Therapeutic value ofalpha-4 integrin blockade in inflammatory bowel disease:the role of natalizumab
Lymphocyte infiltration into the intestinal tract in Crohn’s disease is mediated by the interaction between α4 integrin, expressed on lymphocytes, and its ligand mucosal vascular addressin cell-adhesion molecule-1 (MADCAM-1), expressed on the endothelial cells of the microvasculature in inflamed intestinal tract. Natalizumab, a recombinant, humanised monoclonal antibody against α4 integrin was effective in Crohn’s disease in a Phase II, randomised, controlled trial. The highest response rate and remission rates were 71 and 44% at 6 weeks, respectively, after two infusions of 3 mg of natalizumab administered 4 weeks apart. Natalizumab was well-tolerated in this study. The Phase III trial results are awaited.

title: Management of Crohn’s disease from efficacy, quality of life and health economic perspectives
abstract: The expectations of the physician and patient in the modern management of Crohn’s disease have moved on from alleviation of symptoms whenever relapses occur, to sustained maintenance of remission and mucosal healing. The majority of treatment costs are as a consequence of hospitalizations and surgery. These heightened therapeutic goals are leading to expectations that management costs will decrease. Health-related quality of life can be measured by validated disease-specific tools in Crohn’s disease, and such measurements are now forming common outcome measures in clinical trials of new therapies in Crohn’s disease. This review gives an overview of patient-focused management of Crohn’s disease and the rapid evolution in therapy, with pharmacoeconomic analysis where available.

title: Bioactive natural compounds for the treatment of gastrointestinal disorders.
abstract: Many healthy subjects and patients are taking natural bioactive products for the prevention and treatment of multiple conditions, including gastrointestinal disorders. Based on current evidence, the scientific validity of the use of many of these commercial compounds by the general public is severely limited, with quality control and regulatory issues continuing to be a concern. Nevertheless, there is sufficient preliminary data to warrant further research of these products in order to identify novel compounds for potential clinical use in addition to performing formal randomized controlled clinical trials of the commercial preparations.

title: Natalizumab for active Crohn's disease.
abstract: In chronic inflammatory conditions such as Crohn's disease, the migration of leukocytes from the circulation into the parenchyma and their activation within inflammatory sites are mediated in part by alpha4 integrins.



We conducted a double-blind, placebo-controlled trial of the alpha4 integrin-specific humanized monoclonal antibody natalizumab in 248 patients with moderate-to-severe Crohn's disease. Patients were randomly assigned to receive one of four treatments: two infusions of placebo; one infusion of 3 mg of natalizumab per kilogram of body weight, followed by placebo; two infusions of 3 mg of natalizumab per kilogram; or two infusions of 6 mg of natalizumab per kilogram. Infusions were given four weeks apart. Outcomes included changes in scores for the Crohn's Disease Activity Index (higher scores indicate more severe disease), the health-related quality of life, and C-reactive protein levels.



The group given two infusions of 6 mg of natalizumab per kilogram did not have a significantly higher rate of clinical remission (defined by a score of less than 150 on the Crohn's Disease Activity Index) than the placebo group at week 6 (the prospectively defined primary end point in the efficacy analysis). However, both groups that received two infusions of natalizumab had higher remission rates than the placebo group at multiple time points. Natalizumab also produced a significant improvement in response rates (defined by a reduction of at least 70 points in the score on the Crohn's Disease Activity Index). The highest remission rate was 44 percent and the highest response rate was 71 percent (at week 6 in the group given two infusions of 3 mg per kilogram). Overall, the two infusions of 6 mg of natalizumab per kilogram and of 3 mg per kilogram had similar effects. The quality of life improved in all natalizumab groups; C-reactive protein levels improved in groups receiving two infusions of natalizumab. The rates of adverse events were similar in all four groups.



