Corneal Vascularization Associated With a Novel PDGFRB Variant.

Purpose: The purpose of this study was to identify the genetic cause of aggressive corneal vascularization in otherwise healthy children in one family. Further, to study molecular consequences associated with the identified variant and implications for possible treatment. Methods: Exome sequencing was performed in affected individuals. HeLa cells were transduced with the identified c.1643C>A, p.(Ser548Tyr) variant in the platelet-derived growth factor receptor beta gene (PDGFRB) or wild-type PDGFRB. ELISA and immunoblot analysis were used to detect the phosphorylation levels of PDGFRß and downstream signaling proteins in untreated and ligand-stimulated cells. Sensitivity to various receptor tyrosine kinase inhibitors (TKIs) was determined. Results: A novel c.1643C>A, p.(Ser548Tyr) PDGFRB variant was found in affected family members. HeLa cells transduced with this variant did not have increased baseline levels of phosphorylated PDGFRß. However, upon stimulation with ligand, excessive activation of PDGFRß was observed compared to cells transduced with the wild-type variant. PDGFRß with the p.(Ser548Tyr) amino acid substitution was successfully inhibited with tyrosine kinase inhibitors (axitinib, dasatinib, imatinib, and sunitinib) in vitro. Conclusions: A novel c.1643C>A, p.(Ser548Tyr) PDGFRB variant was found in family members with isolated corneal vascularization. Cells transduced with the newly identified variant showed increased phosphorylation of PDGFRß upon ligand stimulation. This suggests that PDGF-PDGFRß signaling in these patients leads to overactivation of PDGFRß, which could lead to abnormal wound healing of the cornea. The examined TKIs prevented such overactivation, introducing the possibility for targeted treatment in these patients.

A Pellino-2 variant is associated with constitutive NLRP3 inflammasome activation in a family with ocular pterygium-digital keloid dysplasia.

Ocular pterygium-digital keloid dysplasia (OPDKD) is a rare hereditary disease characterized by corneal ingrowth of vascularized conjunctival tissue early in life. Later, patients develop keloids on fingers and toes but are otherwise healthy. In a recently described family with OPDKD, we report the presence of a de novo c.770C > T, p.(Thr257Ile) variant in PELI2 in the affected individual. PELI2 encodes for the E3 ubiquitin ligase Pellino-2. In transgenic U87MG cells overexpressing Pellino-2 with the p.(Thr257Ile) amino acid substitution, constitutive activation of the NLRP3 inflammasome was observed. However, the Thr257Ile variant did not affect Pellino-2 intracellular localization, its binding to known interaction partners, nor its stability. Our findings indicate that constitutive autoactivation of the NLRP3 inflammasome contributes to the development of PELI2-associated OPDKD.

Activity monitoring and patient-reported outcome measures in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome patients.

INTRODUCTION: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disease with no validated specific and sensitive biomarker, and no standard approved treatment. In this observational study with no intervention, participants used a Fitbit activity tracker. The aims were to explore natural symptom variation, feasibility of continuous activity monitoring, and to compare activity data with patient reported outcome measures (PROMs). MATERIALS AND METHODS: In this pilot study, 27 patients with mild to severe ME/CFS, of mean age 42.3 years, used the Fitbit Charge 3 continuously for six months. Patients wore a SenseWear activity bracelet for 7 days at baseline, at 3 and 6 months. At baseline and follow-up they completed the Short Form 36 Health Survey (SF-36) and the DePaul Symptom Questionnaire-Short Form (DSQ-SF). RESULTS: The mean number of steps per day decreased with increasing ME/CFS severity; mild 5566, moderate 4991 and severe 1998. The day-by-day variation was mean 47% (range 25%-79%). Mean steps per day increased from the first to the second three-month period, 4341 vs 4781 steps, p = 0.022. The maximum differences in outcome measures between 4-week periods (highest vs lowest), were more evident in a group of eight patients with milder disease (baseline SF-36 PF > 50 or DSQ-SF < 55) as compared to 19 patients with higher symptom burden (SF-36 PF < 50 and DSQ-SF > 55), for SF-36 PF raw scores: 16.9 vs 3.4 points, and for steps per day: 958 versus 479 steps. The correlations between steps per day and self-reported SF-36 Physical function, SF-36 Social function, and DSQ-SF were significant. Fitbit recorded significantly higher number of steps than SenseWear. Resting heart rates were stable during six months. CONCLUSION: Continuous activity registration with Fitbit Charge 3 trackers is feasible and useful in studies with ME/CFS patients to monitor steps and resting heart rate, in addition to self-reported outcome measures. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov: NCT04195815.

