Gas chromatography coupled with ion trap mass spectrometry (GC-MS and GC-MS-MS) - for arson debris analysis

Gas chromatography - mass spectrometry (GC-MS) is an established instrumental technique used for the analysis of fire debris for accelerant detection. However, matrix problems, such as pyrolysis product interference, are still encountered. These interferences often lead to inconclusive interpretation of the chromatographic results. This study describes methods for analysing arson accelerants using gas chromatography coupled with ion trap mass spectrometry. The latter technique lends itself to both conventional (GC-MS) as well as tandem mass spectrometry (GC-MS-MS). Since petrol (gasoline) is one of the more common distillate blends used by arsonists, especially in South Africa, the identification of petrol in fire debris samples was investigated. In order to overcome pyrolysis product interference and improve detection selectivity of the aromatic hydrocarbons in petrol residues, tandem mass spectrometry was used in combination with capillary gas chromatography. The added parameter of the third dimension of selectivity proved to be superior to conventional GC-MS in obtaining characteristic aromatic hydrocarbon profiles for petrol without interference from pyrolysis artefacts.Dissertation (MSc (Applied Chemistry))--University of Pretoria, 2007.Chemistryunrestricte

Design and characterization of Squalene-Gusperimus nanoparticles for modulation of innate immunity

Immunosuppressive drugs are widely used for the treatment of autoimmune diseases and to prevent rejection in organ transplantation. Gusperimus is a relatively safe immunosuppressive drug with low cytotoxicity and reversible side effects. It is highly hydrophilic and unstable. Therefore, it requires administration in high doses which increases its side effects. To overcome this, here we encapsulated gusperimus as squalene-gusperimus nanoparticles (Sq-GusNPs). These nanoparticles (NPs) were obtained from nanoassembly of the squalene gusperimus (Sq-Gus) bioconjugate in water, which was synthesized starting from squalene. The size, charge, and dispersity of the Sq-GusNPs were optimized using the response surface methodology (RSM). The colloidal stability of the Sq-GusNPs was tested using an experimental block design at different storage temperatures after preparing them at different pH conditions. Sq-GusNPs showed to be colloidally stable, non-cytotoxic, readily taken up by cells, and with an anti-inflammatory effect sustained over time. We demonstrate that gusperimus was stabilized through its conjugation with squalene and subsequent formation of NPs allowing its controlled release. Overall, the Sq-GusNPs have the potential to be used as an alternative in approaches for the treatment of different pathologies where a controlled release of gusperimus could be required

A survey of diseases among 100 free-ranging baboons (Papio ursinus) from the Kruger National Park

