Granulocyte Research Articles

A Prospective Randomized Interventional Study to Establish the Role of Bovine Lactoferrin in Helicobacter Pylori Infection

Helicobacter pylori (H. pylori) are gram-negative spiral bacterium that surrounds itself in the epithelial surface of the stomach. It is found to be the most common bacterial infection affecting the population worldwide approximately 50%. The elimination of H. pylori is not only an important component in the healing of peptic ulcers, but also to decreases their recurrence and reduces the rate of gastric carcinoma. Eradication of H. pylori is currently recommended in numerous guidelines for the treatment of all these associated diseases. In the last two decades, attention has focused on the potential role of bovine lactoferrin (bLf) in the therapy of various gut disorders. bLf, a multifunctional iron-binding glycoprotein that is found in the milk, mucosal secretions (e.g., saliva, tears, bile), pancreatic and seminal fluids, and specific granules of the polymorphonuclear leukocytes in humans. Proton-pumpbased triple therapy (PpTT) is the most commonly accepted H. pylori treatment, which consists of a twice-daily proton pump inhibitor (PPI) such as esomeprazole or pantoprazole or omeprazole; clarithromycin or amoxicillin with tinidazole for 7-14 days. The aim of our study was to evaluate the efficacy of Bovine lactoferrin, administered orally along with triple therapy on H. pylori colonization in humans by a prospective randomized controlled study. 75 Patients with H. pylori positive were recruited and treated as per the protocol for 2 weeks. The subjects were randomized into 2 groups based on inclusion and exclusion criteria, interventional group received bovine lactoferrin at a dose of 100mg twice daily along with triple therapy for 14 days and the control group received, triple therapy for the same period of 14 days. The reduction in symptoms were analyzed by using a set of questionnaire and scored by using Global Overall Symptom Scale (GOS) both prior and post treatment. The eradication of H. pylori was confirmed by (13c) Urea breath test (UBT) that has shown the eradication rate of 76.2% in interventional group and 61.1% in control group. This study results had indicated that the group received bovine lactoferrin had shown significant improvement in the H. pylori eradication. This result can be used to conclude that the role of bovine lactoferrin in the reduction of symptoms and eradication of H. pylori infection by focusing on the relatively high cure rate, good compliance to the treatment and higher quality of life.

A new acyl derivative of sulfadimethoxine inhibits phagocyte oxidative burst and ameliorates inflammation in a mice model of zymosan-induced generalised inflammation.

Chronic inflammation and oxidative stress play a pivotal role in the pathophysiology of most challenging illnesses, including cancer, Alzheimer's, cardiovascular and autoimmune diseases. The present study aimed to investigate the anti-inflammatory potential of a new sulfadimethoxine derivative N-(4-(N-(2,6-dimethoxypyrimidin-4-yl) sulfamoyl) phenyl) dodecanamide (MHH-II-32). The compound was characterised by applying 1H-, 13C-NMR, EI-MS and HRFAB-MS spectroscopic techniques. The compound inhibited zymosan-induced oxidative bursts from whole blood phagocytes and isolated polymorphonuclear cells with an IC50 value of (2.5 ± 0.4 and 3.4 ± 0.3µg/mL), respectively. Furthermore, the inhibition of nitric oxide with an IC50 (3.6 ± 2.2µg/mL) from lipopolysaccharide-induced J774.2 macrophages indicates its in vitro anti-inflammatory efficacy. The compound did not show toxicity towards normal fibroblast cells. The observational findings, gross anatomical analysis of visceral organs and serological tests revealed the non-toxicity of the compound at the highest tested intraperitoneal (IP) dose of 100mg/kg in acute toxicological studies in Balb/c mice. The compound treatment (100mg/kg) (SC) significantly (P < 0.001) downregulated the mRNA expression of inflammatory markers TNF-α, IL-1β, IL-2, IL-13, and NF-κB, which were elevated in zymosan-induced generalised inflammation (IP) in Balb/c mice while upregulated the expression of anti-inflammatory cytokine IL-10, which was reduced in zymosan-treated mice. No suppressive effect was observed at the dose of 25mg/kg. Ibuprofen was taken as a standard drug. The results revealed that the new acyl derivative of sulfadimethoxine has an immunomodulatory effect against generalised inflammatory response with non-toxicity both in vitro and in vivo, and has therapeutic potential for various chronic inflammatory illnesses.

