VSMC3 migration from media to intima plays a determinant role in atherosclerosis and restenosis (1-3). Arachidonic acid (AA) and its oxygenated metabolites, collectively known as eicosanoids, are involved in the maintenance of vascular tone (4, 5). Lipoxygenases (LOXs) are non-heme iron dioxygenases that stereospecifically introduce molecular oxygen into polyunsaturated fatty acids, resulting in the formation of hydroperoxyeicosatetraenoic acids, which are subsequently converted to hydroxyeicosatetraenoic acids (HETEs) (6). Two 15-LOXs, namely, 15-LOX1 and 15-LOX2, have been shown to be expressed in humans (7,8). Both enzymes metabolize linoleic acid to 13(S)-hydroperoxyoctadecadienoic acid and AA to 15(S)-hydroperoxyeicosatetraenoic acid preferentially (9, 10). In regard to their tissue distribution, whereas 15-LOX1 shows a narrow cell-specific expression, including human reticulocytes and airway epithelial cells, 15-LOX2 appears to be expressed in epithelial cell types in cornea, lung, prostate, and skin (11). The studies from our laboratory showed that both 15-LOX1 and 15-LOX2 are expressed in human retinal microvascular endothelial cells (12). Although the presence of 15-LOX2 in VSMC has yet to be reported, these cells express 15-LOX1 (also known as 12/15-LOX in murines) and, when exposed to AA, produce both 15(S)-HETE and 12(S)-HETE (13,14). It is also known that 15-LOX1 and 12/15-LOX are involved in the oxidation of low density lipoprotein, a contributing factor in the pathogenesis of atherosclerosis (15, 16). Furthermore, using either transgenic or knock-out mice models, a number of studies have demonstrated that 15-LOX1 and its murine ortholog 12/15-LOX play a role in atherosclerosis and restenosis (17)(18)(19). In addition, human atheroma homogenates upon incubation with AA converted it mainly to .In recent years LOX products of polyunsaturated fatty acids have also been shown to be potent chemoattractants for resi-* This work was supported, in whole or in part, by National Institutes of Health Grant HL064165 (NHLBI; to G. N. R.

The lupus-prone NZM2410 mice present an expanded B1a cell population that we have mapped to the Sle2c1 lupus susceptibility locus. The expression of Cdkn2c, a gene encoding for cyclin-dependent kinase inhibitor p18Ink4c and located within Sle2c1, is significantly lower in B6.Sle2c1 B cells than in B6 B cells. To test the hypothesis that the B1a cell expansion in B6.Sle2c1 mice was due to a defective p18 expression, we analyzed the B1a cell phenotypes of p18-deficient C57BL/6 mice. We found a dose-dependent negative correlation between the number of B1a cells and p18 expression in B cells, with p18-deficient mice showing an early expansion of the peritoneal B1a cell pool. p18-deficiency enhanced the homeostatic expansion of B1a cells but not of splenic conventional B cells, and the elevated number of B6.Sle2c1 B1a cells was normalized by cyclin D2 deficiency. This data demonstrated that p18 is a key regulator of the size of the B1a cell pool. B6.p18-/- mice produced significant amounts of anti-DNA IgM and IgG, indicating that p18-deficiency contributes to humoral autoimmunity. Finally, we have shown that Sle2c1 increases lpr-associated lymphadenopathy and T cell-mediated pathology. B6.p18-/-.lpr mice showed a greater lymphadenopathy than B6.Sle2c1.lpr mice, but their renal pathology was intermediate between that of B6.lpr and B6.Sle2c1.lpr mice. This indicated that p18-deficiency synergizes, at least partially, with lpr-mediated pathology. Taken together, these results show that Cdkn2c contributes to lupus susceptibility by regulating the size of the B1a cell compartment and hence their contribution to autoimmunity.

