Ove Bruland

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/m2 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.

NATH, a novel gene overexpressed in papillary thyroid carcinomas.

In this study a replica cDNA screening (RCS) approach to identify genes differentially expressed in papillary thyroid carcinomas (PTC) was used, as compared to non-neoplastic thyroid tissues. RCS is based on hybridization of radioactively labeled cDNA probes made from the biopsies to replica membranes with 15 000 clones from a PTC cDNA library. Among the genes overexpressed in PTC, and especially in clinically aggressive tumors with histologic evidence of poorly differentiated or undifferentiated areas, a novel gene named NATH was found. NATH has two mRNA species, 4.6 and 5.8 kb, both harboring the same open reading frame encoding a putative protein of 866 amino acids. The NATH protein is homologous to yeast N-acetyltransferase (NAT)1 and to mouse NARG1 (mNAT1) and contains four tetratricopeptide repeat (TPR) domains, suggesting that NATH may be part of a multiprotein complex. Overlapping RT–PCR fragments from several PTC biopsies confirmed the NATH mRNA sequence. Northern blots, semiquantitative RT–PCR experiments, TaqMan real-time RT–PCR experiments, and in situ hybridization verified the overexpression of NATH mRNA localized to tumor cells in PTC biopsies. NATH was expressed at a low level in most human adult tissues, including the normal thyroid gland. Increased NATH expression was seen especially in a Burkitt lymphoma cell line and in adult human testis. Recombinant in vitro expression showed that NATH protein was located mainly in the cytoplasm, and was present as a single protein band of the expected 105 kDa molecular weight. Heterologous expression of NATH in the papillary carcinoma cell line (NPA) and 293 cells did not alter the cellular proliferation rate. The biological function of NATH remains to be elucidated, but the overexpression in classic PTC and especially in poorly differentiated or undifferentiated components may indicate a function in the progression of papillary thyroid carcinomas.

Serum/glucocorticoid-regulated kinase 1 (SGK1) is a prominent target gene of the transcriptional response to cytokines in multiple myeloma and supports the growth of myeloma cells.

Multiple myeloma (MM) is a paradigm for a malignant disease that exploits external stimuli of the microenvironment for growth and survival. A thorough understanding of the complex interactions between malignant plasma cells and their surrounding requires a detailed analysis of the transcriptional response of myeloma cells to environmental signals. We determined the changes in gene expression induced by interleukin (IL)-6, tumor necrosis factor-α, IL-21 or co-culture with bone marrow stromal cells in myeloma cell lines. Among a limited set of genes that were consistently activated in response to growth factors, a prominent transcriptional target of cytokine-induced signaling in myeloma cells was the gene encoding the serine/threonine kinase serum/glucocorticoid-regulated kinase 1 (SGK1), which is a down-stream effector of PI3-kinase. We could demonstrate a rapid, strong and sustained induction of SGK1 in the cell lines INA-6, ANBL-6, IH-1, OH-2 and MM.1S as well as in primary myeloma cells. Pharmacologic inhibition of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway abolished STAT3 phosphorylation and SGK1 induction. In addition, small hairpin RNA (shRNA)-mediated knock-down of STAT3 reduced basal and induced SGK1 levels. Furthermore, downregulation of SGK1 by shRNAs resulted in decreased proliferation of myeloma cell lines and reduced cell numbers. On the molecular level, this was reflected by the induction of cell cycle inhibitory genes, for example, CDKNA1/p21, whereas positively acting factors such as CDK6 and RBL2/p130 were downregulated. Our results indicate that SGK1 is a highly cytokine-responsive gene in myeloma cells promoting their malignant growth.

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 regulates the invasive potential of oral squamous cell carcinoma derived cell-lines in vitro by modulating expression of matrix metalloproteinases, MMP1 and MMP9

Despite the differential expression of S100A14 (a newly identified S100 member) in various human cancers including oral squamous cell carcinomas (OSCCs), its biological role in tumour invasion has not been characterised. The aim of this study was thus to investigate the possible role of S100A14 in OSCC cell invasion. Using immunohistochemistry in normal (n=13), dysplastic (n=10) and OSCC (n=16) archival tissues, S100A14 protein was found to be down-regulated/lost with concomitant membrane to cytoplasmic translocation in OSCCs, especially in the invading tumour islands. These expression data were corroborated by profiling S100A14 mRNA expression using quantitative RT-PCR (qRT-PCR) in an in vitro human OSCC progression model consisting of cell-lines derived from normal (n=3), dysplastic (n=3) and OSCC (n=8) tissues. Employing in vitro Matrigel invasion assay, we demonstrated that retroviral vector mediated over-expression of S100A14 resulted in significant decrease in the invasive potential of OSCC derived CaLH3 and H357 cell-lines whereas siRNA mediated knockdown resulted in significant increase in the invasive potential of CaLH3 cell-line. Pathway focused PCR array and validation using qRT-PCR revealed that S100A14 over-expression was associated with down-regulation of MMP1 and MMP9 mRNAs in both CaLH3 and H357 cell-lines. Further, S100A14 over-expression was found to be associated with suppression of MMP9 gelatinolytic activity in CaLH3 cell-line. Additionally, an inverse correlation between mRNA expression levels of MMP1 and MMP9 with S100A14 was found in 19 cases of OSCCs. Collectively, these data provide the first evidence for a role of S100A14 protein in regulation of OSCC cell invasion by modulating expression of MMP1 and MMP9.

