Mice Of Haplotypes Research Articles

Epigenetic mechanisms differentially regulate blood pressure and renal dysfunction in male and female Npr1 haplotype mice.

We determined the epigenetic mechanisms regulating mean arterial pressure (MAP) and renal dysfunction in guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) gene-targeted mice. The Npr1 (encoding NPRA) gene-targeted mice were treated with class 1 specific histone deacetylase inhibitor (HDACi) mocetinostat (MGCD) to determine the epigenetic changes in a sex-specific manner. Adult male and female Npr1 haplotype (1-copy; Npr1+/-), wild-type (2-copy; Npr1+/+), and gene-duplicated heterozygous (3-copy; Npr1++/+) mice were intraperitoneally injected with MGCD (2 mg/kg) for 14 days. BP, renal function, histopathology, and epigenetic changes were measured. One-copy male mice showed significantly increased MAP, renal dysfunction, and fibrosis than 2-copy and 3-copy mice. Furthermore, HDAC1/2, collagen1alpha-2 (Col1α-2), and alpha smooth muscle actin (α-SMA) were significantly increased in 1-copy mice compared with 2-copy controls. The expression of antifibrotic microRNA-133a was attenuated in 1-copy mice but to a greater extent in males than females. NF-κB was localized at significantly lower levels in cytoplasm than in the nucleus with stronger DNA binding activity in 1-copy mice. MGCD significantly lowered BP, improved creatinine clearance, and repaired renal histopathology. The inhibition of class I HDACs led to a sex-dependent distinctive stimulation of acetylated positive histone marks and inhibition of methylated repressive histone marks in Npr1 1-copy mice; however, it epigenetically lowered MAP, repaired renal fibrosis, and proteinuria and suppressed NF-kB differentially in males versus females. Our results suggest a role for epigenetic targets affecting hypertension and renal dysfunction in a sex-specific manner.

Colony-forming potential of murine bone marrow-derived progenitor cells in experimental neurodegenerative pathology and under the impact of melatonin in vivo or in vitro

Cell therapy is a promising direction in the treatment of Parkinson’s disease/parkinsonism and multiple sclerosis, the effectiveness of which can be increased with the help of biologically active factors, in particular melatonin. The purpose of the study: to investigate the colony-forming ability of stromal and hematopoietic cells-progenitors of the bone marrow of mice with experimentally induced neurodegenerative pathology and reveal the possibility to change it under the influence of melatonin in vivo or in vitro. Materials and methods: In adult (6-7 months) male FVB/N (haplotype H-2q) and 129/Sv (haplotype H-2b) mice, the parkinsonism model was reproduced by a single subcutaneous injection of the neurotoxin 1-methyl-4-phenyl -1,2,3,6-tetrahydropyridine (MPTP) at a dose of 30 mg/kg 129/Sv mice also received the neurotoxin cuprizone daily with food (at the rate of 0.2 % of the mass of the pure substance from the daily feed) for three weeks (multiple sclerosis model). Melatonin was administered to the 129/Sv mice intraperitoneally at a dose of 1 mg/kg, daily, at 17.00, from the 8th day of receiving cuprizone, or was added in vitro to the culture of bone marrow cells of mice of both strains with parkinsonism at a dose of 0.1 μg/0.1 mL. In the bone marrow, the absolute number of nucleated cells was counted, the relative content of CD4+ lymphocytes was determined by flow cytometry, and the number of colony-forming precursor cells for fibroblasts (CFU-F) and for granulocyte-macrophages (CFU-GM) in culture in vitro was estimated. Results. In the bone marrow of 129/Sv mice with a model of multiple sclerosis, the number of nucleated cells, CFU-GM and CD4+ cells decrease, while under the influence of exogenous melatonin applied in vivo, the values of these indicators increase to the level of intact animals. After the administration of MPTP in the bone marrow of FVB/N mice, in contrast to 129/Sv mice, the number of nucleated cells and CFU-F decreases. After the incubation with melatonin of bone marrow cells of 129/Sv mice with a model of parkinsonism, an increase in the amount of CFU-F was observed compared to control values, and there were no changes in the indicator values in FVB/N mice. Conclusions. In the bone marrow of mice with the MPTP-model of parkinsonism, a decrease in the absolute amount of CFU-F was found, and in mice with the cuprizone model of multiple sclerosis – CFU-GM. The application of melatonin in vitro increases the colony-forming potential of progenitor cells for fibroblasts in the bone marrow of mice with the MPTP model of parkinsonism. The use of melatonin in vivo increases the colony-forming ability of progenitor cells for granulocyte-macrophages and the relative content of CD4+ cells in the bone marrow of mice with the cuprizone model of multiple sclerosis. Changes in the amount of CFU-F in the bone marrow in parkinsonism and under the influence of melatonin in vitro depend on the H-2 haplotype mice, which can be the basis for the development of personalized cell therapy for this pathology.

