Anti-HLA class I antibody-mediated activation of the PI3K/Akt signaling pathway and induction of Bcl-2 and Bcl-xL expression in endothelial cells

Anti-human leukocyte antigen (HLA) antibodies (Ab) have long been implicated in the process of acute and chronic allograft rejection, yet their mechanism(s) of action is not well understood. The aim of this study was to determine whether ligation of HLA class I molecules by anti-HLA Ab on the surface of human endothelial cells (EC) activates the PI3 Kinase (PI3K)/Akt signaling pathway and downstream target proteins of the cell death apparatus. We report that Ab ligation of major histocompatibility complex (MHC) class I molecules on the surface of EC triggers phosphorylation of Akt, PI3K, and recruitment of PI3K and Akt into a signaling unit with focal adhesion kinase. Signaling through class I also stimulated phosphorylation of Bad and upregulated expression of Bcl-2 and Bcl-xL. Pretreatment of EC with the PI3K inhibitor wortmannin blocked class I-mediated expression of Bcl-2, but not Bcl-xL, suggesting a role for the PI3K/Akt signaling pathway in regulation of class I-induced Bcl-2 expression. The intracellular events initiated by class I ligation were influenced by the concentration of the anti-HLA Ab with the lowest tested concentrations of Ab stimulating the highest level of Akt phosphorylation, Bcl-xL and Bcl-2 expression. Consistent with the in vitro experiments, analysis of biopsy samples from heart transplant recipients with evidence of Ab-mediated rejection exhibited increased Bcl-2 expression on the vascular endothelium. These results suggest that exposure of the graft endothelium to low concentrations of anti-HLA Ab may promote cell survival by transducing signals resulting in upregulation of cell survival genes.     

Immunization with a polyprotein vaccine consisting of the T-Cell antigens thiol-specific antioxidant, Leishmania major stress-inducible protein 1, and Leishmania elongation initiation factor protects against leishmaniasis

Development of an effective vaccine against Leishmania infection is a priority of tropical disease research. We have recently demonstrated protection against Leishmania major in the murine and nonhuman primate models with individual or combinations of purified leishmanial recombinant antigens delivered as plasmid DNA constructs or formulated with recombinant interleukin-12 (IL-12) as adjuvant. In the present study, we immunized BALB/c mice with a recombinant polyprotein comprising a tandem fusion of the leishmanial antigens thiol-specific antioxidant, L. major stress-inducible protein 1 (LmSTI1), and Leishmania elongation initiation factor (LeIF) delivered with adjuvants suitable for human use. Aspects of the safety, immunogenicity, and vaccine efficacy of formulations with each individual component, as well as the polyprotein referred to as Leish-111f, were assessed by using the L. major challenge model with BALB/c mice. No adverse reactions were observed when three subcutaneous injections of the Leish-111f polyprotein formulated with either MPL-squalene (SE) or Ribi 529-SE were given to BALB/c mice. A predominant Th1 immune response characterized by in vitro lymphocyte proliferation, gamma interferon production, and immunoglobulin G2A antibodies was observed with little, if any, IL-4. Moreover, Leish-111f formulated with MPL-SE conferred immunity to leishmaniasis for at least 3 months. These data demonstrate success at designing and developing a prophylactic leishmaniasis vaccine that proved effective in a preclinical model using multiple leishmanial antigens produced as a single protein delivered with a powerful Th1 adjuvant suitable for human use.     

Cloning and characterization of a gene encoding an immunoglobulin-binding receptor on the cell surface of some members of the family Trypanosomatidae

Several members of the Trypanosomatidae family, when freshly isolated from their mammalian hosts, have immunoglobulins adsorbed to their cell surfaces. However, a significant portion of these antibody molecules is not parasite specific, i.e., the immunoglobulins are bound to the parasite's cell surface molecules via noncognitive interactions. It has been proposed that this noncognitive adsorption of immunoglobulins to the parasite is mediated by an Fc-like receptor present in several members of the Trypanosomatidae family. However, the molecular identification of this receptor has never been defined. Here, we describe the cloning of a gene encoding a protein that might represent this molecule. The gene, named Lmsp1, was cloned by screening a Leishmania major cDNA expression library using a rabbit antiserum. Lmsp1 is present in both Leishmania and Trypanosoma and is expressed in all developmental stages of these parasites. The predicted protein has a molecular mass of 16.6 kDa and contains an RGD sequence starting at residue 104 and three cysteine residues at positions 55, 74, and 116. The purified recombinant protein strongly binds to normal immunoglobulins of various animal species (humans, rabbits, sheep, goats, guinea pigs, donkeys, rats, and mice) and the binding to human immunoglobulins appears to be immunoglobulin G (IgG) and IgM isotype specific. Moreover, Lmsp1 binds to both purified Fc and Fab fragments of IgG from both humans and rabbits. The mapping of the Lmsp1 epitopes that bind human IgG revealed that different sequences of the molecule bind to Fc or Fab. In addition, fluorescence-activated cell sorter analyses with a specific rabbit anti-Lmsp1 antiserum showed that Lmsp1 is associated with the parasite's cell surface. Finally, inhibition experiments point to an active role of this molecule in the immunoglobulin-mediated attachment and penetration of Trypanosoma cruzi in its macrophage host cells, thus suggesting that Lmsp1 is a putative Trypanosomatidae immunoglobulin receptor.     