Lyme disease caused by the Borrelia burgdorferi (Bb or B. burgdorferi) is the most common vector-borne, multi-systemic disease in the USA. Although most Lyme disease patients can be cured with a course of the first line of antibiotic treatment, some patients are intolerant to currently available antibiotics, necessitating the development of more effective therapeutics. We previously found several drugs, including disulfiram, that exhibited effective activity against B. burgdorferi. In the current study, we evaluated the potential of repurposing the FDA-approved drug, disulfiram for its borreliacidal activity. Our results indicate disulfiram has excellent borreliacidal activity against both the log and stationary phase B. burgdorferi sensu stricto B31 MI. Treatment of mice with disulfiram eliminated the B. burgdorferi sensu stricto B31 MI completely from the hearts and urinary bladder by day 28 post infection. Moreover, disulfiram-treated mice showed reduced expressions of inflammatory markers, and thus they were protected from histopathology and cardiac organ damage. Furthermore, disulfiram-treated mice showed significantly lower amounts of total antibody titers (IgM and IgG) at day 21 and total IgG2b at day 28 post infection. FACS analysis of lymph nodes revealed a decrease in the percentage of CD19+ B cells and an increase in total percentage of CD3+ T cells, CD3+ CD4+ T helpers, and naive and effector memory cells in disulfiram-treated mice. Together, our findings suggest that disulfiram has the potential to be repurposed as an effective antibiotic for treating Lyme disease.

Lyme disease is one of most common vector-borne diseases, reporting more than 300,000 cases annually in the United States. treating Lyme disease during its initial stages with traditional tetracycline antibiotics is effective. However, 10-20% of patients treated with antibiotic therapy still shows prolonged symptoms of fatigue, musculoskeletal pain, and perceived cognitive impairment. When these symptoms persists for more than 6 months to years after completing conventional antibiotics treatment are called post-treatment Lyme disease syndrome (ptLDS). though the exact reason for the prolongation of post treatment symptoms are not known, the growing evidence from recent studies suggests it might be due to the existence of drug-tolerant persisters. In order to identify effective drug molecules that kill drug-tolerant borrelia we have tested two antibiotics, azlocillin and cefotaxime that were identified by us earlier. The in vitro efficacy studies of azlocillin and cefotaxime on drug-tolerant persisters were done by semisolid plating method. the results obtained were compared with one of the currently prescribed antibiotic doxycycline. We found that azlocillin completely kills late log phase and 7-10 days old stationary phase B. burgdorferi. our results also demonstrate that azlocillin and cefotaxime can effectively kill in vitro doxycycline-tolerant B. burgdorferi. Moreover, the combination drug treatment of azlocillin and cefotaxime effectively killed doxycycline-tolerant B. burgdorferi. furthermore, when tested in vivo, azlocillin has shown good efficacy against B. burgdorferi in mice model. These seminal findings strongly suggests that azlocillin can be effective in treating B. burgdorferi sensu stricto JLB31 infection and furthermore in depth research is necessary to evaluate its potential use for Lyme disease therapy. Lyme disease is a major vector-borne disease in the United States caused by Borrelia burgdorferi sensu lato which affects more than 300,000 people annually 1-3. Lyme disease affects various organs such as brain, skin, heart, joints, and nervous systems 4-6. The symptoms of Lyme disease are erythema migrans, fatigue, fever, headache, chills, muscle and joint pain 7,8. Current antibiotic treatment for Lyme disease is effective during early stages of disease and cures the infection in most patients 9. However, 10-20% of patients undergone antibiotic treatment still experience symptoms like pain, fatigue, arthralgia, and cognitive problems. If these symptoms prolong more than 6 months after treatment, it is referred to as Post-treatment Lyme Disease Syndrome (PTLDS) 9-12. Though the exact root cause for PTLDS is not known, research evidences suggests it might be presence of persister forms of B. burgdorferi or due to impaired immunological response 10,13,14. Many research studies has shown that B.

We have previously shown that a novel −74 C to T mutation in the promoter of the cyclin-dependent kinase inhibitor p18 gene was associated with a reduced p18 expression in B cells from mice carrying the Sle2c1 lupus susceptibility locus. To determine the function of the −74 C/T SNP, we have characterized the proximal promoter of the mouse p18 gene. Functional analysis of the 5' flanking region by sequential deletions revealed crucial elements between −300 and +1, confirming the in silico prediction that the −74 T allele created a novel YY-1 binding site adjacent to an existing one common to both alleles. Moreover, we found that YY-1, E2F1 and Sp-1 can synergistically enhance the activity of the p18 promoter. Mutational inactivation revealed that YY-1 binding regulates the p18 activity in an allele-dependent fashion. EMSAs with splenic B cell extracts directly demonstrated that YY-1 binds to the p18 promoter with differences between the C and the T alleles. We also determined in vivo by chromatin immunoprecipitation that the T allele resulted in increased YY-1 and decreased Nrf-2 binding to the p18 promoter as compared to the C allele in B cells. Thus, YY-1 is a direct regulator of p18 gene expression in an allele-dependent fashion that is consistent with the lupus-associated T allele inducing a lower p18 transcriptional activity by increasing YY-1 binding. These results establish the p18 −74 C/T mutation as the leading causal variant for the B1a cell expansion that characterizes the NZB and NZM2410 lupus-prone strains.

