Safedin Beqaj

Stretch-induced alternative splicing of serum response factor promotes bronchial myogenesis and is defective in lung hypoplasia

Smooth muscle (SM) develops only in organs and sites that sustain mechanical tensions. Therefore, we determined the role of stretch in mouse and human bronchial myogenesis. Sustained stretch induced expression of SM proteins in undifferentiated mesenchymal cells and accelerated the differentiation of cells undergoing myogenesis. Moreover, bronchial myogenesis was entirely controlled in lung organ cultures by the airway intraluminal pressure. Serum response factor (SRF) is a transcription factor critical for the induction of muscle-specific gene expression. Recently, a SRF-truncated isoform produced by alternative splicing of exon 5 has been identified (SRFDelta5). Here we show that undifferentiated mesenchymal cells synthesize both SRF and SRFDelta5 but that SRFDelta5 synthesis is suppressed during bronchial myogenesis in favor of increased SRF production. Stretch induces the same change in SRF alternative splicing, and its myogenic effect is abrogated by overexpressing SRFDelta5. Furthermore, human hypoplastic lungs related to conditions that hinder cell stretching continue to synthesize SRFDelta5 and show a marked decrease in bronchial and interstitial SM cells and their ECM product, tropoelastin. Taken together, our findings indicate that stretch plays a critical role in SM myogenesis and suggest that its decrease precludes normal bronchial muscle development.

High RhoA activity maintains the undifferentiated mesenchymal cell phenotype, whereas RhoA down-regulation by laminin-2 induces smooth muscle myogenesis.

Round embryonic mesenchymal cells have the potential to differentiate into smooth muscle (SM) cells upon spreading/elongation (Yang, Y., K.C. Palmer, N. Relan, C. Diglio, and L. Schuger. 1998. Development. 125:2621–2629; Yang, Y., N.K. Relan, D.A. Przywara, and L. Schuger. 1999. Development. 126:3027–3033; Yang, Y., S. Beqaj, P. Kemp, I. Ariel, and L. Schuger. 2000. J. Clin. Invest. 106:1321–1330). In the developing lung, this process is stimulated by peribronchial accumulation of laminin (LN)-2 (Relan, N.K., Y. Yang, S. Beqaj, J.H. Miner, and L. Schuger. 1999. J. Cell Biol. 147:1341–1350). Here we show that LN-2 stimulates bronchial myogenesis by down-regulating RhoA activity. Immunohistochemistry, immunoblotting, and reverse transcriptase–PCR indicated that RhoA, a small GTPase signaling protein, is abundant in undifferentiated embryonic mesenchymal cells and that its levels decrease along with SM myogenesis. Functional studies using agonists and antagonists of RhoA activation and dominant positive and negative plasmid constructs demonstrated that high RhoA activity was required to maintain the round undifferentiated mesenchymal cell phenotype. This was in part achieved by restricting the localization of the myogenic transcription factor serum response factor (SRF) mostly to the mesenchymal cell cytoplasm. Upon spreading on LN-2 but not on other main components of the extracellular matrix, the activity and level of RhoA decreased rapidly, resulting in translocation of SRF to the nucleus. Both cell elongation and SRF translocation were prevented by overexpression of dominant positive RhoA. Once the cells underwent SM differentiation, up-regulation of RhoA activity induced rather than inhibited SM gene expression. Therefore, our studies suggest a novel mechanism whereby LN-2 and RhoA modulate SM myogenesis.

Heterogeneous nuclear ribonucleoprotein-H plays a suppressive role in visceral myogenesis.