Genetic alterations associated with malignant transformation of sporadic vestibular schwannoma.

INTRODUCTION: Malignant peripheral nerve sheath tumor of the vestibulocochlear nerve (VN-MPNST) is exceedingly rare and carries a poor prognosis. Little is known about its underlying genetics and in particular the process of malignant transformation. There is an ongoing debate on whether the transformation is initiated by ionizing radiation. We present here the analysis and comparison of two post-radiation VN-MPNST and one undergoing spontaneous transformation. METHODS: Four tumors from three patients (radiation-naïve vestibular schwannoma before (VS) and after (VN-MPNST) malignant transformation in addition to two post-radiation VN-MPNST) were subjected to DNA whole-genome microarray and whole-exome sequencing and tumor-specific mutations were called. Mutational signatures were characterized using MuSiCa. RESULTS: The tumor genomes were characterized predominantly by copy-number aberrations with 36-81% of the genome affected. Even the VS genome was grossly aberrated. The spontaneous malignant transformation was characterized by a near-total whole-genome doubling, disappearance of NF2 mutation and new mutations in three cancer-related genes (GNAQ, FOXO4 and PDGFRB). All tumors had homozygous loss of the tumor suppressor CDKN2A. Neither mutational signature nor copy number profile was associated with ionizing radiation. CONCLUSION: The VN-MPNST genome in our cases is characterized by large copy-number aberrations and homozygous deletion of CDKN2A. Our study demonstrates a VS with genetic alterations similar to its malignant counterpart, suggesting the existence of premalignant VS. No consistent mutational signature was associated with ionizing radiation.

Pellino-2 in nonimmune cells: novel interaction partners and intracellular localization.

Pellino-2 is an E3 ubiquitin ligase that mediates intracellular signaling in innate immune pathways. Most studies of endogenous Pellino-2 have been performed in macrophages, but none in nonimmune cells. Using yeast two-hybrid screening and co-immunoprecipitation, we identified six novel interaction partners of Pellino-2, with various localizations: insulin receptor substrate 1, NIMA-related kinase 9, tumor necrosis factor receptor-associated factor 7, cyclin-F, roundabout homolog 1, and disheveled homolog 2. Pellino-2 showed cytoplasmic localization in a wide range of nonimmune cells under physiological potassium concentrations. Treatment with the potassium ionophore nigericin resulted in nuclear localization of Pellino-2, which was reversed by the potassium channel blocker tetraethylammonium. Live-cell imaging revealed intracellular migration of GFP-tagged Pellino-2. In summary, Pellino-2 interacts with proteins at different cellular locations, taking part in dynamic processes that change its intracellular localization influenced by potassium efflux.

The SH3PXD2A-HTRA1 fusion transcript is extremely rare in Norwegian sporadic vestibular schwannoma patients.

