Development and characterisation of mgTHP-1, a novel in vitro model for neural macrophages with microglial characteristics.

Neuroinflammation is primarily characterised by activation of the brain's resident macrophages - the microglia. However, other central nervous system (CNS) cells also contribute to this response, including the astrocytes and endothelial cells. In addition, there is infiltration into the CNS of peripherally derived immune cells. Together these cells mediate inflammation by the production of cytokines, chemokines, reactive oxygen species, and secondary messengers, and enacting of the appropriate response to those signals. However, deciphering the specific contributions of each cell type has been challenging. Studying CNS cell biology is often challenging, as the isolation of primary cells is not always feasible, and differentiation towards microglia-like cells is complex. Here, we demonstrate a novel method whereby THP-1 monocytic cells are differentiated into neural macrophage cells with microglia-like cell characteristics. The cells, designated mgTHP-1, show typical morphological and gene expression patterns of resident CNS macrophages and functionally respond to inflammatory stimuli by producing inflammatory cytokines. Furthermore, with the addition of Vicenin-2 (an anti-inflammatory flavonoid) such responses can be reversed. This novel cell model will allow further investigations, and hence insights, into the neuroinflammatory mechanisms associated with CNS diseases.

Oral Lipid Nanocrystal Amphotericin B for Cryptococcal Meningitis: A Randomized Clinical Trial.

BACKGROUND: Amphotericin B is the gold standard treatment for severe mycoses. A new orally delivered, less-toxic formulation of amphotericin has been developed. METHODS: In our randomized clinical trial, we tested oral lipid nanocrystal (LNC) amphotericin B (MAT2203, Matinas Biopharma) vs intravenous (IV) amphotericin for human immunodeficiency virus-associated cryptococcal meningitis in 4 sequential cohorts. Two pilot cohorts assessed safety and tolerability (n = 10 each), and 2 cohorts assessed efficacy with/without 2 IV loading doses (n = 40 each). The experimental arm received 1.8 g/d oral LNC amphotericin through 2 weeks with 100 mg/kg/d flucytosine, then 1.2 g/d LNC amphotericin through 6 weeks. The randomized control arm (n = 41) received 7 days of IV amphotericin with flucytosine, then 7 days of fluconazole 1200 mg/d. The primary end point was cerebrospinal fluid (CSF) early fungicidal activity (EFA). RESULTS: We randomized 80 participants to oral LNC amphotericin + flucytosine with (n = 40) and without (n = 40) 2 IV loading doses and 41 control participants to IV amphotericin + flucytosine. Mean EFA was 0.40 log10 colony-forming units (CFU)/mL/d for all-oral LNC amphotericin, 0.42 log10  Cryptococcus CFU/mL/d for oral LNC amphotericin with IV loading doses, and 0.46 log10 CFU/mL/d for IV amphotericin controls. LNC amphotericin groups achieved 2-week CSF sterility in 63% (44 of 70) vs 68% (23 of 34) of controls. The 18-week survival was 85% (34 of 40) with all-oral LNC amphotericin, 90% (36 of 40) with oral LNC amphotericin given IV loading doses, and 85% (35 of 41) with IV amphotericin.Grade 3-4 laboratory adverse events occurred less frequently in LNC amphotericin groups (41%) than the IV amphotericin group (61%, P = .05), particularly for anemia (21% vs 44%; P = .01) and potassium (5% vs 17%; P = .04). CONCLUSIONS: This new oral amphotericin B LNC formulation appears promising for cryptococcal meningitis with antifungal activity, similar survival, and less toxicity than IV amphotericin. CLINICAL TRIALS REGISTRATION: NCT04031833.

Outpatient Cryptococcal Antigen Screening Is Associated With Favorable Baseline Characteristics and Improved Survival in Persons With Cryptococcal Meningitis in Uganda.

