Migraine and gasdermin D: a new perspective on the inflammatory basis of migraine.

Migraine is a common and disabling primary headache disorder and inflammation is a proposed factor in the complex ethiology of the disease. Gasdermin D (GSDMD) is a membrane pore-forming protein acting through the caspase system. End result is cell death caused by leakage of intracellular components to extracellular space which also results in inflammation. Stemming from this knowledge, the potential role of GSDMD in migraine was investigated in this prospective study. This prospective study was conducted between September 2022 to April 2023. 47 patients with migraine were designated as the patient group, whereas 47 healthy volunteers were designated as the control group. Serum GSDMD levels of both groups were compared, with an additional comparison between migraine patients during symptom-free and attack periods. Migraine related characteristics of the patients were also included in the study. Median GSDMD levels of the patient and control group did not reveal a significant difference. Nausea, vomiting and severity of headache were found to be correlated with GSDMD levels in migraine patients. Patients with nausea revealed a higher GSDMD level compared to patients without nausea during both symptom-free and attack periods (p = 0.021 and p = 0.01, respectively). Nausea was correlated to higher GSDMD levels in the patient population during symptom-free period (p = 0.030). The severity of pain was positively correlated with GSDMD levels during the attack period (p < 0.001). Gasdermin family and GSDMD in particular are promising prospects for therapy in a wide spectrum of disorders. Gasdermin proteins are candidates to be the focus for future studies both related to pathogenesis and drug therapy in migraine and varying inflammatory-driven clinical pictures.

The expression and clinical significance of CD59 and FLAER in Chinese adult AML patients.

BACKGROUND: The role of CD59 and fluorescently labeled aerolysin (FLAER) in acute myeloid leukemia (AML) remains unclear and requires further investigation. To explore the relationship between CD59, FLAER, and AML, we investigated CD59 and FLAER expression in AML and analyzed their relationship with clinical characteristics of AML patients. METHODS: We employed flow cytometry (FCM) to analyze CD59 and FLAER expression in 161 AML patients at Tianjin Medical University General Hospital and evaluated its association with sex, white blood cell (WBC) count, platelet (PLT) count, thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), D-Dimer(D-D), and lactate dehydrogenase (LDH), followed by analyzing its connection with disease progression and complete remission (CR). RESULTS: CD59 and FLAER deficiencies were identified in AML patients. Compared with CR group, non-CR group patients revealed more CD59 and FLAER deficiency. Compared with non-acute promyelocytic leukemia (M3) group, M3 group patients had more CD59 and FLAER deficiency. CD59- level in primordial cells of M3 patients was positively correlated with primordial cell ratio (r = 0.660, p = 0.003). Additionally, we discovered that the decline in CD59 and FLAER levels might be linked to higher D-D and LDH in AML patients. The difference was statistically significant (p < 0.05). CONCLUSIONS: We demonstrated that the decline in CD59 and FLAER levels was associated with leukemia cell proliferation and abnormal coagulation function in AML, suggesting that they could serve as a predictor of AML coagulation dysfunction, particularly in M3.

Circulating extracellular vesicles activate the pyroptosis pathway in the brain following ventilation-induced lung injury.

BACKGROUND: Mechanical ventilation of preterm newborns causes lung injury and is associated with poor neurodevelopmental outcomes. However, the mechanistic links between ventilation-induced lung injury (VILI) and brain injury is not well defined. Since circulating extracellular vesicles (EVs) are known to link distant organs by transferring their cargos, we hypothesized that EVs mediate inflammatory brain injury associated with VILI. METHODS: Neonatal rats were mechanically ventilated with low (10 mL/kg) or high (25 mL/kg) tidal volume for 1 h on post-natal day 7 followed by recovery for 2 weeks. Exosomes were isolated from the plasma of these rats and adoptively transferred into normal newborn rats. We assessed the effect of mechanical ventilation or exosome transfer on brain inflammation and activation of the pyroptosis pathway by western blot and histology. RESULTS: Injurious mechanical ventilation induced similar markers of inflammation and pyroptosis, such as increased IL-1ß and activated caspase-1/gasdermin D (GSDMD) in both lung and brain, in addition to inducing microglial activation and cell death in the brain. Isolated EVs were enriched for the exosomal markers CD9 and CD81, suggesting enrichment for exosomes. EVs isolated from neonatal rats with VILI had increased caspase-1 but not GSDMD. Adoptive transfer of these EVs led to neuroinflammation with microglial activation and activation of caspase-1 and GSDMD in the brain similar to that observed in neonatal rats that were mechanically ventilated. CONCLUSIONS: These findings suggest that circulating EVs can contribute to the brain injury and poor neurodevelopmental outcomes in preterm infants with VILI through activation of GSDMD.

