Dipak Sapkota

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

Prevalence of viral (HPV, EBV, HSV) infections in oral submucous fibrosis and oral cancer from India

Abstract Conclusion: There is a high prevalence of human papilloma viruses (HPV) in oral submucous fibrosis (OSMF) and the etiologic implication of this finding warrants further studies. Objective: The prevalence of oral squamous cell carcinoma (OSCC) and OSMF is high in India, and the diseases are partly attributed to high consumption of betel quid containing areca nut and tobacco. This study investigated the prevalence of HPV, herpes simplex virus (HSV), and Epstein–Barr virus (EBV) DNA in two groups of patients using betel quid with tobacco, those with OSMF (n = 12) and those with OSCC (n = 62). Methods: DNA was extracted from all the samples and viral genome was examined by PCR/DNA sequencing. HPV-positive samples were analyzed separately for the high-risk types HPV 16 and 18. Results: HPV DNA, HSV DNA, and EBV DNA were detected in 11 (91%), 1 (8%), and 3 (25%) of the 12 samples from patients with OSMF compared with 15 (24%), 3 (5%), and 18 (29%), respectively, from 62 patients with OSCC. HPV 16 and 18 DNA was detected in 8/12 (67%) in the OSMF group and 10/62 (16%) in the OSCC group. The difference between presence of HPV DNA in OSMF and OSCC groups was statistically significant, while the difference between HSV and EBV DNA content in OSMF and OSCC groups was insignificant.

Presence of human papilloma virus, herpes simplex virus and Epstein-Barr virus DNA in oral biopsies from Sudanese patients with regard to toombak use

Using PCR/DNA sequencing, we investigated the prevalence of human papillomavirus (HPV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) DNA in brush biopsies obtained from 150 users of Sudanese snuff (toombak) and 25 non-users of toombak in formalin-fixed paraffin-embedded tissue samples obtained from 31 patients with oral dysplasias (25 toombak users and 6 non-users), and from 217 patients with oral cancers (145 toombak users and 72 non-users). In the brush tissue samples from toombak users, HPV was detected in 60 (40%), HSV in 44 (29%) and EBV in 97 (65%) of the samples. The corresponding figures for the 25 samples from non-users were 17 (68%) positive for HPV, 6 (24%) positive for HSV and 21 (84%) for EBV. The formalin-fixed samples with oral dysplasias were all negative for HPV. In the 145 oral cancer samples from toombak users, HPV was detected in 39 (27%), HSV in 15 (10%) and EBV in 53 (37%) of the samples. The corresponding figures for the samples from non-users were 15 (21%) positive for HPV, 5 (7%) for HSV and 16 (22%) for EBV. These findings illustrate that prevalence of HSV, HPV and EBV infections are common and may influence oral health and cancer development. It is not obvious that cancer risk is increased in infected toombak users. These observations warrant further studies involving toombak-associated oral lesions, to uncover the possible mechanisms of these viral infections in the development of oral cancer, and the influence of toombak on these viruses.

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.

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.

Metabolic profiling indicates impaired pyruvate dehydrogenase function in myalgic encephalopathy/chronic fatigue syndrome

Myalgic encephalopathy/chronic fatigue syndrome (ME/CFS) is a debilitating disease of unknown etiology, with hallmark symptoms including postexertional malaise and poor recovery. Metabolic dysfunction is a plausible contributing factor. We hypothesized that changes in serum amino acids may disclose specific defects in energy metabolism in ME/CFS. Analysis in 200 ME/CFS patients and 102 healthy individuals showed a specific reduction of amino acids that fuel oxidative metabolism via the TCA cycle, mainly in female ME/CFS patients. Serum 3-methylhistidine, a marker of endogenous protein catabolism, was significantly increased in male patients. The amino acid pattern suggested functional impairment of pyruvate dehydrogenase (PDH), supported by increased mRNA expression of the inhibitory PDH kinases 1, 2, and 4; sirtuin 4; and PPARδ in peripheral blood mononuclear cells from both sexes. Myoblasts grown in presence of serum from patients with severe ME/CFS showed metabolic adaptations, including increased mitochondrial respiration and excessive lactate secretion. The amino acid changes could not be explained by symptom severity, disease duration, age, BMI, or physical activity level among patients. These findings are in agreement with the clinical disease presentation of ME/CFS, with inadequate ATP generation by oxidative phosphorylation and excessive lactate generation upon exertion.

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.

MicroRNAs as Important Players and Biomarkers in Oral Carcinogenesis.

Oral cancer, represented mainly by oral squamous cell carcinoma (OSCC), is the eighth most common type of human cancer worldwide. The number of new OSCC cases is increasing worldwide, especially in the low-income countries, and the prognosis remains poor in spite of recent advances in the diagnostic and therapeutic modalities. MicroRNAs (miRNAs), 18–25 nucleotides long noncoding RNA molecules, have recently gained significant attention as potential regulators and biomarkers for carcinogenesis. Recent data show that several miRNAs are deregulated in OSCC, and they have either a tumor suppressive or an oncogenic role in oral carcinogenesis. This review summarizes current knowledge on the role of miRNAs as tumor promotors or tumor suppressors in OSCC development and discusses their potential value as diagnostic and prognostic markers in OSCC.

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.