70-kDa Band Research Articles

Alterations in immunized antigens of Anisakis pegreffii by ampicillin-induced gut microbiome changes in mice.

The gut microbiome plays an essential role in host immune responses, including allergic reactions. However, commensal gut microbiota is extremely sensitive to antibiotics and excessive usage can cause microbial dysbiosis. Herein, we investigated how changes in the gut microbiome induced by ampicillin affected the production of IgG1 and IgG2a antibodies in mice subsequently exposed to Anisakis pegreffii antigens. Ampicillin treatment caused a notable change in the gut microbiome as shown by changes in both alpha and beta diversity indexes. In a 1-dimensional immunoblot using Anisakis-specific anti-mouse IgG1, a 56-kDa band corresponding to an unnamed Anisakis protein was detected using mass spectrometry analysis only in ampicillin-treated mice. In the Anisakis-specific anti-mouse IgG2a-probed immunoblot, a 70-kDa band corresponding to heat shock protein 70 (HSP70) was only detected in ampicillin-treated and Anisakis-immunized mice. A 2-dimensional immunoblot against Anisakis extract with immunized mouse sera demonstrated altered spot patterns in both groups. Our results showed that ampicillin treatment altered the gut microbiome composition in mice, changing the immunization response to antigens from A. pegreffii. This research could serve as a basis for developing vaccines or allergy immunotherapies against parasitic infections.

Identification of the Beta Subunit Fas1p of Fatty Acid Synthetase as an Interacting Partner of Yeast Calcium/Calmodulin-Dependent Protein Kinase Cmk2p Through Mass Spectrometry Analysis.

The calcium/calmodulin-dependent protein kinase II (CaMKII) is a mediator of calcium signals and regulates fatty acid metabolism in mammalian cells. Cmk2p is a yeast homolog of CaMKII and functions as a negative regulator of calcium signaling. However, its substrates remain to be identified. Combination of immunoprecipitation (IP) and mass spectrometry has been proven to be very useful for identification of interacting partner proteins and interactome. In this study, through these approaches, we have identified 65 and 110 potential Cmk2p-interacting proteins in yeast cells in the absence or presence of calcium stress, respectively. In yeast cells expressing both CMK2-HA and FAS1-GFP fusion proteins, in the absence or presence of calcium stress, less amounts of FAS1-GFP proteins are present in cell lysates after IP with anti-HA antibody than cell lysates before IP, while FAS1-GFP proteins are detected on both types of IP beads. However, as an internal control, similar amounts of Pgk1p proteins were detected in both after-IP and before-IP cell lysates but not on the IP beads. Therefore, our biochemical analysis demonstrates that the β subunit Fas1p of fatty acid synthetase interacts with Cmk2p in yeast cells independent of calcium stress. It is also interesting to note that, in addition to the expected 52-kDa CMK2-HA band, a faster-moving 48-kDa CMK2-HA band is present in the calcium-stressed cell lysate but not in the cell lysate without calcium stress. Our data would provide important clues for understanding the functions of CaMKII in the regulation of fatty acid metabolism as well as related diseases such as cancers, diabetes, and obesity.

Does flux through the hexosamine biosynthesis pathway differ between endothelial cells from African American and Caucasian donors?