MicroRNA-15b is induced with E2F-controlled genes in HPV-related cancer.

Background:MicroRNAs (miRNAs) are important regulators of cellular processes and are found to be deregulated in many cancers. We here analysed the miRNA expression in anal carcinomas. In a previous study, we found that our anal carcinoma tumours were divided into two groups based on the expression of E2F-regulated genes. Therefore, we searched for miRNAs that could reproduce this grouping.Methods:A global screen of the miRNA population was performed using real-time quantitative PCR (RT–qPCR) array methods and differentially expressed miRNAs were identified. Real-time–qPCR was used to verify the expression levels of selected miRNAs and genes in a larger collection of biopsies. A siRNA-mediated knockdown of human papilloma virus (HPV)16 E7 in a cervical cell line was performed to assess the effect of E7 on miR-15b.Results:The grouping of tumours into two groups based on the expression of E2F-controlled genes was confirmed in a larger collection of anal carcinoma tumours. The expression of miR-15b was shown to be highly correlated with that of five selected E2F-induced genes (CCNA2, CCNB1, CCNB2, MSH6 and MCM7). A knockdown of HPV16 E7 resulted in decreased levels of miR-15b in Ca Ski cells.Conclusion:MiR-15b expression correlates with E2F-regulated genes in anal carcinoma and appears to be part of the E2F-regulatory network.

Expression profile of the S100 gene family members in oral squamous cell carcinomas

BACKGROUND Several of the S100 gene members have been reported to be differentially expressed in many human pathological conditions, in particular, the malignancies. Identification and quantification of the differentially expressed S100 gene members in oral squamous cell carcinoma (OSCC) might facilitate their use as potential diagnostic and/or prognostic markers or targets for therapy. METHODS we examined the expression profile of 16 members of the S100 gene family at the mRNA level by semiquantitative reverse transcription-polymerase chain reaction (sRT-PCR) in 27 cases of OSCCs/their pair-wised normal controls obtained from Sudanese patients, and confirmed the sRT-PCR results by performing quantitative real time-polymerase chain reaction (qRT-PCR) for 6 of the 16 genes examined. RESULTS With sRT-PCR, 4 (25%; S100A4, S100A6, S100A8, S100A14) out of the 16 S100 gene members examined were found to be significantly down-regulated (P < 0.05) in the tumors compared to the normal controls. None of the S100 gene members examined were found to be significantly up-regulated in the tumors. qRT-PCR results confirmed the significant down-regulation of the S100A4, S100A6, and S100A14 genes in the tumors examined. CONCLUSION S100 gene family members might play an important role in the pathogenesis of the OSCCs examined. Findings of the present work warrant in-depth studies of the S100 gene family members, in particular, the S100A4, S100A6, S100A8, and S100A14 to further understand their possible role(s) in OSCC tumorigenesis.

Accurate determination of the number of CAG repeats in the Huntington disease gene using a sequence-specific internal DNA standard

We have developed a sequence‐specific internal DNA size standard for the accurate determination of the number of CAG repeats in the Huntington disease (HD) gene by cloning key fragments (between 15 and 64 CAG repeats) of the HD gene. These fragments, pooled to produce a sequence‐specific DNA ladder, enabled us to observe the true number of CAG repeats directly, with no need for calculations. Comparison of the calculated numbers of CAG repeats in the HD gene using this sequence‐specific DNA standard with a commercially available standard (GENESCAN‐500 TAMRA) showed that the latter underestimated the number of CAG repeats by three when analyzed by capillary electrophoresis on the ABI 310 Genetic Analyzer (POP4 polymer). In contrast, the use of the same standard overestimated the number of CAG repeats by one when the samples were analyzed by denaturing polyacrylamide electrophoresis on ABI 377 DNA Sequencer (6% denaturing polyacrylamide gel). This suggests that our sequence‐specific standard provides greater accuracy for the determination of the true number of CAG repeats in the HD gene than commercially available standards. The sequence‐specific standard can be radioactively labeled and successfully replace conventional DNA size standards when analyzing polymerase chain reaction (PCR)‐amplified HD alleles by denaturing polyacrylamide electrophoresis.

AIRE variations in Addison's disease and autoimmune polyendocrine syndromes (APS): partial gene deletions contribute to APS I

Autoimmune Addisons disease (AAD) is often associated with other components in autoimmune polyendocrine syndromes (APS). Whereas APS I is caused by mutations in the AIRE gene, the susceptibility genes for AAD and APS II are unclear. In the present study, we investigated whether polymorphisms or copy number variations in the AIRE gene were associated with AAD and APS II. First, nine SNPs in the AIRE gene were analyzed in 311 patients with AAD and APS II and 521 healthy controls, identifying no associated risk. Second, in a subgroup of 25 of these patients, AIRE sequencing revealed three novel polymorphisms. Finally, the AIRE copy number was determined by duplex quantitative PCR in 14 patients with APS I, 161 patients with AAD and APS II and in 39 healthy subjects. In two Scandinavian APS I patients previously reported to be homozygous for common AIRE mutations, we identified large deletions of the AIRE gene covering at least exon 2 to exon 8. We conclude that polymorphisms in the AIRE gene are not associated with AAD and APS II. We further suggest that DNA analysis of the parents of patients found to be homozygous for mutations in AIRE, always should be performed.