The effect of structural modification of antimicrobial peptides on their antimicrobial activity, hemolytic activity, and plasma stability.

In this article, a series of modifications were made on an antimicrobial peptide F2,5,12 W, including altering the amino acid sequence, introducing cysteine and other typical amino acids, developing peptide dimers via disulfide bonds, and conjugating with mPEG, in order to enhance the antimicrobial activity, plasma stability, and reduce the hemolytic activity of peptides. The results showed that mPEG conjugation could significantly improve the plasma stability and reduce the hemolytic activity of peptides, while the antimicrobial activity decreased meanwhile. However, altering the sequence of the peptide without changing its amino acid composition had little impact on its antimicrobial activity and plasma stability. The introduction of cysteine enhanced the plasma stability of peptides conspicuously, but at the same time, the increased hydrophobicity of peptides increased their hemolysis. The antimicrobial mechanism and cytotoxicity of the peptides with relatively high antimicrobial activity were also studied. In general, this study provided some ideas for the rational design and structure optimization of antimicrobial peptides.

Role of Platelets in Detection and Regulation of Infection.

Platelets are classically known as essential mediators of hemostasis and thrombosis. However, in recent years, platelets have gained recognition for their inflammatory functions, which modulate the immune response during infectious diseases. Platelets contain various immunoreceptors that enable them to act as sentinels to recognize intravascular pathogens. Upon activation, platelets directly limit pathogen growth through the release of AMPs (antimicrobial proteins) and ensure pathogen clearance through activation of immune cells. However, aberrant platelet activation can lead to inflammation and thrombotic events.

Diminished systemic levels of antimicrobial peptides in tuberculous lymphadenitis and their reversal after anti-tuberculosis treatment.

Pulmonary tuberculosis is associated with higher plasma levels of antimicrobial peptides (AMPs) and lower granulysin levels. However, the association of AMPs with tuberculous lymphadenitis (TBL) is not well studied. Hence, we measured the plasma levels of human beta defensin-2 (HBD2), granulysin, human neutrophil peptides 1-3 (HNP1-3) and cathelicidin (LL37) in TBL compared to latent tuberculosis (LTB) and healthy controls (HC) and in TBL individuals upon completion of anti-tuberculosis treatment (ATT). We examined the association of AMPs with TBL lymph node culture grade or lymph node involvement. Finally, the discriminatory potential of these proteins was assessed using receiver operating characteristic (ROC) analysis. TBL individuals display significantly diminished circulating levels of AMPs (granulysin and HNP1-3) but not HBD-2 and LL-37 in comparison to LTB and HCs. Similarly, after ATT, both HBD-2 and HNP1-3 were significantly elevated and LL-37 was significantly reduced in TBL individuals. Granulysin and HNP1-3 discriminates TBL from LTB and HC individuals upon ROC analysis. AMPs did not exhibit significant correlation either with lymph node culture grades or lymph node involvement. Overall, TBL individuals show decreased AMPs and their reversal after ATT suggesting their association with underlying immune alteration in this poorly studied form of TB disease.

Hemoglobin Reassembly of Antimicrobial Fragments from the Midgut of Triatoma infestans.

Hemoglobin is one of the most important molecules of the human body. Beyond its physiological activity, hemoglobins are able to inhibit the growth of several microorganisms. Since 1999, studies have reported that antimicrobial peptides can be produced by blood-feeding insects through hemoglobin digestion, and it has been reported that Triatoma infestans can generate an antimicrobial fragment from human fibrinopeptide. Thus T. infestans intestinal content was analyzed through Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), the eluted fractions were tested against Micrococcus luteus, Escherichia coli and Staphylococcus aureus, and the active fractions submitted to mass spectrometry. The data obtained were compared to hemoglobin databases to verify the presence of hemoglobin-derived fragments. Ten fractions eluted from chromatography presented antimicrobial activity, and when analyzed through mass spectrometry revealed the presence of 8 murine hemoglobin α-chain fragments and 24 fragments from murine hemoglobin ß fragments. Through the compilation of the fragments is possible to obtain over 67% coverage of both sequences. Part of the amino acid sequences corresponds to the sequences already identified on other intestinal contents of arthropods, and are highly conserved between the blood of other wild animals that are the most common intermediate hosts of Chagas' disease in Brazil and some of the main natural blood source for triatomines.

