Clinical and molecular significance of flow cytometric analysis for reactive oxygen species production and residual p67phox expression in p67phox-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by molecular defects in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. p67phox-CGD is an autosomal recessive CGD, which is caused by a defect in the cytosolic components of NADPH oxidase, p67phox, encoded by NCF2. We previously established a flow cytometric analysis for p67phox expression, which allows accurate assessment of residual protein expression in p67phox-CGD. We evaluated the correlation between oxidase function and p67phox expression, and assessed the relevancy to genotypes and clinical phenotypes in 11 patients with p67phox-CGD. Reactive oxygen species (ROS) production by granulocytes was evaluated using dihydrorhodamine-1,2,3 (DHR) assays. p67phox expression was evaluated in the monocyte population. DHR activity and p67phox expression were significantly correlated (r = 0.718, p < 0.0162). Additionally, DHR activity and p67phox expression were significantly higher in patients carrying one missense variant in combination with one nonsense or frameshift variant in the NCF2 gene than in patients with only null variants. The available clinical parameters of our patients (i.e., age at disease onset, number of infectious episodes, and each infection complication) were not linked with DHR activity or p67phox expression levels. In summary, our flow cytometric analysis revealed a significant correlation between residual ROS production and p67phox expression. More deleterious NCF2 genotypes were associated with lower levels of DHR activity and p67phox expression. DHR assays and protein expression analysis by using flow cytometry may be relevant strategies for predicting the genotypes of p67phox-CGD.

Functional NADPH oxidase 2 in T cells amplifies salt-sensitive hypertension and associated renal damage.

Infiltrating T cells in the kidney amplify salt-sensitive (SS) hypertension and renal damage, but the mechanisms are not known. Genetic deletion of T cells (SSCD247-/-) or of the p67phox subunit of NADPH oxidase 2 (NOX2; SSp67phox-/-) attenuates SS hypertension in the Dahl SS rat. We hypothesized that reactive oxygen species produced by NOX2 in T cells drive the SS phenotype and renal damage. T cells were reconstituted by adoptively transferring splenocytes (∼10 million) from the Dahl SS (SS→CD247) rat, the SSp67phox-/- rat (p67phox→CD247), or only PBS (PBS→CD247) into the SSCD247-/- rat on postnatal day 5. Animals were instrumented with radiotelemeters and studied at 8 wk of age. There were no detectable differences in mean arterial pressure (MAP) or albuminuria between groups when rats were maintained on a low-salt (0.4% NaCl) diet. After 21 days of high-salt diet (4.0% NaCl), MAP and albuminuria were significantly greater in SS→CD247 rats compared with p67phox→CD247 and PBS→CD247 rats. Interestingly, there was no difference between p67phox→CD247 and PBS→CD247 rats in albuminuria or MAP after 21 days. The lack of CD3+ cells in PBS→CD247 rats and the presence of CD3+ cells in rats that received the T cell transfer demonstrated the effectiveness of the adoptive transfer. No differences in the number of CD3+, CD4+, or CD8+ cells were observed in the kidneys of SS→CD247 and p67phox→CD247 rats. These results indicate that reactive oxygen species produced by NOX2 in T cells participates in the amplification of SS hypertension and renal damage.NEW & NOTEWORTHY Our current work used the adoptive transfer of T cells that lack functional NADPH oxidase 2 into a genetically T cell-deficient Dahl salt-sensitive (SS) rat model. The results demonstrated that reactive oxygen species produced by NADPH oxidase 2 in T cells participate in the amplification of SS hypertension and associated renal damage and identifies a potential mechanism that exacerbates the salt-sensitive phenotype.

Isoform-Directed Control of c-Myc Functions: Understanding the Balance from Proliferation to Growth Arrest.

