Biomedical subjects

A Fontana

Publications and source records attributed to A Fontana.

At least 109 records · Page 6Linked to original sources

PubMed1999

Incorporation of noncoded amino acids into the N-terminal domain 1-47 of hirudin yields a highly potent and selective thrombin inhibitor.

Hirudin is an anticoagulant polypeptide isolated from a medicinal leech that inhibits thrombin with extraordinary potency (Kd = 0.2-1.0 pM) and selectivity. Hirudin is composed of a compact N-terminal region (residues 1-47, cross-linked by three disulfide bridges) that binds to the active site of thrombin, and a flexible C-terminal tail (residues 48-64) that interacts with the exosite I of the enzyme. To minimize the sequence of hirudin able to bind thrombin and also to improve its therapeutic profile, several N-terminal fragments have been prepared as potential anticoagulants. However, the practical use of these fragments has been impaired by their relatively poor affinity for the enzyme, as given by the increased value of the dissociation constant (Kd) of the corresponding thrombin complexes (Kd = 30-400 nM). The aim of the present study is to obtain a derivative of the N-terminal domain 1-47 of hirudin displaying enhanced inhibitory potency for thrombin compared to the natural product. In this view, we have synthesized an analogue of fragment 1-47 of hirudin HM2 in which Val1 has been replaced by tert-butylglycine, Ser2 by Arg, and Tyr3 by beta-naphthylalanine, to give the BugArgNal analogue. The results of chemical and conformational characterization indicate that the synthetic peptide is able to fold efficiently with the correct disulfide topology (Cys6-Cys14, Cys16-Cys28, Cys22-Cys37), while retaining the conformational properties of the natural fragment. Thrombin inhibition data indicate that the effects of amino acid replacements are perfectly additive if compared to the singly substituted analogues (De Filippis V, Quarzago D, Vindigni A, Di Cera E, Fontana A, 1998, Biochemistry 37:13507-13515), yielding a molecule that inhibits the fast or slow form of thrombin by 2,670- and 6,818-fold more effectively than the natural fragment, and that binds exclusively at the active site of the enzyme with an affinity (Kd,fast = 15.4 pM, Kd,slow = 220 pM) comparable to that of full-length hirudin (Kd,fast = 0.2 pM, Kd,slow = 5.5 pM). Moreover, BugArgNal displays absolute selectivity for thrombin over the other physiologically important serine proteases trypsin, plasmin, factor Xa, and tissue plasminogen activator, up to the highest concentration of inhibitor tested (10 microM).

Amino Acid Sequence↗

PubMed1999

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains.

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.

Amino Acid Sequence↗

PubMed1999

Occludin proteolysis and increased permeability in endothelial cells through tyrosine phosphatase inhibition.

Regulation of epithelial and endothelial permeability is essential for proper function of compartmentalized organisms, and tyrosine phosphorylation plays an important role in this process. We analyzed the impact of protein tyrosine phosphatase (PTP) inhibition on the structure of endothelial junctional proteins. In human umbilical vein endothelial cells (HUVECs) the PTP inhibitors phenylarsine oxide (PAO) and pervanadate induced proteolysis of the tight junction protein occludin. Occludin proteolysis was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline (PHEN), but not by inhibitors against other types of proteases. The junctional proteins ZO-1, cadherin and beta-catenin were not cleaved. Under conditions of occludin proteolysis, PAO treatment elevated permeability for FITC-dextran. Simultaneous incubation of HUVECs with PAO and PHEN inhibited the rise in permeability by more than 60%. PAO treatment lead to progressive disappearance of occludin from the cell periphery. In contrast, ZO-1, cadherin and beta-catenin retained their positions at the sites of intercellular contact. Simultaneous administration of PAO and PHEN greatly prevented the redistribution of occludin. These results demonstrate a selective cleavage of occludin by a metalloproteinase and suggest that this process can contribute to the control of paracellular permeability in endothelial cells.

Arsenicals↗

PubMed1999

Role of IkappaBalpha and IkappaBbeta in the biphasic nuclear translocation of NF-kappaB in TNFalpha-stimulated astrocytes and in neuroblastoma cells.