People with Type 2 Diabetes (T2D) often exhibit endothelial dysfunction (EndoDys), which underlies the initiation of many diseases. African Americans (AA) have a greater prevalence of T2D and EndoDys than their Caucasian (CA) counterparts. To understand these differences, we examined glucose metabolism within the endothelium. Specifically, we sought to determine whether there are differences between AAs and CAs in the Hexosamine Biosynthesis Pathway (HBP), a route of glucose utilization that is upregulated in T2D. We hypothesized that endothelial cells (EC) from AA donors will show a greater flux through the HBP and express higher markers of EndoDys compared to CA donors. Human umbilical vein ECs (HUVECs), n=2-3/group, were incubated for 24hrs with high (HG, 30mM) or normal (5.5mM) glucose. Protein expression was measured via western blot for the: 1) HBP rate-limiting enzyme (Glutamine: fructose-6-phosphate amidotransferase, GFAT); 2) enzyme catalyzing the addition of HBP end-products to various proteins (O-GlcNAc transferase, OGT); and 3) attachment of O-linked N-acetylglucosamine moieties to various proteins (O-GlcNAcylation, O-GlcNAc), in addition to markers of EndoDys. Hyperglycemia did not differentially impact the expression of HBP markers GFAT or OGT between ECs from CA and AA donors (glycemic status [GS], p=0.6723; race, p=0.3807 and GS, p=0.8695; race, p=0.3536, respectively). Although total O-GlcNAc did not significantly increase with HG, there was a trend toward higher O-GlcNAcylation in AA ECs (p=0.0897). Upon further examination of O-GlcNAcylation, there were 3 prominent bands of highly O-GlcNAcylated proteins at >220kDa (band 1), ~150-200kDa (band 2), and ~85kDa (band 3). There was significantly higher O-GlcNAcylation of protein in band 2 for AA donors compared to CA donors (p=0.0362). The apoptotic marker, Caspase-3, did not change with HG (p=0.9219) nor differ in expression between races (p=0.2060). The inflammatory marker, VCAM, was significantly higher in cells from AA donors (p=0.002) but did not differ with GS (p=0.3468). There was no GS by race interaction found. EndoDys links T2D and other pathologies. We examined the HBP pathway of glucose utilization in ECs from CA and AA donors. There was a main effect of race related to inflammation and total O-GlcNAcylation, without differences in the expression of key proteins within the pathway. Our results suggest that the HBP may contribute to the racial differences in EndoDys, however, this relationship is not straightforward and does not seem to be exacerbated in the context of HG. Future research is needed to examine these unexpected discrepancies and interrogate the link between HBP flux within the endothelium, along with its connection to various pathologies This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Proteomic analysis of the excretory-secretory products from Strongyloides venezuelensis infective larvae: new insights for the immunodiagnosis of human strongyloidiasis.

Serodiagnosis of human strongyloidiasis is a practical alternative to parasitological methods due to its high sensitivity. However, cross-reactivity with other helminth infections limits its utility, and this problem is due to the use of homologous or heterologous somatic extracts of the parasite as an antigen source. Excretory-secretory (E/S) products from Strongyloides infective larvae can be used to improve the serodiagnosis. The combined use of western blot and proteomics became an interesting strategy to identify immunological markers for the serodiagnosis of strongyloidiasis. The present study describes the proteomic analysis of the antigenic components from E/S products of S. venezuelensis infective larvae that were recognized by IgG antibodies from patients with strongyloidiasis. Our results showed that IgG antibodies from patients with strongyloidiasis recognized between 15 and 16 antigenic bands in the E/S products from S. venezuelensis that were incubated in PBS or in RPMI culture medium, respectively. Overall, antigenic bands of low and high molecular weight were more specific than those of intermediate molecular weight, which were cross-reactive. A 36-kDa antigenic band was 93% sensitive and 100% specific (a probably arginine kinase of 37kDa), while other antigenic bands were highly sensitive but low specific. Proteomic analysis revealed differences between the protein profiles from E/S-RPMI and E/S-PBS since only one-third of all proteins identified were common in both types of E/S products. Bioinformatic analysis showed that more than 50% of the proteins from E/S products are secreted within extracellular vesicles and only a small percentage of them are actually released by the classical secretory pathway. Several components from the E/S products were identified as plasminogen-binding proteins, probably used as an immune evasion mechanism. The data provided here provide valuable information to increase understanding of E/S products from S. venezuelensis infective larvae. This may help us to find new targets for the immunodiagnosis of human strongyloidiasis.

The Vibrio parahaemolyticus subunit toxin PirBvp recognizes glycoproteins on the epithelium of the Penaeus vannamei hepatopancreas.

Vibrio parahaemolyticus toxin PirABvp is the major virulence factor exotoxin that contributes to the disruption of the hepatopancreatic epithelium in acute hepatopancreatic necrosis disease in shrimp. The PirBvp subunit is a lectin that recognizes amino sugars; however, its potential role in recognition of the hepatopancreas has not been identified. In the present work, we identified the cellular receptor for PirBvp in the shrimp hepatopancreas. A ligand blot assay of hepatopancreas lysate showed that the PirBvp subunit recognizes two glycoprotein bands of 60 and 70kDa (Gc60 and Gc70). The hepatopancreas lysate was fractionated by anion-exchange chromatography, and the three main fractions obtained contained the recognized Gc60 and Gc70 protein bands. LC-MS/MS indicated that beta-hexosaminidases subunit beta and mucin-like 5AC corresponded to the 60 and 70kDa bands, respectively, which seem to be expressed in the epithelial cells of the hepatopancreas. Endoglycosidase treatment of hepatopancreas lysate with the O-glycosidase from Enterococcus faecalis, inhibits the binding of PirBvp. Altogether, these results suggest the relevance of the interaction of PirBvp with the hepatopancreas in the pathogenesis of acute hepatopancreatic necrosis disease in shrimp.

