STRO-1, HOP-26 (CD63), CD49a and SB-10 (CD166) as markers of primitive human marrow stromal cells and their more differentiated progeny: a comparative investigation in vitro

There is widespread interest in the use of bone marrow stromal cells (BMSC) for tissue reconstruction and repair and for gene therapy. BMSC represent the differentiated progeny of CFU-F, which however comprise a developmentally heterogeneous population as is reflected in the cellular heterogeneity of the cell populations to which they give rise. We have compared the efficacy of monoclonal antibodies recognising a series of stromal antigens, viz. STRO-1, HOP-26, CD49a and SB-10/CD166, as tools for the enrichment of CFU-F prior to culture and as developmental markers for culture-expanded BMSC. In freshly isolated bone marrow mononuclear cells (BMMNC), the proportion of antigen-positive cells was 27%, 46%, 5% and 19% for STRO-1, HOP-26, CD49a and CD166, respectively. All CD49a⁺ cells co-expressed STRO-1. The degree of CFU-F enrichment obtained with anti-CD49a (~18-fold) by a one-pass immunoselection strategy was significantly greater than that of all other antibodies tested. BMSC expressed higher levels of all antigens investigated (except for HOP-26) compared with BMMNC. Expression of STRO-1 and CD49a remained restricted to a subset of BMSC, whereas all BMSC were SB-10/CD166 positive. Treatment with dexamethasone (10 nM), which promotes the differentiation and further maturation of cells of the osteogenic lineage in this cell culture system, increased the expression of CD49a and HOP-26. The CD49a⁺ and HOP-26⁺ fractions of BMSC were further subdivided by dual-labelling with anti-STRO-1 and B4–78 (an antibody recognising the B/L/K isoform of the enzyme alkaline phosphatase), respectively. By using a variety of criteria, the HOP-26 antigen was identified as CD63, a member of the tetraspanin family of proteins thought to modulate integrin compartmentalisation and signalling.K.S., S.W., C.M.J. and J.A.L. gratefully acknowledge the financial support of the University Bath, the Arthritis Research Campaign and the Wellcome Trust     

Early development and quorum sensing in bacterial biofilms

 We develop mathematical models to examine the formation, growth and quorum sensing activity of bacterial biofilms. The growth aspects of the model are based on the assumption of a continuum of bacterial cells whose growth generates movement, within the developing biofilm, described by a velocity field. A model proposed in Ward et al. (2001) to describe quorum sensing, a process by which bacteria monitor their own population density by the use of quorum sensing molecules (QSMs), is coupled with the growth model. The resulting system of nonlinear partial differential equations is solved numerically, revealing results which are qualitatively consistent with experimental ones. Analytical solutions derived by assuming uniform initial conditions demonstrate that, for large time, a biofilm grows algebraically with time; criteria for linear growth of the biofilm biomass, consistent with experimental data, are established. The analysis reveals, for a biologically realistic limit, the existence of a bifurcation between non-active and active quorum sensing in the biofilm. The model also predicts that travelling waves of quorum sensing behaviour can occur within a certain time frame; while the travelling wave analysis reveals a range of possible travelling wave speeds, numerical solutions suggest that the minimum wave speed, determined by linearisation, is realised for a wide class of initial conditions. Received: 10 February 2002 / Revised version: 29 October 2002 / Published online: 19 March 2003 Key words or phrases: Bacterial biofilm – Quorum sensing – Mathematical modelling – Numerical solution – Asymptotic analysis – Travelling wave analysis     

Mutations in the calcium-binding motifs of CDH23 and the 35delG mutation in GJB2 cause hearing loss in one family

We have ascertained a multi-generation family with apparent autosomal recessive non-syndromic childhood hearing loss (DFNB). Failure to demonstrate linkage in a genome-wide scan with 300 polymorphic markers has suggested genetic heterogeneity for the hearing loss in this family. This heterogeneity could be demonstrated by analysis of candidate loci and genes for DFNB. Patients in one branch of the family (branch C) are homozygous for the 35delG mutation in the GJB2 gene (DFNB1). Patients in two other branches (A and B) carry two new mutations in the cadherin 23 ( CDH23) gene (DFNB12). A homozygous CDH23 c.6442G-->A (D2148N) mutation is present in branch A. Patients in branch B are compound heterozygous for this mutation and the c.4021G-->A (D1341N) mutation. The substituted aspartic acid residues are highly conserved and are part of the calcium-binding sites of the extracellular cadherin (EC) domains. Molecular modeling of the mutated EC domains of CDH23 based on the structure of E-cadherin indicates that calcium-binding is impaired. In addition, other aspartic and glutamic acid residue substitutions in the highly conserved calcium-binding sites reported to cause DFNB12 are also likely to result in a decreased affinity for calcium. Since calcium provides rigidity to the elongated structure of cadherin molecules enabling homophilic lateral interaction, these mutations are likely to impair interactions of CDH23 molecules either with CDH23 or with other proteins. DFNB12 is the first human disorder that can be attributed to inherited missense mutations in the highly conserved residues of the extracellular calcium-binding domain of a cadherin.     

