Growth of Murine Splenic Tissue Is Suppressed by Lymphotoxin ß-Receptor Signaling (LTßR) Originating from Splenic and Non-Splenic Tissues.

Development and maintenance of secondary lymphoid organs such as lymph nodes and spleen essentially depend on lymphotoxin ß-receptor (LTßR) signaling. It is unclear, however, by which molecular mechanism their size is limited. Here, we investigate whether the LTßR pathway is also growth suppressing. By using splenic tissue transplantation it is possible to analyze a potential contribution of LTßR signaling inside and outside of the implanted tissue. We show that LTßR signaling within the endogenous spleen and within non-splenic tissues both significantly suppressed the regeneration of implanted splenic tissue. The suppressive activity positively correlated with the total number of LTßR expressing cells in the animal (regenerate weights of 115 ± 8 mg in LTßR deficient recipients and of 12 ± 9 mg in wild-type recipients), affected also developed splenic tissue, and was induced but not executed via LTßR signaling. Two-dimensional differential gel electrophoresis and subsequent mass spectrometry of stromal splenic tissue was applied to screen for potential factors mediating the LTßR dependent suppressive activity. Thus, LTßR dependent growth suppression is involved in regulating the size of secondary lymphoid organs, and might be therapeutically used to eradicate tertiary lymphoid tissues during autoimmune diseases.

The role of microfibrillar-associated protein 4 (MFAP4) in the formation and function of splenic compartments during embryonic and adult life.

Microfibrillar-associated protein 4 (MFAP4) is an extracellular protein belonging to the fibrinogen-related protein superfamily and is recognized as an integrin ligand with suggested functions in pulmonary and vascular tissue homeostasis. MFAP4 expression in the spleen is increased during infections; however, the significance of MFAP4 for the function of the spleen is unknown. Immunohistochemistry, morphometry and real-time RT-PCR were used to analyze wild-type and MFAP4-deficient spleens. In addition, they were compared with splenic tissue, which was newly formed 8 weeks after avascular implantation into adult mice in order to obtain information about the role of MFAP4 in the formation of splenic tissue during ontogeny and adult life. The present study shows that MFAP4 is co-localized with laminin in the B- and T-cell zones of the spleen, in addition to capsular and trabecular expression. MFAP4 is most likely produced by fibroblastic reticulum cells and follicular dendritic cells of the spleen but can also be imported via the blood from other tissues. The development of splenic tissue is not disturbed in MFAP4-deficient mice. However, in splenic tissue regenerating under MFAP4-deficient conditions, the number of FDCs is significantly decreased but is corrected by MFAP4 imported from other tissues. No differences were observed for lymphocyte numbers or splenic structure. The data indicate that MFAP4 promotes FDC development in regenerating splenic tissue and warrant further investigations regarding the MFAP4 dependency of splenic B-cell maturation.

The role of conserved PEX3 regions in PEX19-binding and peroxisome biogenesis.

The human peroxins PEX3 and PEX19 are essential for peroxisome biogenesis. They mediate the import of membrane proteins as well as the de novo formation of peroxisomes. PEX19 binds newly synthesized peroxisomal membrane proteins post-translationally and directs them to peroxisomes by engaging PEX3, a protein anchored in the peroxisomal membrane. After protein insertion into the lipid bilayer, PEX19 is released back to the cytosol. Crystallographic analysis provided detailed insights into the PEX3-PEX19 interaction and identified three highly conserved regions, the PEX19-binding region, a hydrophobic groove and an acidic cluster, on the surface of PEX3. Here, we used site-directed mutagenesis and biochemical and functional assays to determine the role of these regions in PEX19-binding and peroxisome biogenesis. Mutations in the PEX19-binding region reduce the affinity for PEX19 and destabilize PEX3. Furthermore, we provide evidence for a crucial function of the PEX3-PEX19 complex during de novo formation of peroxisomes in peroxisome-deficient cells, pointing to a dual function of the PEX3-PEX19 interaction in peroxisome biogenesis. The maturation of preperoxisomes appears to require the hydrophobic groove near the base of PEX3, presumably by its involvement in peroxisomal membrane protein insertion, while the acidic cluster does not appear to be functionally relevant.

