Delayed-type Hypersensitivity Research Articles

Phase II trial of vaccination with autologous, irradiated melanoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor in patients with stage III and IV melanoma

BackgroundIn the era of immune checkpoint blockade, the role of cancer vaccines in immune priming has provided additional potential for therapeutic improvements. Prior studies have demonstrated delayed type hypersensitivity and anti-tumor immunity with vaccines engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). The safety, efficacy and anti-tumor immunity of GM-CSF secreting vaccine in patients with previously treated stage III or IV melanoma needs further investigation.MethodsIn this phase II trial, excised lymph node metastases were processed to single cells, transduced with an adenoviral vector encoding GM-CSF, irradiated, and cryopreserved. Individual vaccines were composed of 1x106, 4x106, or 1x107 tumor cells, and were injected intradermally and subcutaneously at weekly and biweekly intervals. The primary endpoints were feasibility of producing vaccine in stage III patients and determining the proportion of patients alive at two years in stage IV patients.ResultsGM-CSF vaccine was successfully developed and administered in all 61 patients. Toxicities were restricted to grade 1-2 local skin reactions. The median OS for stage III patients (n = 20) was 71.1 (95% CI, 43.7 to NR) months and 14.9 (95%CI, 12.1 to 39.7) months for stage IV patients. The median PFS in stage III patients was 50.7 (95%CI, 36.3 to NR) months and 4.1 (95% CI, 3.0-6.3) months in stage IV patients. In the overall population, the disease control rate was 39.3% (95%CI, 27.1 to 52.7%). In stage III patients, higher pre-treatment plasma cytokine levels of MMP-1, TRAIL, CXCL-11, CXCL-13 were associated with improved PFS (p<0.05 for all). An increase in post-vaccination levels of IL-15 and TRAIL for stage III patients was associated with improved PFS (p=0.03 for both). Similarly, an increase in post-vaccination IL-16 level for stage IV patients was associated with improved PFS (p=0.02) and clinical benefit.ConclusionsVaccination with autologous melanoma cells secreting GM-CSF augments antitumor immunity in stage III and IV patients with melanoma, is safe, and demonstrates disease control. Luminex data suggests that changes in inflammatory cytokines and immune cell infiltration promote tumor antigen presentation and subsequent tumor cell destruction. Additional investigation to administer this vaccine in combination with immune checkpoint inhibitors is needed.

ALLERGIC CONTACT DERMATITIS AND ATOPIC DERMATITIS: HIGHLIGHTS OF THE OVERLAP SYNDROME

Introduction. The combination of atopic dermatitis (AD) with allergic contact dermatitis (ACD) or the occurrence of ACD on the background of atopic dermatitis is called the overlap syndrome. Studies have demonstrated several reasons why patients with AD have a similar or even increased risk of developing ACD compared to those without AD. Allergens and haptens are trigger factors in a group of patients with AD and ACD overlap syndrome. The aim of the study. To confirm the diagnosis of ACD in a group of patients with AD – diagnose the overlap syndrome and analyze which allergens and haptens were the trigger factors in this group. Materials and methods. To confirm IgE-dependent sensitization in atopic dermatitis, skin prick tests or determination of specific IgE in blood serum were performed. Skin patch tests (European series S-1000) were performed to determine the mechanisms of delayed-type hypersensitivity. Results. It was found that the highest specific weight of positive allergic reactions has been recorded in response to the following allergens: ticks, ticks/ambrosia, birch and mold. The absolute majority of patients demonstrated positive specific IgE-dependent sensitization to Dermatophagoides pteronyssinus and Dermatophagoides farinae – 24 (50%), in turn, on Ambrósia – 14 (29.2%), and on Alternaria alternata – 8 (16.7%). Also, the reaction was most often recorded to haptens: cobalt, nickel, formaldehyde, PPD, textile dyes. Deterioration of the clinical course and shortening of AD remission periods were observed due to the formation of ACD against the background of impaired skin barrier function and the presence of chronic immune inflammation. Conclusions. Patients with AD are more often diagnosed with ACD, which predictably worsens the course of AD. Patients with confirmed overlap syndrome "AD + ACD" most often show reactions to haptens: Cobalt, Nikel, Formaldehyde, PPD, Textile dye mix – and in the vast majority to 2 haptens or more in one patient.

Yogurt Alleviates Cyclophosphamide-Induced Immunosuppression in Mice through D-Lactate.

Numerous studies have investigated the immunomodulatory effects of yogurt, but the underlying mechanism remained elusive. This study aimed to elucidate the alleviating properties of yogurt on immunosuppression and proposed the underlying mechanism was related to the metabolite D-lactate. In the healthy mice, we validated the safety of daily yogurt consumption (600 μL) or D-lactate (300 mg/kg). In immunosuppressed mice induced by cyclophosphamide (CTX), we evaluated the immune regulation of yogurt and D-lactate. The result showed that yogurt restored body weight, boosted immune organ index, repaired splenic tissue, recovered the severity of delayed-type hypersensitivity reactions and increased serum cytokines (IgA, IgG, IL-6, IFN-γ). Additionally, yogurt enhanced intestinal immune function by restoring the intestinal barrier and upregulating the abundance of Bifidobacterium and Lactobacillus. Further studies showed that D-lactate alleviated immunosuppression in mice mainly by promoting cellular immunity. D-lactate recovered body weight and organ development, elevated serum cytokines (IgA, IgG, IL-6, IFN-γ), enhanced splenic lymphocyte proliferation and increased the mRNA level of T-bet in splenic lymphocyte to bolster Th1 differentiation. Finally, CTX is a chemotherapeutic drug, thus, the application of yogurt and D-lactate in the tumor-bearing mouse model was initially explored. The results showed that both yogurt (600 μL) and D-lactate (300 mg/kg) reduced cyclophosphamide-induced immunosuppression without promoting tumor growth. Overall, this study evaluated the safety, immune efficacy and applicability of yogurt and D-lactate in regulating immunosuppression. It emphasized the potential of yogurt as a functional food for immune regulation, with D-lactate playing a crucial role in its immunomodulatory effects.