Identification of cathepsin C mutations in ethnically diverse papillon-Lefèvre syndrome patients

INTRODUCTION—Papillon-Lefèvre syndrome (PLS) is an autosomal recessive disorder characterised by palmoplantar keratoderma and severe, early onset periodontitis, which results from deficiency of cathepsin C activity secondary to mutations in the cathepsin C gene. To date, 13 different cathepsin C mutations have been reported in PLS patients, all of which are homozygous for a given mutation, reflecting consanguinity.
AIM—To evaluate the generality of cathepsin C mutations in PLS, we studied an ethnically diverse group of 20 unrelated families.
METHODS—Mutations were identified by direct automated sequencing of genomic DNA amplified for exonic regions and associated splice site junctions of the cathepsin C gene. Long range PCR was performed to determine the genomic structure of the cathepsin C gene.
RESULTS—The cathepsin C gene spans over 46 kb, with six introns ranging in size from 1.6 to 22.4 kb. Eleven novel mutations and four previously reported mutations were identified in affected subjects from 14 families. Missense mutations were most common (9/15), followed by nonsense mutations (3/15), insertions (2/15), and deletions (1/15). Among these 14 probands, two were compound heterozygotes. Affected subjects with transgressions of the dermal lesions onto the knees or elbows or both had mutations in both the pro- and mature regions of the enzyme, although most were in the mature region.
CONCLUSION—Mutations in the mature region of cathepsin C were more likely to be associated with the transgressions of the dermatological lesions, although the results were not statistically significant. A comprehensive list of all cathepsin C mutations described to date, representing 25 mutations from 32 families with PLS and related conditions, is also presented.


Keywords: cathepsin C; genetics; severe early onset periodontitis; hyperkeratosis     

Genetic similarity of Candida albicans strains from vaginitis patients and their partners.

The moderately repetitive sequence Ca3 was used to fingerprint strains of Candida albicans isolated from vulvovaginal infections of 10 women and strains isolated from their male partners. The Dendron software package was then used to compare the DNA fingerprints of these strains with those of vaginal commensals from women from the same geographical locale, vaginal commensals from women from a different geographical locale, and commensals from male partners of asymptomatic women from the same geographical locale. The results demonstrate that, in the majority of cases (8 of 10), strains from symptomatic patients and their partners are either identical or more similar to each other than to other strains, infecting strains do not represent a group genetically distinguishable from vaginal commensal isolates from women from the same geographical locale, and both infecting strains and commensals from individuals in the test locale can be distinguished from commensals obtained in another geographical locale. The results also suggest that women with vaginal infections are responsible for strain replacement in their male partners.     

Corticosteroids, Serum, and Phagocytosis: In Vitro and In Vivo Studies

The phagocytic-bactericidal capacity (PBC) of human polymorphonuclear leukocytes (PMNs) for strains of Klebsiella (K) and of Enterococcus (E) was unaffected in vitro by the presence of 100 mug of either hydrocortisone (HC) or of methylprednisolone (MP) per ml in the medium. At higher concentrations (500 to 2,000 mug/ml) both compounds impaired PBC-K and PBC-E, but the latter was less sensitive to steroid-induced inhibition. In addition to interfering with intracellular killing of both organisms by PMNs, 2,000 mug of HC per ml also inhibited ingestion of E, although not of K. Steroid-induced inhibition of PBC-K in vitro was completely abolished by increasing the concentration of serum used as opsonin. The PBC-K of human PMNs obtained 30 min after intravenous injection of 1 g of MP was unimpaired in vitro in the presence of 10 to 90% simultaneously obtained autologous serum containing 42 mug of MP per ml. These findings suggest that short-term, high-dosage administration of MP is unlikely to produce clinically significant impairment of intraleukocytic bacterial killing.     