Insights into peroxisome function from the structure of PEX3 in complex with a soluble fragment of PEX19.

The human peroxins PEX3 and PEX19 play a central role in peroxisomal membrane biogenesis. The membrane-anchored PEX3 serves as the receptor for cytosolic PEX19, which in turn recognizes newly synthesized peroxisomal membrane proteins. After delivering these proteins to the peroxisomal membrane, PEX19 is recycled to the cytosol. The molecular mechanisms underlying these processes are not well understood. Here, we report the crystal structure of the cytosolic domain of PEX3 in complex with a PEX19-derived peptide. PEX3 adopts a novel fold that is best described as a large helical bundle. A hydrophobic groove at the membrane-distal end of PEX3 engages the PEX19 peptide with nanomolar affinity. Mutagenesis experiments identify phenylalanine 29 in PEX19 as critical for this interaction. Because key PEX3 residues involved in complex formation are highly conserved across species, the observed binding mechanism is of general biological relevance.

SC68896, a novel small molecule proteasome inhibitor, exerts antiglioma activity in vitro and in vivo.

PURPOSE: Glioblastomas are among the most lethal neoplasms, with a median survival of <1 year. Modulation of the proteasome function has emerged as a novel approach to cancer pharmacotherapy. Here, we characterized the antitumor properties of SC68896, a novel small molecule proteasome inhibitor. EXPERIMENTAL DESIGN: Different tumor cell lines were tested by crystal violet staining for sensitivity to SC68896, given alone or in combination with death ligands. The molecular mechanisms mediating SC68896-induced cell death and changes in cell cycle progression were assessed by immunoblot and flow cytometry. An orthotopic human glioma xenograft model in nude mice was used to examine the in vivo activity of SC68896. RESULTS: SC68896 inhibits the proliferation of cell lines of different types of cancer, including malignant glioma. Exposure of LNT-229 glioma cells to SC68896 results in a concentration- and time-dependent inhibition of the proteasome, with a consequent accumulation of p21 and p27 proteins, cell cycle arrest, caspase cleavage, and induction of apoptosis. Using RNA interference, we show that the effect of SC68896 on glioma cells is facilitated by wild-type p53. SC68896 sensitizes glioma cells to tumor necrosis factor-related apoptosis-inducing ligand and CD95 ligand and up-regulates the cell surface expression of the tumor necrosis factor-related apoptosis-inducing ligand receptor cell death receptors 4 and 5, which may contribute to this sensitization. Intracerebral glioma-bearing nude mice treated either i.p. or intratumorally with SC68896 experience prolonged survival. CONCLUSIONS: SC68896 is the first proteasome inhibitor that exerts antiglioma activity in vivo. It may represent a novel prototype agent for the treatment of malignant gliomas and warrants clinical evaluation.

Anticoagulation for radiation-induced neurotoxicity revisited.

No effective treatment for delayed radiation-induced neurotoxicity has been established. Its natural course is highly variable, but spontaneous recovery has been well documented. Here we report our experience with therapeutic anticoagulation in patients with cerebral lesions (n = 3), cranial nerve lesions (n = 1) or myelopathy (n = 4) attributed to irradiation. Two of three patients with cerebral lesions and the patient with cranial nerve lesions showed a minor improvement of clinical symptoms. In contrast, none of the patients with radiation myelopathy improved. No patient suffered hemorrhage or other adverse effects of anticoagulation. Overall, anticoagulation therapy demonstrates only modest activity for delayed radiation-induced neurotoxicity in this small case series.

The topoisomerase II inhibitor, genistein, induces G2/M arrest and apoptosis in human malignant glioma cell lines.