BACKGROUND: It is unknown whether persons with symptomatic cryptococcal meningitis detected during routine blood cryptococcal antigen (CrAg) screening have better survival than persons presenting with overt meningitis. METHODS: We prospectively enrolled Ugandans with HIV and cryptocococcal meningitis from December 2018 to December 2021. Participants were treated with amphotericin-based combination therapy. We compared outcomes between persons who were CrAg screened then referred to hospital with those presenting directly to the hospital with symptomatic meningitis. RESULTS: Among 489 participants with cryptococcal meningitis, 40% (194/489) received blood CrAg screening and were referred to hospital (median time to referral 2 days; interquartile range [IQR], 1-6). CrAg-screened persons referred to hospital had lower 14-day mortality than non-CrAg-screened persons who presented directly to hospital with symptomatic meningitis (12% vs 21%; hazard ratio, .51; 95% confidence interval, .32-.83; P = .006). Fewer CrAg-screened participants had altered mental status versus non-CrAg-screened participants (29% vs 41%; P = .03). CrAg-screened persons had lower quantitative cerebrospinal fluid (CSF) culture burden (median [IQR], 4570 [11-100 000] vs 26 900 [182-324 000] CFU/mL; P = .01) and lower CSF opening pressures (median [IQR], 190 [120-270] vs 225 [140-340] mmH2O; P = .004) compared with non-CrAg-screened persons. CONCLUSIONS: Survival from cryptococcal meningitis was higher in persons with prior CrAg screening than those without CrAg screening. Altered mental status was the most potent predictor for mortality in a multivariate model. We suggest that CrAg screening detects cryptococcal meningitis at an earlier stage, as evidenced by a favorable baseline risk profile and notably fewer persons with altered mental status.

Distal jejunal obstruction due to Cryptococcus neoformans and rifampicin-resistant Mycobacterium tuberculosis co-infection: A case report.

Jejunal obstruction secondary to Cryptococcus neoformans and rifampicin-resistant Mycobacterium tuberculosis co-infection in HIV is not previously reported. This is a case of a 30-year-old HIV-positive male with severe headaches, a positive cerebrospinal fluid cryptococcal antigen assay, and elevated intracranial pressure requiring serial lumbar punctures and opioids. He developed constipation and abdominal distension, had partial jejunectomy and histopathology revealed Cryptococcus yeasts and caseous granulomas with Mycobacterium tuberculosis (TB). Post-operatively, rifampicin-resistant TB was detected in urine.

Evaluation of the initial 12 months of a routine cryptococcal antigen screening program in reduction of HIV-associated cryptococcal meningitis in Uganda.

BACKGROUND: Asymptomatic Cryptococcal Antigenemia (CrAg) patients develop meningitis within a month of testing positive. Pre-emptive antifungal therapy can prevent progression to Cryptococcal meningitis (CM). In April 2016, a national CrAg screening program was initiated in 206 high-volume health facilities that provide antiretroviral therapy in Uganda. We report the evaluation of the CrAg screening cascade focusing on linkage to care, fluconazole therapy for 10 weeks and 6 months follow up, and ART initiation in a subset of facilities. METHODS: We conducted a retrospective, cross-sectional survey of patients with CD4 < 100 at seven urban and seven rural facilities after 1 year of program implementation. We quantified the number of patients who transitioned through the steps of the CrAg screening cascade over six-months follow-up. We defined cascade completion as a pre-emptive fluconazole prescription for the first 10 weeks. We conducted semi-structured interviews with lab personnel and clinic staff to assess functionality of the CrAg screening program. Data was collected using REDCap. RESULTS: We evaluated 359 patient records between April 2016 to March 2017; the majority (358/359, 99.7%) were from government owned health facilities and just over half (193/359, 53.8%) had a median baseline CD4 cell count of < 50 cell/µL. Overall, CrAg screening had been performed in 255/359 (71.0, 95% CI, 66.0-75.7) of patients' records reviewed, with a higher proportion among urban facilities (170/209 (81.3, 95% CI, 75.4-86.4)) than rural facilities (85/150 (56.7, 95% CI, 48.3-64.7)). Among those who were CrAg screened, 56/255 (22.0, 95% CI, 17.0-27.5%) had cryptococcal antigenemia, of whom 47/56 (83.9, 95% CI, 71.7-92.4%) were initiated on pre-emptive therapy with fluconazole and 8/47 (17.0, 95% CI, 7.6-30.8%) of these were still receiving antifungal therapy at 6 months follow up. At least one CNS symptom was present in 70% (39/56) of those with antigenemia. In patients who had started ART, almost 40% initiated ART prior to CrAg screening. Inadequacy of equipment/supplies was reported by 15/26 (58%) of personnel as a program barrier, while 13/26 (50%) reported a need for training about CM and CrAg screening. CONCLUSION: There was a critical gap in the follow-up of patients after initiation on fluconazole therapy. ART had been initiated in almost 40% of patients prior to CrAg screening.. Higher antigenemia patients presenting with CNS symptoms could be related to late presentation. There is need to address these gaps after a more thorough evaluation.