SAT264 Effects Of Coenzymes In Gene Therapy For Congenital Adrenal Hyperplasia With AAV Vectors Into Model Mice

Abstract Disclosure: Y. Naiki: None. M. Miyado: None. R. Horikawa: None. N. Katsumata: None. S. Takada: None. M. Fukami: None. Congenital adrenal hyperplasia (CAH) is due to defects of steroid synthetic enzymes, which includes microsomal and mitochondrial P450s and they need coenzymes respectively. Microsomal P450s include 21-hydroxylase need P450 oxidoreductase which coded by POR and ferredoxin and ferredoxin reductase, which coded by FDX and FDXR, respectively, work for mitochondrial P450s include 11β-hydroxylase. 21-hydroxylase deficiency (21-OHD), in which CYP21A2 is mutated or deleted, and 11β-hydroxylase deficiency (11β-OHD), which caused by mutated CYP11B1 are the top two cause of CAH. CAH are treated with oral steroid supplementation, but optimal control remains difficult. Thus, new therapeutic approaches are still needed. We succeeded gene therapy for model mice of CAH and iPS cell from CAH patients (1). In this study, we examined the effects of induction of coenzyme genes with an AAV vector into a model mouse. A 21OHD mouse were made by breeding with H-2aw18 haplotype mice and treated with subcutaneous corticosteroid injection until 3 weeks of age. A 11β-OHD mouse were made by using the CRISPR/Cas9 method. A type 9 AAV (AAV9) containing Cyp21a cDNA (AAV9-Cyp21a1) was constructed with pAAV-CMV-shuttle. AAV9 containing Por cDNA (AAV9-Por), Cyp11b1 cDNA (AAV9-Cyp11b1), Fdx (AAV9-Fdx), Fdxr (AAV9-Fdxr) were also made. An AAV9-Cyp21a1 and AAV9-Por were mixed and injected into limbs muscles of a 21-OHD mouse and AAV9-Cyp11b1 mixed with AAV9-Fdx and AAV9-Fdxr injected into limbs muscles of 11β-OHD mouse. Serum progesterone (P4) and deoxycorticosterone (DOC) concentrations for 21-OHD mouse and DOC and corticosterone (B) concentrations for 11β-OHD mouse were measured before and after injection. P4 and DOC concentrations were measured by LC-MS/MS. An AAV9-Cyp21a1 and AAV9-Por injected mouse showed improved steroid synthesis up to 7 months after AAV9 induction. Comparing with a mouse injected only AAV9-Cyp21a1, significant reduction of P4/DOC was observed after 4 weeks of injection but there was no difference in the effectiveness of Cyp21a1 and Por combined treatment at the end of observation. Instead of a 21-OHD mouse, a 11β-OHD mouse did not show significant improvement by AAV mixture injected. These results indicate that extra-adrenal induction of Cyp21a1 and Por may ameliorate steroid metabolism in 21-OHD model mice. Although this coenzyme combined therapy showed some effects, but no long-term improvement of the efficacy exhibited. Instead of microsomal P450s, extra-adrenal induction with coenzyme genes did not work enough for mitochondrial P450s defects. Coenzymes of P450s are necessary for their activity, but our results suggest that the coenzyme combined gene therapeutic strategies are needed to be improved. Reference: (1) Naiki et al., Hum Gene Ther. 2022 Aug;33(15-16):801-809. Presentation: Saturday, June 17, 2023

Abstract 134: Role Of Epigenetic Mechanisms Regulating Renal Pathology And Disorders In Global Npr1 Gene-targeted Mutant Mice In Sex-dependent Manner

The objective of this study was to investigate the effect of class I-specific histone deacetylase inhibitor, mocetinostat (MGCD0103; MGCD) in the regulation of epigenetic mechanisms and renal pathology and disorders in Npr1 (coding for natriuretic peptide receptor A; NPRA) gene-disrupted haplotype mice. Male and female Npr1 gene-disrupted (1-copy; +/- ), wild-type (2-copy; +/+ ) and gene-duplicated (3-copy; ++/+) mice were administrated with MGCD (2 mg/kg) by intraperitoneal injection at alternate days for 2-weeks. The renal NPRA protein levels and guanylyl cyclase (GC) activity were significantly lower in 1-copy mice and higher in 3-copy than in 2-copy mice; however, MGCD increased NPRA protein levels and GC activity. The vehicle-treated females showed lower SBP than male mice, whereas both sexes of 1-copy mice had higher SBPs, and 3-copy mice had lower SBPs as compared with 2-copy mice. Treatment with MGCD distinctly reduced SBP in male (1-copy, Δ-21±2.0 mmHg, p<0.001; 2-copy, Δ-5±0.6 mmHg, p<0.01; and 3-copy, Δ-4±0.5 mmHg, p<0.05) and female mice (1-copy, Δ-17±2.1 mmHg, p<0.01; 2-copy, Δ-3±0.5 mmHg, p<0.01; and 3-copy, Δ-3±0.8 mmHg). Hematoxylin and eosin staining showed pronounced renal morphological damages in 1-copy male than female mice as compared to 2-copy and 3-copy mice. One-copy male mice exhibited a greater degree of mesangial matrix expansion and interstitial nephritis as compared with 2-copy and 3-copy mice, which was significantly (p<0.1) decreased with MGCD treatment as compared with vehicle-treated mice. Creatinine clearance was significantly reduced in 1-copy male mice (52%; p<0.05) than female mice (35%; p<0.05) compared with 2-copy mice. The renal HDAC activity and HDAC1 and 2 protein levels in 1-copy male mice were significantly (p<0.05) higher than female mice as compared with 2-copy mice. MGCD markedly increased acetylation of histone marks (H3-K9, H3-K27, and H3-K18) and greatly reduced the renal pathologies as compared with controls. The present results demonstrate that MGCD upregulates NPRA activity and repairs renal pathology epigenetically in 1-copy haplotype mice. These findings will have important implications for treatment of hypertension and renal injury in humans in both sexes.

