Characterization of cytochrome P450 4F subfamily: Functional role and response in inflammation

CYP4F subfamily comprises a group of enzymes that metabolize LTB4 to biologically less active metabolites. These inactive hydroxy products are incapable of chemotaxis and recruitment of inflammatory cells. This has led to a hypothesis that CYP4Fs may modulate inflammatory conditions serving as a signal of resolution. ^ We investigated the regulation of rat CYP4F gene expression under various inflammatory prompts including a bacterial lipopolysaccharide (LPS) treated model system, controlled traumatic brain injury (TBI) model as well as using direct cytokine challenges. CYP4Fs showed an isoform specific response to LPS. The pro-inflammatory cytokines IL-1β, IL-6 and TNF-α produced an overall inductive CYP4F response whereas IL-10, an anti-inflammatory cytokine, suppressed CYP4F gene expression in primary hepatocytes. The molecular mechanism behind IL-6 mediated CYP4F induction was partially STAT3 dependent. ^ An alternate avenue of triggering the inflammatory cascade is TBI, which is known to cause several secondary effects leading to multiorgan dysfunction syndrome. The results from this study elicited that trauma to the brain can produce acute inflammatory changes in organs distant from the injury site. Local production of LTB4 after CNS injury caused mobilization of inflammatory cells such as neutrophils to the lung. In the resolution phase, CYP4F expression increased with time along with the associated activity causing a decline in LTB4 concentration. This marked a significant reduction in neutrophil recruitment to the lung which led to subsequent recovery and repair. In addition, we showed that CYP4Fs are localized primarily in pulmonary endothelium. We speculate that the temporally regulated LTB4 clearance in the endothelium may be a novel target for treatment of pulmonary inflammation following injury. ^ In humans, several CYP4F isoforms have been identified and shown to metabolize LTB4 and other endogenous eicosanoids. However, the specific activity of the recently cloned human CYP4F11 is unknown. In the final part of this thesis, CYP4F11 protein was expressed in yeast in parallel to CYP4F3A. To our surprise, CYP4F11 displayed a different substrate profile than CYP4F3A. CYP4F3A metabolized eicosanoids while CYP4F11 was a better catalyst for therapeutic drugs. Thus, besides their endogenous function in clearing inflammation, CYP4Fs also may play a part in drug metabolism. ^

Reduced neutrophil count in people of African descent is due to a regulatory variant in the Duffy antigen receptor for chemokines gene.

Persistently low white blood cell count (WBC) and neutrophil count is a well-described phenomenon in persons of African ancestry, whose etiology remains unknown. We recently used admixture mapping to identify an approximately 1-megabase region on chromosome 1, where ancestry status (African or European) almost entirely accounted for the difference in WBC between African Americans and European Americans. To identify the specific genetic change responsible for this association, we analyzed genotype and phenotype data from 6,005 African Americans from the Jackson Heart Study (JHS), the Health, Aging and Body Composition (Health ABC) Study, and the Atherosclerosis Risk in Communities (ARIC) Study. We demonstrate that the causal variant must be at least 91% different in frequency between West Africans and European Americans. An excellent candidate is the Duffy Null polymorphism (SNP rs2814778 at chromosome 1q23.2), which is the only polymorphism in the region known to be so differentiated in frequency and is already known to protect against Plasmodium vivax malaria. We confirm that rs2814778 is predictive of WBC and neutrophil count in African Americans above beyond the previously described admixture association (P = 3.8 x 10(-5)), establishing a novel phenotype for this genetic variant.

STAT3 CONTROLS THE NEUTROPHIL MIGRATORY RESPONSE TO CXCR2 AND ITS LIGAND MIP-2 (CXCL2)

Among the first white blood cells to respond to bacterial and fungal infections, neutrophils are produced in the bone marrow, released into circulating blood, and recruited to inflamed tissue. The cytokine granulocyte colony-stimulating factor (G