The transcription factor c-Myc, a key regulator of cellular processes, has long been associated with roles in cell proliferation and apoptosis. This review analyses the multiple functions of c-Myc by examining the different c-Myc isoforms in detail. The impact of different c-Myc isoforms, in particular p64 and p67, on fundamental biological processes remains controversial. It is necessary to investigate the different isoforms in the context of proto-oncogenesis. The current knowledge base suggests that neoplastic lesions may possess the means for self-destruction via increased c-Myc activity. This review presents the most relevant information on the c-Myc locus and focuses on a number of isoforms, including p64 and p67. This compilation provides a basis for the development of therapeutic approaches that target the potent growth arresting and pro-apoptotic functions of c-Myc. This information can then be used to develop targeted interventions against specific isoforms with the aim of shifting the oncogenic effects of c-Myc from pro-proliferative to pro-apoptotic. The research summarised in this review can deepen our understanding of how c-Myc activity contributes to different cellular responses, which will be crucial in developing effective therapeutic strategies; for example, isoform-specific approaches may allow for precise modulation of c-Myc function.

p67: a cryptic lysosomal hydrolase in Trypanosoma brucei?

p67 is a type I transmembrane glycoprotein of the terminal lysosome of African trypanosomes. Its biosynthesis involves transport of an initial gp100 ER precursor to the lysosome, followed by cleavage to N-terminal (gp32) and C-terminal (gp42) subunits that remain non-covalently associated. p67 knockdown is lethal, but the only overt phenotype is an enlarged lysosome (~250 to >1000 nm). Orthologues have been characterized in Dictyostelium and mammals. These have processing pathways similar to p67, and are thought to have phospholipase B-like (PLBL) activity. The mouse PLBD2 crystal structure revealed that the PLBLs represent a subgroup of the larger N-terminal nucleophile (NTN) superfamily, all of which are hydrolases. NTNs activate by internal autocleavage mediated by a nucleophilic residue, i.e. Cys, Ser or Thr, on the upstream peptide bond to form N-terminal α (gp32) and C-terminal ß (gp42) subunits that remain non-covalently associated. The N-terminal residue of the ß subunit is then catalytic in subsequent hydrolysis reactions. All PLBLs have a conserved Cys/Ser dipeptide at the α/ß junction (Cys241/Ser242 in p67), mutation of which renders p67 non-functional in RNAi rescue assays. p67 orthologues are found in many clades of parasitic protozoa, thus p67 is the founding member of a group of hydrolases that likely play a role broadly in the pathogenesis of parasitic infections.

Role of NADPH oxidase in MAPK signaling activation by a 50 Hz magnetic field in human neuroblastoma cells.

Our previous studies have shown that intermittent exposure to a 50-Hz, 100-µT sine wave magnetic field (MF) promotes human NB69 cell proliferation, mediated by activation of the epidermal growth factor receptor (EGFR) and pathways MAPK-ERK1/2 and p38; being the effects on proliferation and p38 activation blocked by the chelator N-acetylcysteine. The present work investigates the MF effects on free radical (FR) production, and the potential involvement of NADPH oxidase, the main source of reactive oxygen species (ROS), in the MF-induced activation of MAPK pathways. To this end, the field effects on MAPK-ERK1/2, -p38 and -JNK activation in the presence or absence of the NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI), as well as the expression of the p67phox subunit, were analyzed. The results revealed that field exposure increases FR production and induces early, transient expression of the cytosolic component of the NADPH oxidase, p67phox. Also, the MF-induced activation of the MAPK-JNK pathway, but not that of -ERK1/2 or -p38 pathways, was prevented in the presence of the DPI, which has been shown to significantly reduce p67phox expression. These data, together with those from previous studies, identify various, FR-dependent or -independent mechanisms, involved in the MF-induced proliferative response mediated by MAPK signaling activation.

Novel NCF2 Mutation Causing Chronic Granulomatous Disease.

Chronic granulomatous disease (CGD) is a rare primary immunodeficiency disorder caused by defects in the NADPH oxidase complex. Mutations in NCF2 encoding the cytosolic factor p67phox result in autosomal recessive CGD. We describe three patients with a novel c.855G>C NCF2 mutation presenting with diverse clinical phenotype. Two siblings were heterozygous for the novel mutation and for a previously described exon 8-9 duplication, while a third unrelated patient was homozygous for the novel mutation. Mutation pathogenicity was confirmed by abnormal DHR123 assay and absent p67phox production and by sequencing of cDNA which showed abnormal RNA splicing. Clinically, the homozygous patient presented with suspected early onset interstitial lung disease and NCF2 mutation was found on genetic testing performed in search for surfactant-related defects. The two siblings also had variable presentation with one having history of severe pneumonia, lymphadenitis, and recurrent skin abscesses and the other presenting in his 30s with discoid lupus erythematosus and without significant infectious history. We therefore identified a novel pathogenic NCF2 mutation causing diverse and unusual clinical phenotype.