Immunization of chickens with Salmonella gallinarium ghosts expressing Salmonella Enteritidis NFliC-FimAC and CD40LC fusion antigen enhances cell-mediated immune responses and protects against wild-type challenges with both species

This study describes the construction and immunological characterization of a novel Salmonella gallinarium ghost vaccine to protect against S. gallinarium (SG) and S. Enteritidis (SE) serotypes. The SG ghost was designed to express N-terminus FliC (D0-D1 domain) and FimA retrieved from the SE genome, and the receptor-binding domain (RBD) of CD40L from the chicken as a single fusion construct. The construct was built in pJHL184, a phage lysis gene E-mediated ghost plasmid and the expression was confirmed by western blot resulting in an 85-kDa band. Chicken immunization was conducted by intramuscular route with SG ghost FliC-FimA-CD40L, vector control, or PBS alone in a prime-boost schedule. Antibody responses, cell-mediated immune responses (CMI), and cytokine induction was assessed in chicken demonstrating significantly high levels of IgY, CMI, cytokine responses in ghost immunized group delivering partial protection against SG wild type challenge and near complete protection against SE challenge wild type challenge.

AtPng1 knockout mutant of Arabidopsis thaliana shows a juvenile phenotype, morpho-functional changes, altered stress response and cell wall modifications

In order to ascertain the role of plant transglutaminases (TGase) in growth and abiotic stress response, the AtPng1 knock out (KO) line of A. thaliana has been analyzed during plant development and under heat and wound stress. Comparing wild type (WT) and KO lines a 58-kDa band was immunodetected by anti-AtPng1p antibody in the cell wall and chloroplasts only in the WT line. A residual TGase activity, not showing correlation with development nor stress response, was still present in the KO line. The KO line was less developed, with a juvenile phenotype characterized by fewer, smaller and less differentiated cells. Chloroplast TGase activity was insensitive to mutation. Data on stressed plants showed that (i) KO plants under heat stress were more juvenile compared to WT, (ii) different responses between WT and KO lines after wounding took place. TGase activity was not completely absent in the KO line, presenting high activity in the plastidial fraction. In general, the mutation affected A. thaliana growth and development, causing less differentiated cytological and anatomical features.

Crosstalk between vrille transcripts, proteins, and regulatory elements controlling circadian rhythms and development in Drosophila.

SummaryThe vrille (vri) gene encodes a transcriptional repressor required for Drosophila development as well as circadian behavior in adults. Alternate first exons produce vri transcripts predicted to produce a short VRI isoform during development and long VRI in adults. A vri mutant (vriΔ679) lacking long VRI transcripts is viable, confirming that short VRI is sufficient for developmental functions, yet behavioral rhythms in vriΔ679 flies persist, showing that short VRI is sufficient for clock output. E-box regulatory elements that drive rhythmic long VRI transcript expression are required for developmental expression of short VRI transcripts. Surprisingly, long VRI transcripts primarily produce short VRI in adults, apparently due to a poor Kozak sequence context, demonstrating that short VRI drives circadian behavior. Thus, E-box-driven long VRI transcripts primarily control circadian rhythms via short VRI, whereas the same E-boxes drive short VRI transcripts that control developmental functions using short VRI.

Identification of a HSP14-3-3 in Setaria cervi and its cross-reactivity with Wbancrofti-infected human sera.

Identification of a 29kDa heat stress protein in filarial parasite Setaria cervi and evaluation of its diagnostic potential against lymphatic filariasis. The Heat shock proteins (HSPs) were induced in filarial parasite Scervi by incubated at 42°C for 2hours. The 10% SDS-PAGE of cytosolic extract showed several over-expressed bands. The MALDI-LC/MS analysis of 29kDa band showed 100% similarity with Bm14-3-3 like protein 2. Multiple sequence alignment of Bm14-3-3 like protein 2 sequence with Wbancrofti, Caenorhabditis elegans; Loa loa and Homo sapiens showed 100%, 86%, 83% and 78%, sequence similarity respectively. The antigenic efficacy of Sc14-3-3 protein was evaluated with different filarial sera using ELISA which showed cross-reactivity in order to Endemic Normal (EN)<Microfilaraemic (MF)<Chronic(CH) with IgG1 and EN<CH<MF in IgG4 ELISA. IgG1- and IgG4-specific immunoblotting with CH and MF sera further explicated its specific antigenic cross-reactivity. A 29kDa heat shock protein of Scervi was identified as 14-3-3 protein having 100% homology to human filarial parasite Bmalayi. It showed strong reactivity with IgG1 and IgG4 subclass antibodies of Wbancrofti-infected human sera suggesting that 14-3-3 protein could be used as a vaccine/ diagnostic marker.

