Ultrasound Evaluation of Uterine Cavity Changes After Stimulation for In Vitro Fertilization.

OBJECTIVES: Mock embryo transfer (ET) before in vitro fertilization (IVF) allows for the clinical determination of uterine cavity length (UCL) to optimize embryo placement during clinical ET. Most studies have shown that optimal pregnancy rates occur with clinical ET at a depth of 15 mm from the uterine fundus. In our study, we sought to determine the effect of ovarian stimulation and endometrial preparation on UCL using 2D transabdominal ultrasound. METHODS: We performed a retrospective cohort study comparing documented 2D transabdominal ultrasound measurements of UCL at the time of mock ET and clinical ET. Statistical analyses were performed with SPSS v. 26 with paired sample t-test and significance determined with P < .05. RESULTS: Seventy patients who underwent 91 IVF-ET cycles between 2015 and 2018 at our academic center met inclusion criteria. Patient's demographics include a median age of 34 (interquartile range [IQR]: 31, 37), gravida 1 (IQR: 0, 2), parity 0 (IQR: 0, 0), and body mass index 25.87 (IQR: 21.78, 30.01). There was a statistically significant increase in UCL by 11.9 mm after IVF stimulation (P < .001), compared to mock ET. Mean UCL at the time of mock ET was 7.66 cm (±0.98 cm) and at clinical ET was 8.85 cm (±0.98 cm). CONCLUSIONS: The uterine cavity undergoes a significant length change during ovarian stimulation and endometrial preparation. These findings confirm the remarkable uterine plasticity in response to hormonal stimulation even before pregnancy ensues. These changes in UCL should be considered during ultrasound-guided clinical ET to ensure optimal embryo placement.

Long-Term Proton Pump Inhibitor-Acid Suppressive Treatment Can Cause Vitamin B12 Deficiency in Zollinger-Ellison Syndrome (ZES) Patients.

Whether the long-term treatment of patients with proton pump inhibitors (PPIs) with different diseases [GERD, Zollinger-Ellison syndrome (ZES), etc.] can result in vitamin B12 (VB12) deficiency is controversial. In this study, in 175 patients undergoing long-term ZES treatment with anti-acid therapies, drug-induced control acid secretory rates were correlated with the presence/absence of VB12 deficiency, determined by assessing serum VB12 levels, measurements of VB12 body stores (blood methylmalonic acid (MMA) and total homocysteine[tHYC]), and other features of ZES. After a mean of 10.2 yrs. of any acid treatment (5.6 yrs. with PPIs), 21% had VB12 deficiency with significantly lower serum and body VB12 levels (p < 0.0001). The presence of VB12 deficiency did not correlate with any feature of ZES but was associated with a 12-fold lower acid control rate, a 2-fold higher acid control pH (6.4 vs. 3.7), and acid control secretory rates below those required for the activation of pepsin (pH > 3.5). Over a 5-yr period, the patients with VB12 deficiency had a higher rate of achlorhydria (73% vs. 24%) and a lower rate of normal acid secretion (0% vs. 49%). In conclusion, in ZES patients, chronic long-term PPI treatment results in marked acid hyposecretion, resulting in decreased serum VB12 levels and decreased VB12-body stores, which can result in VB12 deficiency.

The Nonpeptide Agonist MK-5046 Functions As an Allosteric Agonist for the Bombesin Receptor Subtype-3.

