Induction Of Cytokines Research Articles

Inflammatory & Apoptotic Factor Fluctuations Associated with Japanese Encephalitis Virus Infection in Transgenic IFNAR1-/- Mice.

Japanese encephalitis virus (JEV) is an orthoflavivirus that causes Japanese encephalitis, a mosquito-borne viral infection that primarily affects humans and animals. JEV is a major cause of encephalitis in many parts of Asia, particularly in rural and agricultural areas. In this study, we used the IFNAR1-/- mice model to investigate alterations in cytokine and apoptotic factor levels in IFNAR1-/- mice upon JEV infection. A 5-week-adult female C57BL/6 IFN-α/β receptor knockout (IFNAR1-/-) transgenic mice were intramuscularly inoculated with several viral titers and monitored within 10 dpi. The weight changes and survival rates were evaluated during the study period. Gene expression analysis was performed using RT-qPCR, targeting genes related to specific cytokines and apoptotic factors, to identify the inflammatory factors fluctuations associated with JEV strain KBPV-VR-27 infection in IFNAR1-/- mice. The expression of cytokine genes was enhanced in IFNAR1-/- mice infected with JEV KBPV-VR-27. Notably, a significant induction of cytokines, such as IL-13, IL-17α, IFN-β, and IFN-γ, was observed in the brain, while upregulation of IL-6, IFN-β, and IFN-γ was exhibited in the lung. In addition, among the targeted apoptotic factors, only significant induction of Bak was observed in the brain. We also found that the spleen exhibited a higher viral load compared to the brain and lungs. In conclusion, the findings of this study shed light on the varying viral loads across targeted organs, with the brain exhibiting a lower viral load but pronounced expression of targeted pro-inflammatory cytokines in IFNAR1-/- mice.

Baricitinib ameliorates inflammatory and neuropathic pain in collagen antibody-induced arthritis mice by modulating the IL-6/JAK/STAT3 pathway and CSF-1 expression in dorsal root ganglion neurons

BackgroundJanus kinase (JAK) inhibitors, such as baricitinib, are widely used to treat rheumatoid arthritis (RA). Clinical studies show that baricitinib is more effective at reducing pain than other similar drugs. Here, we aimed to elucidate the molecular mechanisms underlying the pain relief conferred by baricitinib, using a mouse model of arthritis.MethodsWe treated collagen antibody-induced arthritis (CAIA) model mice with baricitinib, celecoxib, or vehicle, and evaluated the severity of arthritis, histological findings of the spinal cord, and pain-related behaviours. We also conducted RNA sequencing (RNA-seq) to identify alterations in gene expression in the dorsal root ganglion (DRG) following baricitinib treatment. Finally, we conducted in vitro experiments to investigate the direct effects of baricitinib on neuronal cells.ResultsBoth baricitinib and celecoxib significantly decreased CAIA and improved arthritis-dependent grip-strength deficit, while only baricitinib notably suppressed residual tactile allodynia as determined by the von Frey test. CAIA induction of inflammatory cytokines in ankle synovium, including interleukin (IL)-1β and IL-6, was suppressed by treatment with either baricitinib or celecoxib. In contrast, RNA-seq analysis of the DRG revealed that baricitinib, but not celecoxib, restored gene expression alterations induced by CAIA to the control condition. Among many pathways changed by CAIA and baricitinib treatment, the interferon-alpha/gamma, JAK-signal transducer and activator of transcription 3 (STAT3), and nuclear factor kappa B (NF-κB) pathways were considerably decreased in the baricitinib group compared with the celecoxib group. Notably, only baricitinib decreased the expression of colony-stimulating factor 1 (CSF-1), a potent cytokine that causes neuropathic pain through activation of the microglia–astrocyte axis in the spinal cord. Accordingly, baricitinib prevented increases in microglia and astrocytes caused by CAIA. Baricitinib also suppressed JAK/STAT3 pathway activity and Csf1 expression in cultured neuronal cells.ConclusionsOur findings demonstrate the effects baricitinib has on the DRG in relation to ameliorating both inflammatory and neuropathic pain.

From Skin and Gut to the Brain: The Infectious Journey of the Human Commensal Fungus Malassezia and Its Neurological Consequences.