Systematically analysis of decompensated cirrhotic patients with spontaneous bacterial peritonitis to identify diagnostic and prognostic indexes

BackgroundSpontaneous bacterial peritonitis (SBP) is a common complication in patients with cirrhosis. The diagnosis of SBP is still mostly based on ascites cultures and absolute ascites polymorphonuclear (PMN) cell count, which restricts the widely application in clinical settings. This study aimed to identify reliable and easy-to-use biomarkers for both diagnosis and prognosis of cirrhotic patients with SBP.MethodsWe conducted a retrospective study including 413 cirrhotic patients from March 2013 to July 2022 in the First Affiliated Hospital of Guangxi Medical University. Patients’ clinical characteristics and laboratory indices were collected and analyzed. Two machine learning methods (Xgboost and LASSO algorithms) and a logistic regression analysis were adopted to screen and validate the indices associated with the risk of SBP. A predictive model was constructed and validated using the estimated area under curve (AUC). The indices related to the survival of cirrhotic patients were also analyzed.ResultsA total of 413 cirrhotic patients were enrolled in the study, of whom 329 were decompensated and 84 were compensated. 52 patients complicated and patients with SBP had a poorer Child–Pugh score (P < 0.05). Patients with SBP had a greater proportion of malignancies than those without SBP(P < 0.05). The majority of laboratory test indicators differed significantly between patients with and without SBP (P < 0.05). Albumin, neutrophil-to-lymphocyte ratio (NLR), and ferritin-to-neutrophil ratio (FNR) were found to be independently associated with SBP in decompensated cirrhotic patients using LASSO algorithms, and logistic regression analysis. The model established by the three indices showed a high predictive value with an AUC of 0.808. Furthermore, increased neutrophils, ALP, and C-reactive protein-to-albumin ratio (CAR) were associated with the shorter survival time of patients with decompensated cirrhosis, and the combination of these indices showed a greater predictive value for cirrhotic patients.ConclusionsThe present study identified FNR as a novel index in the diagnosis of SBP in decompensated patients with cirrhosis. A model based on neutrophils, ALP and CAR showed high performance in predicting the prognosis of patients with decompensated cirrhosis.

CD20 + cells blockage by rituximab delays wound healing in oral traumatic ulcers in rats

ObjectiveWound healing of oral traumatic ulcers (OTU) is strongly associated with cytokines and inflammatory cells, and the reduction of anti-inflammatory cells, such as lymphocyte B, may interfere with OTU repair. We aimed to evaluate the role of CD20 + cells in the healing process of OTU in rats. DesignWistar male rats were divided into four groups: a control group (treated with 0.1 mL/kg of saline) and three groups treated with anti-CD20 rituximab (RTX) at 2.5, 10, or 40 mg/kg 24 h before OTU production. The animals were weighed (day 0) and euthanized on days 3, 7, 14, and 21 after ulceration. With Blood cells (hematological analysis) and the traumatically induced ulcers were clinically measured. The mucosal samples were histologically (scores 0–4), histochemically (collagen assay (picrosirius)), histomorphometrically (cell counting), and immunohistochemically (CD20+, Tumor Necrosis Factor alpha(TNF-α), Interleukin(IL)− 1β, IL-6 and α-smooth-muscle-actin (α-SMA)) analyzed. ANOVA-1–2-way/Bonferroni, Kruskal-Wallis/Dunn, and correlation analyses were performed (GraphPad Prism 5.0, p < 0.05). ResultsRTX leads to leukopenia, lymphocytopenia, and neutropenia (p < 0.001), and high doses reduced the OTU area (p = 0.001), impaired histologic scores (p < 0.05), and delayed polymorphonuclear (p < 0.001) and mononuclear (p < 0.001) cells, and total (p = 0.011), type-I (p = 0.008), and type-III (p = 0.021) collagen. ConclusionRTX treatment reduced CD20+ cells in OTU (p = 0.001), TNF-α (p = 0.006), and α-SMA (p = 0.022) immunostaining and delayed IL-6 reduction (p = 0.006), with no influence in IL-1β immunostaining. CD20 + cell blockage by RTX reduced cell migration, acute inflammation, and wound healing in OTU.

Kidney Transplant Recipients Show Limited Lung Diffusion Capacity but Similar Hydrogen Peroxide Exhalation as Healthy Matched Volunteers: A Pilot Study.

