Matrix metalloproteinase-9 is increased and correlates with severity in Guillain-Barré syndrome.

OBJECTIVE: To study the expression and activity of matrix metalloproteinases (MMPs) MMP-2 (72-kd type IV collagenase, gelatinase A), MMP-3 (58-kd stromelysin-1), and MMP-9 (92-kd type IV collagenase, gelatinase B) and tissue inhibitors of MPs (TIMP) in patients with Guillain-Barre syndrome (GBS). BACKGROUND: MMPs are able to proteolysis of basement membranes and other matrix components, promoting transmigration of inflammatory cells from circulation to nerve tissue. METHODS: Twenty-five patients with GBS were analyzed according to the phase of the disease, i.e., progression, plateau, early recovery, and late recovery. Determinations of MMP-2, MMP-3, MMP-9, and TIMP-1 were performed using ELISA, zymography, and immunocytochemistry in circulation or peripheral nerve. RESULTS: MMP-9 plasma levels were increased in 67% of patients on admission and decreased from progression to late recovery (p < 0.002). During the course of GBS, MMP-9 was progressively balanced by its inhibitor TIMP-1, as assessed by the MMP-9/TIMP-1 ratio. MMP-9 and TIMP-1 plasma levels and the MMP-9/TIMP-1 ratio correlated positively with disability. MMP-2 expression was similar to controls. MMP-3 activity was not detected, and plasma levels were not different from those in controls. Positive MMP-9 immunolabeling was 51 +/- 11% of circulating lymphocytes. It was observed in some endothelial cells and mononuclear cells adherent to the endothelium and close to myelinated fibers. CONCLUSIONS: Circulating matrix metalloproteinases (MMP-9) correlates with disease severity in Guillain-Barré syndrome (GBS). MMP-9 likely represents an important molecule in the pathogenesis of GBS and therefore could represent an interesting therapeutic target.

Differential expression of the IL-1 system components during in vitro myogenesis: implication of IL-1beta in induction of myogenic cell apoptosis.

We evaluated the expression of IL-1 system by normal human myogenic cells during in vitro myogenesis and the effect of exogenous IL-1beta. Expression of IL-1alpha and beta, IL-1 receptor antagonist (IL-1Ra), IL-1RI and II, IL-1R accessory protein (IL-1RAcP) and IL-1beta converting enzyme (ICE) was studied by immunocytochemistry, immunoblotting, ELISA and RT - PCR. Cell proliferation was evaluated by [3H]thymidine incorporation, cell fusion by flow cytometry and cell death by in situ end-labelling. Human normal myogenic cells constitutively produced IL-1beta and ICE, with a maximum expression at time of cell fusion. IL-1Rs and IL-1RAcP expression reached a peak at time of commitment to fusion. Myogenic cells produced small amounts of IL-1Ra at latest stages of culture, and only the intracellular isoform. Exposure of cultures to exogenous IL-1beta (1-5 ng/ml) induced myogenic cell apoptosis, without effect on cell proliferation or fusion. IL-1beta-induced cell death was associated with morphological changes including spreading appearance of cells and alteration of cell alignment. We conclude that (1) human myogenic cells constitutively produce IL-1beta; (2) IL-1 system components are differentially expressed during in vitro myogenesis; (3) IL-1 system participates to the coordinated regulation of cell density during normal myogenesis, which could serve to control the muscle mass in vivo.

Primary brain lymphoma cell turnover differs in patients with and without AIDS: relationships to bcl-2 expression and host cell reaction.

Primary central nervous system lymphomas (PCNSLs) are more resistant to radiotherapy and chemotherapy in AIDS (A-PCNSLs) than in non-AIDS patients (NA-PCNSLs). We investigated 23 A-PCNSLs and 24 NA-PCNSLs. Lymphoma cell kinetics (i.e. proliferation [mitotic index, MIB-1 and PCNA labeling indices], apoptosis and turnover) were determined and compared with bcl-2 and LMP-1 expression, and to the percentage of tumor-infiltrating T-lymphocytes (T-TILs) and macrophages. A-PCNSLs showed lower proliferation (p < 0.005), less apoptosis (p < 0.0001) and slower cell-turnover (p < 0.0001) than NA-PCNSLs. LMP-1 was detected in 90% of A-PCNSLs and 5% of NA-PCNSLs, a finding correlating positively with bcl-2 expression (p < 0.0007). In contrast, T-TIL counts and CD4/CD8 T-TIL ratios were similar in A-PCNSLs and NA-PCNSLs. T-TIL counts correlated negatively with proliferation indices (from p < 0.05 to p < 0.0005) in NA-PCNSLs, but not in A-PCNSLs. Macrophage counts correlated positively with apoptosis in both groups. We concluded the following: (i) A-PCNSLs are characterized by accumulation of slow-cycling, long-lived cells that might be protected from apoptosis by LMP-1 induced bcl-2 expression, and independently from the host response; (ii) NA-PCNSLs are characterized by a faster cell turnover associated with an insufficient antiproliferative host response; and (iii) A-PCNSLs and NA-PCNSLs constitute 2 entities with distinctive morphology and different kinetic profiles that could account for different responses to therapy.