INTRODUCTION: Vestibular schwannoma (VS) is a benign intracranial tumor in which the underlying genetics is largely uncertain, apart from mutations in the tumor suppressor gene NF2. Alternative tumorigenic mechanisms have been proposed, including a recurrent in-frame fusion transcript of the HTRA1 and SH3PXD2A genes. The gene product of the SH3PXD2A-HTRA1 fusion has been shown to promote proliferation, invasion and resistance to cell death in vitro and tumor growth in vivo. The aim of this study was to replicate the findings and to investigate the frequency of this fusion gene in another cohort of vestibular schwannoma patients. METHODS: The SH3PXD2A-HTRA1 transcript was synthesized in vitro using PCR and used as a positive control to assess the sensitivity of a real-time PCR assay. This real-time PCR assay was used to search for the presence of the fusion transcript in 121 Norwegian sporadic VS patients. RESULTS: The real-time PCR assay showed a high sensitivity and was able to detect as low as ~ 5 copies of the fusion transcript. Out of the 121 investigated tumors, only 1 harbored the SH3PXD2A-HTRA1 fusion. CONCLUSION: Even though the SH3PXD2A-HTRA1 fusion has been shown to be a driver of tumorigenesis, our results suggest that it is a rare event in our VS patients. Further investigation is warranted in order to elucidate whether our results represent an extreme, and if the fusion is present also in other neoplasms.

Temperature-dependent autoactivation associated with clinical variability of PDGFRB Asn666 substitutions.

Ocular pterygium-digital keloid dysplasia (OPDKD) presents in childhood with ingrowth of vascularized connective tissue on the cornea leading to severely reduced vision. Later the patients develop keloids on digits but are otherwise healthy. The overgrowth in OPDKD affects body parts that typically have lower temperature than 37°C. We present evidence that OPDKD is associated with a temperature sensitive, activating substitution, p.(Asn666Tyr), in PDGFRB. Phosphorylation levels of PDGFRB and downstream targets were higher in OPDKD fibroblasts at 37°C but were further greatly increased at the average corneal temperature of 32°C. This suggests that the substitution cause significant constitutive autoactivation mainly at lower temperature. In contrast, a different substitution in the same codon, p.(Asn666Ser), is associated with Penttinen type of premature aging syndrome. This devastating condition is characterized by widespread tissue degeneration, including pronounced chronic ulcers and osteolytic resorption in distal limbs. In Penttinen syndrome fibroblasts, equal and high levels of phosphorylated PDGFRB was present at both 32°C and 37°C. This indicates that this substitution causes severe constitutive autoactivation of PDGFRB regardless of temperature. In line with this, most downstream targets were not affected by lower temperature. However, STAT1, important for tissue wasting, did show further increased phosphorylation at 32°C. Temperature-dependent autoactivation offers an explanation to the strikingly different clinical outcomes of substitutions in the Asn666 codon of PDGFRB.

Gamma Knife Radiosurgery does not alter the copy number aberration profile in sporadic vestibular schwannoma.

INTRODUCTION: Ionizing radiation is a known etiologic factor in tumorigenesis and its role in inducing malignancy in the treatment of vestibular schwannoma has been debated. The purpose of this study was to identify a copy number aberration (CNA) profile or specific CNAs associated with radiation exposure which could either implicate an increased risk of malignancy or elucidate a mechanism of treatment resistance. METHODS: 55 sporadic VS, including 18 treated with Gamma Knife Radiosurgery (GKRS), were subjected to DNA whole-genome microarray and/or whole-exome sequencing. CNAs were called and statistical tests were performed to identify any association with radiation exposure. Hierarchical clustering was used to identify CNA profiles associated with radiation exposure. RESULTS: A median of 7 (0-58) CNAs were identified across the 55 VS. Chromosome 22 aberration was the only recurrent event. A median aberrant cell fraction of 0.59 (0.25-0.94) was observed, indicating several genetic clones in VS. No CNA or CNA profile was associated with GKRS. CONCLUSION: GKRS is not associated with an increase in CNAs or alteration of the CNA profile in VS, lending support to its low risk. This also implies that there is no major issue with GKRS treatment failure being due to CNAs. In agreement with previous studies, chromosome 22 aberration is the only recurrent CNA. VS consist of several genetic clones, addressing the need for further studies on the composition of cells in this tumor.

