Randomized, double-blinded, placebo-controlled pilot study: efficacy of faecal microbiota transplantation on chronic fatigue syndrome.

BACKGROUND: Chronic fatigue syndrome (CFS) is a disabling illness of unknown aetiology. Disruption of gut microbiota may play a role in several neurological disorders. In this study, the effect of faecal microbiota transplantation (FMT) on fatigue severity and health-related quality of life (HRQOL) in patients with CFS was evaluated. METHODS: Randomized, placebo-controlled pilot trial. Patients and researchers were blinded to treatment assignment. 11 patients with CFS (10 female and 1 male, mean age 42.2 years and mean duration of CFS 6.3 years) were randomly assigned to receive either FMT from a universal donor (n = 5) or autologous FMT (n = 6) via colonoscopy. Patients' HRQOL was assessed by using visual analog scale (VAS) and self-reporting questionnaires Modified Fatigue Impact Scale (MFIS), 15D and EQ-5D-3L. Patients' HRQOL was evaluated at baseline, and 1 and 6 months after the FMT. RESULTS: The baseline VAS scores in the FMT and placebo groups were 62.4 and 76.0 (p = 0.29). 1-month scores were 60.0 and 73.7 and 6-months scores 72.8 and 69.5, respectively. Total MFIS scores in the FMT and placebo groups were 59.6 and 61.0 at the baseline (p = 0.80), 53.5 and 62.0 at 1 month and 58.6 and 56.2 at 6 months. Compared to the baseline scores, differences at 1 and 6 months were statistically insignificant both in VAS and in MFIS. The 15D and EQ-5D-3L profiles did not change after the FMT or placebo. FMT-related adverse events were not reported. CONCLUSION: FMT was safe but did not relieve symptoms or improve the HRQOL of patients with CFS. Small number of study subjects limits the generalizability of these results. Trial Registration ClinicalTrials.gov Identifier NCT04158427, https://register. CLINICALTRIALS: gov , date of registration 08/08/2019.

Omentum-derived matrix enables the study of metastatic ovarian cancer and stromal cell functions in a physiologically relevant environment.

High-grade serous (HGS) ovarian cancer is the most lethal gynaecological disease in the world and metastases is a major cause. The omentum is the preferential metastatic site in HGS ovarian cancer patients and in vitro models that recapitulate the original environment of this organ at cellular and molecular level are being developed to study basic mechanisms that underpin this disease. The tumour extracellular matrix (ECM) plays active roles in HGS ovarian cancer pathology and response to therapy. However, most of the current in vitro models use matrices of animal origin and that do not recapitulate the complexity of the tumour ECM in patients. Here, we have developed omentum gel (OmGel), a matrix made from tumour-associated omental tissue of HGS ovarian cancer patients that has unprecedented similarity to the ECM of HGS omental tumours and is simple to prepare. When used in 2D and 3D in vitro assays to assess cancer cell functions relevant to metastatic ovarian cancer, OmGel performs as well as or better than the widely use Matrigel and does not induce additional phenotypic changes to ovarian cancer cells. Surprisingly, OmGel promotes pronounced morphological changes in cancer associated fibroblasts (CAFs). These changes were associated with the upregulation of proteins that define subsets of CAFs in tumour patient samples, highlighting the importance of using clinically and physiologically relevant matrices for in vitro studies. Hence, OmGel provides a step forward to study the biology of HGS omental metastasis. Metastasis in the omentum are also typical of other cancer types, particularly gastric cancer, implying the relevance of OmGel to study the biology of other highly lethal cancers.

Clozapine-Related Diarrhea and Colitis: Report of 4 Cases.