In infectious diseases of the central nervous system astrocytes respond to inflammatory cytokines like tumor necrosis factor alpha (TNFalpha) by activation of the transcription factor NF-kappaB, mediated by the proteolysis of its inhibitors IkappaBalpha and IkappaBbeta. We studied the kinetics of NF-kappaB induction by TNFalpha in primary astrocytes, and in the neuroblastoma cell line Neuro2A, and compared it to fibroblasts. In the latter, NF-kappaB DNA binding activity was induced at 30 min and remained constant up to 4 h. In contrast, in astrocytes and in Neuro2A cells NF-kappaB DNA binding activity followed a biphasic pattern: it was induced after 30 min (early phase), declined after 1 h, and increased again at 2 to 4 h (late phase). The early phase was due to rapid degradation of IkappaBalpha. After 1 h IkappaBalpha was resynthesized to levels exceeding the amounts present in unstimulated cells. This paralleled the low levels of nuclear NF-kappaB binding activity. The decrease was not observed when IkappaBalpha resynthesis was inhibited by cycloheximide. Degradation of both IkappaBalpha and IkappaBbeta contributed to the late phase of induction. However, the second peak occurred also in the absence of IkappaBbeta proteolysis, demonstrating the importance of IkappaBalpha in the formation of the biphasic nuclear translocation of NF-kappaB.

Animals↗

PubMed1999

[Thrombospondins, tumor angiogenesis and breast cancer].

Thrombospondin-1 and -2 are extracellular matrix proteins that are overexpressed in breast cancer tissue. Their role in breast cancer remains unknown. This article reviews the potential effects of thrombospondin-1 and -2 in breast cancer tumori genesis and metastatic dissemination.

Breast Neoplasms↗

PubMed1999

Changing patterns of care for war-related post-traumatic stress disorder at Department of Veterans Affairs medical centers: the use of performance data to guide program development.

This study traces the development of services for war-related post-traumatic stress disorder (PTSD) provided at Department of Veterans Affairs (VA) medical centers. During the 1980s, long-stay inpatient programs were the major source of specialized VA treatment for PTSD, and an initial effort at development of specialized outpatient clinics resulted in incomplete implementation. In 1988, a full continuum of inpatient and outpatient services was designed and a national program of performance monitoring and outcome assessment was implemented to standardize program structure, monitor delivery, and evaluate outcomes. A series of multisite outcome studies showed significant but modest improvement in association with specialized outpatient treatment; they also showed that traditional long-term inpatient programs were no more effective and were far more costly than short-term specialized inpatient programs. Since 1995, the VA has shifted the emphasis of care substantially from inpatient to outpatient settings. National monitoring efforts have documented maintenance of specialized PTSD treatment capacity, increased access, improvement on available administrative measures of quality of care, and improved inpatient outcomes. Although there have been major changes in the treatment of mental illness in most health care systems in recent years, change in the treatment of PTSD at VA medical centers is unique in that it has been guided by the results of multisite outcome studies conducted in a "real-world" setting and has been supported by ongoing nationwide performance monitoring.

Ambulatory Care↗

PubMed1999

[Chemotherapy in the treatment of ovarian carcinosarcoma].

Carcinosarcoma of the ovary is a rare neoplasm representing 1% of this organ malignancies. The disease appears almost exclusively in advanced stage having an unfavourable prognosis. Three patients affected by carcinosarcoma (MMMT) of the ovary admitted to our Operative Unit have been treated. All patients underwent surgery and subsequently chemotherapy. Two patients were affected by heterologous MMMT and were stage IV and IIIc respectively, the other one, affected by homologous MMMT, was stage IIIc. Stage IV patient was submitted to 6 cycles of CARBO + IFX + CDDP, second look and further 6 cycles of TAX. After 23 months she was submitted to colostomy for intestine occlusion. At the 35 months she died for cachexia and intestine occlusion. Stage IIIc heterologous patient was submitted to 6 cycles of CDDP + EPI + IFX + MESNA for 3 days; at 6 months from diagnosis she did not present any sign of disease. Stage IIIc homologous patient, affected by chronic renal insufficiency and submitted to dialysis, underwent 5 cycles of TAX and at 11 months from diagnosis presented partial response. Carcinosarcoma of the ovary, because of its rarity, and of the poor record of cases in the literature, is a much debated topic in particularitis complementary therapy. Opinions of the several authors are in contrast regarding the use of CHT + RT at the same time. Only RT after surgery does not seem to improve the survival of these patients. Personal experience, with the reported outlines, compared with survival, seems to confirm the use of CDDP and of IFX and to give new horizons to TAX, waiting for further findings.

Antineoplastic Agents, Alkylating↗

PubMed1999

[Acute biliary pancreatitis. Therapeutic trends].