Effect of maternal feed restriction during pregnancy on glucose tolerance in the adult guinea pig

Maternal nutrient restriction and impaired fetal growth are associated with postnatal insulin resistance, hyperinsulinemia, and glucose intolerance in humans but not consistently in other species, such as the rat or sheep. We therefore determined the effect of mild (85% ad libitum intake/kg body wt) or moderate (70% ad libitum intake/kg body wt) maternal feed restriction throughout pregnancy on glucose and insulin responses to an intravenous glucose tolerance test (IVGTT) in the young adult guinea pig. Maternal feed restriction reduced birth weight (mild and moderate: both P < 0.02) in male offspring. Moderate restriction increased plasma glucose area under the curve (P < 0.04) and decreased the glucose tolerance index (K(G)) (P < 0.02) during the IVGTT in male offspring compared with those of mildly restricted but not of ad libitum-fed mothers. Moderate restriction increased fasting plasma insulin (P < 0.04, adjusted for litter size) and the insulin response to IVGTT (P < 0.001), and both moderate and mild restriction increased the insulin-to-glucose ratio during the IVGTT (P < 0.003 and P < 0.02) in male offspring. When offspring were classed into tertiles according to birth weight, glucose tolerance was not altered, but fasting insulin concentrations were increased in low compared with medium birth weight males (P < 0.03). The insulin-to-glucose ratio throughout the IVGTT was increased in low compared with medium (P < 0.01) or high (P < 0.05) birth weight males. Thus maternal feed restriction in the guinea pig restricts fetal growth and causes hyperinsulinemia in young adult male offspring, suggestive of insulin resistance. These findings suggest that mild to moderate prenatal perturbation programs postnatal glucose homeostasis adversely in the guinea pig, as in the human.     

Expression of multiple connexins in the rat epididymis indicates a complex regulation of gap junctional communication

In theepididymis, Cx43 forms gap junctions between principal and basal cellsbut not between adjacent principal cells. Cx30.3, 31.1, and 32 wereidentified in adult rat epididymis by RT-PCR, whereas Cx26 was presentin young rats. Postnatal development studies indicate that Cx26 mRNAwas detectable only in the caput-corpus region of the epididymis andthat levels increased by fivefold during the first 4 wk postnatally,when epithelial cells differentiate, and decrease to nondetectablelevels thereafter. Cx31.1 and Cx32 mRNA levels were low throughout theepididymis in young rats and began to increase in the second and thirdweeks postnatally, when Cx26 levels are decreasing. Both Cx26 and Cx32were localized to the lateral plasma membranes between adjacentepithelial cells of the epididymis. Colocalization studies indicatethat Cx26 and Cx32 exist either independently of one another or cancolocalize along the lateral plasma membrane of epithelial cells inyoung rats or between principal cells in the adult rat epididymis. The presence of multiple connexins (Cxs) and their differential regulation suggest that these play different roles in epididymal development.

     

Copper-induced trafficking of the Cu-ATPases: A key mechanism for copper homeostasis

The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (ccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement ccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.     

De novo design,synthesis and evaluation of a non-steroidal diphenylnaphthyl propylene ligand for the estrogen receptor

There is still a strong need for additional diversity and new chemical scaffolds to allow for the exploration of improved tissue selectivity and finding better selective estrogen receptor modulators (SERMs). Using a de novo design technology a diphenylnaphthyl propylene scaffold, exemplified by (E)-9b, with ER antagonist activity has been generated. It was prepared by alkylating 1-[4-methoxyphenyl)-2-(4-(2-chloroethoxy)phenyl]-1-propanone under metal halogen exchange conditions with 1-iodo-6-methoxy-naphthalene. Following dehydration and cleavage of the methoxy groups, (E)-9b was formed by displacement of the chloro group with pyrrolidine. (E)-9b binding to ER generated calculated K(i) values of 3.7 nM for hER(alpha) and 72 nM for hER(beta). The antagonism of (E)-9b was demonstrated in cell transfection assays using the ERE from the vitA2 promotor and the natural ER-responsive pS2 promotor. With increasing concentrations of (E)-9b, the E(2)-dependent response was efficiently inhibited demonstrating that (E)-9b could function as an anti-estrogen in these assays. Interestingly, ER(alpha) activity was inhibited even below basal levels suggesting that ligand-independent activity of ER(alpha) was also inhibited. Computational docking studies suggest that the placement of the hydroxyl group on the naphthalene group may not be optimal and we are currently exploring additional analogues.     