Quantification of low abundance Yersinia pestis markers in dried blood spots by immuno-capture and quantitative high-resolution targeted mass spectrometry.

Plague, caused by the bacterium Yersinia pestis, is still present in several countries worldwide. Besides, Y. pestis has been designated as Tier 1 agent, the highest rank of bioterrorism agents. In this context, reliable diagnostic methods are of great importance. Here, we have developed an original workflow based upon dried blood spot for simplified sampling of clinical specimens, and specific immuno-mass spectrometry monitoring of Y. pestis biomarkers. Targeted proteins were selectively enriched from dried blood spot extracts by multiplex immunocapture using antibody-coated magnetic beads. After accelerated on-beads digestion, proteotypic peptides were monitored by multiplex LC-MS/MS through the parallel reaction monitoring mode. The DBS-IC-MS assay was designed to quantify both F1 and LcrV antigens, although 10-fold lower sensitivity was observed with LcrV. The assay was successfully validated for F1 with a lower limit of quantification at 5 ng·mL-1 in spiked blood, corresponding to only 0.1 ng on spots. In vivo quantification of F1 in blood and organ samples was demonstrated in the mouse model of pneumonic plague. The new assay could help to simplify the laboratory confirmation of positive point of care F1 dipstick.

Placental ischemia-stimulated T-helper 17 cells induce preeclampsia-associated cytolytic natural killer cells during pregnancy.

Previous studies have demonstrated that T-helper 17 (TH17) cells and cytolytic natural killer (cNK) cells are increased in women with preeclampsia. In this study we investigated the role of placental ischemia-stimulated TH17 cells in induction of cNK cells in pregnancy. We further assessed the role of TH17 cell-mediated oxidative stress in facilitation of cNK cell activation in pregnancy by treating rats with the SOD mimetic tempol. CD4+/CD25- cells were isolated from reduced uterine perfusion pressure (RUPP) rats and differentiated into TH17 cells in vitro. On day 12 of gestation ( GD12), 1 × 106 placental ischemia-stimulated TH17 cells were injected into normal pregnant (NP) rats (NP + RUPP TH17 rats), and a subset of rats were treated with tempol (30 mg·kg-1·day-1) from GD12 to GD19 (NP + RUPP TH17 + tempol rats). On GD19, cNK cells, mean arterial pressure, fetal weight, and cNK cell-associated cytokines and proteins were measured. Placental cNK cells were 2.9 ± 1, 14.9 ± 4, and 2.8 ± 1.0% gated in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively. Mean arterial pressure increased from 96 ± 5 mmHg in NP rats to 118 ± 2 mmHg in NP + RUPP TH17 rats and was 102 ± 3 mmHg in NP + RUPP TH17 + tempol rats. Fetal weight was 2.37 ± 0.04, 1.95 ± 0.14, and 2.3 ± 0.05 g in NP, NP + RUPP TH17, and NP + RUPP TH17 + tempol rats, respectively. Placental IFNγ increased from 1.1 ± 0.6 pg/mg in NP rats to 3.9 ± 0.6 pg/mg in NP + RUPP TH17 rats. Placental perforin increased from 0.18 ± 0.18 pg/mg in NP rats to 2.4 ± 0.6 pg/mg in NP + RUPP TH17 rats. Placental levels of granzymes A and B followed a similar pattern. Treatment with tempol did not lower placental cNK cytokines or proteins. The results of the present study identify TH17 cells as a mediator of aberrant NK cell activation that is associated with preeclampsia.

Epidemiological analysis of serum anti-Pseudomonas aeruginosa PcrV titers in adults.

Of the various virulence mechanisms of the opportunistic pathogen Pseudomonas aeruginosa, the type III secretion system (TTSS) has been characterized as a major factor associated with acute lung injury, bacteremia and mortality. In addition, PcrV, a component protein of the TTSS, has been characterized as a protective antigen against infection with P. aeruginosa. This study comprised an epidemiological analysis of serum anti-PcrV titers in a cohort of Japanese adults. From April 2012 to March 2013, serum anti-PcrV titers of 198 volunteer participants undergoing anesthesia for scheduled surgeries were measured. The median, minimum and maximum serum anti-PcrV titers among the 198 participants were 4.09 nM, 1.01 nM and 113.81 nM, respectively. The maximum peaks in the histogram were within the anti-PcrV 2.00-4.99 nM titer range; values for 115 participants (58.1%) were within this range. Anti-PcrV titers were more than approximately three-fold greater (>12 nM) than the median value in 21 participants (10.6%). Ten-year interval age increases, history of treatment for traffic trauma, and a history of past surgery each showed statistically significant associations with higher anti-PcrV titers (i.e., >10 nM) than did the other factors assessed by binomial analysis. This study revealed a considerable variation in anti-PcrV titers in adult subjects without any obvious histories of infection with P. aeruginosa.

