Impact of hydrocortisone and of CRH infusion on the hypothalamus-pituitary-adrenocortical axis of septic male mice

Purpose Sepsis is hallmarked by high plasma cortisol/corticosterone (CORT), low adrenocorticotropic hormone (ACTH) and high pro-opiomelanocortin (POMC). While corticotropin-releasing hormone-(CRH) and arginine-vasopressin-(AVP) driven pituitary POMC expression remains active, POMC processing into ACTH becomes impaired. Low ACTH is accompanied by loss of adrenocortical structure, although steroidogenic enzymes remain expressed. We hypothesized that treatment of sepsis with hydrocortisone (HC) aggravates this phenotype whereas CRH infusion safeguards ACTH-driven adrenocortical structure. Methods In a fluid-resuscitated, antibiotics-treated mouse model of prolonged sepsis, we compared the effects of HC and CRH infusion with placebo, on plasma ACTH, POMC and CORT and on markers of hypothalamic CRH and AVP signaling and pituitary POMC processing, and on the adrenocortical structure and markers of steroidogenesis. In adrenal explants, we studied the steroidogenic capacity of POMC. Results During sepsis, HC further suppressed plasma ACTH, but not POMC, predominantly by suppressing sepsis-activated CRH/AVP-signaling pathways. In contrast, in CRH-treated sepsis, plasma ACTH was normalized following restoration of pituitary POMC processing. The sepsis-induced rise in markers of adrenocortical steroidogenesis was unaltered by CRH and suppressed partially by HC which also increased adrenal markers of inflammation. Ex vivo stimulation of adrenal explants with POMC increased CORT as effectively as an equimolar dose of ACTH. Conclusions Treatment of sepsis with HC impaired integrity and function of the HPA axis at the level of the pituitary and the adrenal cortex while CRH restored pituitary POMC processing without affecting the adrenal cortex. Sepsis-induced high circulating POMC may be responsible for ongoing adrenocortical steroidogenesis despite low ACTH.

Soluble Antigen Arrays Efficiently Deliver Peptides and Arrest Spontaneous Autoimmune Diabetes

Antigen-specific immunotherapy (ASIT) offers a targeted treatment of autoimmune diseases that selectively inhibits autoreactive lymphocytes, but there remains an unmet need for approaches that address their limited clinical efficacy. Soluble Antigen Arrays (SAgAs) deliver antigenic peptides or proteins in multivalent form, attached to a hyaluronic acid backbone using hydrolysable linkers (hSAgA) or stable “click” chemistry linkers (cSAgA). They were evaluated for the ability to block the spontaneous development of disease in the non-obese diabetic mouse model of Type 1 diabetes (T1D). Two peptides, a hybrid insulin peptide and a mimotope, efficiently prevented the onset of T1D when delivered in combination as SAgAs, but not individually. Relative to free peptides administered at equimolar dose, SAgAs (particularly cSAgA) enabled a more effective engagement of antigen-specific T cells with greater persistence and induction of tolerance markers such as CD73, IL-10, PD-1, KLRG-1. Anaphylaxis caused by the free peptides was attenuated using hSAgA and obviated using cSAgA platforms. Despite similarities, the two peptides elicited largely non-overlapping and possibly complementary responses among endogenous T cells in treated mice. Thus, SAgAs offer a novel and promising ASIT platform superior to soluble peptides in inducing tolerance while mitigating risks of anaphylaxis for the treatment of T1D.

Soluble Antigen Arrays Efficiently Deliver Peptides and Arrest Spontaneous Autoimmune Diabetes

Antigen-specific immunotherapy (ASIT) offers a targeted treatment of autoimmune diseases that selectively inhibits autoreactive lymphocytes, but there remains an unmet need for approaches that address their limited clinical efficacy. Soluble Antigen Arrays (SAgAs) deliver antigenic peptides or proteins in multivalent form, attached to a hyaluronic acid backbone using hydrolysable linkers (hSAgA) or stable “click” chemistry linkers (cSAgA). They were evaluated for the ability to block the spontaneous development of disease in the non-obese diabetic mouse model of Type 1 diabetes (T1D). Two peptides, a hybrid insulin peptide and a mimotope, efficiently prevented the onset of T1D when delivered in combination as SAgAs, but not individually. Relative to free peptides administered at equimolar dose, SAgAs (particularly cSAgA) enabled a more effective engagement of antigen-specific T cells with greater persistence and induction of tolerance markers such as CD73, IL-10, PD-1, KLRG-1. Anaphylaxis caused by the free peptides was attenuated using hSAgA and obviated using cSAgA platforms. Despite similarities, the two peptides elicited largely non-overlapping and possibly complementary responses among endogenous T cells in treated mice. Thus, SAgAs offer a novel and promising ASIT platform superior to soluble peptides in inducing tolerance while mitigating risks of anaphylaxis for the treatment of T1D.