Patients with end-stage chronic kidney disease show higher systemic oxidative stress and exhale more hydrogen peroxide (H2O2) than healthy controls. Kidney transplantation reduces oxidative stress and H2O2 production by blood polymorphonuclear leukocytes (PMNs). Kidney transplant recipients (KTRs) may be predisposed to an impairment of lung diffusing capacity due to chronic inflammation. Lung function and H2O2 concentration in the exhaled breath condensate (EBC) were compared in 20 KTRs with stable allograft function to 20 healthy matched controls. Serum interleukin eight (IL-8) and C-reactive protein (CRP), blood cell counts, and spirometry parameters did not differ between groups. However, KTRs showed lower total lung diffusing capacity for carbon monoxide, corrected for hemoglobin concentration (TLCOc), in comparison to healthy controls (92.1 ± 11.5% vs. 102.3 ± 11.9% of predicted, p = 0.009), but similar EBC H2O2 concentration (1.63 ± 0.52 vs. 1.77 ± 0.50 µmol/L, p = 0.30). The modality of pre-transplant renal replacement therapy had no effect on TLCOc and EBC H2O2. TLCOc did not correlate with time after transplantation. In this study, TLCOc was less reduced in KTRs in comparison to previous reports. We suggest this fact and the non-elevated H2O2 exhalation exhibited by KTRs, may result perhaps from the evolution of the immunosuppressive therapy.

Primary Spontaneous Splenic Rupture: A Rare and Life-Threatening Condition

This case report outlines the presentation, diagnosis, and management of a 26-year-old male with primary spontaneous splenic rupture, a highly uncommon clinical condition. Primary spontaneous splenic rupture necessitates a high index of suspicion for accurate diagnosis. Diagnostic Imaging, particularly abdominal CT scans, is pivotal in its detection. This case underscores the critical importance of timely recognition and intervention. Clinicians should consider various inflammatory, neoplastic, and infectious etiologies in the differential diagnosis of splenic rupture. Physical examination revealed tenderness and guarding, predominantly in the left upper quadrant and left flank. Laboratory findings indicated an elevated white blood cell count, predominantly polymorphonuclear leukocytes. Contrast-enhanced CT scan showed substantial free fluid in the acute abdomen, with high density observed around the liver and spleen. Exploratory laparotomy confirmed hemoperitoneum and spontaneous splenic rupture. This case report highlights the rarity of primary spontaneous splenic rupture and emphasises the significance of early detection and intervention. Through examination and advanced imaging techniques, clinicians can accurately diagnose and promptly manage this life-threatening condition.

The Response of Paracoccidioides lutzii to the Interaction with Human Neutrophils.

The fungal pathogen Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.

Correlations of Long COVID Symptoms and Inflammatory Markers of Complete Blood Count (CBC): A Cross-sectional Study.

Long-COVID refers to lasting unspecific symptoms like fatigue, decreased concentration and sleep issues after infection which persist for at least three months and cannot be attributed to other causes. Previous studies surveyed the association between inflammatory markers like C - reactive protein (CRP) at hospital admission and long-COVID symptoms in the preceding months. Post-COVID syndrome can affect one-third of patients. Thus early diagnosis can assist in reducing burdens on public health. We attempted to see any correlations between complete blood count (CBC) markers (like red blood cell (RBC), white blood cell (WBC), Neutrophil to lymphocyte ratio (NLR), etc.) at hospital admission and long COVID symptoms at a 6-month follow-up. 167 patients (44.9% females, mean age 49 years old) answered semi-structural interviews through telemedicine which focused on the three prominent symptoms: fatigue, loss of concentration and decreased libido. Results: Two third of patients have symptoms of long COVID and others do not have. NLR in the symptomatic group was statically higher. Patients who underwent decreased libido at a 6-month follow-up had significantly more severe lymphopenia (p = 0.028) and higher NLR values (p-value = 0.007). Poor mental concentration is associated with high WBC in numbers and polymorphonuclear (PMN) count. Other symptoms do not correlate with blood markers. Utilizing available data like CBC can help predict the upcoming symptoms of previously hospitalized patients and further measures like rehabilitation. Additional investigations should be done on the effect of COVID vaccination on converting long COVID. Different variants of the virus may have different results.

Spatial Dissection of the Bone Marrow Microenvironment in Multiple Myeloma By High Dimensional Multiplex Tissue Imaging