The human mycobiome encompasses diverse communities of fungal organisms residing within the body and has emerged as a critical player in shaping health and disease. While extensive research has focused on the skin and gut mycobiome, recent investigations have pointed toward the potential role of fungal organisms in neurological disorders. Among those fungal organisms, the presence of the commensal fungus Malassezia in the brain has created curiosity because of its commensal nature and primary association with the human skin and gut. This budding yeast is responsible for several diseases, such as Seborrheic dermatitis, Atopic dermatitis, Pityriasis versicolor, Malassezia folliculitis, dandruff, and others. However recent findings surprisingly show the presence of Malassezia DNA in the brain and have been linked to diseases like Alzheimer's disease, Parkinson's disease, Multiple sclerosis, and Amyotrophic lateral sclerosis. The exact role of Malassezia in these disorders is unknown, but its ability to infect human cells, travel through the bloodstream, cross the blood-brain barrier, and reside along with the lipid-rich neuronal cells are potential mechanisms responsible for pathogenesis. This also includes the induction of pro-inflammatory cytokines, disruption of the blood-brain barrier, gut-microbe interaction, and accumulation of metabolic changes in the brain environment. In this review, we discuss these key findings from studies linking Malassezia to neurological disorders, emphasizing the complex and multifaceted nature of these cases. Furthermore, we discuss potential mechanisms through which Malassezia might contribute to the development of neurological conditions. Future investigations will open up new avenues for our understanding of the fungal gut-brain axis and how it influences human behavior. Collaborative research efforts among microbiologists, neuroscientists, immunologists, and clinicians hold promise for unraveling the enigmatic connections between human commensal Malassezia and neurological disorders.

Phase II trial of BXCL701 and pembrolizumab in patients with metastatic pancreatic ductal adenocarcinoma (EXPEL-PANC): Preliminary findings.

LBA4132 Background: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options and is thought to be a “cold” tumor that does not respond to immunotherapy, due to a tumor microenvironment (TME) consisting of a desmoplastic stroma and poor T cell infiltrate. BXCL701 is an oral synthetic dipeptide that competitively inhibits dipeptidyl peptidases DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). BXCL701 exerts antitumor activity via inhibition of DPP8/9, which is associated with induction of proinflammatory cytokines, as well as inhibition of FAP, which disrupts tumor-stromal interactions. Preclinical xenograft models demonstrate synergy between BXCL701 and PD-1 blockade, reducing tumor growth and promoting an increase in intratumoral CD4+ and CD8+ T cells, macrophages and NK cells, with induction of host-protective immunity. Methods: This is a phase II trial of BXCL701 in patients with metastatic PDAC (mPDAC) following progression on 1 line of treatment for advanced disease and amenable to serial biopsies. BXCL701 is administered at 0.2 mg PO BID days 1-7 and 0.3 mg BID days 8-14 during cycle 1 (21 days) followed by 0.3 mg BID days 1-14 every 21 days in all other cycles, given with pembrolizumab 200 mg IV every 21 days (all cycles). The primary objective is to determine the 18-week progression-free survival rate (PFS18weeks). We estimate that historical 2nd-line PFS18weeks is 30% or less; using a Simon’s two-stage (minimax) design, a type I error rate of 0.05 and power of 80% if the true rate is 50%, we will need 19 patients in stage 1 and 20 in stage 2 (39 total). There is a safety lead-in phase of 6 patients. We plan to enroll 43 patients to account for a predicted 10% drop out of unevaluable patients. Correlative pharmacodynamic studies include imaging mass cytometry to examine 36 markers of the PDAC TME in tissue biopsies, as well as blood-based analyses of KRAS circulating tumor DNA, circulating markers of fibrosis, and IL-6. Enrollment began in Q3 2023 (NCT05558982). Results: Six patients have enrolled, 3 women and 3 men, median age 57.5 (range 37-80). One patient was progression-free at 18 weeks and 1 patient had stable disease (SD) at 9 weeks, not yet evaluable for the 18-week landmark. Objective response rate is 16% and disease control rate is 50% (RECIST: 1 partial response, -41%, and 2 patients with SD, -18% and 0%). Three patients had significant reductions in CA19-9 from baseline (-100%, -73%, and -97%). Median PFS and overall survival have not been reached (NR, 95% CI 1.45 months-NR and 0.92 months-NR, respectively). There have been no serious treatment-related safety events. The safety lead-in will complete after the next patient completes the 6-week safety window (1 patient was unevaluable). Conclusions: BXCL701 plus pembrolizumab is well-tolerated and shows early signs of potential clinical activity in patients with mPDAC refractory to chemotherapy. Clinical trial information: NCT05558982 .

Cutting Edge: ATP13A2 Is an Endolysosomal Regulator of TLR9/7 Activation in Human Plasmacytoid Dendritic Cells.

ATPase cation transporting 13A2 (ATP13A2) is an endolysosomal P-type ATPase known to be a polyamine transporter, explored mostly in neurons. As endolysosomal functions are also crucial in innate immune cells, we aimed to explore the potential role of ATP13A2 in the human immunocellular compartment. We found that human plasmacytoid dendritic cells (pDCs), the professional type I IFN-producing immune cells, especially have a prominent enrichment of ATP13A2 expression in endolysosomal compartments. ATP13A2 knockdown in human pDCs interferes with cytokine induction in response to TLR9/7 activation in response to bona fide ligands. ATP13A2 plays this crucial role in TLR9/7 activation in human pDCs by regulating endolysosomal pH and mitochondrial reactive oxygen generation. This (to our knowledge) hitherto unknown regulatory mechanism in pDCs involving ATP13A2 opens up a new avenue of research, given the crucial role of pDC-derived type I IFNs in protective immunity against infections as well as in the immunopathogenesis of myriad contexts of autoreactive inflammation.