The role of STAT3 in the emergency neutrophil response

Neutrophils are an essential component of innate immunity, serving to provide an immediate response to microbial invasion. In response to emergency situations such as an infection, serum levels of granulocyte colony-stimulating factor (G-CSF) are induced, causing a boost in neutrophil production and a rapid mobilization of bone marrow neutrophils to the blood, where they can circulate to clear foreign pathogens. Signal transducer and activator of transcription 3 (STAT3) is a principal downstream signaling intermediate of the G-CSF receptor. Mice null for STAT3 are embryonic lethal; therefore, to examine the role that STAT3 has in granulocytic development and function in vivo, we utilized a conditional knockout mouse that deletes functional STAT3 in the hematopoietic system (referred to herein as STAT3-deficient). Using this model, we show that STAT3 is required for G-CSF-induced expansion of granulocytic progenitor cells within the bone marrow and for acute G-CSF-dependent neutrophil mobilization into the blood. Thus, STAT3 has a critical role in the immediate G-CSF-response in vivo. Sustained G-CSF exposure causes skewed granulocytic production and mobilization in STAT3-deficient mice, suggesting an atypical granulocytic developmental pathway. To determine if STAT3-deficient neutrophils were functional, we examined neutrophil chemotaxis, since neutrophil function relies on proper chemoattractant-induced migration to infected tissue sites. STAT3-deficient neutrophils have impaired chemotaxis in response to the potent neutrophil chemoattractants MIP-2 and KC, both ligands for the chemokine receptor CXCR2. Additionally, STAT3-deficient mice have a defect in NIIP-2-induced acute neutrophil mobilization in vivo. Chemotaxis in response to fMLP and SDF-1, which utilize distinct seven-transmembrane chemokine receptors, was similar between wild type and STAT3-deficient neutrophils, suggesting that STAT3 specifically regulates CXCR2-mediated migration. MIP-2-induced activation of the Raf/MEK/ERK signaling cascade, which we show is required for MIP-2-dependent neutrophil chemotaxis, was impaired in STAT3-deficient neutrophils. Interestingly, acute G-CSF administration induced CXCR2 expression and Raf/MEK/ERK activation in neutrophils from wild type mice, whereas these responses were abrogated in neutrophils from STAT3-deficient mice. Thus, STAT3 regulation of CXCR2 functions may also contribute to STAT3's control of the acute G-CSF mobilization response. These combined results place STAT3 as a critical intermediate in neutrophil migration and G-CSF-induced neutrophil production responses required for emergency granulopoiesis. ^

Association of ALLHAT investigator characteristics with participant recruitment

Clinical trials are often not successful because of the inability to recruit a sufficient number of patients. The Antihypertensive and Lipid-Lowering Treatment to Prevent Heart Attack Trial (ALLHAT), the largest antihypertensive trial ever conducted, provided highly generalized results and successful recruitment of over 42,000 participants. The overall purpose of this study was to examine the association of investigator characteristics with anti-hypertensive (AHT) participant recruitment in ALLHAT. This secondary data analyses collected data from the ALLHAT investigator profile survey and related investigator characteristics to recruitment success. The sample size was 502 investigators, with recruitment data from 37,947AHT participants. Recruitment was dichotomized by categorizing all sites with recruitment numbers at or above the overall median recruitment number of 46 as "Successful Recruitment". Frequency distributions and univariate and multivariate logistic regression were conducted. When adjusting for all other factors, Hispanic ethnicity, suburban setting, Department of Veterans Affairs Medical Centers (VAMC) site type, number of clinical site staff working on the trial, study coordinator hours per week, medical conference sessions attended, the investigator's primary goal and the likelihood that a physician will convince a patient to continue on randomized treatment, have significant impacts on the recruitment success of ALLHAT investigators. Most of the ALLHAT investigators described their primary commitment as being towards their patients and not to scientific knowledge alone. However, investigators that distinguished themselves as leaders in research had greater recruitment success than investigators who were leaders in clinical practice. ALLHAT was a highly successful trial that proved that community based cardiovascular trials can be implemented on a large scale. Exploring characteristics of ALLHAT investigators provides data that can be generalized to sponsors, sites, and others interested in maximizing clinical trial recruitment numbers. Future studies should further evaluate investigator and study coordinator factors that impact cardiovascular clinical trial recruitment success.^

Binding of the HIV-1 gp120 -V3 to cellular glycosphingolipids is necessary for CXCR4 recruitment and infection

Infection by human immunodeficiency virus type 1 (HIV-1) is a multi-step process, and detailed analyses of the various events critical for productive infection are necessary to clearly understanding the infection process and identifying novel targets for therapeutic interventions. Evidence from this study reveals binding of the viral envelope protein to host cell glycosphingolipids (GSLs) as a novel event necessary for the orderly progression of the host cell-entry and productive infection by HIV-1. Data obtained from co-immunoprecipitation analyses and confocal microscopy showed that the ability of viral envelope to interact with the co-receptor CXCR4 and productive infection of HIV-1 were inhibited in cells rendered GSL-deficient, while both these activities were restored after reconstitution of the cells with specific GSLs like GM3. Furthermore, evidence was obtained using peptide-inhibitors of HIV-1 infection to show that binding of a specific region within the V3-loop of the envelope protein gp120 to the host cell GSLs is the trigger necessary for the CD4-bound gp120 to recruit the CXCR4 co-receptor. Infection-inhibitory activity of the V3 peptides was compromised in GSL-deficient cells, but could be restored by reconstitution of GSLs. Based on these findings, a revised model for HIV-1 infection is proposed that accounts for the established interactions between the viral envelope and host cell receptors while enumerating the importance of the new findings that fill the gap in the current knowledge of the sequential events for the HIV-1 entry. According to this model, post-CD4 binding of the HIV-1 envelope surface protein gp120 to host cell GSLs, mediated by the gp120-V3 region, enables formation of the gp120-CD4-GSL-CXCR4 immune-complex and productive infection. The identification of cellular GSLs as an additional class of co-factors necessary for HIV-1 infection is important for enhancing the basic knowledge of the HIV-1 entry that can be exploited for developing novel antiviral therapeutic strategies. ^