Allosteric ligands of various G-protein-coupled receptors are being increasingly described and are providing important advances in the development of ligands with novel selectivity and efficacy. These unusual properties allow expanded opportunities for pharmacologic studies and treatment. Unfortunately, no allosteric ligands are yet described for the bombesin receptor family (BnRs), which are proposed to be involved in numerous physiologic/pathophysiological processes in both the central nervous system and peripheral tissues. In this study, we investigate the possibility that the bombesin receptor subtype-3 (BRS-3) specific nonpeptide receptor agonist MK-5046 [(2S)-1,1,1-trifluoro-2-[4-(1H-pyrazol-1-yl)phenyl]-3-(4-[[1-(trifluoromethyl)cyclopropyl]methyl]-1H-imidazol-2-yl)propan-2-ol] functions as a BRS-3 allosteric receptor ligand. We find that in BRS-3 cells, MK-5046 only partially inhibits iodine-125 radionuclide (125I)-Bantag-1 [Boc-Phe-His-4-amino-5-cyclohexyl-2,4,5-trideoxypentonyl-Leu-(3-dimethylamino) benzylamide N-methylammonium trifluoroacetate] binding and that both peptide-1 (a universal BnR-agonist) and MK-5046 activate phospholipase C; however, the specific BRS-3 peptide antagonist Bantag-1 inhibits the action of peptide-1 competitively, whereas for MK-5046 the inhibition is noncompetitive and yields a curvilinear Schild plot. Furthermore, MK-5046 shows other allosteric behaviors, including slowing dissociation of the BRS-3 receptor ligand 125I-Bantag-1, dose-inhibition curves being markedly affected by increasing ligand concentration, and MK-5046 leftward shifting the peptide-1 agonist dose-response curve. Lastly, receptor chimeric studies and site-directed mutagenesis provide evidence that MK-5046 and Bantag-1 have different binding sites determining their receptor high affinity/selectivity. These results provide evidence that MK-5046 is functioning as an allosteric agonist at the BRS-3 receptor, which is the first allosteric ligand described for this family of receptors. SIGNIFICANCE STATEMENT: G-protein-coupled receptor allosteric ligands providing higher selectivity, selective efficacy, and safety that cannot be obtained using usual orthosteric receptor-based strategies are being increasingly described, resulting in enhanced usefulness in exploring receptor function and in treatment. No allosteric ligands exist for any of the mammalian bombesin receptor (BnR) family. Here we provide evidence for the first such example of a BnR allosteric ligand by showing that MK-5046, a nonpeptide agonist for bombesin receptor subtype-3, is functioning as an allosteric agonist.

Endometrial Abnormalities: Correlation Between Different Diagnostic Modalities.

OBJECTIVES: We sought to evaluate the diagnostic accuracy of transvaginal ultrasound (TVUS) saline infusion sonohysterogram (SIS), and hysteroscopy for diagnosing endometrial abnormalities and their correlation with histological findings. In addition, we sought to validate the subsistence of a more subtle abnormality called "redundant endometrium" (RE). METHODS: Retrospective cohort study of patients presenting with infertility and diagnosed with endometrial abnormalities who underwent hysteroscopy and pathology evaluation. Each patient underwent TVUS at the first visit regardless of the cycle phase, followed by SIS during proliferative phase, and then hysteroscopy, which was performed when abnormal SIS findings were diagnosed. Endometrial abnormalities were categorized as polyps (EP), RE, or normal. Frequencies of the abnormalities were recorded for the 3 imaging modalities and their sensitivity, specificity, positive predictive value, and negative predictive value were calculated for each. RESULTS: A total of 105 TVUS and 73 SIS were performed. Because of the frequent association of EP with RE, when all endometrial pathologies were combined, the three diagnostic modalities showed high sensitivity (TVUS 88.9%, SIS 100%, hysteroscopy 82.9%, respectively), but they also showed low specificity (TVUS 56.7%, SIS 56.1%, hysteroscopy 58.2%, respectively). Pathological evaluation showed isolated RE (26 cases), to harbor polyps (19.2%), disordered endometrium (19.2%), and complex atypical hyperplasia (3.8%). CONCLUSIONS: The three diagnostic modalities showed high sensitivity in diagnosing endometrial abnormalities. We identified RE as an independent endometrial abnormality. Benign endometrium is the predominant histology in RE, however, a small proportion harbors endometrial hyperplasia. Based on these results, we advocate further evaluation when this condition is diagnosed with TVUS, or SIS.

Anti-Müllerian Hormone (AMH) regulates BRCA1 and BRCA2 gene expression after ovarian cortex transplantation.