The protein tyrosine kinase inhibitor, genistein, has been reported to inhibit proliferation and to induce cell death in various non-solid and solid cancer cell lines. Herein, we examined the effects of genistein in several human malignant glioma cell lines. We found that genistein inhibited the proliferation of LN-18, LNT-229, LN-308 and T98G cells at EC50 concentrations of 25-80 microM (72 h of exposure). The growth of a non-neoplastic immortalized human astrocyte cell line, SV-FHAS, was inhibited at similar concentrations. There was a reduction in [3H]-methylthymidine incorporation and a moderate lactate dehydrogenase release as a sign of cell death in genistein-treated glioma cells. Electron microscopy showed morphological changes with mitochondrial swelling and apoptosis in glioma cells treated with high concentrations of genistein. Genistein-induced cytotoxicity was associated with an increased DNA/topoisomerase II complex formation. Furthermore, genistein induced cell cycle arrest in G2/M. There was an increase in the p53 and p21 levels in response to genistein. However, there was no difference in genistein sensitivity between p21-deficient colon carcinoma cells and isogenic control cells. Genistein-induced cell death in LN-18 and LNT-229 was unaffected by the ectopic expression of the preferential caspase 1/8 inhibitor, crm-A, or co-exposure to the pan-specific pseudosubstrate caspase inhibitor, zVAD-fmk. The ectopic expression of the anti-apoptotic BCL-2 protein attenuated the cytotoxic effects of genistein. Moreover, the ectopic expression of temperature-sensitive p53V135A, which acts as a dominant-negative p53 mutant at 38.5 degrees C but assumes p53 wild-type properties at 32.5 degrees C, in LN-18 or LNT-229 cells, had no effect on genistein cytotoxicity at either temperature. Genistein did not act in synergy with CD95 ligand-induced apoptosis or various cancer chemotherapy drugs in cytotoxic or clonogenic cell death assays. Thus, genistein-like protein kinase inhibitors are promising agents for the experimental treatment of malignant gliomas.

Target lesion ulceration and arch calcification are associated with increased incidence of carotid stenting-associated ischemic lesions in octogenarians.

PURPOSE: Although evidence is accumulating that advanced age is a risk factor for carotid angioplasty and stenting (CAS), the reason for this finding is incompletely understood. The aims of this study were to compare the prevalence of anatomic risk factors in patients <80 years with those in patients > or =80 years and to determine the effect of these risk factors on the incidence of new lesions seen on diffusion-weighted imaging (DWI) after protected CAS as surrogate markers for stroke. METHODS: Various potential anatomic risk factors for CAS were analyzed in 62 symptomatic patients (49 aged <80 years; 13 aged > or =80 years) by using preprocedural digital subtraction angiograms and extracranial contrast-enhanced magnetic resonance angiographies. DWI was performed immediately before and or =80 years) were not significant, the proportion of patients with any new ipsilateral DWI lesions, as well as the total number of these lesions, was higher in octogenarians than in patients aged <80 years (85% vs 47%, P < .05), with a median of 2 (interquartile range [IQR], 1 to 5) vs 0 (IQR, 0 to 3; P = .07). Similarly, the proportion of patients with any new DWI lesions outside the vascular territory of the target vessel as well as the total number of these lesions was significantly higher in octogenarians compared with patients aged <80 years (54% vs 10%, P < .01), with a median of 1.5 (IQR, 0.25 to 10.75) vs 0 (IQR, 0 to 1; P < .05). The presence of an ulcerated lesion was an independent predictor of any new ipsilateral DWI lesion (odds ratio [OR], 4.3; 95% confidence interval [CI], 1.06 to 17.1; P < .05), whereas a severe aortic arch calcification tended to be a predictor of new DWI lesions outside the territory of the treated artery (OR, 1.8; 95% CI, 0.99 to 3335; P = .05). CONCLUSIONS: Increased prevalences of severe aortic arch calcifications and target lesion ulceration are associated with an increased risk for magnetic resonance DWI-detected embolic events during CAS. Because in our study arch calcification and target lesion ulceration were more prevalent in octogenarians, this association may explain the increased risk of CAS in the elderly.