BACKGROUND: During clozapine treatment, diarrhea is a rare but clinically relevant adverse effect. Cases of microscopic colitis and eosinophilic colitis have been previously reported. PROCEDURES: We present 4 patients who developed severe diarrhea in early weeks of clozapine therapy. FINDINGS: Two patients had significant peripheral eosinophilia 1 week after diarrhea symptoms. One of these patients also had Charcot-Leyden crystals in stool afterward, confirming the presence of eosinophils in the gut lumen. One of our patients had a confirmed microscopic colitis and later also neutropenia, which required treatment. CONCLUSIONS: Charcot-Leyden crystals in stool may be associated with concurrent diarrhea and eosinophilia during clozapine treatment, which is a previously unreported finding. Occurrence of blood dyscrasias with diarrhea symptoms during clozapine treatment needs further investigation to understand the possible shared mechanisms.

Blocking Angiopoietin-2 Promotes Vascular Damage and Growth Inhibition in Mouse Tumors Treated with Small Doses of Radiation.

Abnormal vasculature in tumors leads to poor tissue perfusion and cytostatic drug delivery. Although drugs inducing vascular normalization, for example, angiopoietin-2 (Ang2)-blocking antibodies, have shown promising results in preclinical tumor models, clinical studies have so far shown only little efficacy. Because Ang2 is known to play a protective role in stressed endothelial cells, we tested here whether Ang2 blocking could enhance radiation-induced tumor vascular damage. Tumor-bearing mice were treated with anti-Ang2 antibodies every 3 or 4 days starting 3 days before 3 × 2 Gy or 4 × 0.5 Gy whole-body or tumor-focused radiation. Combination treatment with anti-Ang2 and radiation improved tumor growth inhibition and extended the survival of mice with melanoma or colorectal tumors. Single-cell RNA-sequencing revealed that Ang2 blocking rescued radiation-induced decreases in T cells and cells of the monocyte/macrophage lineage. In addition, anti-Ang2 enhanced radiation-induced apoptosis in cultured endothelial cells. In vivo, combination treatment decreased tumor vasculature and increased tumor necrosis in comparison with tumors treated with monotherapies. These results suggest that a combination of Ang2-blocking antibodies with radiation increases tumor growth inhibition and extends the survival of tumor-bearing mice. SIGNIFICANCE: These findings offer a preclinical rationale for further testing of the use of radiation in combination with Ang2-blocking antibodies to improve the overall outcome of cancer treatment.

Spa type distribution in MRSA and MSSA bacteremias and association of spa clonal complexes with the clinical characteristics of bacteremia.

The genetic distribution of invasive methicillin-susceptible (MSSA) and resistant S. aureus (MRSA) strains has to be addressed in order to target infection control strategies. A large MRSA epidemic caused by a certain MRSA strain (spa type 067) broke out in 2001 in our health district. We wanted to investigate the current spa type distribution in MRSA and MSSA bacteremias and assess the potential association of spa clonal complexes (spaCC) with the clinical characteristics of S. aureus bacteremia. One hundred nine invasive MRSA isolates and 353 invasive MSSA isolates were spa typed and grouped into clonal complexes (spaCC). Spa type distribution was compared to that of colonizing MRSA strains. Spa type and spaCC data linked to clinical information on the course of bacteremic cases was used to search for differences in virulence between strains. Spa type distribution in MRSA is less heterogenic than in MSSA. t067 dominates both in MRSA colonisations and in invasive findings. Among MSSA, no such dominating strains were found. Of spaCCs, mortality was the highest in spaCC 067 (25.6%). SpaCC 008 was more often associated with endocarditis than other CCs (22.7 vs 5.8%, p = 0.013), spaCC 2133 with skin infections (68.4 vs 36.4%, p = 0.007), and spaCC 012 with foreign body infections (25.0 vs 9.3%, p = 0.029) than other clonal complexes. A single successful strain can explain the major proportion of MRSA among S. aureus bacteremias. Certain spaCCs showed association with certain clinical characteristics. These findings suggest that S. aureus strains differ in their virulence and invasiveness.

Trends in incidence and resistance patterns of Staphylococcus aureus bacteremia.