B-Lymphocyte Depletion in Patients With Myalgic Encephalomyelitis/Chronic Fatigue Syndrome: A Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Previous phase 2 trials indicated benefit from B-lymphocyte depletion in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Objective: To evaluate the effect of the monoclonal anti-CD20 antibody rituximab versus placebo in patients with ME/CFS. Design: Randomized, placebo-controlled, double-blind, multicenter trial. (ClinicalTrials.gov: NCT02229942). Setting: 4 university hospitals and 1 general hospital in Norway. Patients: 151 patients aged 18 to 65 years who had ME/CFS according to Canadian consensus criteria and had had the disease for 2 to 15 years. Intervention: Treatment induction with 2 infusions of rituximab, 500 mg/m2 of body surface area, 2 weeks apart, followed by 4 maintenance infusions with a fixed dose of 500 mg at 3, 6, 9, and 12 months (n = 77), or placebo (n = 74). Measurements: Primary outcomes were overall response rate (fatigue score ≥4.5 for ≥8 consecutive weeks) and repeated measurements of fatigue score over 24 months. Secondary outcomes included repeated measurements of self-reported function over 24 months, components of the Short Form-36 Health Survey and Fatigue Severity Scale over 24 months, and changes from baseline to 18 months in these measures and physical activity level. Between-group differences in outcome measures over time were assessed by general linear models for repeated measures. Results: Overall response rates were 35.1% in the placebo group and 26.0% in the rituximab group (difference, 9.2 percentage points [95% CI, -5.5 to 23.3 percentage points]; P = 0.22). The treatment groups did not differ in fatigue score over 24 months (difference in average score, 0.02 [CI, -0.27 to 0.31]; P = 0.80) or any of the secondary end points. Twenty patients (26.0%) in the rituximab group and 14 (18.9%) in the placebo group had serious adverse events. Limitation: Self-reported primary outcome measures and possible recall bias. Conclusion: B-cell depletion using several infusions of rituximab over 12 months was not associated with clinical improvement in patients with ME/CFS. Primary Funding Source: The Norwegian Research Council, Norwegian Regional Health Trusts, Kavli Trust, MEandYou Foundation, and Norwegian ME Association.

A tyrosine kinase-activating variant Asn666Ser in PDGFRB causes a progeria-like condition in the severe end of Penttinen syndrome.

Missense variants located to the "molecular brake" in the tyrosine kinase hinge region of platelet-derived growth factor receptor-ß, encoded by PFGFRB, can cause Penttinen-type (Val665Ala) and Penttinen-like (Asn666His) premature ageing syndromes, as well as infantile myofibromatosis (Asn666Lys and Pro660Thr). We have found the same de novo PDGFRB c.1997A>G p.(Asn666Ser) variants in two patients with lipodystrophy, acro-osteolysis and severely reduced vision due to corneal neovascularisation, reminiscent of a severe form of Penttinen syndrome with more pronounced connective tissue destruction. In line with this phenotype, patient skin fibroblasts were prone to apoptosis. Both in patient fibroblasts and stably transduced HeLa and HEK293 cells, autophosphorylation of PDGFRß was observed, as well as increased phosphorylation of downstream signalling proteins such as STAT1, PLCγ1, PTPN11/SHP2-Tyr580 and AKT. Phosphorylation of MAPK3 (ERK1) and PTPN11/SHP2-Tyr542 appeared unaffected. This suggests that this missense change not only weakens tyrosine kinase autoinhibition, but also influences substrate binding, as both PTPN11 tyrosines (Tyr542 and Tyr580) usually are phosphorylated upon PDGFR activation. Imatinib was a strong inhibitor of phosphorylation of all these targets, suggesting an option for precision medicine based treatment.

Screening for viral nucleic acids in vestibular schwannoma.