Changes in lipid biology during ovarian development in farmed beluga sturgeon, Huso huso L.

The present study was conducted to understand key biochemical, physiological, and molecular changes associated with ovarian growth and with lipid transfer and/or accumulation into the ovary during oogenesis in captive beluga sturgeon. Plasma levels of triacylglycerides, cholesterol, phospholipid, and sex steroid hormones were determined and all were found to increase notably throughout development from the perinucleolar to the tertiary yolk stage. Using fast protein liquid chromatography, we recognized three major lipoprotein peaks in chromatograms from all samples. These peaks were characterized as containing very low-density lipoprotein (Vldl), low-density lipoprotein/high-density lipoprotein (Ldl/Hdl), and plasma proteins. While Ldl/Hdl represented the most abundant lipoprotein fraction, the relative abundance of different lipoprotein classes did not change with the stage of oogenesis. Eluted lipoproteins were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis and sequenced. The peptide sequence spectra for 66-kDa, 205-kDa, 29-kDa, and 70-kDa bands matched with albumin, vitellogenin (Vtg) AB2b, immunoglobulin light-chain precursor, and immunoglobulin heavy-chain, respectively. The large amount of albumin in the plasma protein peak and the confined presence of Vtg AB2b to within Ldl/Hdl reinforce the lipoprotein classification. Lastly, transcript levels of genes encoding ovarian lipoprotein lipase (lpl), apolipoprotein E (apoe), very low-density lipoprotein receptors (vldlr), and low-density lipoprotein receptor-related protein 8-like (lrp8) were estimated using quantitative RT-PCR. The high mRNA levels of lpl, apoe, and lipoprotein receptors vldlr and lrp8 in previtellogenic females suggest that sturgeon oocytes need to be prepared to accept and traffic Vtg and lipids internally, before the start of vitellogenesis.

Evaluation of the Toxicity of Supernatant Cultures and Spore-Crystal Mixtures of Bacillus thuringiensis Strains Isolated from Algeria.

Bacillus thuringiensis (Bt) is the most used technology for biological control of insect pathogens worldwide. In order to select new Bt candidates challenging the emergence of insect's resistance, a mass bioassay and molecular screening was performed on an autochthonous collection. Toxicity assays against neonate larvae of three lepidopteran species (Mamestra brassicae, Grapholita molesta, and Spodoptera exigua) were conducted using spore-crystal mixtures and supernatant cultures of 49 Bt isolates harboring at least one gene coding for a lepidopteran-specific insecticidal protein. A threshold of 30% of "functional mortality" was used to discriminate between "nontoxic" and "toxic" isolates. The toxicity of many Bt isolates competed with that of Btk-HD1. However, only three of them (Bl4NA, Bl5NA, and Bl9NA) showed high toxicity in both spore-crystal mixtures and supernatant cultures against the three lepidopteran species. The Bt isolates Bl4NA and Bl9NA express a protein of 130kDa whereas the Bt isolate Bl5NA expresses a protein of 65-70kDa. The LC-MS/MS results indicate that the major peptides in the 130kDa band of Bl9NA were Cry1Da, Cry1Ca, Cry1Ab, and Cry1Aa, and those in the 70kDa band of Bl5NA were Cry1Aa and Cry1Ca. The evaluation of the protein content of the supernatants by comparison to Btk-HD1 indicates the overproduction of Vip3 proteins in these strains (most likely Vip3Aa in Bl4NA and Bl9NA and Vip3Ca in Bl5NA). In addition, these three Bt strains do not produce β-exotoxins. Based on our results, the three selected strains could be considered promising candidates to be used in insect pest control.

Antigen Recognition Patterns of Intestinal Capillariasis Using Immunoblot-Based Serodiagnosis.

