A synthetic nanofibrillar matrix promotes in vivo-like organization and morphogenesis for cells in culture

The purpose of this study was to design a synthetic nanofibrillar matrix that more accurately models the porosity and fibrillar geometry of cell attachment surfaces in tissues. The synthetic nanofibrillar matrices are composed of nanofibers prepared by electrospinning a polymer solution of polyamide onto glass coverslips. Scanning electron and atomic force microscopy showed that the nanofibers were organized into fibrillar networks reminiscent of the architecture of basement membrane, a structurally compact form of the extracellular matrix (ECM). NIH 3T3 fibroblasts and normal rat kidney (NRK) cells, when grown on nanofibers in the presence of serum, displayed the morphology and characteristics of their counterparts in vivo. Breast epithelial cells underwent morphogenesis to form multicellular spheroids containing lumens. Hence the synthetic nanofibrillar matrix described herein provides a physically and chemically stable three-dimensional surface for ex vivo growth of cells. Nanofiber-based synthetic matrices could have considerable value for applications in tissue engineering, cell-based therapies, and studies of cell/tissue function and pathology.     

A biological context for the self-nonself discrimination and the regulation of effector class by the immune system

An effective immune response to an antigen requires two sets of decisions: Decision 1, the sorting of the repertoire, and Decision 2, the regulation of effector class. The repertoire, because it is somatically generated, large, and random, must be sorted by a somatic mechanism that subtracts those specificities (anti-self) that, if expressed, would debilitate the host, leaving a residue (anti-nonself) that, if not expressed, would result in the death of the host by infection. The self-nonself discrimination is the metaphor used to describe Decision 1, the sorting of the repertoire. In order to be functional, the sorted repertoire must be coupled to a set of biodestructive and ridding effector functions, such that the response to each antigen is treated in a coherent and independent manner. Although a reasonably complete framework for Decision 1 exists, Decision 2 lacks conceptualization. The questions that must be considered to arrive at a proper framework are posed. It should be emphasized that manipulation at the level of Decision 2 is where clinical applications are likely to be found.     

Apparent replication of suggestive linkage on chromosome 16 in the NIMH genetics initiative bipolar pedigrees

Analyses of a replication sample of families collected as part of the National Institute of Mental Health (NIMH) Genetics Initiative for bipolar disorder provide further evidence for linkage to a region of chromosome 16. Families who had a bipolar I (BPI) proband and at least one BPI or schizoaffective, bipolar type (SABP) first-degree relative were ascertained for the purpose of identifying genes involved in bipolar affective disorder. A series of hierarchical models of affected status was used in linkage analyses. Initial genetic analyses of chromosomes 3, 5, 15, 16, 17, and 22, completed at Indiana University in 540 subjects from 97 families, suggested evidence of linkage to chromosomes 5, 16, and 22 [Edenberg et al., 1997: Am J Med Genet 74:238-246]. Genotyping was subsequently performed on these chromosomes in a replication sample of 353 individuals from 56 families. Nonparametric linkage analyses were performed using both affected relative and sibling pair methods. Analyses in the new sample on chromosome 16, using the broadest model of affected status, corroborate previously reported suggestive linkage to the marker D16S2619. Combining the initial and replication samples further increased the evidence of linkage to this region, with a peak lod score of 2.8.     

Immunohistochemistry of neurone specific enolase with gamma subunit specific anti-peptide monoclonal antibodies.

AIMS--To investigate the application in immunohistochemistry of gamma-subunit specific anti-peptide monoclonal antibodies to human neurone specific enolase (NSE); and to determine their reactivity with formalin fixed, wax embedded sections of normal tissue and neuroendocrine tumours. METHODS--Immunohistochemical staining was performed on sections of formalin fixed, wax embedded tissue with two monoclonal antibodies (NSE-P1 and NSE-P2) raised against different synthetic peptides specific for the gamma subunit of human enolase (neurone specific enolase). RESULTS--Both antibodies gave strong immunostaining in normal tissues and cells known to contain NSE. There was no immunoreactivity in tissues containing either the alpha alpha or beta beta isozymes of enolase. The reactivity of the antibodies with a range of neuroendocrine tumours was also studied and both antibodies gave strong immunostaining of tumour cells in the different tumours. CONCLUSIONS--The use of synthetic peptides from defined regions of a molecule as immunogenes provides antibodies of high specificity. These monoclonal antibodies to NSE are ideally suited for immunohistochemical studies and they should be particularly useful in histopathology as they react with epitopes which are resistant to formalin fixation and wax embedding.     