Treatment with the selective adhesion-molecule inhibitor natalizumab increased the rates of clinical remission and response, improved the quality of life and C-reactive protein levels, and was well tolerated in patients with active Crohn's disease.

title: Mechanism of intestinal entry of infectious prion protein in the pathogenesis of variant Creutzfeldt-Jakob disease.
abstract: The pathogenesis of variant Creutzfeldt-Jakob disease (vCJD) is most likely to be dependent on intestinal entry of orally ingested infectious prion proteins, though tonsils or other oral portals of entry are possible. The exact route of entry of infectious prion proteins is uncertain but receptors for prion proteins such as laminin receptor precursor (LRP) may be expressed on intestinal brush border. Cellular prion protein (PrP(c)) is expressed on intestinal enteric nervous system and is separated by a single layer of epithelial cells from ingested infectious prion proteins. Intestinal M cells in the Peyer's patches may also transcytose prion proteins which may be transported to the lymphatic system by migrating dendritic cells underlying the M cells. The relative importance of the several potential portals of intestinal entry of infectious prion proteins is uncertain but may determine susceptibility of the population and also potential preventive strategies.

title: Intestinal entry of prions.
abstract: Variant Creutzfeldt-Jakob disease is thought to be caused by infected prion protein via ingestion of contaminated beef. After ingestion of infected prion proteins, uptake by small intestine may be by either M-cell dependent or M-cell independent routes. A receptor for prion protein, laminin receptor precursor is expressed on the brush border of small intestinal epithelium in 40 % of subjects. The cellular prion protein expressed on the enteric nervous system might serve as the target for conversion to infectious prion protein and a route for spread to the central nervous system.

title: Idiopathic intracranial hypertension in a child after hemodialysis.
abstract: Idiopathic intracranial hypertension remains a disease of unknown etiology. Epidemiology, associations, features, and prognosis in children have changed over the years. The main clinical complaint of pediatric idiopathic intracranial hypertension is headache with visual disturbance. We report on a patient with childhood idiopathic intracranial hypertension, who presented without headache after hemodialysis for acute renal failure. Idiopathic intracranial hypertension without headache is believed to have a poor prognosis. However, this child demonstrated a favorable outcome with medical therapy. The child was followed with serial optical coherence tomography of retinal nerve fiber layer thickness around the optic nerve head. In the absence of a reliable procedure to evaluate the therapeutic efficacy in the follow-up of patients with idiopathic intracranial hypertension, the importance of optical coherence tomography, a noninvasive, objective, and reproducible procedure, is highlighted as a useful adjunct in the management of idiopathic intracranial hypertension.

title: Cloning of the p50 DNA binding subunit of NF-kappa B: homology to rel and dorsal.
abstract: The DNA binding subunit of the transcription factor NF-kappa B, p50, has been cloned. p50 appears to be synthesized as a larger protein that is then processed to its functional size. Sequence analysis reveals remarkable homology for over 300 amino acids at the amino-terminal end to the oncogene v-rel, its cellular homolog c-rel, and the Drosophila maternal effect gene dorsal. This establishes NF-kappa B as a member of the rel family of proteins, all of which display nuclear-cytosolic translocation. Protein sequence from the p65 polypeptide has established that it is not encoded in the same mRNA as p50. However, p65 appears homologous to c-rel, suggesting that c-rel may form heterodimers with p50 and rel may include a homodimerization motif.

title: Memory B Cell Function in HIV-Infected Children—Decreased Memory B Cells Despite ART
abstract: B cell dysfunction is a well-studied complication of HIV infection in adults. Data on B cell differentiation in normal and HIV-infected children are lacking. We show the distribution of B cell subsets and immunoglobulin levels in HIV-infected children compared with controls. Furthermore, we observe the long-term B cell reconstitution of vaccine-specific immunity after antiretroviral therapy (ART). Phenotype of B cells (naive, non-switched memory, switched memory) was analyzed in 48 infected children and 62 controls. In nine HIV-infected children, functional reconstitution was quantified by tetanus-specific antibodies and by performing a lymphocyte transformation test (LTT) in a longitudinal approach. Switched memory B cells are significantly reduced in HIV-infected children. Vaccine-specific antibodies and response to LTT increase after initiation of ART. Our data indicate a significant dysfunction in the B cell system, despite effective ART. Partial reconstitution of humoral immunity may have therapeutic implications in a subset of HIV-infected children.