Introduction Multiple myeloma (MM) is shaped by interactions between immune, stromal and tumor cells in the bone marrow (BM) microenvironment (BMME). Our understanding of these processes is based on high parametric flow cytometry and single cell transcriptomics, which lack spatial resolution. Thus, crucial aspects of the in situ tumor ecosystem, including cell-cell interactions, can only be predicted. Novel high dimensional, multiplex tissue imaging methods are able to capture similar granularity while also preserving spatial organization. By optimizing antibody selection, as well as staining, imaging and data processing protocols we have established a BMME-targeted CODEX workflow based on a 60 marker panel. Here, we conducted the first comprehensive multiplex protein imaging survey of the BMME across the spectrum of MM and precursor stages and leveraged paired FISH and whole genome sequencing (WGS) to elucidate the interplay of tumor intrinsic features and the BMME. Methods We imaged 489 BM trephine biopsies in tissue microarray format on the CODEX (Akoya Biosciences) platform. After quality control (≥750 cells per core, &amp;lt;25% unclassifiable cells) we retained data from 12 non-malignant controls, 13 MGUS, 20 smoldering MM, 349 uniformly treated newly diagnosed MM (NDMM) patients from a phase III clinical trial, as well as 22 relapsed MM samples with a median of 3 (range 1-6) tissue cores per sample, comprising a total of 3.1 million single cells. CODEX data was processed and analyzed using custom scripts. Cellular neighborhoods were computed by clustering the cell type composition of sliding windows (n=10 nearest neighboring cells) across the tissue. WGS (n=212) and FISH (n=309) were performed on CD138-purified BM mononuclear cells and analyzed using in-house pipelines. Results We identified 40 cell types, encompassing the relevant lineages (myeloid, lymphoid, stromal, endothelial, tumor), subtypes (e.g. T, B, NK cells) and functional states (e.g GRZB, Ki67, and PD1) of the BMME. The degree of plasma cell infiltration determined by CODEX correlated with the clinical pathologist's quantification based on classical immunohistology (r=0.77, p&amp;lt;0.0001), and the kappa/lambda status of MM cells matched the reported light chain restriction, supporting the validity of our data. At the compositional level, we observed shifts in relative cell type abundance from precursor to MM stages. Major changes in the BMME included enriched stromal, endothelial and CD8+ T cells as well as depleted polymorphonuclear (PMN), erythropoietic and CD4+ T cells in advanced stages. The highest proportion of monocytoid dendritic cells, monocytes, mast cells and cytotoxic CD8+ T cells was seen in relapsed MM. Leveraging the spatial resolution of the CODEX data, we identified multiple distinct cellular neighborhoods (CNs) defined by recurrent local cell type composition. CNs enriched for plasma cells in combination with different immune cell subsets including exhausted T cells increased towards the MM stage, while CNs dominated by PMNs, erythropoietic cells and CD206 negative macrophages decreased. To investigate links between MM subtypes and BMME patterns, we correlated CODEX data with MM-initiating and driver events. While patients with t(4;14) showed a significant increase in eosinophils, patients with gain1q had higher levels of endothelial and stromal subsets colocalizing in the same CN. Distinct types of stromal cells were also enriched in patients with double hits (≥2 FISH high risk features). RAS mutations and biallelic inactivation of tumor suppressor genes were associated with altered T cell subset composition. Further corroborating the importance of BMME architecture, a CN with colocalization of plasma and exhausted CD8 T cells was associated with unfavorable prognosis in NDMM patients. Conclusions High dimensional, multiplex tissue imaging of the BMME enables interrogation of cellular interactions and tissue architecture in situ in clinical samples at single cell resolution. To our knowledge this is the first highly multiplexed spatial study of the human BMME in a large clinical cohort and the first such study in multiple myeloma. We track characteristic shifts in tissue composition associated with disease progression, reveal genotype-phenotype associations in the BMME as a correlate of tumor-microenvironment co-evolution and detect prognostically relevant tissue architectural features.

Impact of gold nanoparticles (AuNPs) in human neutrophils in vitro and in leukocytes attraction in vivo: A sex-based analysis

Some differences exist between the male and female immune systems. Despite this, a sex-based analysis is not frequently performed in most studies. Knowing that inflammation is a common undesired effect observed resulting from nanoparticle (NP) exposure, we investigate here how gold NPs with a primary size of 20 (AuNP20) and 70 nm (AuNP70) will alter the biology of polymorphonuclear neutrophil cells (PMNs) isolated from men and women as well as their potential pro-inflammatory effect in vivo in male and female mice. We found that AuNP20 significantly delay apoptosis only in PMN isolated from men. The production of interleukin (IL)− 8 by PMNs was increased by both AuNPs regardless of sex although significance was only observed in AuNP20-induced PMNs. Using the murine air pouch model of inflammation, AuNPs did not induce a neutrophilic infiltration regardless of sex. In conclusion, AuNPs could differently alter the biology of PMNs according to sex.