To investigate if viruses are involved in the pathogenesis of vestibular schwannomas (VS), we have screened biopsies from VS patients using different molecular techniques. Screening for the presence of known viruses using a pan-viral microarray assay (ViroChip) indicated the presence of several viruses including human endogenous retrovirus K (HERV-K) and human herpes virus 2 (HHV2). But with the exception of HERV-K, none of the findings could be verified by other methods. Whole transcriptome sequencing showed only the presence of HERV-K transcripts and whole genome sequencing showed only the presence of Epstein-Barr virus, most likely originating from infiltration of lymphocytes. We therefore conclude that it is less likely that viruses are involved in the pathogenesis of vestibular schwannomas.

Genetic landscape of sporadic vestibular schwannoma.

OBJECTIVE Vestibular schwannoma (VS) is a benign tumor with associated morbidities and reduced quality of life. Except for mutations in NF2, the genetic landscape of VS remains to be elucidated. Little is known about the effect of Gamma Knife radiosurgery (GKRS) on the VS genome. The aim of this study was to characterize mutations occurring in this tumor to identify new genes and signaling pathways important for the development of VS. In addition, the authors sought to evaluate whether GKRS resulted in an increase in the number of mutations. METHODS Forty-six sporadic VSs, including 8 GKRS-treated tumors and corresponding blood samples, were subjected to whole-exome sequencing and tumor-specific DNA variants were called. Pathway analysis was performed using the Ingenuity Pathway Analysis software. In addition, multiplex ligation-dependent probe amplification was performed to characterize copy number variations in the NF2 gene, and microsatellite instability testing was done to investigate for DNA replication error. RESULTS With the exception of a single sample with an aggressive phenotype that harbored a large number of mutations, most samples showed a relatively low number of mutations. A median of 14 tumor-specific mutations in each sample were identified. The GKRS-treated tumors harbored no more mutations than the rest of the group. A clustering of mutations in the cancer-related axonal guidance pathway was identified (25 patients), as well as mutations in the CDC27 (5 patients) and USP8 (3 patients) genes. Thirty-five tumors harbored mutations in NF2 and 16 tumors had 2 mutational hits. The samples without detectable NF2 mutations harbored mutations in genes that could be linked to NF2 or to NF2-related functions. None of the tumors showed microsatellite instability. CONCLUSIONS The genetic landscape of VS seems to be quite heterogeneous; however, most samples had mutations in NF2 or in genes that could be linked to NF2. The results of this study do not link GKRS to an increased number of mutations.

Serum BAFF and APRIL Levels, T-Lymphocyte Subsets, and Immunoglobulins after B-Cell Depletion Using the Monoclonal Anti-CD20 Antibody Rituximab in Myalgic Encephalopathy/Chronic Fatigue Syndrome.

Myalgic Encephalopathy/Chronic Fatigue Syndrome (ME/CFS) is a disease of unknown etiology. We have previously suggested clinical benefit from B-cell depletion using the monoclonal anti-CD20 antibody rituximab in a randomized and placebo-controlled study. Prolonged responses were then demonstrated in an open-label phase-II study with maintenance rituximab treatment. Using blood samples from patients in the previous two clinical trials, we investigated quantitative changes in T-lymphocyte subsets, in immunoglobulins, and in serum levels of two B-cell regulating cytokines during follow-up. B-lymphocyte activating factor of the tumor necrosis family (BAFF) in baseline serum samples was elevated in 70 ME/CFS patients as compared to 56 healthy controls (p = 0.011). There were no significant differences in baseline serum BAFF levels between patients with mild, moderate, or severe ME/CFS, or between responders and non-responders to rituximab. A proliferation-inducing ligand (APRIL) serum levels were not significantly different in ME/CFS patients compared to healthy controls at baseline, and no changes in serum levels were seen during follow-up. Immunophenotyping of peripheral blood T-lymphocyte subsets and T-cell activation markers at multiple time points during follow-up showed no significant differences over time, between rituximab and placebo groups, or between responders and non-responders to rituximab. Baseline serum IgG levels were significantly lower in patients with subsequent response after rituximab therapy compared to non-responders (p = 0.03). In the maintenance study, slight but significant reductions in mean serum immunoglobulin levels were observed at 24 months compared to baseline; IgG 10.6-9.5 g/L, IgA 1.8-1.5 g/L, and IgM 0.97-0.70 g/L. Although no functional assays were performed, the lack of significant associations of T- and NK-cell subset numbers with B-cell depletion, as well as the lack of associations to clinical responses, suggest that B-cell regulatory effects on T-cell or NK-cell subsets are not the main mechanisms for the observed improvements in ME/CFS symptoms observed in the two previous trials. The modest increase in serum BAFF levels at baseline may indicate an activated B-lymphocyte system in a subgroup of ME/CFS patients.

Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

S100A16 promotes differentiation and contributes to a less aggressive tumor phenotype in oral squamous cell carcinoma.

BACKGROUND: Altered expression of S100A16 has been reported in human cancers, but its biological role in tumorigenesis is not fully understood. This study aimed to investigate the clinical significance and functional role of S100A16 in oral squamous cell carcinoma (OSCC) suppression. METHODS: S100A16 mRNA and/or protein levels were examined by quantitative RT-PCR and immunohistochemistry in whole- and laser microdissected-specimens of normal human oral mucosa (NHOM, n = 65), oral dysplastic lesions (ODL, n = 21), OSCCs (n = 132) and positive cervical nodes (n = 17). S100A16 protein expression in OSCC was examined for correlations with clinicopathological variables and patient survival. S100A16 was over-expressed and knocked-down in OSCC-derived (CaLH3 and H357) cells by employing retroviral constructs to investigate its effects on cell proliferation, sphere formation and three dimensional (3D)-organotypic invasive abilities in vitro and tumorigenesis in a mouse xenograft model. RESULTS: Both S100A16 mRNA and protein levels were found to be progressively down-regulated from NHOM to ODL and OSCC. Low S100A16 protein levels in OSCC significantly correlated with reduced 10-year overall survival and poor tumor differentiation. Analysis of two external OSCC microarray datasets showed a positive correlation between the mRNA expression levels of S100A16 and keratinocyte differentiation markers. CaLH3 and H357 cell fractions enriched for differentiated cells either by lack of adherence to collagen IV or FACS sorting for low p75NTR expression expressed significantly higher S100A16 mRNA levels than the subpopulations enriched for less differentiated cells. Corroborating these findings, retroviral mediated S100A16 over-expression and knock-down in CaLH3 and H357 cells led to respective up- and down-regulation of differentiation markers. In vitro functional studies showed significant reduction in cell proliferation, sphere formation and 3D-invasive abilities of CaLH3 and H357 cells upon S100A16 over-expression. These functional effects were associated with concomitant down-regulation of self-renewal (Bmi-1 and Oct 4A) and invasion related (MMP1 and MMP9) molecules. S100A16 over-expression also suppressed tumorigenesis of H357 cells in a mouse xenograft model and the resulting tumor xenografts displayed features/expression of increased differentiation and reduced proliferation/self-renewal. CONCLUSIONS: These results indicate that S100A16 is a differentiation promoting protein and might function as a tumor suppressor in OSCC.

B-Lymphocyte Depletion in Myalgic Encephalopathy/ Chronic Fatigue Syndrome. An Open-Label Phase II Study with Rituximab Maintenance Treatment.

