Reduction of Pertussis Inflammatory Pathology by Therapeutic Treatment With Sphingosine-1-Phosphate Receptor Ligands by a Pertussis Toxin-Insensitive Mechanism.

Recent data have demonstrated the potential of sphingosine 1-phosphate (S1P) receptor (S1PR) agonism in the treatment of infectious diseases. A previous study used a murine model of Bordetella pertussis infection to demonstrate that treatment with the S1PR agonist AAL-R reduces pulmonary inflammation during infection. In the current study, we showed that this effect is mediated via the S1PR1 on LysM+ (myeloid) cells. Signaling via this receptor results in reduced lung inflammation and cellular recruitment as well as reduced morbidity and mortality in a neonatal mouse model of disease. Despite the fact that S1PRs are pertussis toxin-sensitive G protein-coupled receptors, the effects of AAL-R were pertussis toxin insensitive in our model. Furthermore, our data demonstrate that S1PR agonist administration may be effective at therapeutic time points. These results indicate a role for S1P signaling in B. pertussis-mediated pathology and highlight the possibility of host-targeted therapy for pertussis.

Determining the isotopic abundance of a labeled compound by mass spectrometry and how correcting for natural abundance distribution using analogous data from the unlabeled compound leads to a systematic error.

When the isotopic abundance or specific activity of a labeled compound is determined by mass spectrometry (MS), it is necessary to correct the raw MS data to eliminate ion intensity contributions, which arise from the presence of heavy isotopes at natural abundance (e.g., a typical carbon compound contains ~1.1% (13) C per carbon atom). The most common approach is to employ a correction in which the mass-to-charge distribution of the corresponding unlabeled compound is used to subtract the natural abundance contributions from the raw mass-to-charge distribution pattern of the labeled compound. Following this correction, the residual intensities should be due to the presence of the newly introduced labeled atoms only. However, this will only be the case when the natural abundance mass isotopomer distribution of the unlabeled compound is the same as that of the labeled species. Although this may be a good approximation, it cannot be accurate in all cases. The implications of this approximation for the determination of isotopic abundance and specific activity have been examined in practice. Isotopically mixed stable-atom labeled valine batches were produced, and both these and [(14) C6 ]carbamazepine were analyzed by MS to determine the extent of the error introduced by the approach. Our studies revealed that significant errors are possible for small highly-labeled compounds, such as valine, under some circumstances. In the case with [(14) C6 ]carbamazepine, the errors introduced were minor but could be significant for (14) C-labeled compounds with particular isotopic distributions. This source of systematic error can be minimized, although not eliminated, by the selection of an appropriate isotopic correction pattern or by the use of a program that varies the natural abundance distribution throughout the correction.

CD27 stimulation promotes the frequency of IL-7 receptor-expressing memory precursors and prevents IL-12-mediated loss of CD8(+) T cell memory in the absence of CD4(+) T cell help.

Fully functional CD8(+) T cell memory is highly dependent upon CD4(+) T cell support. CD4(+) T cells play a critical role in inducing the expression of CD70, the ligand for CD27, on dendritic cells. In this study, we demonstrate that CD27 stimulation during primary CD8(+) T cell responses regulates the ability to mount secondary CD8(+) T cell responses. CD27 stimulation during vaccinia and dendritic cell immunization controls the expression of the IL-7R (CD127), which has been shown to be necessary for memory CD8(+) T cell survival. Furthermore, CD27 stimulation during primary CD8(+) T cell responses to vaccinia virus restrained the late expression on memory precursor cells of cytokine receptors that support terminal differentiation. The formation of CD8(+) T cell memory precursors and secondary CD8(+) T cell responses was restored in the absence of CD27 costimulation when endogenous IL-12 was not available. Similarly, the lesion in CD8(+) T cell memory that occurs in the absence of CD4(+) T cells did not occur in mice lacking IL-12. These data indicate that CD4(+) T cell help and, by extension, CD27 stimulation support CD8(+) T cell memory by modulating the expression of cytokine receptors that influence the differentiation and survival of memory CD8(+) T cells.

Validating the American Association for the Surgery of Trauma Pancreas Injury Grade Using Trauma Quality Improvement Program Data.