Deciphering the genetic interaction between klotho and APOE with novel mouse models

AbstractBackgroundApolipoprotein E4 (APOE4), a common variant of APOE, is a major genetic risk factor for LOAD, but APOE4 carriers do not always develop LOAD. Several large‐scale genetic studies have identified a common haplotype of the aging factor klotho that modify disease risk in APOE4 carriers. In humans, klotho harbors two common missense variants (rs9536314, p.F352V; rs9527025, p.C370S) that define the klotho V/S (KL‐V/S) haplotype, which is protective against LOAD in APOE4 carriers, and the klotho F/C (KL‐F/C) haplotype, which is not. We sought to understand this genetic interaction using novel mouse models.MethodsWe introduced the human klotho variants into the mouse. Mouse klotho differs at the key amino acid position p.C370S rendering the native mouse haplotype as “KL‐F/S”, matching neither of the observed human haplotypes. We have replaced this native haplotype with the homozygous protective KL‐V/S and common KL‐F/C haplotypes using CRISPR/Cas9. To validate the effects of these klotho haplotypes on klotho secretion, soluble p‐KL serum levels of four month old mice were compared after cheek bleeding using ELISA. We also measured p‐KL in eight month old amyloidogenic App SAA mutant mice, which have amyloid plaques in the brain.ResultsGenomic modifications in mouse klotho were confirmed by PCR and Sanger sequencing. Secreted p‐KL was decreased in KL‐F/C mice harboring the novel mouse risk haplotype when compared to age matched B6 controls and KL‐V/S mice. In contrast, KL‐V/S mice showed no significant differences in soluble p‐KL serum levels when compared to B6 controls. Secreted p‐KL levels in KL‐V/S mice were significantly elevated when compared to the App SAA mice, whereas young KL‐F/C mice showed similar levels to the aged App SAA model.ConclusionsTaken together, these data suggest the native mouse klotho allele may acts similarly to the protective KL‐V/S haplotype, while the KL‐F/C haplotype leads to lower secreted klotho and potentially greater disease risk. These models are therefore suitable for experiments to elucidate how klotho variants protect from LOAD pathologies, and will enable detailed studies of how this protection is specific to the APOE4 genotype.

Molecular Determinants Regulating the Plasticity of the MHC Class II Immunopeptidome.

In the last few years, advancement in the analysis of the MHC class II (MHC-II) ligandome in several mouse and human haplotypes has increased our understanding of the molecular components that regulate the range and selection of the MHC-II presented peptides, from MHC class II molecule polymorphisms to the recognition of different conformers, functional differences in endosomal processing along the endocytic tract, and the interplay between the MHC class II chaperones DM and DO. The sum of all these variables contributes, qualitatively and quantitatively, to the composition of the MHC II ligandome, altogether ensuring that the immunopeptidome landscape is highly sensitive to any changes in the composition of the intra- and extracellular proteome for a comprehensive survey of the microenvironment for MHC II presentation to CD4 T cells.

Glucose homeostasis is impaired in Guanylyl Cyclase/Natriuretic Peptide Receptor‐A Gene‐Knockout and Gene‐Duplication Mutant Mice

Atrial and brain natriuretic peptides (ANP and BNP) are produced in the heart, regulate blood pressure (BP) and fluid homeostasis through vasodilatory and diuretic actions. Both ANP and BNP act by binding to guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPR‐A). Systemic disruption of Npr1(encoding GC‐A/NPRA) leads to volume overload, high BP, and congestive heart failure. However, the underlying mechanisms are not yet precisely determined. The aim of this study was to investigate whether Npr1plays a critical role in regulating glucose homeostasis in Npr1 gene‐disrupted mice. The adult male and female (16‐18 wk) Npr1 gene‐knockout haplotype (Npr1+/‐, 1‐copy), wild‐type (Npr1+/+, 2‐copy), and gene‐duplicated (Npr1+ +/+ +, 4‐copy) mice were fasted for 16 h and given free access to water. The glucose administration was done both orally and intraperitoneally (IP: 2 g/kg body weight) in mice to determine oral glucose tolerance test (OGTT) and IP glucose tolerance test (IPGTT). The glucose levels in blood were determined by performing tail bleeds at 0, 15, 30, 60, 90, and 120 min using the AlphaTRAK blood glucose monitoring system (Zoetis Inc, Kalamazoo, MI). Systolic BP (SBP) was determined by non‐invasive computerized tail‐cuff method (Visitech 2000). The results showed that the blood glucose level was elevated to the maximum value at 15 min after glucose administration. Glucose levels were declined to near base line at 120 min in 2 copy mice (OGTT: 102 ± 3 mg/dL in male and 97 ± 4 mg/dL in female, IPGT: 101 ± 4 mg/dL in male and 96 ± 3 mg/dL in female). However, in 1‐copy mice, the blood glucose levels remained elevated even after 120 min (OGTT: 248 ± 5 mg/dL in male and 226 ± 3 mg/dL in female, IPGT: 254 ± 4 mg/dL in male and 229 ± 5 mg/dL in female) compared with 2‐copy mice. The blood glucose levels were also significantly lower at 120 min in 4‐copy mice (OGTT: 77 ± 4 mg/dL in male and 74 ± 3 mg/dL in female, IPGT: 75 ± 3 mg/dL in male and 71 ± 2 mg/dL in female) compared with 2‐copy mice. SBP was significantly greater in 1‐copy mice (137 + 4 mmHg in male and 126 + 3 mmHg in female) than 2‐copy mice (100 + 2 mmHg in male and 91 + 2 mmHg in female). SBP was significantly lower in 4‐copy mice (83 + 2 mmHg in male and 77 + 2 mmHg in female) than 2‐copy mice. OGTT showed a significantly lower level of maximal blood glucose compared with IPGTT. The findings of the present study suggest that Npr1 plays a critical role in the regulation of glucose homeostasis and the loss of Npr1 significantly increases the glucose levels in mutant animals.