Variant analysis of the sporozoite surface antigen gene reveals that asymptomatic cattle from wildlife-livestock interface areas in northern Tanzania harbour buffalo-derived T. parva.

Buffalo-derived Theileria parva can 'break through' the immunity induced by the infection and treatment vaccination method (ITM) in cattle. However, no such 'breakthroughs' have been reported in northern Tanzania where there has been long and widespread ITM use in pastoralist cattle, and the Cape buffalo (Syncerus caffer) is also present. We studied the exposure of vaccinated and unvaccinated cattle in northern Tanzania to buffalo-derived T. parva using p67 gene polymorphisms and compared this to its distribution in vaccinated cattle exposed to buffalo-derived T. parva in central Kenya, where vaccine 'breakthroughs' have been reported. Additionally, we analysed the CD8+ T cell target antigen Tp2 for positive selection. Our results showed that 10% of the p67 sequences from Tanzanian cattle (n = 39) had a buffalo type p67 (allele 4), an allele that is rare among East African isolates studied so far. The percentage of buffalo-derived p67 alleles observed in Kenyan cattle comprised 19% of the parasites (n = 36), with two different p67 alleles (2 and 3) of presumptive buffalo origin. The Tp2 protein was generally conserved with only three Tp2 variants from Tanzania (n = 33) and five from Kenya (n = 40). Two Tanzanian Tp2 variants and two Kenyan Tp2 variants were identical to variants present in the trivalent Muguga vaccine. Tp2 evolutionary analysis did not show evidence for positive selection within previously mapped epitope coding sites. The p67 data indicates that some ITM-vaccinated cattle are protected against disease induced by a buffalo-derived T. parva challenge in northern Tanzania and suggests that the parasite genotype may represent one factor explaining this.

NADPH oxidase activation in neutrophils: Role of the phosphorylation of its subunits.

Neutrophils are key cells of innate immunity and during inflammation. Upon activation, they produce large amounts of superoxide anion (O2 -. ) and ensuing reactive oxygen species (ROS) to kill phagocytized microbes. The enzyme responsible for O2 -. production is called the phagocyte NADPH oxidase. This is a multicomponent enzyme system that becomes active after assembly of four cytosolic proteins (p47phox , p67phox , p40phox and Rac2) with the transmembrane proteins (p22phox and gp91phox , which form the cytochrome b558 ). gp91phox represents the catalytic subunit of the NADPH oxidase and is also called NOX2. NADPH oxidase-derived ROS are essential for microbial killing and innate immunity; however, excessive ROS production induces tissue injury and prolonged inflammatory reactions that contribute to inflammatory diseases. Thus, NADPH oxidase activation must be tightly regulated in time and space to limit ROS production. NADPH oxidase activation is regulated by several processes such as phosphorylation of its components, exchange of GDP/GTP on Rac2 and binding of p47phox and p40phox to phospholipids. This review aims to provide new insights into the role of the phosphorylation of the NADPH oxidase components, that is gp91phox , p22phox , p47phox , p67phox and p40phox , in the activation of this enzyme.

Chronic Granulomatous Disease Due to Neutrophil Cytosolic Factor (NCF2) Gene Mutations in Three Unrelated Families.

Chronic granulomatous disease (CGD) is an inheritable and genetically heterogeneous disease resulting from mutations in different subcomponents of the NADPH oxidase system. Mutations in the NCF2 gene account for <5% of all cases of CGD. We analyzed the clinical and laboratory findings of CGD with mutations in the NCF2 gene from amongst our cohort of CGD patients. A homozygous mutation (c.835_836delAC, p.T279fsX294), a deletion in NCF2 gene was found in two cases. In the third case, two heterozygous mutations were detected, IVS13-2A>T on one allele and c.1099C>T (p.) on the other allele. The mother of this child was a carrier for the IVS13-2A>T mutation. All three cases had colitis, and it was the initial symptom in two patients. One of the patients also developed a lung abscess due to Nocardia cyriacigeorgica.

