Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity.

BACKGROUND: As the primary immune response cell in the central nervous system, microglia constantly monitor the microenvironment and respond rapidly to stress, infection, and injury, making them important modulators of neuroinflammatory responses. In diseases such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, and human immunodeficiency virus-induced dementia, activation of microglia precedes astrogliosis and overt neuronal loss. Although microgliosis is implicated in manganese (Mn) neurotoxicity, the role of microglia and glial crosstalk in Mn-induced neurodegeneration is poorly understood. METHODS: Experiments utilized immunopurified murine microglia and astrocytes using column-free magnetic separation. The effect of Mn on microglia was investigated using gene expression analysis, Mn uptake measurements, protein production, and changes in morphology. Additionally, gene expression analysis was used to determine the effect Mn-treated microglia had on inflammatory responses in Mn-exposed astrocytes. RESULTS: Immunofluorescence and flow cytometric analysis of immunopurified microglia and astrocytes indicated cultures were 97 and 90% pure, respectively. Mn treatment in microglia resulted in a dose-dependent increase in pro-inflammatory gene expression, transition to a mixed M1/M2 phenotype, and a de-ramified morphology. Conditioned media from Mn-exposed microglia (MCM) dramatically enhanced expression of mRNA for Tnf, Il-1ß, Il-6, Ccl2, and Ccl5 in astrocytes, as did exposure to Mn in the presence of co-cultured microglia. MCM had increased levels of cytokines and chemokines including IL-6, TNF, CCL2, and CCL5. Pharmacological inhibition of NF-κB in microglia using Bay 11-7082 completely blocked microglial-induced astrocyte activation, whereas siRNA knockdown of Tnf in primary microglia only partially inhibited neuroinflammatory responses in astrocytes. CONCLUSIONS: These results provide evidence that NF-κB signaling in microglia plays an essential role in inflammatory responses in Mn toxicity by regulating cytokines and chemokines that amplify the activation of astrocytes.

Threshold of toxicological concern (TTC) for developmental and reproductive toxicity of anticancer compounds.

Pharmaceutical companies develop specialized therapies to treat late stage cancer. In order to accelerate life-saving treatments and reduce animal testing, compounds to treat life-threatening malignancies are allowed modified requirements for preclinical toxicology testing. Limited data packages in early drug development can present product quality challenges at multi-product manufacturing facilities. The present analysis established an endpoint-specific threshold of toxicological concern (TTC) for developmental and reproductive toxicity (DART) for anticancer compounds. A comprehensive database was created consisting of over 300 no-observed adverse effect levels (NOAELs) for DART of 108 anticancer compounds. The 5th percentile NOAEL for developmental and reproductive toxicity was 0.005 mg/kg/day (300 µg/day), resulting in a human exposure threshold of 3 µg/day assuming standard uncertainty factors and a 60 kg human bodyweight. The analysis shows this threshold is protective for developmental and reproductive toxicity of highly potent groups of anticancer compounds. There were similar TTC values calculated for direct-acting and indirect-acting anticancer compounds.

Trends and Clinical Outcomes of Fungal Keratitis in Canada: A 20-year Retrospective Multicentre Study.

PURPOSE: An increase in fungal and particularly filamentous keratitis has been observed in many geographic areas, mostly in contact lens wearers. This study seeks to characterize long-term trends in fungal keratitis in a continental climate area to provide guidance for diagnosis and treatment. DESIGN: Retrospective multicentric case series. METHODS: Cases of microbiology-confirmed fungal keratitis from 2003 to 2022 presenting to tertiary care centers across Canada were included. Charts were reviewed for patient demographics, risk factors, visual acuity, and treatments undertaken. RESULTS: A total of 138 patients were identified: 75 had yeast keratitis while 63 had filamentous keratitis. Patients with yeast keratitis had more ocular surface disease (79% vs 28%) while patients with filamentous keratitis wore more refractive contact lenses (78% vs 19%). Candida species accounted for 96% of all yeast identified, while Aspergillus (32%) and Fusarium (26%) were the most common filamentous fungi species. The mean duration of treatment was 81 ± 96 days. Patients with yeast keratitis did not have significantly improved visual acuity with medical treatment (1.8 ± 1 LogMAR to 1.9 ± 1.5 LogMAR, P = .9980), in contrast to patients with filamentous keratitis (1.4 ± 1.2 LogMAR to 1.1 ± 1.3 LogMAR, P = .0093). CONCLUSIONS: Fungal keratitis is increasing in incidence, with contact lenses emerging as one of the leading risk factors. Significant differences in the risk factors and visual outcomes exist between yeast keratitis and filamentous keratitis which may guide diagnosis and treatment.