Abstract PO3-05-12: Randomized Phase 2 of Bria-IMT, an Allogenic Human Cell Line with Antigen Presenting Activity, in Heavily Pretreated Metastatic Breast Cancer

Abstract Introduction Despite progress in MBC, too many continue to succumb due to treatment resistance or intolerance. SV-BR-1-GM is an allogenic human breast cancer cell line with antigen presenting cell activity designed to overcome the immune-suppressive tumor microenvironment while being acceptable to extensively pretreated contemporary MBC patients. We report interim exploratory analysis of the ongoing RCT of the Bria-IMT regimens. Methods An interim exploratory analysis using preliminary partially available evaluable data of the ongoing prospective, randomized phase 2 (NCT03328026; 2018-present) of Bria-IMT (irradiated SV-BR-1-GM) with a PD-1check point inhibitor (CPI) every 3 weeks (25 patients dosed to date). Both regimens include cyclophosphamide 300 mg/m2 48-72 hours prior to intradermal Bria-IMT (~20 x 106 cells) followed by interferon-alpha at the inoculation sites 2 days later. The control arm used the original sequence with simultaneous start of Bria-IMT and CPI in cycle 1. The experimental arm started CPI in cycle 2 after the 2nd inoculation. Candida skin test was performed before cycle 1 to determine anergy status while delayed-type hypersensitivity skin test (DTH), defined as >5mm erythema & induration, was done at every cycle by intradermal injection of a small test dose of Bria-IMT prior to full dose inoculation. Results Median age: 58 (range 37-81); median prior therapies: 8 lines (range 4-18); 89% were grade 2/3 (n=18); 68% hormone receptor positive; 61% HER2 negative, 33% HER2 low, and 6% HER2 high; 33% triple negative. Overall, 53% of subjects had a clinical benefit as best response by BICR, PI or target lesion measurement. The two treatment arms were similar with a trend showing longer median durations on therapy (mDOT) of 3.7 mo in the experimental delayed CPI arm compared with mDOT = 2.9 for original sequence control arm. 47% of patients were anergic to candida. Among all anergic subjects, the mDOT was 3.5 compared to 2.5 mo for non-anergic patients. However, among anergic subjects, the alternative treatment arm had a lower risk of treatment discontinuation (improved HR) with mDOT of 3.7 compared to the 3.3 mo mDOT of the original treatment. 50% of anergic subjects eventually developed DTH to SV-BR-1-GM. mDOT was 4.0 mo for DTH+ pts vs 3.0 mo for those who did not mount DTH. Median OS was not yet reached; mean OS was higher in non-anergic vs anergic pts. Among those who developed an inoculation site reaction to Bria-IMT, OS was greater than for those who did not. Time on the Bria-IMT regimen exceeded the time on penultimate treatment in 22% of subjects while 33% of subjects with prior CPI use exceeded last therapy. AEs were mild with 28% of subjects (n=7) having > grade 3 events. The most common grade 3+ AE was lipase increase (n=3). One subject with encouraging changes in peripheral blood tumor markers and CTCs completed a PET study using Zr-89 crefmirlimab berdoxam, a zirconium-89 labelled truncated antibody specific to human CD8α (CD8 Immuno-PET) and demonstrated increased uptake in some metastatic lesions and draining lymph nodes. Updated results will be provided at the meeting. Conclusion Sequencing of CPI with Bria-IMT is associated with differential clinical outcome trends but not statistically significant in this interim preliminary exploratory analysis regardless of prior CPI use. Bria-IMT is immunogenic with clinical benefit demonstrated for both treatment schedules among patients with inducible DTH reactions. Patients who develop an immune response to Bria-IMT had an increase in OS compared to those who did not. The nodal localization of CD8+ cells on PET may indicate a systemic activation of CD8 positive lymphoid cells. It also provides support that the Bria-IMT + CPI combination immune-based therapy can result in an increase of CD8+ tumor infiltrating lymphocytes in breast cancer metastatic sites. These preliminary results will be evaluated in the ongoing randomized phase 3 pivotal registrational trial. Citation Format: Carmen Calfa, Chaitali Nangia, Minal Barve, John Knecht, Jarrod Holmes, Kendrith Roland, Ralph Boccia, Frances Valdes-Albini, Zach Gostout, Mingjin Chang, William Williams, Charles Wiseman, Bonnie Guerin, Shaker Dakhil, Giuseppe Del Priore, Saranya Chumsri. Randomized Phase 2 of Bria-IMT, an Allogenic Human Cell Line with Antigen Presenting Activity, in Heavily Pretreated Metastatic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-05-12.

