recovery of ALI by improving the structure of intestinal flora and enhancing metabolic function of intestinal flora.

Up-regulation of inflammatory, oxidative stress, and apoptotic mediators via inflammatory, oxidative stress, and apoptosis-associated pathways in bovine endometritis

Endometritis is the inflammation of the endothelial lining of the uterine lumen and is multifactorial in etiology. Escherichia (E.) coli is a Gram-negative bacteria, generally considered as a primary causative agent for bovine endometritis. Bovine endometritis is characterized by the activation of Toll-like receptors (TLRs) by E. coli, which in turn triggers inflammation, oxidative stress, and apoptosis. The objective of this study was to investigate the gene expression of inflammatory, oxidative stress, and apoptotic markers related to endometritis in the uteri of cows. Twenty uterine tissues were collected from the abattoir. Histologically, congestion, edema, hyperemia, and hemorrhagic lesions with massive infiltration of neutrophil and cell necrosis were detected markedly (P < 0.05) in infected uterine samples. Additionally, we identify E. coli using the ybbW gene (177 base pairs; E. coli-specific gene) from infected uterine samples. Moreover, qPCR and western blot results indicated that TLR2, TLR4, proinflammatory mediators, and apoptosis-mediated genes upregulated except Bcl-2, which is antiapoptotic, and there were downregulations of oxidative stress-related genes in the infected uterine tissue. The results of our study suggested that different gene expression regimes related to the immune system reflex were activated in infected uteri. This research gives a novel understanding of active immunological response in bovine endometritis.

Vascular growth factors of the placenta, apoptosis of blood neutrophils in pregnant women with post-COVID syndrome

Research objectives: to determine the activity of vascular placental growth factors, apoptosis and necrosis of neutrophils in the blood of pregnant women with post-COVID syndrome.Methods and materials. 30 pregnant women (the main group) with SARS-CoV-2, positive Ig A, M or G to SARS-CoV-2 at 30–34 weeks of pregnancy took part in the study. The control group consisted of 30 women with physiological pregnancy.Blood neutrophils were studied by flow cytofluorometry. The proportion of neutrophils that were at the stage of apoptosis and necrosis was determined. The correlation of these indicators with the uncomplicated course of the gestational process during the physiological course of pregnancy and with the development of gestational complications against the background of post-COVID syndrome was determined.In the third trimester of pregnancy, we collected blood samples to determine the effect of SARS-CoV-2 infection on placental angiogenesis. We evaluated a panel of biomarkers: vascular endothelial growth factor (VEGF), placental growth factor PlGF, and interleukin-32α (IL-32α).Results. In pregnant women of the main group with a complicated course of pregnancy accompanied by post-COVID syndrome, apoptosis was at the level of 24.30 ± 0.50% of neutrophil cells, which was significantly different from the control group (4.45 ± 0.25%) (р &lt; 0.001). The level of late apoptosis, necrosis of neutrophils increased 4 times – from 4.20 ± 0.65 to 16.80 ± 0.54% (р &lt; 0.001). The concentration of IL-32α in pregnant women of the control group was 67.27 ± 5.63 pg/ml. Post-COVID syndrome caused an increase in this indicator in the main group by 2.8 times compared to the control group (188.36 ± 25.22 pg/ml) (p &lt; 0.001). In the III trimester, the concentration of VEGF reached maximum values in the main group and was 192.20 ± 10.02 pg/ml, which is 2 times higher than in the control group at the same time (95.30 ± 5.65 pg/ml), (p &lt; 0.001). In the control group the level of PIGF at full-term pregnancy was 144.53 ± 15.55 pg/ml. In women with postpartum syndrome, PIGF significantly decreased and was 43.92 ± 4.81 pg/ml, which was only 30% of the PIGF value in women with an uncomplicated pregnancy (p &lt; 0.001).Conclusions. The development of metabolic disorders and apoptotic changes in the placental tissue are confirmed at the morphological level in the form of destructive and necrotic changes in the microcirculatory channel of the placenta. An increase in the level of annexin-positive neutrophils and activation of the degree of neutrophil necrosis is accompanied by fetoplacental dysfunction, by violation of the balance of placental growth factors and is an important marker for predicting of fetal growth retardation in pregnant women with post-COVID syndrome.

Human Neutrophil Response to Pseudomonas Bacteriophage PAK_P1, a Therapeutic Candidate.