Effect of epitope variant co-delivery on the depth of CD8 T cell responses induced by HIV-1 conserved mosaic vaccines

To stop the HIV-1 pandemic, vaccines must induce responses capable of controlling vast HIV-1 variants circulating in the population as well as those evolved in each individual following transmission. Numerous strategies have been proposed, of which the most promising include focusing responses on the vulnerable sites of HIV-1 displaying the least entropy among global isolates and using algorithms that maximize vaccine match to circulating HIV-1 variants by vaccine cocktails of optimized complementing sequences. In this study, we investigated CD8 T cell responses induced by a bi-valent mosaic of highly conserved HIVconsvX regions delivered by a combination of simian adenovirus ChAdOx1 and poxvirus MVA. We compared partially and fully mono- and bi-valent prime-boost regimens and their ability to elicit T cells recognizing natural epitope variants using an interferon-γ enzyme-linked immunospot (ELISPOT) assay. We used 11 well-defined CD8 T cell epitopes in two mouse haplotypes and, for each epitope, assessed recognition of the two vaccine forms together with the other most frequent epitope variants in the HIV-1 database. We conclude that for the magnitude and depth of epitope recognition, CD8 T cell responses benefitted in most comparisons from the combined bi-valent mosaic and envisage the main advantage of the bi-valent vaccine during its deployment to diverse populations.

Regulation of Blood Pressure and Renal Fibrosis by Class I Specific HDAC Inhibitor, Mocetinostat, in Npr1 Gene‐targeted Male and Female Mice

Mice lacking Npr1 (encoding guanylyl cyclase/natriuretic peptide receptor-A; GC-A/NPRA) exhibit hypertension, kidney disease, and heart failure; however, the epigenetic determinants regulating Npr1 expression and renal function are not well understood. The objective of this study was to investigate the effect of class I-specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103; MGCD) on NPRA expression and regulation of mean arterial pressure (MAP) and renal pathology. Adult male and female Npr1 haplotype (1-copy, Npr1+/-), wild-type (2-copy, Npr1+/+), and gene-duplicated (3-copy, Npr1++/+) mice were injected intraperitoneally with MGCD (2 mg/kg) at alternate days for 2-weeks. Treatment with MGCD significantly enhanced NPRA protein levels and GC activity in male and female mice in all three genotypes. MAP was monitored by telemetry recording. Sex-differences were observed in MAP as female mice had lower MAP (p < 0.01) than male mice, in all genotypes; whereas, 1-copy mice exhibited higher MAP than 2-copy mice. Treatments with MGCD distinctly reduced MAP in 1-copy and 2-copy male mice (untreated 1-copy, 125 ± 3 vs. treated 1-copy, 108 ± 3 mmHg; p < 0.001; untreated 2-copy, 98 ± 2 vs. treated 2-copy, 90 ± 2 mmHg; p < 0.01) and 1-copy and 2-copy female mice (untreated 1-copy, 117 ± 3 vs. treated 1-copy, 105 ± 2 mmHg; p < 0.001; untreated 2-copy, 90 ± 2 vs. treated 2-copy, 84 ± 2 mmHg; p < 0.01). Treatment with MGCD attenuated HDAC activity by 30% in male and 40% in female animals (p < 0.05). The Western blot analyses indicated that 1-copy male and female mice showed upregulation of alpha-smooth muscle actin (α-SMA; 39% and 29%, p < 0.05) and collagen 1 alpha 2 (COL1α2; 49% and 20%, p < 0.05) proteins, respectively, compared with 2-copy mice. Picrosirius red staining in renal sections showed significant collagen deposition in 1-copy mice compared with 2-copy animals of both sexes. MGCD reduced fibrosis by 30%-45% (p < 0.05) in treated 1-copy mice compared with untreated control mice in both sexes. The present results indicate that MGCD lowers MAP, and repairs renal fibrosis in male and female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in both genders.

B cell expression patterns before and after Nippostrongylus brasiliensis infection; location matters over lineage.