Isolation of a fastidious Bergeyella species associated with cellulitis after a cat bite and a phylogenetic comparison with Bergeyella zoohelcum strains

Bergeyella zoohelcum is an uncommon zoonotic pathogen typically associated with cat or dog bites. Previously, only five cases of B. zoohelcum infection have been reported. We report the isolation and characterization of a fastidious Bergeyella species from acute cellulitis in the upper extremity of a 60-year-old woman. The organism was too fastidious for identification and susceptibility testing with traditional culture methods. The isolate was characterized further by PCR amplification and sequencing of the 16S rRNA gene with broad-range eubacterial primers. Phylogenetic analysis of the 16S ribosomal DNA sequence indicated that this isolate was a member of the species B. zoohelcum (previously Weeksella zoohelcum), a gram-negative bacillus that is rarely associated with infections in humans. Despite sharing a close genetic relationship with other B. zoohelcum strains, this isolate was extremely fastidious in nature, raising the possibility that similar strains from cat or dog bite wound infections have been underreported.     

Decorin inhibition of PDGF-stimulated vascular smooth muscle cell function: potential mechanism for inhibition of intimal hyperplasia after balloon angioplasty

Decorin is a small proteoglycan that binds to transforming growth factor-beta (TGF-beta) and inhibits its activity. However, its interaction with platelet-derived growth factor (PDGF), involved in arterial repair after injury, is not well characterized. The objectives of this study were to assess decorin-PDGF and decorin-PDGF receptor (PDGFR) interactions, the in vitro effects of decorin on PDGF-stimulated smooth muscle cell (SMC) functions and the in vivo effects of decorin overexpression on arterial repair in a rabbit carotid balloon-injury model. Decorin binding to PDGF was demonstrated by solid-phase binding and affinity cross-linking assays. Decorin potently inhibited PDGF-stimulated PDGFR phosphorylation. Pretreatment of rabbit aortic SMC with decorin significantly inhibited PDGF-stimulated cell migration, proliferation, and collagen synthesis. Decorin overexpression by adenoviral-mediated gene transfection in balloon-injured carotid arteries significantly decreased intimal cross-sectional area and collagen content by approximately 50% at 10 weeks compared to beta-galactosidase-transfected or balloon-injured, non-transfected controls. This study shows that decorin binds to PDGF and inhibits its stimulatory activity on SMCs by preventing PDGFR phosphorylation. Decorin overexpression reduces intimal hyperplasia and collagen content after arterial injury. Decorin may be an effective therapy for the prevention of intimal hyperplasia after balloon angioplasty.     

Influences of chorion type on measurements of the corpus callosum in adult monozygotic male twins?

MRI imaging was used to estimate volumes of corpus callosum structure in 45 pairs of identical (monozygotic, MZ) twins from the National Heart, Lung, and Blood Institute (NHLBI) twin study. Age range of the study subjects was from 68–78 years. Finger, palm, and footprint data (dermatoglyphics) collected at previous examinations of the NHLBI twin study were available for 39 pairs. The dermatoglyphics were scored for an index to retrospectively assess chorion type in MZ twin‐pairs. The results indicated an association between variability in various structures of the corpus callosum with some of these dermatoglyphic traits, suggesting greater structural variation within pairs with dichorionic placentas. In contrast, total intracranial volume, which has similar heritability estimates as a result of shared genetic effects with the corpus callosum, was unrelated to the dermatoglyphic traits. The results provide indirect evidence that the intrauterine environment may influence twin‐pair similarity of corpus callosum measures in adults. Am. J. Hum. Biol. 14:338–346, 2002. © 2002 Wiley‐Liss, Inc.     

The effects of somatostatin and metiamide on tachyphylaxis of pentagastrin stimulated gastric acid and pepsin secretion in the conscious cat.

1. Pentagastrin stimulated gastric acid and pepsin secretions show parallel rates of tachyphylaxis in the conscious cat. The responses to histamine show only slight tachyphylaxis. 2. Somatostatin 10 microng.kg(-1).hr(-1) inhibits pentagastrin but not histamine stimulated acid secretion and inhibits pentagastrin stimulated pepsin secretion. 3. The inhibition of pentagastrin stimulated acid and pepsin secretion by Somatostatin delays the tachyphylaxis of these responses, but the rates of tachyphylaxis when they do subsequently occur are identical. 4. Metiamide 10 mg-kg(-1)-hr(-1) equally inhibits histamine and pentagastrin stimulated acid secretion but does not inhibit pentagastrin stimulated pepsin secretion. 5. Inhibiton of acid secretion during metiamide infusion neither prevents nor delays acid nor pepsin tachyphylaxis. 6. It is suggested that tachyphylaxis of acid and pepsin secretion is a gastrin receptor phenomenon and that Somatostatin occupies or modifies the behaviour of these receptors, preventing tachyphylaxis. Metiamide, however, exerts its action only on the histmine H2-receptor and not the gastrin receptor mechanism, and this apparently does not prevent or delay acid tachyphylaxis.