Phase 1 clinical trial of AMG 340, a prostate-specific membrane antigen (PSMA)-targeted T-cell engager with a novel low-affinity CD3 binding domain designed to mitigate toxicity for the treatment of metastatic castration-resistant prostate cancer (mCRPC).

e14587 Background: PSMA, a transmembrane protein highly expressed on the surface of prostate cancer cells, is a validated target in prostate cancer; however, achieving a favorable therapeutic index has been challenging for PSMA-directed T-cell engagers (TCE’s). AMG 340, a PSMA-targeted TCE with a novel low-affinity CD3 binding domain specifically engineered to mitigate cytokine release syndrome (CRS) toxicity and maintain efficacy was investigated in a phase 1, open-label, dose-escalation study (NCT04740034). Methods: Patients (pts) with mCRPC who had received ≥2 prior lines of therapy were treated with AMG 340 monotherapy (1.5–800 mg intravenously every 3 weeks [Q3W]) during dose escalation. The primary outcome measures were dose-limiting toxicities, treatment-emergent adverse events (TEAEs), and pharmacokinetic profile. Secondary outcomes included prostate antigen specific (PSA) response and objective response per response evaluation criteria in solid tumors (RECIST). Results: As of September 6, 2023, a total of 42 pts (mean ± SD age, 68.5±7.8 years) received AMG 340 step up dosing across 11 cohorts. At doses up to 800 mg Q3W, AMG 340 had a manageable side effect profile. Any grade TEAEs reported in ≥30% of pts included CRS (52%), nausea (52%), vomiting (52%), fatigue (48%), diarrhea (38%), increased aspartate aminotransferase (36%), increased alanine aminotransferase (33%), and hypocalcemia (31%). Treatment-related toxicities included CRS (52%; Grade 1, 26%; Grade 2, 24%; Grade ≥3, 2%) and thrombocytopenia (24%; Grade 2, 10%; Grade ≥3, 14%). Step dosing was implemented at doses >40 mg and successfully mitigated CRS incidence and severity after the first dose. The maximal tolerated dose was not reached, as increasing the dose beyond 800 mg was predicted to minimally increase the potential AMG 340 activity. Exposure increased in an approximately dose-proportional manner with no evidence of clinically significant anti-drug antibodies. AMG 340 demonstrated evidence for pharmacodynamic engagement as shown by cytokine induction. Of the 41 PSA evaluable pts, PSA responses were seen across dose levels (PSA50, n=4), but were not dose dependent. Eight of 42 pts remained on treatment after 6 months. Among 27 RECIST evaluable pts, no RECIST responses were observed (n=14 stable disease, n=0 partial or complete response). Conclusions: Despite reaching high doses, no dose response was observed, and AMG 340 did not generate sufficient clinical activity to warrant further exploration. However, AMG 340 demonstrated manageable CRS toxicity showing that decreased CD3-binding strength mitigates CRS, the main toxicity of TCEs. Clinical studies are ongoing evaluating the PSMA protein as a molecular target for prostate cancer. Clinical trial information: NCT04740034 .

Unraveling the role of human microglia in tick-borne encephalitis virus infection: insights into neuroinflammation and viral pathogenesis

Tick-borne encephalitis virus (TBEV) is a neurotropic orthoflavivirus responsible for severe infections of the central nervous system. Although neurons are predominantly targeted, specific involvement of microglia in pathogenesis of TBE is not yet fully understood. In this study, the susceptibility of human microglia to TBEV is investigated, focusing on productive infection and different immune responses of different viral strains. We investigated primary human microglia and two immortalized microglial cell lines exposed to three TBEV strains (Hypr, Neudörfl and 280), each differing in virulence. Our results show that all microglia cultures tested support long-term productive infections, regardless of the viral strain. In particular, immune response varied significantly with the viral strain, as shown by the differential secretion of cytokines and chemokines such as IP-10, MCP-1, IL-8 and IL-6, quantified using a Luminex 48-plex assay. The most virulent strain triggered the highest cytokine induction. Electron tomography revealed substantial ultrastructural changes in the infected microglia, despite the absence of cytopathic effects. These findings underscore the susceptibility of human microglia to TBEV and reveal strain-dependent variations in viral replication and immune responses, highlighting the complex role of microglia in TBEV-induced neuropathology and contribute to a deeper understanding of TBE pathogenesis and neuroinflammation.

Induction of the antiviral factors APOBEC3A and RSAD2 upon CCL2 neutralization in primary human macrophages involves NF-κB, JAK/STAT, and gp130 signaling.