BACKGROUND: Treatment of biliary pancreatitis includes suppression of the biliary cause by cholecystectomy and common bile duct clearance. Endoscopic retrograde cholangiopancreatography (ERCP) and endoscopic sphincterotomy for eradication of biliary stones and laparoscopic cholecystectomy (L.C.) for residual gallbladder stones would be ideal but were once considered to be contraindicated by most surgeons. The timing of definitive biliary tract surgery and the role of ERCP have been the focus of discussion in recent years. METHODS: During a two-year study period 51 patients with acute biliary pancreatitis were studied. Seven patients (14%) underwent emergency laparotomy, necrosectomy, cholecystectomy, exploration of the common bile duct and T-tube insertion, because unstable clinical conditions, with evidence of pancreatic and peripancreatic necrosis on CT-scan. Elective open cholecystectomy and CBD exploration were performed in 7 patients after the resolution of acute pancreatitis during the same hospital admission. RESULTS: Early ERCP and L.C. were associated with favourable outcomes. 33 patients underwent ERCP preoperatively: 17 within 72 hours of admission and 16 after signs of clinical improvement. Laparoscopic cholecystectomy performed 3-25 days after admission was successful in 27 of 29 patients. Postsphincterectomy bleeding occurred in one patient and was treated successfully by endoscopic epinephrine injection. For median hospital stay and recurrence there were statistical differences between early and delayed ERCP. CONCLUSIONS: ERCP and sphincterectomy have a certain role in conjunction with laparoscopic cholecystectomy in the management of patients with acute biliary pancreatitis, particularly in institutions where there is easy access to expert interventional endoscopic techniques. This policy should reduce the risk of cholangitis and recurrent pancreatitis.

Acute Disease↗

PubMed1998

Synthesis and characterization of more potent analogues of hirudin fragment 1-47 containing non-natural amino acids.

Hirudin is the most potent and specific inhibitor of thrombin, a key enzyme in the coagulation process existing in equilibrium between its procoagulant (fast) and anticoagulant (slow) form. In a previous study, we described the solid-phase synthesis of a Trp3 analogue of fragment 1-47 of hirudin HM2, which displayed approximately 5-fold higher thrombin inhibitory potency relative to that of the natural product [De Filippis, V., et al. (1995) Biochemistry 34, 9552-9564]. By combining automated and manual peptide synthesis, here we have produced in high yields seven analogues of fragment 1-47 containing natural and non-natural amino acids. In particular, we have replaced Val1 with tert-butylglycine (tBug), Ser2 with Arg, and Tyr3 with Phe, cyclohexylalanine (Cha), Trp, alpha-naphthylalanine (alphaNal), and beta-naphthylalanine (betaNal). The crude reduced peptides are able to fold almost quantitatively into the disulfide-cross-linked species, whose unique alignment (Cys6-Cys14, Cys16-Cys28, and Cys22-Cys37) has been shown to be identical to that of the natural fragment. The results of conformational characterization provide evidence that synthetic peptides retain the structural features of the natural species, whereas thrombin inhibition data indicate that the synthetic analogues are all more potent inhibitors of thrombin. In particular, Val --> tBug exchange leads to a 3-fold increase in binding, interpreted as arising from a favorable reduction of the entropy of binding, due to the presence of the more symmetric side chain of tBug relative to that of Val. The S2R analogue binds 24- and 125-fold more tightly than the natural fragment to the fast or slow form of thrombin. These results are explained by considering that Arg2 may favorably couple to Glu192, a key residue involved in the slow to fast transition, thus stabilizing the slow form. Replacement of Tyr3 with more hydrophobic residues having different side chain orientations and electronic structures improves binding by 2-40-fold, suggesting that nonpolar interactions and shape-dependent packing effects strongly influence binding at this position. Overall, these results provide new insights for elucidating the mechanism of hirudin-thrombin recognition at the molecular level and highlight new strategies for designing more potent and selective inhibitors of thrombin.

Alanine↗

PubMed1998

Ex vivo malignant glioma cells are sensitive to Fas (CD95/APO-1) ligand-mediated apoptosis.