Diagnostic evaluation of leukocyte esterase reagent strip in bedside rapid diagnosis of spontaneous bacterial peritonitis in cirrhotic patients

Background: Spontaneous bacterial peritonitis (SBP) is the most common and serious infection of liver cirrhosis patients with ascites. If not intervened with early antibiotic treatment leads to high morbidity and mortality. Thus, early diagnosis and treatment of SBP are needed for survival. Leukocyte esterase reagent can aid in early diagnosis. Aims and Objectives: This study aimed to assess the diagnostic accuracy of the leukocyte esterase dipstick test for the diagnosis of SBP in resource poor settings. Materials and Methods: This cross-sectional study was conducted during August 2018–January 2019 on patients with cirrhotic liver disease and ascites in a tertiary care medical college hospital, Puducherry. All patients underwent abdominal paracentesis, and ascitic fluid was subjected to cell counts, biochemistry tests, culture, and leukocyte esterase test (Multistrix SG8- Seimens) at 120 s and graded with five levels. Grade 3 was positive (125 polymorphonuclear leukocytes [PMNL]/mL) and grade 4 (&lt;500/mL), which exceeds the threshold for SBP (&lt;250 PMNL/mL). Sensitivity, specificity, positive, and negative predictive value (PPV and NPV) was calculated. Results: A total of 50 ascitic fluids were analyzed. Nine out of 50 (18%), 16 (32%), and 4 (8%) were positive for cell count method, Leucocyte esterase reagent (LER) test and culture, respectively. The sensitivity, specificity, PPV, NPV, and diagnostic accuracy for LER test were 6.25%, 91.18%, 25%, 67.39%, and 64%, respectively. Conclusion: The LER strip test is useful for SBP diagnosis in an emergency and resource poor setting. It provides good diagnostic accuracy and high NPV and it is cost effective and better bedside tool for SBP diagnosis.

How Reliable Is the Absolute Synovial Polymorphonuclear Neutrophil Cell Count in Diagnosing Periprosthetic Joint Infection?

BackgroundDiagnosing periprosthetic joint infection (PJI) is a daunting task for even the most experienced orthopedic surgeons, as there is currently no test available that can provide absolute accuracy. Utilizing an established synovial indicator for detecting PJI without incurring additional costs or resources would be the optimal solution for predicting the presence of infection. Therefore, we hypothesized that synovial absolute neutrophil count (ANC) would improve the diagnostic accuracy of chronic knee and hip PJI. MethodsThe study included 260 patients (134 men and 126 women, mean age of 70 years [range, 26 to 89]) who underwent aspiration during preoperative workup. Of these, 109 patients (41.9%) were diagnosed with chronic PJI (50 knees, 59 hips), and 151 patients (58.1%) were diagnosed as aseptic (94 knees, 57 hips). Data obtained from all patients included age, sex, procedure type (total hip or total knee arthroplasty), operation side, synovial white blood cell count (cells/μL), synovial polymorphonuclear cells percentage, and synovial α-defensin immunoassay value at the admission were retrieved from the electronic medical record. ResultsThe calculated optimal threshold for synovial ANC of 1,415.5 cells/μL was associated with an area under the receiver operating characteristic curve (AUC) of 0.930 for chronic knee PJI diagnosis. The calculated optimal threshold for synovial ANC of 2,247 cells/μL was associated with an AUC of 0.905 for chronic hip PJI diagnosis. ConclusionsThis study has conclusively shown that the synovial ANC serves as a valuable marker in the complicated diagnosis of PJI. This highly effective and efficient approach should be utilized for obtaining further information through standard tests, thereby ruling out the possibility of PJI. Level of evidenceIII.

Exploring uterine inflammation in postpartum primiparous precocious and conventional and multiparous Bos indicus beef cows.

This study aimed to compare the postpartum uterine dynamics of primiparous precocious (PP), primiparous conventional (PC) and multiparous conventional (MC) Bos indicus beef cows. For this purpose, PP (n = 8), PC (n = 18) and MC (n = 12) cows were enrolled in this study. These cows were evaluated at 20 and 10 days prepartum and weekly from parturition to 42 days postpartum (DPP). During this period, body weight (BW), subcutaneous fat thickness (SFT) and serum concentrations of glucose, β-hydroxybutyrate, albumin and haptoglobin were measured. Proportion of polymorphonuclear (PMN) cells, and abundance of mRNA transcripts of genes involved in uterine inflammation and uterine health were evaluated. The PP cows had lower (p < .05) BW and SFT than that for PC and MC cows during the study period. The serum concentration of albumin after 35 DPP was lower (p < .05) in PP cows. The PP cows had the highest proportion of PMN on 28 and 35 DPP compared to PC and MC cows. The relative mRNA abundance of IL-1β and IL-8 increased after 21 DPP in PP cows compared to the other groups. The PC had the highest, MC had an intermediate, and PP cows had the lowest relative abundance of IL10 mRNA. Overall, these findings indicated that uterine inflammation was more pronounced in PP cows. Moreover, based on the proportion of PMN and abundance of transcripts associated with inflammation in the uterus, PP cows may require a longer period to recover their uterine health after calving.

