The purpose of this study was to evaluate the impact of real-time PCR reporting both on timely identification of clustered Grampositive cocci (GPC) in blood cultures and on appropriate antibiotic treatment. This retrospective, interventional cohort study evaluated inpatients with blood cultures positive for GPC in the pre-PCR (15 January 2009 to 14 January 2010) and post-PCR (15 January 2010 to 14 January 2011) periods. Post-PCR implementation, laboratory services completed batched PCR; results other than methicillin-resistant Staphylococcus aureus (MRSA) were reported in the electronic medical record without additional interventions. The assay's sensitivity and specificity, time to identification of staphylococcal bacteremia, and clinically relevant outcomes, including time to optimal antibiotic therapy, were evaluated. Demographic information was also collected and analyzed. Sixty-eight and 58 patients with Staphylococcus aureus bacteremia from the pre-and post-PCR periods, respectively, met inclusion criteria. Similar numbers of consecutive patients with coagulase-negative staphylococci were analyzed for comparison. The time to identification was significantly reduced post-PCR implementation (mean, 13.2 h; 95% confidence interval [95% CI], 10.5 to 15.9 h; P < 0.0001). However, the time to optimal antibiotic therapy was not significantly reduced. We conclude that implementation of a PCR assay demonstrated the potential to improve appropriate antibiotic use based on clinically meaningful and statistically significant reductions in the time to microbiologic identification. However, in order to realize this potential benefit, processes must be optimized and additional interventions initiated to facilitate providers' use of the PCR result.

Multipotent, self-renewing neural stem cells and their progeny [collectively referred to as neural precursor cells (NPCs)] represent a population of cells with great promise for CNS repair. To effectively harness their potential for therapeutic applications, the factors that regulate NPC behavior and/or fate must be well understood. The ability of immunomodulatory molecules to affect NPC behavior is of interest because of recent work elucidating the complex interactions between the immune system and nervous system. Herein, we examined the effects of cyclosporin A, a commonly used immunosuppressive molecule, on NPC proliferation kinetics, survival, and fate using in vitro assays at the population level and at the single-cell level. The use of pure populations of NPCs revealed a direct effect of cyclosporin A on cell survival, resulting in increased numbers and larger colonies, with no effect on proliferation kinetics. Cyclosporin A did not alter the differentiation profile of NPC colonies, indicating that it did not promote selective survival of a particular neural lineage. Additionally, we observed decreased cell-cell adhesions in developing cyclosporin A-treated NPC colonies. Consistent with the in vitro observations, in vivo administration of cyclosporin A to adult animals increased the numbers of NPCs within the neurogenic niche lining the lateral ventricles. Together, our findings establish that cyclosporin A has direct effects on NPCs both in vitro and in vivo, making it a promising candidate molecule for developing clinically relevant strategies to stimulate NPCs for brain repair.

Cyclosporin A (CsA) has direct effects on neural stem and progenitor cells (together termed neural precursor cells; NPCs) in the adult central nervous system. Administration of CsA in vitro or in vivo promotes the survival of NPCs and expands the pools of NPCs in mice. Moreover, CsA administration is effective in promoting NPC activation, tissue repair and functional recovery in a mouse model of cortical stroke. The mechanism(s) by which CsA mediates this cell survival effect remains unknown. Herein, we examined both calcineurin-dependent and calcineurin-independent pathways through which CsA might mediate NPC survival. To examine calcineurin-dependent pathways, we utilized FK506 (Tacrolimus), an immunosuppressive molecule that inhibits calcineurin, as well as drugs that inhibit cyclophilin A-mediated activation of calcineurin. To evaluate the calcineurin-independent pathway, we utilized NIM811, a non-immunosuppressive CsA analog that functions independently of calcineurin by blocking mitochondrial permeability transition pore formation. We found that only NIM811 can entirely account for the pro-survival effects of CsA on NPCs. Indeed, blocking signaling pathways downstream of calcineurin activation using nNOS mice did not inhibit CsA-mediated cell survival, which supports the proposal that the effects are calcinuerin-independent. In vivo studies revealed that NIM811 administration mimics the pro-survival effects of CsA on NPCs and promotes functional recovery in a model of cortical stroke, identical to the effects seen with CsA administration. We conclude that CsA mediates its effect on NPC survival through calcineurin-independent inhibition of mitochondrial permeability transition pore formation and suggest that this pathway has potential therapeutic benefits for developing NPC-mediated cell replacement strategies.