Synthesis of a [6-pyridinyl-18F]-labelled fluoro derivative of WAY-100635 as a candidate radioligand for brain 5-HT1A receptor imaging with PET

In recent years, considerable effort has been spent on the design, synthesis and pharmacological characterization of radiofluorinated derivatives of the 5-HT(1A) receptor antagonist, WAY-100635, for the in vivo study of these receptors in human brain with PET. (Pyridinyl-6)-fluoro- and (pyridinyl-5)-fluoro-analogues of WAY-100635 (6-fluoro and 5-fluoro-WAY-100635, 5a/6a) were synthesized as well as the corresponding chloro-, bromo- and nitro-derivatives as precursors for labelling (5b-d and 6b-d). Comparative radiolabelling of these precursors with fluorine-18 (positron-emitting isotope, 109.8 min half-life) clearly demonstrated that only ortho-fluorination in this pyridine series, and not meta-fluorination, is of interest for the preparation of a radioligand by nucleophilic heteroaromatic substitution. 6-[(18)F]Fluoro-WAY-100635 ([(18)F]5a) can be efficiently synthesized in one step, either from the corresponding 6-bromo precursor (using conventional heating at 145 degrees C for 10 min) or from the corresponding 6-nitro precursor (using microwave activation at 100 W for 1 min). Typically, 15-25 mCi (0.55-0.92 GBq) of 6-[(18)F]fluoro-WAY-100635 ([(18)F]5a, 1-2 Ci/micromol or 37-72 GBq/micromol) were obtained in 50-70 min starting from a 100 mCi (3.7 GBq) aliquot of a batch of cyclotron-produced [(18)F]fluoride. This (18)F-labelled radioligand is now being evaluated in PET studies.     

Differential role of leptin receptors at the hypothalamic paraventricular nucleus in tonic regulation of food intake and cardiovascular functions

Leptin plays an important role in the central regulation of body weight and arterial pressure via activation of leptin receptors (Ob-Rs) in the hypothalamic area, including the hypothalamic paraventricular nucleus (PVN). The present study was undertaken to investigate whether endogenous leptin in the PVN plays a dual role in the tonic regulation of body weight and arterial pressure. Adult, male normal-weight Sprague-Dawley rats, which were anesthetized and maintained with propofol, were used. A direct bilateral microinjection into the PVN of an antisense oligonucleotide against Ob-R mRNA (ASON1, 50 pmol) significantly increased the daily food intake and body weight gain, effects which lasted for at least 14 days. The same treatment, on the other hand, had no appreciable effect on the basal mean systemic arterial pressure (SAP), heart rate (HR), or power density of the vasomotor components of SAP signals, the experimental index of neurogenic sympathetic vasomotor tone. ASON1 treatment also exerted an insignificant effect on the baroreceptor reflex control of HR. Western blot analysis revealed that a bilateral microinjection into the PVN of ASON1 (50 pmol) significantly decreased the expression of the Ob-R protein in the hypothalamus. The same treatment also attenuated hypertension, tachycardia, and the increase in the power density of the vasomotor components of the SAP signals induced by exogenous bilateral application of leptin (5 or 50 ng) into the PVN. Control application of sense (SON, 50 pmol) or a scrambled antisense Ob-R oligonucleotide (ASON2, 50 pmol) into the bilateral PVN promoted no discernible effect on Ob-R protein expression in the hypothalamus, on daily food intake, or on cardiovascular performance. Our results indicate that whereas the Ob-Rs in the PVN are involved in the tonic regulation of food intake, they might not be actively involved in the tonic regulation of cardiovascular functions.     

CD49d overexpression and T cell autoimmunity

D10.G4.1 (D10) cells, a murine conalbumin-reactive Th2 cell line, made to overexpress the beta(2) integrin LFA-1 by pharmacological manipulation or by transfection become autoreactive and are capable of inducing in vivo autoimmunity. However, whether this is specific to LFA-1 and whether overexpression of other T cell integrin molecules has the same effect are unknown. We examined the functional consequences of T cell CD49d (alpha(4) integrin) overexpression by transfecting murine CD49d cDNA into D10 cells. Similar to the LFA-1-transfected cells, the CD49d-overexpressing T cells are autoreactive and proliferate in response to APCs in an MHC class II-dependent manner in the absence of nominal Ag. Additionally, CD49d overexpression is associated with increased in vitro adhesion to endothelial cells and increased in vivo splenic homing. However, in contrast to LFA-1 overexpression, increased T cell CD49d expression is not associated with autoreactive cytotoxicity or the ability to induce in vivo autoimmunity. In addition to the novel observation that CD49d overexpression is sufficient to induce T cell autoreactivity, our results also support the hypothesis that the ability to induce in vivo autoimmunity is related to T cell cytotoxicity and not to T cell proliferation function in the D10 murine adoptive transfer model of autoimmunity.