Perception of visual outcomes in patients after delayed compared with immediately sequential bilateral cataract surgery.

PURPOSE: To evaluate patient-reported visual outcomes of immediately sequential bilateral cataract surgery (ISBCS) compared with delayed sequential bilateral cataract surgery (DSBCS). SETTING: Tertiary university teaching hospital of Laval University, Quebec City, Canada. DESIGN: Prospective observational cohort study. METHODS: The Catquest-9SF questionnaire was administered on the day of surgery for ISBCS patients and on the day of first-eye surgery for DSBCS patients who underwent cataract surgery between August and September 2021. The questionnaire was administered again 1 month postoperatively for ISBCS patients and 1 month postoperatively after each surgery for DSBCS patients. RESULTS: 186 patients (ISBCS: n = 152 vs DSBCS: n = 34) were included. At 1 month postoperatively, the Catquest-9SF score of ISBCS patients was significantly lower than that of DSBCS patients after first-eye surgery ( P < .001). Furthermore, the ISBCS group achieved significantly better scores on multiple tasks of the Catquest-9SF, such as reading text in the newspaper ( P < .001) or reading text on television ( P < .001). In multiple linear regression analysis, the type of surgery was the factor most associated with a lower Catquest-9SF score (ß = -0.391, P < .001). 1 month after the second-eye surgery, DSBCS patients had achieved similar Catquest-9SF scores compared with ISBCS patients. CONCLUSIONS: Compared with DSBCS patients, ISBCS patients had significantly greater perceived visual function and fewer vision-related limitations in their daily activities at 1 month postoperatively. This difference lost significance 1 month after the second-eye surgery of DSBCS patients. This patient-reported outcome study did not find evidence of perceived bilateral visual impairment in the early postoperative period after ISBCS.

Perception of Canadian ophthalmologists on immediately sequential bilateral cataract surgery: insights and implications.

OBJECTIVE: To evaluate the perception of immediately sequential bilateral cataract surgery (ISBCS) among Canadian ophthalmologists. DESIGN: An anonymous survey was sent to all active members of the Canadian Ophthalmological Society. METHODS: Basic demographic information, cataract surgery practice patterns, and perceived advantages, disadvantages, and concerns regarding ISBCS were collected from respondents. RESULTS: A total of 352 ophthalmologists answered the survey. Among these, 94 respondents (27%) practice ISBCS routinely, 123 (35%) practice ISBCS in exceptional cases, and 131 (37%) do not practice ISBCS. ISBCS practitioners were significantly younger than nonpractitioners (p < 0.001) and had a shorter duration of practice (p < 0.001). The prevalence of ISBCS practitioners also varied significantly by province (p < 0.001): most practitioners who routinely practice ISBCS were from Quebec (n = 44; 48%), where financial disincentives are lowest in the country. The main work setting of ISBCS practitioners was academic centres (n = 39; 42%) as opposed to private or community settings (p < 0.001). The main reason for performing ISBCS was more efficient operating theatre use (n = 142; 65%). The main concerns regarding ISBCS were the risk of bilateral complications (n = 193; 57%) and the lack of refractive outcomes for second-eye surgery (n = 184; 52%). The COVID-19 pandemic positively influenced the view of 152 respondents (43%), but this was mostly among practitioners who already performed ISBCS routinely (n = 77; 84%). CONCLUSIONS: ISBCS practitioners are more likely younger ophthalmologists working in academic centres. Quebec has the highest prevalence of ISBCS practitioners. ISBCS practitioners were positively influenced by the COVID-19 pandemic to offer ISBCS more often compared with non-ISBCS practitioners.

Intraocular methotrexate for epithelial downgrowth: long-term outcomes in a multicentre case series.