Abstract Our lab has previously shown that B1 B cells are important in the response to Nippostrongylus brasiliensus. We next wanted to examine the transcriptional changes between B1 and B2 B cells before and after N. brasiliensis infection. After examination of peritoneal cavity (PerC) B1 B cells and B2 B cells compared to mesenteric lymph node (MLN) B2 B cells by RNAseq we showed that there was more transcriptional similarity between PerC B cell populations than PerC B2 B cells and MLN B2 B cells. Due to this finding, we proceeded to determine the lineage of these B cells populations before and after helminth infection. To do this, one day postnatal, all B cells were depleted from CD45.1 IgMa haplotype mice using anti-IgMa antibody (DS.1). The following day the entire PerC lavage of an adult CD45.2 IgMb mouse was reconstituted, intraperitoneally. DS.1 was administered twice weekly until the mice were 6 weeks of age. After resting the mice to allow host bone marrow (BM) derived B cells to recover, the resulting mice had distinguishable B cell populations based on lineage. Interestingly, we found that regardless of whether the B cells were BM or fetal liver derived, both lineages were found in classically defined B1 B cell and B2 B cell population based on surfaces marker patterns by flow cytometry. In conclusion, this work suggests that anatomic location plays a larger role in terminal function than lineage, and that PerC B cell populations have a more ambiguous lineage than previously thought.

The Impact of the Hybridoma Technology on the R&D of Idiotypic Antibodies.

The PubMed data set was scanned with the title and abstract term "Idiotype" followed by secondary searches with "Vaccine" and "Clinical trial." The retrieved references were analyzed from the period before and after hybridoma technology (1975). In 1963, Oudin and Kunkel discovered that antibodies against antibodies can be raised to identify determinants unique to an antibody termed idiotype or individual antigenic determinant. Two laboratories reported that anti-idiotypic antibodies can suppress specific antibody responses in mice. In 1974, Jerne proposed a network of idiotypes and anti-idiotypes and the functionality of the idiotype network was confirmed. This prompted the proposal of a symmetrical regulatory immune response. By 1989, the concept and the functional parameters of the immune idiotype network were established in the prehybridoma period. It was not until 1981 that monoclonal anti-idiotypic antibodies were used as tools to study the expression of idiotypic determinants on antibodies and to categorize functional properties in the immune network as network antigens in 1989. Hybridoma-generated monoclonal anti-idiotypic antibodies provided the tools to precisely identify different idiotypic regions on antibodies and test these as targets to induce network cascades. The initial distinction of Ab2s as alpha and beta were expanded to include gamma and delta. The initial concept of Ab2beta being an antigen internal image, used as vaccine, was challenged showing that targeting all idiotopes on B cell receptors can induce specific antibodies. After the discovery of the hybridoma technology a wave of idiotype topic publications occurred, that declined by 2015. In 1985, in this wave of reports on anti-idiotypes, their importance to vaccines dominated. These vaccines targeted in animal models parasite, bacterial, and viral diseases, and cancer. The reported data indicated a therapeutic response in inbred mice. The issue of idiotype matching between mouse haplotypes of vaccine origin and treated mice were raised. In 1995, the human clinical trials in different cancers using anti-Id vaccines were reported. Only one such vaccine received conditional approval in Argentina and Cuba, whereas the other trials failed in phase II and III. The reasons for this failure were subsequently discussed. Although the use of the Milstein and Kohler hybridoma technology and subsequently alternative methods to produce monoclonal animal and human antibodies created a new class of drugs, commonly referred as "Biological," it failed on the promise therapeutic of anti-Id vaccines.

MHC Restriction: Where Are We Now?

MHC Restriction: Where Are We Now?

Class I HDAC Differentially Regulates Blood Pressure and Renal Injury in Npr1 Gene‐targeted Male and Female Mutant Mice

Atrial and brain natriuretic peptides (ANP, BNP) activate guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA), which plays a critical role in the regulation of blood pressure (BP) and fluid volume homeostasis, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 gene (coding for GC‐A/NPRA) exhibit pathophysiology of hypertension, kidney disease, and heart failure; however, the mechanisms regulating Npr1 expression are not well understood. The objective of the present study was to gain insight into the effect of class I‐specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103) on Npr1 gene expression and regulation of BP and renal injury in gene‐targeted mutant mice. We utilized 16‐ to 20‐weeks old male and female Npr1 gene‐knockout haplotype (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+), and gene‐duplicated heterozygous (3‐copy; Npr1++/+) mice. All mice genotypes were injected intraperitoneally with MGCD0103 (2 mg/kg) at an alternate day for 2‐weeks. The Western blot results showed that MGCD0103 significantly increased the renal NPRA protein levels in male and female mice compared with vehicle‐treated (olive oil) controls. After MGCD treatment, the renal GC‐A activity was significantly increased in both male and female mice. Male mice exhibited significantly higher systolic BP (SBP) than female mice and treatment with MGCD0103 decreased SBP in both 1‐copy male (106 ± 0.6 vs. vehicle‐treated, 129 ± 1.9; mmHg) and female (97 ± 2.2 vs. vehicle‐treated, 110 ± 2.1 mmHg, p &lt; 0.001) mice. Renal HDAC activity was considerably higher (p &lt; 0.05) in male mice than female mice; however, treatment with MGCD0103 attenuated (p &lt; 0.05) HDAC activity by 40% in male and ~50% in female animals. Significantly higher urinary albumin to creatinine ratio was detected in male mice compared with female mice, which was attenuated by MGCD0103 treatment in male mice. The present results demonstrate that class I HDAC inhibitor, MGCD0103 upregulates NPRA expression in vivo, lowers SBP, and repairs renal injury differentially in male vs. female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in a gender‐related manner.Support or Funding InformationThis work was supported by NIH grant HL062147.