The immune system offers several mechanisms of response to harmful microbes that invade the human body. As a first line of defense, neutrophils can remove pathogens by phagocytosis, inactivate them by the release of reactive oxygen species (ROS) or immobilize them by neutrophil extracellular traps (NETs). Although recent studies have shown that bacteriophages (phages) make up a large portion of human microbiomes and are currently being explored as antibacterial therapeutics, neutrophilic responses to phages are still elusive. Here, we show that exposure of isolated human resting neutrophils to a high concentration of the Pseudomonas phage PAK_P1 led to a 2-fold increase in interleukin-8 (IL-8) secretion. Importantly, phage exposure did not induce neutrophil apoptosis or necrosis and did not further affect activation marker expression, oxidative burst, and NETs formation. Similarly, inflammatory stimuli-activated neutrophil effector responses were unaffected by phage exposure. Our work suggests that phages are unlikely to inadvertently cause excessive neutrophil responses that could damage tissues and worsen disease. Because IL-8 functions as a chemoattractant, directing immune cells to sites of infection and inflammation, phage-stimulated IL-8 production may modulate some host immune responses.

Macrophages polarize to the pro-inflammatory phenotype and delay neutrophil efferocytosis to augment Aggregatibacter actinomycetemcomitance JP2 clearance.

The study examines how neutrophils cross-talk with macrophages during JP2 Aggregatibacter actinomycetemcomitance infection and factors that are involved in inflammatory resolution and efferocytosis. Although sub-gingival bacteria constitute the primary initiating factor in the pathogenesis of molar-incisor pattern periodontitis (MIPP), the non-resolved host response has a major role in tissue destruction. While evidence links neutrophils to MIPP pathogenesis, their clearance during inflammatory resolution, governed by macrophages, is poorly understood. Human neutrophils (differentiated from HL60 cells) and macrophages (differentiated from THP1 cells) were inoculated with JP2. The supernatants were collected and exposed to naïve neutrophils or macrophages with or without exposure to JP2. Reactive oxygen species (ROS) were measured with 2'-7'-dichlorofluorescein-diacetate and a fluorescent plate reader. Immunofluorescence labeling of CD47 and cell vitality were examined using flow cytometry. Macrophage polarization was tested by immunofluorescence staining for CD163 and CD68 and a fluorescent microscope, and TNFα and IL-10 secretion was tested using ELISA and RT-PCR. Efferocytosis was examined by pHrodo and carboxyfluorescein succinimidyl ester staining and fluorescent microscopy. In vivo, macrophages were depleted from C57Bl/6 mice and neutrophil CD47 levels were tested using the subcutaneous chamber model. Neutrophils exposed to macrophage supernatant show increased ROS, mainly extracellularly, that increased during JP2 infection. Macrophages showed pro-inflammatory M1 phenotype polarization during JP2 infection, and their supernatants prolonged neutrophil survival by inhibiting CD47 down-expression and reducing neutrophil necrosis and apoptosis. Also, the macrophages delay neutrophil efferocytosis during JP2 infection which, in turn, enhanced JP2 clearance. Depletion of macrophages in mice mildly prevented neutrophils CD47 reduction and reduced JP2 clearance. The JP2 infection in mice also led to macrophage M1 polarization similar to the in vitro results. As shown in this study, neutrophil efferocytosis potentially may be reduced during JP2 infection, promoting JP2 clearance, which may contribute to the inflammatory-mediated periodontal tissue damage.

The effect of silymarin given before partial hepatectomy on liver regeneration in rats with ischemic preconditioning during liver resection

Background: The regeneration capacity of liver tissue after partial hepatectomy closely affects morbidity and mortality. We aimed to investigate the effect of silymarin and ischemic preconditioning (IPC) before partial hepatectomy on liver regeneration.Methods: Thirty rats were randomly divided into 5 groups (n=6). Serum AST, bilirubin, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) values were studied 48 hours after hepatectomies. Mitotic count, congestion, necrosis, cytoplasmic vacuolization, and presence of neutrophils were evaluated histopathologically. Proliferating cell nuclear antigen (PCNA) antibody was studied immunohistochemically.Results: The AST value (2071.5±938.4) was the highest in the partial hepatectomy (PH) group. The Ischemic Preconditioning Partial Hepatectomy Group (IPC) (1535.5±204.8) and the Silymarin+Partial Hepatectomy Group (Silymarin group) (1192.3±526.3) had lower levels of AST values; however, the AST values were the lowest in the Silymarin+Ischemic Preconditioning+ Partial Hepatectomy Group (IPC+ Silymarin group) (1002.8±348.9). Likewise, the highest improvement in bilirubin levels was observed in the IPC+ Silymarin group (0.33±0.11). IL-6 (11.54±2.89) and TNF-α (39.2±22.73) values were the lowest in the IPC group, and interestingly, both were higher in the silymarin-treated groups. It was observed that these values increased in Silymarin group and IPC+ Silymarin group who received silymarin treatment compared to PH group and IPC group who did not receive silymarin. In histopathological analysis, it was found that the mitosis rate and PCNA percentage were higher in the Silymarin and IPC+ Silymarin groups.Conclusions: The use of silymarin before hepatectomy and IPC during partial hepatectomy increase liver regeneration.