Pharmacokinetics of Sodium and Calcium Salts of (6S)-5-Methyltetrahydrofolic Acid Compared to Folic Acid and Indirect Comparison of the Two Salts

(6S)-5-Methyltetrahydrofolic acid ((6S)-5-Methyl-THF) salts and folic acid may differ in their abilities to raise plasma (6S)-5-Methyl-THF levels. We compared the area under the curve (AUC), Cmax, and Tmax of plasma (6S)-5-Methyl-THF after intakes of (6S)-5-Methyl-THF-Na salt (Arcofolin®) and folic acid. Moreover, we compared the AUCs after intakes of (6S)-5-Methyl-THF-Na and the calcium salt, (6S)-5-Methyl-THF-Ca, that were tested against folic acid in two independent studies. The study was randomized, double blind, and cross over. Twenty-four adults (12 men and 12 women) received a single oral dose of 436 µg (6S)-5-Methyl-THF-Na and an equimolar dose of folic acid (400 µg) on two kinetic days with two weeks washout period in between. The plasma concentrations of (6S)-5-Methyl-THF were measured at 9 time points between 0 and 8 h. We found that the AUC0–8 h of plasma (6S)-5-Methyl-THF (mean (SD) = 126.0 (33.6) vs. 56.0 (25.3) nmol/L*h) and Cmax (36.8 (10.8) vs. 11.1 (4.1) nmol/L) were higher after administration of (6S)-5-Methyl-THF-Na than after the administration of folic acid (p < 0.001 for both). These differences were present in men and women. Only administration of folic acid resulted in a transient increase in plasma unmetabolized folic acid (2.5 (2.0) nmol/L after 0.5 h and 4.7 (2.9) nmol/L after 1 h). Intake of (6S)-5-Methyl-THF-Na was safe. The ratios of the AUC0–8 h for (6S)-5-Methyl-THF-Na and (6S)-5-Methyl-THF-Ca to the corresponding folic acid reference group and the delta of these AUC0–8 h did not differ between the studies. In conclusion, a single oral dose of (6S)-5-Methyl-THF-Na caused higher AUC0–8 h and Cmax of plasma (6S)-5-Methyl-THF compared to folic acid. The Na- and Ca- salts of (6S)-5-Methyl-THF are not likely to differ in their pharmacokinetics. Further studies may investigate whether supplementation of the compounds for a longer time will lead to differences in circulating or intracellular/tissue folate concentrations.

Effectivity of Two Cell Proliferation Markers in Brain of a Songbird Zebra Finch

There are two most heavily used markers of cell proliferation, thymidine analogues 5-bromo-2′-deoxyuridine (BrdU) and 5-ethynyl-2′-deoxyuridine (EdU) that are incorporated into the DNA during its synthesis. In neurosciences, they are often used consecutively in the same animal to detect neuronal populations arising at multiple time points, their migration and incorporation. The effectivity of these markers, however, is not well established. Here, we studied the effectivity of equimolar doses of BrdU and EdU to label new cells and looked for the dose that will label the highest number of proliferating cells in the neurogenic ventricular zone (VZ) of adult songbirds. We found that, in male zebra finches (Taeniopygia guttata), the equimolar doses of BrdU and EdU did not label the same number of cells, with BrdU being more effective than EdU. Similarly, in liver, BrdU was more effective. The saturation of the detected brain cells occurred at 50 mg/kg BrdU and above 41 mg/kg EdU. Higher dose of 225 mg/kg BrdU or the equimolar dose of EdU did not result in any further significant increases. These results show that both markers are reliable for the detection of proliferating cells in birds, but the numbers obtained with BrdU and EdU should not be compared.