Determinants of cryptococcal antigen (CrAg) screening uptake in Kampala, Uganda: An assessment of health center characteristics.

Cryptococcal antigen (CrAg) screening and pre-emptive antifungal therapy for people with CD4 cell counts <100 cells/µl are recommended by the World Health Organization and several national HIV guidelines. We sought to evaluate CrAg screening program implementation across Uganda, in relation to health center level and distance from the capital. We conducted a cross-sectional study of 22 health centers across southern Uganda from April to June 2019. We reviewed laboratory records regarding number of CD4 cell count tests performed, proportion of outpatients with CD4 counts <200 cells/µl, and number of CrAg screening tests performed. We administered surveys to health center staff to understand barriers to advanced HIV care. We observed no significant difference in health center level and performance of CrAg screening; with each subsequent health center level, there was 1.17-fold (95% CI: 0.92-1.41) higher odds of CrAg screening performed per level. CrAg screening uptake was not associated with distance from the capital city (odds ratio = 0.96, 95% CI: 0.89-1.04). Qualitative data from surveys indicated that limitations to uptake of CrAg screening were secondary to dysfunctional CD4 machines, lack of provider awareness of CrAg screening guidelines, and inadequate/intermittent supply of CrAg tests. There were no significant associations between CrAg screening uptake and level of health center or distance of health center from the capital city. We identified systemic barriers to CrAg screening related to inadequate CD4 testing, insufficient knowledge regarding national screening guidelines, and irregular laboratory testing supplies. LAY SUMMARY: The objective of this study was to evaluate cryptococcal antigen (CrAg) screening program implementation in Uganda, by type of healthcare center and by distance from the capital city. CrAg screening uptake was not associated with distance from the capital city, or the type of healthcare center.

Single-Dose Liposomal Amphotericin B Treatment for Cryptococcal Meningitis.