CD18 Mediates Neutrophil Imperviousness to the Aggregatibacter actinomycetemcomitans JP2 Clone in Molar-Incisor Pattern Periodontitis.

IntroductionMolar-incisor pattern periodontitis (MIPP) in the absence of significant local risk factors or systemic disease, is a rare, early onset periodontal disease phenotype, with 0.5% to 2.5% global prevalence. The condition is characterized by impaired neutrophil function and persistent Aggregatibacter actinomycetemcomitans (JP2 clone) infection. The aim of this study was to characterize neutrophil functional responses to JP2 and to investigate the neutrophil receptors involved.Materials and MethodsNeutrophils were obtained from whole blood samples of periodontally healthy and MIPP subjects and incubated with the JP2 clone or a non-JP2 clone of A. actinomycetemcomitans. Bacterial survival was tested by blood agar culture; neutrophil death was tested with propidium iodide and flow cytometry; Reactive oxygen production (ROS) was measured with 2′,7′-dichlorofluorescein diacetate and a fluorescence plate reader; the cytokinome was analysed using an array profiler, ELISA and RT-PCR. Receptors binding to JP2 were isolated using a novel immunoprecipitation assay and validated functionally using specific blocking antibodies.ResultsJP2 and non-JP2 survival was comparable between all the neutrophil groups. Resistance to neutrophil necrosis following exposure to JP2 was significantly lower in the MIPP group, than in all the other groups (p<0.0001). Conversely, MIPP neutrophils showed lower levels of ROS production in response to JP2 infection compared with that of healthy neutrophils (p<0.001). Furthermore, significantly lower levels of cytokines, such as IL8, IL10 and TNFα, were observed during JP2 incubation with MIPP neutrophils than upon incubation with periodontally healthy neutrophils. Various proteins expressed on neutrophils bind to JP2. Of these, CD18 was found to mediate neutrophil necrosis. The CD18 receptor on MIPP neutrophils acts differently from that on periodontally healthy patients neutrophils, and appears to reflect differential neutrophil reactions to JP2.ConclusionThis study portrays a fundamental difference in neutrophil response to JP2 infection between periodontally healthy and MIPP patients. This was evident in the resistance to necrosis, and lower ROS and cytokine production, despite the persistent presence of viable JP2. Whilst in periodontally healthy neutrophils, JP2 binds to CD18 on cell surfaces, this is not the case in MIPP neutrophils, suggesting a potential role for CD18 in the periodontal susceptibility of MIPP patients.

Loss of Autotaxin inhibits Toll‐like receptor4‐mediated immune mechanisms and promote spontaneous colitis in Il10‐ko mice