Fas (also known as CD95/APO-1) is a cell surface receptor and member of the tumor necrosis factor receptor superfamily which mediates apoptosis in sensitive cells upon oligomerization by specific antibodies or by its ligand (FasL). Recently, human glioma cell lines were found to be susceptible to Fas-mediated apoptosis triggered by alpha-Fas antibodies. However, whether the Fas system can also be targeted in ex vivo high grade gliomas is at present unknown. In the present investigation, alpha-Fas antibodies and FasL were tested in short-term monolayer cultures or in colony forming assays established from freshly resected tumors of patients with anaplastic astrocytomas (WHO grade III) and glioblastoma multiforme (WHO grade IV). Anti-Fas antibodies induced only moderate apoptosis in four of the 19 tested glioma cell cultures. This contrasts FasL which induced apoptosis in all of the 19 tumor cell cultures analyzed. Mean cytotoxicity of glioma cell cultures treated for 48 h with alpha-Fas antibodies or FasL was 9.6% and 44.3%, respectively. Irrespective of whether alpha-Fas antibodies or FasL were used, pretreatment with recombinant hu (rhu) IFN-gamma and rhuTNF-alpha for 48 h did not sensitize glioma cells to Fas-mediated cytotoxicity. The long-term effect by FasL on tumor colony formation was more striking. FasL treatment resulted in more than 90% inhibition of clonal tumor cell growth of all the eight high grade gliomas analyzed. These results suggest that Fas targeting by FasL but not by alpha-Fas antibodies may provide a promising approach for locoregional glioma treatment.

Antibodies↗

PubMed1998

Chemokines and chemotaxis of leukocytes in infectious meningitis.

Chemokines constitute a constantly growing family of small inflammatory cytokines. They have been implied in many different diseases of the CNS including trauma, stroke and inflammation, e.g., multiple sclerosis. In this review we focus on the role of chemokines in infectious meningitis of bacterial or viral origin. In experimental bacterial meningitis induced by Listeria monocytogeneses both CXC and CC chemokines namely MIP-1alpha, MIP-1beta and MIP-2 are produced intrathecally by meningeal macrophages and leukocytes which infiltrate into the CNS. In patients with bacterial meningitis, IL-8, GROalpha, MCP-1, MIP-1alpha and MIP-1beta are detectable in the CSF. These chemokines contribute to CSF mediated chemotaxis on neutrophils and PBMC in vitro. In viral meningitis IL-8, IP-10 and MCP-1 are identified in the CSF to be responsible for chemotactic activity on neutrophils, PBMC and activated T cells. Taken collectively these data indicate that the recruitment of leukocytes in infectious meningitis involves the intrathecal production of chemokines.

Chemokines↗

PubMed1998

Conversion of membrane-bound Fas(CD95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity.

Human Fas ligand (L) (CD95L) and tumor necrosis factor (TNF)-alpha undergo metalloproteinase-mediated proteolytic processing in their extracellular domains resulting in the release of soluble trimeric ligands (soluble [s]FasL, sTNF-alpha) which, in the case of sFasL, is thought to be implicated in diseases such as hepatitis and AIDS. Here we show that the processing of sFasL occurs between Ser126 and Leu127. The apoptotic-inducing capacity of naturally processed sFasL was reduced by >1,000-fold compared with membrane-bound FasL, and injection of high doses of recombinant sFasL in mice did not induce liver failure. However, soluble FasL retained its capacity to interact with Fas, and restoration of its cytotoxic activity was achieved both in vitro and in vivo with the addition of cross-linking antibodies. Similarly, the marginal apoptotic activity of recombinant soluble TNF-related apoptosis-inducing ligand (sTRAIL), another member of the TNF ligand family, was greatly increased upon cross-linking. These results indicate that the mere trimerization of the Fas and TRAIL receptors may not be sufficient to trigger death signals. Thus, the observation that sFasL is less cytotoxic than membrane-bound FasL may explain why in certain types of cancer, systemic tissue damage is not detected, even though the levels of circulating sFasL are high.

Amino Acid Sequence↗

PubMed1998

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in viral meningitis: upregulation of MMP-9 and TIMP-1 in cerebrospinal fluid.