Expression of the transforming growth factor beta isoforms in inflammatory cells of nasal polyps.

OBJECTIVE: To determine the expression and the potential role of transforming growth factor beta (TGF-beta) in nasal polyposis. DESIGN: Comparison of TGF-beta expression between normal and inflammatory nasal mucosa and polyps; in inflammatory nasal polyps, characterization of the TGF-beta isoforms expression and their potential location in macrophages and eosinophils. SETTING: Patients and samples were selected at the Hôpital Intercommunal, Créteil, France, and immunohistochemistry and immunoblots were performed at the Institut National de la Sante et de la Recherche Medicale U296 (Universite Paris XII, France). SUBJECTS: Nasal polyps and nasal mucosa were sampled in 21 patients during ethmoidectomy, and muscosa was sampled in 6 healthy patients during rhinoplasty. METHODS: Immunohistochemistry and Western blot analysis were performed using specific antibodies to TGF-beta1-3, TGF-beta1, TGF-beta2, and TGF-beta3 isoforms. Double labeling was also performed using anti-TGF-beta1 antibody together with macrophages or eosinophil-specific antibodies. RESULTS: The expression of TGF-beta(1-3) was significantly higher in inflammatory nasal polyps than in inflammatory nasal mucosa and higher in inflammatory nasal mucosa than in nasal mucosa from healthy patients. Transforming growth factor beta1 was the main isoform detected in inflammatory nasal polyps, and it was present in numerous macrophages and in some eosinophils. CONCLUSIONS: Transforming growth factor beta, mainly TGF-beta1, is strongly expressed in inflammatory nasal mucosa, where it could be produced by macrophages and eosinophils. Transforming growth factor beta could induce epithelium and connective tissue modifications and therefore be involved in the pathogenesis of nasal polyposis.

Incidence of primary ciliary dyskinesia in children with recurrent respiratory diseases.

The goal of the study was to evaluate the incidence of primary ciliary dyskinesia (PCD) in children suffering from recurrent respiratory tract infections (RRIs) by means of noninvasive method. Respiratory ciliated cells were collected by nasal brushing in 118 children (4.6 +/- 2.5 years) with RRIs. The ciliary beat frequency (CBF) was measured with a stroboscopic method, and when the CBF was abnormal, the ciliary ultrastructure was analyzed by a quantitative method. The CBF could be measured in 106 patients (90%) and was abnormal in 15 patients. The ciliary ultrastructure was found to be abnormal in 11 of 15 patients: PCD was diagnosed in 6 cases, and acquired ciliary defects were observed in the remaining 5 patients. Our conclusion, that PCD is rare but net exceptional (5.6%) in children with RRIs, justifies the systematic investigation of ciliated cells in such patients. For this purpose, nasal brushing can be used to sample ciliated cells even in young children.

Interleukin-1 expression in normal motor endplates and muscle fibers showing neurogenic changes.

Histological features of neurogenic muscle involvement include type grouping, muscle fiber atrophy and target fibers. In zidovudine-induced myopathy and dermatomyositis, immunoreactivity for interleukin (IL)-1 has been reported in diseased muscle fibers involving myofibrillar breakdown and atrophy. Since IL-1 is a signal for muscle proteolysis, we studied myofiber expression of IL-1 in neurogenic muscle involvement, specially in atrophic myofibers and target fibers which are associated with myofilament breakdown. Muscle biopsy samples from patients with normal (5 cases) or neurogenic muscle involvement (25 cases) were studied by enzyme histochemistry and immunohistochemistry. In normal muscles, immunoreactivity for IL-1beta was restricted to the postsynaptic domain of motor endplates and that for IL-1alpha had a similar localization but was faint. Immunoreactivity for IL-1alpha and -beta was observed, respectively, in 42.5% and 75.5% of target fibers, in 8.5% and 10.4% of dark angulated fibers, in 0% and 0.3% of non-atrophic type-grouped fibers, in 14.2% and 16.5% of moderately atrophic fibers, and in 65% and 20.9% of severely atrophic fibers. Immunoblot study showed the presence of both proIL-1 (31 kDa) and mature IL-1 (17.5 kDa). From this study, we conclude that IL-1 is normally expressed in the muscular domain of neuromuscular junctions; that IL-1 is mainly expressed in neurogenic target fibers; and that IL-1 expression by muscle fibers in pathological conditions seems to be associated with myofibrillar protein breakdown and regeneration.