Abstract 306: Alamandine but not angiotensin-(1-7) produces cardiovascular effects in the Insular Cortex

In the course of experiments aimed to evaluate the immunofluorescence distribution of MrgD receptors we observed the presence of immunoreactivity for the MrgD protein in the Insular Cortex. In order to evaluate the functional significance of this finding, we investigated the cardiovascular effects produced by the endogenous ligand of MrgD, alamandine, in this brain region. Urethane (1.4g/kg) anesthetized rats were instrumented for measurement of MAP, HR and renal sympathetic nerve activity (RSNA). Unilateral microinjection of alamandine (40 pmol/100nl), Angiotensin-(1-7) (40pmol/100nl), Mas/MrgD antagonista D-Pro7-Ang-1-7 (50pmol/100nl), Mas agonist A779 (100 pmol/100nl) or vehicle (0,9% NaCl) were made in different rats (N=4-6 per group) into posterior insular cortex (+1.5mm rostral to the bregma). Microinjection of alamandine in this region produced a long-lasting (> 18 min) increase in MAP (Δ saline= -2±1 vs. alamandine= 12±2 mmHg, p< 0.05) associated to increases in HR (Δ saline= 2±2 vs. alamandine= 35±5 bpm; p< 0.05) and in the amplitude of renal nerve discharges (Δ saline = -2±1 vs. alamandine= 35±5.5 % of the baseline; p< 0.05). Strikingly, an equimolar dose of angiotensin-(1-7) did not produce any change in MAP or HR (Δ MAP=-0.5±0.3 mmHg and +2.7±1.2 bpm, respectively; p> 0.05) and only a slight increase in RSNA (Δ =7.3±3.2 %) . In keeping with this observation the effects of alamandine were not significantly influenced by A-779 (Δ MAP=+13± 2.5 mmHg, Δ HR= +26±3.6 bpm; Δ RSNA = 25± 3.4%) but completely blocked by the Mas/MrgD antagonist D-Pro7-Ang-(1-7) (Δ MAP=+0 ± 1 mmHg Δ HR= +4±2.6 bpm; Δ RSNA = 0.5± 2.2 %). Therefore, we have identified a brain region in which alamandine/MrgD receptors but not Ang-(1-7)/Mas could be involved in the modulation of cardiovascular-related neuronal activity. This observation also suggests that alamandine might possess unique effects unrelated to Ang-(1-7) in the brain.

Importance of apical membrane delivery of 1,25-dihydroxyvitamin D3 to vitamin D-responsive gene expression in the colon

Synthetic conjugation of a glucuronide to 1,25-dihydroxyvitamin D3 (1,25D3) to produce β-25-monoglucuronide-1,25D3 (βGluc-1,25D3) renders the hormone biologically inactive and resistant to mammalian digestive enzymes. However, β-glucuronidase produced by bacteria in the lower intestinal tract can cleave off the glucuronide, releasing the active hormone. In mice given a single oral dose of 1,25D3, 24-hydroxylase (Cyp24a1) gene expression was strongly enhanced in the duodenum, but not in the colon, despite circulating concentrations of 1,25D3 that peaked at ∼3.0 nmol/l. In contrast, in mice treated with an equimolar dose of βGluc-1,25D3, Cyp24a1 gene expression increased 700-fold in the colon but was significantly weaker in the duodenum compared with mice treated with 1,25D3. Similar results were observed with another vitamin D-dependent gene. When administered subcutaneously, 1,25D3 weakly stimulated colon Cyp24a1 gene expression while βGluc-1,25D3 again resulted in strong enhancement. Surgical ligation to block passage of ingesta beyond the upper intestinal tract abolished upregulation of colon Cyp24a1 gene expression by orally and subcutaneously administered βGluc-1,25D3. Feeding βGluc-1,25D3 for 5 days revealed a linear, dose-dependent increase in colon Cyp24a1 gene expression but did not significantly increase plasma 1,25D3 or calcium concentrations. This study indicates that the colon is relatively insensitive to circulating concentrations of 1,25D3 and that the strongest gene enhancement occurs when the hormone reaches the colon via the lumen of the intestinal tract. These findings have broad implications for the use of vitamin D compounds in colon disorders and set the stage for future therapeutic studies utilizing βGluc-1,25D3 in their treatment.

Abstract 2495: Pharmacodynamic Profile of a Novel Chimeric Natriuretic Peptide, CD-NP, as Compared to C-Type Natriuretic Peptide

