In vitro activation of specific helper and suppressor T cells by the type 2 antigen polyvinylpyrrolidone (PVP)

Although type 2 antigens, such as PVP, generally do not activate specific TH, previous studies have established that low doses of PVP (0.0025 microgram) can activate TH in vivo which provide help in primed B cells for PVP-specific IgG responses. Doses of PVP that are optimally immunogenic for IgM antibody production (0.25 to 25 micrograms) preferentially activate PVP-specific TS, which suppress IgG antibody production. In the studies reported here, TH and TS that regulate PVP-specific IgG antibody responses were activated in vitro by culturing normal spleen cells for 4 days with PVP. Induction of the TH and TS is dependent upon the amount of PVP in culture: 10(-4) micrograms PVP activates TH, whereas 10(-2) micrograms PVP preferentially activates TS. TH induced in vitro express Thy-1, L3T4, and I-A determinants and help provided by these TH is similar in magnitude to that provided by TH from mice primed with 0.0025 microgram PVP in vivo. TH can also be activated in vitro if donor mice are treated with Cy before culture of their spleen cells with 10(-2) micrograms PVP. Cy pretreatment prevents TS activation, and TH are then induced in these cultures. The presence of TS does not prevent activation of TH by 10(-2) micrograms PVP, because removal of TS by treatment of T cells with anti-Lyt-2 + complement at the end of culture uncovers TH activity. This TH activity is comparable with that of TH obtained after culture with 10(-4) micrograms PVP. The ability to activate PVP-specific TH and TS in vitro should allow determination of the mechanisms involved in activation of T cells by type 2 antigens and the mechanisms by which TS and TH interact with one another.     

Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.     

Disruption of microfilaments alters laminin synthesis but not laminin trafficking in NHEKin vitro

Summary Laminin synthesis and deposition are concomitant with the development of a basal lamina between the human epidermis and the underlying dermis. One of the challenges in tissue engineering of human epidermal models is to develop substrates and conditions that encourage the development of a basement membrane. The purpose of this study was to determine if actin filaments and/or microtubules are involved in the synthesis/secretion of laminin by normal human epidermal keratinocytes (NHEK)in vitro. NHEK synthesize and secrete laminin subunits B1, B2, and M but little, if any, of laminin subunit A. Data indicate that disruption of microfilaments by the destabilizing agent, cytochalasin D, had no apparent effect on the relative synthesis rates of most cytosolic proteins as, revealed by one-dimensional sodium dodecyl sulfate (SDS) gel electrophoresis. This drug, however, increased laminin B2 synthesis several fold over untreated controls. This enhanced synthetic rate was independent of the type of collagen, matrix on which the NHEK were grown. Similar increases in synthesis of the M and B1 laminin chains were not observed. To determine if this increase in synthesis lead to increases in laminin B2 secretion, laminin B2 was immunoprecipitated from both the apical and basal domains of NHEK cells grown on microporous membranes. While more laminin B1, B2, and M were secreted basally than apically, an observation consistent with laminin’s role in basal lamina formation, cytochalasin D had no apparent effect on either basal or apical laminin B2 secretion. Experiments with the microtubule destabilizer, nocodazole, showed no similar effects on laminin synthesis and/or secretion. We conclude that (a) disruption of the actin network in NHEK selectively increases the synthesis of laminin B2, (b) the secretion of laminin B2 from NHEK cells is not governed by either the microfilamentous cytoskeleton or the amount of laminin synthesized by NHEK, and (c) disruption of the microtubular network does not alter laminin synthesis or secretion.     

Modeling the biomechanics of the mandible: a three-dimensional finite element study.

Three-dimensional finite element models of a partially edentulated human mandible were generated to calculate the mechanical response to simulated isometric biting and mastication loads. The level of mesh refinement was established via a convergence test and showed that a model with over 30,000 degrees of freedom was required to obtain analysis accuracy. The functional loading cases included muscle loading based on an algorithm that assigns muscle forces in accordance with muscle cross-sectional area, while maintaining static equilibrium. Results were found for isometric application of unilateral and bilateral bite and mastication loading, and two different sets of displacement boundary conditions were imposed at the condyles. The mechanical response is shown in terms of displacements, principal strains, and a new measure called the 'mechanical intensity scalar'. For each load case studied, there was substantial bending in the molar region of the corpus and high tensile strains in the anterior portion of the ramus.     

A molecular phylogenetic analysis of Speyeria and its implications for the management of the threatened Speyeria zerene hippolyta

The genetic structure of lineages can provide important information for delineating “evolutionarily significant units” (ESUs) for conservation, and for planning actions to protect and restore taxa threatened with extinction. Speyeria zerene hippolyta, the Oregon silverspot butterfly, is a U.S.A. federally threatened subspecies that is the focus of considerable conservation effort, but whose evolutionary relationships with other Speyeria taxa are not well-understood. We conducted a genetic analysis of nine Speyeria species and 25 subspecies from western U.S.A., using both mitochondrial and nuclear markers. Our goal was to determine whether such data supported (a) S. z. hippolyta’s designation as an ESU, and (b) the current morphologically-based taxonomy of Speyeria spp. Our data for S. z. hippolyta were equivocal; while nuclear markers resolved all these individuals into a single clade, mtDNA data suggested the existence of two clades. Aside from S. cybele, which was consistently supported as monophyletic, our data provided little support for most of the species currently recognized for western U.S. Speyeria, including S. zerene, and even less for the many subspecies designations. These genetic findings stand in contrast to the morphological differences recognized by experts, and suggest a relatively recent origin for many of these taxa. Two of 66 individuals screened for Wolbachia infection tested positive for this symbiont. Our results provide no persuasive evidence that S. z. hippolyta should lose its status as an ESU, but they have important implications for ongoing management actions such as population augmentation.