Impact of intranasal budesonide on immune inflammatory responses and epithelial remodeling in chronic upper airway inflammation

BACKGROUND: Histologic and immunohistologic features of nasal polyps (NP) are similar to those observed in asthma, thus suggesting a similar immunopathology. OBJECTIVE: The primary objective of this study was to further understand the anti-inflammatory and immunoregulatory effects of locally delivered corticosteroids. To this end, the effect of intranasal budesonide on the expression of specific cytokines, lymphocyte subsets, and epithelial remodeling in this model of airway tissue inflammation were studied. METHODS: We used immunohistochemical techniques to examine nasal mucosae (NM) from healthy individuals and nasal polyp (NP) tissues from patients with nasal polyposis obtained before and after intranasal budesonide treatment. RESULTS: First, the density of CD8(+) cells was markedly increased in NP tissues after intranasal budesonide treatment from 16.1 +/- 8.4 (M +/- SEM) per mm(2) to 39.9 +/- 24.1. Second, the density of cells immunoreactive for IL-4, IL-5, IFN-gamma, IL-12, and TGF-beta in NP was significantly greater than in control NM tissues. The density of IL-4(+) and IL-5(+) cells in NP tissues significantly decreased after budesonide treatment from 40 +/- 12 to 17.8 +/- 8 and from 19.3 +/- 11 to 10.4 +/- 7, respectively. In contrast, the density of IFN-gamma(+) and IL-12(+) cells remained unchanged. In addition, we found that the density of TGF-beta(+) cells significantly increased after intranasal budesonide from 18 +/- 5 to 41 +/- 9. Third, damage to the entire length of the NP epithelium was quantified using a grading system. The epithelium of untreated NP was substantially damaged; remarkable epithelial restitution with no apparent changes in stromal collagen deposition was observed after intranasal budesonide treatment. CONCLUSIONS: These findings demonstrate that intranasal budesonide induced an increase in CD8 population and a selective regulatory effect on tissue cytokine expression. Furthermore, intranasal budesonide promoted epithelial remodeling. We hypothesize that these immunoregulatory and remodeling effects elicited by steroids might be, at least in part, mediated by the induction of TGF-beta.     

Clinical and molecular study in congenital muscular dystrophy with partial laminin alpha 2 (LAMA2) deficiency

Complete laminin alpha2 (LAMA2) deficiency causes approximately half of congenital muscular dystrophy (CMD) cases. Many loss-of-function mutations have been reported in these severe, neonatal-onset patients, but only single missense mutations have been found in milder CMD with partial laminin alpha2 deficiency. Here, we studied nine patients diagnosed with CMD who showed abnormal white-matter signal at brain MRI and partial deficiency of laminin alpha2 on immunofluorescence of muscle biopsy. We screened the entire 9.5 kb laminin alpha2 mRNA from patient muscle biopsy by direct capillary automated sequencing, single strand conformational polymorphism (SSCP), or denaturing high performance liquid chromatography (DHPLC) of overlapping RT-PCR products followed by direct sequencing of heteroduplexes. We identified laminin alpha2 sequence changes in six of nine CMD patients. Each of the gene changes identified, except one, was novel, including three missense changes and two splice-site mutations. The finding of partial laminin alpha2 deficiency by immunostaining is not specific for laminin alpha2 gene mutation carriers, with only two patients (22%) showing clear causative mutations, and an additional three patients (33%) showing possible mutations. The clinical presentation and disease progression was homogeneous in the laminin alpha2-mutation positive and negative CMD patients.     

