MINocyclinE to Reduce inflammation and blood-brain barrier leakage in small Vessel diseAse (MINERVA): A phase II, randomized, double-blind, placebo-controlled experimental medicine trial.

INTRODUCTION: Cerebral small vessel disease (SVD) is a common cause of stroke/vascular dementia with few effective treatments. Neuroinflammation and increased blood-brain barrier (BBB) permeability may influence pathogenesis. In rodent models, minocycline reduced inflammation/BBB permeability. We determined whether minocycline had a similar effect in patients with SVD. METHODS: MINERVA was a single-center, phase II, randomized, double-blind, placebo-controlled trial. Forty-four participants with moderate-to-severe SVD took minocycline or placebo for 3 months. Co-primary outcomes were microglial signal (determined using 11C-PK11195 positron emission tomography) and BBB permeability (using dynamic contrast-enhanced MRI). RESULTS: Forty-four participants were recruited between September 2019 and June 2022. Minocycline had no effect on 11C-PK11195 binding (relative risk [RR] 1.01, 95% confidence interval [CI] 0.98-1.04), or BBB permeability (RR 0.97, 95% CI 0.91-1.03). Serum inflammatory markers were not affected. DISCUSSION: 11C-PK11195 binding and increased BBB permeability are present in SVD; minocycline did not reduce either process. Whether these pathophysiological mechanisms are disease-causing remains unclear. INTERNATIONAL CLINICAL TRIALS REGISTRY PORTAL IDENTIFIER: ISRCTN15483452 HIGHLIGHTS: We found focal areas of increased microglial signal and increased blood-brain barrier permeability in patients with small vessel disease. Minocycline treatment was not associated with a change in these processes measured using advanced neuroimaging. Blood-brain barrier permeability was dynamic but MRI-derived measurements correlated well with CSF/serum albumin ratio. Advanced neuroimaging is a feasible outcome measure for mechanistic clinical trials.

Prevalence of, and risk factors for, cognitive impairment in lacunar stroke.

BACKGROUND: Small vessel disease (SVD) is associated with vascular cognitive impairment (VCI) but why VCI occurs in some, but not other patients, is uncertain. We determined the prevalence of, and risk factors for, VCI in a large cohort of patients with lacunar stroke. METHODS: Participants with magnetic resonance imaging (MRI)-confirmed lacunar stroke were recruited in the multicenter DNA Lacunar 2 study and compared with healthy controls. A logistic regression model was used to determine which vascular risk factors and MRI parameters were independent predictors of VCI, assessed using the Brief Memory and Executive Test (BMET). RESULTS: A total of 912 lacunar stroke patients and 425 controls were included, with mean (SD) age of 64.6 (12.26) and 64.7 (12.29) years, respectively. VCI was detected in 38.8% lacunar patients and 13.4% controls. In a logistic regression model, diabetes mellitus (odds ratio (OR) = 1.98 (95% confidence interval (CI) = 1.40-2.80), p < 0.001) and higher body mass index (BMI) (OR = 1.03 (95% CI = 1.00-1.05), p = 0.029) were independently associated with increased risk of VCI, and years of full-time education with lower risk (OR = 0.92 (95% CI = 0.86-0.99), p = 0.018). When entering both lacune count and white matter hyperintensity (WMH) in the same logistic regression model, only WMH grade was significantly associated with VCI (OR = 1.46 (95% CI = 1.24-1.72), p < 0.001). CONCLUSION: VCI is common in lacunar stroke patients, affecting almost 40%. This prevalence suggests that it should be routinely screened for in clinical practice. Risk factors for VCI in patients with lacunar stroke include diabetes mellitus, depressive symptoms, higher BMI, and WMH severity, while education is protective.

MINocyclinE to Reduce inflammation and blood brain barrier leakage in small Vessel diseAse (MINERVA) trial study protocol.

Background: Cerebral small vessel disease (SVD) is a common cause of stroke and cognitive impairment. Recent data has implicated neuroinflammation and increased blood-brain barrier (BBB) permeability in its pathogenesis, but whether such processes are causal and can be therapeutically modified is uncertain. In a rodent model of SVD, minocycline was associated with reduced white matter lesions, inflammation and BBB permeability. Aims: To determine whether blood-brain barrier permeability (measured using dynamic contrast-enhanced MRI) and microglial activation (measured by positron emission tomography using the radioligand 11C-PK11195) can be modified in SVD. Design: Phase II randomised double blind, placebo-controlled trial of minocycline 100 mg twice daily for 3 months in 44 participants with moderate to severe SVD defined as a clinical lacunar stroke and confluent white matter hyperintensities. Outcomes: Primary outcome measures are volume and intensity of focal increases of blood-brain barrier permeability and microglial activation determined using PET-MRI imaging. Secondary outcome measures include inflammatory biomarkers in serum, and change in conventional MRI markers and cognitive performance over 1 year follow up. Discussion: The MINERVA trial aims to test whether minocycline can influence novel pathological processes thought to be involved in SVD progression, and will provide insights into whether central nervous system inflammation in SVD can be therapeutically modulated.