Highly enantioselective epoxidation of 2-methylnaphthoquinone (vitamin K3) mediated by new cinchona alkaloid phase-transfer catalysts.

In the area of catalytic asymmetric epoxidation, the highly enantioselective transformation of cyclic enones and quinones is an extremely challenging target. With the aim to develop new and highly effective phase-transfer catalysts for this purpose, we conducted a systematic structural variation of PTCs based on quinine and quinidine. In the total of 15 new quaternary ammonium PTCs, modifications included, for example, the exchange of the quinine methoxy group for a free hydroxyl or other alkoxy substituents, and the introduction of additional elements of chirality through alkylation of the alkaloid quinuclidine nitrogen atom by chiral electrophiles. For example, the well-established 9- anthracenylmethyl group was exchanged for a "chiral" anthracene in the form of 9-chloromethyl-[(1,8-S;4,5-R)-1,2,3,4,5,6,7,8-octahydro-1,4:5,8-dimethanoanthracene. The asymmetric epoxidation of vitamin K(3) was used as the test reaction for our novel PTCs. The readily available PTC 10 (derived from quinine in three convenient and high-yielding steps) proved to be the most enantioselective catalyst for this purpose known to date: At a catalyst loading of only 2.50 mol %, the quinone epoxide was obtained in 76 % yield and with 85 % ee (previously: < or =34 % ee), using commercial bleach (aqueous sodium hypochlorite) as the oxidant. To rationalize the sense of induction effected by our novel phase-transfer catalysts, a computational analysis of steric interactions in the intermediate chlorooxy enolate-PTC ion pair was conducted. Based on this analysis, the sense of induction for all 15 novel PTCs could be consistently explained.

Dramatic enhancement of enone epoxidation rates in nonionic microemulsions.

The ability of microemulsions to dissolve polar and non-polar components with a huge internal interface can overcome the reagent incompatibilities frequently encountered in organic reactions. We investigated model epoxidation reactions of alpha,beta-unsaturated enones and alkaline hydrogen peroxide in different nonionic microemulsions, both in the presence and absence of a phase-transfer agent (PTA). The obtained reaction profiles were compared with those for the corresponding surfactant-free two-phase systems. In addition, we defined a time constant tau as a measure for the rate of turnover. The epoxidation of trans-chalcone using an n-alkyl-polyoxyethylene surfactant based microemulsion was fastest in the system with the PTA (tau=66 min) and slightly slower without the PTA (tau=77 min). It was still slower in the two-phase system with a PTA (tau=114 min) and extremely sluggish without a phase-transfer agent. With n-alkyl beta-D-glucopyranoside as the surfactant the conversion was twice as fast than in the former microemulsion systems, but the PTA did not accelerate the reaction further (tau=35 and 33 min). The epoxidation of vitamin K(3), the second model system, was extremely accelerated. It proceeded a factor of approximately 35 faster in the microemulsion (tau=1.44 min) than in the corresponding two-phase system (tau=57 min).

Incidence of new brain lesions after carotid stenting with and without cerebral protection.

BACKGROUND AND PURPOSE: Diffusion-weighted imaging (DWI) may be a useful tool to evaluate the efficacy of cerebral protection devices in preventing thromboembolic complications during carotid angioplasty and stenting (CAS). The goals of this study were (1) to compare the frequency, number, and size of new DWI lesions after unprotected and protected CAS; and (2) to determine the clinical significance of these lesions. METHODS: DWI was performed immediately before and within 48 hours after unprotected or protected CAS. Clinical outcome measures were stroke and death within 30 days. RESULTS: The proportion of patients with any new ipsilateral DWI lesion (49% versus 67%; P<0.05) as well as the number of new ipsilateral DWI lesions (median=0; interquartile range [IQR]=0 to 3 versus median=1; IQR=0 to 4; P<0.05) were significantly lower after protected (n=139) than unprotected (n=67) CAS. The great majority of these lesions were asymptomatic and less than 10 mm in diameter. Although there were no significant differences in clinical outcome between patients treated and not treated with protection devices (7.5% versus 4.3%, not significant), the number of new DWI lesions was significantly higher in patients who developed a stroke (median=7.5; IQR=1.5 to 17) than in patients who did not (median=0; IQR=1 to 3.25; P<0.01). CONCLUSIONS: The use of cerebral protection devices significantly reduces the incidence of new DWI lesions after CAS of which the majority are asymptomatic and less than 10 mm in diameter. The frequent occurrence of these lesions and their close correlation with the clinical outcome indicates that DWI could become a sensitive surrogate end point in future randomized trials of unprotected versus protected CAS.

