IntroductionVisceral smooth muscle (SM) originates from local mesenchymal cells that in early-midgestation begin to synthesize SM proteins, including SM α-actin, desmin, SM myosin, SM22, and calponin in a specific periairway distribution (1-5). In the mouse developing respiratory system, cells expressing SM proteins are first detected in the trachea on day 11 of gestation (3, 4), and then SM differentiation proceeds in a cranial-tocaudal fashion to form the bronchial musculature (1-5). The other type of visceral SM cells found in the lung are interstitial SM cells, also known as interstitial contractile cells, or myofibroblasts. Interstitial SM cells are originally located at the sites of future alveolar septae, and, in the mature organ, they form part of the septae tips (6). Except for the aorta, the development of the vascular musculature lags behind that of visceral SM by several days (4, 7-9).Unlike striated muscle differentiation, on which considerable information was gathered over the years, the mechanisms and genetic program that control SM myogenesis remain, for the most part, unknown. We and others have observed that lung mesenchymal cell precursors change their shape from round to elongated before undergoing bronchial SM differentiation (ref.3; Y. Yang and L. Schuger, unpublished observations). Based on this observation we recently examined whether changes in cell shape might play a role in airway myogenesis. Unexpectedly, our studies demonstrated that essentially all undifferentiated embryonic mesenchymal cells are potential SM precursors (10-12). These studies also confirmed the critical role of cell shape in myogenesis. Specifically, we found that cell rounding prezvents myogenesis, regardless of the normal fate of the cell in vivo, whereas cell spreading/elongation induces SM differentiation, even in mesenchymal cells from nonmuscular organs (10-12).Developing tubular tissues, such as those of the respiratory, gastrointestinal, and urinary systems, are filled with liquid. As a consequence, the periluminal mesenchymal cells are subjected to mechanical tension/stretch exerted by the liquid's hydrostatic pressure (13). These forces likely represent a significant factor in determining the periluminal mesenchymal cell shape. In the developing lung, cells are additionally subjected to repeated stretch caused by intrauterine breathing (13). The fact that mechanical stretch causes cell elongation and that cell elongation is likely to be sensed by the cell as a mechanical stimulus suggested to us that cell tension/stretch may play an important role in the process of visceral myogenesis.Here we used a combination of lung cell and organ cultures from fetal mouse and human origin to determine the effect of mechanical stretch upon SM myogenesis. 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 acc...

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.

Bronchial smooth muscle (SM) mesenchymal cell precursors change their shape from round to spread/elongated while undergoing differentiation. Here we show that this change in cell shape induces the expression of laminin (LM) α2 chain not present in round mesenchymal cells. LM α2 expression is reversible and switched on and off by altering the cell's shape in culture. In comparison, the expression of LM β1 and γ1 remains unchanged. Functional studies showed that mesenchymal cell spreading and further differentiation into SM are inhibited by an antibody against LM α2. Dy/dy mice express very low levels of LM α2 and exhibit congenital muscular dystrophy. Lung SM cells isolated from adult dy/dy mice spread defectively and synthesized less SM α-actin, desmin, and SM-myosin than controls. These deficiencies were completely corrected by exogenous LM-2. On histological examination, dy/dy mouse airways and gastrointestinal tract had shorter SM cells, and lungs from dy/dy mice contained less SM-specific protein. The intestine, however, showed compensatory hyperplasia, perhaps related to its higher contractile activity. This study therefore demonstrated a novel role for the LM α2 chain in SM myogenesis and showed that its decrease in dy/dy mice results in abnormal SM.

BackgroundA 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 Cronbach's alpha.MethodsAntibody 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.ResultsAntibodies 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.ConclusionsThere 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.

A new integrated extraction and real-time PCR-based system for the detection of group B streptococci in antepartum screening samples enriched in Lim broth was compared to the CDC-recommended culture method. The BD Max GBS assay exhibited acceptable sensitivity (95%) and specificity (96.7%) compared to those of the culture method in this multisite evaluation.The number of neonatal group B streptococcus (GBS) infections has decreased significantly over the past 4 decades; however, GBS still remains one of the most common causes of neonatal sepsis in the United States (3). Current management guidelines recommend that all pregnant women be screened for vaginal/rectal GBS colonization at 35 to 37 weeks of gestation, with those found to be colonized receiving intrapartum antibiotic prophylaxis.While culture-based methods have historically been the gold standard for demonstrating GBS colonization, several recent studies have demonstrated the utility of PCR-based detection as a sensitive and specific alternative (1, 2, 4-6, 8, 9). The BD Max GBS assay (BDM) implemented using the BD Max system (previously known as the HandyLab Jaguar system; BDHandyLab, Ann Arbor, MI) is one such PCR-based alternative. The BD Max system is a benchtop molecular diagnostic system, which provides fully automated clinical sample preparation, cell lysis, nucleic acid extraction, and mixing of nucleic acid with master mix reagents. With no user intervention, the system then dispenses the sample into a microfluidic chamber where real-time PCR amplification and detection are performed.The goal of this three-site investigational study was to compare the results obtained by BDM to those obtained by the CDC-recommended culture procedure, which served as the reference method (3). This study was designed to generate the data necessary for 510(k) submission to the FDA, so the study design, reference method, and evaluation criteria were performed as required by the FDA. Performance characteristics of the assay were derived from the results of 601 compliant specimens collected from antepartum women presenting for TRL obtained broth from Becton-Dickinson, Sparks, MD) and incubated for 18 to 24 h (as per protocol) prior to testing by either method. The growth obtained with each Lim broth was subcultured onto a sheep blood agar plate and incubated for up to 48 h. Colonies with morphology and color suggestive of GBS (both hemolytic and nonhemolytic) were Gram stained, tested for catalase production, and confirmed as GBS using latex agglutination (DCL and TRL used PathoDX Strep Grouping reagents, Thermo Fisher-Remel; UM used Slidex, bioMérieux, Durham, NC) and/or CAMP testing. BDM (cfb gene target and limit of detection of 200 CFU of GBS/ml of sample preparation reagent) (data not shown) was performed using a residual 15-l aliquot of Lim broth, and when possible, an alternate, validated PCR method was performed at each site (at DCL, the IDI-Strep B assay was performed on the Cepheid SmartCycler system with a cfb gene target; at TRL, the Roche analyte-specifi...