Role of Nox4 and p67phox subunit of Nox2 in ROS production in response to increased tubular flow in the mTAL of Dahl salt-sensitive rats.

Nox4 and Nox2 are the most abundant NADPH oxidases (Nox) in the kidney and have been shown to contribute to hypertension, renal oxidative stress, and injury in Dahl salt-sensitive (SS) hypertensive rats. The present study focused on the role of Nox4 and p67phox/Nox2 in the generation of H2O2 and O2 (·-) in the renal medullary thick ascending limb of Henle (mTAL) of SS rats in response to increasing luminal flow (from 5 to 20 nl/min). Nox4 and p67phox/Nox2 genes were found to be expressed in the mTAL of SS rats. Responses of SS rats were compared with those of SS rats with knockout of Nox4 (SS(Nox4-/-)) or functional mutation of p67phox (SS(p67phox-/-)). Nox4 was the dominant source of increased intracellular H2O2 production in response to increased luminal flow as determined using the fluorescent dye peroxyfluor 6-AM (PF6-AM). The rate of mitochondrial H2O2 production [as determined by mitochondria peroxy yellow 1 (mitoPY1)] was also significantly reduced in SS(Nox4-/-) compared with SS rats, but not in SS(p67phox-/-) rats. In contrast, intracellular superoxide (O2 (·-)) production (the ratio of ethidium to dihydroethidium) in the mTAL of SS(Nox4-/-) rats was nearly identical to that of SS rats in response to luminal flow, indicating that Nox4 made no measurable contribution. mTAL O2 (·-) production was reduced in SS(p67phox-/-) compared with SS rats at the lower luminal flow of 5 nl/min and progressively increased when perfusion was changed to 20 nl/min. We conclude that increased mTAL luminal flow results in increases in intracellular and mitochondrial H2O2, which are dependent on the presence of Nox4, and that p67phox/Nox2 accounts solely for increases in O2 (·-) production.

Regulation of the NADPH oxidase and associated ion fluxes during phagocytosis.

The production of reactive oxygen species (ROS) within immune cell phagosomes is critical for antimicrobial activity and for correct antigen processing, and influences signaling pathways that direct host responses to infection and inflammation. Because excess oxidants can cause tissue damage and oxidative stress, phagocytes must precisely control both the location and timing of NADPH oxidase activity. How differential regulation is achieved at phagosomes is not well understood. Recent studies have revealed that the PI(3)P phosphoinositide plays an important role in locally boosting phagosomal NADPH oxidase activity through its binding to the p40(phox) NADPH oxidase subunit. Furthermore, phox subunit dynamics at phagosomes may regulate the timing of the oxidative burst. Novel elements regulating catalytic core trafficking include Rab27 and SNAP-23. In addition to trafficking events, the activity of the electrogenic oxidase is also governed by ionic fluxes, which are constrained at phagosomes owing to low intraphagosomal volume and dynamic display of channels, transporters, and pumps. New insights on the interdependence of phagosomal pH and ROS have been recently elucidated, and chloride channels important for microbicidal functions, including CFTR, and CLIC family channels, have been identified. Finally, periphagosomal calcium microdomains and calcium-dependent S100A8/9 protein recruitment may help fine-tune spatiotemporal regulation of NADPH oxidase activation for an effective immune response.

Arachidonic acid induces direct interaction of the p67(phox)-Rac complex with the phagocyte oxidase Nox2, leading to superoxide production.