BACKGROUND: Cryptococcal meningitis is a leading cause of human immunodeficiency virus (HIV)-related death in sub-Saharan Africa. Whether a treatment regimen that includes a single high dose of liposomal amphotericin B would be efficacious is not known. METHODS: In this phase 3 randomized, controlled, noninferiority trial conducted in five African countries, we assigned HIV-positive adults with cryptococcal meningitis in a 1:1 ratio to receive either a single high dose of liposomal amphotericin B (10 mg per kilogram of body weight) on day 1 plus 14 days of flucytosine (100 mg per kilogram per day) and fluconazole (1200 mg per day) or the current World Health Organization-recommended treatment, which includes amphotericin B deoxycholate (1 mg per kilogram per day) plus flucytosine (100 mg per kilogram per day) for 7 days, followed by fluconazole (1200 mg per day) for 7 days (control). The primary end point was death from any cause at 10 weeks; the trial was powered to show noninferiority at a 10-percentage-point margin. RESULTS: A total of 844 participants underwent randomization; 814 were included in the intention-to-treat population. At 10 weeks, deaths were reported in 101 participants (24.8%; 95% confidence interval [CI], 20.7 to 29.3) in the liposomal amphotericin B group and 117 (28.7%; 95% CI, 24.4 to 33.4) in the control group (difference, -3.9 percentage points); the upper boundary of the one-sided 95% confidence interval was 1.2 percentage points (within the noninferiority margin; P<0.001 for noninferiority). Fungal clearance from cerebrospinal fluid was -0.40 log10 colony-forming units (CFU) per milliliter per day in the liposomal amphotericin B group and -0.42 log10 CFU per milliliter per day in the control group. Fewer participants had grade 3 or 4 adverse events in the liposomal amphotericin B group than in the control group (50.0% vs. 62.3%). CONCLUSIONS: Single-dose liposomal amphotericin B combined with flucytosine and fluconazole was noninferior to the WHO-recommended treatment for HIV-associated cryptococcal meningitis and was associated with fewer adverse events. (Funded by the European and Developing Countries Clinical Trials Partnership and others; Ambition ISRCTN number, ISRCTN72509687.).

Evaluation of the Dynamiker Cryptococcal Antigen Lateral Flow Assay for the Diagnosis of HIV-Associated Cryptococcosis.

Cryptococcal meningitis is a leading cause of meningitis in sub-Saharan Africa. Given the need for rapid point-of-care testing, we evaluated the diagnostic performance of the Dynamiker cryptococcal antigen (CrAg) lateral flow assay (LFA). We assessed the diagnostic performance of the Dynamiker CrAg LFA compared to the IMMY CrAg LFA as the reference standard. We tested 150 serum, 115 plasma, and 100 cerebrospinal fluid (CSF) samples from HIV patients with symptomatic meningitis and 113 serum samples from patients with suspected asymptomatic cryptococcal antigenemia. Compared to the IMMY CrAg LFA, sensitivity of Dynamiker CrAg LFA was 98% in serum, 100% in plasma, 100% in CSF from symptomatic patients and 96% in serum from asymptomatic patients. Specificity was 66% in serum, 61% in plasma, and 91% in CSF from symptomatic patients, and 86% in serum from asymptomatic patients. The positive predictive value was 85% in serum, 82% in plasma, and 96% in CSF from symptomatic patients, and 69% in serum from asymptomatic patients. The negative predictive value was 94% in serum, 100% in plasma, and 100% in CSF from symptomatic patients, and 99% in serum from asymptomatic patients. The interassay reproducibility was 100% across the four sample types with no observed discordant results when Dynamiker CrAg LFA was tested in duplicate. However, a high number of false positives were observed on serum of symptomatic patients (11%), serum of asymptomatic patients (11%) and plasma of symptomatic patients (14%). The Dynamiker CrAg LFA had excellent sensitivity but poor specificity, particularly when tested on serum and plasma.

Cerebrospinal Fluid Bacillary Load by Xpert MTB/RIF Ultra Polymerase Chain Reaction Cycle Threshold Value Predicts 2-Week Mortality in Human Immunodeficiency Virus-Associated Tuberculous Meningitis.