Diagnosis of intestinal capillariasis depending on microscopic detection of parasitic stages is of low sensitivity, especially in cases with low worm burden. There is a necessity to develop sensitive and specific diagnostic tools of capillariasis for early treatment to avoid complications. Western blot (WB) technique showed promising results for antigen recognition patterns in several parasitic infections. This study is directed to identify and evaluate relevant proteins of intestinal capillariasis crude worm antigens using WB immunodiagnosis. Capillaria crude worm antigens were extracted and analyzed using SDS-PAGE. Sixty serum samples belonging to 3 groups (20 individuals each) were included; Group I (shedding Capillaria in feces), Group II (infected with other parasites) and Group III (negative parasitological results). Reactivity of the resulting bands of Capillaria with serum samples was analyzed using WB technique. Thirty-two immunoreactive bands were detected in WB analysis representing recognition of proteins with molecular weights (MW) varying from 19 to 110kDa. Immunodominant proteins of 23.5, 31, 36.5, 40.5 and 44kDa were recognized, respectively, in 35%, 30%, 85%, 95% and 75% of sera from patients with confirmed capillariasis and in 30%, 25%, 35%, 25%, and 20% of sera from those infected with other parasitic infections. One serum sample from group III gave reaction with 31kDa band. Immunodiagnosis of intestinal capillariasis using WB proved that 23.5, 31, 36.5, 40.5 and 44kDa bands could be considered useful tools for diagnosis of capillariasis.

Plasmodium falciparum evades immunity of anopheline mosquitoes by interacting with a Pfs47 midgut receptor

The surface protein Pfs47 allows Plasmodium falciparum parasites to survive and be transmitted by making them "undetectable" to the mosquito immune system. P. falciparum parasites express Pfs47 haplotypes compatible with their sympatric vectors, while those with incompatible haplotypes are eliminated by the mosquito. We proposed that Pfs47 serves as a "key" that mediates immune evasion by interacting with a mosquito receptor "the lock," which differs in evolutionarily divergent anopheline mosquitoes. Recombinant Pfs47 (rPfs47) was used to identify the mosquito Pfs47 receptor protein (P47Rec) using far-Western analysis. rPfs47 bound to a single 31-kDa band and the identity of this protein was determined by mass spectrometry. The mosquito P47Rec has two natterin-like domains and binds to Pfs47 with high affinity (17 to 32 nM). P47Rec is a highly conserved protein with submicrovillar localization in midgut cells. It has structural homology to a cytoskeleton-interacting protein and accumulates at the site of ookinete invasion. Silencing P47Rec expression reduced P. falciparum infection, indicating that the interaction of Pfs47 with the receptor is critical for parasite survival. The binding specificity of P47Rec from distant anophelines (Anopheles gambiae, Anopheles dirus, and Anopheles albimanus) with Pfs47-Africa (GB4) and Pfs47-South America (7G8) haplotypes was evaluated, and it is in agreement with the previously documented compatibility between P. falciparum parasites expressing different Pfs47 haplotypes and these three anopheline species. Our findings give further support to the role of Pfs47 in the adaptation of P. falciparum to different vectors.

Biochemical properties and ultrastructure of mesophyll and bundle sheath thylakoids from maize (Zea mays) chloroplasts.

A characteristic feature of C4 plants is the differentiation of the photosynthetic leaf tissues into two distinct cell types: mesophyll (M) and bundle sheath (BS) cells. We have investigated several biochemical parameters, including pigment composition, polypeptide patterns, fluorescence at 77K, the activity of photosystems and ultrastructure of mesophyll and bundle sheath chloroplasts of maize (Zea mays L.) plants. It is shown that the BS chloroplasts have ~2-fold higher chlorophyll a/b ratio than M chloroplasts, 6.15 and 3.12 respectively. The PSI apoprotein (68 kDa) was more abundant in BS than in M thylakoids. Polypeptides belonging to PSII core antenna, are in similar amounts in both types of membranes, but the 45kDa band is more intensive in M thylakoids. Polypeptides in the region of 28-24 kDa of the light-harvesting complex of PSII (LHCII) are also present in both types of chloroplasts, though their amounts are reduced in BS thylakoids. The chlorophyll fluorescence emission spectra in M cells showed the presence of three bands at 686, 695 and 735 nm characteristics of LHCII, PSII core and PSI complexes, respectively. However, in the fluorescence spectrum of agranal plastids, there are almost traces of the band at 695 nm, which belongs to the PSII core complex. The research results revealed that the photochemical activity of PSII in BS chloroplasts is ~5 times less than in the chloroplasts of M cells. The highest PSI activity was found in maize BS chloroplasts.