Mouse embryonic mesenchymal cells undergo spontaneous smooth muscle (SM) differentiation upon spreading/elongation in culture (Relan et al., J. Cell Biol. 147 (1999) 1341; Yang et al., Development 125 (1998) 2621; Yang et al., Development 126 (1999) 3027). Using these cells we generated a subtracted cDNA library to identify potential suppressors of SM myogenesis. One of the differentially expressed genes was heterogeneous nuclear ribonucleoprotein-H (hnRNP-H), which is involved in pre-mRNA alternative splicing. hnRNP-H was highly expressed in mesenchymal cells prior to the onset of SM differentiation, but its expression rapidly decreased in mesenchymal cells undergoing SM myogenesis. In vivo, the drop in hnRNP-H expression was restricted to visceral SM cells. Antisense oligodeoxynucleotide and antisense RNA were used to inhibit hnRNP-H synthesis in SM-differentiating mesenchymal cells and in embryonic lung explants. A decrease in hnRNP-H levels resulted in upregulation of SM-specific gene expression and increased bronchial SM development in lung explants. hnRNP-H overexpression in cell cultures had the opposite effect. These studies, therefore, indicate a novel role for hnRNP-H in the control of visceral myogenesis.

A Small, Randomized, Placebo-Controlled Trial of the Use of Antiviral Therapy for Patients with Chronic Fatigue Syndrome

Sir—We have presented controlled and observational data that are consistent with the hypothesis that subsets of cases of chronic fatigue syndrome (CFS) result from cardiac disease due to a single, persisting infection caused by Epstein-Barr virus (EBV) or, in turn, to a single, persisting infection caused by human cytomegalovirus (HCMV) in immunocompetent patients [1]. Patients who have a separate subset of CFS have simultaneous coinfection with EBV and HCMV. Cardiomyopathic changes are observed in right ventricular endomyocardial biopsy specimens obtained from such patients, and abnormal findings on Holter monitoring (e.g., oscillating abnormal T-wave flattenings and T-wave inversions) are “uniformly” present [2–4]. Left ventricular dysfunction is manifested by sinus tachycardia at rest, abnormal cardiac-wall motion, and decreased left ventricular ejection fractions (rest/stress) in those patients with CFS who are most ill [5]. These findings belie the relatively normal findings observed on standard 12-lead electrocardiograms [6]. In January 1995, a double-blinded, placebo-controlled, phase III crossover study of patients with CFS was initiated. Eleven patients who had CFS (10 of whom were women) were each followed for 18 consecutive months. The mean patient age was 42.7 years, and the mean duration of CFS was 35.1 months. Before antiviral nucleosides were administered, endomyocardial biopsies were performed. Cardiac tissues and blood samples tested negative for isolation of HCMV in cultures of human fibroblast tissues. Two cardiac biopsy specimens that were obtained from patients who had CFS tested positive for HCMV nucleic acids by means of PCR. No cardiac specimen that was obtained from a patient with CFS tested positive for EBV nucleic acids. (Cardiac tissue samples that were obtained from 4 of 21 control patients who had coronary artery disease but who did not have CFS also tested positive for HCMV nucleic acids.) Cardiomyopathic degenerative findings (e.g., myofiber disarray, interstitial fibrosis, increased intracellular granules, and interstitial fat) were noted in patients who had CFS. One patient who had CFS had myocarditis with focal lymphocytic infiltrates. Patients with CFS had negative antistreptolysin O titers and no Lyme disease antibodies, IgM, or IgG, according to both ELISA and Western blot analysis. At the time of entry into the study, patients had positive HCMV IgG antibody titers with or without HCMV IgM antibody titers; both types of titer were tested by use of ELISA. Antibody titers to EBV, viral capsid antigen (VCA)–IgM, and EBV–diffuse early antigen (EBV-EA) were also tested. The patients in the treatment group received iv ganciclovir, 5 mg/ kg given q12h for 30 days. Treatment with iv ganciclovir was followed by administration of oral ganciclovir, 1 g given q8h. At the completion of a 6-month period of observation after discontinuation of treatment with iv ganciclovir, if no improvement was observed and if elevated EBV antibody titers suggested the presence of coinfections, oral valacyclovir, 1 g given q6h, was added to oral ganciclovir treatment. Oral administration of antiviral nucleosides then continued for the remainder of the 18 months of the study. When 2 patients with CFS who were undergoing right ventricular endomyocardial biopsies experienced serious pericardial bleeding, the study was ended prematurely. Measurement of HCMV and EBV antibody titers was repeated at 3and 6month intervals. Unchanging, high positive titers of HCMV IgG antibody were noted throughout the 18-month trial. At baseline, 1 of the 11 patients who had CFS had a positive HCMV IgM antibody titer. This HCMV IgM antibody titer was absent after the patient received 30 days of treatment with iv ganciclovir. Four of the 11 patients with CFS had coinfection with EBV, which was indicated by positive EBV-EA antibody titers. After administration of valacyclovir, EBV-EA antibody titers decreased or became negative in 3 of these patients who had CFS. The findings with regard to these serologic titers are consistent with incomplete herpesvirus multiplication of HCMV and, when present, EBV in patients who have CFS [7]. Energy index point scores (EIs) and symptom scores were assessed at 30-day intervals [3]. A series of questions was used to calculate EIs (A.M.L. and R.G.D, unpublished data). An EI of 0 denoted a bedridden patient, whereas an EI of 10 denoted a healthy patient. At baseline, the mean EI for the entire group was 3.5. After 6 months, 4 patients with CFS who were receiving iv placebo had a mean EI of 3.9. When they were assessed 6 months after initiation of treatment with iv ganciclovir, they had a mean EI of 4.4. At this point in the study, as indicated by the presence of positive EBV, VCA-IgM, and/or EBV-EA antibody titers, valacyclovir was added to treatment and administration of oral ganciclovir was continued. At month 12 of the study, the mean EI for the 11 patients was 5.8, and at month 18, the mean EI was 6.1. Symptom scores (e.g., chest pain, wooziness, palpitations, and muscle aches) were assessed at 30-day intervals. A symptom score of 1 denoted the presence of all 4 symptoms tested, whereas a symptom score of 0 denoted the absence of all 4 specific symptoms. At baseline, the mean symptom score was 0.81 for the 11 patients who had CFS. After 6

