Comparison of immune activation of the COVID vaccines: ChAdOx1, BNT162b2, mRNA-1273, BBIBP-CorV, and Gam-COVID-Vac from serological human samples in Hungary showed higher protection after mRNA-based immunization.

OBJECTIVE: To gain insight into the different protective mechanisms of approved vaccines, this study focuses on the comparison of humoral and cellular immune responses of five widely used vaccines including ChAdOx1 (AZD1222, AstraZeneca), BNT162b2 (Pfizer), mRNA-1273 (Moderna), BBIBP-CorV (Sinopharm), and Gam-COVID-Vac (Sputnik V). MATERIALS AND METHODS: Isolated plasma from 95 volunteers' blood samples was used to measure anti-SARS-CoV-2 humoral and cellular immune responses. Positive controls were recovered patients from COVID-19 (unvaccinated). Specific quantification kits for anti-nucleocapsid IgG, anti-Spike protein IgG, neutralizing antibodies as well as specific SARS-CoV-2 antigens for T-cell activation were used and Spearman correlation and matrix analyses were performed to compare overall immune responses. RESULTS: Nucleocapsid antibodies were significantly higher for the BBIBP-CorV and convalescent group when compared to other vaccines. In contrast, subjects vaccinated with BNT162b2 and mRNA-1273 presented significantly higher anti-spike IgG. In fact, 9.1% of convalescent, 4.5% of Gam-COVID-Vac, 28.6% of ChAdOx1, and 12.5% of BBIBP-CorV volunteers did not generate anti-spike IgG. Similarly, a positive correlation was observed after the neutralization assay. T-cell activation studies showed that mRNA-based vaccines induced a T-cell driven immune response in all cases, while 55% of convalescents,  8% of BNT162b1,  12,5% of mRNA-1273, 9% of Gam-COVID-Vac,  57% of ChAdOx1,  and  56% of BBIBP-CorV subjects presented no cellular response. Further correlation matrix analyses indicated that anti-spike IgG and neutralizing antibodies production, and T-cell activation follow the same trend after immunization. CONCLUSIONS: RNA-based vaccines induced the most robust adaptive immune activation against SARS-CoV-2 by promoting a significantly higher T-cell response, anti-spike IgG and neutralization levels. Vector-based vaccines protected against the virus at a comparable level to convalescent patients.

Nuclear factor κB (NF-κB) suppresses food intake and energy expenditure in mice by directly activating the Pomc promoter.

AIMS/HYPOTHESIS: While chronic low-grade inflammation is associated with obesity, acute inflammation reduces food intake and leads to negative energy balance. Although both types of inflammation activate nuclear factor κB (NF-κB) signalling, it remains unclear how NF-κB activation results in opposite physiological responses in the two types of inflammation. The goal of this study was to address this question, and to understand the link between inflammation and leptin signalling. METHODS: We studied the ability of NF-κB to modulate Pomc transcription, and how it impinges on signal transducer and activator of transcription 3 (STAT3)-mediated leptin signalling by using a combination of animal models, biochemical assays and molecular biology. RESULTS: We report that suppression of food intake and physical movement with acute inflammation is not dependent on STAT3 activation in pro-opiomelanocortin (POMC) neurons. Under these conditions, activated NF-κB independently leads to increased Pomc transcription. Electrophoretic mobility shift assay and chromatin immunoprecipitation (ChIP) experiments reveal that NF-κB v-rel reticuloendotheliosis viral oncogene homologue A (avian) (RELA [also known as p65]) binds to the Pomc promoter region between -138 and -88 bp, which also harbours the trans-acting transcription factor 1 (SP1) binding site. We found significant changes in the methylation pattern at this region and reduced Pomc activation under chronic inflammation induced by a high-fat diet. Furthermore, RELA is unable to bind and activate transcription when the Pomc promoter is methylated. Finally, RELA binds to STAT3 and inhibits STAT3-mediated promoter activity, suggesting that RELA, possibly together with forkhead box-containing protein 1 (FOXO1), may prevent STAT3-mediated leptin activation of the Pomc promoter. CONCLUSIONS/INTERPRETATION: Our study provides a mechanism for the involvement of RELA in the divergent regulation of energy homeostasis in acute and chronic inflammation.

Muscle regeneration is undisturbed by repeated polytraumatic injury.