The CCL2/CC chemokine receptor 2 axis plays key roles in the pathogenesis of HIV-1 infection. We previously reported that exposure of monocyte-derived macrophages to CCL2 neutralizing antibody (αCCL2 Ab) restricted HIV-1 replication at postentry steps of the viral life cycle. This effect was associated with induction of transcripts coding for innate antiviral proteins, including APOBEC3A and RSAD2. This study aimed at identifying the signaling pathways involved in induction of these factors by CCL2 blocking in monocyte-derived macrophages. Through a combination of pharmacologic inhibition, quantitative reverse transcription polymerase chain reaction, Western blotting, and confocal laser-scanning microscopy, we demonstrated that CCL2 neutralization activates the canonical NF-κB and JAK/STAT pathways, as assessed by time-dependent phosphorylation of IκB, STAT1, and STAT3 and p65 nuclear translocation. Furthermore, pharmacologic inhibition of IκB kinase and JAKs strongly reduced APOBEC3A and RSAD2 transcript accumulation elicited by αCCL2 Ab treatment. Interestingly, exposure of monocyte-derived macrophages to αCCL2 Ab resulted in induction of IL-6 family cytokines, and interference with glycoprotein 130, the common signal-transducing receptor subunit shared by these cytokines, inhibited APOBEC3A and RSAD2 upregulation triggered by CCL2 neutralization. These results provide novel insights into the signal transduction pathways underlying the activation of innate responses triggered by CCL2 neutralization in macrophages. Since this response was found to be associated with protective antiviral effects, the new findings may help design innovative therapeutic approaches targeting CCL2 to strengthen host innate immunity.

Biofilm-derived membrane vesicles exhibit potent immunomodulatory activity in Pseudomonas aeruginosa PAO1.

Pathogenic bacteria form biofilms on epithelial cells, and most bacterial biofilms show increased production of membrane vesicles (MVs), also known as outer membrane vesiclesin Gram-negative bacteria. Numerous studies have investigated the MVs released under planktonic conditions; however, the impact of MVs released from biofilms on immune responses remains unclear. This study aimed to investigate the characteristics and immunomodulatory activity of MVs obtained from both planktonic and biofilm cultures of Pseudomonas aeruginosa PAO1. The innate immune responses of macrophages to planktonic-derived MVs (p-MVs) and biofilm-derived MVs (b-MVs) were investigated by measuring the mRNA expression of proinflammatory cytokines. Our results showed that b-MVs induced a higher expression of inflammatory cytokines, including Il1b, Il6, and Il12p40, than p-MVs. The mRNA expression levels of Toll-like receptor 4 (Tlr4) differed between the two types of MVs, but not Tlr2. Polymyxin B significantly neutralized b-MV-mediated cytokine induction, suggesting that lipopolysaccharideof native b-MVs is the origin of the immune response. In addition, heat-treated or homogenized b-MVs induced the mRNA expression of cytokines, including Tnfa, Il1b, Il6, and Il12p40. Heat treatment of MVs led to increased expression of Tlr2 but not Tlr4, suggesting that TLR2 ligands play a role in detecting the pathogen-associated molecular patternsin lysed MVs. Taken together, our data indicate that potent immunomodulatory MVs are produced in P. aeruginosa biofilms and that this behavior could be a strategy for the bacteria to infect host cells. Furthermore, our findings would contribute to developing novel vaccines using MVs.

Comparison of the transcriptome, lipidome, and c-di-GMP production between BCGΔBCG1419c and BCG, with Mincle- and Myd88-dependent induction of proinflammatory cytokines in murine macrophages

We have previously reported the transcriptomic and lipidomic profile of the first-generation, hygromycin-resistant (HygR) version of the BCGΔBCG1419c vaccine candidate, under biofilm conditions. We recently constructed and characterized the efficacy, safety, whole genome sequence, and proteomic profile of a second-generation version of BCGΔBCG1419c, a strain lacking the BCG1419c gene and devoid of antibiotic markers. Here, we compared the antibiotic-less BCGΔBCG1419c with BCG. We assessed their colonial and ultrastructural morphology, biofilm, c-di-GMP production in vitro, as well as their transcriptomic and lipidomic profiles, including their capacity to activate macrophages via Mincle and Myd88. Our results show that BCGΔBCG1419c colonial and ultrastructural morphology, c-di-GMP, and biofilm production differed from parental BCG, whereas we found no significant changes in its lipidomic profile either in biofilm or planktonic growth conditions. Transcriptomic profiling suggests changes in BCGΔBCG1419c cell wall and showed reduced transcription of some members of the DosR, MtrA, and ArgR regulons. Finally, induction of TNF-α, IL-6 or G-CSF by bone-marrow derived macrophages infected with either BCGΔBCG1419c or BCG required Mincle and Myd88. Our results confirm that some differences already found to occur in HygR BCGΔBCG1419c compared with BCG are maintained in the antibiotic-less version of this vaccine candidate except changes in production of PDIM. Comparison with previous characterizations conducted by OMICs show that some differences observed in BCGΔBCG1419c compared with BCG are maintained whereas others are dependent on the growth condition employed to culture them.