Abstract PO1-20-12: CD8+ Tumor Infiltrating Lymphocytes Turn a Cold Tumor Hot in Metastatic Breast Cancer

Abstract Introduction: Turning “cold” tumors “hot” is considered essential for immune-oncology therapies to be effective. Bria-IMT is an allogenic human cancer cell line with antigen presenting cell activity (SV-BR-1-GM) designed to overcome the immune-suppressive tumor microenvironment. However, until now there has not been proof that “cold” tumors can become “hot” with this therapy. Using Zr-89 crefmirlimab berdoxam, a zirconium-89 labelled truncated antibody specific to human CD8α and developed for CD8 ImmunoPET, we image before and after Bria-IMT therapy administration to assess the baseline status as well as changes in CD8 cell presence after initiation of therapy. Here we present the first evidence that cold tumors can become hot on treatment with Bria-IMT combined with an anti-PD-1 checkpoint inhibitor (CPI), showing CD8+ cells increased over baseline using CD8 ImmunoPET. Case: After informed consent, a 61 year-old Hispanic female, enrolled in the ongoing randomized phase 2 Bria-IMT trial in advanced heavily pretreated metastatic breast cancer (MBC) (NCT03328026). Her history included MBC in May 2020, ductal type, grade 3, triple negative breast cancer (TNBC) with 6 prior lines of therapy including a Trop-2-directed antibody-drug conjugate and 6 lines of chemotherapy. Precision medicine testing identified no actionable targets. All prior therapies had progressive disease as their best response. The subject was randomized to start the CPI after 2 cycles of Bria-IMT to “train” the host immune system before proceeding with combination immune-oncology therapy. Baseline testing revealed the subject to be anergic to candida and SV-BR-1-GM inoculation. However, by cycle 3 delayed-type hypersensitivity to SV-BR-1GM developed. Interestingly the subject matched SV-BR-1-GM at 2 HLA loci: DRB3*02:02 and DRB1*11:04 which in previous publications is associated with greater clinical benefit. Tumor markers were stable or decreased slightly including CEA (25% decrease) and CA 15-3 (43% decrease). Circulating Tumor Cells (CTCs) and Cancer Associated Macrophage Like cells (CAMLs) also improved. Standard imaging at baseline showed 3 main sites of disease including a large right breast mass, multiple right axillary lymph nodes and a right lower lobe mass with CD8 ImmunoPET uptake in these areas similar to the blood pool. On the follow-up CT imaging, all of these lesions increased in size with newly appreciated pulmonary nodules. The right lower lobe mass showed increased CD8 ImmunoPET accumulation with an SUV of 5.8 (close to the liver background) as compared to an SUV of 2.2 at baseline. The lesions in the right breast and right axilla remained at blood pool levels. There was an increase in CD8 ImmunoPET uptake in bilateral inguinal lymph nodes reaching an SUV of 4.8 as compared to SUV 2.2 at baseline. In addition, there were non-enlarged lymph nodes in bilateral axillary regions that also showed increased radio-tracer accumulation reaching an SUV of 9.5 as compared to SUV 4.9 at baseline. Conclusion: This subject demonstrated a mixed response of metastatic lesions on CD8 ImmunoPET with encouraging changes in peripheral blood tumor markers and CTCs. The nonspecific nodal localization of CD8 ImmunoPET may indicate a systemic activation of CD8 positive lymphoid cells. This shows the value of CD8 ImmunoPET in identifying lesions that are progressing on treatment versus pseudo progression. It also provides support that the Bria-IMT + CPI combination immune-based therapy can result in an increase of CD8+ tumor infiltrating lymphocytes in breast cancer metastatic sites as well as an increase in lymphoid organs. This advance may aid in triaging patients, adjudicating pseudo-progression and predicting clinical benefit of immune based therapies. Citation Format: Russ Kuker, Frances Valdes-Albini, Giuseppe Del Priore, Aazim Arif, Kristin Schmiedehausen, Carmen Calfa. CD8+ Tumor Infiltrating Lymphocytes Turn a Cold Tumor Hot in Metastatic Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-20-12.

Effect of feeding Moringa oleifera based complete pellet feed on milk yield, composition, fatty acids, somatic cell count and cell mediated immunityin lactating goats

Lactation cum feeding trial was conducted on 12 female Barbari goats (Age approx. 2-3 years and mean body weight 34.72 ±1.28 kg) for 120 days after approximately one month of kidding and effect on milk production, milk constituents, somatic cell count, milk fatty acid profile and cell mediated immunity was studied. Animals were divided into two groups (Gr G and Gr M) of six each as per completely randomized design. Two types of complete pellet (Moringa oleifera and Cicer arietinum) based was formulated containing sundried Moringa biomass and Gram straw along with conventional feed ingredients. Lactating goats of Gr G was fed with gram straw based complete pellet feed while goats of Gr M was fed with Moringa based complete pellet feed. Both the complete pellet feeds were iso nitrogenous. No significant difference in body weight changes was recorded between groups. The milk production (g/day) was higher (Avg. 14.54%) in Gr M as compared to Gr G at different fortnight. No significant difference was reported in the milk composition. Milk fat, SNF, Protein and lactose was similar in both the groups. Milk Somatic cell count (×10 3/ml) was significantly (P<0.05) lower in lactating goats fed with Moringa based feed. Cell-mediated immune response assessed through delayed-type hypersensitivity (DTH) reaction against phyto haemagglutinin (PHA-P) was more pronounced in Gr M at 72 and 96 hrs.Higher concentration of unsaturated fatty acids (about 16.19%) and lower saturated fatty acids (about 5.49%) was reported in Gr M. Milk fatty acid profile indicators like Index of atherogenicity (IA) was lower while hypo/hyper cholesteremic ratio and health promoting index was higher in milk of moringa based pellet fed goats. Present study concluded that Barbari goats fed with Moringa based complete pellet feed produced higher milk yield with healthier fatty acid profile with low somatic cell counts.