Background: Cytomegalovirus (CMV) infection is extremely common after lung transplant and can be associated with significant morbidity and mortality. Current practice suggests the use of 900 mg daily of valganciclovir for CMV prophylaxis, but there is no literature assessing whether 450 mg daily of valganciclovir is sufficient in intermediate CMV risk lung transplant recipients. Therefore, we sought to assess the role of low-dose valganciclovir (LDV) versus high-dose valganciclovir (HDV) prophylaxis in intermediate-risk (R+) recipients.Methods: We conducted a retrospective analysis on lung transplant recipients at the Norton Thoracic Institute in Phoenix, Arizona looking at intermediate-risk patients that received either valganciclovir 450 mg per day (LDV) or 900 mg/day (HDV). All patients were followed for 1 year posttransplant for incidence of CMV viremia. The primary outcome was the rate of CMV viremia as determined by a positive CMV polymerase chain reaction ([PCR] >2.7 log copies/mL). Secondary outcomes included rate of adverse events, acute cellular rejection, and mortality.Results: The primary analysis included 103 patients (55 in the LDV group, 48 in the HDV group). In the LDV group, 9 patients (16.4%) developed CMV viremia compared to 4 (8.3%) in the HDV group (p=0.221) with no difference observed in adverse event rates between groups.Conclusion: There was no statistical difference between groups for the primary outcome. However, the effect size demonstrated in this analysis may be of clinical relevance and valganciclovir 450 mg daily would not be recommended in intermediate risk lung transplant recipients at this time. To confirm our results, further prospective studies enrolling larger patient populations are necessary.

We describe the induction of a hiatal hernia via a transthoracic approach in domestic swine. The hiatal hernia is amenable to subsequent laparoscopic repair, enabling surgeons to acquire the technical skills required to correct this defect in the laboratory. To our knowledge, this is the first report of a reproducible model of a transthoracically induced hiatal hernia that allows subsequent laparoscopic repair. We suggest that in addition to refinement of surgical skills, our model may provide new information to researchers regarding the potential indications for antireflux procedures, as well as the natural history and appropriate management of hiatal hernias.

The advent of cutting edge technology has paved the way for the identification of new players for sodium homeostasis and blood pressure (BP) control. The role of gut microbiome in metabolic disorders has been the subject of a growing number of metagenomic and metabolomic studies. Dysbiosis, or the alteration in the gut microbiota composition and function, affects the development of hypertension. Gut microbiota, comprised of mainly of Firmicutes (F) and Bacteroidetes (B), constantly adapt to lifestyle modifications. An increased F/B is observed in rodent and human hypertension. Minocycline selectively inhibits M1 polarization of macrophages, which results in anti‐inflammatory and anti‐hypertensive effects and normalizes the microbiome ratio and thus, the BP. We reported that the hormone gastrin responds to increased dietary sodium load and promotes the excretion of excess sodium through the interaction of renal dopamine receptors (D1R and D5R), and cholecystokinin B receptor (CCKBR), the so‐called “gastrorenal axis”. Gast−/− mice on a salt‐resistant background develop hypertension while wild‐type littermates have normal BP. Gast−/− mice have negligible gastric acid production, which results in increased bacterial survival and dysbiosis of the gut microbiota. Therefore, we tested the hypothesis that minocycline alleviates the high BP of Gast−/− mice. Gast−/− mice and their wild‐type littermates fed a high salt diet (4% NaCl) were given minocycline (50 mg/kg/day, p.o., 28 days). Results showed that minocycline decreased the high BP of conscious Gast−/− mice both at nighttime (130.7±0.4 vs. 123.2±0.3, P<0.05, Dunn's test, 4/group) and daytime (126.1±0.5 vs. 117.5±0.4 P<0.05), and the nighttime (122.4±0.4 vs. 118.4±0.5 P<0.001, Dunn's test, 4/group), but not the daytime BP of the wild‐type littermates. These results imply that inhibition of M1 macrophages decreases BP. In another set of Gast−/− mice, we tested the effect of doxycycline (43 mg/kg/day, p.o., 28 days) which selectively inhibits M2 polarization of macrophages leading to pro‐inflammatory and pro‐hypertensive effects. Doxycycline decreased the BP of conscious Gast−/− mice at nighttime (134.7±0.7 vs. 123.8±8.0 mm Hg, P<0.05, Dunn's test) and at daytime (120.7±0.6 vs. 113.1±2.8 mm Hg), conceivably by normalizing the dysbiosis expected in the knockout mice. By contrast, doxycycline increased BP in wild‐type littermates at nighttime (97.6±1.2 vs. 110.5±5.1 mm Hg) but not at daytime, which can be explained by the inhibition of the M2 macrophage polarization. Thus, the resultant hypertensive phenotype is influenced by the dynamic interplay among genetics, state of inflammation, and gut microbiota.Support or Funding InformationR01DK039308, R01HL092196, and P01HL074940This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dopamine receptor D2 (D2R) plays a significant role in kidney function by maintaining normal blood pressure (BP) and preventing renal inflammation and injury. D2R silencing in the mouse kidney increases BP and increases renal injury. To study the effects of D2R in the renal proximal tubule we generated Drd2 fl/fl , P SGLT2 ::Cre+ mice (D2R PT-/- ) that lack D2R only in the renal proximal tubule and Drd2 fl/fl , P SGLT2 ::Cre- mice (D2R PT+/+ ) that do not have the deletion. We studied male and female mice on normal salt (NS; 0.4% NaCl) and high salt (HS; 4% NaCl) diets. Mice were genotyped for Drd2 fl/fl and a smaller amplicon representing the Cre deletion mutant. On NS diet, male D2R PT-/- had higher systolic BP (SBP) measured under anesthesia than male D2R PT+/+ (113±1 vs 102±3 mmHg, n=5/group; P<0.05) and female D2R PT-/- (104±5 mmHg) or female D2R PT+/+ (106±3 mmHg). SBPs on HS diet were similar in D2R PT-/- females and males. On NS diet renal mRNA expressions of TNF-α, TGFβ1, Fn1, and Col1a1 were higher (P<0.01) in female D2R PT+/+ than in male D2R PT+/+ ; conversely the expression of the kidney injury marker, Kim-1, was higher in male than female D2R PT+/+ (P<0.01). Male D2R PT-/- expressed less (P<0.01) renal TNF-α, TGFβ1, Col1a1, cell proliferation marker Mki-67, and Fn1 than female D2R PT-/- . However, the renal expression of Kim-1 was less (P<0.01) in female than male D2R PT-/- . On HS diet, renal mRNA expressions of TNF-α, TGFβ1, and Fn-1 were similar in both male and female D2R PT+/+ and D2R PT-/- . HS diet increased (P<0.05) the expression of Col1a1 and Kim-1 in male D2R PT+/+ and D2R PT-/- but not in females of the two genotypes; HS diet also increased the expression of Mki-67 only in D2R PT-/- males. Our data show striking differences in the inflammatory response, cell proliferation, and kidney injury between males and females, with females expressing more inflammatory and proliferation markers but less injury markers than males. Deletion of the D2R in the proximal tubule increases the renal expression of extracellular matrix proteins and inflammatory and proliferation markers, effects that were more pronounced in females, except for Kim-1. The differences between males and females are maintained on HS diet, indicating that female mice have a “healthier” response to renal injury.