BACKGROUND: Staphylococcus aureus bacteremia (SAB) causes a significant burden on the population. Several infection control measures have been implemented in Pirkanmaa county to combat a local epidemic with methicillin-resistant Staphylococcus aureus (MRSA). We aimed to study the epidemiology of SAB and antibiotic resistance of S. aureus and the possible influence of improved infection control. METHODS: Register data from 2005 to 2015 were retrospectively analysed to study the antimicrobial susceptibility, the incidence and mortality in SAB in a population-based setting. RESULTS: The incidence of SAB increased during the study period from 21.6 to 35.8/100,000 population. The number of both health care-associated (HA) and community-associated (CA) cases has increased. The incidence of MSSA bacteremia increased from 19.9 to 35.2/100,000 population in Pirkanmaa in parallel to other parts of Finland. The incidence of MRSA bacteremia was 10-fold (4.5/100,000 population) higher in 2011 than in other parts of the country, but sank to the national level (0.59/100,000 population) in 2015. The fatality rate decreased from 22% to 17%. The proportion of penicillin-susceptible Staphylococcus aureus (PSSA) increased from 23.9% in 2008 to 43.1% in 2015. CONCLUSION: The incidence of both HA and CA SAB has increased since 2005. Conversely, the proportion of MRSA and PRSA bacteremia has decreased. Promotion of infection control measures may have reduced the incidence of MRSA bacteremia but not the overall incidence of SAB. The rising proportion of PSSA enables the use of targeted, narrow spectrum antimicrobials.

Comparison of outcome and clinical characteristics of bacteremia caused by methicillin-resistant, penicillin-resistant and penicillin-susceptible Staphylococcus aureus strains.

BACKGROUND: The aim of this study was to assess the association of methicillin resistance and penicillinase production with clinical characteristics and outcome of Staphylococcus aureus bacteremia. METHODS: For 126 patients with methicillin-resistant Staphylococcus aureus (MRSA) bacteremia, 378 age- and gender-matched controls with methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia were selected. Of controls, 126 had bacteremia caused by penicillin-susceptible strains (PSSA) and 252 by penicillinase-producing strains (PRSA). Underlying diseases, clinical course and mortality were retrospectively assessed. RESULTS: Patients with MRSA bacteremia were more often smokers than patients with MSSA bacteremia (OR 2.34, 95% CI 1.27-4.32). MRSA bacteremia was more often healthcare-associated (OR 4.23, 95% CI 2.47-7.24), associated with central venous catheters (OR 2.09, 95% CI 1.27-3.47), glucocorticoid therapy (OR 1.82, 95% CI 1.12-2.93) and prior surgery (OR 2.32, 95% CI 1.43-3.76). Patients with MRSA bacteremia received appropriate empiric antibiotic (31%) less often than controls (98%). Mortality within 28 days was higher in MRSA bacteremia (26.8%) than in MSSA bacteremia (15.5%) (OR 2.00, 95% CI 1.20-3.34), PRSA bacteremia (17.0%) (OR 1.79 95% CI 1.04-3.09) or PSSA bacteremia (12.5%) (OR 2.56 95% CI 1.27-5.15). The difference remained after adjusting for underlying diseases and foci. There was no significant difference in clinical course between PRSA and PSSA bacteremias. CONCLUSIONS: MRSA bacteremia was associated with poorer outcome than either PRSA or PSSA bacteremia. We corroborated several risk factors found in previous studies.

Nrf2/Keap1 Pathway and Expression of Oxidative Stress Lesions 8-hydroxy-2'-deoxyguanosine and Nitrotyrosine in Melanoma.