Neurofilament light chain predicts future dementia risk in cerebral small vessel disease.

OBJECTIVES: Serum neurofilament light chain (NfL) has been proposed as prognostic markers in neurogenerative disease. A cross-sectional study in cerebral small vessel disease (SVD) reported an association with cognition and disability. If NfL is to be used to predict outcome, studies are required to demonstrate baseline NfL predicts future dementia risk. Furthermore, if it is to be used as a surrogate marker in clinical trials, change in NfL over time periods typical of a clinical trial must be linked to clinical progression. In a longitudinal study of patients with lacunar stroke and confluent white matter hyperintensities, we determined whether both baseline, and change, in NfL levels were linked to changes in MRI markers, cognitive decline and dementia risk. METHODS: Patients underwent MRI, cognitive testing and blood taking at baseline and annually for 3 years. Clinical and cognitive follow-up continued for 5 years. RESULTS: NfL data were available for 113 subjects for baseline analysis, and 90 patients for the longitudinal analysis. Baseline NfL predicted cognitive decline (global cognition ß=-0.335, SE=0.094, p=0.001) and risk of converting to dementia (HR=1.676 (95% CI 1.183 to 2.373), p=0.004). In contrast to imaging, there was no change in NfL values over the follow-up period. CONCLUSIONS: Baseline NfL predicts changes in MRI markers, cognitive decline and dementia rate over a 5 years follow-up period in SVD, suggesting NfL may be a useful prognostic marker. However, change in NfL values was not detected, and therefore NfL may not be a useful surrogate marker in clinical trials in SVD.

Radiofrequency-induced Thermo-chemotherapy Effect Versus a Second Course of Bacillus Calmette-Guérin or Institutional Standard in Patients with Recurrence of Non-muscle-invasive Bladder Cancer Following Induction or Maintenance Bacillus Calmette-Guérin Therapy (HYMN): A Phase III, Open-label, Randomised Controlled Trial.

BACKGROUND: There is no effective intravesical second-line therapy for non-muscle-invasive bladder cancer (NMIBC) when bacillus Calmette-Guérin (BCG) fails. OBJECTIVE: To compare disease-free survival time (DFS) between radiofrequency-induced thermo-chemotherapy effect (RITE) and institutional standard second-line therapy (control) in NMIBC patients with recurrence following induction/maintenance BCG. DESIGN, SETTINGS, AND PARTICIPANTS: Open-label, phase III randomised controlled trial accrued across 14 centres between May 2010 and July 2013 (HYMN [ClinicalTrials.gov: NCT01094964]). INTERVENTION: Patients were randomly assigned (1:1) to RITE (60min, 40mg mitomycin-C, 42±2°C) or control following stratification for carcinoma in situ (CIS) status (present/absent), therapy history (failure of previous induction/maintenance BCG), and treatment centre. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Primary outcome measures were DFS and complete response (CR) at 3 mo for the CIS at randomisation subgroup. Analysis was based on intention-to-treat. RESULTS AND LIMITATIONS: A total of 104 patients were randomised (48 RITE: 56 control). Median follow-up for the 31 patients without a DFS event was 36 mo. There was no significant difference in DFS between treatment arms (hazard ratio [HR] 1.33, 95% confidence interval [CI] 0.84-2.10, p=0.23) or in 3-mo CR rate in CIS patients (n=71; RITE: 30% vs control: 47%, p=0.15). There was no significant difference in DFS between treatment arms in non-CIS patients (n=33; RITE: 53% vs control: 24% at 24 mo, HR 0.50, 95% CI 0.22-1.17, p=0.11). DFS was significantly lower in RITE than in control in CIS with/without papillary patients (n=71; HR 2.06, 95% CI 1.17-3.62, p=0.01; treatment-subgroup interaction p=0.007). Disease progression was observed in four patients in each treatment arm. Adverse events and health-related quality of life between treatment arms were comparable. CONCLUSIONS: DFS was similar between RITE and control. RITE may be a second-line therapy for non-CIS recurrence following BCG failure; however, confirmatory trials are needed. RITE patients with CIS with/without papillary had lower DFS than control. HYMN highlights the importance of the control arm when evaluating novel therapies. PATIENT SUMMARY: This study did not show a difference in bladder cancer outcomes between microwave-heated chemotherapy and standard of care treatment. Papillary bladder lesions may benefit from microwave-heated chemotherapy treatment; however, more research is needed. Both treatments are similarly well tolerated.