BACKGROUND: Myalgic Encephalopathy/Chronic Fatigue Syndrome (ME/CFS) is a disease of unknown etiology. We previously reported a pilot case series followed by a small, randomized, placebo-controlled phase II study, suggesting that B-cell depletion using the monoclonal anti-CD20 antibody rituximab can yield clinical benefit in ME/CFS. METHODS: In this single-center, open-label, one-armed phase II study (NCT01156909), 29 patients were included for treatment with rituximab (500 mg/m2) two infusions two weeks apart, followed by maintenance rituximab infusions after 3, 6, 10 and 15 months, and with follow-up for 36 months. FINDINGS: Major or moderate responses, predefined as lasting improvements in self-reported Fatigue score, were detected in 18 out of 29 patients (intention to treat). Clinically significant responses were seen in 18 out of 28 patients (64%) receiving rituximab maintenance treatment. For these 18 patients, the mean response durations within the 156 weeks study period were 105 weeks in 14 major responders, and 69 weeks in four moderate responders. At end of follow-up (36 months), 11 out of 18 responding patients were still in ongoing clinical remission. For major responders, the mean lag time from first rituximab infusion until start of clinical response was 23 weeks (range 8-66). Among the nine patients from the placebo group in the previous randomized study with no significant improvement during 12 months follow-up after saline infusions, six achieved a clinical response before 12 months after rituximab maintenance infusions in the present study. Two patients had an allergic reaction to rituximab and two had an episode of uncomplicated late-onset neutropenia. Eight patients experienced one or more transient symptom flares after rituximab infusions. There was no unexpected toxicity. CONCLUSION: In a subgroup of ME/CFS patients, prolonged B-cell depletion with rituximab maintenance infusions was associated with sustained clinical responses. The observed patterns of delayed responses and relapse after B-cell depletion and regeneration, a three times higher disease prevalence in women than in men, and a previously demonstrated increase in B-cell lymphoma risk for elderly ME/CFS patients, suggest that ME/CFS may be a variant of an autoimmune disease. TRIAL REGISTRATION: ClinicalTrials.gov NCT01156909.

S100A14 interacts with S100A16 and regulates its expression in human cancer cells.

Both S100A14 and S100A16 are members of the multifunctional S100 protein family. Formation of homo/heterodimers is considered to be one of the major mechanisms for S100 proteins to execute their diverse cellular functions. By employing a classical Yeast two hybrid (Y-2 H) screen, we identified S100A16 as the single interaction partner of S100A14. This interaction was verified by co-immunoprecipitation, double indirect immunofluorescence and double immunostaining in specimens of oral squamous cell carcinoma and normal oral mucosa. The functional significance of this interaction was examined by employing retroviral mediated over-expression and knock-down of these proteins in several cancer cell-lines. Over-expression and knock-down of S100A14 led to concomitant up- and down-regulation of S100A16 protein in the cell-lines examined. However, there was no up-regulation of S100A16 mRNA upon S100A14 over-expression, indicating that modulation of S100A16 expression was not due to enhanced transcriptional activity but possibly by post-transcriptional regulation. In contrary, over-expression of S100A16 was associated neither with the up-regulation of S100A14 mRNA nor its protein, suggesting a unidirectional regulation between S100A14 and S100A16. Cellular treatment with protein synthesis inhibitor cycloheximide demonstrated a time-dependent intracellular degradation of both S100A16 and S100A14 proteins. Additionally, regulation of S100A16 and S100A14 degradation was found to be independent of the classical proteasomal and lysosomal pathways of protein degradation. Further studies will therefore be necessary to understand the functional significance of this interaction and the mechanisms on how S100A14 is involved in the regulation of S100A16 expression.

S100A14 inhibits proliferation of oral carcinoma derived cells through G1-arrest.

Altered expression of S100A14 has been reported in various human cancers including oral squamous cell carcinomas (OSCCs). Its biological functions in carcinogenesis, however, are largely unknown. This study aimed to investigate the functional role of S100A14 in tumor cell proliferation and its possible functional association with p53. S100A14 protein was found to be gradually down-regulated during the transition from normal to dysplastic and carcinoma cells in an in vitro human OSCC progression model. When over-expressed by employing retroviral expression vector, S100A14 inhibited proliferation of CaLH3 and OSCC1, OSCC cell-lines harboring wild type (wt) p53, by inducing G1-arrest. This G1-arrest correlated with up-regulation of p21 both in the CaLH3 and OSCC1 cell-lines. shRNA mediated silencing of p53 led to partial suppression of p21 in S100A14 over-expressing CaLH3 cells, indicating that p21 up-regulation was, at least, partly dependent on p53. We further demonstrated that nuclear accumulation of p53 occurred with over-expression of S100A14 in CaLH3 cells. Our data suggest a novel role of S100A14 in OSCC cell proliferation by inducing G1-arrest and also indicate a functional link between S100A14 and the tumor suppressor protein p53.