Immunotoxicity of 2-Acetyl-4-tetrahydroxybutylimidazole in BALB/c mice with different vitamin B6 nutritional statuses

Caramel color is a widely used food pigment, and 2-Acetyl-4-tetrahydroxybutylimidazole (THI) is a by-products of Class III caramel color. Some studies have shown that THI can reduce the number of peripheral blood lymphocytes. However, the comprehensive mechanism of THI immunotoxicity requires further study. In this study, the effects of THI on lymphocyte count, humoral immunity, cellular immunity and nonspecific immunity were determined and the effect of the nutritional status of VB6 on THI immunotoxicity was evaluated. Female BALB/c mice were divided into 3 groups and fed chow containing different doses of VB6: VB6-normal (6mg/kg VB6), VB6-deprived (0.5mg/kg VB6) or VB6-enhanced (12mg/kg VB6) feed. Each group was further divided into 4 subgroups and treated with THI (0.5, 2.5 or 12.5mg/kg bw) or the solvent control by gavage for 30 days. The thymic cortical thickness was measured with ViewPoint; the proportions of major immune cells and T cells in peripheral blood and tissues were detected via flow cytometry; the transformation and proliferation abilities of T and B cells were detected via T and B lymphocyte proliferation assays; NK cell activity was assessed via lactate dehydrogenase assays; humoral immune function was assessed via plaque-forming cell assays; and the immune function of T lymphocytes was assessed via delayed type hypersensitivity assays. The results showed that compared with those in the corresponding control group, the white blood cell count and lymphocyte count decreased significantly in all the VB6-deprived groups, in the 2.5 and 12.5mg/kg VB6 groups, and in the 12.5mg/kg VB6-enhanced group. With increasing THI dose, the thymic cortical layer became thinner. In the thymus, THI increased the proportions of CD3+ T cells and mature CD8+ T cells and decreased the proportions of immature double-positive, double-negative T cells and CD69-expressing lymphocytes. The proportions of naïve T cells and Tcm (central memory T) cells related to homing decreased. The proportion of mature T cells in the spleen decreased significantly. The proliferation of T cells stimulated by ConA decreased after THI exposure. VB6-deficient mice were more sensitive to THI immunotoxicity, and supplementation with VB6 had a certain protective effect on these mice. The results of the PFC and NK cell activity assays indicated that THI exposure might not affect humoral immune or innate immune function.

Safety and immunogenicity of a polyvalent DNA–protein HIV vaccine with matched Env immunogens delivered as a prime–boost regimen or coadministered in HIV-uninfected adults in the USA (HVTN 124): a phase 1, placebo-controlled, double-blind randomised controlled trial

An effective HIV vaccine will most likely need to have potent immunogenicity and broad cross-subtype coverage. The aim of the HIV Vaccine Trials Network (HVTN) 124 was to evaluate safety and immunogenicity of a unique polyvalent DNA-protein HIV vaccine with matching envelope (Env) immunogens. HVTN 124 was a randomised, phase 1, placebo-controlled, double-blind study, including participants who were HIV seronegative and aged 18-50 years at low risk for infection. The DNA vaccine comprised five plasmids: four copies expressing Env gp120 (clades A, B, C, and AE) and one gag p55 (clade C). The protein vaccine included four DNA vaccine-matched GLA-SE-adjuvanted recombinant gp120 proteins. Participants were enrolled across six clinical sites in the USA and were randomly assigned to placebo or one of two vaccine groups (ie, prime-boost or coadministration) in a 5:1 ratio in part A and a 7:1 ratio in part B. Vaccines were delivered via intramuscular needle injection. The primary outcomes were safety and tolerability, assessed via frequency, severity, and attributability of local and systemic reactogenicity and adverse events, laboratory safety measures, and early discontinuations. Part A evaluated safety. Part B evaluated safety and immunogenicity of two regimens: DNA prime (administered at months 0, 1, and 3) with protein boost (months 6 and 8), and DNA-protein coadministration (months 0, 1, 3, 6, and 8). All randomly assigned participants who received at least one dose were included in the safety analysis. The study is registered with ClinicalTrials.gov (NCT03409276) and is closed to new participants. Between April 19, 2018 and Feb 13, 2019, 60 participants (12 in part A [five men and seven women] and 48 in part B [21 men and 27 women]) were enrolled. All 60 participants received at least one dose, and 14 did not complete follow-up (six of 21 in the prime-boost group and eight of 21 in the coadminstration group). 11 clinical adverse events deemed by investigators as study-related occurred in seven of 48 participants in part B (eight of 21 in the prime-boost group and three of 21 in the coadministration group). Local reactogenicity in the vaccine groups was common, but the frequency and severity of reactogenicity signs or symptoms did not differ between the prime-boost and coadministration groups (eg, 20 [95%] of 21 in the prime-boost group vs 21 [100%] of 21 in the coadministration group had either local pain or tenderness of any severity [p=1·00], and seven [33%] vs nine [43%] had either erythema or induration [p=0·97]), nor did laboratory safety measures. There were no delayed-type hypersensitivity reactions or vasculitis or any severe clinical adverse events related to vaccination. The most frequently reported systemic reactogenicity symptoms in the active vaccine groups were malaise or fatigue (five [50%] of ten in part A and 17 [81%] of 21 in the prime-boost group vs 15 [71%] of 21 in the coadministration group in part B), headache (five [50%] and 18 [86%] vs 12 [57%]), and myalgia (four [40%] and 13 [62%] vs ten [48%]), mostly of mild or moderate severity. Both vaccine regimens were safe, warranting evaluation in larger trials. US National Institutes of Health and US National Institute of Allergy and Infectious Diseases.