Synergistic effects of Smac mimetic APG-1387 with anti-PD-1 antibody are attributed to increased CD3 + NK1.1 + cell recruitment secondary to induction of cytokines from tumor cells

BackgroundImmune checkpoint inhibitors are approved for the treatment of various tumors, but the response rate is not satisfactory in certain malignancies. Inhibitor of apoptosis proteins (IAP) ubiquitin-E3 ligase activity is involved in the regulation of immune responses. APG-1387 is a novel second mitochondria-derived activator of caspase (Smac) mimetic IAP inhibitor. The aim of this study was to explore the synergistic effect of APG-1387 when combined with anti-PD-1 antibody in a preclinical setting.MethodsWe utilized syngeneic mouse models of ovarian cancer (ID8), colon cancer (MC38), malignant melanoma (B16), and liver cancer (Hepa1-6) to assess the combination effect of APG-1387 and anti-PD-1 antibody, including immune-related factors, tumor growth, and survival. MSD V-PLEX validated assays were used to measure in vitro and in vivo cytokine release.ResultsIn ID8 ovarian cancer and MC38 colon cancer models, APG-1387 and anti-PD1 antibody had synergistic antitumor effects. In the MC38 model, the combination of APG-1387 and anti-PD-1 antibody significantly inhibited tumor growth (P < 0.0001) and increased the survival rate of tumor-bearing animals (P < 0.001). Moreover, we found that APG-1387 upregulated tumor-infiltrating CD3 + NK1.1 + cells by nearly 2-fold, by promoting tumor cell secretion of IL-12. Blocking IL-12 secretion abrogated the synergistic effects of APG-1387 and anti-PD-1 antibody in both MC38 and ID8 models.ConclusionsAPG-1387 has the potential to turn “cold tumors” into hot ones by recruiting more CD3 + NK1.1 + cells into certain tumors. Based on these and other data, the safety and therapeutic effect of this combination will be investigated in a phase 1/2 trial in patients with advanced solid tumors or hematologic malignancies (NCT03386526).Graphic abstract

Lipid A modification of colistin-resistant Klebsiella pneumoniae does not alter innate immune response in a mouse model of pneumonia.

Polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae bacteria is associated with high morbidity and mortality in vulnerable populations throughout the world. Ineffective antimicrobial activity by these last resort therapeutics can occur by transfer of mcr-1, a plasmid-mediated resistance gene, causing modification of the lipid A portion of lipopolysaccharide (LPS) and disruption of the interactions between polymyxins and lipid A. Whether this modification alters the innate host immune response or carries a high fitness cost in the bacteria is not well established. To investigate this, we studied infection with K. pneumoniae (KP) ATCC 13883 harboring either the mcr-1 plasmid (pmcr-1) or the vector control (pBCSK) ATCC 13883. Bacterial fitness characteristics of mcr-1 acquisition were evaluated. Differentiated human monocytes (THP-1s) were stimulated with KP bacterial strains or purified LPS from both parent isolates and isolates harboring mcr-1. Cell culture supernatants were analyzed for cytokine production. A bacterial pneumonia model in WT C57/BL6J mice was used to monitor immune cell recruitment, cytokine induction, and bacterial clearance in the bronchoalveolar lavage fluid (BALF). Isolates harboring mcr-1 had increased colistin MIC compared to the parent isolates but did not alter bacterial fitness. Few differences in cytokines were observed with purified LPS from mcr-1 expressing bacteria in vitro. However, in a mouse pneumonia model, no bacterial clearance defect was observed between pmcr-1-harboring KP and parent isolates. Consistently, no differences in cytokine production or immune cell recruitment in the BALF were observed, suggesting that other mechanisms outweigh the effect of these lipid A mutations in LPS.

Glycyrrhizic Acid Alleviates Semen Strychni-Induced Neurotoxicity Through the Inhibition of HMGB1 Phosphorylation and Inflammatory Responses

The neurotoxicity of Semen Strychni has been reported recently in several clinical cases. Therefore, this study was conducted to investigate the role of HMGB1 in a model of neurotoxicity induced by Semen Strychni and to assess the potential alleviating effects of glycyrrhizic acid (GA), which is associated with the regulation of HMGB1 release. Forty-eight SD rats were intraperitoneally injected with Semen Strychni extract (175 mg/kg), followed by oral administration of GA (50 mg/kg) for four days. After treatment of SS and GA, neuronal degeneration, apoptosis, and necrosis were observed via histopathological examination. Inflammatory cytokines (TNF-α and IL-1β), neurotransmitter associated enzymes (MAO and AChE), serum HMGB1, nuclear and cytoplasmic HMGB1/ph-HMGB1, and the interaction between PP2A, PKC, and HMGB1 were evaluated. The influence of the MAPK pathway was also examined. As a result, this neurotoxicity was characterized by neuronal degeneration and apoptosis, the induction of pro-inflammatory cytokines, and a reduction in neurotransmitter-metabolizing enzymes. In contrast, GA treatment significantly ameliorated the abovementioned effects and alleviated nerve injury. Furthermore, Semen Strychni promoted HMGB1 phosphorylation and its translocation between the nucleus and cytoplasm, thereby activating the NF-κB and MAPK pathways, initiating various inflammatory responses. Our experiments demonstrated that GA could partially reverse these effects. In summary, GA acid alleviated Semen Strychni-induced neurotoxicity, possibly by inhibiting HMGB1 phosphorylation and preventing its release from the cell.