BACKGROUND/AIMS: Sheet-like type of epithelial downgrowth (EDG) is not easily amenable to surgical excision. We describe long-term outcomes in patients with EDG treated with intraocular methotrexate (MTX). METHODS: This is a retrospective, multicentric case series including 10 eyes (nine patients) treated with intraocular MTX for sheet-like EDG. Relevant ocular history, previous EDG treatments, MTX injection regimen, long-term outcomes and complications are reported. RESULTS: All cases were associated with intraocular surgery. Most patients were treated with 400 µm/0.1 mL MTX injections with a starting frequency of two times per week or weekly injections. Mean and SD number of injections per eye was 16±13 injections and duration of follow-up was 54±36 months (range: 7-120 months). Eradication of EDG was achieved in seven eyes of which one required a second MTX treatment course to achieve eradication, while clinical resolution with recurrence was observed in two. One treatment failure occurred despite eight weekly injections which slowed but did not halt EDG progression; the patient later requested that treatments be stopped given difficulty to come to follow-ups. Surface epitheliopathy developed in eight patients and was used to titrate MTX treatment. Six patients also developed endothelial failure. CONCLUSION: We report the largest case series of diffuse, sheet-like EDG treated with intraocular MTX with follow-ups up to 10 years. Intraocular MTX may be used effectively to achieve eradication of EDG in cases where surgery is not amenable. However, further recommendations to guide treatment remain warranted.

Conjunctival Infiltration in a Child as a Rare Manifestation of IgG4-Related Disease.

PURPOSE: Ocular manifestations of immunoglobulin G4 (IgG4)-related disease are common in children although remain ill-defined because of the disease's rarity. We describe a pediatric case of IgG4-related orbital disease (IgG4-ROD) who presented with persistent conjunctival infiltration before developing lacrimal gland enlargement 3 years later. METHODS: This was a case report. RESULTS: An 8-year-old girl developed forniceal salmon-patch-like conjunctival lesions in her left eye that were refractory to topical corticosteroids. Investigations, including an orbital MRI and 2 conjunctival biopsies, were negative for lymphoma. She was treated with topical corticosteroids and then nonsteroidal antiinflammatory drops. The lesions decreased mildly, and no new lesion emerged. After 3 years, the patient developed a ptosis, new salmon-patch conjunctival lesions, and papillae. Vision deteriorated to 20/80 because of severe punctate epithelial erosions in the left eye, and the Schirmer test was significantly reduced. A repeat MRI revealed an enlarged left lacrimal gland. A biopsy was performed and was compatible with IgG4-ROD. An elevated IgG4 level of 4.61 g/L was also found. The patient was successfully treated with oral prednisone but flared on tapering the dosage. Rituximab was therefore initiated with excellent clinical response, and prednisone was discontinued. Vision returned to 20/20 after aggressive lubrification, punctal plugs, and autologous serum eye drops. Tear function came back to normal, and local treatments were stopped. CONCLUSIONS: This case describes a pediatric case of IgG4-ROD presenting initially with conjunctival follicular reaction, later developing lacrimal gland involvement. Therefore, it is important to consider IgG4-ROD in chronic atypical follicular conjunctival lesions in children, even in the absence of orbital disease.

Immediately sequential bilateral cataract surgery: an academic teaching center's experience.

PURPOSE: To evaluate the safety and outcomes of immediately sequential bilateral cataract surgery (ISBCS) at a Canadian academic teaching center. SETTING: Tertiary university teaching hospital of Laval University, Quebec City, Canada. DESIGN: Retrospective cohort study. METHODS: 2003 consecutive patients (4006 eyes) who underwent ISBCS under topical anesthesia from January 2019 to December 2019 were included. All charts were retrospectively reviewed. Outcome measures included intraoperative and postoperative complications, postoperative uncorrected distance (UCVA) and pinhole (PHVA) visual acuities, and autorefraction measurements. RESULTS: 4006 eyes from 1218 (60.8%) female and 785 (39.2%) male patients with a mean age of 74 ± 8 years had a mean preoperative visual acuity of 0.503 logMAR (Snellen 20/63). The mean axial length was 23.53 ± 1.37 mm. Most eyes had monofocal intraocular lenses (IOLs) implanted (n = 3738, 93.3%) followed by toric (n = 226, 5.6%), multifocal (n = 25, 0.6%), and multifocal toric (n = 17, 0.4%) IOLs. Intraoperative complications included 14 (0.3%) posterior capsule ruptures with 5 (0.1%) requiring sulcus IOL placement, and 7 (0.2%) partial zonulysis, with 3 requiring capsular tension rings (0.07%). There were no cases of endophthalmitis or toxic anterior segment syndrome. Mean 5-week postoperative UCVA was 0.223 (Snellen 20/33), PHVA was 0.153 (Snellen 20/28) with a mean spherical equivalent of -0.21 diopters. CONCLUSIONS: ISBCS performed following International Society of Bilateral Cataract Surgeons recommended guidelines is a safe procedure. This cohort of 4006 eyes had very few complications, with none attributable to the surgery being done bilaterally. The UCVA, PHVA, and refractive outcomes were good.