MCT8 expression in human fetal cerebral cortex is reduced in severe intrauterine growth restriction.

The importance of the thyroid hormone (TH) transporter, monocarboxylate transporter 8 (MCT8), to human neurodevelopment is highlighted by findings of severe global neurological impairment in subjects with MCT8 (SLC16A2) mutations. Intrauterine growth restriction (IUGR), usually due to uteroplacental failure, is associated with milder neurodevelopmental deficits, which have been partly attributed to dysregulated TH action in utero secondary to reduced circulating fetal TH concentrations and decreased cerebral thyroid hormone receptor expression. We postulate that altered MCT8 expression is implicated in this pathophysiology; therefore, in this study, we sought to quantify changes in cortical MCT8 expression with IUGR. First, MCT8 immunohistochemistry was performed on occipital and parietal cerebral cortex sections obtained from appropriately grown for gestational age (AGA) human fetuses between 19 weeks of gestation and term. Secondly, MCT8 immunostaining in the occipital cortex of stillborn IUGR human fetuses at 24-28 weeks of gestation was objectively compared with that in the occipital cortex of gestationally matched AGA fetuses. Fetuses demonstrated widespread MCT8 expression in neurons within the cortical plate and subplate, in the ventricular and subventricular zones, in the epithelium of the choroid plexus and ependyma, and in microvessel wall. When complicated by IUGR, fetuses showed a significant fivefold reduction in the percentage area of cortical plate immunostained for MCT8 compared with AGA fetuses (P<0.05), but there was no significant difference in the proportion of subplate microvessels immunostained. Cortical MCT8 expression was negatively correlated with the severity of IUGR indicated by the brain:liver weight ratios (r(2)=0.28; P<0.05) at post-mortem. Our results support the hypothesis that a reduction in MCT8 expression in the IUGR fetal brain could further compromise TH-dependent brain development.

Monocarboxylate transporter 8 modulates the viability and invasive capacity of human placental cells and fetoplacental growth in mice.

Monocarboxylate transporter 8 (MCT8) is a well-established thyroid hormone (TH) transporter. In humans, MCT8 mutations result in changes in circulating TH concentrations and X-linked severe global neurodevelopmental delay. MCT8 is expressed in the human placenta throughout gestation, with increased expression in trophoblast cells from growth-restricted pregnancies. We postulate that MCT8 plays an important role in placental development and transplacental TH transport. We investigated the effect of altering MCT8 expression in human trophoblast in vitro and in a Mct8 knockout mouse model. Silencing of endogenous MCT8 reduced T3 uptake into human extravillous trophoblast-like cells (SGHPL-4; 40%, P<0.05) and primary cytotrophoblast (15%, P<0.05). MCT8 over-expression transiently increased T3 uptake (SGHPL-4∶30%, P<0.05; cytotrophoblast: 15%, P<0.05). Silencing MCT8 did not significantly affect SGHPL-4 invasion, but with MCT8 over-expression T3 treatment promoted invasion compared with no T3 (3.3-fold; P<0.05). Furthermore, MCT8 silencing increased cytotrophoblast viability (∼20%, P<0.05) and MCT8 over-expression reduced cytotrophoblast viability independently of T3 (∼20%, P<0.05). In vivo, Mct8 knockout reduced fetal:placental weight ratios compared with wild-type controls at gestational day 18 (25%, P<0.05) but absolute fetal and placental weights were not significantly different. The volume fraction of the labyrinthine zone of the placenta, which facilitates maternal-fetal exchange, was reduced in Mct8 knockout placentae (10%, P<0.05). However, there was no effect on mouse placental cell proliferation in vivo. We conclude that MCT8 makes a significant contribution to T3 uptake into human trophoblast cells and has a role in modulating human trophoblast cell invasion and viability. In mice, Mct8 knockout has subtle effects upon fetoplacental growth and does not significantly affect placental cell viability probably due to compensatory mechanisms in vivo.

Pituitary tumor transforming gene binding factor: a new gene in breast cancer.