BACKGROUND/AIM: Increased expression and prognostic significance of major redox regulator nuclear factor erythroid-2-related factor (Nrf2) is recognized in many cancers. Our aim was to investigate the role of oxidative stress markers in melanoma. MATERIALS AND METHODS: We characterized the immunohistochemical expression of Nrf2, kelch-like ECH-associated protein 1 (Keap1), BRAF(V600E), 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine in 36 nevi, 14 lentigo maligna and 71 malignant melanomas. We measured Nrf2 expression in melanoma cell lines and conducted cytotoxicity assays combining BRAF/NRAS ablation and H2O2treatment. RESULTS: Nuclear Nrf2 expression in melanoma correlated with deeper Breslow (p<0.0005), invasive phenotype (Clark III-V) (p=0.011), nodular growth (p=0.001) and worse melanoma-specific survival (p=0.008). Absence of 8-OHdG in the endothelium was a greater significant predictor of poor prognosis (p=0.024) than ulceration (p=0.17) and had a similar impact on prognosis as Breslow (p=0.024). A decrease of Nrf2 followed the BRAF/NRAS inhibition, but combination of inhibitor with H2O2did not increase cytotoxicity. CONCLUSION: Nrf2 and 8-OHdG influence prognosis in melanoma.

Combined interventions are effective in MRSA control.

BACKGROUND: A large healthcare-associated epidemic mainly caused by one methicillin-resistant Staphylococcus aureus (MRSA) strain broke out in Pirkanmaa County, Finland, in 2001. This study describes the impact of infection control and screening practices on the epidemic. METHODS: The number of hospital-acquired (HA)-MRSA findings obtained from clinical and screening samples during the epidemic was calculated. Strains were typed by pulsed-field electrophoresis (PFGE) or spa typing. Strain type distribution was studied in relation to sample type, year of the epidemic and site of transmission. Several infection control interventions were launched stepwise and screening protocols were expanded. RESULTS: A total of 4118 cases were identified during 2001-2014, of which 3527 were classified as HA. One strain (spa t067) dominated in the epidemic. HA-MRSA cases decreased constantly from the year 2011. The number of new HA-MRSA cases was 57% less in the year 2014 (n = 171) as compared with the year 2011 (n = 399). The proportion of the epidemic strain declined significantly over the years. Screening samples comprised 71% (2439/3527) and clinical samples 29% (1034/3527) of HA-MRSA findings. The number of HA-MRSA cases found from clinical samples started to decrease when screening was expanded. An increase in hand-rub consumption was associated with a decrease in transmissions in Tampere University Hospital (TAUH). CONCLUSION: Implementation of universal screening together with several other interventions is effective in containing an MRSA epidemic. The proportion of other than Pirkanmaa epidemic (PE)-MRSA strain findings increased throughout the period, indicating the changing epidemiology of MRSA.

Bcl-xl and Mcl-1 are the major determinants of the apoptotic response to dual PI3K and MEK blockage.

The dual targeting of PI3K-AKT-mTOR and Ras-Raf-MEK-ERK pathways is a potential anticancer therapy, but unfortunately, the response rate has been low in early phase clinical trials. Pre-clinical models have suggested that an apoptotic response to dual PI3K and MEK targeting is relatively rare and understanding apoptotic avoidance could lead to increased clinical efficiency. This study investigated solid cancer cell lines, which are known to be sensitive to dual PI3K and MEK inhibition but to have a limited apoptotic response. The cells were exposed to dual PI3K and MEK blockage in combination with a panel of additional pharmacological agents and cytotoxicity and apoptosis were analyzed. Our results indicated that the BH3 mimetic ABT-263, the HDAC inhibitor entinostat and the multikinase inhibitor dasatinib increased the cytotoxicity and apoptotic response of dual PI3K and MEK targeting. Furthermore, ABT-263 and entinostat was able to induce apoptosis in combination with single agent PI3K and MEK inhibitors. Protein expression, immunoprecipitation and siRNA knockdown models suggested that Bcl-xl and Mcl-1 were the most important factors circumventing PI3K and/or MEK inhibition-mediated apoptosis. The results suggest that the cytotoxicity of PI3K and/or MEK inhibitor treatments can be augmented by combinatory approaches targeting anti-apoptotic mediators Bcl-xl and Mcl-1.

