Digital Pathology Identifies Associations between Tissue Inflammatory Biomarkers and Multiple Sclerosis Outcomes.

BACKGROUND: Multiple sclerosis (MS) is a clinically heterogeneous disease underpinned by inflammatory, demyelinating and neurodegenerative processes, the extent of which varies between individuals and over the course of the disease. Recognising the clinicopathological features that most strongly associate with disease outcomes will inform future efforts at patient phenotyping. AIMS: We used a digital pathology workflow, involving high-resolution image acquisition of immunostained slides and opensource software for quantification, to investigate the relationship between clinical and neuropathological features in an autopsy cohort of progressive MS. METHODS: Sequential sections of frontal, cingulate and occipital cortex, thalamus, brain stem (pons) and cerebellum including dentate nucleus (n = 35 progressive MS, females = 28, males = 7; age died = 53.5 years; range 38-98 years) were immunostained for myelin (anti-MOG), neurons (anti-HuC/D) and microglia/macrophages (anti-HLA). The extent of demyelination, neurodegeneration, the presence of active and/or chronic active lesions and quantification of brain and leptomeningeal inflammation was captured by digital pathology. RESULTS: Digital analysis of tissue sections revealed the variable extent of pathology that characterises progressive MS. Microglia/macrophage activation, if found at a higher level in a single block, was typically elevated across all sampled blocks. Compartmentalised (perivascular/leptomeningeal) inflammation was associated with age-related measures of disease severity and an earlier death. CONCLUSION: Digital pathology identified prognostically important clinicopathological correlations in MS. This methodology can be used to prioritise the principal pathological processes that need to be captured by future MS biomarkers.

Metabolic profiling in serum, cerebrospinal fluid, and brain of patients with cerebrotendinous xanthomatosis.

Cerebrotendinous xanthomatosis (CTX) is caused by autosomal recessive loss-of-function mutations in CYP27A1, a gene encoding cytochrome p450 oxidase essential for bile acid synthesis, resulting in altered bile acid and lipid metabolism. Here, we aimed to identify metabolic aberrations that drive ongoing neurodegeneration in some patients with CTX despite chenodeoxycholic acid (CDCA) supplementation, the standard treatment in CTX. Using chromatographic separation techniques coupled to mass spectrometry, we analyzed 26 sterol metabolites in serum and cerebrospinal fluid (CSF) of patients with CTX and in one CTX brain. Comparing samples of drug naive patients to patients treated with CDCA and healthy controls, we identified 7α,12α-dihydroxycholest-4-en-3-one as the most prominently elevated metabolite in serum and CSF of drug naive patients. CDCA treatment substantially reduced or even normalized levels of all metabolites increased in untreated patients with CTX. Independent of CDCA treatment, metabolites of the 27-hydroxylation pathway were nearly absent in all patients with CTX. 27-hydroxylated metabolites accounted for ∼45% of total free sterol content in CSF of healthy controls but <2% in patients with CTX. Metabolic changes in brain tissue corresponded well with findings in CSF. Interestingly, 7α,12α-dihydroxycholest-4-en-3-one and 5α-cholestanol did not exert toxicity in neuronal cell culture. In conclusion, we propose that increased 7α,12α-dihydroxycholest-4-en-3-one and lack of 27-hydroxycholesterol may be highly sensitive metabolic biomarkers of CTX. As CDCA cannot reliably prevent disease progression despite reduction of most accumulated metabolites, supplementation of 27-hydroxylated bile acid intermediates or replacement of CYP27A1 might be required to counter neurodegeneration in patients with progressive disease despite CDCA treatment.

Deep mining of oxysterols and cholestenoic acids in human plasma and cerebrospinal fluid: Quantification using isotope dilution mass spectrometry.

Both plasma and cerebrospinal fluid (CSF) are rich in cholesterol and its metabolites. Here we describe in detail a methodology for the identification and quantification of multiple sterols including oxysterols and sterol-acids found in these fluids. The method is translatable to any laboratory with access to liquid chromatography - tandem mass spectrometry. The method exploits isotope-dilution mass spectrometry for absolute quantification of target metabolites. The method is applicable for semi-quantification of other sterols for which isotope labelled surrogates are not available and approximate quantification of partially identified sterols. Values are reported for non-esterified sterols in the absence of saponification and total sterols following saponification. In this way absolute quantification data is reported for 17 sterols in the NIST SRM 1950 plasma along with semi-quantitative data for 8 additional sterols and approximate quantification for one further sterol. In a pooled (CSF) sample used for internal quality control, absolute quantification was performed on 10 sterols, semi-quantification on 9 sterols and approximate quantification on a further three partially identified sterols. The value of the method is illustrated by confirming the sterol phenotype of a patient suffering from ACOX2 deficiency, a rare disorder of bile acid biosynthesis, and in a plasma sample from a patient suffering from cerebrotendinous xanthomatosis, where cholesterol 27-hydroxylase is deficient.

Meningeal inflammation and cortical demyelination in acute multiple sclerosis.

OBJECTIVE: Cortical gray matter (GM) pathology, involving demyelination and neurodegeneration, associated with meningeal inflammation, could be important in determining disability progression in multiple sclerosis (MS). However, we need to know more about how cortical demyelination, neurodegeneration, and meningeal inflammation contribute to pathology at early stages of MS to better predict long-term outcome. METHODS: Tissue blocks from short disease duration MS (n = 12, median disease duration = 2 years), progressive MS (n = 21, disease duration = 25 years), non-diseased controls (n = 11), and other neurological inflammatory disease controls (n = 6) were quantitatively analyzed by immunohistochemistry, immunofluorescence, and in situ hybridization. RESULTS: Cortical GM demyelination was extensive in some cases of acute MS (range = 1-48% of total cortical GM), and subpial lesions were the most common type (62%). The numbers of activated (CD68+ ) microglia/macrophages were increased in cases with subpial lesions, and the density of neurons was significantly reduced in acute MS normal appearing and lesion GM, compared to controls (p < 0.005). Significant meningeal inflammation and lymphoid-like structures were seen in 4 of 12 acute MS cases. The extent of meningeal inflammation correlated with microglial/macrophage activation (p < 0.05), but not the area of cortical demyelination, reflecting the finding that lymphoid-like structures were seen adjacent to GM lesions as well as areas of partially demyelinated/remyelinated, cortical GM. INTERPRETATION: Our findings demonstrate that cortical demyelination, neuronal loss, and meningeal inflammation are notable pathological hallmarks of acute MS and support the need to identify early biomarkers of this pathology to better predict outcome. Ann Neurol 2018;84:829-842.

Working in a medicalised world: the experiences of palliative care nurse specialists and midwives.

AIM: This paper reports a study that explored birth and death care experiences from the perspectives of midwives and palliative care clinical nurse specialists (CNSs). METHODS: A grounded theory approach illuminated the social processes associated with caring during birth and death. Data was collected during 2011 through in-depth semi-structured interviews with eight participants: four midwives and four CNSs from UK NHS Trusts. FINDINGS: Four categories were identified through thematic analysis: memories, one act, advocate, and medical event. These integrated to create a core category: working to counter the pathologisation of birth and death. CONCLUSION: The participants illustrate the impact of medicalisation on the experience of birth and death and struggle with the negative effects of this. Health professionals should work toward practice that emphasises the importance of holistic care.