A new histone deacetylase inhibitor remodels the tumor microenvironment by deletion of polymorphonuclear myeloid-derived suppressor cells and sensitizes prostate cancer to immunotherapy

BackgroundProstate cancer (PCa) is the most common malignancy diagnosed in men. Immune checkpoint blockade (ICB) alone showed disappointing results in PCa. It is partly due to the formation of immunosuppressive tumor microenvironment (TME) could not be reversed effectively by ICB alone.MethodsWe used PCa cell lines to evaluate the combined effects of CN133 and anti-PD-1 in the subcutaneous and osseous PCa mice models, as well as the underlying mechanisms.ResultsWe found that CN133 could reduce the infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), and CN133 combination with anti-PD-1 could augment antitumor effects in the subcutaneous PCa of allograft models. However, anti-PD-1 combination with CN133 failed to elicit an anti-tumor response to the bone metastatic PCa mice. Mechanistically, CN133 could inhibit the infiltration of PMN-MDSCs in the TME of soft tissues by downregulation gene expression of PMN-MDSC recruitment but not change the gene expression involved in PMN-MDSC activation in the CN133 and anti-PD-1 co-treatment group relative to the anti-PD-1 alone in the bone metastatic mice model.ConclusionsTaken together, our work firstly demonstrated that combination of CN133 with anti-PD-1 therapy may increase the therapeutic efficacy to PCa by reactivation of the positive immune microenvironment in the TME of soft tissue PCa.

Correlations between different protein species of oral rinse MMP-8 and patient-related factors.

The aim of this study is to examine correlations between different oral rinse matrix metalloproteinase (MMP)-8 protein species in western blot (WB) analysis, quantitative MMP-8 measurements, and patient-related factors. Elevated activated MMP-8 (aMMP-8) associate with periodontitis and a diagnostic point-of-care technology has been developed based on aMMP-8. In WB, different MMP-8 protein species can be analyzed. Relative abundancyof fragmented 20-25 kDa forms in WB has been associated with and reflects MMP-8 activation and related fragmentation and elevated quantitative aMMP-8 measurements. A random sample of 192 participants from a periodontal disease screening study was used for this study. Oral rinse samples for biomarker analyses were collected before clinical periodontal examinations. aMMP-8 immunofluorometric (IFMA) and WB analysis (utilizing the same monoclonal antibody, 8708), polymorphonuclear leukocyte (PMN) elastase activity test and tissue inhibitor of metalloproteinases (TIMP)-1 ELISA levels were performed from the oral rinse samples. Distinct MMP-8 protein species were differentiated in the WB analysis. Principal component (PC) analysis was conducted to explore correlation patterns between the different species. Adjusted correlation analysis between the extracted PCs of WB and aMMP-8 IFMA levels and multilevel regression analysis were conducted to explore if the other periodontal disease-related biomarkers and clinical surrogate measures and patient-related factors are co-variating with the extracted components. Distinct correlation patterns between the MMP-8 protein species were observed. The first four PCs explained 89% of the whole variance in PC analysis. Statistically significant correlation (p < 0.05) were observed as follows: PC1 positively with 21 kDa (r = .69) and 25 kDa fragments (r = .55) and negatively with 150 kDa complexes (r = -.46). PC2 correlated with 45 (r = .70) and 55 kDa (r = .65) activated forms, PC3 with 70-80 kDa latent proforms (r = .63) and 90-100 kDa complexes (r = .67), and PC4 with 35 kDa fragments (r = .81). There were significant correlations between quantitative (IFMA) aMMP-8 measurements and PC1 (p < 0.001), PC2 (<0.05) and PC3 (<0.05) but not with PC4. In multilevel regression models age, PMN elastase activity, TIMP-1 levels, and a number of 4-5 mm periodontal pockets were associated with PC1, nonsmoking with PC2, ageand PMN elastase activity with PC3, and age and smoking with PC4. Relative abundancy of fragmented 21-25 kDa protein species was correlated with the quantitative aMMP-8 (IFMA) measurements, which is in line with previous results. Different patient-related factors (smoking, age, proteolytic activity) may modify the formation of different MMP-8 protein species in oral rinse samples and may cause variability in quantitative aMMP-8 measurement.

Efficacy of Transdermal Delivery Nano Ethosomal Gel from Ashitaba Leaves on In-vivo Burn Wound Healing in Albino Rats