The phagocyte NADPH oxidase Nox2, heterodimerized with p22(phox) in the membrane, is dormant in resting cells but becomes activated upon cell stimulation to produce superoxide, a precursor of microbicidal oxidants. Nox2 activation requires two switches to be turned on simultaneously: a conformational change of the cytosolic protein p47(phox) and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22(phox) and p67(phox), leading to productive oxidase assembly at the membrane. Although arachidonic acid (AA) efficiently activates Nox2 both in vivo and in vitro, the mechanism has not been fully understood, except that AA induces p47(phox) conformational change. Here we show that AA elicits GDP-to-GTP exchange on Rac at the cellular level, consistent with its role as a potent Nox2 activator. However, even when constitutively active forms of p47(phox) and Rac1 are both expressed in HeLa cells, superoxide production by Nox2 is scarcely induced in the absence of AA. These active proteins also fail to effectively activate Nox2 in a cell-free reconstituted system without AA. Without affecting Rac-GTP binding to p67(phox), AA induces the direct interaction of Rac-GTP-bound p67(phox) with the C-terminal cytosolic region of Nox2. p67(phox)-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67(phox) activation domain that localizes the C-terminal to the Rac-binding domain. Thus the "third" switch (AA-inducible interaction of p67(phox)·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.

Effects of p67phox on the mitochondrial oxidative state in the kidney of Dahl salt-sensitive rats: optical fluorescence 3-D cryoimaging.

The goal of the present study was to quantify and correlate the contribution of the cytosolic p67(phox) subunit of NADPH oxidase 2 to mitochondrial oxidative stress in the kidneys of the Dahl salt-sensitive (SS) hypertensive rat. Whole kidney redox states were uniquely assessed using a custom-designed optical fluorescence three-dimensional cryoimager to acquire multichannel signals of the intrinsic fluorophores NADH and FAD. SS rats were compared with SS rats in which the cytosolic subunit p67(phox) was rendered functionally inactive by zinc finger nuclease mutation of the gene (SS(p67phox)-null rats). Kidneys of SS rats fed a 0.4% NaCl diet exhibited significantly (P = 0.023) lower tissue redox ratio (NADH/FAD; 1.42 ± 0.06, n = 5) than SS(p67phox)-null rats (1.64 ± 0.07, n = 5), indicating reduced levels of mitochondrial electron transport chain metabolic activity and enhanced oxidative stress in SS rats. When fed a 4.0% salt diet for 21 days, both strains exhibited significantly lower tissue redox ratios (P < 0.001; SS rats: 1.03 ± 0.05, n = 9, vs. SS(p67phox)-null rats: 1.46 ± 0.04, n = 7) than when fed a 0.4% salt, but the ratio was still significantly higher in SS(p67phox) rats at the same salt level as SS rats. These results are consistent with results from previous studies that found elevated medullary interstitial fluid concentrations of superoxide and H2O2 in the medulla of SS rats. We conclude that the p67(phox) subunit of NADPH oxidase 2 plays an important role in the excess production of ROS from mitochondria in the renal medulla of the SS rat.

p67 gene alleles sequence analysis reveals Theileria parva parasites associated with East Coast fever and Corridor disease in buffalo from Zambia.

Theileriosis caused by Theileria parva infections is responsible for high cattle mortalities in Zambia. Although infected buffalo are a risk to cattle, the characterization of T. parva parasites occurring in this host in Zambia has not been reported. Furthermore, considering the advances in the development of a p67 subunit vaccine, the knowledge of p67 genetic and antigenic diversity in both cattle and buffalo associated T. parva is crucial. Therefore, blood samples from buffalo (n=43) from Central, Eastern and Southern provinces, and cattle (n=834) from Central, Copperbelt, Eastern, Lusaka, and Southern provinces, were tested for T. parva infection and the parasites characterized by sequencing the gene encoding the p67 antigen. About 76.7 % of buffalo and 19.3 % of cattle samples were PCR positive for T. parva. Three of the four known p67 allele types (1, 2 and 3) were identified in parasites from buffalo, of which two (allele types 2 and 3) are associated with T. parva parasites responsible for Corridor disease. Only allele type 1, associated with East Coast fever, was identified from cattle samples, consistent with previous reports from Zambia. Phylogenetic analysis revealed segregation between allele type 1 sequences from cattle and buffalo samples as they grouped separately within the same sub-clade. The high occurrence of T. parva infection in buffalo samples investigated demonstrates the risk of Corridor disease infection, or even outbreaks, should naïve cattle co-graze with infected buffalo in the presence of the tick vector. In view of a subunit vaccine, the antigenic diversity in buffalo associated T. parva should be considered to ensure broad protection. The current disease control measures in Zambia may require re-evaluation to ensure that cattle are protected against buffalo-derived T. parva infections. Parasite stocks used in 'infection and treatment' immunization in Zambia, have not been evaluated for protection against buffalo-derived T. parva parasites currently circulating in the buffalo population.