27Lyme disease caused by the Borrelia burgdorferi (Bb or B. burgdorferi) is a most common 28 vector-borne, multi-systemic disease in USA. Although, most Lyme disease patients can be 29 cured with a course of antibiotic treatment, a significant percent of patient population fail to be 30 disease-free post-treatment, necessitating the development of more effective therapeutics. We 31 previously found several drugs including disulfiram having with good activity against B. 32 burgdorferi. In current study, we evaluated the potential of repurposing the FDA approved 33 disulfiram drug for its borreliacidal activity. Our in vitro results indicate disulfiram shows 34 excellent borreliacidal activity against both the log and stationary phase B. burgdorferi. 35 Subsequent mice studies have determined that the disulfiram eliminated B. burgdorferi 36 completely from hearts and urinary bladder by day 28 post infection, demonstrating the practical 37 application and efficacy of disulfiram against B. burgdorferi in vivo. Moreover, disulfiram 38 treated mice showed reduced expression of inflammatory markers and protected against 39 histopathology and organ damage. Furthermore, disulfiram treated mice showed significantly 40 lower amounts of total antibody titers (IgM and IgG) at day 21 and total IgG2b at day 28 post 41 infection. Mechanistically, cellular analysis of lymph nodes revealed a decrease in percentage of 42 CD19+ B cells and increase in total percentage of CD3+ T cells, CD3+ CD4+ T helpers, and 43 naïve and effector memory cells in disulfiram-treated mice. Together, we demonstrate that 44 disulfiram has the potential and could be repurposed as an effective antibiotic for treating Lyme 45 disease in near future. 46 47 48 49 50 51

BackgroundLyme disease accounts for >90% of all vector-borne disease cases in the United States and affect ~300,000 persons annually in North America. Though traditional tetracycline antibiotic therapy is generally prescribed for Lyme disease, still 10%–20% of patients treated with current antibiotic therapy still show lingering symptoms.MethodsIn order to identify new drugs, we have evaluated four cephalosporins as a therapeutic alternative to commonly used antibiotics for the treatment of Lyme disease by using microdilution techniques like minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). We have determined the MIC and MBC of four drugs for three Borrelia burgdorferi s.s strains namely CA8, JLB31 and NP40. The binding studies were performed using in silico analysis.ResultsThe MIC order of the four drugs tested is cefoxitin (1.25 µM/mL) > cefamandole (2.5 µM/mL), > cefuroxime (5 µM/mL) > cefapirin (10 µM/mL). Among the drugs that are tested in this study using in vivo C3H/HeN mouse model, cefoxitin effectively kills B. burgdorferi. The in silico analysis revealed that all four cephalosporins studied binds effectively to B. burgdorferi proteins, SecA subunit penicillin-binding protein (PBP) and Outer surface protein E (OspE).ConclusionBased on the data obtained, cefoxitin has shown high efficacy killing B. burgdorferi at concentration of 1.25 µM/mL. In addition to it, cefoxitin cleared B. burgdorferi infection in C3H/HeN mice model at 20 mg/kg.

Sle2c1 is a sublocus from the NZB-derived Sle2 locus found in the NZM2410 lupus-prone mouse. In addition to expanding the peritoneal cavity B1a cells, Sle2c1 significantly reduced IL-2 production by activated CD4+ T cells in vitro and is associated with a reduction of the Treg compartment, especially in Fas-deficient mice. The characterization of three Sle2c1 recombinants mapped the IL-2 deficiency to a short interval on chromosome 4 that contains the Cdkn2c gene. Cdkn2c encodes for p18INK4c (p18), a cyclin-dependent kinase inhibitor that regulates G1 cell cycle arrest. We have found a novel SNP in the Cdkn2c promoter that is associated with a significantly reduced expression of this gene in CD4+ T cells. Activated CD4+ T cells from B6.p18-/- mice produced significantly less IL-2 than B6 controls, demonstrating that a dysregulated cell cycle interferes with IL-2 production. Interestingly, we have also found that p18 reduced expression, either in B6.Sle2c1 or B6.p18-/- mice, is also associated with an expansion of the B1a cells compartment. Overall these results show that a dysregulated cell cycle affects CD4+ T cell and B cell homeostasis and is associated with lupus susceptibility, either through an expansion of the b1a cells, a reduced IL-2 production, or both.