INTRODUCTION: Clinical observations suggest that repeated injury within a week after a traumatic event impairs the regeneration of tissues. Our aim was to investigate the effect of repeated trauma on the proliferation of satellite cells in skeletal muscle tissue. MATERIALS AND METHODS: Cold lesion injury was performed in the soleus muscle and in the motor cortex of anesthetized male Wistar rats 0, 1, or 2 times with 7 day intervals between the interventions. Following the last operation, 5-bromo-2'-deoxyuridine was injected i.p. for 6 or 12 days to label dividing cells. Gut epithelium was used as positive control. Immunohistochemistry was performed 1 and 5 weeks after the last injury and the sections were analyzed with confocal microscopy. RESULTS: In the case of repeated trauma, the percentage of proliferating cells remained the same compared to single hit animals after 1 week (28.0 ± 2.5% and 29.6 ± 3.0%) as well as after 5 weeks (13.9 ± 1.8% and 14.5 ± 2.2%). CONCLUSION: The second hit phenomenon is probably due to systemic factors rather than to a diminished regenerating potential of injured soft tissues.

Nitrative stress and poly(ADP-ribose) polymerase activation in healthy and gestational diabetic pregnancies.

AIMS/HYPOTHESIS: Increased oxidative-nitrosative stress, poly(ADP-ribose) polymerase (PARP) activation and subsequent cellular damage play important roles in the complications of both diabetes mellitus and pregnancy. Our aim was to investigate nitrative stress and PARP activity levels during normal and gestational diabetic (GDM) pregnancy in both maternal and fetal tissues. METHODS: Blood samples were collected during pregnancy (weeks 16-29 and 36-40), and placental and umbilical cord tissues were harvested after delivery from healthy volunteers and GDM patients subjected to a carbohydrate-restricted diet or insulin treatment. Immunohistochemical staining was performed on leucocytes and tissue sections using anti-nitrotyrosine (NT), anti-poly(ADP-ribose) (PAR) and anti-apoptosis inducing factor antibodies. RESULTS: In healthy pregnancies the intensity of NT and PAR staining of leucocytes correlated positively with gestational week (R (2) = 0.43, p < 0.01 and R (2) = 0.49, p < 0.001, respectively). In patients on a carbohydrate-restricted diet PAR staining was already strong in weeks 16-29 (p < 0.001 vs control) and did not increase further. In weeks 16-29 there was a correlation between PAR staining and the 2 h value of the oral glucose tolerance test (R (2) = 0.49, p < 0.001). Patients with the highest level of leucocyte PARP activity later required insulin therapy, which decreased the intensity of NT and PAR staining. Placental and umbilical cord tissues also had a higher level of nitrative stress markers in GDM pregnancies, but the highest level of PARP activity was observed after insulin therapy. CONCLUSIONS/INTERPRETATION: Continuous elevation of tyrosine nitration and PARP activation may be considered physiological during pregnancy. However, the high level of PARP activity in early pregnancy may signal the subsequent development of severe GDM.

Reactive oxygen species mediate a cellular 'memory' of high glucose stress signalling.

AIMS/HYPOTHESIS: A long-term 'memory' of hyperglycaemic stress, even when glycaemia is normalised, has been previously reported in endothelial cells. In this report we sought to duplicate and extend this finding. MATERIALS AND METHODS: HUVECs and ARPE-19 retinal cells were incubated in 5 or in 30 mmol/l glucose for 3 weeks or subjected to 1 week of normal glucose after being exposed for 2 weeks to continuous high glucose. HUVECs were also treated in this last condition with several antioxidants. Similarly, four groups of rats were studied for 3 weeks: (1) normal rats; (2) diabetic rats not treated with insulin; (3) diabetic rats treated with insulin during the last week; and (4) diabetic rats treated with insulin plus alpha-lipoic acid in the last week. RESULTS: In human endothelial cells and ARPE-19 retinal cells in culture, as well as in the retina of diabetic rats, levels of the following markers of high glucose stress remained induced for 1 week after levels of glucose had normalised: protein kinase C-beta, NAD(P)H oxidase subunit p47phox, BCL-2-associated X protein, 3-nitrotyrosine, fibronectin, poly(ADP-ribose) Blockade of reactive species using different approaches, i.e. the mitochondrial antioxidant alpha-lipoic acid, overexpression of uncoupling protein 2, oxypurinol, apocynin and the poly(ADP-ribose) polymerase inhibitor PJ34, interrupted the induction both of high glucose stress markers and of the fluorescent reactive oxygen species (ROS) probe CM-H(2)DCFDA in human endothelial cells. Similar results were obtained in the retina of diabetic rats with alpha-lipoic acid added to the last week of normalised glucose. CONCLUSIONS/INTERPRETATION: These results provide proof-of-principle of a ROS-mediated cellular persistence of vascular stress after glucose normalisation.