Abstract 034: Differential Regulation of Renal Injury in Npr1 Gene-Targeted Male and Female Mice: Role of Class I-Specific HDAC Inhibitor

The objective of the present study was to gain insight into the effect of class I-specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103; MGCD) on Npr1 (coding for natriuretic peptide receptor-A; NPRA) gene expression and regulation of renal pathology. Adult male and female Npr1 haplotype (1-copy; Npr1 +/- ), wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were injected intraperitoneally with MGCD (2 mg/kg) at alternate days for 2-weeks. The renal NPRA protein levels were significantly increased in the treated male (1-copy, 2.1-fold; 2-copy, 2.3-fold; and 3-copy, 1.5-fold; p &lt; 0.05) and female mice (1-copy, 3.6-fold; 2-copy, 2.1-fold; and 3-copy, 1.7-fold; p &lt; 0.05) compared with untreated animals. After MGCD treatment, the renal guanylyl cyclase-A (GC-A) activity significantly increased in male mice (1-copy, 77.4 ± 0.5 vs. control 30.7 ± 0.5; 2-copy 126.1 ± 5.0 vs. control 76.3 ± 6.3; and 3-copy, 182.7 ± 2.8 vs. control 141.2 ± 6.4; p &lt; 0.05; cGMP/mg protein/ 30 min) and female mice (1-copy, 108.2 ± 6.8 vs. control 51.9 ± 3.0; p &lt; 0.05). Male mice exhibited significantly higher systolic blood pressure (SBP) than females and treatment with MGCD decreased SBP in both 1-copy male (106 ± 0.6 vs. control, 129 ± 1.9; mmHg) and female mice (97 ± 2.2 vs. control, 110 ± 2.1, p &lt; 0.001). Higher urinary albumin to creatinine ratio was detected in male mice compared with female mice, which was attenuated by MGCD treatment in male mice (1-copy, 0.39 ± 0.04 vs. untreated 0.71 ± 0.05; 2-copy 0.31 ± 0.01 vs. untreated 0.46 ± 0.05; and 3-copy, 0.2 ± 0.02 vs. untreated 0.21 ± 0.02; p &lt; 0.05). HDAC activity was significantly higher in male mice (1-copy, 24.4 ± 2.8; 2-copy, 15.9 ± 1.2; and 3-copy, 6.4 ± 0.7; ng/min/mg protein) than female mice (1-copy, 13.7 ± 1.4; 2-copy, 8.5 ± 0.9; and 3-copy, 4.7 ± 0.9, p &lt; 0.05); however, MGCD treatment significantly attenuated HDAC activity in both male and female animals (p &lt; 0.05). The present results indicate MGCD upregulates Npr1 expression in 1-copy haplotype mice via inhibition of HDAC activity, lowers SBP and repairs renal injury differentially in male vs. female mice. These findings will have important implications for treatment of hypertension and renal injury in humans in a sex-related manner.

SAT-046 Modulation of Histone 3 Acetylation and Methylation Enhances the Expression and Function of Npr1 in Haplotype Mice

Atrial and brain natriuretic peptides (ANP, BNP) activate guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA), which plays a critical role in the regulation of blood pressure and fluid volume homeostasis, and inhibits the cell proliferation and fibrosis. Mice lacking functional Npr1 (coding for GC-A/NPRA) gene exhibit hypertension and renal disorders; however, the mechanisms regulating Npr1 expression and function are not well understood. The objective of the present study was to gain insight into the epigenetic regulatory mechanisms involved in Npr1 gene expression modulated by class I specific histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103). We utilized 16- to 20-weeks old male and female Npr1 gene-knockout haplotype (1-copy; Npr1+/-), wild-type (2-copy; Npr1+/+) and gene-duplicated heterozygous (3-copy; Npr1++/+) mice. Mice were injected intraperitoneally with MGCD0103 (2 mg/kg) at an alternate days for 2-weeks. The Western blot analysis revealed that MGCD0103 significantly increased the renal NPRA protein levels in all male and female mice groups compared with vehicle-treated controls. After MGCD0103 treatment, the renal Npr1 mRNA expression increased in Npr1 haplotype male and female mice. Renal HDAC activity was significantly higher (p < 0.05) in all genotypes of male mice than female mice; however, treatment with MGCD0103 markedly attenuated HDAC activity in both male (~40%, p < 0.05) and female (~50%, p < 0.05) animals. The histone acetyltransferase (HAT) activity was lower in untreated 1-copy male mice compared with wild type mice and MGCD0103 treatment significantly increased the HAT activity whereas no significant differences were observed in drug- or vehicle-treated female mice. The drug treatment significantly enhanced the acetylation levels of H3-K9 and H3-K27 in 1-copy male mice and H3-K18 in 1-copy female mice. There was substantial increase in protein levels of active gene transcription mark H3-K4 tri-methylation and decrease in suppressive marks H3-K9 and H3-K27 methylation in MGCD0103-treated 1-copy male and female mice. The present results suggest that MGCD0103 epigenetically regulates Npr1 expression in vivo in 1-copy haplotype male and female mice, via inhibition of HDAC activity, upregulation of the H3 acetylation and attenuation of H3 methylation levels. These present findings will have important implications in prevention and treatment of hypertension-related renal pathophysiological conditions under reduced NPRA signaling. This work was supported by NIH grants (HL057531 and HL062147) and a partial support from Tulane Carol Lavin Bernick grant award.