OBJECTIVE: To test whether recombinant anti-Müllerian hormone (rAMH) could exert an inhibitory function on BRCA1/2 expression in human ovarian cortex. METHODS: Pilot study on ovariectomized nude mice xenotransplanted with human vitrified/warmed ovarian cortex and treated with rAMH via infusion pump. Twelve nude mice were ovariectomized and Alzet pumps delivering 1.23 mcg rAMH/day to reach a serum concentration of 17.5 ng/mL, or placebo (controls), were inserted intraabdominally. Previously vitrified/warmed 2x2 mm ovarian cortex fragments were transplanted on day 7 and then harvested on day 14 after pump placement. PCR analyses determined mRNA levels for BRCA1 and BRCA2 in the human ovarian cortex. RESULTS: In mice treated with rAMH, BRCA1 expression was significantly lower (0.196 fg/µg RNA, IQR 0.158, 0.236) than in controls (0.544 fg/µg RNA, IQR 0.458, 0.554; p = .030), while BRCA2 expression remained similar in rAMH mice (5.355 fg/µg RNA, IQR 4.479, 6.230) and in controls (4.011 fg/µg RNA, IQR 3.650, 4.182; p = .327). CONCLUSION: Administration of rAMH in the peri-transplant period caused downregulation of BRCA1, but not of BRCA2 expression, in human ovarian cortex. These results help our understanding of DNA repair mechanism in the ovarian cortex and identify AMH's possible protective effect on ovarian reserve in BRCA1 mutation carriers.

Group II p21-activated kinase, PAK4, is needed for activation of focal adhesion kinases, MAPK, GSK3, and ß-catenin in rat pancreatic acinar cells.

PAK4 is the only member of the Group II p21-activated kinases (PAKs) present in rat pancreatic acinar cells and is activated by gastrointestinal hormones/neurotransmitters stimulating PLC/cAMP and by various pancreatic growth factors. However, little is known of the role of PAK4 activation in cellular signaling cascades in pancreatic acinar cells. In the present study, we examined the role of PAK4's participation in five different cholecystokinin-8 (CCK-8)-stimulated signaling pathways (PI3K/Akt, MAPK, focal adhesion kinase, GSK3, and ß-catenin), which mediate many of its physiological acinar-cell effects, as well as effects in pathophysiological conditions. To define PAK4's role, the effect of two different PAK4 inhibitors, PF-3758309 and LCH-7749944, was examined under experimental conditions that only inhibited PAK4 activation and not activation of the other pancreatic PAK, Group I PAK2. The inhibitors' effects on activation of these five signaling cascades by both physiological and pathophysiological concentrations of CCK, as well as by 12-O-tetradecanoylphobol-13-acetate (TPA), a PKC-activator, were examined. CCK/TPA activation of focal adhesion kinases(PYK2/p125FAK) and the accompanying adapter proteins (paxillin/p130CAS), Mek1/2, and p44/42, but not c-Raf or other MAPKs (JNK/p38), were mediated by PAK4. Activation of PI3K/Akt/p70s6K was independent of PAK4, whereas GSK3 and ß-catenin stimulation was PAK4-dependent. These results, coupled with recent studies showing PAK4 is important in pancreatic fluid/electrolyte/enzyme secretion and acinar cell growth, show that PAK4 plays an important role in different cellular signaling cascades, which have been shown to mediate numerous physiological and pathophysiological processes in pancreatic acinar cells.NEW & NOTEWORTHY In pancreatic acinar cells, cholecystokinin (CCK) or 12-O-tetradecanoylphobol-13-acetate (TPA) activation of focal adhesion kinases (p125FAK,PYK2) and its accompanying adapter proteins, p130CAS/paxillin; Mek1/2, p44/42, GSK3, and ß-catenin are mediated by PAK4. PI3K/Akt/p70s6K, c-Raf, JNK, or p38 pathways are independent of PAK4 activation.

Serum VEGF Predicts Clinical Improvement Induced by Cerebrolysin Plus Donepezil in Patients With Advanced Alzheimer's Disease.

Serum vascular endothelial growth factor (VEGF) increases with Alzheimer's disease (AD) severity and may prevent cognitive decline. However, information on the influence of AD drug therapy on circulating VEGF is limited. This study assessed changes in serum VEGF levels and its association with clinical and functional responses in mild to moderate AD patients who were treated with Cerebrolysin, donepezil, or the combined therapy in a randomized, controlled trial. Treatment with Cerebrolysin plus donepezil reduced elevated serum VEGF levels and improved functioning and cognition significantly compared with donepezil alone in patients with advanced AD, and treatment differences were more pronounced in patients with higher VEGF levels. Our results indicate that the combined therapy reversed the increase of serum VEGF in advanced AD, which was associated with cognitive and functional responses, particularly in patients with high baseline VEGF.