Autotaxin (Atx) is a secreted enzyme converting lysophosphatidylcholine (LPC) and sphingosyl‐phosphorylcholine into lysophosphatidic acid and sphingosine 1‐phosphate, respectively. Given the high affinity of LPC to cholesterol and the enrichment of cholesterol and sphingolipids in lipid rafts wherein LPS sensor Toll‐like receptor 4 (TLR4) and its co‐receptor CD14 reside, we hypothesized that Atx deficiency inhibits TLR4‐mediated innate and adaptive immunity; thereby, accelerating the susceptibility to microbe‐induced intestinal inflammation.To study this hypothesis, we generated myeloid cell lineage‐restricted Atx‐knockout (ko) mice (Atx‐dME/dME) to investigate TLR4‐mediated immune and inflammatory responses and examined LPS‐receptor complex formation through fluorescence resonance energy transfer technique, confocal microscopy, and flow cytometry. Phagocytic activity of macrophages was assessed. Lamina propria CD4+ T cells and bacterial load in the intestinal mucosa were examined. An impact of Atx deficiency in inflammatory bowel diseases (IBD) was investigated using Atx‐dME/dME;Il10‐/‐ mice that have both Atx‐deletion in myeloid cells and a global Il10 deletion. We examined the serum samples from IBD patients.With peritoneal macrophages from Atx‐dME/dME mice, we identified that Atx‐ko disrupted the integrity of lipid rafts at the plasma membrane, resulting in the inhibition of TLR4 complex formation. Accordingly, the recruitment of adaptor molecules to TLR4 was suppressed, and TLR4‐mediated responses were substantially reduced in Atx‐ko macrophages. TLR4‐induced innate immunity such as phagocytosis was attenuated in Atx‐ko macrophages. The activation of CD4+ effector T cells and regulatory T cells was diminished in the lamina propria lymphocytes of Atx‐dME/dME mice compared to that of Atx‐wt littermates. Consequently, Atx‐dME/dME mice had a higher bacterial prevalence in the intestinal mucosa compared to controls. Just like the notion that commensal microbes translocated from the lumen into the intestinal mucosa can elicit spontaneous colitis in Il10‐/‐ mice, combining Atx‐dME/dME with Il10‐/‐ mice (Atx‐dME/dME;Il10‐/‐) did accelerate spontaneous colitis development. Atx‐dME/dME;Il10‐/‐ mice had gross inflammation occurring throughout the colon, massive neutrophil infiltration and necrosis in the colonic mucosa, and increased mortality (Log‐rank P=0.0046), while Atx‐wt;Il10‐/‐ littermates are normal. Notably, ATX serum protein level was lower in UC (n=26) and CD (n=34) patients compared to the level in normal subjects (n=26) (P&lt;0.001), indicating an association of reduced ATX levels with intestinal inflammation.Collectively, we found that Atx deficiency suppresses TLR4‐mediated innate and adaptive immune mechanisms; thereby accelerating the susceptibility to microbe‐induced gut inflammation.

Anti-inflammatory and anti-necrotic effects of lectins from Canavalia ensiformis and Canavalia brasiliensis in experimental acute pancreatitis.

Lectins isolated from Canavalia ensiformis (ConA) and Canavalia brasiliensis (ConBr) are promising molecules to prevent cell death. Acute pancreatitis, characterized by acinar cell necrosis and inflammation, presents significant morbidity and mortality. This study has investigated the effects of ConA and ConBr in experimental acute pancreatitis and pancreatic acinar cell death induced by bile acid. Pancreatitis was induced by retrograde pancreatic ductal injection of 3% sodium taurocholate (Na-TC) in male Swiss mice. ConA or ConBr (0.1, 1 or 10mg/kg) were intravenously applied to mice 1h and 12h after induction. After 24h, the severity of pancreatitis was evaluated by serum amylase and lipase, histopathological changes and myeloperoxidase assay. Pancreatic acinar cells were incubated with ConA (200µg/ml) or ConBr (200µg/ml) and taurolithocholic acid 3-sulfate (TLCS; 500µM). Necrosis and changes in mitochondrial membrane potential (ΔѰm) were detected by fluorescence confocal microscopy. Treatment (post-insult) with ConA and ConBr decreased pancreatic damage caused by retrograde injection of Na-TC in mice, reducing pancreatic neutrophil infiltration, edema and necrosis. In addition, ConA and ConBr decreased pancreatic acinar cell necrosis and depolarization of ΔѰm caused by TLCS. The inhibition of necrosis was prevented by the lectin domain blockade. In conclusion, ConA and ConBr markedly inhibited in vitro and in vivo damage, effects partly dependent on the interaction with mannose residues on acinar cells. These data support the potential application of these proteins for treatment of acute pancreatitis.

Diacerein counteracts acetaminophen-induced hepatotoxicity in mice via targeting NLRP3/caspase-1/IL-1\u03b2 and IL-4/MCP-1 signaling pathways

The current study aims at repurposing the anti-arthritic drug diacerein (DCN) for the treatment of acetaminophen hepatotoxicity and investigating the potential underlying mechanisms. Mice were randomly divided into six groups receiving either no treatment (control group), 20 mg/kg DCN i.p, 400 mg/kg acetaminophen i.p, DCN 4 h before acetaminophen, DCN 2 h after acetaminophen, or 400 mg/kg N-acetylcysteine (NAC) i.p, 2 h after acetaminophen. Biomarkers of liver dysfunction, oxidative stress, and apoptosis were assessed. Hepatic necroinflammatory changes were evaluated along with hepatic expression of NF-κB and caspase-1. The levels of NLRP3, IL-1β, IL-4, MCP-1, and TNF-α in the liver, as well as CYP2E1 mRNA expression, were measured. Diacerein significantly reduced biomarkers of liver dysfunction, oxidative stress, hepatocyte necrosis, and infiltration of neutrophils and macrophages whether administered 4 h before or 2 h after acetaminophen. Further, the effects were comparable to those of NAC. Diacerein also counteracted acetaminophen-induced hepatocellular apoptosis by increasing Bcl-2 and decreasing Bax and caspase-3 expression levels. Moreover, DCN normalized hepatic TNF-α and significantly decreased NF-κB p65 expression. Accordingly, DCN can prevent or reverse acetaminophen hepatotoxicity in mice, suggesting potential utility as a repurposed drug for clinical treatment.