NAT10 promotes synovial aggression by increasing the stability and translation of N4-acetylated PTX3 mRNA in rheumatoid arthritis

ObjectiveRecent studies indicate that N-acetyltransferase 10 (NAT10)-mediated ac4C modification plays unique roles in tumour metastasis and immune infiltration. This study aimed to uncover the role of NAT10-mediated ac4C in fibroblast-like...

Immune Regulation by TNF Receptor-associated Factor 5

The tumor necrosis factor receptor (TNFR)-associated factor (TRAF) family of molecules are intracellular adaptors that regulate cellular signaling through members of the TNFR and Toll-like receptor superfamily. Mammals have seven TRAF molecules numbered sequentially from TRAF1 to TRAF7. Although TRAF5 was identified as a potential regulator of TNFR superfamily members, the in vivo function of TRAF5 has not yet been fully elucidated. We identified an unconventional role of TRAF5 in interleukin-6 (IL-6) receptor signaling involving CD4+ T cells. Moreover, TRAF5 binds to the signal-transducing glycoprotein 130 (gp130) receptor for IL-6 and inhibits the activity of the janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway. In addition, Traf5-deficient CD4+ T cells exhibit significantly enhanced IL-6-driven differentiation of T helper 17 (Th17) cells, which exacerbates neuroinflammation in experimental autoimmune encephalomyelitis. Furthermore, TRAF5 demonstrates a similar activity to gp130 for IL-27, another cytokine of the IL-6 family. Additionally, Traf5-deficient CD4+ T cells display significantly increased IL-27-mediated differentiation of Th1 cells, which increases footpad swelling in delayed-type hypersensitivity response. Thus, TRAF5 functions as a negative regulator of gp130 in CD4+ T cells. This review aimed to explain how TRAF5 controls the differentiation of CD4+ T cells and discuss how the expression of TRAF5 in T cells and other cell types can influence the development and progression of autoimmune and inflammatory diseases.

Unconventional activation of PRKDC by TNF-α: deciphering its crucial role in Th1-mediated inflammation beyond DNA repair as part of the DNA-PK complex

BackgroundThe DNA-dependent protein kinase (DNA-PK) complex comprises a catalytic (PRKDC) and two requisite DNA-binding (Ku70/Ku80) subunits. The role of the complex in repairing double-stranded DNA breaks (DSBs) is established, but its role in inflammation, as a complex or individual subunits, remains elusive. While only ~ 1% of PRKDC is necessary for DNA repair, we reported that partial inhibition blocks asthma in mice without causing SCID.MethodsWe investigated the central role of PRKDC in inflammation and its potential association with DNA repair. We also elucidated the relationship between inflammatory cytokines (e.g., TNF-α) and PRKDC by analyzing its connections to inflammatory kinases. Human cell lines, primary human endothelial cells, and mouse fibroblasts were used to conduct the in vitro studies. For animal studies, LPS- and oxazolone-induced mouse models of acute lung injury (ALI) and delayed-type hypersensitivity (DHT) were used. Wild-type, PRKDC+/−, or Ku70+/− mice used in this study.ResultsA ~ 50% reduction in PRKDC markedly blocked TNF-α-induced expression of inflammatory factors (e.g., ICAM-1/VCAM-1). PRKDC regulates Th1-mediated inflammation, such as DHT and ALI, and its role is highly sensitive to inhibition achieved by gene heterozygosity or pharmacologically. In endothelial or epithelial cells, TNF-α promoted rapid PRKDC phosphorylation in a fashion resembling that induced by, but independent of, DSBs. Ku70 heterozygosity exerted little to no effect on ALI in mice, and whatever effect it had was associated with a specific increase in MCP-1 in the lungs and systemically. While Ku70 knockout blocked VP-16-induced PRKDC phosphorylation, it did not prevent TNF-α − induced phosphorylation of the kinase, suggesting Ku70 dispensability. Immunoprecipitation studies revealed that PRKDC transiently interacts with p38MAPK. Inhibition of p38MAPK blocked TNF-α-induced PRKDC phosphorylation. Direct phosphorylation of PRKDC by p38MAPK was demonstrated using a cell-free system.ConclusionsThis study presents compelling evidence that PRKDC functions independently of the DNA-PK complex, emphasizing its central role in Th1-mediated inflammation. The distinct functionality of PRKDC as an individual enzyme, its remarkable sensitivity to inhibition, and its phosphorylation by p38MAPK offer promising therapeutic opportunities to mitigate inflammation while sparing DNA repair processes. These findings expand our understanding of PRKDC biology and open new avenues for targeted anti-inflammatory interventions.