Antibody to Epstein-Barr Virus Deoxyuridine Triphosphate Nucleotidohydrolase and Deoxyribonucleotide Polymerase in a Chronic Fatigue Syndrome Subset

Background A defined diagnostic panel differentiated patients who had been diagnosed with chronic fatigue syndrome (CFS), based upon Fukuda/Carruthers criteria. This diagnostic panel identified an Epstein-Barr virus (EBV) subset of patients (6), excluding for the first time other similar “clinical” conditions such as cytomegalovirus (CMV), human herpesvirus 6 (HHV6), babesiosis, ehrlichiosis, borreliosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, and adult rheumatic fever, which may be mistakenly called CFS. CFS patients were treated with valacyclovir (14.3 mg/kg q6h) for ≥12 months. Each patient improved, based upon the Functional Activity Appraisal: Energy Index Score Healthcare Worker Assessment (EIPS), which is a validated (FSS-9), item scale with high degree of internal consistency measured by Cronbachs alpha. Methods Antibody to EBV viral capsid antigen (VCA) IgM, EBV Diffuse Early Antigen EA(D), and neutralizing antibodies against EBV-encoded DNA polymerase and EBV-encoded dUTPase were assayed serially approximately every three months for 13–16 months from sera obtained from patients with CFS (6) and from sera obtained from twenty patients who had no history of CFS. Results Antibodies to EBV EA(D) and neutralizing antibodies against the encoded-proteins EBV DNA polymerase and deoxyuridine triphosphate nucleotidohydrolase (dUTPase) were present in the EBV subset CFS patients. Of the sera samples obtained from patients with CFS 93.9% were positive for EA(D), while 31.6% of the control patients were positive for EBV EA(D). Serum samples were positive for neutralizing antibodies against the EBV-encoded dUTPase (23/52; 44.2%) and DNA polymerase (41/52; 78.8%) in EBV subset CFS patients, but negative in sera of controls. Conclusions There is prolonged elevated antibody level against the encoded proteins EBV dUTPase and EBV DNA polymerase in a subset of CFS patients, suggesting that this antibody panel could be used to identify these patients, if these preliminary findings are corroborated by studies with a larger number of EBV subset CFS patients.