This study aimed to evaluate the in-vivo burn wound healing process in albino rats treated using transdermal delivery nano ethosomal gel from ashitaba leaves. Ethosomal vesicles were formulated using soy lecithin, cholesterol, ethanol, water, and ashitaba leaf nanoparticles using the cold method. A total of 25 male rats were randomly divided into 5 groups, i.e., (C-) treated without nanoparticle extract, (C+) treated using 1% Silver Sulfadiazine®, treatment group treated nano ethosomal gel from ashitaba leaves with the respective doses were (T1) 1%, (T2) 2,5%, and (T3) 5%. Therapy was initiated on day 1 or after being induced by a burn wound for 14 days, twice a day. On the 15th day, a termination was carried out to take skin tissue from burn scars, then histopathology preparations were made with routine staining. Microscopic observations with an optical microscope on collagen, polymorphonuclear cell infiltration, angiogenesis, and re-epithelialization. The T3 group that was treated using 5% nano ethosomal gel showed the best burn wound healing, this may be caused by compounds in ashitaba leaf nanoparticles which have antioxidant, anti-inflammatory, and antibacterial effects, thus the use of transdermal delivery therapy of 5% ashitaba leaf nano ethosomal gel was effective for wound treatment burn on rat skin.

GPC3-IL7-CCL19-CAR-T primes immune microenvironment reconstitution for hepatocellular carcinoma therapy.

Chimeric antigen receptor (CAR)-T-cell therapy is a revolutionary treatment that has become a mainstay of advanced cancer treatment. Conventional glypican-3 (GPC3)-CAR-T cells have not produced ideal clinical outcomes in advanced hepatocellular carcinoma (HCC), and the mechanism is unclear. This study aims to investigate the clinical utility of novel GPC3-7-19-CAR-T cells constructed by our team and to explore the mechanisms underlying their antitumor effects. We engineered a novel GPC3-targeting CAR including an anti-GPC3 scFv, CD3ζ, CD28 and 4-1BB that induces co-expression of IL-7 at a moderate level (500pg/mL) and CCL19 at a high level (15000pg /mL) and transduced it into human T cells. In vitro, cell killing efficacy was validated by the xCELLigence RTCA system, LDH nonradioactive cytotoxicity assay and was confirmed in primary HCC organoid models employing a 3D microfluid chip. In vivo, the antitumor capacity was assessed in a humanized NSG mouse xenograft model. Finally, we initiated a phase I clinical trial to evaluate the safety and effect of GPC3-7-19-CAR-T cells in the clinic. GPC3-7-19-CAR-T cells had 1.5-2 times higher killing efficiency than GPC3-CAR-T cells. The tumor formation rates in GPC3-7-19-CAR-T cells treated model were reduced (3/5vs.5/5), and the average tumor volumes were 0.74 cm3 ± 1.17 vs. 0.34 cm3 ± 0.25. Of note, increased proportion of CD4+ TEM and CD8+ TCM cells was infiltrated in GPC3-7-19-CAR-T cells group. GPC3-7-19-CAR-T cells obviously reversed the immunosuppressive tumor microenvironment (TME) by reducing polymorphonuclear (PMN)-myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells infiltration and recruiting more dendritic cells (DCs) to HCC xenograft tumor tissues. In one patient with advanced HCC, GPC3-7-19-CAR-T-cell treatment resulted in tumor reduction 56days after intravenous infusion. In conclusion, GPC3-7-19-CAR-T cells achieved antitumor effects superior to those of conventional GPC3-CAR-T cells by reconstructing the TME induced by the dominant CD4+ TEM and CD8+ TCM cell subsets. Most importantly, GPC3-7-19-CAR-T cells exhibited good safety and antitumor efficacy in HCC patients in the clinic. ► Novel GPC3-7-19-CAR-T cells designed with mediate level of IL-7 secretion and high level of CCL19 secretion, which could recruit more mature DCs to assist killing on GPC3+HCCs. ►DC cells recruited by CCL19 could interact with CD4+ T cells and promote the differentiation of CD4+TEFF cells into CD4+TEM and CD8+TCM subsets, leading a better anti-tumor effect on GPC3+HCCs. ►Compared with conventional GPC3-CAR-T, GPC3-7-CCL19-CAR-T cells could reverse tumor immunosuppressive microenvironment by reducing PMN-MDSC and Treg cell infiltration.

Delta Neutrophil Index as a New Marker of Purulent Inflammation in Men With Non-odontogenic Abscesses of the Neck.