Troponin 1 of human filarial parasite Brugia malayi: cDNA cloning, expression, purification, and its immunoprophylactic potential

In the search for immunoprophylactics for the control of human lymphatic filariasis, we recently identified troponin 1 (Tn1) in Brugia malayi adult worms. The present study reports the cloning and expression of the B. malayi Tn1 (Tn1bm), its immunoprophylactic efficacy against B. malayi infection, and the immunological responses of the host. The Tn1bm gene was cloned (Acc no. JF912447) and expressed, and the purified recombinant Tn1bm (rTn1bm) presented a single ~ 27kDa band. Parasite load in rTn1bm-immunized BALB/c mice challenged with B. malayi infective larvae (L3) was assessed. In rTn1bm-immunized animals, IgE, IgG, and IgG subclasses in the serum, cell proliferative response, Th1 and Th2 cytokine secretion (from splenocytes), and NO release (from peritoneal macrophages) were determined. Antibody-dependent cell-mediated cytotoxicity (ADCC) to L3 was assayed using rTn1bm-immune serum. The innate immune response markers MHC class-I, MHC class-II, TLR2, TLR4, and TLR6 expression in peritoneal macrophages and CD3+, CD4+, CD8+, and CD19+ in the splenocyte population were determined in Tn1bm-exposed cells from naïve mice. rTn1bm-immunized L3-challenged animals showed a 60% reduction in parasite burden. Immunization upregulated cellular proliferation, cytokine (IFN-γ, TNF-α, IL-1β, IL-4, IL-6, and IL-10) secretion, NO release, and antigen-specific IgG, IgG1, and IgG2b antibody levels. rTn1bm-immune serum killed > 65% of L3 in the ADCC assay. Increased MHC class-II, TLR2, and TLR6 expression and the relative CD4+ and CD19+ cell populations of naïve animal cells indicated the ability of rTn1bm to mobilize innate immune responses. This is the first report of the immunoprophylactic potential of rTn1bm against B. malayi.

Inducible renal‐tubule insulin receptor (IR) knockout affects sodium transporter/channel expression and activity in a sex‐dependent manner

Insulin has been demonstrated to be natriferic in thick ascending limb (TAL), distal convoluted tubule (DCT), and collecting duct (CD). Direct activation of sodium (Na+) transporters and channels by the insulin receptor (IR) likely underlies this effect. To elucidate the role of tubular IR on blood pressure (BP) and apical transporter/channel activity at these sites, we knocked out (KO) IR from the renal tubule, in male (M) and female (F) mice (C57Bl6 background) using a tetracycline‐inducible system. Doxycycline (DOX) was fed to all mice ad libitum (625 mg/kg·diet) for two weeks to induce KO in homozygously IR floxed mice harboring both inducible Cre and paired box gene 8 (PAX8) promoter transgenes. WT mice were negative for Cre, PAX8 or both transgenes. BP and sensitivity to select diuretics was assessed in the pre‐DOX and post‐DOX states. Four weeks post‐DOX, mice were euthanized 4 hours after an acute injection of insulin (0.5 U/kg·bw) and blood and kidneys collected for analysis. Two‐way ANOVA (sex X genotype) revealed DOX marginally reduced kidney weight (p = 0.059) and increased blood potassium (K+, p = 0.02). These differences were abrogated in F. Final blood glucose was also lower in F (p = 0.001) and in KO mice (p = 0.016). Pre‐Dox BP (mm Hg) measured by tail‐cuff plethysmography was similar in M (103.1 ± 3.2) and F (100.2 ± 6.0). DOX caused a fall in MAP (~ 5 mm Hg) in F, but not M KO mice. Natriuretic responses to furosemide (FUR), thiazide (TZD), and benzamil (BNZ) were used as indexes of Na‐K‐2Cl cotransporter (NKCC2), Na‐Cl cotransporter (NCC), and epithelial sodium channel (ENaC) activity, respectively, and were all marginally‐to‐significantly higher in F versus M mice in the pre‐Dox state (18, 300, and 44% higher, respectively), suggesting greater basal activity of these Na+ reabsorptive routes in F (n = 12–17/sex). After DOX, there was a significant interaction (p = 0.010, sex X genotype) for the response to FUR, with M KO responding with an increase, and F KO, with a decrease. The response to TZD tended to increase in the M KO, but decrease in the F KO (relative to same sex WT). The response to BNZ remained higher in the F, and was slightly increased in both sexes by KO (~20–50%). In cortex homogenates (CTXH), western blotting revealed band density for the β‐subunit of IR decreased on average 60% with no sex difference. F had significantly higher band density for α‐ENaC (90‐kDa band), as compared to M (p &lt; 0.0001), and KO led to a significant interactive term in that band density was decreased in F, but increased in M. Both sexes showed reduced band densities for β‐ and γ‐ENaC (85 kDa) and outer medullary NKCC2 in KO. NCC band density was not affected by KO, but significantly higher in F (p &lt; 0.0001). Taken together these results demonstrate differential sensitivity between the sexes to renal tubular IR KO in regard to homeostatic control of Na+ and K+ handling and BP. KO led to modest reductions in kidney weight and transporter/channel expression levels, supporting the role for insulin in growth and maintenance of cell size and differentiation. Little to no reduction in measurable activity could result from compensation, which appeared more effective in males.Support or Funding InformationAmerican Diabetes Association (MF)Georgetown University Medical CenterThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Epitope recognized by anti-glomerular basement membrane (GBM) antibody in a patient with repeated relapse of anti-GBM disease