Combining targeted drugs to overcome and prevent resistance of solid cancers with some stem-like cell features.

Treatment resistance significantly inhibits the efficiency of targeted cancer therapies in drug-sensitive genotypes. In the current work, we studied mechanisms for rapidly occurring, adaptive resistance in targeted therapy-sensitive lung, breast, and melanoma cancer cell lines. The results show that in ALK translocated lung cancer lines H3122 and H2228, cells with cancer stem-like cell features characterized by high expression of cancer stem cell markers and/or in vivo tumorigenesis can mediate adaptive resistance to oncogene ablative therapy. When pharmacological ablation of ALK oncogene was accompanied with PI3K inhibitor or salinomycin therapy, cancer stem-like cell features were reversed which was accompanied with decreased colony formation. Furthermore, co-targeting was able to block the formation of acquired resistance in H3122 line. The results suggest that cells with cancer stem-like cell features can mediate adaptive resistance to targeted therapies. Since these cells follow the stochastic model, concurrent therapy with an oncogene ablating agent and a stem-like cell-targeting drug is needed for maximal therapeutic efficiency.

Alternative dosing of dual PI3K and MEK inhibition in cancer therapy.

BACKGROUND: PI3K/AKT/mTOR and RAS/RAF/MEK/ERK pathways are thought to be the central transducers of oncogenic signals in solid malignancies, and there has been a lot of enthusiasm for developing inhibitors of these pathways for cancer therapy. Some preclinical models have suggested that combining inhibitors of both parallel pathways may be more efficacious, but it remains unknown whether dual inhibition with high enough concentrations of the drugs to achieve meaningful target inhibition is tolerable in a clinical setting. Furthermore, the predictive factors for dual inhibition are unknown. METHODS: Non-small cell lung cancer (NSCLC) cell lines (n=12) with the most frequent oncogenic backgrounds (K-Ras mut n=3, EGFR mut n=3, ALK translocated n=3, and triple-negative n=3) were exposed to PI3K inhibitors (ZSTK474, PI-103) or MEK inhibitor (CI-1040) alone or in combination and analysed with an MTS growth/cytotoxicity assay and statistically by combination index analysis. The activity of the intracellular signaling pathways in response to the inhibitor treatments was analysed with a western blot using phospho-specific antibodies to AKT, ERK1/2, S6, and 4E-BPI. For the differential dosing schedule experiments, additional breast and colon cancer cell lines known to be sensitive to dual inhibition were included. RESULTS: Two of the 12 NSCLC cell lines tested, H3122 (ALK translocated) and H1437 (triple-negative), showed increased cytotoxicity upon dual MEK and PI3K inhibition. Furthermore, MDA-MB231 (breast) and HCT116 (colon), showed increased cytotoxicity upon dual inhibition, as in previous studies. Activation of parallel pathways in the dual inhibition-sensitive lines was also noted in response to single inhibitor treatment. Otherwise, no significant differences in downstream intracellular pathway activity (S6 and 4E-BPI) were noted between PI3K alone and dual inhibition other than the increased cytotoxicity of the latter. In the alternative dosing schedules two out of the four dual inhibition-sensitive cell lines showed similar cytotoxicity to continuous PI3K and short (15min) MEK inhibition treatment. CONCLUSIONS: Therapy with a dual PI3K and MEK inhibitor combination is more efficient than either inhibitor alone in some NSCLC cell lines. Responses to dual inhibition were not associated with any specific oncogenic genotype and no other predictive factors for dual inhibition were noted. The maximal effect of the dual PI3K and MEK inhibition can be achieved with alternative dosing schedules which are potentially more tolerable clinically.

A controlled register-based study of 460 neurofibromatosis 1 patients: increased fracture risk in children and adults over 41 years of age.