BACKGROUND: The World Health Organization recommends GeneXpert MTB/RIF Ultra (Xpert Ultra), a fully automated polymerase chain reaction (PCR) assay, as the initial tuberculous meningitis (TBM) diagnostic test. The assay's PCR cycle threshold (Ct) values represent the number of PCR cycles required for probe signal to be detected (low Ct value = high bacillary load) and may approximate tuberculosis (TB) bacillary load. We measured the relationship between cerebrospinal fluid (CSF) TB bacillary load with mortality. METHODS: We prospectively enrolled 102 human immunodeficiency virus (HIV)-positive Ugandans with probable or definite TBM from April 2015 to August 2019. Xpert Ultra Ct tertiles and semi-quantitative categories were separately analyzed as predictors of 2-week mortality. We investigated associations between Ct and baseline clinical and CSF parameters. RESULTS: Subjects with Ct values in the low tertile (ie, high bacillary load) had 57% 2-week mortality-worse than the intermediate (17%) and high (25%) Ct tertiles and Xpert Ultra-negative (30%) probable TBM cases (P = .01). In contrast, the reported semi-quantitative Xpert Ultra categorization was less precise; with the medium to low category trending toward worse 2-week survival (42%) compared with very low (28%), trace (26%), and negative (30%) categories (P = .48). Ct tertile was significantly associated with baseline CSF lactate (P = .03). CONCLUSIONS: High CSF TB bacillary load, as measured by Xpert Ultra Ct tertile, is associated with an almost 2-fold higher 2-week mortality in HIV-associated TBM and is a better predictor than the reported Xpert Ultra semi-quantitative category. Xpert Ultra Ct values could identify TBM patients at increased risk of death who may benefit from enhanced supportive care.

Adjunctive sertraline for asymptomatic cryptococcal antigenemia: A randomized clinical trial.

Cryptococcal antigen (CrAg) screening in HIV-infected persons with CD4 < 100 cells/µl can reduce meningitis and death, yet preemptive fluconazole therapy fails in ∼25%. Sertraline has in vitro and in vivo activity against Cryptococcus and is synergistic with fluconazole in mice. We evaluated the efficacy and safety of sertraline in asymptomatic cryptococcal antigenemia. We conducted a randomized trial of asymptomatic CrAg-positive Ugandans from November 2017 to February 2018. All subjects received WHO standard therapy of fluconazole 800 mg for 2 weeks, then 400 mg for 10 weeks, then 200 mg through 24 weeks. Participants were randomized to receive adjunctive sertraline or placebo, given in once-weekly escalating 100 mg/day doses up to 400 mg/day, which was then given for 8 weeks, then tapered. The primary endpoint was meningitis-free 6-month survival. The data and safety monitoring board halted the trial after 21 subjects were enrolled due to safety concerns. Meningitis-free 6-month survival occurred in 9 of 11 of placebo participants and 10 of 10 of sertraline participants. However, seven serious adverse events (SAEs) occurred (n = 4 sertraline group; n = 3 placebo group). Three SAEs in the sertraline group presented with psychosis and aggressive behavioral changes with one meeting Hunter's criteria for serotonin syndrome while receiving 200 mg/day sertraline. Two transient psychoses were associated with antecedent fluconazole and sertraline interruption. The serotonin syndrome resolved within 1 day, but psychosis persisted for 4 months after sertraline discontinuation. Sertraline was associated with excess SAEs of psychosis. Due to early stopping, we were unable to determine any efficacy for cryptococcal antigenemia.

The effect of the acute inflammatory response of burns and its treatment on clot characteristics and quality: A prospective case controlled study.

INTRODUCTION: Burns are known to have an effect on coagulation in the early period after burn. Current coagulation tests have been criticised in acute burns due to their inherent limitations. This study aims to investigate the potential for a new quantitative functional biomarker of clot quality, fractal dimension, to identify changes in clot microstructure as a result of the burn inflammatory response and its treatment. METHODS: A total of fifty-eight burn patients were included in this prospective case-controlled study. The control group (29 patients mean TBSA 1%), and case group (29 patients mean TBSA 30%) were compared at baseline and the case group investigated further over four time points (baseline, 12h, 24h and 5-7 days). Fractal analysis was performed, as well as current markers of coagulation, inflammatory markers and point-of-care tests, Thromboelastography and Multiplate analysis. RESULTS: Fractal dimension did not differ between groups at admission (1.73±0.06 and 1.72±0.1), and fell within the healthy index normal range (1.74±0.7), suggesting a normal clot microstructure in the early period after burn. Fractal dimension significantly reduced from baseline over the first 24h following injury (1.59±0.03 p<0.005), indicating a significant reduction in mechanical clot strength and functionality consistent with a hypocoagulable state, not identified with other markers. CONCLUSIONS: This is the first study to quantify the changes in clot microstructure following burn injury. This study confirms clot microstructure is significantly altered during the first 24h after burn, with the production of a weaker, more porous fibrin clot, consistent with a hypocoagulable state.