Benefit from B-lymphocyte depletion using the anti-CD20 antibody rituximab in chronic fatigue syndrome. A double-blind and placebo-controlled study.

BACKGROUND: Chronic fatigue syndrome (CFS) is a disease of unknown aetiology. Major CFS symptom relief during cancer chemotherapy in a patient with synchronous CFS and lymphoma spurred a pilot study of B-lymphocyte depletion using the anti-CD20 antibody Rituximab, which demonstrated significant clinical response in three CFS patients. METHODS AND FINDINGS: In this double-blind, placebo-controlled phase II study (NCT00848692), 30 CFS patients were randomised to either Rituximab 500 mg/m(2) or saline, given twice two weeks apart, with follow-up for 12 months. Xenotropic murine leukemia virus-related virus (XMRV) was not detected in any of the patients. The responses generally affected all CFS symptoms. Major or moderate overall response, defined as lasting improvements in self-reported Fatigue score during follow-up, was seen in 10 out of 15 patients (67%) in the Rituximab group and in two out of 15 patients (13%) in the Placebo group (p = 0.003). Mean response duration within the follow-up period for the 10 responders to Rituximab was 25 weeks (range 8-44). Four Rituximab patients had clinical response durations past the study period. General linear models for repeated measures of Fatigue scores during follow-up showed a significant interaction between time and intervention group (p = 0.018 for self-reported, and p = 0.024 for physician-assessed), with differences between the Rituximab and Placebo groups between 6-10 months after intervention. The primary end-point, defined as effect on self-reported Fatigue score 3 months after intervention, was negative. There were no serious adverse events. Two patients in the Rituximab group with pre-existing psoriasis experienced moderate psoriasis worsening. CONCLUSION: The delayed responses starting from 2-7 months after Rituximab treatment, in spite of rapid B-cell depletion, suggests that CFS is an autoimmune disease and may be consistent with the gradual elimination of autoantibodies preceding clinical responses. The present findings will impact future research efforts in CFS. TRIAL REGISTRATION: ClinicalTrials.gov NCT00848692.

Disruption of a long distance regulatory region upstream of SOX9 in isolated disorders of sex development.

BACKGROUND: The early gonad is bipotential and can differentiate into either a testis or an ovary. In XY embryos, the SRY gene triggers testicular differentiation and subsequent male development via its action on a single gene, SOX9. The supporting cell lineage of the bipotential gonad will differentiate as testicular Sertoli cells if SOX9 is expressed and conversely will differentiate as ovarian granulosa cells when SOX9 expression is switched off. RESULTS: Through copy number variation mapping this study identified duplications upstream of the SOX9 gene in three families with an isolated 46,XX disorder of sex development (DSD) and an overlapping deletion in one family with two probands with an isolated 46,XY DSD. The region of overlap between these genomic alterations, and previously reported deletions and duplications at the SOX9 locus associated with syndromic and isolated cases of 46,XX and 46,XY DSD, reveal a minimal non-coding 78 kb sex determining region located in a gene desert 517-595 kb upstream of the SOX9 promoter. CONCLUSIONS: These data indicate that a non-coding regulatory region critical for gonadal SOX9 expression and subsequent normal sex development is located far upstream of the SOX9 promoter. Its copy number variations are the genetic basis of isolated 46,XX and 46,XY DSDs of variable severity (ranging from mild to complete sex reversal). It is proposed that this region contains a gonad specific SOX9 transcriptional enhancer(s), the gain or loss of which results in genomic imbalance sufficient to activate or inactivate SOX9 gonadal expression in a tissue specific manner, switch sex determination, and result in isolated DSD.