The major epitopes recognized by autoantibodies in anti-glomerular basement membrane (GBM) disease are found in the α3-subunit non-collagenous domain of type IV collagen [α3(IV)NC1], which is present in the glomerular and alveolar basement membranes. These epitopes are structurally cryptic, owing to the hexamer formation of the non-collagenous domain of α3, α4, and α5 subunits and are expressed by the dissociation of the hexamer. Anti-GBM disease usually manifests as a single attack (SA), and we rarely see patients who repeatedly relapse. We recently treated a patient with anti-GBM disease who exhibited repeated relapse (RR). Here, we conducted immunohistochemistry of formalin-fixed paraffin-embedded normal kidney sections and immunoblotting using recombinant human α3(IV)NC1 to compare the epitopes recognized by anti-GBM antibodies in the RR patient and SA patients. Although a clear staining of GBM especially in the connecting basement membrane of Bowman's capsule was observed when IgGs of SA patients were used as primary antibodies, such staining was not obtained when IgG of the RR patient was employed. In immunoblotting of α3(IV)NC1 using the IgG of the RR patient as a primary antibody, an 18-kDa band was detected besides the 56.8-kDa band corresponding to the whole-size α3(IV)NC1. Whereas the 56.8-kDa band disappeared after digestion of the recombinant α3(IV)NC1 by protease, the 18-kDa band remained. Furthermore, the 18-kDa band was not detected by a commercially available anti-α3(IV)NC1 monoclonal antibody. These findings suggest that the IgG of the RR patient recognizes the epitope distinct from that recognized by the anti-α3(IV)NC1 monoclonal antibody.

Characterization of a Synovial B Cell–Derived Recombinant Monoclonal Antibody Targeting Stromal Calreticulin in the Rheumatoid Joints

Rheumatoid arthritis (RA) is characterized by formation of synovial ectopic lymphoid structures (ELS) supporting B cell autoreactivity toward locally generated citrullinated (cit) antigens, including those contained in neutrophil extracellular traps (NETs). However, only a minority of RA-rmAbs from B cells isolated from ELS+ RA tissues react against NETs. Thus, alternative cellular sources of other potential autoantigens targeted by locally differentiated B cells remain undefined. RA fibroblast–like synoviocytes (FLS) have been implicated in the release of RA-associated autoantigens. In this study, we aimed to define stromal-derived autoantigens from RA-FLS targeted by RA-rmAbs. Seventy-one RA-rmAbs were screened toward RA-FLS by living-cell immunofluorescence (IF). Western blotting was used to identify potential autoantigens from RA-FLS protein extracts. Putative candidates were validated using colocalization immunofluorescence confocal microscopy, ELISA, immunoprecipitation assay, and surface plasmon resonance on unmodified/cit proteins. Serum immunoreactivity was tested in anti-citrullinated peptide/protein Abs (ACPA)+ versus ACPA− RA patients. Ten out of 71 RA-rmAbs showed clear reactivity toward RA-FLS in immunofluorescence with no binding to NETs. One stromal-reactive RA-rmAb (RA057/11.89.1) decorated a ∼58-kDa band that mass spectrometry and Western blotting with a commercial Ab identified as calreticulin (CRT). Confocal microscopy demonstrated significant cellular colocalization between anti-CRT RA057/11.89.1 in RA-FLS. RA057/11.89.1 was able to immunoprecipitate rCRT. Deimination of CRT to cit-CRT moderately increased RA057/11.89.1 immunoreactivity. cit-CRT displayed increased blocking capacity compared with unmodified CRT in competitive binding assays. Finally, anti–cit-CRT Abs were preferentially detected in ACPA+ versus ACPA− RA sera. We identified a synovial B cell–derived RA-rmAb locally differentiated within the ELS+ RA synovium reacting toward CRT, a putative novel autoantigen recently described in RA patients, suggesting that FLS-derived CRT may contribute to fuel the local autoimmune response.