The pathological and parasitological findings from 100 free-ranging chacma baboons are described. One of the most striking discoveries was a heretofore unknown coccidian parasite, Isospora papionis, and even more important than its presence in the small intestine was its occurrence in skeletal muscles. Serological and histopathological evidence of toxoplasmosis was found in several animals. Other previously unknown parasites encountered included two mites- Rhinophaga elongata, an unusually long mite that produced small granulomas in the nasal mucosae, and Pneumonyssus vocalis, a mite found in the laryngeal ventricles. A new species of filarid, Tetrapetalonema papionis was found in the subcutis and intermuscular fascia. New records for the chacma baboon of known parasites were R. papionis, a mite found only in the maxillary recess, where it stimulated a polyplike growth, and, in the skeletal muscles, cysticerci of Taenia crocutae, a tapeworm of hyenas (Crocuta crocuta and Hyaena brunnea). Apart from the pathological changes associated with the above parasites, another important finding was numerous cases of "capture myopathy", a syndrome that resembles Meyer-Betz disease of man. One of the most severe diseases encountered was pulmonary acariasis (P. mossambicensis), which at times caused large foci of supportive pneumonia and diffuse pleuritis. The mite pigment was also found in draining lymph nodes. The most serious diseases of the liver were cytomegaly, which was similar to that produced by mycotoxins in other animal species, multiple granulomatous foci caused by Hepatocystis simiae and microgranulomas caused by ova of Schistosoma mattheei. Adult schistosomes were also found in the mesenteric vessels. The most important lesion in the central nervous system was an axonal hamartoma, which was found in two cases and involved a large portion of the brain stem. Also of note were a meningioma in the falx cerebelli, a few examples of non-suppurativc encephalitis and several cases in which neurons in the medulla oblongata had been replaced by a globular eosinophilic mass. Other tumours found were a fibroma in the subcutis of the face and a basal cell carcinoma in the skin on the back. Both were of local importance only. Developmental anomalies included an accessory spleen, ectopic pancreatic tissue in the duodenum, thymic tissue embedded in the thyroid and parathyroid and microcysts in the thymus, parathyroid and adenohypophysis. Arteriosclerosis of limited severity was found in the aorta and coronary and renal arteries of many of the older baboons (males and females). Another vascular change related to previous pregnancy was sclerosis of the ovarian and uterine vessels. Degenerative changes were found in the central arteries of germinal follicles in various lymph nodes and the spleen. Other noteworthy findings included the presence of spargana in the skeletal muscles; ranula formation of the ducts of the glands of Ebner; para-ovarian crystals; large intranuclear inclusions in the submandibular salivary gland compatible with those produced by cytomegalovirus and intranuclear inclusions in the epididymis. Various gastro-intestinal parasites were found and their corresponding lesions are described. Selected bacterial studies for shigellae and salmonellae were negative, as were intradermal tests for tuberculosis and serological tests for leptospirosis and brucellosis. The brain, heart, spleen, liver, lungs and kidneys were mass measured and were compared to the body mass. In all age groups the heart varied the least when expressed as percent body mass. The brain was the most variable in this regard but changed the least in total mass.The articles have been scanned in colour with a HP Scanjet 5590; 300dpi. Adobe Acrobat XI Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format

In Vitro Studies of Squalene-Gusperimus Nanoparticles in Islet-Containing Alginate Microcapsules to Regulate the Immune Response in the Immediate Posttransplant Period

Grafting of microencapsulated pancreatic islets has been proposed as an alternative to exogenous insulin for the treatment of type 1 diabetes mellitus. Microencapsulated islets are protected from direct contact with immune cells and larger immune‐active molecules such as immunoglobulins. Unfortunately, many islet cells in the microcapsules are lost in the immediate period after transplantation due to an early host immune response limiting long‐term function of the graft. Gusperimus has shown to reduce the inflammatory responses to grafted encapsulated islets, but it cannot be appropriately used because it is easily hydrolyzed leading to loss of activity. To temporarily modulate the inflammatory response directly after implantation and stabilize gusperimus, squalene‐gusperimus nanoparticles (Sq‐GusNPs) are developed and incorporated in human islets‐containing alginate‐based microcapsules. A prolonged and continuous release of gusperimus is achieved. This offers an anti‐inflammatory microenvironment in the vicinity of the microcapsules and a reduction of cytokine secretion by lipopolysaccharides‐activated human macrophages. Release of gusperimus from Sq‐GusNPs does not affect the in vitro viability or function of human pancreatic islets. The data illustrate that incorporation of Sq‐GusNPs in alginate microcapsules offers an opportunity to temporarily modulate the immediate immune response after the grafting procedure of encapsulated islets cells and reduce loss of islet cells

In vitro determination of the immunosuppressive effect, internalization, and release mechanism of squalene-gusperimus nanoparticles for managing inflammatory responses

Gusperimus is an anti-inflammatory drug that has shown to be effective in managing autoimmunity and preventing graft rejection. This is unstable and easily broken down into cytotoxic components. We encapsulated gusperimus binding it covalently to squalene obtaining squalene-gusperimus nanoparticles (Sq-GusNPs). These nanoparticles enhanced the immunosuppressive effect of gusperimus in both mouse macrophages and T cells. The half-maximal inhibitory concentration in macrophages was 9-fold lower for Sq-GusNPs compared with the free drug. The anti-inflammatory effect of the Sq-GusNPs was maintained over time without cytotoxicity. By studying nanoparticles uptake by cells with flow cytometry, we demonstrated that Sq-GusNPs are endocytosed by macrophages after binding to low-density lipoprotein receptors (LDLR). In presence of cathepsin B or D release of gusperimus is increased demonstrating the participation of proteases in the release process. Our approach may allow the application of Sq-GusNPs for effective management of inflammatory disorders including autoimmunity and graft rejection

CSF Proteomic Alzheimer's Disease-Predictive Subtypes in Cognitively Intact Amyloid Negative Individuals.