Leukocytospermia and/or Bacteriospermia: Impact on Male Infertility.

Infertility is a globally underestimated public health concern affecting almost 190 million people, i.e., about 17.5% of people during their lifetime, while the prevalence of male factor infertility is about 7%. Among numerous other causes, the prevalence of male genital tract infections reportedly ranges between 10% and 35%. Leukocytospermia is found in 30% of infertile men and up to 20% in fertile men. Bacterial infections cause an inflammatory response attracting leukocytes, which produce reactive oxygen species (ROS) and release cytokines, both of which can cause damage to sperm, rendering them dysfunctional. Although leukocytospermia and bacteriospermia are both clinical conditions that can negatively affect male fertility, there is still debate about their impact on assisted reproduction outcomes and management. According to World Health Organization (WHO) guidelines, leukocytes should be determined by means of the Endtz test or with monoclonal antibodies against CD15, CD68 or CD22. The cut-off value proposed by the WHO is 1 × 106 peroxidase-positive cells/mL. For bacteria, Gram staining and semen culture are regarded as the "gold standard", while modern techniques such as PCR and next-generation sequencing (NGS) are allowing clinicians to detect a wider range of pathogens. Whereas the WHO manual does not specify a specific value as a cut-off for bacterial contamination, several studies consider semen samples with more than 103 colony-forming units (cfu)/mL as bacteriospermic. The pathogenic mechanisms leading to sperm dysfunction include direct interaction of bacteria with the male germ cells, bacterial release of spermatotoxic substances, induction of pro-inflammatory cytokines and ROS, all of which lead to oxidative stress. Clinically, bacterial infections, including "silent" infections, are treatable, with antibiotics being the treatment of choice. Yet, non-steroidal antiphlogistics or antioxidants should also be considered to alleviate inflammatory lesions and improve semen quality. In an assisted reproduction set up, sperm separation techniques significantly reduce the bacterial load in the semen. Nonetheless, contamination of the semen sample with skin commensals should be prevented by applying relevant hygiene techniques. In patients where leukocytospermia is detected, the causes (e.g. infection, inflammation, varicocele, smoking, etc.) of the leukocyte infiltration have to be identified and addressed with antibiotics, anti-inflammatories or antioxidants in cases where high oxidative stress levels are detected. However, no specific strategy is available for the management of leukocytospermia. Therefore, the relationship between bacteriospermia and leukocytospermia as well as their specific impact on functional sperm parameters and reproductive outcome variables such as fertilization or clinical pregnancy must be further investigated. The aim of this narrative review is to provide an update on the current knowledge on leukocytospermia and bacteriospermia and their impact on male fertility.

(S)-(-)-blebbistatin O-benzoate has the potential to improve atopic dermatitis symptoms in NC/Nga mice by upregulating epidermal barrier function and inhibiting type 2 alarmin cytokine induction.

Atopic dermatitis is a multi-pathogenic disease characterized by chronic skin inflammation and barrier dysfunction. Therefore, improving the skin's ability to form an epidermal barrier and suppressing the production of cytokines that induce type 2 inflammatory responses are important for controlling atopic dermatitis symptoms. (-)-Blebbistatin, a non-muscle myosin II inhibitor, has been suggested to improve pulmonary endothelial barrier function and control inflammation by suppressing immune cell migration; however, its efficacy in atopic dermatitis is unknown. In this study, we investigated whether (S)-(-)-blebbistatin O-benzoate, a derivative of (-)-blebbistatin, improves dermatitis symptoms in a mite antigen-induced atopic dermatitis model using NC/Nga mice. The efficacy of the compound was confirmed using dermatitis scores, ear thickness measurements, serum IgE levels, histological analysis of lesions, and filaggrin expression analysis, which is important for barrier function. (S)-(-)-Blebbistatin O-benzoate treatment significantly reduced the dermatitis score and serum IgE levels compared to those in the vehicle group (p < 0.05). Furthermore, the histological analysis revealed enhanced filaggrin production and a decreased number of mast cells (p < 0.05), indicating that (S)-(-)-blebbistatin O-benzoate improved atopic dermatitis symptoms in a pathological model. In vitro analysis using cultured keratinocytes revealed increased expression of filaggrin, loricrin, involucrin, and ceramide production pathway-related genes, suggesting that (S)-(-)-blebbistatin O-benzoate promotes epidermal barrier formation. Furthermore, the effect of (S)-(-)-blebbistatin O-benzoate on type 2 alarmin cytokines, which are secreted from epidermal cells upon scratching or allergen stimulation and are involved in the pathogenesis of atopic dermatitis, was evaluated using antigens derived from mite feces. The results showed that (S)-(-)-blebbistatin O-benzoate inhibited the upregulation of these cytokines. Based on the above, (S)-(-)-blebbistatin O-benzoate has the potential to be developed as an atopic dermatitis treatment option that controls dermatitis symptoms by suppressing inflammation and improving barrier function by acting on multiple aspects of the pathogenesis of atopic dermatitis.