Experimental and Theoretical Approaches To Investigate the Immunogenicity of Taenia solium-Derived KE7 Antigen.

Taenia solium cysticercosis, a parasitic disease that affects human health in various regions of the world, is preventable by vaccination. Both the 97-amino-acid-long KETc7 peptide and its carboxyl-terminal, 18-amino-acid-long sequence (GK-1) are found in Taenia crassiceps Both peptides have proven protective capacity against cysticercosis and are part of the highly conserved, cestode-native, 264-amino-acid long protein KE7. KE7 belongs to a ubiquitously distributed family of proteins associated with membrane processes and may participate in several vital cell pathways. The aim of this study was to identify the T. solium KE7 (TsKE7) full-length protein and to determine its immunogenic properties. Recombinant TsKE7 (rTsKE7) was expressed in Escherichia coli Rosetta2 cells and used to obtain mouse polyclonal antibodies. Anti-rTsKE7 antibodies detected the expected native protein among the 350 spots developed from T. solium cyst vesicular fluid in a mass spectrometry-coupled immune proteomic analysis. These antibodies were then used to screen a phage-displayed 7-random-peptide library to map B-cell epitopes. The recognized phages displayed 9 peptides, with the consensus motif Y(F/Y)PS sequence, which includes YYYPS (named GK-1M, for being a GK-1 mimotope), exactly matching a part of GK-1. GK-1M was recognized by 58% of serum samples from cysticercotic pigs with 100% specificity but induced weak protection against murine cysticercosis. In silico analysis revealed a universal T-cell epitope(s) in native TsKE7 potentially capable of stimulating cytotoxic T lymphocytes and helper T lymphocytes under different major histocompatibility complex class I and class II mouse haplotypes. Altogether, these results provide a rationale for the efficacy of the KETc7, rTsKE7, and GK-1 peptides as vaccines.

Abstract P161: Inhibition of Class I HDAC Enhances the Expression of Npr1 and Repairs Renal Pathology in Gene-targeted Haplotype Mice

The objective of the present study was to determine the effect of class I histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103) on the expression of Npr1 (coding for natriuretic peptide receptor-A; NPRA) and renal pathology in Npr1 gene-disrupted haplotype (50% of normal) mice. Adult male Npr1 haplotype (1-copy; Npr1 +/- ), wild-type (2-copy; Npr1 +/+ ), and gene-duplicated (3-copy; Npr1 ++/+ ) mice were injected intraperitonealy with MGCD0103 (2 mg/kg) at alternate days for 2-weeks. After MGCD0103 treatment, the renal Npr1 mRNA was increased in Npr1 +/- mice by 7.3-fold (p &lt; 0.01), Npr1 +/+ mice by 4.4-fold (p &lt; 0.05) and Npr1 ++/+ mice by 3.6-fold (p &lt; 0.05) compared with vehicle-treated controls. The MGCD0103 also enhanced renal cGMP levels (pmol/mg protein) in Npr1 +/- (57 ± 7 vs. control, 12 ± 1; p &lt; 0.01), Npr1 +/+ (125 ± 9 vs. control, 66 ± 5; p &lt; 0.01), and Npr1 ++/+ (202 ± 7 vs. control, 127 ± 15; p &lt; 0.05) mice. An increased HDAC activity (ng/min/mg protein) was observed in Npr1 +/- mice (24.4 ± 2.8; p &lt; 0.05); however, Npr1 ++/+ mice showed lower HDAC activity (6.4 ± 0.7; p &lt; 0.05); compared with Npr1 +/+ mice (15.9 ± 1.3). Treatment with MGCD0103 significantly attenuated HDAC activity by almost 50% (15.3 ± 1.2; p &lt; 0.01) in Npr1 haplotype mice. A significant decrease in systolic blood pressure was observed in MGCD0103-treated Npr1 +/- mice (106 ± 0.6 vs. control, 131 ± 1.9, p &lt; 0.001). Significantly lower creatinine clearance (μl/min) was observed in Npr1 +/- (51 ± 11; p &lt; 0.05) vs. Npr1 +/+ mice (130 ± 14.0; p &lt; 0.05) and treatment with MGCD0103 increased creatinine clearance in Npr1 +/- mice (105 ± 13; p &lt; 0.05) vs. controls. Higher urinary albumin to creatinine ratio was detected in Npr1 +/- mice (0.7 ± 0.05) vs. Npr1 +/+ animals (0.5 ± 0.05; p &lt; 0.05) and a complete reversal was observed in drug-treated Npr1 +/- mice (0.4 ± 0.04). The present results provide direct evidence that class I HDAC inhibition upregulates NPRA expression in vivo and repairs the renal pathology in Npr1 +/- haplotype mice. The present findings will have important implications in the treatment and prevention of hypertension and renal pathophysiological conditions.