Efficacy of the spray-drying treatment to inactivate the bovine leukemia virus in bovine colostrum.

Previous studies have shown the presence of bovine leukemia virus (BLV) in colostrum and milk of naturally infected cows. The relationship between virus or provirus and specific antibodies in these secretions is particular to each infected cow and will probably determine whether the consumption of colostrum or milk from these naturally infected dams provides an infective or a protective effect in recipient calves. Our recent findings suggest that this issue is a key point in BLV transmission in very young calves. Based on this, the aim of the present study was to determine the effect of the spray-drying treatment of colostrum on BLV infectivity. The treatment was done on scale-down conditions, using fresh colostrum from BLV-negative cows spiked with infective BLV. Residual infectivity was tested in susceptible lambs. Lambs inoculated with colostrum spiked with BLV-infected cells or cell-free BLV showed evidence of infection 60 d after inoculation, whereas none of the lambs inoculated with spray-dried colostrum showed evidence of infection 60 d after inoculation. These results provide direct evidence that the experimental spray-drying process used in this study was effective in inactivating infectious BLV in colostrum. These findings suggest that the risk for BLV transmission could be reduced if milk and colostrum were treated by spray-drying prior to consumption in dairy facilities. The effect of spray-drying on the functional properties and stability of the antibodies present in colostrum under long-term storage should be further investigated.

Pilot study establishing a nomogram of yolk sac growth during the first trimester of pregnancy.

AIM: The yolk sac (YS) has been reported as a reliable predictor of adverse pregnancy outcomes, however, it has always been evaluated cross-sectionally with a single ultrasound per patient. We sought to validate the use of YS dimensions in serial ultrasounds throughout the first 10 weeks of singleton and multiple gestations. METHODS: This was a prospective cohort study where YS diameters were serially obtained with 2D ultrasound in singleton and multiple gestations from 5 to 11 weeks. Nonparametric test were used for comparisons with P < 0.05 indicating significance. RESULTS: One hundred ninety-three patients were included, 42 twins (3 monochorionic and 39 dichorionic), 2 triplets (monochorionic twins plus a singleton) and 148 singleton pregnancies (238 total fetuses). There was no difference in YS dimensions in singleton versus multiple pregnancies. Starting at 5 weeks' gestation, the YS increased 0.4 mm (95% CI 0.3-0.5 mm) per week until 10 weeks' gestation. Forty-five fetuses were lost in the first trimester. The risk of pregnancy loss was higher with a large YS until 8 weeks (P ≤ 0.001), while after 8 weeks it was higher with a small YS (P < 0.005). CONCLUSION: We established a nomogram of YS development during the first 10 weeks of pregnancy. The YS reliably detected pregnancies that ended in loss as early as 6 weeks' gestation. The YS was either smaller or larger than in ongoing pregnancies. While all pregnancies with large YS were lost within 10 weeks, those with smaller YS were lost beyond the first 10 weeks.

BLV: lessons on vaccine development.

Vaccination against retroviruses is a challenge because of their ability to stably integrate into the host genome, undergo long-term latency in a proportion of infected cells and thereby escape immune response. Since clearance of the virus is almost impossible once infection is established, the primary goal is to achieve sterilizing immunity. Besides efficacy, safety is the major issue since vaccination has been associated with increased infection or reversion to pathogenicity. In this review, we discuss the different issues that we faced during the development of an efficient vaccine against bovine leukemia virus (BLV). We summarize the historical failures of inactivated vaccines, the efficacy and safety of a live-attenuated vaccine and the economical constraints of further industrial development.

Cyclic AMP-dependent protein kinase A and EPAC mediate VIP and secretin stimulation of PAK4 and activation of Na+,K+-ATPase in pancreatic acinar cells.