Incidence and risk factors for medical complications after carotid artery stenting.

OBJECTIVE: Carotid angioplasty and stenting (CAS) is being evaluated as an alternative to carotid endarterectomy for the treatment of carotid artery stenosis; however, to date little is known about the incidence of medical complications after CAS. The goal of this study was to determine the frequency of, and to identify potential clinical risk factors for, the development of medical complications after CAS. METHODS: Medical complications that occurred < or = 30 days after CAS in 327 consecutive patients (241 men, 86 women; mean age, 69 +/- 9 years; range, 45 to 90 years) treated for symptomatic (n = 182, 56%) or asymptomatic (n = 145, 44%) carotid artery stenosis were recorded. The effect of clinical characteristics on the subsequent development of medical complications was analyzed by logistic regression. RESULTS: Fifty-one patients (15%) had 62 medical complications: 3 (0.9%) myocardial infarctions, 3 (0.9%) cardiac arrhythmias, 4 (1.2%) episodes of angina pectoris, 3 (0.9%) episodes of symptomatic hypertension, 16 (4.9%) episodes of symptomatic hypotension, 10 (3.1%) chest infections, 9 (2.7%) had periods of confusion, 5 (1.5%) had urinary retention, and 9 (2.7%) urinary tract infections. One chest infection was fatal and 16 complications prolonged the intensive care unit monitoring period > 24 hours. Advanced age (odds ratio [OR], 1.1; 95% confidence interval [CI], 1.05 to 1.14) and a symptomatic carotid stenosis (OR, 2.1; 95% CI, 1.07 to 4.1) independently predicted the occurrence of medical complications. CONCLUSION: Although life-threatening or fatal non-neurologic events were uncommon in this series, the overall incidence of medical complications after CAS might be higher than currently anticipated. Older and symptomatic patients are at the highest risk, and these subgroups should be monitored closely.

Adenoviral natural born killer gene therapy for malignant glioma.

Glioblastoma is a lethal neoplasm resistant to conventional radiotherapy and chemotherapy. Natural born killer (NBK), also known as Bcl-2-interacting killer (BIK), is a death-promoting Bcl-2 family protein sharing with Bcl-2 only the Bcl homology 3 (BH3) domain. We here report that an adenoviral vector encoding NBK (Ad-NBK) uniformly induces cell death in 12 human malignant glioma cell lines. Ad-NBK-induced cell death involves neither quantitative mitochondrial cytochrome c release nor caspase 8, 9, 7, or 3 processing and is unaffected by the viral caspase inhibitor, cytokine response modifier A (CRM-A), or selective caspase 8 or 9 inhibitors. In contrast, Ad-NBK-induced cell death is inhibited by the broad-range caspase inhibitor, zVAD-fmk, or by adenoviral gene transfer of the X-linked inhibitor of apoptosis protein (XIAP). Further, Ad-NBK-induced cell death is inhibited by Bcl-2 or Bcl-xL gene transfer. Interestingly, Bcl-2- and Bcl-xL-transfected glioma cells, which are partially protected from Ad-NBK-induced cell death, accumulate much higher levels of NBK than are ever observed in control-infected cells. This indicates that complex formation with Bcl-2 or Bcl-xL sequesters NBK in an inactive form and that free NBK, rather than an NBK-mediated depletion of free antiapoptotic Bcl-2 family proteins, is the proximate mediator of Ad-NBK-induced cell death. Conversely, proteasome inhibition-mediated accumulation of NBK strongly enhances Ad-NBK-induced cell death. Finally, Ad-NBK-infected LN-229 glioma cells are not tumorigenic in nude mice. Thus Ad-NBK triggers an XIAP- and zVAD-fmk-sensitive cell death pathway in glioma cells with potential therapeutic value, provided that NBK expression can be selectively targeted to cancer cells.