DNA methylation dynamics in blood after hematopoietic cell transplant.

Epigenetic deregulation is considered a common hallmark of cancer. Nevertheless, recent publications have demonstrated its association with a large array of human diseases. Here, we explore the DNA methylation dynamics in blood samples during hematopoietic cell transplant and how they are affected by pathophysiological events during transplant evolution. We analyzed global DNA methylation in a cohort of 47 patients with allogenic transplant up to 12 months post-transplant. Recipients stably maintained the donor's global methylation levels after transplant. Nonetheless, global methylation is affected by chimerism status. Methylation analysis of promoters revealed that methylation in more than 200 genes is altered 1 month post-transplant when compared with non-pathological methylation levels in the donor. This number decreased by 6 months post-transplant. Finally, we analyzed methylation in IFN-γ, FASL, IL-10, and PRF1 and found association with the severity of the acute graft-versus-host disease. Our results provide strong evidence that methylation changes in blood are linked to underlying physiological events and demonstrate that DNA methylation analysis is a viable strategy for the study of transplantation and for development of biomarkers.

BY55/CD160 cannot be considered a cytotoxic marker in cytomegalovirus-specific human CD8(+) T cells.

CD160/BY55 is a glucosyl-phosphatidylinositol (GPI)-anchored cell membrane receptor that is expressed primarily in natural killer (NK) cells. Its presence in CD8(+) T lymphocytes is considered to be a marker of cytotoxic activity, although there are few data in this regard. In the present work, we analysed the expression of CD160 in subpopulations of cytomegalovirus (CMV)-specific CD8(+) T cells. Subpopulations were defined by CD28 and CD57 expression and exhibited varying degrees of differentiation and cytotoxic potential, as evaluated by the expression of perforin, interferon (IFN)-gamma and interleukin (IL)-7Ralpha/CD127. We included subjects with different intensities of anti-viral immune response. Results showed that the terminally differentiated CD28(-) CD57(+) subset displaying the highest level of perforin expressed CD160 at a level similar to that of memory CD28(+) CD57(-)perforin(-) cells. A comparison of the expression of perforin in CD160(+) cells versus CD160(-) cells showed that expression was significantly higher in the absence of CD160. Interestingly, the CMV-specific CD8(+) T cell subset from a patient with ongoing CMV reactivation did not begin to express CD160 until day +92 of the follow-up period. Taken together, our data show that CD160 cannot be considered a cytotoxic marker in CMV-specific CD8(+) T cells.

Analysis of perforin expression in peripheral blood and lesions in severe and mild psoriasis.

BACKGROUND: Perforin is a membrane-disrupting protein that allows the entry of granzymes into a target cell inducing degradation of target substances in the cytoplasm and nucleus thus leading to programmed cell death or apoptosis. Recent work demonstrated a possible involvement of perforin mediated cytotoxicity in immunopathogenesis of psoriasis. OBJECTIVES: To investigate a difference in systemic (peripheral blood) and local (lesions) expression and distribution of perforin in psoriatic patients with severe and mild disease. METHODS: Flow cytometry was used for simultaneous detection of intracellular (perforin) and cell surface antigens in peripheral blood lymphocytes. The expression of perforin in skin lesions was evaluated by immunohistochemistry. RESULTS: Significant increase of perforin expression in T lymphocytes, especially cytotoxic CD8+ cells was found in severe psoriasis compared to mild disease (p<0.01 and p<0.05, respectively). There was also an increase of CD56+P+ NK cells (p<0.05) in severe compared to mild psoriasis. The psoriatic plaque of both, severe and mild disease were abundant with perforin showing no significant difference on local level. CONCLUSION: Based on our results we suggest the association between perforin expression and disease severity.

Diagnosis of acute renal allograft rejection by analyzing whole blood mRNA expression of lymphocyte marker molecules.