The American Association for the Surgery of Trauma (AAST) Organ Injury Scale (OIS) for the pancreas was created in 1990. Our aim was to validate the ability of the AAST-OIS pancreas grade to predict adjuncts to operative management, including endoscopic retrograde cholangiopancreatography (ERCP) and percutaneous drain placement. We analyzed the Trauma Quality Improvement Program (TQIP) database from 2017 to 2019, including all patients with a pancreas injury. Outcomes included the rates of mortality, laparotomy, ERCP, and peri-pancreatic or hepatobiliary percutaneous drain placement. Outcomes were analyzed by AAST-OIS, and odds ratios (ORs) and 95 confidence intervals (CIs) were calculated for each. 3571 patients were included in the analysis. The AAST grade was associated with increased rates of mortality and laparotomy at every level (P < .05). Endoscopic retrograde cholangiopancreatography rates increased from grade 2 to 3 (OR 4.685, 95% CI 3.254-6.745), were similar between grades 3 and 4 (P > .05), and decreased from grades 4 to 5 (OR .443, CI .250-.788). Likewise, rates of percutaneous drain placement increased from grade 2 to 3 (OR 1.999, CI 1.192-3.353), were similar between grades 3 and 4 (P > .05), and decreased from grades 4 to 5 (OR .266, .076-.934). Increasing pancreatic injury grade is associated with increased mortality and laparotomy rates at all levels. Endoscopic retrograde cholangiopancreatography and percutaneous drainage procedures are used most in mid-grade (3-4) pancreatic trauma. The decrease in nonsurgical procedures in grade 5 pancreatic trauma is likely secondary to increased rates of surgical management (resection and/or wide drainage). The AAST-OIS for pancreatic injury is associated with mortality and interventions.

The American Association for the Surgery of Trauma Organ Injury Scale is Associated With Cystoscopic and Percutaneous Urologic Procedures in Renal Injuries.

BACKGROUND: The American Association for the Surgery of Trauma Organ Injury Scale for the kidney was created in 1989. It has been validated to various outcomes including operations. It was updated in 2018 to better predict endourologic interventions, but this change has not been validated. In addition, the AAST-OIS does not consider mechanism of trauma in its interpretation. METHODS: We analyzed 3 years of the Trauma Quality Improvement Program database including all patients with a kidney injury. We recorded rates of mortality, operation, renal operation, nephrectomy, renal embolization, cystoscopic intervention, and percutaneous urologic procedures. RESULTS: 26294 patients were included. In penetrating trauma, mortality, operation, renal-specific operation, and nephrectomy rates increased at every grade. Renal embolization and cystoscopy rates peaked in grade IV. Percutaneous interventions were rare across all grades. In blunt trauma, mortality and nephrectomy rates increased only in grades IV and V. Operation, renal operation, and renal embolization rates increased at every grade level. Cystoscopy rates peaked in grade IV. Percutaneous procedure rates only increased between grades III and IV. Penetrating injuries are more likely to require nephrectomy in grades III-V, cystoscopic procedures in grade III, and percutaneous procedures in grades I-III. DISCUSSION: Endourologic procedures are most utilized in grade IV injuries, which are in part defined by injuries with damage to the central collecting system. Despite penetrating injuries more frequently requiring nephrectomy, they also more frequently require nonsurgical procedures. Mechanism of trauma should be considered when interpreting the AAST-OIS for kidney injuries.

The American Association for the Surgery of Trauma Organ Injury Scale for Spleen Does Not Equally Predict Interventions in Penetrating and Blunt Trauma.