Intracorneal ring segments implantation followed by same-day photorefractive keratectomy and corneal collagen cross-linking in keratoconus.

PURPOSE: To evaluate the efficacy of intrastromal corneal ring segments implantation followed by same-day photorefractive keratectomy (PRK) and ultraviolet-A/riboflavin collagen cross-linking (CXL) in patients with keratoconus. METHODS: Four patients (five eyes) were included in the study. All patients first underwent femtosecond laser-enabled placement of intracorneal ring segments (Intacs, Addition Technology). Uncorrected (UDVA) and corrected distance visual acuity (CDVA) and keratometry readings remained stable for 6 months. Same-day PRK and CXL were subsequently performed in all patients. RESULTS: Six months after Intacs plus PRK/CXL, significant improvements were noted for UDVA, CDVA, spherical equivalent refraction, keratometry, and total aberrations. No patient lost lines of CDVA or developed haze. CONCLUSIONS: The combination of intracorneal ring segments implantation followed by sequential same-day PRK/CXL may be a reasonable option for improving visual acuity in select patients with keratoconus.

A retrospective study of adverse effects of mycophenolate mofetil administration to dogs with immune-mediated disease.

BACKGROUND: Information regarding adverse events (AEs) of mycophenolate mofetil (MMF) is limited. OBJECTIVES: To evaluate the types and frequency of potential AEs of MMF in dogs with immune-mediated disease. ANIMALS: One hundred thirty-one dogs treated with MMF for management of suspected immune-mediated disease. METHODS: Retrospective study. Medical records were reviewed to find and group suspect AEs in gastrointestinal (GI), hematologic, and other categories. Age, dosage, body weight, and sex were analyzed between dogs with and without AEs by using the Mann-Whitney U-test and chi-squared test. RESULTS: The median starting dosage of MMF was 17.5 mg/kg/day (interquartile range [IQR] = 15.1-20.6 mg/kg/day) and the median treatment duration was 56 days (IQR = 14-236 days). Mycophenolate mofetil was prescribed for immune-mediated hemolytic anemia (n = 31), immune-mediated thrombocytopenia (n = 31), pemphigus foliaceus (n = 15), immune-mediated polyarthritis (n = 12), and others (n = 42). Overall, potential AEs of MMF were observed in 34 of 131 dogs (GI 24.4% [31/127], neutropenia 4% [3/76], anemia 4% [1/25], thrombocytopenia 4.0% [1/25], and dermatologic 1.5% [2/131]). There were no significant differences among dogs with (n = 37) or without potential AEs (n = 94) in regards to sex, age, body weight, or dosage of MMF (P = .06, .13, .24, and .26, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: In the dogs administered MMF, GI AEs were most common. Since potential hematologic and dermatologic AEs developed in a few dogs, clinicians should be aware of these when prescribing MMF to dogs with immune-mediated disease.

Analysis of targeted mutation in DJ-1 on cellular function in primary astrocytes.

DJ-1 mutation induces early-onset Parkinson's disease, and conversely over-expression of DJ-1 is associated with cancer in numerous tissues. A gene-trap screening library conducted in embryonic stem cells was utilized for generation of a DJ-1 mutant mouse. Real-time PCR and immunoblotting were utilized to confirm functional mutation of the DJ-1 gene. Normal DJ-1 protein expression in adult mouse tissue was characterized and demonstrates high expression in brain tissue with wide systemic distribution. Primary astrocytes isolated from DJ-1(-/-) mice reveal a decreased nuclear localization of DJ-1 protein in response to rotenone or LPS, with a concomitant increase in mitochondrial localization of DJ-1 found only in the rotenone exposure. Resting mitochondrial membrane potential was significantly lower in DJ-1(-/-) astrocytes, as compared to controls. Our DJ-1 knockout mouse provides an exciting tool for exploring the molecular and physiological roles of DJ-1 to further explicate its functions in neurodegeneration.

Hemolytic-uremic syndrome in a postpartum mare concurrent with encephalopathy in the neonatal foal.

A postpartum mare and foal were presented for evaluation of fever and lethargy in the mare. The mare was diagnosed with endometritis and initially responded well to treatment. On the second day of hospitalization, the mare developed renal insufficiency characterized by oliguria, azotemia, hemolysis, and thrombocytopenia. Concurrently, the foal developed rapidly progressive central nervous system signs culminating in refractory seizures. Both animals failed to respond to treatment and were euthanized. Thrombotic microangiopathy involving glomeruli was evident on microscopic examination of the mare's kidneys. Microscopic evidence of brain edema was the principal postmortem finding in the foal. No specific etiology was confirmed in either case. Notably, Escherichia coli 0103:H2 was isolated from the mare's uterus and the gastrointestinal tracts of both animals. To the authors' knowledge, this is the first report in which an organism implicated as a cause of hemolytic-uremic syndrome was isolated from an animal with clinical signs and postmortem findings consistent with the disease.