Development of a dual vaccine against East Coast fever and lumpy skin disease.

East Coast fever is an acute bovine disease caused by the apicomplexan parasite Theileria parva and is regarded as one of the most important tick-vectored diseases in Africa. The current vaccination procedure has many drawbacks, as it involves the use of live T. parva sporozoites. As a novel vaccination strategy, we have constructed the recombinant lumpy skin disease virus (LSDV) named LSDV-SODis-p67HA-BLV-Gag, encoding a modified form of the T. parva p67 surface antigen (p67HA), as well as the bovine leukemia virus (BLV) gag gene for the formation of virus-like particles (VLPs) to potentially enhance p67 immunogenicity. In place of the native sequence, the chimeric p67HA antigen has the human tissue plasminogen activator signal sequence and the influenza hemagglutinin A2 transmembrane domain and cytoplasmic tail. p67HA was detected on the surface of infected cells, and VLPs comprising BLV Gag and p67HA were produced. We also show that higher multiple bands observed in western blot analysis are due to glycosylation of p67. The two vaccines, pMExT-p67HA (DNA) and LSDV-SODis-p67HA-BLV-Gag, were tested for immunogenicity in mice. p67-binding antibodies were produced by vaccinated animals, with higher titers detected in mice vaccinated with the recombinant LSDV. This candidate dual vaccine warrants further testing in cattle.

RNA-binding domain of SARS-CoV2 nucleocapsid: MD simulation study of the effect of the proline substitutions P67S and P80R on the structure of the protein.

The nucleocapsid component of SARS-CoV2 is involved in the viral genome packaging. GammaP.1(Brazil) and the 20 C-US(USA) variants had a high frequency of the P80R and P67S mutations respectively in the RNA-binding domain of the nucleocapsid. Since RNA-binding domain participates in the electrostatic interactions with the viral genome, the study of the effects of proline substitutions on the flexibility of the protein will be meaningful. It evinced that the trajectory of the wildtype and mutants was stable during the simulation and exhibited distinct changes in the flexibility of the protein. Moreover, the beta-hairpin loop region of the protein structures exhibited high amplitude fluctuations and dominant motions. Additionally, modulations were detected in the drug binding site. Besides, the extent of correlation and anti-correlation motions involving the protruding region, helix, and the other RNA binding sites differed between the wildtype and mutants. The secondary structure analysis disclosed the variation in the occurrence pattern of the secondary structure elements between the proteins. Protein-ssRNA interaction analysis was also done to detect the amino acid contacts with ssRNA. R44, R59, and Y61 residues of the wildtype and P80R mutant exhibited different duration contacts with the ssRNA. It was also noticed that R44, R59, and Y61 of the wildtype and P80R formed hydrogen bonds with the ssRNA. However in P67S, residues T43, R44, R45, R40, R59, and R41 displayed contacts and formed hydrogen bonds with ssRNA. Binding free energy was also calculated and was lowest for P67S than wildtype andP80R. Thus, proline substitutions influence the structure of the RNA-binding domain and may modulate viral genome packaging besides the host-immune response.Communicated by Ramaswamy H. Sarma.

[Bacillus Calmette-Guérin infection and chronic granulomatous disease due to new pathogenic variants in the NCF2 gene in the Mayan ethnic group. Report of two cases.] / Infección por bacilo de Calmette-Guérin y enfermedad granulomatosa crónica por nuevas variantes patogénicas del gen NCF2 en la etnia maya. Reporte de dos casos.