Rat pancreatic acinar cells possess only the p21-activated kinase (PAKs), PAK4 of the group II PAK, and it is activated by gastrointestinal hormones/neurotransmitters stimulating PLC and by a number of growth factors. However, little is known generally of cAMP agents causing PAK4 activation, and there are no studies with gastrointestinal hormones/neurotransmitters activating cAMP cascades. In the present study, we examined the ability of VIP and secretin, which stimulate cAMP generation in pancreatic acini, to stimulate PAK4 activation, the signaling cascades involved, and their possible role in activating sodium-potassium adenosine triphosphatase (Na+,K+-ATPase). PAK4 activation was compared with activation of the well-established cAMP target, cyclic AMP response element binding protein (CREB). Secretin-stimulated PAK4 activation was inhibited by KT-5720 and PKA Type II inhibitor (PKI), protein kinase A (PKA) inhibitors, whereas VIP activation was inhibited by ESI-09 and HJC0197, exchange protein directly activated by cAMP (EPAC) inhibitors. In contrast, both VIP/secretin-stimulated phosphorylation of CREB (pCREB) via EPAC activation; however, it was inhibited by the p44/42 inhibitor PD98059 and the p38 inhibitor SB202190. The specific EPAC agonist 8-CPT-2- O-Me-cAMP as well 8-Br-cAMP and forskolin stimulated PAK4 activation. Secretin/VIP activation of Na+,K+-ATPase, was inhibited by PAK4 inhibitors (PF-3758309, LCH-7749944). These results demonstrate PAK4 is activated in pancreatic acini by stimulation of both VIP-/secretin-preferring receptors, as is CREB. However, they differ in their signaling cascades. Furthermore, PAK4 activation is needed for Na+,K+ATPase activation, which mediates pancreatic fluid secretion. These results, coupled with recent studies reporting PAKs are involved in both pancreatitis/pancreatic cancer growth/enzyme secretion, show that PAK4, similar to PAK2, likely plays an important role in both pancreatic physiological/pathological responses. NEW & NOTEWORTHY Pancreatic acini possess only the group II p21-activated kinase, PAK4, which is activated by PLC-stimulating agents/growth factors and is important in enzyme-secretion/growth/pancreatitis. Little information exists on cAMP-activating agents stimulating group II PAKs. We studied ability/effect of cyclic AMP-stimulating agents [vasoactive intestinal polypeptide (VIP), secretin] on PAK4 activity in rat pancreatic-acini. Both VIP/secretin activated PAK4/CREB, but the cAMP signaling cascades differed for EPAC, MAPK, and PKA pathways. Both hormones require PAK4 activation to stimulate sodium-potassium adenosine triphosphatase activity. This study shows PAK4 plays an important role in VIP-/secretin-stimulated pancreatic fluid secretion and suggests it plays important roles in pancreatic acinar physiological/pathophysiological responses mediated by cAMP-activating agents.

Development and Characterization of a Novel, High-Affinity, Specific, Radiolabeled Ligand for BRS-3 Receptors.

Bombesin (Bn) receptor subtype 3(BRS-3) is an orphan G-protein-coupled receptor of the Bn family, which does not bind any natural Bn peptide with high affinity. Receptor knockout studies show that the animals develop diabetes, obesity, altered temperature control, and other central nervous system (CNS)/endocrine/gastrointestinal changes. It is present in CNS, peripheral tissues, and tumors; however, its role in normal physiology/pathophysiology, as well as its receptor localization/pharmacology is largely unknown, in part due to the lack of a convenient, specific, direct radiolabeled ligand. This study was designed to address this problem and to develop and characterize a specific radiolabeled ligand for BRS-3. The peptide antagonist Bantag-1 had >10,000-fold selectivity for human BRS-3 (hBRS-3) over other mammalian Bn receptors (BnRs) [i.e., gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR)]. Using iodogen and basic conditions, it was radiolabeled to high specific activity (2200 Ci/mmol) and found to bind with high affinity/specificity to hBRS-3. Binding was saturable, rapid, and reversible. The ligand only interacted with known BRS-3 ligands, and not with other specific GRPR/NMBR ligands or ligands for unrelated receptors. The magnitude of 125I-Bantag-1 binding correlated with BRS-3 mRNA expression and the magnitude of activation of phospholipase C in lung cancer cells, as well as readily identifying BRS-3 in lung cancer cells and normal tissues, allowing the direct assessment of BRS-3 receptor pharmacology/numbers on cells containing BRS-3 with other BnRs, which is usually the case. This circumvents the need for subtraction assays, which are now frequently used to assess BRS-3 indirectly using radiolabeled pan-ligands, which interact with all BnRs.