A novel diindolylmethane analog, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane, inhibits the tumor necrosis factor-induced inflammatory response in primary murine synovial fibroblasts through a Nurr1-dependent mechanism.

The progression of rheumatoid arthritis involves the thickening of the synovial lining due to the proliferation of fibroblast-like synoviocytes (FLS) and infiltration by inflammatory cells. Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine involved in progression of the disease. Under rheumatoid conditions, FLS express the tumor necrosis factor (TNF)-recognition complex (TNFR1, TNFR2, VCAM-1 and ICAM-1), which induces local macrophage activation and leads to downstream nuclear factor κB (NF-κB) signaling. The NF-κB-regulated inflammatory gene, cyclooxygenase (COX), increases synthesis of prostaglandins that contribute to the propagation of inflammatory damage within the joint. Because the nuclear orphan receptor, NR4A2 (Nurr1), can negatively regulate NF-κB-dependent inflammatory gene expression in macrophages, we postulated that activation of this receptor by the Nurr1 ligand 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) would modulate inflammatory gene expression in synovial fibroblasts by inhibiting NF-κB. Treatment with C-DIM12 suppressed TNFα-induced expression of adhesion molecules and NF-κB regulated genes in primary synovial fibroblasts including vascular adhesion molecule 1 (VCAM-1), PGE2 and COX-2. Immunofluorescence studies indicated that C-DIM12 did not prevent translocation of p65 and stabilized nuclear localization of Nurr1 in synovial fibroblasts. Knockdown of Nurr1 expression by RNA interference prevented the inhibitory effects of C-DIM12 on inflammatory gene expression, indicating that the anti-inflammatory effects of this compound are Nurr1-dependent. Collectively, these data suggest that this receptor may be a viable therapeutic target in RA.

The aryl hydrocarbon receptor agonist 3,3',4,4',5-pentachlorobiphenyl induces distinct patterns of gene expression between hepatoma and glioma cells: chromatin remodeling as a mechanism for selective effects.

Genome-wide oligonucleotide DNA microarrays and real time RT-PCR were used to assess differential gene expression in rat glioma and hepatoma cell lines after exposure to the aryl hydrocarbon receptor (AhR) agonist 3,3',4,4',5-pentachlorobiphenyl (penta-CB). Under maximal inducing concentrations for cytochrome P450 1A1 (CYP1A1) in H4IIE rat hepatoma cells, both H4IIE and C6 rat glioma cells were exposed to sub-micromolar concentrations of penta-CB for 24h. Differential gene expression for approximately 28,000 gene probes were computationally analyzed and compared. As expected, penta-CB potently activated CYP1A1/2 transcription in liver-derived H4IIE hepatoma cells yet did not do so in brain-derived C6 glioma cells. Additionally, we show that penta-CB causes: (1) distinct patterns of gene expression between tumor cells derived from liver or brain; (2) robust transcriptional activation of select C6 glioma gene ontologies; (3) over-expression of H4IIE hepatoma genes associated with tumor progression in liver; (4) greater than 100-fold over-expression of C6 glioma genes associated with protein processing and programmed cell death and/or metastasis; (5) tissue-selective histone deacetylase inhibition in C6 glioma, but not H4IIE hepatoma cells as signaled by galectin-1 over-expression.

Manganese-induced neurotoxicity: the role of astroglial-derived nitric oxide in striatal interneuron degeneration.