Stable transformation of Spirulina (Arthrospira) platensis: a promising microalga for production of edible vaccines.

The planktonic blue-green microalga Spirulina (Arthrospira) platensis possesses important features (e.g., high protein and vital lipids contents as well as essential vitamins) and can be consumed by humans and animals. Accordingly, this microalga gained growing attention as a new platform for producing edible-based pharmaceutical proteins. However, there are limited successful strategies for the transformation of S. platensis, in part because of an efficient expression of strong endonucleases in its cytoplasm. In the current work, as a pilot step for the expression of therapeutic proteins, an Agrobacterium-based system was established to transfer gfp:gus and hygromycin resistance (hygr) genes into the genome of S. platensis. The presence of acetosyringone in the transfection medium significantly reduced the transformation efficiency. The PCR and real-time RT-PCR data confirmed the successful integration and transcription of the genes. Flow cytometry and β-glucuronidase (GUS) activity experiments confirmed the successful production of GFP and the enzyme. Moreover, the western blot analysis showed a ~ 90kDa band in the transformed cells, indicating the successful production of the GFP:GUS protein. Three months after the transformation, the gene expression stability was validated by histochemical, flow cytometry, and hygromycin B resistance analyses.

Abstract 362: Plasminogen Activator Inhibitor 1 Forms a Complex With FXIa on the Surface of Endothelial Cells, Blocking Its Activity and Inducing the Clearance, Internalization and Degradation of FXIa

Background: The activation of coagulation factor (F) XI by activated coagulation FXII (FXIIa) is a prothrombotic process. The endothelium is known to play an antithrombotic role by preventing thrombin generation and platelet activation. It is unknown whether the antithrombotic role of the endothelium includes regulation of activated FXI (FXIa) activity. Hypothesis: Endothelial cells (ECs) express anticoagulant properties that block the procoagulant activity of FXIa. Methods: We used a chromogenic assay to measure FXIa, kallikrein or FXIIa activity on cultured human umbilical vein ECs (HUVECs). To detect FXIa-inhibitor complexes in either the supernatant (SN) or cell lysate (CL), HUVECs were exposed to FXIa for 2 hrs at 4°C, then washed and lysed, followed by immunoprecipitation and western blot (WB) using an anti-FXIa mAb to the catalytic domain. To assess FXIa binding and internalization, HUVECs were exposed to FXIa for 2 hrs a 4C, washed and incubated at 37C for 5 min to 2 hrs, followed by fixation, and immunostaining with an anti-FXI antibody. Results: HUVECs block the activity of FXIa but not kallikrein or FXIIa activity. The catalytic domain of FXIa (30 kDa) formed a covalent bond with one inhibitor generating one band at 80 kDa. The formation of FXIa-inhibitor complexes appeared in the SN when ECs were transferred to 37C. Mass spectrometry analysis of the 80kDa band revealed that FXIa forms a complex with plasminogen activator inhibitor 1 (PAI-1). WB for PAI-1 from the SN and CL detected a band at 80 kDa, while the expression of PAI-1 on the HUVEC surface decreased after incubation with FXIa. FXIa bound to the EC surface at 4C. After 5 mins at 37C, FXIa was internalized and localized beneath the plasma membrane, and after 30 mins to 2 hr at 37C, FXIa accumulated in the early and late endosomes and lysosomes. The serine protease inhibitor PPACK blocked FXIa-PAI-1 complex formation and internalization of FXIa. A blocking anti-PAI-1 antibody increased the cleavage of the chromogenic substrate by FXIa and the capacity of FXIa to promote fibrin formation on ECs in recalcified plasma. Summary: FXIa forms a complex with PAI-1 on the surface of ECs blocking its activity. Inhibition of FXIa on the endothelium may support internalization and clearance of FXIa from the circulation.