Insights into Effects/Risks of Chronic Hypergastrinemia and Lifelong PPI Treatment in Man Based on Studies of Patients with Zollinger-Ellison Syndrome.

The use of proton pump inhibitors (PPIs) over the last 30 years has rapidly increased both in the United States and worldwide. PPIs are not only very widely used both for approved indications (peptic ulcer disease, gastroesophageal reflux disease (GERD), Helicobacter pylori eradication regimens, stress ulcer prevention), but are also one of the most frequently off-label used drugs (25-70% of total). An increasing number of patients with moderate to advanced gastroesophageal reflux disease are remaining on PPI indefinitely. Whereas numerous studies show PPIs remain effective and safe, most of these studies are <5 years of duration and little data exist for >10 years of treatment. Recently, based primarily on observational/epidemiological studies, there have been an increasing number of reports raising issues about safety and side-effects with very long-term chronic treatment. Some of these safety issues are related to the possible long-term effects of chronic hypergastrinemia, which occurs in all patients taking chronic PPIs, others are related to the hypo-/achlorhydria that frequently occurs with chronic PPI treatment, and in others the mechanisms are unclear. These issues have raised considerable controversy in large part because of lack of long-term PPI treatment data (>10-20 years). Zollinger-Ellison syndrome (ZES) is caused by ectopic secretion of gastrin from a neuroendocrine tumor resulting in severe acid hypersecretion requiring life-long antisecretory treatment with PPIs, which are the drugs of choice. Because in <30% of patients with ZES, a long-term cure is not possible, these patients have life-long hypergastrinemia and require life-long treatment with PPIs. Therefore, ZES patients have been proposed as a good model of the long-term effects of hypergastrinemia in man as well as the effects/side-effects of very long-term PPI treatment. In this article, the insights from studies on ZES into these controversial issues with pertinence to chronic PPI use in non-ZES patients is reviewed, primarily concentrating on data from the prospective long-term studies of ZES patients at NIH.

Experimental infection of sheep with Bovine leukemia virus (BLV): Minimum dose of BLV-FLK cells and cell-free BLV and neutralization activity of natural antibodies.

Bovine leukemia virus (BLV) is an important cattle pathogen that causes major economic losses worldwide, especially in dairy farms. The use of animal models provides valuable insight into the pathogenesis of viral infections. Experimental infections of sheep have been conducted using blood from BLV-infected cattle, infectious BLV molecular clones or tumor-derived cells. The Fetal Lamb Kidney cell line, persistently infected with BLV (FLK-BLV), is one of the most commonly used long-term culture available for the permanent production of virus. FLK-BLV cells or the viral particles obtained from the cell-free culture supernatant could be used as a source of provirus or virus to experimentally infect sheep. In this report, we aimed to determine the minimum amount of FLK-BLV cells or cell-free supernatant containing BLV needed to produce infection in sheep. We also evaluated the amount of antibodies obtained from a naturally-infected cow required to neutralize this infection. We observed that both sheep experimentally inoculated with 5000 FLK-BLV cells became infected, as well as one of the sheep receiving 500 FLK-BLV cells. None of the animals inoculated with 50 FLK-BLV cells showed evidence of infection. The cell-free FLK-BLV supernatant proved to be infective in sheep up to a 1:1000 dilution. Specific BLV antibodies showed neutralizing activity as none of the sheep became infected. Conversely, the animals receiving a BLV-negative serum showed signs of BLV infection. These results contribute to the optimization of a sheep bioassay which could be useful to further characterize BLV infection.

Genome-wide scan for commons SNPs affecting bovine leukemia virus infection level in dairy cattle.