Establishment of Anoxia/cytokine-induced Injury and Regeneration in Colonoids

Intestinal epithelial cells (IECs) play key roles in nutrient absorption and fluid secretion, providing a barrier to protect cells. IECs, derived from intestinal stem cells (ISCs), are crucial in maintaining intestinal homeostasis and regeneration, which is crucial in gastrointestinal diseases such as inflammatory bowel disease (IBD). To study epithelial injury/regeneration, primary cultured colonoids were utilized as a model system to mimic injury. They retain the composition and differentiation of the tissue, making it a suitable model for studying homeostasis and regeneration of ISCs in vitro. Anoxia and proinflammatory cytokines frequently coexist and exacerbate one another during intestinal injury. However, anoxic conditions have been mainly ignored in previous research. Thus, we developed a regeneration model using cytokines/anoxia injury to better recapitulate intestinal inflammation. Colonoids were treated with cytokines TNF-α and IFN-γ (5 ng/μL) in anoxic conditions for 24 hours to provoke an injury. After 24 hours of injury, the colonoids were exposed to normal oxygen and media conditions for regeneration. Regeneration was observed for 24, 48, and 72 hours and documented using photomicrographs. Morphological analysis detailed that injury was most severe 24 hours post-injury with the combined induction of cytokines and anoxia resulting in decreased colonoid area and number of buds compared to control groups. RT-PCR analysis detailed mRNA expression of stem cell markers ( Lgr5, Bmi1, and Hopx), Wnt ( Axin2, Myc2, and Ascl2), and YAP target genes ( Sca1 and Ctgf) were increased at peak injury and returned to pre-injury levels at 72 hours of recovery. Regeneration of the colonoids is consistent with in vivo observations, thus confirming the validity of the model. This versatile model can be used to study responses to injury and regeneration of intestinal epithelium and the function of ISCs with applications in intestinal disease therapeutics. University of Maryland College of Agriculture and Natural Resources and the Maryland Agricultural Experiment Station Undergraduate Research Grant Grant #4307572 from the Maryland Stem Cell Research Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3018: Expression Of The Aim2 Inflammasome In Skeletal Is Modulated By Surgical Bypass And Is A Target To Reduce Ischemic Myopathy In Pad

Introduction: Peripheral arterial disease (PAD) causes skeletal muscle myopathy and impairment, yet relevant pathways are understudied. We reported a new role for the absent in melanoma 2 (AIM2) inflammasome in PAD myocyte function. We hypothesize that AIM2 is an important mediator of cell death pathways in PAD muscle and is reduced by revascularization. Methods: Myocytes were obtained commercially or from ischemic and perfused PAD muscle using cell sorting. mRNA libraries underwent next generation sequencing (NGS) and differential gene expression (DGE) mapped to the GRCh368 human genome. Ingenuity Pathway Analysis (IPA) identified relevant biological pathways filtered by both fold change (fc) &gt;2 and false discovery rate of p&lt; 0.05. Sections were procured from human gastrocnemius before and after vascular bypass and analyzed for AIM2 with immunofluorescence (IF) and western blot (WB). Inflammatory caspase-1 activity was measured +/- siRNA against AIM2. Statistical analysis included t- test and ANOVA + post-hoc analysis. P&lt;0.05 was considered significant. Results: DGE and IPA on isolated myocytes highlighted Neutrophil Degranulation and Pathogen Induced Cytokine Storm Signaling Pathways to be significantly altered in ischemic muscle cells (Fig1, p &lt; 1xE-30 each, N=4) emphasizing AIM2 inflammasome activation (CASP1, fc 30.73; NLRP3/ASC/CASP1, fc 27.23; PYCARD fc 40.63; IL1B fc 63.49). AIM2 protein was high in PAD by IF and WB. Caspase-1 activity was attenuated in the presence of AIM2 inhibition (N=3; ****p&lt;0.0001 in PAD). AIM2 expression was significantly reduced after surgery for PAD (p&lt;0.05, N=6/group, t-test). Conclusion: AIM2 inflammasome expression in PAD induces pyroptotic caspase activity and is attenuated by surgical bypass, directly demonstrating a unique biologic benefit of muscle revascularization in PAD. Targeting AIM2 pathways either pharmacologically or by surgery may help to reduce myopathic damage in ischemic muscle from PAD.