Prognostic value of circulating tumour cells in limited-stage small-cell lung cancer: analysis of the concurrent once-daily versus twice-daily radiotherapy (CONVERT) randomised controlled trial.

BACKGROUND: The clinical significance of circulating tumour cells (CTCs) in limited-stage small-cell lung cancer (LS-SCLC) is not well defined. We report a planned exploratory analysis of the prevalence and prognostic value of CTCs in LS-SCLC patients enrolled within the phase III randomised CONVERT (concurrent once-daily versus twice-daily chemoradiotherapy) trial. PATIENTS AND METHODS: Baseline blood samples were enumerated for CTCs using CellSearch in 75 patients with LS-SCLC who were enrolled in the CONVERT trial and randomised between twice- and once-daily concurrent chemoradiation. Standard statistical methods were used for correlations of CTCs with clinical factors. Log-rank test and Cox regression analyses were applied to establish the associations of 2, 15 and 50 CTC thresholds with progression-free survival (PFS) and overall survival (OS). An optimal CTC count threshold for LS-SCLC was established. RESULTS: CTCs were detected in 60% (45/75) of patients (range 0-3750). CTC count thresholds of 2, 15 and 50 CTCs all significantly correlate with PFS and OS. An optimal CTC count threshold in LS-SCLC was established at 15 CTCs, defining 'favourable' and 'unfavourable' prognostic risk groups. The median OS in <15 versus ≥15 CTCs was 26.7 versus 5.9 m (P = 0.001). The presence of ≥15 CTCs at baseline independently predicted ≤1 year survival in 70% and ≤2 years survival in 100% of patients. CONCLUSION: We report the prognostic value of baseline CTC count in an exclusive LS-SCLC population at thresholds of 2, 15 and 50 CTCs. Specific to LS-SCLC, ≥15 CTCs was associated with worse PFS and OS independent of all other factors and predicted ≤2 years survival. These results may improve disease stratification in future clinical trial designs and aid clinical decision making. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT00433563.

Bone turnover markers in relation to vitamin D status and disease activity in adults with systemic lupus erythematosus.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have altered bone metabolism and are at risk of osteoporosis. The aim of this study was to examine bone turnover markers in relation to vitamin D, disease activity, and clinical risk factors in patients with established SLE. METHODS: Clinical registry and biorepository data of 42 SLE patients were assessed. Serum samples were analyzed for osteocalcin as a marker of bone formation, C-terminal telopeptide of type 1 collagen (CTX) as a marker for bone resorption, and 25-hydroxy vitamin D. RESULTS: Patients with a Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI) score of 3 or greater had a lower median osteocalcin level ( P = 0.02) and lower 25-hydroxy vitamin D levels ( P = 0.03) than those with a score of less than 3. No significant differences in bone turnover markers were observed between patients dichotomized into subgroups using a 25-hydroxy vitamin D cut-off of 30 ng/mL or by a daily prednisone dose greater than or 5 mg or less. Osteocalcin levels were negatively correlated with SLEDAI scores ( P = 0.034), and were positively correlated with the CTX index (a ratio of measured CTX value to the upper limit of the normal value for age and gender) ( P < 0.01). No association between the CTX index and SLEDAI scores was found. CONCLUSION: SLE disease activity may have direct effects on bone formation, but no effects on bone resorption in this cohort of established SLE patients, probably related to the inflammation-suppressing effects of glucocorticoids, thereby inhibiting cytokine-induced osteoclast activity. A fine balance exists between disease control and the use of glucocorticoids with regard to bone health.