Neurofibromatosis 1 (NF1, von Recklinghausen's disease) is an autosomal dominant neurocutaneous-skeletal syndrome in which low bone mineral density (BMD) and osteoporosis are common. Low BMD is, however, not the sole component of fracture risk. In the current study, 460 Finnish patients with NF1 were identified from the hospital medical records and their fracture risk was evaluated. The control population included 3988 appendectomy patients whose age and gender distribution was similar to that of the NF1 patients. Medical records of NF1 and control cohorts were screened for fractures according to the International Statistical Classification of Diseases and Related Health Problems, 10th Revision (ICD-10) between January 2000 and October 2011. The results show that patients with NF1 had increased age-dependent fracture risk compared to controls. Specifically, patients with NF1 aged 41 years and older had a risk ratio of ×5.2 for fractures compared to controls, and children with NF1 had a ×3.4 risk ratio for fractures compared to children without NF1. In contrast, the fracture risk was not increased in NF1 patients aged 17 to 40 years. When fractures not traditionally related to osteoporosis such as fractures of fingers, toes, and skull were excluded, the results were essentially the same. No gender related differences were observed. In conclusion, patients with NF1 have increased fracture risk depending on age. We recommend considering prophylactic measures, such as lifestyle advice, to prevent fractures from occurring.

A novel treatment strategy for EGFR mutant NSCLC with T790M-mediated acquired resistance.

The purpose of our study was to identify novel kinase inhibitors for the treatment of genetic subsets of non-small cell lung cancer (NSCLC). NSCLC cell lines (n = 8) with known oncogenic backgrounds (K-Ras, EGFR and EML4-ALK) were exposed to several kinase inhibitors and analyzed for cell growth/cytotoxicity and signaling. Gö6976, a classical protein kinase C inhibitor, showed high potency against mutated EGFR and was further validated in vitro using additional NSCLC lines (n = 4) and Ba/F3 models and in vivo using a xenograft model. Gö6976 was identified to be a potent inhibitor of mutated EGFR with IC50 values from 0.033 nM to 3.3 µM and down regulating phosphorylation of EGFR, AKT and ERK1/2 at concentrations in the range of the IC(50) values. Gö6976 has only a minor effect on wild-type EGFR and cell lines independent of signaling from the mutant EGFR. Most importantly, the activity of Gö6976 remains unchanged despite the presence of the T790M-mediated resistance, and it prevents the occurrence of this resistance in vitro. Gö6976 was also shown to significantly reduce tumor growth in an in vivo xenograft model with a EGFR-mutated NSCLC cell line containing T790M. These findings demonstrate that Gö6976 acts as a potent inhibitor of mutant EGFR despite the presence of T790M, the most important mechanism of acquired resistance for EGFR tyrosine kinase inhibitors, in both in vitro and in vivo models.

The development of cutaneous neurofibromas.

Cutaneous neurofibromas are the hallmarks of neurofibromatosis type 1 (NF1). They are composed of multiple cell types, and traditionally they are believed to arise from small nerve tributaries of the skin. A key finding in the context of this view has been that subpopulations of tumor Schwann cells harbor biallelic inactivation of the NF1 gene (NF1(-/-)). In the present study, our aim was to clarify further the pathogenesis of cutaneous neurofibromas. First, we detected cells expressing multipotency-associated biomarkers in cutaneous neurofibromas. Second, we developed a method for isolating and expanding multipotent neurofibroma-derived precursor cells (NFPs) from dissociated human cutaneous neurofibromas and used it to analyze their growth and differentiation potential. In analogy to solitary cells resident in neurofibromas, NFPs were found to express nestin and had the potential to differentiate to, at least, Schwann cells, neurons, epithelial cells, and adipocytes. Mutation analysis of the NFPs revealed that their genotype was NF1(+/-). The results led us to speculate that the development of cutaneous neurofibromas includes the recruitment of multipotent NF1(+/-) precursor cells. These cells may be derived from the multipotent cells of the hair roots, which often are intimately associated with microscopic neurofibromas.