We recently discovered three distinct pathophysiological subtypes in Alzheimer's disease (AD) using cerebrospinal fluid (CSF) proteomics: one with neuronal hyperplasticity, a second with innate immune system activation, and a third subtype with blood-brain barrier dysfunction. It remains unclear whether AD proteomic subtype profiles are a consequence of amyloid aggregation, or might exist upstream from aggregated amyloid. We studied this question in 127 older individuals with intact cognition and normal AD biomarkers in two independent cohorts (EMIF-AD MBD and ADNI). We clustered 705 proteins measured in CSF that were previously related to AD. We identified in these cognitively intact individuals without AD pathology three subtypes: two subtypes were seen in both cohorts (n = 49 with neuronal hyperplasticity and n = 44 with blood-brain barrier dysfunction), and one only in ADNI (n = 12 with innate immune activation). The proteins specific for these subtypes strongly overlapped with AD subtype protein profiles (overlap coefficients 92%-71%). Longitudinal p <sub>181</sub> -tau and amyloid β 1-42 (Aβ42) CSF analysis showed that in the hyperplasticity subtype p <sub>181</sub> -tau increased (β = 2.6 pg/mL per year, p = 0.01) and Aβ42 decreased over time (β = -4.4 pg/mL per year, p = 0.03), in the innate immune activation subtype p <sub>181</sub> -tau increased (β = 3.1 pg/mL per year, p = 0.01) while in the blood-brain barrier dysfunction subtype Aβ42 decreased (β = -3.7 pg/mL per year, p = 0.009). These findings suggest that AD proteomic subtypes might already manifest in cognitively normal individuals and may predispose for AD before amyloid has reached abnormal levels

Effects of age, amyloid, sex, and APOE ε4 on the CSF proteome in normal cognition

Introduction: It is important to understand which biological processes change with aging, and how such changes are associated with increased Alzheimer's disease (AD) risk. We studied how cerebrospinal fluid (CSF) proteomics changed with age and tested if associations depended on amyloid status, sex, and apolipoprotein E Ɛ4 genotype. Methods: We included 277 cognitively intact individuals aged 46 to 89 years from Alzheimer's Disease Neuroimaging Initiative, European Medical Information Framework for Alzheimer's Disease Multimodal Biomarker Discovery, and Metabolic Syndrome in Men. In total, 1149 proteins were measured with liquid chromatography mass spectrometry with multiple reaction monitoring/Rules-Based Medicine, tandem mass tag mass spectrometry, and SOMAscan. We tested associations between age and protein levels in linear models and tested enrichment for Reactome pathways. Results: Levels of 252 proteins increased with age independently of amyloid status. These proteins were associated with immune and signaling processes. Levels of 21 proteins decreased with older age exclusively in amyloid abnormal participants and these were enriched for extracellular matrix organization. Discussion: We found amyloid-independent and -dependent CSF proteome changes with older age, perhaps representing physiological aging and early AD pathology

Pathophysiological subtypes of Alzheimer's disease based on cerebrospinal fluid proteomics.