Persistent histone modifications at the BDNF and Cdk-5 promoters following extinction of nicotine-seeking in rats.

Drugs of addiction lead to a wide range of epigenetic changes at the promoter regions of genes directly implicated in learning and memory processes. We have previously shown that the histone deactylase inhibitor, sodium butyrate (NaB), accelerates the extinction of nicotine-seeking and provides resistance to relapse. Here, we explore the potential molecular mechanisms underlying this effect. Rats received intravenous nicotine or saline self-administration, followed by 6 days of extinction training, with each extinction session followed immediately by treatment with NaB or vehicle. On the last day of extinction, rats were killed and the medial ventral prefrontal cortex retained for chromatin immunoprecipitation and quantitative polymerase chain reaction (qPCR). A history of nicotine exposure significantly decreased H3K14 acetylation at the brain-derived neurotrophic factor (BDNF) exon IV promoter, and this effect was abolished with NaB treatment. In contrast, nicotine self-administration alone, resulted in a significant decrease in histone methylation at the H3K27me3 and H3K9me2 marks in the promoter regions of BDNF exon IV and cyclin-dependent kinase 5 (Cdk-5). Quantitative PCR-identified changes in several genes associated with NaB treatment that were independent of nicotine exposure; however, an interaction of nicotine history and NaB treatment was detected only in the expression of BDNF IV and BDNF IX. Together these results suggest that nicotine self-administration leads to a number of epigenetic changes at both the BDNF and Cdk-5 promoters, and that these changes may contribute to the enhanced extinction of nicotine-seeking by NaB.

Characterisation of the porcine cytokines which activate the CD131ßc common sub-unit, for potential immune-augmentation.

Early acting cytokines and growth factors such as those of the CD131 ßc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.

Use of maxillofacial laboratory materials to construct a tissue-equivalent head phantom with removable titanium implantable devices for use in verification of the dose of intensity-modulated radiotherapy.

The dose of radiotherapy is often verified by measuring the dose of radiation at specific points within a phantom. The presence of high-density implant materials such as titanium, however, may cause complications both during calculation and delivery of the dose. Numerous studies have reported photon/electron backscatter and alteration of the dose by high-density implants, but we know of no evidence of a dosimetry phantom that incorporates high density implants or fixtures. The aim of the study was to design and manufacture a tissue-equivalent head phantom for use in verification of the dose in radiotherapy using a combination of traditional laboratory materials and techniques and 3-dimensional technology that can incorporate titanium maxillofacial devices. Digital designs were used together with Mimics® 18.0 (Materialise NV) and FreeForm® software. DICOM data were downloaded and manipulated into the final pieces of the phantom mould. Three-dimensional digital objects were converted into STL files and exported for additional stereolithography. Phantoms were constructed in four stages: material testing and selection, design of a 3-dimensional mould, manufacture of implants, and final fabrication of the phantom using traditional laboratory techniques. Three tissue-equivalent materials were found and used to successfully manufacture a suitable phantom with interchangeable sections that contained three versions of titanium maxillofacial implants. Maxillofacial and other materials can be used to successfully construct a head phantom with interchangeable titanium implant sections for use in verification of doses of radiotherapy.

Evaluation of the novel artus C. difficile QS-RGQ, VanR QS-RGQ and MRSA/SA QS-RGQ assays for the laboratory diagnosis of Clostridium difficile infection (CDI), and for vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) screening.

Clostridium difficile, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) are worldwide prevalent healthcare-associated pathogens. We have evaluated three Qiagen artus QS-RGQ assays for the detection of these pathogens. We examined 200 stool samples previously tested for C. difficile infection (CDI), 94 rectal swabs previously screened for VRE and 200 MRSA screening nasal swabs. With the routine diagnostic laboratory results being adopted as the gold standard, the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the artus C. difficile assay were 100%, for the artus VanR QS-RGQ assay, 95, 68, 44 and 98%, and for the artus MRSA/SA assay, 80, 94, 93 and 83%, respectively. The artus VanR assay detected the vanA and/or vanB genes in 32% of culture-negative VRE screens; in 71% of these cases, only vanB was detected. An over-estimation of the rate of faecal VRE colonisation could be due to a patient population with high rates of faecal carriage of non-enterococcal species carrying vanB. Based on our findings, we conclude that all three artus QS-RGQ assays could be a useful addition to a diagnostic laboratory, and that the optimal choice of assay should be determined according to user needs.