Evaluation of Myeloperoxidase as Target for Host-Directed Therapy in Tuberculosis In Vivo.

Due to the rise of tuberculosis cases infected with multi and extensively drug-resistant Mycobacterium tuberculosis strains and the emergence of isolates resistant to antibiotics newly in clinical use, host-directed therapies targeting pathogenesis-associated immune pathways adjunct to antibiotics may ameliorate disease and bacterial clearance. Active tuberculosis is characterized by neutrophil-mediated lung pathology and tissue destruction. Previously, we showed that preventing M. tuberculosis induced necrosis in human neutrophils by inhibition of myeloperoxidase (MPO) promoted default apoptosis and subsequent control of mycobacteria by macrophages taking up the mycobacteria-infected neutrophils. To translate our findings in an in vivo model, we tested the MPO inhibitor 4-aminobenzoic acid hydrazide (ABAH) in C3HeB/FeJ mice, which are highly susceptible to M. tuberculosis infection manifesting in neutrophil-associated necrotic granulomas. MPO inhibition alone or as co-treatment with isoniazid, a first-line antibiotic in tuberculosis treatment, did not result in reduced bacterial burden, improved pathology, or altered infiltrating immune cell compositions. MPO inhibition failed to prevent M. tuberculosis induced neutrophil necrosis in C3Heb/FeJ mice in vivo as well as in murine neutrophils in vitro. In contrast to human neutrophils, murine neutrophils do not respond to M. tuberculosis infection in an MPO-dependent manner. Thus, the murine C3HeB/FeJ model does not fully resemble the pathomechanisms in active human tuberculosis. Consequently, murine infection models of tuberculosis are not necessarily adequate to evaluate host-directed therapies targeting neutrophils in vivo.

Pseudomonas aeruginosamexR and mexEF Antibiotic Efflux Pump Variants Exhibit Increased Virulence.

Antibiotic-resistant Pseudomonas aeruginosa infections are the primary cause of mortality in people with cystic fibrosis (CF). Yet, it has only recently become appreciated that resistance mutations can also increase P. aeruginosa virulence, even in the absence of antibiotics. Moreover, the mechanisms by which resistance mutations increase virulence are poorly understood. In this study we tested the hypothesis that mutations affecting efflux pumps can directly increase P. aeruginosa virulence. Using genetics, physiological assays, and model infections, we show that efflux pump mutations can increase virulence. Mutations of the mexEF efflux pump system increased swarming, rhamnolipid production, and lethality in a mouse infection model, while mutations in mexR that increased expression of the mexAB-oprM efflux system increased virulence during an acute murine lung infection without affecting swarming or rhamnolipid gene expression. Finally, we show that an efflux pump inhibitor, which represents a proposed novel treatment approach for P. aeruginosa, increased rhamnolipid gene expression in a dose-dependent manner. This finding is important because rhamnolipids are key virulence factors involved in dissemination through epithelial barriers and cause neutrophil necrosis. Together, these data show how current and proposed future anti-Pseudomonal treatments may unintentionally make infections worse by increasing virulence. Therefore, treatments that target efflux should be pursued with caution.