Human Th1 cells preferentially induce interleukin (IL)-1β while Th2 cells induce IL-1 receptor antagonist production upon cell/cell contact with monocytes

The role of human T cells in the induction and regulation, upon cell/cell contact, of inflammatory responses by monocytic cells was investigated. The production of interleukin (IL)-1β and IL-1 receptor antagonist (IL-1Ra) by the monocytic THP-1 cell line was measured upon contact with either Th1 or Th2 cell clones. CD4⁺ T cell clones specific for purified protein derivative of Mycobacterium tuberculosis, predominantly Th1 [high interferon (IFN)-γ and low IL-4 producers], or tetanus toxoid, predominantly Th2 (low IFN-γ and high IL-4 producers), were generated. Cell membranes from antigen-stimulated, but not from resting T cell clones induced dose-dependent cytokine production by THP-1 cells. Th1 clones induced higher levels of IL-1β production (484–806 pg/ml) than did Th2 clones (21–114 pg/ml). In contrast, Th1 clones induced lower levels of IL-1Ra (0.9–7.8 ng/ml) than did Th2 clones (7.0–49.6 ng/ml). Similar results were obtained when T cell clones were activated by cross-linked CD3 and CD28. IL-1β production by THP-1 cells correlated with IFN-γ production by T cell clones but was unaffected by IFN-γ neutralization. IL-1Ra production by THP-1 cells correlated with IL-4 production by T cells and was partially inhibited by IL-4 neutralization. These data indicate that activated Th1 and Th2 cells express different molecules on the cell surface able to induce distinct pro-inflammatory (IL-1β) or anti-inflammatory (IL-1Ra) responses in monocytes. This differential induction of molecules with opposite effects on inflammation stresses the functional heterogeneity in CD4⁺ T cells.     

Predominant and stable T cell responses to regions of myelin basic protein can be detected in individual patients with multiple sclerosis

T lymphocytes from patients with multiple sclerosis (MS) recognize multiple myelin basic protein (MBP) epitopes. This situation complicates the design of specific immunotherapies. We investigated to which extent the T cell response to MBP is heterogeneous in single subjects in terms of preferentially recognized regions of the molecule, major histocompatibility complex (MHC) restriction, and stability over time. From each of nine patients with MS, a minimum of six MBP-specific T lymphocyte lines (TLL) were assayed for the proliferative response to a panel of overlapping peptides, encompassing the whole MBP. Predominant Tcell recognitions of distinct MBP regions were present in three patients, all HLA-DR2⁺, independently of the clinical features of their disease. Tcell reactivity was preferentially directed to residues 16-38 in one patient. In this case the response was also stable over time, during different phases of the disease. Predominant reactivity to residues 86-99 was detected in the two other DR2⁺ patients. In each of the patients with other HLA-DR haplotypes (DR2^?), as well as in three DR2⁺ non-MS donors, the Tcell response to MBP appeared to be considerably more heterogeneous. The HLA restriction element varied among TLL recognizing the same MBP region, even when raised from the same individual. The genomic HLA typing, performed on the DRB1 and DRB5 genes in the DR2⁺ subjects, showed no obvious correspondence between preferential responses to regions of MBP and HLA-DR2 subtypes. In this context, a simple, new method for the genomic typing of the HLA-DRB1 gene in individuals with the HLA-DR2 serological specificity is also described. We conclude that predominant and stable T cell responses to a single MBP region can be detected in some patients with MS. In these individuals, the MHC restriction of the T cell recognition of predominant regions appears to be variable. Polymorphisms of the HLA-DR2 gene products alone do not account for the selection of the dominant MBP Tcell epitope.     

MEG Characterization of Spontaneous Alpha Rhythm in the Human Brain

The present piece of research studied the spontaneous alpha rhythm of the human brain by combining the use of a whole-cortex neuromagnetometer and Magnetic Resonance Imaging. Single trials of spontaneous brain activity were recorded from ten human subjects asked to rest, with their eyes either closed or open, in relaxed wakefulness. MEG measurements were conducted over a period of one and a half years. The replicability of the results was confirmed for eight subjects out of ten. For three subjects, the alpha rhythm did not show any reductions due to the opening of the eyes. Both field map pattern and location of the estimated source were persistently stationary during each of the bursts of oscillations of the alpha rhythm. Dipoles were concentrated in clusters, indicating the existence of several spatially distributed sources. The calcarine fissure, the parieto-occipital sulcus and the surrounding occipital and parieto-occipital areas were identified as cortical sites of the brain where the alpha rhythm may originate. For four subjects, the majority of the sources were located near or in the calcarine fissure, while for five subjects, they were located near or in the parieto-occipital sulcus and for the remaining subject they were equally divided between the two generation sites.     