Subset-directed antiviral treatment of 142 herpesvirus patients with chronic fatigue syndrome

correspondence: A Martin Lerner 32804 Pierce road, Beverly hills, Mi 48025, UsA Tel +1 248 540 9866 Fax +1 248 540 0139 email amartinlerner@yahoo.com Purpose: We hypothesized that chronic fatigue syndrome (CFS) may be caused by single or multiple Epstein–Barr virus (EBV), cytomegalovirus (HCMV), or human herpesvirus 6 (HHV6) infection. To determine if CFS life-altering fatigue and associated findings including muscle aches, tachycardia at rest, chest aches, left ventricular dysfunction, syncope, and elevated herpesvirus serum antibody titers are reversed by long-term subset-directed valacyclovir and/or valganciclovir. Patients and methods: Data were collected at physician visits every 4–6 weeks from 142 CFS patients at one clinic from 2001 to 2007. To be included in this study, patients had to be followed for at least six months. The data captured included over 7000 patient visits and over 35,000 fields of information. Severity of fatigue was monitored by a validated Energy Index Point Score (EIPS). Baseline and follow-up serum antibody titers to EBV, HCMV, and HHV6, as well as coinfections with Borrelia burgdorferi, Anaplasma phagocytophila, Babesia microti, and antistreptolysin O, 24-hour ECG Holter monitors, 2D echocardiograms, cardiac dynamic studies, symptoms, and toxicity were captured and monitored. International criteria for CFS plus a specifically designed CFS diagnostic panel were used. Results and conclusions: The Group A herpesvirus CFS patients (no coinfections) returned to a near-normal to normal life (P = 0.0001). The long-term EIPS value increased (primary endpoint, P , 0.0001) with subset-directed long-term valacyclovir and/or valganciclovir therapy. Secondary endpoints (cardiac, immunologic, and neurocognitive abnormalities) improved or disappeared. Group B CFS patients (herpesvirus plus coinfections) continued to have CFS.

A paradigm linking herpesvirus immediate-early gene expression apoptosis and myalgic encephalomyelitis chronic fatigue syndrome

correspondence: A Martin Lerner 32804 Pierce rd, Beverly hills, Mi 48025, UsA Tel +1 248 540 9866 Fax +1 248 540 0139 email amartinlerner@yahoo.com Abstract: There is no accepted science to relate herpesviruses (Epstein–Barr virus [EBV], human cytomegalovirus [HCMV], and human herpesvirus 6 [HHV6]) as causes of myalgic encephalomyelitis (ME)/chronic fatigue syndrome (CFS). ME/CFS patients have elevated serum immunoglobulin (Ig)G serum antibody titers to EBV, HCMV, and HHV6, but there is no herpesvirus DNA-emia, herpesvirus antigenemia, or uniformly elevated IgM serum antibody titers to the complete virions. We propose that herpesvirus EBV, HCMV, and HHV6 immediate-early gene expression in ME/CFS patients leads to host cell dysregulation and host cell apoptosis without lytic herpesvirus replication. Specific antiviral nucleosides, which alleviate ME/CFS, namely valacyclovir for EBV ME/CFS and valganciclovir for HCMV/ HHV6 ME/CFS, inhibit herpesvirus DNA polymerases and/or thymidine kinase functions, thus inhibiting lytic virus replication. New host cell recruitment thus ceases. In the absence of new herpesvirus, nonpermissive herpesvirus replication stops, and ME/CFS recovery ensues.