title: Mobilization of cytoplasmic CE droplets by overexpression of human macrophage cholesteryl ester hydrolase Published, JLR Papers in Press, July 1, 2003. DOI 10.1194/jlr.M300162-JLR200
abstract: The obligatory first step in the removal of cholesterol from foam cells is the hydrolysis of stored cholesteryl esters (CEs) to release free cholesterol (FC). Neutral cholesteryl ester hydrolase (CEH) catalyzes this hydrolysis, and limiting levels of CEH could play a role in determining the susceptibility to atherosclerosis. We have recently reported the first identification and cloning of cDNA for human macrophage CEH. In the present study, we tested the hypothesis that systematically varied levels of overexpression of human macrophage CEH results in a proportional degree of reduction in cellular CE content in a cell system with known and reproducible amounts of CE accumulation. CEH expression was confirmed by demonstrating the presence of CEH mRNA and protein with an increase in CEH activity. A significant reduction in intracellular lipid droplets was observed in CEH-expressing cells, together with a decrease in cellular CE mass and a 2-fold increase in FC efflux. These results demonstrate that when human macrophage CEH is expressed in lipid-laden cells, hydrolysis and mobilization of CE (stored as lipid droplets) occur. These data establish the possibility that increased CE hydrolysis, mediated by CEH up-regulation, could represent an important mechanism to reduce the cholesterol burden of foam cells.

title: Resolution of the spectroscopy versus crystallography issue for NO intermediates of nitrite reductase from Rhodobacter sphaeroides.
abstract: Copper nitrite reductase (NiR) is a homotrimeric enzyme, containing a T1 copper site, which transfers electrons to the T2 catalytic site, where nitrite is reduced by one electron to nitric oxide (NO2− + 2H+ + e− → NO + H2O).1–3 Recently a side-on bound copper nitrosyl complex of NiR has been crystallized by reacting the reduced enzyme with excess NO.4 Based on the EPR spectrum of the species generated by the reaction of reduced NiR with saturated NO in solution, it has been assigned as an η 2-NO−Cu2+species. However, spectroscopic data on several Cu-nitrosyl model complexes5 and an NO adduct formed by reacting reduced NiR with nitrite6 and DFT calculations on an η2-NOCu species7 describe these complexes as Cu+NO•. In this study, we define the species generated in solution by reacting reduced NiR with NO, using EPR, MCD and ENDOR spectroscopy and correlate these with that observed by crystallography. 
 
Reduced NiR reacted with saturated NO solution generates the EPR spectrum in Figure 1A red, which is equivalent to the spectrum reported by Tocheva et al.4 This species has an EPR signal characteristic of an oxidized T2 Cu (g|| = 2.30 and A|| = 117 G). The MCD spectrum  has low energy d-d bands, also indicative of a T2 cupric complex. Both EPR and MCD spectra show a negligible T1 contribution indicating that this site is reduced . 
 
 
 
Figure 1 
 
Overlay of reduced NiR with excess NO (red) and nitrite bound NiR (blue) A) EPR B) MCD, C) N14 ENDOR, D) H1 ENDOR, with exchangeable features with 8.5 MHz coupling. Parts A and B have nitrite bound to T2 site with the T1 Cu reduced to remove intense overlaying ... 
 
 
 
Nitrite binds to the T2 site of the resting WT enzyme,8,9 but the EPR and, in particular, the MCD features of the nitrite bound T2 site are obscured by the dominant T1 Cu signals. However under these conditions (nitrite bound), the T1 Cu could be selectively reduced with acsorbate. The EPR spectrum of the nitrite bound T2 species  is identical to that produced above by reacting reduced NiR with excess NO. The MCD spectrum  of the T1 reduced T2-nitrite bound form is also identical to that generated by the reduced enzyme with NO. The T2 EPR spectrum and the MCD d-d bands of the resting WT enzyme are very sensitive to nitrite binding . Thus the EPR and MCD data strongly indicate that the species generated by the reaction of excess NO with reduced NiR has T1 reduced and T2 oxidized with nitrite bound. 
 
Further N14 and H1 ENDOR data for this species generated by the reaction of reduced NiR with an NO solution  and those for a form with T1+T2 oxidized and nitrite bound (collected at g=2.266 where there is no contribution from T1, Fig. 1C, D, blue) have the same features. The nitrogen features  from Cu2+NO2− bound T2 are from its histidines, and these features all show lower ENDOR frequencies and smaller hyperfine couplings due to the change of the T2 axial ligand from aquo to nitrite.10 
 
From the above results the reaction of reduced NiR with excess NO gives T1 reduced T2 Cu2+-NO2−. However, the reaction of the reduced enzyme with limited NO has an EPR signal , characteristic of a Cu+NO•  This species lacks d-d bands in the MCD spectrum , further confirming that the Cu is reduced. Therefore, reduced NiR forms a Cu+NO• species at low concentrations of NO. 
 