BACKGROUND: Currently, the diagnosis of acute rejection after kidney transplantation is based on a kidney biopsy taken after clinical rejection suspicion. A robust, noninvasive diagnostic method would allow easier and more frequent monitoring of the patient and the graft. Potentially, a straightforward method would be the analysis of lymphocyte marker molecule expression from whole blood samples. METHODS: Whole blood samples were collected prospectively in a single kidney transplantation center from 50 adult kidney recipients transplanted between 2001 and 2005. The mRNA expression of granzyme B, perforin, FasL, granulysin, CD154, ICOS, CTLA4 and PD-1 were analyzed with real-time quantitative polymerase chain reaction. RESULTS: The expression of ICOS and CD154 were significantly lower in rejection patients than in control patients (P<0.001). Both genes gave statistically significant area under receiver operating characteristic curve (AUC; 0.87, 0.88) with 84% sensitivity and 100% specificity for CD154 and 76% and 86% for ICOS, respectively. In paired rejection and postrejection therapy samples, the expression of both genes significantly increased during rejection therapy (P<0.001). When rejection patients were compared to patients biopsied because of other reasons of graft dysfunction, both CD154 and ICOS were lower in rejection patients but only CD154 was statistically significant (P=0.028, AUC=0.740, sensitivity 52%, specificity 90%). The other studied genes gave no consistent statistically significant results. CONCLUSIONS: The whole blood gene expression quantities of costimulatory molecules CD154 and ICOS reasonably robustly differentiated rejection patients from control patients. The clinical use of the analysis is limited by poor capability to differentiate patients with rejection from patients with other causes of graft dysfunction.

Healthy lifestyles are associated with higher levels of perforin, granulysin and granzymes A/B-expressing cells in peripheral blood lymphocytes.

OBJECTIVE: It is well documented that natural killer (NK) cells provide host defense against tumors and viruses. We previously showed that lifestyle affects human NK and LAK activities. In order to explore the underlying mechanism, we investigated the effect of lifestyle on intracellular perforin, granulysin, and granzymes A/B in peripheral blood lymphocytes (PBL). METHODS: 114 healthy male subjects, aged 20-59 years, from a large company in Osaka, Japan were selected with informed consent. The subjects were divided into groups reporting good, moderate, and poor lifestyles according to their responses on a questionnaire regarding eight health practices (cigarette smoking, alcohol consumption, sleeping hours, working hours, physical exercise, eating breakfast, balanced nutrition, and mental stress). Peripheral blood was taken, and numbers of NK, T, perforin, granulysin, and granzymes A/B-expressing cells in PBL were measured by flow cytometry. RESULTS: Subjects with good or moderate lifestyle showed significantly higher numbers of NK, and perforin, granulysin, and granzymes A/B-expressing cells and a significantly lower number of T cells in PBL than subjects with poor lifestyle. Among the eight health practices, cigarette smoking, physical exercise, eating breakfast, and balanced nutrition significantly affect the numbers of NK, T cells, perforin, granulysin, and/or granzymes A/B-expressing cells, and alcohol consumption significantly affects the number of granzyme A-expressing cells. On the other hand, mental stress, sleeping, and working hours had no effect on those parameters. CONCLUSIONS: Taken together, these findings indicate that poor lifestyle significantly decreases the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells in PBL.

Differing activation status and immune effector molecule expression profiles of neonatal and maternal lymphocytes in an African population.

Higher susceptibility of newborns to infections has been attributed to the hypo-responsiveness of their cellular immune system. Here we compared the activation status and expression of cytokines and cytotoxic molecules of cord versus maternal peripheral blood mononuclear cells in an African population. Human leucocyte antigen-DR was expressed on a lower percentage of cord compared to maternal gammadelta and CD3(+) T cells. Similarly, a lower proportion of cord versus maternal gammadelta and CD3(+) T cells displayed perforin, granzyme B and cytokine activity either ex vivo or following non-specific stimulation in vitro. In contrast, comparable proportions of cord and maternal CD94(+) CD3(-) natural killer (NK) cells showed perforin and granzyme B expression ex vivo. We conclude that cord blood gammadelta and CD3(+) T cells are functionally hypo-responsive as reflected by reduced numbers of such cells expressing either an activation marker, T helper 1 (Th1) and Th2 cytokines or cytotoxic effector molecules. The similarity in numbers of cord and maternal CD94(+) CD3(-) cells expressing cytotoxic effector molecules suggests that neonatal Africans' NK cells may be functionally mature.