CD40 expression by gingival fibroblasts: correlation of phenotype with function

CD40, a member of the tumor necrosis factor-alpha receptor family, is constitutively expressed by cells of hematopoietic and non- hematopoietic origin, including fibroblasts. Signaling through this receptor molecule regulates inflammatory cytokine secretion by many cell types. Based on the recently described cytokine secretory heterogeneity of fibroblast cell subsets, we hypothesized that secretion of inflammatory cytokines by gingival fibroblast cultures may be dictated by the existence of differential proportions of cytokine- secreting subpopulations which express high levels of CD40. After examining a large number of gingival fibroblast (GF) cultures we find that the frequency of IL-6- and IL-8-secreting cells mirrors the frequency of cells expressing high levels of CD40 in these cultures. In addition, we demonstrate a direct functional relationship between CD40 expression and IL-6 or IL-8 secretion by showing that ligation of this molecule on GF, and CD40+ fibroblast subsets in particular, up- regulates secretion of these cytokines in vitro.      

Description of a novel HLA-DRB1 allele found in a candidate for bone marrow transplantation

We report the identification of a new DRB1* allele in a Spanish Caucasoid family during a search for a histocompatible bone marrow donor. This novel allele, designated as DRB1*1145, differs from DRB1*1123 in one nucleotide at position 199 in exon 2 (A replacing T), leading to one amino acid change from phenylalanine (Phe) to isoleucine (Ile) at codon 67. The propositus's father had identical class II alleles but showed a minor mismatch at locus B (B*4403 by B*4402) and a C-locus mismatch (Cw*1502 by Cw*0501). We discuss the criteria of selecting a non-related bone marrow donor with a minor mismatch on the DRB1* allele or the related father having a minor mismatch at B locus and a C-locus mismatch.     

Characterization of the human jumonji gene

While constructing a cDNA library of human embryos, we have isolated a clone homologous to jumonji, a mouse gene required for neural tube formation. We have determined the complete coding sequence of the human homologue (JMJ) and deduced the amino acid sequence of the putative protein. We show here that human and mouse jumonji putative proteins are homologous and present 90% identity. During human embryogenesis, JMJ mRNAs are predominantly expressed in neurons and particularly in dorsal root ganglion cells. They are also expressed in neurons of human adult cerebral cortex. In view of these observations, we propose JMJ as a candidate gene for developmental defects of the central nervous system in the human. The human JMJ gene maps at position 6p24-6p23.      

Screening for proteins with polyglutamine expansions in autosomal dominant cerebellar ataxias

Expansion of trinucleotide CAG repeats coding for polyglutamine has been implicated in five neurodegenerative disorders, including spinocerebellar ataxia (SCA) 1 and SCA3 or Machado-Joseph disease (SCA3/MJD), two forms of type I autosomal dominant cerebellar ataxias (ADCA). Using the 1C2 antibody which specifically recognizes large polyglutamine tracts, particularly those that are expanded, we recently reported the detection of proteins with pathological glutamine expansions in lymphoblasts from another form of ADCA type I, SCA2, as well as from patients presenting with the distinct phenotype of ADCA type II. We now have screened a large series of patients with ADCA or isolated cases with cerebellar ataxia, for the presence of proteins with polyglutamine expansions. A 150 kDa SCA2 protein was detected in 16 out of 40 families with ADCA type I. This corresponds to 24% of all ADCA type I families, which is much more frequent than SCA1 in this series of patients (13%). The signal intensity of the SCA2 protein was negatively correlated to age at onset, as expected for an expanded and unstable trinucleotide repeat mutation. The disease segregated with markers closely linked to the SCA2 locus in all identified SCA2 families. In addition, a specific 130 kDa protein, which segregated with the disease, was detected in lymphoblasts of patients from nine families with ADCA type II. It was also visualized in the cerebral cortex of one of the patients, demonstrating its translation in the nervous system. Finally, no new disease-related proteins containing expanded polyglutamine tracts could be detected in lymphoblasts from the remaining patients with ADCA or isolated cases with cerebellar ataxia.      

Are isofurans and neuroprostanes increased after subarachnoid hemorrhage and traumatic brain injury?

Current diagnostic tools to assess neurological injury after aneurysmal subarachnoid hemorrhage (aSAH) and traumatic brain injury (TBI) have poor discriminatory abilities. Free radicals are associated with the pathophysiology of secondary damage after brain trauma. We examined cerebrospinal fluid (CSF) lipid markers of oxidative stress, isofurans (IsoFs), F(4)-neuroprostanes (F(4)-NeuroPs), and F(2)-isoprostanes (F(2)-IsoPs), in two case-controlled studies in patients with aSAH or severe TBI. Patients with aSAH (n=18) or TBI (n=18) were age and gender matched with separate control groups. CSF samples were collected from patients within 24?h of the injury. CSF IsoFs and F(4)-NeuroPs were increased in aSAH patients compared with their controls. In TBI patients, IsoFs and F(4)-NeuroPs were increased compared with their controls. F(2)-IsoPs were increased in aSAH patients, but not in TBI patients, compared with their respective controls. CSF IsoFs and F(4)-NeuroPs are consistently increased after a catastrophic central nervous system injury. These results suggest their measurement may enhance the management of unconscious patients in neurological care.     