Structural characterization and immunostimulant activities of polysaccharides fractionated by gradient ethanol precipitation method from Panax ginseng C. A. Meyer.

Panax ginseng C. A. Meyer is a dual-purpose plant for medicine and food, its polysaccharide is considered as an immune enhancer. Four polysaccharides, WGP-20-F, WGP-40-F, WGP-60-F and WGP-80-F were obtained from ginseng via water extraction and gradient ethanol precipitation with different molecular weights (Mw) of 1.720 × 106, 1.434 × 106, 4.225 × 104 and 1.520 × 104Da, respectively. WGP-20-F and WGP-40-F which with higher Mw and a triple-helix structure are glucans composed of 4-ɑ-Glcp, do not show remarkable immunoregulatory effects. WGP-60-F and WGP-80-F are heteropolysaccharides mainly composed of 4-ɑ-Glcp and also contain t-ɑ-Araf, 5-ɑ-Araf and 3,5-ɑ-Araf. They are spherical branched conformations without a triple-helix structure and can effectively increase the index of immune organs, lymphocyte proliferation, activate macrophages to regulate the immune system in mice and further enhance immune functions by improving delayed-type hypersensitivity reaction and antibody response. These results indicated that WGP-60-F and WGP-80-F could be used as potential immune enhancers, and gradient ethanol precipitation can be applied for the preparation of ginseng bioactive polysaccharide.

Antimicrobial and Antiplasmodial Activities of Endophytic Fungi Associated with Psidium guajava

Infections due to antimicrobial-resistant microorganisms have become widespread in recent years. Thus, searching for novel antimicrobial agents to combat such pathogens has become crucial. The current study aimed to evaluate the antimicrobial, antiplasmodial, and immunomodulatory activities of the extracts of endophytic fungi isolated from Psidium guajava. Isolation, identification, fermentation, and extraction of the secondary metabolites of the fungal endophytes were carried out following standard procedures. The extracts were subjected to High-Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) analysis to detect their bioactive components. The Antimicrobial activity and Minimum Inhibitory Concentration of the fungal extracts were evaluated against pure cultures of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Salmonella typhi, and Klebsiella pneumonia using Agar well diffusion and Agar dilution method respectively. The acute toxicity study (LD50) was carried out using Lorke’s method. The extracts were tested in vivo in mice for antiplasmodial activity against Plasmodium berghei and in vitro against Plasmodium falciparum using Peter and Reyley's curative test method and WHO standardized micro-test system with modification respectively. The immunomodulatory activity of the extracts was evaluated by cyclophosphamide-induced myelosuppression (hematological parameters). Active extracts were further subjected to Delayed-Type Hypersensitivity Response (DTHR) and Haemagglutination Inhibition Assay using Sheep Red Blood Cells as antigens. The result showed Alternaria sp. (PGL1, PGL2, PGL3), from P. guajava. The HPLC-DAD analysis revealed the presence of bioactive compounds previously reported to have antimicrobial, antiplasmodial, and immunomodulatory properties. The fungal extracts exhibited varying degrees of antimicrobial activity. The LD50 of the fungi extracts was>5000 mg/kg in mice. The extracts at 100 and 200 mg/kg body weight in mice showed varying degrees of antiplasmodial activity. Growth of P. berghei was significantly (p<0.001) inhibited, curative effect ranges from 59.09 – 100%. Schizont maturation of P. falciparum isolates was inhibited and the highest level of inhibition was observed at 1 mg/ml (p<0.05). The fungal extracts reversed the effect of cyclophosphamide-induced reduction in total white blood cell counts and % neutrophil. This study showed that the tested plant harbors species of endophytic fungi that contain numerous secondary metabolites. The endophytic fungi showed prophylactic, immunostimulatory, and antiplasmodial activities, which can be exploited to develop antimicrobial, antiplasmodial, and immunomodulatory agents.

The scar-reducing effect of a novel chitosan gel: an in vivo study.

Scar tissue formation, as a normal part of wound healing, initiates in the proliferation phase, continues after the remodelling phase, and may cause an unpleasant appearance or disruption in normal functioning. This study investigated the effects of a topical gel on acute wound healing and reducing scars in a rat model. ChitoScar (ChitoTech Company, Iran), a commercial scar-reducing gel based on chitosan, was analysed for antibacterial and antiviral activity through a quantitative suspension test. Its cytotoxic effect was investigated, and then irritation and delayed-type hypersensitivity tests were carried out on rabbits through direct application of the gel. Furthermore, the effect of the chitosan-based gel on wound healing and scar tissue formation was studied in rats with an acute wound in two groups: the treatment group (topical application of the chitosan-based gel); and the control group (without treatment). Histopathological examination was carried out based on the inflammatory cells, collagen fibre, keratinocytes and fibroblasts. Analysis revealed that the chitosan-based gel had no cytotoxicity and caused no erythema, oedema, local or other systemic adverse response. Wound healing occurred earlier in the treatment group, which was a result of a significant increase in re-epithelialisation, angiogenesis, fibroblast population and collagen fibre thickness (p<0.05). In the treatment group, wounds healed completely after 21 days and scars totally disappeared after 28 days, while in the control group, wound healing remained incomplete with distinct scar tissue. The results demonstrated the positive effect of the chitosan-based gel on the duration and quality of the wound healing process, as well as minimising the scar tissue formation in this in vivo study.