Introduction If non-odontogenic abscesses and phlegmons (all purulent inflammations where the etiology is not a diseased tooth) of the neck are not promptly treated, they can lead to serious complications and even end in the death of the affected patient. Classical markers of inflammation such as plasma concentration of leukocytes (WBC), neutrophils (Neu), C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) are elevated in inflammatory neck diseases, but none of them has been proven as a definite marker in the prediction of this type of pathology. This necessitates the search and analysis of new indicators that could be used in the diagnosis, follow-up, and prognosis of patients with purulent neck infections. Potentially, such a marker could be the delta neutrophil index (DNI), which is increasingly entering clinical practice as a prognostic indicator in critically ill patients with life-threatening illnesses, including sepsis and systemic inflammatory response syndrome (SIRS). In the world literature, there are no data that have been studied in patients with purulent diseases of the neck of non-odontogenic origin, which is the aim of this original article. Materials and methods This retrospective study included 40 men with an average age of 46 (18-87) years with non-odontogenic abscesses and phlegmons of the neck who were hospitalized and operated on. In all of them, the concentration of leukocytes, eosinophils (Eo), neutrophils, and mature polymorphonuclear neutrophil leukocytes (PMN) was examined on an automatic 5-Diff hematology analyzer, ADVIA 2120i (SiemensMedical Solutions USA, Malvern, PA). Thus, it was calculated according to the formula for calculating DNI (DNI% = (Neu%+Eo%) - PMN%). Retrospectively, 30 healthy men with an average age of 42 (18-81) years were used as a control group, in which the same indicators were examined during a preventive examination. Results and discussion Comparing the mean values of WBC, Neu, and DNI between the studied patients with non-odontogenic purulent neck infections (n=40) and the healthy male controls (n=30) it was found that all three indicators of inflammation are significantly higher in the ill men, and these differences are statistically significant (p<0.05): 10.19 ±2.68x103/L versus 7.37 ±1.93x103/L for leukocytes, 7.68 ±2, 76x103/L versus 4.13 ±1.48x103/L for neutrophils, and 1.11±0.83% versus -1.07±1.22% for DNI. Therefore, the high mean numbers of measured WBC and Neu in men with non-odontogenic purulent neck infections were associated with an increase in their mean calculated DNI. This gives us reason to think that while WBC and Neualone are not sufficient for definitive diagnosis, treatment follow-up, and prediction of disease outcome, in combination with DNI they become reliable indicators in purulent neck infections. Conclusions The DNI correlates well with other well-known and established indicators of inflammation, such as the concentration of leukocytes and neutrophils in the peripheral blood of patients. Its calculation is fast as an implementation procedure and is economically beneficial. Its independent use in the diagnosis and treatment of these diseases is about to be investigated and analyzed.

Therapeutic management of non-infectious repeat breeder buffaloes by using phytomedicine

The goal of the current study was to assess the efficacy of powder Harbominvit in non-infectious repeat breeder buffaloes with a selective test such as white side test, cervical pH, PMNs cell count, Fern patten and estimation of serum phosphorous. 24 buffaloes that tested negative for white side test was chosen for the current study out of a total of 51 buffaloes that underwent the test from Nanded and Parbhani districts of Marathwada region in Maharashtra under infertility camps organized by NDDB, Mother dairy, and Collage of animal and veterinary science Parbhani. MAFSU. In the Treatment group at day 0 before treatment repeat breeder buffaloes showed a Mean ± SE cervical pH value was 7.38±0.07 while the Control group showed a Mean ± SE cervical pH value was 7.59±0.08. The white side test was 100% negative for both groups. The mean ± SE value for PMN cell count for the Treatment group is 2.67±0.31 while the Control group was 2.08±0.22. The mean ± SE value for serum phosphorus value for the Treatment group before treatment is 3.81a±0.11 and after treatment is 4.937a±0.18 while the Control group shows before 3.68a±0.09 and after treatment 4.17a±0.20 and mean ± SE value for Fern pattern for Treatment group and Control group was 0.750±0.13 and 0.66±0.14 respectively. The conception rates in buffaloes of Group 1 with the treatment of powder Harbominvit and Group 2 with no treatment serve as control were 75% (09/12), and 41.66 % (05/12) respectively (Table 2). The overall conception rate in the present study was 58.33% (14/24) in non-infectious repeat-breeding buffaloes. As an herbal combination with chelated minerals and some vitamins has best conception rate in non-infectious repeat breeder buffaloes having the advantage of reducing the cost of treatment in repeat breeding as compared to other treatment protocols.

The Role of Neutrophil in The Pathophysiology of Frailty Syndrome

Neutrophils, also known as polymorphonuclear leukocytes, play a significant role in fueling systemic inflammation and tissue damage within the context of frailty, a clinical syndrome associated with increased susceptibility to stressors and decreased functional performance typically linked to aging. This literature review delves into the interplay between neutrophils and frailty, shedding light on their contribution to age-related systemic inflammation. The investigation explored the correlation between heightened neutrophil levels, diminished physical activity, increased frailty, and elevated white blood cell counts. These findings underscore the substantial role of neutrophils in the development of frailty, necessitating further research to enhance comprehension of their involvement for more effective prevention and intervention strategies. The implications of this study emphasize the importance of tailored exercise regimens, promoting optimal nutrition practices, efficiently managing chronic inflammation, and improving the control of chronic diseases as essential approaches to address frailty among older individuals.