A controversial role of neutrophils in tuberculosis infection pathogenesis

Tuberculosis (TB) continues to be an important and unresolved medical problem. About a quarter of mankind is infected with Mycobacterium tuberculosis, and about 5–10% of these people eventually develop TB. Macrophages and CD4+ T cells are considered the key cells providing defense against TB infection. The role of neutrophils in TB is less well defined. Neutrophils are short-lived granulocytes among first migrate into the infectious lung tissue and phagocy tose mycobacteria. On the one hand, there is evidence for protective role of neutrophils in TB released via anti-microbial peptides inhibiting mycobacterial growth, up-regulation of CD4+ T-cell activation, and dendritic cell migration in the lymph nodes. On the other hand, infection of genetically TB susceptible animals leads to an overwhelming lung neutrophil inflammation, development of necrotic granulomata, and a rapid death. Neutrophils act directly or indirectly on mycobacteria by different oxidative or other reactions including neutrophil extracellular traps (NETs) formation. Phagocytosis of mycobacteria by neutrophils is accompanied by the production of pro-inflammatory factors, thus making neutrophils active participants of inflammation in all stages of the infectious process. Finally, neutrophils die by apoptosis or necrosis. Necrosis of neutrophils, which is activated by reactive oxygen species, also prolongs the inflammation. In this way, there is strong evidence that neutrophils are the cells involved in the transition of infection to the terminal stage, participating in lung tissue destruction. Although neutrophils evolutionary developed many ways to resist pathogens, it is likely, that neutrophils do not possess sufficient anti-mycobactericidal capacities due to the development of many adaptations allowing mycobacteria to survive inside the neutrophils. Neutrophils effectively phagocytose but poorly kill mycobacteria, thus hiding bacilli from more efficient killers, macrophages, and playing the role of the “Trojan Horse”. In this review, we summarize the data on the involvement of neutrophils in TB inflammation. We discuss their ambiguous role in pathogenesis which depends upon mycobacterial virulence, host genetics, dynamics of migration to inflammatory foci, and persistence during initial and chronic stages of the infectious process.

Investigation of the Oxidative Stress Response of a Green Synthesis Nanoparticle (RP-Ag/ACNPs) in Zebrafish.

Silver nanoparticles (AgNPs) are prominent nanomaterials that are efficiently used in different industries including medical products, water treatment, and cosmetics. However, AgNPs are known to cause adverse effects on the ecosystem and human health. In this study, aqueous extract of Rumex patientia (RP) was used as a reducing and stabilizing agent in AgNP biosynthesis. The obtained activated carbon (AC) from Chenopodium album (CA) plant was combined with RP-AgNPs to synthesize RP-Ag/AC NPs. Next, the effects of these green synthesis RP-Ag/AC NPs on zebrafish (Danio rerio) embryos and larvae were investigated. First, we characterized the RP-Ag/AC NPs by using X-ray diffraction (XRD) and transmission electron microscopy (TEM) and determined LC50 value as 217.23mg/L at 96h. Next, the alterations in survival rate, hatching rate, and morphology of the larvae at 96h were monitored. The survival rates decreased in a dose-dependent manner. Morphological defects such as yolk sac edema, pericardial edema, spinal curvature, and tail malformation in the NP-treated larvae were observed. RP-Ag/AC NPs stimulated the production of neuronal NOS (nNOS) and 8-OHdG in zebrafish brain tissues in a dose-dependent manner and enhanced neutrophil degeneration and necrosis at concentrations of 50 and 100mg/L. Thus, the obtained data suggest that the green synthesis process is not sufficient to reduce the effect of oxidative stress caused by AgNPs on oxidative signaling.

25656 Vulvar discoid lupus erythematosus: An unusual site for cutaneous lupus erythematosus

A 46-year-old woman presented with a 1-month history of a pruritic rash on trunk and face. On examination, she had well-defined erythematous scaly thin plaques photodistributed on the face, chest, ears, and neck. Biopsy demonstrated an interface dermatitis with increased dermal mucin. Laboratory evaluation was remarkable for a positive ANA 1:1280, but negative Ro/SS-A and La/SS-B antibodies. She was diagnosed with subacute cutaneous lupus erythematosus on the basis of clinical-pathologic findings. Tobacco cessation and strict sun protection were recommended, and she was prescribed hydroxychloroquine 200 mg twice daily and topical triamcinolone ointment. She was lost to follow-up during the COVID pandemic but was seen 6 months later with a persistent painful eruption of the vulva of several months’ duration. The patient had been hospitalized for this eruption and surgical debridement was performed which revealed a nonspecific neutrophilic infiltrate and tissue necrosis. Immunohistochemistry stain was negative for herpesvirus. She was treated with intravenous antibiotics and referred back to us. Upon her return visit, she had stopped smoking but was only taking hydroxychloroquine 200 mg daily. On examination, she had scarred erythematous patches on the labia minora and inguinal folds. Biopsy demonstrated a vacuolar interface dermatitis with superficial epidermal necrosis, dermal edema, and superficial and deep perivascular and periappendageal lymphoplasmacytic inflammation, favoring a diagnosis of discoid lupus erythematosus. We recommended increasing the hydroxychloroquine to 200 mg twice daily and continuing topical triamcinolone ointment.

Effects of adipose tissue-derived mesenchymal stem cells and/or sildenafil citrate in experimental colon anastomosis model.