INTRODUCTION: Chronic granulomatous disease (CGD) is an inborn error of immunity, characterized by abnormal susceptibility to bacterial and fungal infections and a lack of systemic inflammatory regulation. Pathogenic variants in the CYBB gene are transmitted in an X-linked pattern of inheritance; while the pathogenic variants present in the EROS, NCF1, NCF2, NCF4, or CYBA genes are transmitted with an autosomal recessive inheritance pattern. OBJETIVES: To describe the clinical, immunological, and genetic characteristics of two patients with CGD and BCG infection. METHODS: In peripheral blood neutrophils, H2O2 production and the expression of NADPH oxidase subunits were measured. Detection of pathogenic variants was by Sanger sequencing of the NCF2 gene. The clinical information was extracted from the records by the treating physicians. RESULTS: We present two male infants from two unrelated families of Mayan ethnicity, with CGD and BCG vaccine infection. Three different pathogenic variants in the NCF2 gene were identified; on the one hand, c.304 C>T (p.Arg102*) has already been reported, on the other hand, c.1369 A>T (p.Lys457*) and c.979 G>T (p.Gly327*) not reported. CONCLUSIONS: In patients with mycobacterial infection with BCG, we should suspect an inborn error of immunity, such as CGD. The diagnosis of CGD is made through the detection of a lack of radical oxygen species in neutrophils. The reported patients had pathogenic variants in the NCF2 gene, two of which have not been previously reported in the literature.

INTRODUCCIÓN: La enfermedad granulomatosa crónica (EGC) es un error innato de la inmunidad, se caracteriza por una susceptibilidad a padecer infecciones bacterianas y fúngicas y a una falta de regulación inflamatoria sistémica. Las variantes patogénicas en el gen CYBB se trasmiten con un patrón de herencia ligada al X; mientras que las variantes patogénicas presentes en los genes EROS, NCF1, NCF2, NCF4 o CYBA se trasmiten con un patrón de herencia autosómico recesivo. OBJETIVOS: Describir las características clínicas, inmunológicas y genéticas de dos pacientes con EGC e infección por BCG. MÉTODOS: En neutrófilos de sangre periférica se midió la producción de H2O2 y la expresión de las subunidades de la NADPH oxidasa. La detección de las variantes patogénicas fue por secuenciación Sanger del gen NCF2. La información clínica fue extraída de los expedientes por los médicos tratantes. RESULTADOS: Presentamos a dos lactantes masculinos de dos familias no relacionadas de la etnia maya, con EGC e infección por la vacuna de BCG. Se identificaron tres diferentes variantes patogénicas en el gen NCF2; por un lado, c.304 C>T (p.Arg102*) ya reportada, por otro lado, c.1369 A>T (p.Lys457*) y c.979 G>T (p.Gly327*) no reportadas. CONCLUSIONES: En pacientes con infección micobacteriana por BCG debemos sospechar en un error innato de la inmunidad, como la EGC. El diagnóstico de EGC se realiza a través de la detección de una falta de producción de radicales libres en los neutrófilos. Los pacientes reportados tuvieron variantes patogénicas en el gen NCF2, dos de ellas no han sido reportadas previamente en la literatura.

Characterization of a Novel Chimeric Theileria parva p67 Antigen Which Incorporates into Virus-like Particles and Is Highly Immunogenic in Mice.

The current method to protect cattle against East Coast Fever (ECF) involves the use of live Theileria parva sporozoites. Although this provides immunity, using live parasites has many disadvantages, such as contributing to the spread of ECF. Subunit vaccines based on the sporozoite surface protein p67 have been investigated as a replacement for the current method. In this study, two DNA vaccines expressing recombinant forms of p67 designed to display on retrovirus-like particles were constructed with the aim of improving immunogenicity. The native leader sequence was replaced with the human tissue plasminogen activator leader in both vaccines. The full-length p67 gene was included in the first DNA vaccine (p67); in the second, the transmembrane domain and cytoplasmic tail were replaced with those of an influenza A virus hemagglutinin 5 (p67HA). Immunofluorescent staining of fixed and live transfected mammalian cells showed that both p67 and p67HA were successfully expressed, and p67HA localised on the cell surface. Furthermore, p67HA was displayed on the surface of both bovine leukaemia virus (BLV) Gag and HIV-1 Gag virus-like particles (VLPs) made in the same cells. Mice vaccinated with DNA vaccines expressing p67 and p67HA alone, or p67HA with BLV or HIV-1 Gag, developed high titres of p67 and BLV Gag-binding antibodies. Here we show that it is possible to integrate a form of p67 containing all known antigenic domains into VLPs. This p67HA-VLP combination has the potential to be incorporated into a vaccine against ECF, as a DNA vaccine or as other vaccine platforms.