S-nitrosoglutathione-containing hydrogel increases dermal blood flow in streptozotocin-induced diabetic rats.

BACKGROUND: Endothelial dysfunction is characterized by decreased vasodilatory capacity of the arterioles mainly due to the reduced release of nitric oxide (NO). Application of NO donors may prevent or even reverse the consequences of endothelial dysfunction, such as diabetic leg ulcers. OBJECTIVES: To investigate the vasodilatory capacity and the possible side-effects of topical application of an NO donor-containing hydrogel in diabetic rats. METHODS: S-nitrosoglutathione (GSNO) was incorporated in Pluronic F127 hydrogel and applied on the foot sole skin of healthy and streptozotocin-induced diabetic rats. Blood flow was monitored using a laser-Doppler probe. Nitrotyrosine formation, a possible side-effect of GSNO action, was evaluated by Western blotting of skin protein extracts. Systemic circulatory side-effects were investigated by monitoring blood pressure and heart rate during the application. RESULTS: The hydrogel alone did not induce any changes in microvascular flow, while GSNO-containing hydrogel caused a twofold increase in perfusion. This effect was similar in diabetic and healthy animals. Topical GSNO application did not increase the nitrotyrosine content of skin proteins, nor did it have any effect on blood pressure or heart rate. CONCLUSIONS: Dermal application of GSNO may be an effective treatment for promoting the local vasodilation in both healthy and diabetic states, without inducing protein nitration or alterations in blood pressure or heart rate.

Inhibition of neuronal nitric oxide synthase-mediated activation of poly(ADP-ribose) polymerase in traumatic brain injury: neuroprotection by 3-aminobenzamide.

Focal traumatic injury to the cerebral cortex is associated with early activation of the neuronal isoform of nitric oxide synthase (nNOS), where high concentrations of nitric oxide-derived free radicals elicit extensive DNA damage. Subsequent activation of the nuclear repair enzyme poly(ADP-ribose) polymerase (PARP) causes a severe energy deficit leading to the ultimate demise of affected neurons. Little is known about the temporal relationship of nNOS and PARP activation and the neuroprotective efficacy of their selective blockade in traumatic brain injury. To determine the relationship of nNOS and PARP activation, brain injury was induced by cryogenic lesion to the somatosensory cortex applying a pre-cooled cylinder after trephination for 6 s to the intact dura mater. Pre-treatment with 3-bromo-7-nitroindazole (BrNI; 25 mg/kg, i.p.), and pre- or combined pre- and post-treatment with 3-aminobenzamide (AB; 10 mg/kg (i.c.v.) or 10 mg/kg/h (i.p.)) were used to inhibit nNOS and PARP, respectively. Cold lesion-induced changes in the somatosensory cortex and neuroprotection by BrNI and AB were determined using immunocytochemistry and immunodot-blot for detection of poly(ADP-ribose; PAR), the end-product of PARP activation, and the triphenyltetrazolium-chloride assay to assess lesion volume. PAR immunoreactivity reached its peak 30 min post-lesion and was followed by gradual reduction of PAR immunolabeling. BrNI pre-treatment significantly decreased the lesion-induced PAR concentration in damaged cerebral cortex. Pre-treatment by i.c.v. infusion of AB markedly diminished cortical PAR immunoreactivity and significantly reduced the lesion volume 24 h post-injury. In contrast, i.p. AB treatment remained largely ineffective. In conclusion, our data indicate early activation of PARP after cold lesion that is, at least in part, related to nNOS induction and supports the relevance of nNOS and/or PARP inhibition to therapeutic approaches of traumatic brain injury.

Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia.

Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K(ATP) channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia.

NO synthase blockade induces chaotic cerebral vasomotion via activation of thromboxane receptors.