Chronic exposure to excessive manganese (Mn) is the cause of a neurodegenerative movement disorder, termed manganism, resulting from degeneration of neurons within the basal ganglia. Pathogenic mechanisms underlying this disorder are not fully understood but involve inflammatory activation of glial cells within the basal ganglia. It was postulated in the present studies that reactive astrocytes are involved in neuronal injury from exposure to Mn through increased release of nitric oxide. C57Bl/6 mice subchronically exposed to Mn by intragastric gavage had increased levels of Mn in the striatum and displayed diminutions in both locomotor activity and striatal DA content. Mn exposure resulted in neuronal injury in the striatum and globus pallidus, particularly in regions proximal to the microvasculature, indicated by histochemical staining with fluorojade and cresyl fast violet. Neuropathological assessment revealed marked perivascular edema, with hypertrophic endothelial cells and diffusion of serum albumin into the perivascular space. Immunofluorescence studies employing terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (DUTP)-biotin nick-end labeling revealed the presence of apoptotic neurons expressing neuronal nitric oxide synthase (NOS), choline acetyltransferase, and enkephalin in both the striatum and globus pallidus. In contrast, soma and terminals of dopaminergic neurons were morphologically unaltered in either the substantia nigra or striatum, as indicated by immunohistochemical staining for tyrosine hydroxylase. Regions with evident neuronal injury also displayed increased numbers of reactive astrocytes that coexpressed inducible NOS2 and localized with areas of increased neuronal staining for 3-nitrotyrosine protein adducts, a marker of NO formation. These data suggest a role for astrocyte-derived NO in injury to striatal-pallidal interneurons from Mn intoxication.

Outcomes of photorefractive keratectomy following laser in situ keratomileusis: a cohort study.

OBJECTIVE: To analyze the outcomes of photorefractive keratectomy (PRK) on residual myopia and hyperopia post-laser in situ keratomileusis (LASIK) and to compare these results with PRK on eyes without previous laser refractive surgery. DESIGN: Retrospective comparative cohort study. PARTICIPANTS: Patients undergoing PRK between 2006 and 2010 were reviewed. METHODS: Patients were divided into 4 groups, myopic or hyperopic PRK post-LASIK (mPRK-PL and hPRK-PL, respectively) and myopic or hyperopic PRK on corneas without previous laser refractive surgery (mPRK and hPRK, respectively). Uncorrected and corrected distance visual acuity, mean refractive spherical equivalent (MRSE), and mean keratometry and aberrations (total, higher order [HOA], coma, trefoil, and spherical aberration) were recorded at months 3 and 6 postoperatively, as were complications and attempted versus achieved MRSE. RESULTS: Thirty-three eyes of 25 patients who underwent PRK post-LASIK (21 eyes of 14 patients for hPRK-PL and 12 eyes of 11 patients for mPRK-PL) and 35 eyes of 21 patients who underwent PRK on virgin eyes (11 eyes of 8 patients for hPRK and 24 eyes of 13 patients for mPRK) were included in the study. The only significant differences in outcomes were found to be HOA at 3 months for hPRK-PL as compared with both hPRK and mPRK. Achieved MRSE was significantly different from expected MRSE for hPRK-PL at 3 months postoperatively. No haze- or flap-related complications were observed. CONCLUSION: Outcomes of PRK were not different in myopic and hyperopic corrections post-LASIK by 6 months or when compared with PRK in virgin eyes. HOA may render hPRK-PL results less predictable early in the postoperative period.

Immune Modulation as an Effective Adjunct Post-exposure Therapeutic for B. pseudomallei.

Melioidosis is caused by the facultative intracellular bacterium Burkholderia pseudomallei and is potentially fatal. Despite a growing global burden and high fatality rate, little is known about the disease. Recent studies demonstrate that cyclooxygenase-2 (COX-2) inhibition is an effective post-exposure therapeutic for pulmonary melioidosis, which works by inhibiting the production of prostaglandin E2 (PGE2). This treatment, while effective, was conducted using an experimental COX-2 inhibitor that is not approved for human or animal use. Therefore, an alternative COX-2 inhibitor needs to be identified for further studies. Tolfenamic acid (TA) is a non-steroidal anti-inflammatory drug (NSAID) COX-2 inhibitor marketed outside of the United States for the treatment of migraines. While this drug was developed for COX-2 inhibition, it has been found to modulate other aspects of inflammation as well. In this study, we used RAW 264.7 cells infected with B pseudomallei to analyze the effect of TA on cell survival, PGE2 production and regulation of COX-2 and nuclear factor- kappaB (NF-ĸB) protein expression. To evaluate the effectiveness of post-exposure treatment with TA, results were compared to Ceftazidime (CZ) treatments alone and the co-treatment of TA with a sub-therapeutic treatment of CZ determined in a study of BALB/c mice. Results revealed an increase in cell viability in vitro with TA and were able to reduce both COX-2 expression and PGE2 production while also decreasing NF-ĸB activation during infection. Co-treatment of orally administered TA and a sub-therapeutic treatment of CZ significantly increased survival outcome and cleared the bacterial load within organ tissue. Additionally, we demonstrated that post-exposure TA treatment with sub-therapeutic CZ is effective to treat melioidosis in BALB/c mice.