BACKGROUND: Bovine leukemia virus (BLV) infection is omnipresent in dairy herds causing direct economic losses due to trade restrictions and lymphosarcoma-related deaths. Milk production drops and increase in the culling rate are also relevant and usually neglected. The BLV provirus persists throughout a lifetime and an inter-individual variation is observed in the level of infection (LI) in vivo. High LI is strongly correlated with disease progression and BLV transmission among herd mates. In a context of high prevalence, classical control strategies are economically prohibitive. Alternatively, host genomics studies aiming to dissect loci associated with LI are potentially useful tools for genetic selection programs tending to abrogate the viral spreading. The LI was measured through the proviral load (PVL) set-point and white blood cells (WBC) counts. The goals of this work were to gain insight into the contribution of SNPs (bovine 50KSNP panel) on LI variability and to identify genomics regions underlying this trait. RESULTS: We quantified anti-p24 response and total leukocytes count in peripheral blood from 1800 cows and used these to select 800 individuals with extreme phenotypes in WBCs and PVL. Two case-control genomic association studies using linear mixed models (LMMs) considering population stratification were performed. The proportion of the variance captured by all QC-passed SNPs represented 0.63 (SE ± 0.14) of the phenotypic variance for PVL and 0.56 (SE ± 0.15) for WBCs. Overall, significant associations (Bonferroni's corrected -log10p > 5.94) were shared for both phenotypes by 24 SNPs within the Bovine MHC. Founder haplotypes were used to measure the linkage disequilibrium (LD) extent (r2 = 0.22 ± 0.27 at inter-SNP distance of 25-50 kb). The SNPs and LD blocks indicated genes potentially associated with LI in infected cows: i.e. relevant immune response related genes (DQA1, DRB3, BOLA-A, LTA, LTB, TNF, IER3, GRP111, CRISP1), several genes involved in cell cytoskeletal reorganization (CD2AP, PKHD1, FLOT1, TUBB5) and modelling of the extracellular matrix (TRAM2, TNXB). Host transcription factors (TFs) were also highlighted (TFAP2D; ABT1, GCM1, PRRC2A). CONCLUSIONS: Data obtained represent a step forward to understand the biology of BLV-bovine interaction, and provide genetic information potentially applicable to selective breeding programs.

P21-activated kinase 4 in pancreatic acinar cells is activated by numerous gastrointestinal hormones/neurotransmitters and growth factors by novel signaling, and its activation stimulates secretory/growth cascades.

p21-activated kinases (PAKs) are highly conserved serine/threonine protein kinases, which are divided into two groups: group-I (PAKs1-3) and group-II (PAKs4-6). In various tissues, Group-II PAKs play important roles in cytoskeletal dynamics and cell growth as well as neoplastic development/progression. However, little is known about Group-II PAK's role in a number of physiological events, including their ability to be activated by gastrointestinal (GI) hormones/neurotransmitters/growth factors (GFs). We used rat pancreatic acini to explore the ability of GI hormones/neurotransmitters/GFs to activate Group-II-PAKs and the signaling cascades involved. Only PAK4 was detected in pancreatic acini. PAK4 was activated by endothelin, secretagogues-stimulating phospholipase C (bombesin, CCK-8, and carbachol), by pancreatic GFs (insulin, insulin-like growth factor 1, hepatocyte growth factor, epidermal growth factor, basic fibroblast growth factor, and platelet-derived growth factor), and by postreceptor stimulants (12-O-tetradecanoylphobol-13-acetate and A23187 ). CCK-8 activation of PAK4 required both high- and low-affinity CCK1-receptor state activation. It was reduced by PKC-, Src-, p44/42-, or p38-inhibition but not with phosphatidylinositol 3-kinase-inhibitors and only minimally by thapsigargin. A protein kinase D (PKD)-inhibitor completely inhibited CCK-8-stimulated PKD-activation; however, stimulated PAK4 phosphorylation was only inhibited by 60%, demonstrating that it is both PKD-dependent and PKD-independent. PF-3758309 and LCH-7749944, inhibitors of PAK4, decreased CCK-8-stimulated PAK4 activation but not PAK2 activation. Each inhibited ERK1/2 activation and amylase release induced by CCK-8 or bombesin. These results show that PAK4 has an important role in modulating signal cascades activated by a number of GI hormones/neurotransmitters/GFs that have been shown to mediate both physiological/pathological responses in acinar cells. Therefore, in addition to the extensive studies on PAK4 in pancreatic cancer, PAK4 should also be considered an important signaling molecule for pancreatic acinar physiological responses and, in the future, should be investigated for a possible role in pancreatic acinar pathophysiological responses, such as in pancreatitis. NEW & NOTEWORTHY This study demonstrates that the only Group-II p21-activated kinase (PAK) in rat pancreatic acinar cells is PAK4, and thus differs from islets/pancreatic cancer. Both gastrointestinal hormones/neurotransmitters stimulating PLC and pancreatic growth factors activate PAK4. With cholecystokinin (CCK), activation is PKC-dependent/-independent, requires both CCK1-R affinity states, Src, p42/44, and p38 activation. PAK4 activation is required for CCK-mediated p42/44 activation/amylase release. These results show PAK4 plays an important role in mediating CCK physiological signal cascades and suggest it may be a target in pancreatic acinar diseases besides cancer.