BACKGROUND: The American Association for the Surgery of Trauma (AAST) Organ Injury Scale (OIS) for the spleen (and other organs) was created in 1989. It has been validated to predict mortality, need for operation, length of stay (LOS), and intensive care unit (ICU) LOS. PURPOSE: We aimed to determine if the Spleen OIS is applied equally to blunt and penetrating trauma. RESEARCH DESIGN/STUDY SAMPLE: We analyzed the Trauma Quality Improvement Program (TQIP) database from 2017-2019, including patients with spleen injuries. DATA COLLECTION: Outcomes included the rates of mortality, operation, spleen-specific operation, splenectomy, and splenic embolization. RESULTS: 60900 patients had a spleen injury with an OIS grade. Mortality rates increased in Grades IV and V for both blunt and penetrating trauma. In blunt trauma, the odds for any operation, spleen-specific operation, and splenectomy increased, for each increase in grade. Penetrating trauma showed similar trends in grades up to grade IV, but were statistically similar between grade IV and V. Splenectomy was higher in penetrating trauma for all grades. Splenic embolization peaked at 25% of grade IV trauma before decreasing in grade V. Rates in penetrating trauma were significantly lower in all grades, peaking at 2.5% of Grade III injuries. CONCLUSIONS: The mechanism of trauma is a significant factor for all outcomes, independent of AAST-OIS. Hemostasis is predominantly surgical in penetrating trauma, achieved with angioembolization more frequently in blunt trauma. Penetrating trauma management is influenced by the potential for injury to peri-splenic organs.

Medicare Part D Plans Rarely Cover Brand-Name Drugs When Generics Are Available.

Recent press reports and other evidence suggest that Medicare Part D plans may be encouraging the use of brand-name drugs instead of generics. However, the scope of such practices is unclear. We examined Medicare Part D formulary coverage and tier placement of matched pairs of brand-name drugs and generics to quantify how often preferred formulary placement of brand-name drugs is occurring within and across Part D plans and to assess the cost implications for Medicare and its beneficiaries. We found that in 2019, 84 percent of 4,176,772 Part D plan-product combinations had generic-only coverage (that is, the brand-name counterparts were not covered). Another 15 percent covered both the brand-name and generic versions of a product. For the small number of products whose brand-name versions were covered preferentially to their generic equivalents, beneficiary and Medicare prices were generally low for both products. Overall, we found that most Part D plan formularies are designed to encourage the use of generics rather than their brand-name counterparts. Policy makers should continue to monitor Part D formulary coverage patterns to ensure consistent and generous coverage for generic drugs, given their important role in reducing prescription drug spending.

Noncanonical Mechanisms for Direct Bone Marrow Generating Ang II (Angiotensin II) Predominate in CD68 Positive Myeloid Lineage Cells.

Bone marrow (BM) Ang II (angiotensin II) is a major participant in the regulation of hematopoiesis and immunity. The novel tissue substrate Ang-(1-12) [angiotensin-(1-12)] and its cleaving enzyme chymase are an essential source of Ang II production in cardiac tissue. We hypothesized this noncanonical chymase-mediated Ang II-producing mechanism exists in the BM tissue. Immunohistostaining and flow cytometry confirmed the presence of Ang-(1-12) immunoreaction in the BM of SD (Sprague Dawley) rats. Chymase-mediated Ang II-producing activity in BM was ≈1000-fold higher than ACE (angiotensin-converting enzyme)-mediated Ang II-producing activity (4531±137 and 4.2±0.3 fmol/min per mg, respectively; n=6; P<0.001) and 280-fold higher than chymase activity in the left ventricle of 16.3±1.7 fmol/min per mg (P<0.001). Adding a selective chymase inhibitor, TEI-F00806, eliminated almost all 125I-Ang II production. Flow cytometry demonstrated that delta median fluorescence intensity of chymase in cluster of differentiation 68 positive cells was significantly higher than that in cluster of differentiation 68 negative cells (1546±157 and 222±48 arbitrary units, respectively; P=0.0021). Cluster of differentiation 68 positive and side scatter low subsets, considered to be myeloid progenitors, express the highest chymase fluorescence intensity in rat BM. Chymase activity and cellular expression was similar in both male and female rats. In conclusion, myeloid lineage cells, especially myeloid progenitors, have an extraordinary Ang II-producing activity by chymase in the BM.

Activation of the Human Angiotensin-(1-12)-Chymase Pathway in Rats With Human Angiotensinogen Gene Transcripts.