Cerebrospinal fluid profile of amyloid β42 (Aβ42), hTau and ubiquitin in North Indian Alzheimer's disease patients

Alzheimer's disease (AD) is the most common form of dementia, and is characterized by the degeneration of neurons and their synapses, and a higher number of amyloid plaques and neurofibrillary tangles (NFTs) compared with that found in non-demented individuals. Amyloid-β-peptides (Aβ) are major components of amyloid plaques in AD brain whereas NFTs are composed of Tau and associated with ubiquitin. The aim of the present study was to analyze the levels of Aβ42, hTau (total Tau) and ubiquitin in CSF of North Indian population. CSF Aβ42, Tau and ubiquitin were measured in CSF of AD patients as well as controls using ELISA assays. Here we report low Aβ42 levels in AD patients (324.24 ± 76.38 pg/ml) as compared to those in non-AD (NAD) (668.34 ± 43.13 pg/ml), neurological controls (NCs) (727.28 ± 46.49 pg/ml) and healthy controls (HCs) (976.47 ± 124.46 pg/ml). In contrast, hTau and ubiquitin levels were significantly high (568.65 ± 48.89 pg/ml and 36.82 ± 4.34 ng/ml, respectively) in AD patients compared to those in NAD, NC and HC. The hTau levels were 267.37 ± 36.64 pg/ml, 167.34 ± 44.27 pg/ml and 107.62 ± 24.27 pg/ml in NAD, NC and HC, respectively. Similarly, ubiquitin levels were 23.57 ± 2.32 ng/ml, 19.76 ± 3.64 ng/ml and 13.24 ± 4.56 ng/ml in NAD, NC and HC, respectively. In conclusion, low Aβ42 and high Tau–ubiquitin levels were found in North Indian AD patients.     

Oxidative stress time course in the rat diaphragm after freezing-thawing cycles

Hyperthermia was shown to induce oxidative stress by uncoupling mitochondrial respiratory chain and to reduce superoxide dismutase (SOD) activity in muscles. Reactive carbonyl groups, malondialdehyde (MDA)-protein adducts, 3-nitrotyrosine immunoreactivity, Mn-SOD, and catalase were detected using immunoblotting in rat diaphragm specimens and homogenates thawed at room temperature (after previous storage at -80 degrees C) for 5, 15, 30, and 60 min, and 3, 6, and 24h to be subsequently and immediately stored at -80 degrees C. Mn-SOD activity was also measured in all muscles. Both total protein carbonylation (reactive carbonyl groups and MDA-protein adducts) and nitration were significantly increased over time, reaching their peaks in the diaphragms of the 60- and 15-min groups, respectively. Mn-SOD expression and activity were significantly reduced over time, while catalase expression showed no significant variation. Protein oxidation was significantly increased in the rat diaphragms exposed to freezing-thawing cycles of different time lengths, while Mn-SOD was substantially reduced in all muscles.     

Dynamics of dominance of a dipeptide insertion in reverse transcriptase of HIV-1 from patients subjected to prolonged therapy

A small proportion (0.8%) of individuals of a cohort of HIV-1 infected patients subjected to prolonged therapy with nucleoside analogues included a recently recognised dipeptide insertion in their RT (Ser-Ser or Ser-Gly between RT codons 69 and 70). To study the dynamics of dominance of genomes with this genetic change, sequential HIV-1 isolates from two patients were analyzed with regard to consensus sequences and complexity of mutant spectra. The two patients displayed completely different, complex evolutionary patterns leading to temporary dominance of dipeptide insertions. In one patient, a virus very closely related to an ancestor virus from the same patient overtook the population at late times, displacing genomes encoding a Ser-Ser insertion. In another patient the sequential dominance of genomes with Ser-Ser insertion-->no insertion-->Ser-Gly insertion was observed. These three types of genomes coexisted in the mutant spectrum of one HIV-1 isolate. Complexity was also reflected in the shape of phylogenetic trees derived with genomes from the mutant spectrum at each time point. The results suggest that HIV-1 genomes encoding a dipeptide insertion between RT codons 69 and 70 do not show a clear selective advantage over other genomes lacking the insertion. Such an absence of a clear selective advantage will favor that such genomes encoding this RT insertion become dominant only in a transient fashion, and following disparate kinetics in different patients.     

A comparative study of pseudorabies virus (PRV) strains with defects in thymidine kinase and glycoprotein genes

In the course of two experiments, an examination was made of the virulence and neuroinvasiveness for pigs of two pseudorabies virus mutants (strain 6C2TK(-), with a defect in thymidine kinase (TK) function; and strain 6C2TK(-), gI(-)/gE(-), with defects in TK and glycoproteins I and E) and of the wild-type parent strain (86/27V). At various times after intranasal inoculation, pigs were killed and samples of tonsil, lung and different levels of the trigeminal and olfactory nervous pathways were examined by methods that included viral isolation, polymerase chain reaction assay and immunohistochemistry. Both mutant viruses were of reduced virulence, as indicated by no more than moderate clinical signs and lesions, and only sporadic isolation of virus; moreover, unlike the wild-type parent strain, the mutant viruses were not reactivated from the latent state by corticosteroid treatment. In addition, migration of the mutant strains to the central nervous system (olfactory and trigeminal nervous pathways) was reduced as compared with that of the wild-type strain. Thus, mutations in the genes encoding the TK enzyme and the gI/gE complex were associated with reduced virulence, reduced replication in peripheral target tissues, and reduced migration to the olfactory and trigeminal pathways.