A hallmark of viral meningitis is the invasion of monocytes, lymphocytes and, in the initial phase of the disease, neutrophils into the subarachnoidal space. By their degradation of different macromolecular components in the extracellular connective tissue, matrix metalloproteinases (MMPs) may be essential for the breakdown of the vessel wall in the meninges and the choroid plexus. In this study, the occurrence of MMP-1, MMP-2, MMP-3 and MMP-9 and the two tissue inhibitors of metalloproteinases, TIMP-1 and TIMP-2, was monitored in the cerebrospinal fluid (CSF) from patients with viral meningitis. Of the proteinases, MMP-9 was found in 13 of 39 (33%) patients, but not in controls; the levels being correlated with the neutrophil cell number in CSF. The CSF concentration of TIMP-1 was increased three-fold compared to the control group (median 233 ng/ml; range 9.4-1252.5 ng/ml) and was correlated to the levels of total protein in CSF. Of the other MMPs and TIMPs assayed, MMP-2 and TIMP-2 were constitutively expressed and not upregulated in viral meningitis. High levels of MMP-9 and MMP-2, as measured by ELISA, was associated with high proteolytic activity detected in CSF by zymography. In conclusion, invasion of the leukocytes into the CSF compartment in viral meningitis may involve MMP-9, its proteolytic effect likely being controlled by expression of TIMP-1.

Adolescent↗

PubMed1998

Enhanced protein thermostability by Ala-->Aib replacement.

The introduction into peptide chains of alpha-aminoisobutyric acid (Aib) has proven to stabilize the helical structure in short peptides by restricting the available range of polypeptide backbone conformations. In order to evaluate the potential stabilizing effect of Aib at the protein level, we have studied the conformational and stability properties of Aib-containing analogs of the carboxy-terminal subdomain 255-316 of thermolysin. Previous NMR studies have shown that this disulfide-free 62-residue fragment forms a dimer in solution and that the global 3D structure of each monomer (3 alpha-helices encompassing residues 260-274, 281-295, and 301-311) is largely coincident with that of the corresponding region in the X-ray structure of intact thermolysin. The Aib analogs of fragment 255-316 were prepared by a semisynthetic approach in which the natural fragment 255-316 was coupled to synthetic analogs of peptide 303-316 using V8-protease in 50% (v/v) aqueous glycerol [De Filippis, V., and Fontana, A. (1990) Int. J. Pept. Protein Res. 35, 219-227]. The Ala residue in position 304, 309, or 312 of fragment 255-316 was replaced by Aib, leading to the singly substituted fragments Ala304Aib, Ala309Aib, and Ala312Aib. Moreover, fragment Ala304Aib/Ala309Aib with a double Ala-->Aib exchange in positions 304 and 309 was produced. Far- and near-UV circular dichroism measurements demonstrated that both secondary and tertiary structures of the natural fragment 255-316 are fully retained upon Ala-->Aib substitution(s). Thermal unfolding measurements, carried out by recording the ellipticity at 222 nm upon heating, showed that the melting temperatures (Tm) of analogs Ala304Aib and Ala309Aib were 2.2 and 5.4 degrees C higher than that of the Ala-containing natural species (Tm = 63.5 degrees C), respectively, whereas the Tm of the Ala312Aib analog was lowered by -0.6 degree C. The enhanced stability of the Ala304Aib analog can be quantitatively explained on the basis of a reduced backbone entropy of unfolding due to the restriction of the conformational space allowed to Aib in respect to Ala, while the larger stabilization observed for the Ala309Aib analog can be accounted for by both entropic and hydrophobic effects. In fact, whereas Ala304 is a surface residue, Ala309 is shielded from the solvent, and thus the enhanced stability of fragment Ala309Aib is also due to the burial of an additional -CH3 group with respect to the natural fragment. The slightly destabilizing effect of the Ala-->Aib exchange in position 312 appears to derive from unfavorable strain energy effects, since phi and psi values for Ala312 are out of the allowed angles for Aib. Of interest, the simultaneous incorporation of Aib at positions 304 and 309 leads to a significant and additive increase of +8 degrees C in Tm. The results of this study indicate that the rational incorporation of Aib into a polypeptide chain can be a general procedure to significantly stabilize proteins.

Alanine↗

PubMed1998

Does participation in psychosocial treatment augment the benefit of clozapine? Department of Veterans Affairs Cooperative Study Group on Clozapine in Refractory Schizophrenia.