Pituitary tumor transforming gene (PTTG) binding factor (PBF; PTTG1IP) is a relatively uncharacterized oncoprotein whose function remains obscure. Because of the presence of putative estrogen response elements (ERE) in its promoter, we assessed PBF regulation by estrogen. PBF mRNA and protein expression were induced by both diethylstilbestrol and 17beta-estradiol in estrogen receptor alpha (ERalpha)-positive MCF-7 cells. Detailed analysis of the PBF promoter showed that the region -399 to -291 relative to the translational start site contains variable repeats of an 18-bp sequence housing a putative ERE half-site (gcccctcGGTCAcgcctc). Sequencing the PBF promoter from 122 normal subjects revealed that subjects may be homozygous or heterozygous for between 1 and 6 repeats of the ERE. Chromatin immunoprecipitation and oligonucleotide pull-down assays revealed ERalpha binding to the PBF promoter. PBF expression was low or absent in normal breast tissue but was highly expressed in breast cancers. Subjects with greater numbers of ERE repeats showed higher PBF mRNA expression, and PBF protein expression positively correlated with ERalpha status. Cell invasion assays revealed that PBF induces invasion through Matrigel, an action that could be abrogated both by siRNA treatment and specific mutation. Furthermore, PBF is a secreted protein, and loss of secretion prevents PBF inducing cell invasion. Given that PBF is a potent transforming gene, we propose that estrogen treatment in postmenopausal women may upregulate PBF expression, leading to PBF secretion and increased cell invasion. Furthermore, the number of ERE half-sites in the PBF promoter may significantly alter the response to estrogen treatment in individual subjects.

Skewed X-chromosome inactivation in scleroderma.

Scleroderma is a female-prevalent autoimmune disease of unclear etiology. Two fundamental gender differences, skewed X-chromosome inactivation (XCI) and pregnancy-related microchimerism, have been implicated in scleroderma. We investigated the XCI patterns of female scleroderma patients and the parental origin of the inactive X chromosome in those patients having skewed XCI patterns (>80%). In addition, we investigated whether a correlation exists between XCI patterns and microchimerism in a well-characterized cohort. About 195 female scleroderma patients and 160 female controls were analyzed for the androgen receptor locus to assess XCI patterns in the DNA extracted from peripheral blood cells. Skewed XCI was observed in 67 (44.9%) of 149 informative patients and in 10 of 124 healthy controls (8.0%) [odds ratio (OR) = 9.3 (95% confidence interval (CI) 4.3-20.6, P < 0.0001)]. Extremely skewed XCI (>90%) was present in 44 of 149 patients (29.5%) but only in 3 of 124 controls (2.4%; OR = 16.9; 95% CI 4.8-70.4, P < 0.0001). Parental origin of the inactive X chromosome was investigated for ten patients for whom maternal DNA was informative, and the inactive X chromosome was of maternal origin in eight patients and of paternal origin in two patients. Skewed XCI mosaicism could be considered as an important risk factor in scleroderma.

Maternal microchimerism in peripheral blood in type 1 diabetes and pancreatic islet beta cell microchimerism.

Maternal cells have recently been found in the circulation and tissues of mothers' immune-competent children, including in adult life, and is referred to as maternal microchimerism (MMc). Whether MMc confers benefits during development or later in life or sometimes has adverse effects is unknown. Type 1 diabetes (T1D) is an autoimmune disease that primarily affects children and young adults. To identify and quantify MMc, we developed a panel of quantitative PCR assays targeting nontransmitted, nonshared maternal-specific HLA alleles. MMc was assayed in peripheral blood from 172 individuals, 94 with T1D, 54 unaffected siblings, and 24 unrelated healthy subjects. MMc levels, expressed as the genome equivalent per 100,000 proband cells, were significantly higher in T1D patients than unaffected siblings and healthy subjects. Medians and ranges, respectively, were 0.09 (0-530), 0 (0-153), and 0 (0-7.9). Differences between groups were evident irrespective of HLA genotypes. However, for patients with the T1D-associated DQB1*0302-DRB1*04 haplotype, MMc was found more often when the haplotype was paternally (70%) rather than maternally transmitted (14%). In other studies, we looked for female islet beta cells in four male pancreases from autopsies, one from a T1D patient, employing FISH for X and Y chromosomes with concomitant CD45 and beta cell insulin staining. Female islet beta cells (presumed maternal) formed 0.39-0.96% of the total, whereas female hematopoietic cells were very rare. Thus, T1D patients have higher levels of MMc in their circulation than unaffected siblings and healthy individuals, and MMc contributes to islet beta cells in a mother's progeny.

Maternal microchimerism in healthy adults in lymphocytes, monocyte/macrophages and NK cells.