Alzheimer's disease is biologically heterogeneous, and detailed understanding of the processes involved in patients is critical for development of treatments. CSF contains hundreds of proteins, with concentrations reflecting ongoing (patho)physiological processes. This provides the opportunity to study many biological processes at the same time in patients. We studied whether Alzheimer's disease biological subtypes can be detected in CSF proteomics using the dual clustering technique non-negative matrix factorization. In two independent cohorts (EMIF-AD MBD and ADNI) we found that 705 (77% of 911 tested) proteins differed between Alzheimer's disease (defined as having abnormal amyloid, n = 425) and controls (defined as having normal CSF amyloid and tau and normal cognition, n = 127). Using these proteins for data-driven clustering, we identified three robust pathophysiological Alzheimer's disease subtypes within each cohort showing (i) hyperplasticity and increased BACE1 levels; (ii) innate immune activation; and (iii) blood-brain barrier dysfunction with low BACE1 levels. In both cohorts, the majority of individuals were labelled as having subtype 1 (80, 36% in EMIF-AD MBD; 117, 59% in ADNI), 71 (32%) in EMIF-AD MBD and 41 (21%) in ADNI were labelled as subtype 2, and 72 (32%) in EMIF-AD MBD and 39 (20%) individuals in ADNI were labelled as subtype 3. Genetic analyses showed that all subtypes had an excess of genetic risk for Alzheimer's disease (all P > 0.01). Additional pathological comparisons that were available for a subset in ADNI suggested that subtypes showed similar severity of Alzheimer's disease pathology, and did not differ in the frequencies of co-pathologies, providing further support that found subtypes truly reflect Alzheimer's disease heterogeneity. Compared to controls, all non-demented Alzheimer's disease individuals had increased risk of showing clinical progression (all P < 0.01). Compared to subtype 1, subtype 2 showed faster clinical progression after correcting for age, sex, level of education and tau levels (hazard ratio = 2.5; 95% confidence interval = 1.2, 5.1; P = 0.01), and subtype 3 at trend level (hazard ratio = 2.1; 95% confidence interval = 1.0, 4.4; P = 0.06). Together, these results demonstrate the value of CSF proteomics in studying the biological heterogeneity in Alzheimer's disease patients, and suggest that subtypes may require tailored therapy

Cerebrospinal fluid tau levels are associated with abnormal neuronal plasticity markers in Alzheimer's disease

BACKGROUND: Increased total tau (t-tau) in cerebrospinal fluid (CSF) is a key characteristic of Alzheimer's disease (AD) and is considered to result from neurodegeneration. T-tau levels, however, can be increased in very early disease stages, when neurodegeneration is limited, and can be normal in advanced disease stages. This suggests that t-tau levels may be driven by other mechanisms as well. Because tau pathophysiology is emerging as treatment target for AD, we aimed to clarify molecular processes associated with CSF t-tau levels. METHODS: We performed a proteomic, genomic, and imaging study in 1380 individuals with AD, in the preclinical, prodromal, and mild dementia stage, and 380 controls from the Alzheimer's Disease Neuroimaging Initiative and EMIF-AD Multimodality Biomarker Discovery study. RESULTS: We found that, relative to controls, AD individuals with increased t-tau had increased CSF concentrations of over 400 proteins enriched for neuronal plasticity processes. In contrast, AD individuals with normal t-tau had decreased levels of these plasticity proteins and showed increased concentrations of proteins indicative of blood-brain barrier and blood-CSF barrier dysfunction, relative to controls. The distinct proteomic profiles were already present in the preclinical AD stage and persisted in prodromal and dementia stages implying that they reflect disease traits rather than disease states. Dysregulated plasticity proteins were associated with SUZ12 and REST signaling, suggesting aberrant gene repression. GWAS analyses contrasting AD individuals with and without increased t-tau highlighted several genes involved in the regulation of gene expression. Targeted analyses of SNP rs9877502 in GMNC, associated with t-tau levels previously, correlated in individuals with AD with CSF concentrations of 591 plasticity associated proteins. The number of APOE-e4 alleles, however, was not associated with the concentration of plasticity related proteins. CONCLUSIONS: CSF t-tau levels in AD are associated with altered levels of proteins involved in neuronal plasticity and blood-brain and blood-CSF barrier dysfunction. Future trials may need to stratify on CSF t-tau status, as AD individuals with increased t-tau and normal t-tau are likely to respond differently to treatment, given their opposite CSF proteomic profiles