Most human isolates of Mycobacterium avium Mav-A and Mav-B are strong producers of hemolysin, a putative virulence factor

Hemolysin was quantified in 58 isolates of Mycobacterium avium from human, animal, and environmental sources. Human Mav-A and Mav-B isolates were the strongest producers; in contrast, animal and environmental Mav-A isolates and human, animal, and environmental Mav-C organisms were low-level producers. Hemolysin production was not restricted to isolates causing invasive infections.     

Role of surface-exposed loops of Haemophilus influenzae protein P2 in the mitogen-activated protein kinase cascade

The outer membrane of gram-negative bacteria contains several proteins, and some of these proteins, the porins, have numerous biological functions in the interaction with the host; porins are involved in the activation of signal transduction pathways and, in particular, in the activation of the Raf/MEK1-MEK2/mitogen-activated protein kinase (MAPK) cascade. The P2 porin is the most abundant outer membrane protein of Haemophilus influenzae type b. A three-dimensional structural model for P2 was constructed based on the crystal structures of Klebsiella pneumoniae OmpK36 and Escherichia coli PhoE and OmpF. The protein was readily assembled into the beta-barrel fold characteristic of porins, despite the low sequence identity with the template proteins. The model provides information on the structural features of P2 and insights relevant for prediction of domains corresponding to surface-exposed loops, which could be involved in the activation of signal transduction pathways. To identify the role of surface-exposed loops, a set of synthetic peptides were synthesized according to the proposed model and were assayed for MEK1-MEK2/MAPK pathway activation. Our results show that synthetic peptides corresponding to surface loops of protein P2 are able to activate the MEK1-MEK2/MAPK pathways like the entire protein, while peptides modeled on internal beta strands are unable to induce significant phosphorylation of the MEK1-MEK2/MAPK pathways. In particular, the peptides corresponding to loops L5 (Lys206 to Gly219), L6B (Ser239 to Lys253), and L7 (Thr280 to Lys287) activate, as the whole protein, essentially JNK and p38.     

Mitochondrial antigens as targets of cellular and humoral auto-immunity in primary biliary cirrhosis

Several factors point toward an auto-immune pathogenesis for primary biliary cirrhosis (PBC), mostly based on the presence of serum auto-antibodies to mitochondrial antigens (AMAs) and autoreactive T cells (both helper and cytotoxic). Interestingly, epitopes recognized by AMA and T-cell clones are located within overlapping areas of the antigens. Moreover, a role for an imbalance in cytokine pattern and for natural-killer lymphocytes has also been proposed. Despite several experimental reports, no clear evidence is available regarding the interaction of these factors leading to bile duct destruction. This article reviews the current reports regarding the auto-immune reaction against mitochondrial auto-antigens in PBC.     

Conformational analysis of heparin binding peptides

A properly engineered biomaterial for dental/orthopaedic applications must induce specific responses from the osteoblasts at the implant site. A most desirable response is an efficient adhesion, as it represents the first phase in the cell/material interaction and the quality of this phase will influence the cell's capacity to organize into a new functional tissue. The four osteoblast-adhesive peptides discussed in this paper are mapped on the 339-364 sequence (339MAPRPSLAKKQRFRHRNRKGYRSQRG364) located in the primary heparin-binding site of human vitronectin (HVP). Adsorbed on a polystyrene scaffold, these peptides display different adhesive activities towards osteoblasts. In this paper we report on the structural analysis in solution of the peptides through NMR and computational techniques. We find that the peptides with the highest adhesive activities display a hydrophobic patch opposite to the charged surface candidate to interact with heparin. These findings suggest that the peptides might adsorb on the polystyrene support in a favourable orientation for their activity. Furthermore, molecular models obtained for the four peptides in solution were used in rigid docking simulations with a heparin model. Assuming that the peptide solution conformations are not very different from the polystyrene-adsorbed structures, the simulations reveal that peptide adhesive activity is also affected by the number of ionic interactions and spacing between charged residues.