Immunization of C57BL/6 Mice with GRA2 Combined with MPL Conferred Partial Immune Protection against Toxoplasma gondii

Background:We have previously reported that immunization with GRA2 antigen of Toxoplasma gondii induces protective immunity in CBA/J (H2k) and BALB/c mice (H2d). We aimed to examine whether immunization of a distinct strain of rodent with recombinant dense granule antigens (GRA2) combined with monophosphorryl lipid A (MPL) adjuvant elicits protective immune response against T. gondii.Methods:C57BL/6 (H2b haplotype) mice were immunized with GRA2, formulated in MPL adjuvant.Results:Strong humoral response, predominantly of IgG1 subclass and cellular response, IFN-γ, was detected at three weeks post immunization. Mice immunized with GRA2 had significantly (p < 0.01) fewer brain cysts than those in the adjuvant group, upon challenge infection. Despite the production of a strong antibody response, IFN-γ production and brain cyst reduction were not significant when the immunized mice were infected four months after the immunization.Conclusions:We can conclude that GRA2 immunization partially protects against T. gondii infection in C57BL/6 mice, though the potency and longevity of this antigen as a standalone vaccine may vary in distinct genetic backgrounds. This observation further emphasizes the utility of GRA2 for incorporation into a multi-antigenic vaccine against T. gondii.

Inhibition of HDAC enhances STAT acetylation, blocks NF-κB, and suppresses the renal inflammation and fibrosis in Npr1 haplotype male mice.

Guanylyl cyclase/natriuretic peptide receptor-A (GC-A/NPRA) plays a critical role in the regulation of blood pressure and fluid volume homeostasis. Mice lacking functional Npr1 (coding for GC-A/NPRA) exhibit hypertension and congestive heart failure. However, the underlying mechanisms remain largely less clear. The objective of the present study was to determine the physiological efficacy and impact of all-trans-retinoic acid (ATRA) and sodium butyrate (NaBu) in ameliorating the renal fibrosis, inflammation, and hypertension in Npr1 gene-disrupted haplotype (1-copy; +/-) mice (50% expression levels of NPRA). Both ATRA and NaBu, either alone or in combination, decreased the elevated levels of renal proinflammatory and profibrotic cytokines and lowered blood pressure in Npr1+/- mice compared with untreated controls. The treatment with ATRA-NaBu facilitated the dissociation of histone deacetylase (HDAC) 1 and 2 from signal transducer and activator of transcription 1 (STAT1) and enhanced its acetylation in the kidneys of Npr1+/- mice. The acetylated STAT1 formed a complex with nuclear factor-κB (NF-κB) p65, thereby inhibiting its DNA-binding activity and downstream proinflammatory and profibrotic signaling cascades. The present results demonstrate that the treatment of the haplotype Npr1+/- mice with ATRA-NaBu significantly lowered blood pressure and reduced the renal inflammation and fibrosis involving the interactive roles of HDAC, NF-κB (p65), and STAT1. The current findings will help in developing the molecular therapeutic targets and new treatment strategies for hypertension and renal dysfunction in humans.

Class I‐Specific HDAC Inhibitor Stimulates the Expression of Npr1 in Haplotype Mice by Enhanced Histone Acetylation at Different Lysine Residues

Guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) signaling activates natriuresis/diuresis, vasodilatation, and inhibits the cell proliferation and fibrosis. The objective of the present study was to gain insight into the mechanisms of Npr1 (coding for GC‐A/NPRA) expression and receptor function involving epigenetic regulatory mechanisms activated by class I histone deacetylase (HDAC) inhibitor, mocetinostat (MGCD0103). We utilized 20‐ to 22‐weeks old male Npr1 gene‐knockout haplotype (1‐copy; Npr1+/−), wild‐type (2‐copy; Npr1+/+) and gene‐duplicated heterozygous (3‐copy; Npr1++/+) mice. The mice were injected intraperitonealy with MGCD0103 (5 mg/kg) at the alternate days for 2‐weeks. The Western blot analysis showed that MGCD0103 significantly increased the renal NPRA mRNA and protein levels in all mice groups compared with vehicle‐treated controls. There was marked increase in renal cGMP levels in MGCD0103‐treated mice groups. The Npr1 haplotype mice exhibited increased HDAC activity and decreased acetylated histone levels; however, 3‐copy mice showed lower HDAC activity compared with 2‐copy mice. Treatment with MGCD0103 significantly attenuated HDAC activity by almost 50% in Npr1 haplotype mice. A substantial global increase in acetylation levels of histone 3 at different lysine positions (H3‐K9/14ac, H3‐K18ac, and H3‐K27ac) and histone 4 lysine 8 (H4‐K8ac) was observed in the kidneys of MGCD0103‐treated mice. The present results suggest that MGCD0103 epigenetically regulates NPRA expression in vivo via HDAC inhibition and histone modifications, which might have important implications in the renal function in disease states.Support or Funding InformationThis work was supported by NIH grants (HL057531 and HL062147) and by an Institutional Development Award (IDeA) from the NIGMS.