Hb FM-Fort Ripley: Confirmation of autosormal dominant inheritance and diagnosis by PCR and direct nucleotide sequencing

We describe a normal neonate who presented at four days of age with asymptomatic cyanosis. There was no evidence of cardiac or pulmonary abnormality and an extended family history included 13 other affected family members with asymptomatic cyanosis lasting one to three months. Polymerase chain reaction (PCR) amplification and direct nucleotide sequencing of the proband's Gγ chain gene revealed the mutation at codon 92 (CAC→TAC) previously shown in haemoglobin FM-Fort Ripley (α₂γ₂^{Gγ 92 (F8) His→Tyr}). This is the first family with Hb FM-Fort Ripley reported so far. It demonstrates autosomal dominant inheritance of this condition and incomplete penetrance. © 1994 Wiley-Liss, Inc.     

One-way positive cellular reactions between two HLA-A, B, C, D/DR genotypically identical brothers following active allogeneic immunization

The HLA-D/DR region in man encodes major determinants which stimulate T lymphocytes to proliferation. The genetic organization of this region is apparently complex and is at present largely unknown. One obstacle is the scarcity and quality of available typing reagents. In an attempt to obtain high quality anti-DR sera, a series of active immunizations was performed between highly selected, healthy unrelated donors and recipients.
One recipient (AR8) was immunized using cells incompatible for HLA-A2, B40 (w60), Cw3 and DIDRw6 and readily developed anti-A2 and B40 antibodies but no anti-C, DR, or other antibodies. When tested against his HLA genotypically fully identical brother using the cellular MLC, PLT, or CML techniques before immunization, results were mutually negative as expected. Following immunization, however, AR8 was able to mount MLC, PLT, and possibly CML responses against lymphocytes from the brother while the reverse combinations remained negative. When tested in the family the trait(s) thus identified seems to be maternally inherited.
These results suggest the existence of minor histocompatibility determinants encoded from regions not closely linked to HLA. The brother of AR8 and the immunizing donor thus seem to share one or more determinants not possessed by AR8.     

Assessment of Coxsackievirus B3 ts Mutants for Induction of Myocarditis in a Murine Model

Ten temperature-sensitive (ts) mutants isolated from a myocarditis-inducing wild-type (WT) coxsackievirus B3 parent did not induce myocarditis in adolescent CD-1 mice. An avirulent prototype ts mutant from one of the three complementation groups adsorbed to murine cardiac tissue, as did WT virus. Heart tissues from mice inoculated with WT virus contained 100- to 1,000-fold more virus than heart tissues from mice inoculated with any of the three prototype ts mutants. WT virus exhibited a greater capsid stability and a higher efficiency of replication at 37 degrees C than any of the three prototype ts mutants. All three prototype ts mutants induced less interferon in vivo than WT virus. Cell-mediated immune responses, assessed by the cell migration inhibition assay, were different in mice inoculated with WT virus when compared to ts 5 mutant virus. Peritoneal exudate cells from mice inoculated with WT but not ts 5 virus reacted specifically against antigens in WT virus HeLa cell lysates and antigens extracted with KCl from cardiac tissues of mice inoculated with WT virus. Cardiac tissues of mice inoculated with WT but not ts 5 virus contained KCl-extractable antigens which were able to specifically inhibit the migration of peritoneal exudate cells taken from mice immunized with WT virus. Therefore, ts 5 neither elicited a measurable cell-mediated immune response nor induced antigens in cardiac tissues which were immunoreactive with sensitized-(WT virus)-peritoneal exudate cells. Of 9 revertant viruses isolated from the 10 ts mutants, 5 showed covariance in ability to replicate at 39.5 degrees C and capacity for induction of myocarditis. Some revertants exhibited a reduced capsid thermostability compared to WT virus but yet retained the capacity for induction of myocarditis. The data suggest that induction of myocarditis by coxsackievirus B3 variants depends on a combination of several variables, including capsid stability, capacity for replication at 37 degrees C, and expression of the three identified genes. All three prototype ts mutants served as vaccine viruses in preventing myocarditis in adolescent mice subsequently challenged with WT virus. However, all three prototype ts mutants and their revertant variants retained partial to complete lethality in CD-1 neonates.     

Disease-specific electrophysiological findings in adult ceroid-lipofuscinosis (Kufs disease)

Two adults with mild dementia and a history of memory loss and disequilibrium were seen in the eye clinic following complaints of acuity loss in the 20/30–20/70 (Snellen) range. Results from the fundus examination of one patient were entirely normal; the other showed minimal vascular attenuation and optic atrophy. Electrophysiology was remarkable: (1) Photopic ERG b-waves were reduced, delayed, and showed pronounced oscillations. (2) EOG light-rise potentials were absent or very small. (3) Binocular pattern-VER signals showed addition of the monocular signal. Scotopic ERG signals were normal. Brain biopsy and microscopy showed intercellular, autofluorescent ceroid deposits which provided a clear diagnosis of Kufs disease. Histology of model animal retinal cells show ceroid deposits in cell classes implicated by the human retinal signals. The cluster of electrophysiological results point toward early changes in the pigment epithelium and inner plexiform layer cells as a means of non-invasive diagnosis.