During pregnancy some maternal cells reach the fetal circulation. Microchimerism (Mc) refers to low levels of genetically disparate cells or DNA. Maternal Mc has recently been found in the peripheral blood of healthy adults. We asked whether healthy women have maternal Mc in T and B lymphocytes, monocyte/macrophages and NK cells and, if so, at what levels. Cellular subsets were isolated after fluorescence activated cell sorting. A panel of HLA-specific real-time quantitative PCR assays was employed targeting maternal-specific HLA sequences. Maternal Mc was expressed as the genome equivalent (gEq) number of microchimeric cells per 100,000 proband cells. Thirty-one healthy adult women probands were studied. Overall 39% (12/31) of probands had maternal Mc in at least one cellular subset. Maternal Mc was found in T lymphocytes in 25% (7/28) and B lymphocytes in 14% (3/21) of probands. Maternal Mc levels ranged from 0.9 to 25.6 and 0.9 to 25.3 gEq/100,000 in T and B lymphocytes, respectively. Monocyte/macrophages had maternal Mc in 16% (4/25) and NK cells in 28% (5/18) of probands with levels from 0.3 to 36 and 1.8 to 3.2 gEq/100,000, respectively. When compared to fetal Mc, as assessed by quantification of male DNA in women with sons, maternal Mc was substantially less prevalent in all cellular subsets; fetal Mc prevalence in T and B lymphocytes, monocyte/macrophages and NK cells was 58, 75, 50 and 62% (P=0.01, P=0.005, P=0.04, P=0.05) respectively. In summary, maternal Mc was identified among lymphoid and myeloid compartments of peripheral blood in healthy adult women. Maternal Mc was less frequent than fetal Mc in all cellular subsets tested. Studies are needed to investigate the immunological effects and function of maternal Mc and to explore whether maternal Mc in cellular subsets has biological effects on her progeny.

Male microchimerism in women without sons: quantitative assessment and correlation with pregnancy history.

PURPOSE: Fetal microchimerism, derived from fetal cells that persist after pregnancy, is usually evaluated by tests for male microchimerism in women who gave birth to sons. We investigated male microchimerism in women without sons and examined correlation with prior pregnancy history. Immunologic consequences of microchimerism are unknown. We studied healthy women and women with rheumatoid arthritis (RA). METHODS: Y-chromosome-specific real-time quantitative polymerase chain reaction was used to test peripheral blood mononuclear cells of 120 women (49 healthy and 71 with RA). Results were expressed as the number of male cells that would be equivalent to the total amount of male DNA detected within a sample containing the equivalent of 100000 female cells. RESULTS: Male microchimerism was found in 21% of women overall. Healthy women and women with RA did not significantly differ (24% vs 18%). Results ranged from the DNA equivalent of 0 to 20.7 male cells per 100000 female cells. Women were categorized into 4 groups according to pregnancy history. Group A had only daughters (n = 26), Group B had spontaneous abortions (n = 23), Group C had induced abortions (n = 23), and Group D were nulligravid (n = 48). Male microchimerism prevalence was significantly greater in Group C than other groups (8%, 22%, 57%, 10%, respectively). Levels were also significantly higher in the induced abortion group. CONCLUSIONS: Male microchimerism was not infrequent in women without sons. Besides known pregnancies, other possible sources of male microchimerism include unrecognized spontaneous abortion, vanished male twin, an older brother transferred by the maternal circulation, or sexual intercourse. Male microchimerism was significantly more frequent and levels were higher in women with induced abortion than in women with other pregnancy histories. Further studies are needed to determine specific origins of male microchimerism in women.

Expressing engineered thymidylate kinase variants in human cells to improve AZT phosphorylation and human immunodeficiency virus inhibition.

The triphosphorylated form of the nucleoside analogue AZT (AZTTP) acts as a chain terminator during reverse transcription of the human immunodeficiency virus (HIV) genome. The bottleneck in the conversion of AZT to AZTTP is the phosphorylation of AZT monophosphate (AZTMP) by cellular thymidylate kinase. Human thymidylate kinase was engineered to exhibit highly improved activity for AZTMP to AZTDP conversion. It was demonstrated here that genetically modified human cells transiently expressing these enzyme variants show more than 10-fold higher intracellular concentrations of AZTDP and AZTTP. Stable clones expressing these enzymes appear to phosphorylate AZTMP less efficiently, but first experiments indicate they are still more potent in HIV inhibition than the parental cells. It was proposed that the concept of introducing into human cells a catalytically improved human enzyme, rather than an enzyme of viral, bacterial or yeast origin, may serve as a paradigm for ameliorating the metabolic activation of an established drug.