 
 
Figure 2 
 
EPR spectra A) following the reaction of reduced NiR with sub-stoichiometric NO solution (10:1 enzyme: NO) trapped at 1 second (green) 4 second (red) and 18 seconds (purple). B) following the reaction of the resting oxidized enzyme (black) with NO gas ... 
 
 
 
The reaction of reduced NiR with excess NO involves two one-electron oxidations; reduced T2 → oxidized T2, and NO → NO2−. A similar reaction has been observed for the coupled binuclear Cu active sites in hemocyanins11 (Scheme S1). Deoxyhemocyanin (2Cu+) reacts with NO generating N2O, and a met (2Cu2+) form, which further reacts with NO to generate the half-met (Cu2+Cu+) nitrite bound form. N2O evolution is in fact detected during the reaction of reduced NiR with excess NO, using nitrous oxide reductase (35 μmols N2O reduced min−1mg−1of enzyme), which requires N2O as its substrate.12 Reaction of the resting WT NiR (analogous to the met form of hemocyanin) with NO generates the T1 reduced, T2 oxidized form of the enzyme (g||=2.30, A||=150G), which converts to the nitrite bound T2 form , indicating NO is oxidized to nitrite and nitrite binding is slow. 
 
 
 
Scheme 1 
 
Proposed mechanism for the formation of nitrite bound T2 NiR from reduced enzyme with excess NO. Red boxes = species observed. 
 
 
 
To evaluate the entire reaction mechanism, reduced NiR was reacted with limited NO solutions and intermediates were trapped at different times. We were able to trap the Cu+NO• form at shorter times, leading to the formation of the T1 reduced, T2 oxidized form, and ultimately generating the T1 reduced, T2 oxidized nitrite bound form  at longer times. A reaction mechanism consistent with these results is presented in Scheme 1. Note that in the scheme, intermediate E is not observed because of rapid ET from T2 → T1.13 The experimental details and kinetic rates are provided in the supporting information. 
 
There is still one issue to resolve on correlating the spectroscopy with crystallography. The Cu-NO species trapped in the crystal structure has a side-on  DFT calculations indicate that this species is best described as a Cu+-NO• species.7 However an end-on Cu-NO species is also trapped after limited exposure to NO gas (B, in scheme 1) which is equivalent to the species generated by reacting reduced NiR with nitrite (η1-CuNO).6 Geometry optimized DFT calculations (B3LYP/6–311g* on Cu, N, O and 6–31g* on C, H)14 of both side-on  and end-on  Cu-NO adducts indicate that both forms have a Cu+NO• electronic configuration and that the end-on structure is more stable by 7 kcal/mol. However the orientation of the 2nd sphere aspartic acid residue (Asp129)9,15 appears to play a key role in determining the geometry of the Cu-NO species. In the side-on adduct this residue is perpendicular to the Cu-N-O plane and H-bonds to the O of N- O  while in the end-on structure the Asp129 residue H-bonds to the O of NO and is parallel to the Cu-N-O plane . Both orientations of the Asp129 residue have been observed in crystal structures.15 The Asp129 residue exists in the perpendicular orientation in the crystal structures of the oxidized and reduced NiR and H-bonds to the axial ligand. However, to form the more stable end-on Cu+NO• adduct this aspartate residue has to rotate 90° which can occur in solution but may not be feasible in a crystal. 
 
 
 
Figure 3 
 
Overlay of the DFT optimized side-on bound NO (red) and end-on bound NO (blue) structures indicating the different orientations of the aspartate residue. 
 
 
 
In summary, the species generated in solution during the reaction of reduced NiR with excess NO is the T1 reduced, T2 oxidized nitrite bound form and not the side-on Cu-NO species observed in the crystal structure. The latter is best described as Cu+NO• rather than Cu2+NO−. The mechanism for the reaction of reduced NiR with excess NO involves its two electron oxidation, generating N2O. This oxidized form of the enzyme oxidizes NO generating nitrite bound to the oxidized T2 site generated by ET from T2 → T1. DFT calculations provide insight into factors which favor the η2-NO•Cu+ complex in the crystal structure.