Comparison of microencapsulation techniques for the water-soluble drugs nitenpyram and clomipramine HCl.

In this investigation, the water-soluble drugs nitenpyram and clomipramine HCl were encapsulated using coacervation, solvent evaporation and film-coating. The effect of different process factors on the encapsulation efficiency and the release profile of the microparticles was evaluated. For coacervation it was shown that the core to wall ratio was the most important factor. For solvent evaporation using an w/o emulsion the type and concentration of the surfactant were the most important parameters for a successful encapsulation. Additionally the coated material was tested for its stability under different conditions as a powder or compressed into tablets. It could clearly be demonstrated that the coated drug substance exhibited a better stability then the uncoated material. The particles prepared by film-coating showed the best stability.

Low molecular weight heparin for deep vein thrombosis in glioma patients.

The treatment and secondary prophylaxis of deep vein thrombosis (DVT) and pulmonary embolism, a common complication in patients with malignant glioma, has remained controversial. We treated 11 patients with malignant glioma and DVT prospectively with low molecular weight heparin (LMWH) at 175 IU/kg for 10 days and then for 3 months at 100 IU/kg. No patient developed bleeding complications or any other severe side effects of LMWH treatment. Two patients had a dose reduction of LMWH to 75 IU/kg because of chemotherapy-induced thrombocytopenia. One patient developed progressive DVT and nonlethal pulmonary embolism on day 14 of LMWH 100 IU/kg. After increasing the dose to 175 IU/kg he had no further recurrence. One patient had recurrence of DVT after a fracture of the leg affected by DVT at 8 months after the diagnosis of DVT and 5 months after the end of LMWH therapy. LMWH therapy may be safe and effective in the treatment and secondary prophylaxis of DVT in patients with malignant glioma.

MRI characteristics of acute and subacute brainstem and thalamic infarctions: value of T2- and diffusion-weighted sequences.

MRI including diffusion-weighted sequences (DW-MRI) has demonstrated its high sensitivity for acute supratentorial ischemic lesions. In this study we examined the sensitivity of different MRI sequences for the detection of acute brainstem and isolated thalamic infarctions. Diffusion- and T2-weighted MRI of 45 consecutive patients with signs and symptoms of infratentorial and thalamic infarction between 6/1997 and 1/2000 were analysed. The time between the onset of symptoms and the first MRI varied between 2 hours to 7 days with a median of 2 days. MRI repeats were performed in 4 patients in whom the clinical brainstem infarction had not been detected initially. Lesion detectability and size were evaluated for different brainstem and thalamic localizations. An acute brainstem or thalamic infarction as defined by the clinical condition could be identified in all patients by comparison of DW-MRI and T2-weighted images. Pons in farctions were the largest, followed by midbrain and thalamic lesions. Medulla oblongata infarctions were small in comparison. Pons, mid-brain and thalamic infarctions were reliably identified beginning 12 hours after the onset of symptoms. In contrast, detectability of medulla oblongata infarctions varied within the first 24 hours and their overall visibility was worse than that of other brainstem infarctions corresponding to their small size. However, regardless of loca tion, none of the 3 infarctions examined within the first 5 hours after the onset of symptoms could be identified. These lesions were demonstrated in follow-up examinations. In conclusion, pontine, midbrain and thalamic infarctions can reliably be visualized by a combination of DW-MRI and T2-weighted images beginning 12 hours after the ischemic attack. However, sensitivity seems to be lower earlier than 12 hours after ischemia and for medulla oblongata lesions.