This study aimed to evaluate the healing effects of adipose tissue-derived mesenchymal stem cells (AT-MSC) and sildenafil citrate alone or in combination of colon anastomosis experimental model. A total of 40 female Wistar rats were randomly distributed to four groups: Control (without any intervention post-anas-tomosis), stem cell (AT-MSC injection on the anastomosis site), SIL (oral gavage of 10 mg/kg sildenafil citrate), and stem cell + SIL (AT-MSC injection and oral administration of sildenafil citrate) groups. Rats were euthanized 5 days post-anastomosis. Intra-abdominal adhesion status and anastomotic burst pressure were measured to assess anastomotic healing. Hydroxyproline and TNF-α level, neu-trophil leukocyte infiltration, epithelial regeneration, and necrosis in the anastomosis tissue were examined. Anastomosis leakage and anastomosis burst pressure were not different among the groups. Treatment with sildenafil, stem cell, and stem cell + SIL reduced the degree of perianastomotic adhesions compared to control (p<0.05). A significant increase was noted in hydroxyproline in the stem cell and stem cell + SIL groups (p=0.001). AT-MSC injection alone or in combination with sildenafil citrate reduced the TNF-α concentration at the anastomosis site (p=0.001). Histopathological examination revealed that all treatments enhanced the clearance of the necrotic debris, reduced leukocytes infiltration, and accelerated the retraction of anastomo-sed ends except control (p=0.001). Epithelial regeneration was more pronounced in the stem cell group than other groups (p=0.001). Macrophage density was lower in groups treated with the SIL or stem cell groups than the control and stem cell + SIL groups (p=0.001). Sildenafil citrate and/or AT-MSC in the anastomosed rats promoted the anastomosis healing that was more pro-nounced in groups receiving stem cell injections.

Влияние хлорида лития на активацию нейтрофилов при развитии синдрома системного воспалительного ответа у пациентов после операций на сердце с использованием аппарата искусственного кровообращения

Цель исследования - изучение in vitro действия хлорида лития на активность нейтрофилов человека при действии сывороток пациентов с синдромом системного воспалительного ответа, развившемся после операций на сердце с искусственным кровообращением. Методика. Исследование проводили in vitro на нейтрофилах, выделенных из крови 6 здоровых доноров. Нейтрофилы активировали при помощи сыворотки пациентов с синдромом системного воспалительного ответа (ССВО), перенесших операции на сердце с искусственным кровообращением (ИК). Активность нейтрофилов оценивали с использованием флуоресцентных антител к маркерам дегрануляции CD11b и CD66b. Уровень апоптоза и некроза нейтрофилов оценивали через 22 ч после выделения из крови здоровых доноров; количественная оценка была проведена с использованием аннексина V и иодистого пропидия на проточном цитофлуориметре. Интактные и активированные нейтрофилы обрабатывали раствором хлорида лития в концентрациях 0,3; 3,0 и 9,0 мМ. Результаты. Инкубация нейтрофилов с сывороткой крови пациентов с ССВО после операций на сердце с ИК увеличивала экспрессию CD11b в 1,5 раза и экспрессию CD66b в 1,4 раза в сравнении с экспрессией на интактных нейтрофилах. Инкубация нейтрофилов с сывороткой крови пациентов с ССВО и раствором хлорида лития в концентрациях 3,0 и 9,0 мМ приводило к статистически значимому снижению уровня экспрессии CD11b CD66b на поверхности нейтрофилов в сравнении с активированными контрольными. Установлено, что хлорид лития в концентрациях 3,0 и 9,0 мМ возвращал уровни экспрессии CD11b и CD66b на активированных нейтрофилах к уровню экспрессии на интактных нейтрофилах. В концентрации 0,3 мМ хлорид лития, используемый при инкубации с активированными нейтрофилами, не вызывал значимого снижения экспрессии CD11b и CD66b относительно контрольных активированных нейтрофилов. Экспрессия CD11b и CD66b на активированных нейтрофилах при их инкубации с хлоридом лития в концентрации 0,3 мМ была значимо выше относительно экспрессии данных молекул на интактных нейтрофилах. Сыворотка пациентов с развившемся ССВО снижала спонтанный апоптоз нейтрофилов, а раствор хлорида лития в концентрации 3,0 или 9,0 мМ, добавленный в среду инкубации, увеличивал способность нейтрофилов к спонтанному апоптозу. Заключение. Хлорид лития оказывал противовоспалительный эффект снижал дегрануляцию и активацию нйтрофилов посредством уменьшения уровня экспрессии молекул CD11b и CD66b на поверхности нейтрофилов, которые предварительно были активированы сыворотками пациентов с ССВО. В концентрации 3,0 мМ и выше хлорид лития индуцировал спонтанный апоптоз нейтрофилов, активированных сыворотками пациентов с ССВО после операций на сердце с ИК. The aim of this work was to study the anti-inflammatory effect of lithium chloride on human neutrophils in vitro under the action of the serum of patients with systemic inflammatory response syndrome (SIRS), which developed after on-pump cardiac surgery. Methods. The study was performed on neutrophils isolated from the blood of five healthy donors, which was activated using serum from patients with SIRS. Neutrophil activity was assessed using fluorescent antibodies to CD11b and CD66b degranulation markers. The level of apoptosis and necrosis of human neutrophils was evaluated 22 hours after isolation. Quantification was performed using annexin V and propidium iodide on a flow cytometer. Intact and activated neutrophils were treated with 0.3, 3.0 аnd 9.0 mM lithium chlorides. Results. Incubation of neutrophils with the blood serum of patients with SIRS after on-pump cardiac surgery increased the expression of CD11b by 1.5 times and the expression of CD66b by 1.4 times compared to expression on intact neutrophils. Incubation of neutrophils with blood serum of patients with SIRS and 3.0 and 9.0 mM lithium chloride solutions led to a statistically significant decrease in the level of expression of CD11b CD66b on the surface of neutrophils in comparison with control activated neutrophils. It was found that 3.0 and 9.0 mM lithium chloride solutions returned the expression levels of CD11b and CD66b on activated neutrophils to the expression level on intact neutrophils. 0.3 mM of lithium chloride, used during incubation with activated neutrophils, did not cause a significant decrease in the expression of CD11b and CD66b relative to control activated neutrophils. The expression of CD11b and CD66b on activated neutrophils during their incubation with 0.3 mM of lithium chloride was significantly higher relative to the expression of these molecules on intact neutrophils. The serum of patients with advanced SIRS decreased the ability of neutrophils to spontaneous apoptosis. 3.0 or 9.0 mM lithium chloride solutions added to the incubation medium increased the ability of neutrophils to spontaneous apoptosis. Conclusion. Lithium chloride reduced the degranulation and activation of neutrophils by reducing the expression level of CD11b and CD66b molecules on the surface of neutrophils that were previously activated by the serum of patients with SIRS. This effect determines the anti-inflammatory influence of lithium chloride. Lithium chloride at 3.0 mM and higher induced spontaneous apoptosis of neutrophils activated by the serum of patients with SIRS after on-pump cardiac surgery.