BACKGROUND: This study examines the role of participation in psychosocial treatment as a mediator of the clinical effectiveness of clozapine. METHODS: Subjects participated in a 12-month double-blind random-assignment trial comparing clozapine and haloperidol in patients hospitalized 30 to 364 days for refractory schizophrenia at 15 Department of Veterans Affairs medical centers. A broker-advocate case management intervention was used to facilitate participation in psychosocial treatments and to document such participation. RESULTS: Between those who continued receiving clozapine (n=122) or a conventional antipsychotic drug (n=169) for 12 months, those receiving clozapine were more likely to participate in psychosocial rehabilitation treatment. Although they were no more likely to receive clinical recommendations for such treatments, they were more likely to both verbally accept recommendations and to act on them. Structural equation modeling shows that participation in psychosocial treatment did not play a mediating role in clozapine's effect on outcomes at 6 months, but was associated with both reduced symptoms and improved quality of life at 12 months. CONCLUSIONS: Clozapine facilitates participation in psychosocial treatment, and such enhanced participation is associated with improved quality-of-life and symptom outcomes. Psychosocial rehabilitation should be offered concomitantly with clozapine.

Adult↗

PubMed1998

Mice with an inactivation of the inducible nitric oxide synthase gene are susceptible to experimental autoimmune encephalomyelitis.

Nitric oxide (NO) generated by the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of experimental autoimmune encephalomyelitis (EAE). In this study mice genetically deficient for iNOS are shown to be susceptible to EAE induced by immunization with myelin oligodendrocyte glycoprotein (MOG). In iNOS (-/-) mice the course of disease was earlier in onset and more aggressive compared to control animals. A disease-relevant compensatory up-regulation of neuronal (n)NOS and endothelial (e)NOS with increased production of NO in iNOS (-/-) mice is excluded by 1) the failure to detect increased nNOS and eNOS mRNA, 2) the absence of detection of nitrosylated tyrosine residues in EAE tissue indicating absence of NO-derived peroxynitrite, and 3) the lack of disease-preventing effects of NG-nitro-L-arginine methyl ester. In conclusion, these results do not support the hypothesis that NO is crucial for the development of EAE.

Animals↗

PubMed1998

IL-6-deficient mice resist myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is induced by immunization with myelin components including myelin oligodendrocyte glycoprotein (MOG). Myelin-specific Th1 cells enter the central nervous system (CNS) via binding of very late antigen 4 (VLA-4) to the endothelial vascular cell adhesion molecule 1 (VCAM-1). In the present study, mice with a homologous disruption of the gene encoding IL-6 are found to be resistant to MOG-induced EAE as evidenced by absence of clinical symptoms, minimal infiltration of CD3+ T cells and monocytes into the CNS and lack of demyelination. The failure to induce EAE in IL-6-/- mice is not due to the absence of priming, since lymphocytes of immunized IL-6-/- mice proliferate in response to MOG and produce pro-inflammatory cytokines including IL-2 and IFN-gamma. However, in MOG-immunized IL-6-/- mice, serum anti-MOG antibody titers were found to be drastically reduced. This observation is unlikely to be responsible for resistance to EAE, because B cell-deficient (microMT) mice proved to be fully susceptible to the disease. A striking difference between MOG-immunized wild-type (wt) and IL-6-/- mice was the expression of endothelial VCAM-1 and ICAM-1, which were dramatically up-regulated in the CNS in wt but not in IL-6-/- mice. Taking into account recent studies on the role of VCAM-1 in the entry of Th1 cells into the CNS, the absence of VCAM-1 on endothelial cells in IL-6-/- mice may explain their resistance to EAE.

Animals↗

PubMed1998

Interleukin-16, produced by synovial fibroblasts, mediates chemoattraction for CD4+ T lymphocytes in rheumatoid arthritis.

The massive infiltration of synovium with CD4+ T cells during the course of rheumatoid arthritis (RA) implies the expression of chemoattractant factors by resident synovial cells. Therefore, we analyzed the expression of IL-16, a potent chemoattractant for CD4+ T cells, to account for the accumulation of CD4+ T cells in RA. Indeed, IL-16 was found to be significantly elevated in synovial fluid (SF) from patients with RA as compared to non-RA arthritis (p < 0.001), osteoarthritis (p < 0.001) and controls (p < 0.001). Chemotaxis studies showed IL-16 to contribute to the strong chemotactic activities of RA-SF. In situ hybridization (ISH) revealed IL-16 mRNA-expressing cells located within the lining layer of rheumatoid synovial tissue. In the sublining area, only scattered IL-16 transcript-positive cells could be detected, mainly adjacent to blood vessels. By a double-labeling technique, combining ISH for IL-16 mRNA and immunohistochemistry for CD68, synovial fibroblast-like, CD68-negative cells were identified as a major source of IL-16 mRNA within RA synovium. This study demonstrates that synovial fibroblasts produce IL-16 in RA and thus mediate chemoattraction of CD4+ cells into synovial tissue.

Adult↗