Xenotransplantation of pre-pubertal ovarian cortex and prevention of follicle depletion with anti-Müllerian hormone (AMH).

OBJECTIVE: To determine whether recombinant AMH (rAMH) could prevent post-transplant follicular depletion by acting on the stemness markers Oct-4, Sox2, and NANOG. MATERIALS AND METHODS: This was an experimental study where 12 ovariectomized nude mice were xenotransplanted with vitrified/warmed ovarian cortex obtained from a pre-pubertal girl and Alzet pumps delivering rAMH, or placebo (control), were inserted intra-abdominally. Previously vitrified/warmed ovarian cortex fragments were transplanted after 7 days and then harvested after 14 days from pump placement. We performed real-time RT-PCR analyses, ELISA for AMH, FSH, and estradiol, histologic measurement of ovarian follicles, and immunohistochemistry for Ki67 and TUNEL. The main outcome measures were serum levels and tissue expression of the parameters under investigation and follicle count. RESULTS: Serum AMH, FSH, and estradiol reflected post-ovariectomy profiles and were mildly influenced by rAMH administration. Ovarian cortex expression of AMH, AMH-R2, VEGF, GDF9, Oct-4, and Sox2 was lower in rAMH mice than in controls, while NANOG was upregulated. There was a non-significant decrease in primordial follicles after vitrification-warming, and xenotransplantation further decreased this number. There were lower cell replication and depressed apoptosis in the rAMH group. CONCLUSIONS: Administration of recombinant AMH in the peri-transplant period did not protect the initial follicular depletion but decreased apoptosis and cellular activation and regulated stem cell markers' tissue expression. These results aid our understanding of the inhibitory effects of AMH on follicular development and show the benefit of administering exogenous AMH at the time of pre-pubertal ovarian cortex transplant to protect the follicles from pre-activation and premature depletion.

The p21-activated kinase, PAK2, is important in the activation of numerous pancreatic acinar cell signaling cascades and in the onset of early pancreatitis events.

In a recent study we explored Group-1-p21-activated kinases (GP.1-PAKs) in rat pancreatic acini. Only PAK2 was present; it was activated by gastrointestinal-hormones/neurotransmitters and growth factors in a PKC-, Src- and small-GTPase-mediated manner. PAK2 was required for enzyme-secretion and ERK/1-2-activation. In the present study we examined PAK2's role in CCK and TPA-activation of important distal signaling cascades mediating their physiological/pathophysiological effects and analyzed its role in pathophysiological processes important in early pancreatitis. In rat pancreatic acini, PAK2-inhibition by the specific, GP.1.PAK-inhibitor, IPA-3-suppressed cholecystokinin (CCK)/TPA-stimulated activation of focal-adhesion kinases and mitogen-activated protein-kinases. PAK2-inhibition reversed the dual stimulatory/inhibitory effect of CCK/TPA on the PI3K/Akt/GSK-3ß pathway. However, its inhibition did not affect PKC activation. PAK2-inhibition protected acini from CCK-induced ROS-generation; caspase/trypsin-activation, important in early pancreatitis; as well as from cell-necrosis. Furthermore, PAK2-inhibition reduced proteolytic-activation of PAK-2p34, which is involved in programmed-cell-death. To ensure that the study did not only rely in the specificity of IPA-3 as a PAK inhibitor, we used two other approaches for PAK inhibition, FRAX597 a ATP-competitive-GP.1-PAKs-inhibitor and infection with a PAK2-dominant negative(DN)-Advirus. Those two approaches confirmed the results obtained with IPA-3. This study demonstrates that PAK2 is important in mediating CCK's effect on the activation of signaling-pathways known to mediate its physiological/pathophysiological responses including several cellular processes linked to the onset of pancreatitis. Our results suggest that PAK2 could be a new, important therapeutic target to consider for the treatment of diseases involving deregulation of pancreatic acinar cells.