The genetic causes of salt sensitivity andhypertension in humans are not completely understood. The kidney plays a preeminent regulatory rolein blood pressure (BP) homeostasis and water and electrolyte balance. The renaldopamine receptors, D1R and D3R, engender natriuresis viathe inhibition of renal Na+ transport, whereas the angiotensin IItype 1 receptor (AT1R) does the opposite. The renal paracrineinhibition of Na+ transport by dopamine is impaired insalt‐sensitive rats, mice, and humans. Agonist activation promotes thephosphorylation of D1R and D3R by the G protein‐coupledreceptor kinase type 4 (GRK4), whose gene variants impair D1R and D3Ractivity. The global expression of GRK4 65R>L in transgenic mice results in salt‐sensitive hypertension, in part, due toincreased endogenous GRK4 and AT1R expression. To demonstrate thespecific renal causal mechanisms in GRK4 65R>L‐mediated hypertension, weheterologously expressed the GRK4 65R>Lvs. GRK4 wild‐type (WT) transgenes in the kidneys of Grk4 knockout mice on normal salt diet. The transgenes were delivered selectively into the renal tubules by thebilateral retrograde ureteral infusion of AAV‐9 vectors. The expression anddistribution along the entire nephron of the GRK4 WT and GRK4 65R>L were similar in both groups. However, the renal tubule‐restricted expression ofGRK4 65R>L increased the BP (117±4vs. 93±1 mm Hg, P<0.05, n=4), while that of the GRK4 WT only tendedto increase the BP (105±6 vs. 96±2 mmHg, n=5), indicating that the presence of the GRK4 variant in the kidney causedthe increase in BP. We next evaluated the renal expression profiles of selectgenes. We found that the pro‐natriuretic D1R (0.81±0.01 vs. 1.28±0.04, P<0.01) and D3R(0.44±0.02 vs. 1.27±0.07, P<0.01)were decreased whereas the anti‐natriuretic Na+/K+‐ ATPase(1.14±0.024 vs. 1.0 ±0.007, P<0.05) and α‐ENaC (1.4±0.14 vs. 1.0±0.11, P<0.05) were increased, demonstrating themechanistic changes that underlie the hypertension in these mice. Interestingly, we also observed that the proximal tubule Na+transporters NaPi2 (0.81±0.02 vs. 1.04±0.02), SGLT2 (0.89±0.03 vs. 1.07±0.05), NBCe2(0.50±0.07 vs. 1.15±0.03), and the AT1R(0.82±0.02 vs 1.02±0.02) were decreased, which may represent insufficientcompensatory mechanisms against the increase in BP. Our results highlight theunderlying and compensatory renal mechanisms for the hypertension thatdeveloped in mice with either kidney‐restricted or globally expressed GRK4 65R>L.Support or Funding InformationR01DK039308, R01HL092196, and P01HL074940This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.