Epithelial cell proliferation in nasal polyps could be up-regulated by platelet-derived growth factor.

The modifications of epithelial differentiation and proliferation observed in nasal polyps (NP) could be related to local secretion of growth factors, among which platelet-derived growth factor (PDGF) could play a key role. We therefore prospectively studied, by immunohistochemistry, proliferating cell nuclear antigen (PCNA, an S-phase cell marker), PDGF, and CD-68 (activated macrophages marker) expression in NP and inferior turbinate mucosa (NM) in 11 patients. Our data show that PCNA and PDGF expression are increased in NP epithelium, while CD-68 expression is increased in NP epithelium and lamina propria when compared to NM. Increased local PDGF secretion by numerous activated macrophages could therefore be involved in epithelial cell proliferation up-regulation in NP. PDGF could also be involved in the pathogenesis of NP via its connective tissue remodeling actions.

Increased epithelial cell proliferation in nasal polyps.

OBJECTIVE: To detect, quantify, and compare respiratory epithelial cell proliferation in nasal mucosa and polyps from patients with nasal polyposis. DESIGN: Cohort study. SETTING: Patients and samples were selected at the Hôpital Intercommunal de Créteil (France). Flow cytofluorometry and immunohistochemistry were performed at Hôpitaux Tenon and Mondor (Université Paris [France] VI et XII). PATIENTS: Twenty-one patients undergoing endoscopic ethmoidectomy for treatment of nasal polyposis. METHODS: In 10 cases, epithelial cells were removed from frozen inferior turbinate mucosa and polyps by mechanical disaggregation and were then analyzed by flow cytofluorometry, providing the cell DNA content (propidium iodide labeling) and the percentage of S-phase cells. In 11 cases, inferior turbinate mucosa and polyps were fixed in formaldehyde and embedded in paraffin. Proliferating cell nuclear antigen expression in the epithelium was quantified by immunohistochemistry; a proliferating cell nuclear antigen index was calculated for each sample in the basal area, suprabasal area, and full height of the epithelium. RESULTS: All cell populations studied were diploid, and percentages of S-phrase cells were significantly higher in nasal polyps than in mucosa. Proliferating cell nuclear antigen indexes were significantly higher in nasal polyps than in the suprabasal area and full height of the mucosal epithelium. CONCLUSION: Cell proliferation is increased in epithelium from nasal polyps. Epithelial damage caused by inflammatory mediators could induce this increased cell proliferation via epithelial repair processes. Inflammatory cells could up-regulate epithelial cell proliferation by secreting growth factors.

Acquired ciliary abnormalities of nasal mucosa in marrow recipients.

Respiratory symptoms are frequent after bone marrow transplantation (BMT). Most studies focus on lesions of the lower respiratory tract. However, sinusitis is also common in this setting, especially after allogeneic BMT. The nasal respiratory epithelium is the first line of airway defense and is very similar to the bronchial epithelium, especially in terms of ciliary beat frequency and ultrastructural pattern of ciliated cells. We have prospectively studied the nasal respiratory epithelium of 20 marrow recipients (four autologous, 16 allogeneic) with or without sinusitis, by brushing and biopsy of the median turbinate between 2.5 and 148 months after transplant. Samples were studied for ciliary beat frequency, cytology, ultrastructural pattern and HLA-DR expression. We found that 17 of our 20 patients had abnormalities of their nasal epithelium, mainly consisting of either squamous metaplasia or heterogeneous axonemal defects of peripheral and central microtubules. No relationship between these findings and the presence of acute or chronic sinus infection, previous irradiation, graft-versus-host disease or immunosuppressive therapy could be demonstrated in this preliminary study. These abnormalities probably have multiple causes. Prospective studies are needed to determine the respective roles of treatments, infections and immune disorders associated with BMT in these abnormalities, and to know their natural evolution over time and their impact on the occurrence of upper or lower respiratory tract infections.