Angiotensin-(1-12) [Ang-(1-12)], an alternate substrate for tissue angiotensin II (Ang II) formation, underscores the importance of alternative renin-independent pathway(s) for the generation of angiotensins. Since renin enzymatic activity is species-specific, a transgenic model of hypertension due to insertion of the human angiotensinogen (AGT) gene in Sprague Dawley rats allowed for characterizing the contribution of a non-renin dependent mechanism for Ang II actions in their blood and heart tissue. With this in mind, we investigated whether TGR(hAGT)L1623 transgenic rats express the human sequence of Ang-(1-12) before and following a 2-week oral therapy with the type I Ang II receptor (AT1-R) antagonist valsartan. Plasma and cardiac expression of angiotensins, plasma renin activity, cardiac angiotensinogen, and chymase protein and the enzymatic activities of chymase, angiotensin converting enzyme (ACE) and ACE2 were determined in TGR(hAGT)L1623 rats given vehicle or valsartan. The antihypertensive effect of valsartan after 14-day treatment was associated with reduced left ventricular wall thickness and augmented plasma concentrations of angiotensin I (Ang I) and Ang II; rat and human concentrations of angiotensinogen or Ang-(1-12) did not change. On the other hand, AT1-R blockade produced a 55% rise in left ventricular content of human Ang-(1-12) concentration and no changes in rat cardiac Ang-(1-12) levels. Mass-Spectroscopy analysis of left ventricular Ang II content confirmed a >4-fold increase in cardiac Ang II content in transgenic rats given vehicle; a tendency for decreased cardiac Ang II content following valsartan treatment did not achieve statistical significance. Cardiac chymase and ACE2 activities, significantly higher than ACE activity in TGR(hAGT)L1623 rats, were not altered by blockade of AT1-R. We conclude that this humanized model of angiotensinogen-dependent hypertension expresses the human sequence of Ang-(1-12) in plasma and cardiac tissue and responds to blockade of AT1-R with further increases in the human form of cardiac Ang-(1-12). Since rat renin has no hydrolytic activity on human angiotensinogen, the study confirms and expands knowledge of the importance of renin-independent mechanisms as a source for Ang II pathological actions.

Angiotensin-(1-12)/chymase axis modulates cardiomyocyte L-type calcium currents in rats expressing human angiotensinogen.

BACKGROUND: Activation of the intracrine renin angiotensin systems (RAS) is increasingly recognized as contributing to human pathologies, yet non-canonical renin-independent mechanisms for angiotensin II (Ang II) biosynthesis remain controversial. Direct Ang II generation from angiotensin-(1-12) [Ang-(1-12)] by chymase is an essential intracrine source for regulation of cardiac function. Using a transgenic rat model that overexpresses the human angiotensinogen gene [TGR(hAGT)L1623] and displays increased cardiac Ang II levels, this study aimed to provide evidence for intracrine activation of L-type calcium currents (ICa-L) mediated by the Ang-(1-12)/chymase axis. METHODS AND RESULTS: On patch clamp, ICa-L density was significantly higher in TGR(hAGT)L1623 (-6.4 ±â€¯0.3 pA/pF) compared to Sprague Dawley (SD) cardiomyocytes (-4.8, ± 0.5 pA/pF). Intracellular administration of Ang II and Ang-(1-12) elicited a ICa-L increase in both SD and TGR(hAGT)L1623 cardiomyocytes, albeit blunted in transgenic cells. ICa-L activation by intracellular Ang II and Ang-(1-12) was abolished by the specific Ang II type 1 receptor blocker E-3174. Co-administration of a chymase inhibitor prevented activation of ICa-L by Ang-(1-12). Confocal micrographs revealed abundant chymase (mast cell protease 5) immunoreactive protein in SD and TGR(hAGT)L1623 cardiomyocytes. CONCLUSIONS: Our data demonstrate the existence in cardiomyocytes of a calcium channel modulatory activity responsive to Ang II generated by the Ang-(1-12)/chymase axis that signals via intracellular receptors. Chronically elevated Ang II in TGR(hAGT)L1623 hearts leading to increased intracellular calcium through ICa-L suggests that activation of this Ang-(1-12)/chymase-governed cardiac intracrine RAS may contribute to the pathological phenotypes observed in the humanized model of chronic hypertension and cardiac hypertrophy.