An update on the management of glandular fever (infectious mononucleosis) and its sequelae caused by Epstein–Barr virus (HHV-4): new and emerging treatment strategies

Correspondence: A Martin Lerner 32804 Pierce Rd, Beverly Hills, MI 48025, USA Tel +1 248 540 9866 Fax +1 248 540 0139 Email amartinlerner@yahoo.com Purpose: Beginning in 1993 at a single chronic fatigue syndrome (CFS) treatment center, we began studies that demonstrate Epstein–Barr virus (EBV) nonpermissive replication. In the most recent study performed, EBV nonpermissive replication is the cause of 28.3% of 106 consecutive CFS cases, and is etiologic with human cytomegalovirus (HCMV) and/or human herpes virus 6 (HHV-6) as a coinfection in an additional 52.8% of CFS cases. Therefore, EBV is causally involved in 81% of cases of CFS. Further, EBV CFS is effectively treated with long-term valacyclovir. Coinfection HCMV and HHV-6 CFS requires valganciclovir with valacyclovir. Patients and results: The validated Energy Index Point Score (EIPS) monitors severity of CFS illness and its recovery. A specific CFS diagnostic panel identifies EBV CFS subsets. Four separate EBV CFS therapeutic studies of several hundred CFS patients describe valacyclovir administration and long-term patient recovery. With valacyclovir, serum EBV titers (EBV, early antigen (diffuse); EBV, viral capsid antigen, immunoglobulin M); 24-hour electrocardiography Holter monitors; and cardiac dynamic studies improve. Conclusion: Nonpermissive EBV infection is causal in a significant proportion of CFS cases. EBV CFS is safely and effectively treated with long-term valacyclovir.

Abortive lytic Epstein–Barr virus replication in tonsil-B lymphocytes in infectious mononucleosis and a subset of the chronic fatigue syndrome

Correspondence: A Martin Lerner 32804 Pierce Road, Beverly Hills, MI, 48025, USA Tel +1 248 540 9866 Fax +1 248 540 0139 Email amartinlerner@yahoo.com Abstract: A systematic 2001–2007 review of 142 chronic fatigue syndrome (CFS) patients identified 106 CFS patients with elevated serum IgG antibodies to the herpesviruses Epstein– Barr virus (EBV), cytomegalovirus, or human herpesvirus (HHV) 6 in single or multiple infections, with no other co-infections detected. We named these 106 patients group-A CFS. Eighty-six of these 106 group-A CFS patients (81%) had elevated EBV early antibody, early antigen (diffuse), serum titers. A small group of six patients in the group-A EBV subset of CFS, additionally, had repetitive elevated-serum titers of antibody to the early lytic replicationencoded proteins, EBV dUTPase, and EBV DNA polymerase. The presence of these serum antibodies to EBV dUTPase and EBV DNA polymerase indicated EBV abortive lytic replication in these 6 CFS patients. None of 20 random control people (ageand sex-matched, with blood drawn at a commercial laboratory) had elevated serum titers of antibody to EBV dUTPase or EBV DNA polymerase (P , 0.01). This finding needs verification in a larger group of EBV CFS subset patients, but if corroborated, it may represent a molecular marker for diagnosing the EBV subset of CFS. We review evidence that EBV abortive lytic replication with unassembled viral proteins in the blood may be the same in infectious mononucleosis (IM) and a subset of CFS. EBV-abortive lytic replication in tonsil plasma cells is dominant in IM. No complete lytic virion is in the blood of IM or CFS patients. Complications of CFS and IM include cardiomyopathy and encephalopathy. Circulating abortive lytic-encoded EBV proteins (eg, EBV dUTPase, EBV DNA polymerase, and others) may be common to IM and CFS. The intensity and duration of the circulating EBV-encoded proteins might differentiate the IM and EBV subsets of CFS. Abortive lytic replication may be a pathogenic mechanism for EBV disease. EBV (HHV4) is a gamma herpesvirus composed of dsDNA about 170 Kb in length. For this discussion, there are early genes (including expressions of encoded proteins EBV dUTPase, DNA polymerase, and nuclear proteins) and late genes (including expressions of capsid and membrane proteins). Abortive infection infers incomplete virion expressions of either early or late proteins, but the virion is incomplete. The lytic virus infers a complete virion. The pathologic consequences of EBV abortive replication are currently being investigated by authors.

Beneficial response of cervical dystonia spasmodic torticollis to cidofovir, an acyclic phosphonate analog (s-1-3-hydroxy-2- phosphonylmethoxypropyl) of cytosine

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