BACKGROUND AND PURPOSE: Instability of the vascular tone (vasomotion) develops in several cerebrovascular diseases associated with endothelial dysfunction. The aim of the present study was to characterize cerebral vasomotion induced by diminished NO production with quantitative evaluation and chaos analysis. We tested the hypothesis that activation of thromboxane receptors mediates chaotic vasomotion after NO synthase (NOS) inhibition. METHODS: Measurements of vascular tension were carried out in isolated rat middle cerebral arteries. The extent of vasomotion was characterized by tension instability, whereas vasomotion complexity was assessed by chaos analysis. RESULTS: Blocking the basal NO release by N(omega)-nitro-L-arginine (L-NA) induced vasomotion, which was further enhanced and became irregular after UTP administration. The NO donor sodium nitroprusside was able to reverse this effect, and stable steady-state conditions reappeared. The guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) or coapplication of ODQ and L-NA had an effect identical to that of L-NA alone. Vasoconstriction by K(+) failed to induce vasomotion in intact vessels or in the presence of L-NA or ODQ. The thromboxane receptor antagonist ICI 192605 dose-dependently attenuated the vasomotion induced by L-NA and UTP, and the thromboxane-receptor agonist U-46619 induced significant vasomotion in intact vessels. CONCLUSIONS: The lack of NO in cerebral vessels provokes vulnerability to chaotic vasomotion, which can be triggered by the administration of UTP, whereas excess NO reverses it to stable conditions. The vasomotion after blockade of the NO-cGMP pathway is mediated by activation of thromboxane receptors.

Involvement of prostanoid release in the mediation of UTP-induced cerebrovascular contraction in the rat.

The interaction between uridine-5'-triphosphate (UTP) and prostanoids was studied in isolated rat middle cerebral arteries (MCAs). The strong contractions in MCA segments induced by UTP were weakened significantly by indomethacin and more markedly by the thromboxane receptor antagonist ICI 192605. Thromboxane A(2) (TXA(2)) release by MCAs was below the detection limit of the chemiluminescence enzyme immunoassay, but increased TXA(2) formation was detected in basilar arteries in the presence of UTP. Prostacyclin (PGI(2)) formation by MCAs also increased in the presence of UTP. These results suggest that UTP stimulates the release of both TXA(2) and PGI(2) from the rat MCA but the vascular effect of TXA(2) is dominant.

Functional importance of neuronal nitric oxide synthase in the endothelium of rat basilar arteries.

The function of the neuronal isoform of nitric oxide synthase (nNOS) was studied by comparing the effects of the specific nNOS blocker 7-nitro indazole monosodium salt (7-NINA) with that of the general NOS inhibitor N(G)-nitro-L-arginine (L-NA) in isolated rat basilar arteries (BAs). 7-NINA had no significant effect on the resting tone of the vessels, while both L-NA and 1H-[1,2,4]oxadiazolo[4, 3-a]quinoxalin-1-one (ODQ), a selective inhibitor of the soluble guanylyl cyclase, induced contraction. The relaxant effect of bradykinin was attenuated in the presence of L-NA but was not changed by 7-NINA. In contrast, 7-NINA markedly reduced the acetylcholine-induced, endothelium-dependent relaxation. These results demonstrate that nNOS contributes significantly to the relaxant effect of acetylcholine, indicating the functional importance of this isoenzyme in the cerebrovascular endothelium.

The cerebrocortical microcirculatory effect of nitric oxide synthase blockade is dependent upon baseline red blood cell flow in the rat.

The effects of nitric oxide synthase (NOS) blockade on the cerebrocortical microcirculation were investigated under physiological conditions in anesthetized ventilated rats using laser-Doppler (LD) flowmetry. LD flow values of the parietal cortex were determined before and after systemic administration of the NOS inhibitor N(G)-nitro-L-arginine-methyl-esther. NOS blockade reduced the LD flow significantly and the magnitude of the reduction was in close correlation with the baseline value. Synchronized sinus-wave-like LD flow oscillations were observed frequently after NOS inhibition and their appearance was also dependent on the high baseline flow values. These results indicate marked, baseline-dependent differences in the cerebrocortical blood flow response to the inhibition of the nitric oxide pathway, and may suggest that areas with high resting red blood cell flow express high NOS activity.

Selective inhibition of neuronal nitric oxide synthase fails to alter the resting tension and the relaxant effect of bradykinin in isolated rat middle cerebral arteries.

The role of the neuronal isoform of the nitric oxide (NO) synthase (nNOS) in the regulation of the cerebrovascular tone was studied in vitro. Selective inhibition of nNOS by 7-nitro indazole monosodium salt (7-NINA) failed to alter the resting tension and the relaxant effect of bradykinin in isolated rat middle cerebral arteries. These results indicate that 1./ 7-NINA is selective for nNOS and 2./ cerebrovascular nNOS is involved neither in the resting NO production nor in the mediation of the relaxant effect of bradykinin. Therefore, nNOS-derived NO that contributes to the maintenance of the resting cerebral blood flow in vivo appears to be released from neurons and/or glial cells.