QSAR Prediction of Passive Permeability in the LLC-PK1 Cell Line: Trends in Molecular Properties and Cross-Prediction of Caco-2 Permeabilities.

A QSAR model for predicting passive permeability (Papp ) was derived from Papp values measured in the LLC-PK1 cell line. The QSAR method and descriptor set that performed best in terms of cross-validation was random forest with a combination of AP, DP, and MOE_2D descriptors. The QSAR model was used to predict the Caco-2 cell permeability for 313 compounds described in the literature with good success. We find that passive permeability for different cell lines can be predicted with similar molecular properties and descriptors. It is shown that the variation in experimental measurements of Papp is smaller than the error in QSAR predictions indicating that predictions are not quantitatively perfect, although qualitatively useful. We get better predictions if the training set is large and diverse, rather than smaller and more internally consistent. This is because prediction accuracy falls off quickly with decreasing similarity to the training set and it is therefore better to have as large a training set as possible. While single physical parameters are not as good as a full QSAR model in predicting Papp , logD seems the most important parameter. Intermediate values of logD are associated with higher Papp .

Control of established melanoma by CD27 stimulation is associated with enhanced effector function and persistence, and reduced PD-1 expression of tumor infiltrating CD8(+) T cells.

The immune response to the tumor can be enhanced by targeting costimulatory molecules on T cells. As the CD70-CD27 costimulatory axis plays an important role in the activation, survival, and differentiation of lymphocytes, we have examined the efficacy of agonistic anti-CD27 antibodies as monotherapies for established melanoma in a murine model. We show that this approach leads to a substantial reduction in the outgrowth of both experimental lung metastases and subcutaneous tumors. Anti-CD27 treatment supports the maintenance of tumor-specific CD8(+) T cells within the tumor, reduces the frequency of FoxP3-expressing CD4(+) T cells within tumors, and potentiates the ability of NK1.1(+) and CD8(+) tumor infiltrating cells to secrete IFNγ upon coculture with tumor cells. The enhanced effector function correlated with lower levels of PD-1 expression on CD8(+) T cells from anti-CD27-treated mice. Despite the modulating effect of anti-CD27 on multiple cell types, only CD8(+) T cells were absolutely required for tumor control. The CD4(+) T cells were dispensable, whereas NK1.1(+) cells were needed during early stages of tumor growth but not for the effectiveness of anti-CD27. Thus, CD27-mediated costimulation provides a potent boost to multiple aspects of the endogenous responses to tumor, and may be exploited to enhance tumor immunity.

Quantitative trait locus analysis of neointimal formation in an intercross between C57BL/6 and C3H/HeJ apolipoprotein E-deficient mice.

BACKGROUND: Inbred mouse strains C57BL/6J (B6) and C3H/HeJ (C3H) exhibit marked differences in neointimal formation after arterial injury when deficient in apolipoprotein E (apoE(-/-)) and fed a Western diet. Quantitative trait locus (QTL) analysis was performed on an intercross between B6.apoE(-/-) and C3H.apoE(-/-) mice to determine genetic factors contributing to the phenotype. METHODS AND RESULTS: Female B6.apoE(-/-) mice were crossed with male C3H.apoE(-/-) mice to generate F(1)s, which were intercrossed to generate 204 male F(2) progeny. At 10 weeks of age, F(2)s underwent endothelium denudation injury to the left common carotid artery. Mice were fed a Western diet for 1 week before and 4 weeks after injury and analyzed for neointimal lesion size, plasma lipid and MCP-1 levels. One significant QTL, named Nih1 (61cM, LOD score: 5.02), on chromosome 12 and a suggestive locus on chromosome 13 (35cM, LOD: 2.67) were identified to influence lesion size. One significant QTL on distal chromosome 1 accounted for major variations in plasma non-HDL cholesterol and triglyceride levels. Four suggestive QTLs on chromosomes 1, 2, and 3 were detected for circulating MCP-1 levels. No correlations were observed between neointimal lesion size and plasma lipid levels or between lesion size and plasma MCP-1 levels. CONCLUSIONS: Neointimal formation is controlled by genetic factors independent of those affecting plasma lipid levels and circulating MCP-1 levels in the B6 and C3H mouse model.