Upregulation of p67phox in response to ischemia/reperfusion is cardioprotective by increasing ZIP2 expression via STAT3.

While the zinc transporter ZIP2 (Slc39a2) is upregulated via STAT3 as an adaptive response to protect the heart from ischemia/reperfusion (I/R) injury, the precise mechanism underlying its upregulation remains unclear. The purpose of this study was to investigate the role of NADPH oxidase (NOX) isoform NOX2-derived ROS in the regulation of ZIP2 expression, focusing on the role of the NOX2 cytosolic factor p67phox. Mouse hearts or H9c2 cells were subjected to I/R. Protein expression was detected with Western blotting. Infarct size was measured with TTC staining. The cardiac-specific p67phox conditional knockout mice (p67phox cKO) were generated by adopting the CRISPR/Cas9 system. I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression was reversed by the ROS scavenger N-acetylcysteine (NAC) and the NOX inhibitor diphenyleneiodonium (DPI). p67phox but not NOX2 expression was increased 30 min after the onset of reperfusion, and downregulation of p67phox by siRNA or cKO invalidated I/R-induced upregulation of STAT3 phosphorylation and ZIP2 expression. Both NAC and DPI prevented upregulation of STAT3 phosphorylation and ZIP2 expression induced by overexpression of p67phox, whereas the STAT3 inhibitor stattic abrogated upregulation ZIP2 expression, indicating that the increase of p67phox at reperfusion is an upstream signaling event responsible for ZIP2 upregulation via STAT3. Experiments also showed that chelation of Zn2+ markedly enhanced p67phox and ZIP2 expression as well as STAT3 phosphorylation, whereas supplementation of Zn2+ had the opposite effects, indicating that cardiac Zn2+ loss upon reperfusion triggers p67phox upregulation. Furthermore, ischemic preconditioning (IPC) upregulated ZIP2 via p67phox, and cKO of p67phox aggravated cardiac injury after I/R, indicating that p67phox upregulation is cardioprotective against I/R injury. In conclusion, an increase of p67phox expression in response to Zn2+ is an intrinsic adaptive response to I/R and leads to cardioprotection against I/R by upregulating ZIP2 via STAT3.

Memantine Promotes Bactericidal Effect of Neutrophils Against Infection with Pseudomonas aeruginosa and Its Drug-Resistant Strain, by Improving Reactive Oxygen Species Generation.

Pseudomonas aeruginosa is an opportunistic pathogen, which usually presents multiple antibiotic resistance. Host-directed therapy involves modulating the host defense system and the interplay between innate and adaptive immunity is a new strategy for designing anti-infection drugs. Memantine (MEM), a drug used to treat Alzheimer's disease, has a good inhibitory effect on neonatal mice with Escherichia coli-associated bacteremia and meningitis; however, the inhibitory effect and mechanisms of MEM against P. aeruginosa infection remain unclear. Here, we investigated whether MEM could inhibit P. aeruginosa infection and explored the potential mechanisms. MEM significantly promoted the bactericidal effect of neutrophils against P. aeruginosa and its drug-resistant strain. The combination index of MEM and amikacin (AMK) was <1. In vivo experiments showed that the bacteremia and inflammation severities in the MEM-treated group were less than those in the untreated group, and the bacterial load in the organs was significantly less than that in the control group. Combining MEM with the reactive oxygen species (ROS) inhibitor, N-acetyl-l-cysteine, weakened the anti-infective effect of MEM. MEM increased the expression of NADPH p67phox and promoted neutrophilic ROS production. Deleting the p67phox gene significantly weakened the effects of MEM on ROS generation and improving bactericidal effect of neutrophils. In conclusion, MEM promoted the bactericidal effect of neutrophils against P. aeruginosa and its drug-resistant strain, and had a synergistic antibacterial effect when combined with AMK. MEM may exert its anti-infective effects by promoting neutrophilic bactericidal activity via increasing the expression level of p67phox and further stimulating ROS generation.