Long-Term Survival and Immune Response Dynamics in Melanoma Patients Undergoing TAPCells-Based Vaccination Therapy.

Cancer vaccines present a promising avenue for treating immune checkpoint blockers (ICBs)-refractory patients, fostering immune responses to modulate the tumor microenvironment. We revisit a phase I/II trial using Tumor Antigen-Presenting Cells (TAPCells) (NCT06152367), an autologous antigen-presenting cell vaccine loaded with heat-shocked allogeneic melanoma cell lysates. Initial findings showcased TAPCells inducing lysate-specific delayed-type hypersensitivity (DTH) reactions, correlating with prolonged survival. Here, we extend our analysis over 15 years, categorizing patients into short-term (<36 months) and long-term (≥36 months) survivors, exploring novel associations between clinical outcomes and demographic, genetic, and immunologic parameters. Notably, DTHpos patients exhibit a 53.1% three-year survival compared to 16.1% in DTHneg patients. Extended remissions are observed in long-term survivors, particularly DTHpos/M1cneg patients. Younger age, stage III disease, and moderate immune events also benefit short-term survivors. Immunomarkers like increased C-type lectin domain family 2 member D on CD4+ T cells and elevated interleukin-17A were detected in long-term survivors. In contrast, toll-like receptor-4 D229G polymorphism and reduced CD32 on B cells are associated with reduced survival. TAPCells achieved stable long remissions in 35.2% of patients, especially M1cneg/DTHpos cases. Conclusions: Our study underscores the potential of vaccine-induced immune responses in melanoma, emphasizing the identification of emerging biological markers and clinical parameters for predicting long-term remission.

A CD25-biased interleukin-2 for autoimmune therapy engineered via a semi-synthetic organism

BackgroundNatural cytokines are poorly suited as therapeutics for systemic administration due to suboptimal pharmacological and pharmacokinetic (PK) properties. Recombinant human interleukin-2 (rhIL-2) has shown promise for treatment of autoimmune (AI) disorders yet exhibits short systemic half-life and opposing immune responses that negate an appropriate therapeutic index.MethodsA semi-synthetic microbial technology platform was used to engineer a site-specifically pegylated form of rhIL-2 with enhanced PK, specificity for induction of immune-suppressive regulatory CD4 + T cells (Tregs), and reduced stimulation of off-target effector T and NK cells. A library of rhIL-2 molecules was constructed with single site-specific, biorthogonal chemistry-compatible non-canonical amino acids installed near the interface where IL-2 engages its cognate receptor βγ (IL-2Rβγ) signaling complex. Biorthogonal site-specific pegylation and functional screening identified variants that retained engagement of the IL-2Rα chain with attenuated potency at the IL-2Rβγ complex.ResultsPhenotypic screening in mouse identifies SAR444336 (SAR’336; formerly known as THOR-809), rhIL-2 pegylated at H16, as a potential development candidate that specifically expands peripheral CD4+ Tregs with upregulation of markers that correlate with their suppressive function including FoxP3, ICOS and Helios, yet minimally expands CD8 + T or NK cells. In non-human primate, administration of SAR’336 also induces dose-dependent expansion of Tregs and upregulated suppressive markers without significant expansion of CD8 + T or NK cells. SAR’336 administration reduces inflammation in a delayed-type hypersensitivity mouse model, potently suppressing CD4+ and CD8 + T cell proliferation.ConclusionSAR’336 is a specific Treg activator, supporting its further development for the treatment of AI diseases.

Cyclosporin A-loaded dissolving microneedles for dermatitis therapy: Development, characterisation and efficacy in a delayed-type hypersensitivity in vivo model.

Several drugs can be used for treating inflammatory skin pathologies like dermatitis and psoriasis. However, for the management of chronic and long-term cases, topical administration is preferred over oral delivery since it prevents certain issues due to systemic side effects from occurring. Cyclosporin A (CsA) has been used for this purpose; however, its high molecular weight (1202Da) restricts the diffusion through the skin structure. Here, we developed a nano-in-micro device combining lipid vesicles (LVs) and dissolving microneedle array patches (DMAPs) for targeted skin delivery. CsA-LVs allowed the effective incorporation of CsA in the hydrophilic DMAP matrix despite the hydrophobicity of the drug. Polymeric matrix composed of poly (vinyl alcohol) (5% w/v), poly (vinyl pyrrolidine) (15% w/v) and CsA-LV dispersion (10% v/v) led to the formation of CsA-LVs@DMAPs with adequate mechanical properties to penetrate the stratum corneum barrier. The safety and biocompatibility were ensured in an in vitro viability test using HaCaT keratinocytes and L929 fibroblast cell lines. Ex vivo permeability studies in a Franz-diffusion cell setup showed effective drug retention in the skin structure. Finally, CsA-LVs@DMAPs were challenged in an in vivo murine model of delayed-type hypersensitivity to corroborate their potential to ameliorate skin inflammatory conditions. Different findings like photon emission reduction in bioluminescence study, normalisation of histological damage and decrease of inflammatory cytokines point out the effectivity of CsA-LVs@DMAPs to treat these conditions. Overall, our study demonstrates that CsA-LVs@DMAPs can downregulate the skin inflammatory environment which paves the way for their clinical translation and their use as an alternative to corticosteroid-based therapies.