Background: C-type natriuretic peptide (CNP), a 22-amino-acid peptide and a ligand of the natriuretic peptide receptor-B (NPR-B), exhibits venodilating, anti-fibrotic, and vascular-regenerating properties but limited renal actions. A Mayo-designed chimeric peptide, CD-NP, which consists of CNP and the C-terminus of Dendroaspis NP, was synthesized to test the hypothesis that CD-NP would possess CNP-like properties with an improved pharmacodynamic profile. Methods: Normal anesthetized dogs were given CD-NP 50 ng/kg/min (n = 10) or an equimolar dose of CNP 29.3 ng/kg/min (n = 9) i.v. for 75 min. Hemodynamic, renal, and hormonal data were obtained pre-infusion (pre-I), at 30 and 60 min I, and post-I, and were compared both within group vs pre-I (mean ± SE, P< 0.05*, < 0.01 † ) and between groups ( P < 0.05 ‡ , < 0.01 § , < 0.001 ¶ ). Plasma BNP was measured by radioimmunoassay. Results: CD-NP resulted in greater increases in plasma cGMP (7±.4 to 25±3 †¶ to 36±3 †¶ to 23±3 †¶ pmol/ml), urinary cGMP excretion (978±145, 3170±205 †¶ , 5919±616 †¶ , 3077±298 †¶ pmol/min) vs CNP (8±.8, 10±.6 † , 11±.7 † , 8±.5 pmol/ml; 976±110, 1104±115, 1317±103, 998±101 pmol/min, respectively). CD-NP also increased urine Na + excretion (19±4, 168±24 †§ , 237±26 †¶ , 96±12 † μEq/min), urine flow (0.2±.1, 1.3±.2 † , 1.8±.3 † , 0.8±.2 † ml/min), and GFR (37±2 ‡ , 48±3 † , 51±3 † , 53±4 † ml/min) vs CNP (36±12, 68±10, 85±26, 81±25 μEq/min; 0.4±.1, 0.9±.2, 1.2±.3*, 0.9±.3 ml/min; 52±5, 53±7, 50±4, 49±6 ml/min, respectively). CD-NP reduced PCWP (5.7±.7, 4.1±1*, 3.2±.7 † , 4.3±.8 mmHg), RAP (1.8±.4, 1.1±.4 † , 0.9±.5 †§ , 1.3±.5 § mmHg), PAP (12±.6, 10±.4*, 10±.6, 11±.7 mmHg) vs CNP (6±.6, 5±.8, 6±.8, 7±.9 † mmHg; 2.6±.3, 2.5±.3 to 2.9±.3 to 3.5±.5 † mmHg; 13±1, 12±1, 12±1, 13±1 mmHg, respectively). Mean blood pressure was unchanged in either group. CD-NP increased circulating BNP during infusion vs CNP (40±4 vs 18±2 pg/ml, P<0.001). Conclusion: This study demonstrates the successful transformation of CNP to a CNP-like peptide with enhanced natriuretic, diuretic, GFR-enhancing, and cardiac-unloading actions. CD-NP may also promote increases in plasma BNP which via the NPR-A receptor may contribute to its biological actions. The therapeutic potential of CD-NP in heart failure warrants further studies.

Des-serine-proline brain natriuretic peptide 3–32 in cardiorenal regulation

Brain natriuretic peptide (BNP 1–32) plays an important physiologic role in cardiorenal homeostasis. Recently, it has been reported that BNP 1–32 is rapidly cleaved by the ubiquitous enzyme dipeptidyl peptidase IV to BNP 3–32, which lacks the two NH2-terminal amino acids of BNP 1–32. The bioactivity of BNP 3–32 in cardiorenal regulation is unknown. We hypothesized that BNP 3–32 has reduced vasodilating and natriuretic bioactivity compared with BNP 1–32 in vivo. Synthetic human BNP 3–32 and BNP 1–32 were administered to eight anesthetized normal canines. After baseline measurements, BNP 1–32 at 30 ng·kg−1·min−1 was administered, followed by a washout, a postinfusion clearance, and a clearance with an equimolar dose of BNP 3–32. In four studies, the sequence of BNP 1–32 and BNP 3–32 infusion was reversed. Peptides were compared by analyzing the changes from the respective preinfusion clearance to the respective infusion clearance. * P < 0.05 between peptides. BNP 3–32, unlike BNP 1–32, did not decrease mean arterial pressure (0 ± 1 vs. −7 ± 2* mmHg, respectively) and did not increase renal blood flow (+12 ± 10 vs. +52 ± 10* ml/min). Effects on heart rate and cardiac output were similar. Urinary sodium excretion increased 128 ± 18 μeq/min with BNP 3–32 and 338 ± 40* μeq/min with BNP 1–32. Urine flow increased 1.1 ± 0.2 ml/min with BNP 3–32 and 2.8 ± 0.4* ml/min with BNP 1–32. Plasma BNP immunoreactivity was lower with BNP 3–32, suggesting accelerated degradation. In this study, BNP 3–32 showed reduced natriuresis and diuresis and a lack of vasodilating actions compared with BNP 1–32.