The Effect of Lithium Chloride on Neutrophil Activation on Exposure to Serum of Patients with Septic Shock

The aim of the study: to examine the anti-inflammatory effect of lithium chloride by exposing the human neutrophils to serum of patients with septic shockin vitro.Material and methods. The study was carried out on neutrophils extracted from the blood of 6 healthy donors, which were activated with serum from patients with septic shock. The neutrophil activity was evaluated with fluorescent antibodies to the CD11b and CD66b markers of degranulation. The level of human neutrophil apoptosis and necrosis was assessed 22 hours after extraction; quantitative assessment was made using annexin V and propidium iodide with flow cytofluorimetry. Intact and activated neutrophils were treated with 0.3, 3.0 and 9.0 mmol lithium chloride solution.Results. The level of CD11b expression on the surface of intact neutrophils (healthy donors) was 3434.50 [3311.0-3799.0] arbitrary fluorescence units (AFU). Incubation of neutrophils with serum of patients with septic shock increased CD11b expression 2.5 times to 8589.0 [7279.0-11258.0] AFU (P=0.005) vs intact leukocytes, and increased CD66b expression 2.7 times up to 27 600.0 [22 999.0-28 989.0] AFU ((P=0.005) vs intact neutrophils. Lithium chloride in concentrations of 0.3, 3.0 and 9.0 mmol in a dose-dependent manner reduced the level of expression of CD11b and CD66b molecules on the surface of activated neutrophils. Septic serum reduced spontaneous neutrophil apoptosis, and 3.0 mmol and higher lithium chloride solution induced spontaneous neutrophil apoptosis.Conclusion. Lithium chloride reduces the activation of neutrophils preactivated by serum of patients with septic shock, reduces expression of CD11b and CD66b molecules on the neutrophil surface, inhibiting the process of their activation (degranulation). Lithium chloride in concentration of 3.0 mmol and higher is able to induce spontaneous apoptosis of neutrophils activated by serum of patients with septic shock.