Epstein-Barr virus-encoded BART9 and BART15 miRNAs are elevated in exosomes of cerebrospinal fluid from relapsing-remitting multiple sclerosis patients

Epstein-Barr virus (EBV) infection is approved as the main environmental trigger of multiple sclerosis (MS). In this path, we quantified ebv-miR-BART9-3p and ebv-miR-BART15 in exosomes of cerebrospinal fluid (CSF) of untreated relapsing-remitting MS (RRMS) patients in comparison with the control group. Interestingly, patients displayed significant upregulation of ebv-miR-BART9-3p (18.4-fold) and ebv-miR-BART15 (3.1-fold) expression in CSF exosomes. Moreover, the expression levels of hsa-miR-21-5p and hsa-miR-146a-5p were found to be significantly elevated in the CSF samples obtained from the patient group compared to those obtained from the HC group. The levels of Interferon-gamma (IFN-γ), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23), transforming growth factor beta (TGF-β), and tumor necrosis factor-alpha (TNF-α) were observed to be significantly elevated in the serum and CSF exosomes of the patients. The highest increase was observed in TGF-β (8.5-fold), followed by IL-23 (3.9-fold) in CSF exosomes. These findings are in agreement with the association between EBV infection and inflammatory cytokines induction. Furthermore, the ratios of TGF-β: TNF-α and TGF-β: IFN-γ attained values of 4 to 16.4 and 1.3 to 3.6, respectively, in the CSF exosomes of the patients, in comparison to those of the control group. These findings show EBV activity in RRMS patients is different from that of healthy ones. Elevation of ebv-miR-BART9-3p, ebv-miR-BART15, and inflammatory cytokines expression in CSF exosomes in RRMS patients provides a substantial link between EBV activity and the onset of the disease, as well as the transition from EBV infection to MS.

Quantitative and qualitative analysis of stability for 16 serum immunoregulators over 50 freeze-thaw cycles.

To evaluate the reliability of data from the assay of bio-archived specimens, a 50-freeze-thaw-cycle (FTC) degradation study of fresh sera was conducted to test the stability of 16 immunoregulators. Twenty de-identified serum specimens were obtained from volunteers at United Health Services-Wilson Memorial Hospital. Specimens were stored at -20°C and underwent daily 1 h thawing and subsequent freezing for each FTC over 50 consecutive days. Immunoregulator concentrations were assessed via enzyme-linked immunosorbent assay (ELISA) in participant samples at 2 FTC (baseline), 25 FTC, and 50 FTC. Specific immunoregulators observed in the study were C-reactive protein (CRP), interleukin (IL)-1α, 4, 6, 8, 10, monocyte chemoattractant protein-1 (MCP-1, CCL2), monocyte chemoattractant protein-2 (MCP-2, CCL8), eotaxin-1, thymus-and-activation-regulated chemokine (TARC, CCL17), regulated on activation normal T-cell expressed and secreted (RANTES, CCL5), growth-regulated oncogene-alpha (GRO-α, CXCL1), small inducible cytokine A1 (I-309, CCL1), interferon-gamma (IFN-γ), interferon-gamma inducible protein-10 (IP-10, CXCL10), and tumor necrosis factor-alpha (TNF-α). Quantitative stability of serum immunoregulators: Serum CRP, IL-8, IL-10, IFN-γ, IP-10, and eotaxin-1 levels appear to be statistically equivalent from baseline to 50 FTC (p ≤ .05). Retention of patterns in serum immunoregulators: patterns across FTC were retained for TARC (age) and CRP, IFN-γ, and MCP-2 (sex). While the effect of multiple FTC on serum immunoregulator levels may not replicate prolonged freezer storage, the results of this study provide valuable information on the robustness of immunoregulators for research using bio-archived sera.

Autonomous circadian rhythms in the human hepatocyte regulate hepatic drug metabolism and inflammatory responses.

Critical aspects of physiology and cell function exhibit self-sustained ~24-hour variations termed circadian rhythms. In the liver, circadian rhythms play fundamental roles in maintaining organ homeostasis. Here, we established and characterized an in vitro liver experimental system in which primary human hepatocytes display self-sustained oscillations. By generating gene expression profiles of these hepatocytes over time, we demonstrated that their transcriptional state is dynamic across 24 hours and identified a set of cycling genes with functions related to inflammation, drug metabolism, and energy homeostasis. We designed and tested a treatment protocol to minimize atorvastatin- and acetaminophen-induced hepatotoxicity. Last, we documented circadian-dependent induction of pro-inflammatory cytokines when triggered by LPS, IFN-β, or Plasmodium infection in human hepatocytes. Collectively, our findings emphasize that the phase of the circadian cycle has a robust impact on the efficacy and toxicity of drugs, and we provide a test bed to study the timing and magnitude of inflammatory responses over the course of infection in human liver.