Safety and effectiveness of a single and repeat intramuscular injection of a GnRH vaccine (GonaCon™) in adult female domestic cats.

Sterilization is a key strategy to reduce the number of domestic cats entering and killed in shelters each year. However, surgical sterilization is expensive and labour-intensive and cannot fully address the 70 million free-roaming cats estimated to exist in the United States. GonaCon™ is a gonadotropin-releasing hormone vaccine originally developed for use as a wildlife immunocontraceptive. An earlier formulation was tested in domestic cats and found to be safe and effective for long-term contraception. However, the current Environmental Protection Agency (EPA)-registered formulation consists of a different antigen-carrier protein and increased antigen concentration and has never been tested in cats. A pilot study was undertaken to evaluate the short-term safety of a single GonaCon immunization, assess the consequences of vaccinated cats receiving an accidental second GonaCon injection and determine the humoral immune response to immunization. During Phase 1, cats in Group A (n = 3) received a single intramuscular injection of GonaCon and Group B (n = 3) received a single intramuscular injection of saline. During Phase 2, Group A received a second GonaCon injection and Group B received their initial GonaCon injection. All cats developed GnRH antibodies within 30 days of vaccine administration. The endpoint titre (1:1,024,000) was similar among all cats, and levels remained high throughout the duration of the study. Four cats developed a sterile, painless, self-limiting mass at the site of injection. The mean number of days to mass development was 110.3 (range, 18-249 days). In conclusion, this preliminary study suggests that the EPA-registered GonaCon formulation is safe for continued testing in domestic cats, an accidental revaccination should not increase the risk of a vaccine reaction and the EPA-registered formulation effectively elicits a strong humoral immune response.

Treatment of aplastic anaemia with lower-dose anti-thymocyte globulin produces similar response rates and survival as per standard dose anti-thymocyte globulin schedules.

BACKGROUND: Aplastic anaemia (AA) is a rare acquired bone marrow failure syndrome resulting from the immune-mediated destruction of haemopoietic stem cells. For adults in whom first-line haemopoietic progenitor cell transplantation is not feasible, combination anti-thymocyte globulin (ATGAM) plus cyclosporine A is standard therapy; however, there are minimal data available regarding the optimal ATGAM dosage in terms of efficacy and survival. AIMS: Our institutions have historically used different dosing protocols of ATGAM in the treatment of AA. We aimed to review the outcome of AA patients treated with these protocols and compare them to the published literature. METHODS: We conducted a retrospective study of 31 adults who received first-line ATGAM for AA and compared response rates and survival between cohorts who received standard (40 mg/kg/day D1-4) versus lower-dose (15 mg/kg/day D1-5) ATGAM schedules. RESULTS: There were similar rates of response (64 vs 71%, P = 1.0), relapse (33 vs 33%, P = 1.0), transformation (14 vs 24%, P = 0.66) or infection (43 vs 47%, P = 1.0), respectively, between standard and lower-dose cohorts. At a median follow up of 24 months, there was no statistical difference between standard and lower-dose cohorts in either event-free (42.2 vs 64.7%, P = 0.91) or overall survival (73.1 vs 88.2%, P = 0.75). CONCLUSION: Our experience suggests that lower-dose ATGAM at 15 mg/kg/day D1-5 as treatment of AA produces similar responses and outcomes as per standard-dose ATGAM schedules. Prospective trials comparing ATGAM dose schedules in AA are warranted.