Cytochrome c oxidase deficiencies in the muscle of patients with inflammatory myopathies.

We studied mitochondrial function in inflammatory myopathies, using cytochrome c oxidase (COX) reaction on muscle biopsy samples from 30 patients (15 with dermatomyositis, 12 with polymyositis, and 3 with inclusion body myositis) and 30 age-matched controls. We also performed immunocytochemistry for COX II and COX IV subunits in 7 of these patients who had COX deficiency. COX-deficient fibers were a constant finding in patients or controls older than 65 years and the percentage of COX-deficient fibers correlated with age in both patients and controls. Focal COX deficiency was found in 24 patients (13 of 15 with dermatomyositis, 8 of 12 with polymyositis, and 3 of 3 with inclusion body myositis) and 18 controls. The percentages of COX-deficient fibers were higher in patients with inflammatory myopathies (range: 0-4.7%; mean: 1.2%) than in age-matched controls (range: 0-1.9%; mean: 0.4%) (P < 0.01). In the subgroup of patients under age 65, COX-deficient fibers were more frequent in dermatomyositis than in polymyositis (mean: 0.8% vs 0.2%, P = 0.02). In patients with dermatomyositis, capillary loss correlated positively with COX deficiency (P < 0.02). Immunocytochemistry for COX II and IV showed that 82% of COX-negative fibers were COX II-negative and 26% were COX IV-negative, suggesting that proteins encoded by mitochondrial DNA are predominantly, but not exclusively, involved in COX deficiency. We conclude that mitochondrial dysfunction and COX deficiency can occur in inflammatory myopathies. Such a mitochondrial dysfunction is not solely related to the aging process. We suggest that muscle ischemia contributes to mitochondrial dysfunction in dermatomyositis.

Neuronal apoptosis in HIV infection in adults.

Productive infection of the central nervous system by HIV predominantly involves the white matter and basal ganglia. Involvement of the cerebral cortex with neuronal loss is also described in AIDS patients but not in asymptomatic HIV-positive patients. The mechanism of neuronal damage is unknown. To enquire whether neuronal loss in AIDS may be due to an apoptotic process, we examined the cerebral cortex from 12 patients who died from AIDS using two different methods: in situ end labelling and gel electrophoresis of DNA to demonstrate DNA fragmentation. None of the patients had cerebral opportunistic infection or tumour. Four patients had no significant neuropathological changes, eight patients had variable cerebral atrophy and four of them also had productive HIV infection of the brain. These patients were compared with four HIV-positive asymptomatic patients, five seronegative asymptomatic controls, and two seronegative patients with Alzheimer's disease. We demonstrated neuronal apoptosis in the cortex in all AIDS patients, as well as in the Alzheimer's patients. Apoptosis was not observed in the asymptomatic cases whether seropositive or seronegative. Neuronal apoptosis was more severe in atrophic brains, and did not directly correlate with productive HIV infection, suggesting an indirect mechanism of neuronal damage is most likely.

Cytokine expression in the muscle of HIV-infected patients: evidence for interleukin-1 alpha accumulation in mitochondria of AZT fibers.

To evaluate the possible role of cytokines in human immunodeficiency virus (HIV)-associated muscular disorders, we performed immunocytochemistry for interleukin-1 alpha, -1 beta, and -6 and tumor necrosis factor-alpha on frozen muscle biopsy specimens from HIV-infected patients with various myopathies (HIV polymyositis in 5, HIV-wasting syndrome in 5, zidovudine myopathy in 10) and from seronegative individuals (normal muscle in 2, mitochondrial cytopathies in 10). The HIV-infected patients showed positive reactivities in vessels (interleukin-1) and in inflammatory cells (mainly interleukin-1 and tumor necrosis factor-alpha), including perivascular hemosiderin-laden macrophages in 5 patients. In zidovudine myopathy, a majority of AZT fibers (i.e., ragged-red fibers with marked myofibrillar changes) showed mild to marked expression of interleukin-1. Expression of interleukin-1 in the other mitochondrial myopathies was much weaker. Interleukin-1 beta messenger RNA was demonstrated in muscle fibers by in situ hybridization, implying that interleukin-1 was produced in muscle cells. Immunoelectron microscopy showed that interleukin-1 alpha was mainly bound to mitochondrial membranes in AZT fibers. Proinflammatory and destructive effects of the studied cytokines might be responsible for several myopathological changes observed in HIV-infected patients, including inflammation and hemosiderin deposits in muscle tissue, and prominent myofibrillar breakdown in AZT fibers.