Polyvinylpyrrolidone hydrogel coating for ureteral stent: Safety and performance evaluation.

Ureteral stents are commonly used in urology. However, complications such as encrustation and infection on the surface of the stent, and injury to the ureteral mucosa can occur after implantation, causing discomfort for patients. We intend to confirm the biosafety of polyvinylpyrrolidone (PVP) hydrophilic coating and its lubrication properties for surface modification of ureteral stents to reduce friction and improve patient comfort. Based on our previous studies, we have developed a PVP hydrophilic coating for surface modification of ureteral stents. We firstly investigated the cytotoxicity, intradermal irritation, delayed type hypersensitivity, and acute systemic reactions of stent coating extracts. We further characterized the break strength, retention strength, and dynamic friction of the stent. The cell survival rate of all experimental groups was greater than 70%. No hypersensitivity reaction, systemic toxicity reaction, or obvious intradermal reaction were observed. The above results indicate that the test results of the modified stent meet the requirements of ISO 10993-5: 2009 (Cytotoxicity); ISO 10993-10:2021 (Sensitization and Irritation); ISO 10993-11:2017 (Acute Systemic Toxicity). After soaking in artificial urine for an extended period, there was no obvious change in its super-slip performance. Our results confirm the safety and lubrication characteristics of PVP hydrophilic coating for ureteral stent surface modification. The performance of this coating has the potential to reduce complications after stent implantation, thereby improving patient comfort, reducing medical burden, and has a good clinical application prospect.

Chicory root powder included as a prebiotic in different cereal-based diets for dogs: Influences on gut health, metabolic and immunological status

The present experiment was conducted to study the influence of chicory root powder as a prebiotic on gut health, metabolic, and immunological status of dogs fed with different cereal-based diets. Sixteen Labrador dogs, fed either a wheat (WT) or sorghum (SG) based diet, each without (C) or with (P) supplemental chicory root powder at 10 g/kg diet, were divided into four equal groups namely WT-C, WT-P, SG-C and SG-P. The digestibility of DM, OM, and total carbohydrates was significantly (p < 0.05) higher with the SG-C diet than with the other diets. Faecal ammonia was reduced (p = 0.002) in the WT-P group; however, there were no differences (p > 0.05) observed in the faecal pH and contents of lactate, acetate, propionate and total short chain fatty acids (SCFAs). The faecal lactobacilli population was higher (p = 0.001) in both the prebiotic-supplemented groups with a similar trend (p = 0.079) for bifidobacteria. Coliform counts were reduced (p = 0.002) in prebiotic-supplemented diets irrespective of cereals. There were significant (p < 0.05) variations in haemoglobin and haematocrit values among the dietary groups. The delayed-type hypersensitivity response to intradermal phytohemagglutinin (PHA-P) was higher (p<0.001) in both the prebiotic-supplemented groups; a similar trend was evident in the humoral immune response measured as antibody response to sheep erythrocytes. The peripheral lymphocyte subpopulations of CD4+ and CD8+ varied with prebiotic supplementation of the sorghum-based diet. Overall, the findings implies that tailoring the dietary composition to include specific cereals may optimize the beneficial effects of prebiotics in supporting canine health.

Effects of Bifidobacterium animalis subsp. lactis IU100 on Immunomodulation and Gut Microbiota in Immunosuppressed Mice.

Probiotics are live microorganisms with immunomodulatory effects in a strain-specific and dose-dependent manner. Bifidobacterium animalis subsp. lactis IU100 is a new probiotic strain isolated from healthy adults. This study aimed to evaluate the effects of IU100 on cyclophosphamide (CTX)-induced immunosuppression in mice. The results showed that IU100 significantly ameliorated CTX-induced decreases in body weight and immune organ indices. The promoted delayed-type hypersensitivity, serum hemolysins and immunoglobulin (IgA, IgG and IgM) levels after IU100 treatment indicated its enhancing role in cellular and humoral immunity. In addition, oral administration of IU100 increased serum cytokine (IL-1β, IL-2, IL-4, IL-6, IFN-γ, TNF-α) levels dose-dependently, which are associated with CTX-induced shifts in the Th1/Th2 balance. The probiotic IU100 also modulated the composition of gut microbiota by reducing the Firmicutes/Bacteroidetes ratio; increasing beneficial Muribaculaceae and the Lachnospiraceae NK4A136 group; and inhibiting harmful Clostridium sensu stricto 1, Faecalibaculum and Staphylococcus at the genus level. The above genera were found to be correlated with serum cytokines and antibody levels. These findings suggest that IU100 effectively enhances the immune function of immunosuppressed mice, induced by CTX, by regulating gut microbiota.