Progression of retinal ganglion cell loss in multiple sclerosis is associated with new lesions in the optic radiations.

BACKGROUND AND PURPOSE: The mechanism of retinal ganglion cell and retinal nerve fiber layer loss in multiple sclerosis (MS) remains unknown. This study aimed to investigate the association between temporal retinal nerve fiber layer (tRNFL) thinning and disease activity in the brain determined by T2 lesions on magnetic resonance imaging (MRI). METHODS: Fifty-five consecutive patients with relapsing-remitting MS and 25 controls were enrolled. All patients underwent annual optical coherence tomography and high-resolution MRI scans for tRNFL thickness and brain lesion volume analysis, respectively. RESULTS: Significant tRNFL thickness reduction was observed over the 3-year follow-up period at a relatively constant rate (1.02 µm/year). Thinning of tRNFL fibers was more prominent in younger patients (P = 0.01). The tRNFL loss was associated with new MRI lesions in the optic radiations (ORs). There was significantly greater tRNFL thinning in patients with new lesional activity in the ORs compared with patients with new lesions outside the ORs (P = 0.009). CONCLUSIONS: This study supports the notion that retrograde transneuronal degeneration caused by OR lesions might play a role in progressive retinal nerve fiber layer loss. In addition, the results of the study also indicate that the disease-related neurodegenerative changes in the retina start much earlier than the clinical diagnosis of MS.

Relationship between chronic demyelination of the optic nerve and short term axonal loss.

BACKGROUND: Axonal loss is a major determinant of disability in multiple sclerosis (MS). While acute inflammatory demyelination is a principal cause of axonal transection and subsequent axonal degeneration in acute disease, the nature of chronic axonal loss is less well understood. In the current study, the relationship between degree of chronic demyelination and axonal degeneration was investigated using optic neuritis (ON) as a model. METHOD: 25 patients with a first episode of unilateral ON, good recovery of visual function and concurrent brain or spinal cord MRI lesions were enrolled. Axonal loss was assessed using change in retinal nerve fibre layer (RNFL) thickness between 1 and 3 years after ON. Optic nerve conduction was evaluated using latency of multifocal visual evoked potentials (mfVEP). The level of mfVEP latency delay at 12 and 36 months was considered indicative of the degree of permanent demyelination. Data from 25 age and gender matched normal controls were used for comparison. RESULTS: RNFL thickness was significantly reduced in ON eyes at 12 months compared with controls but remained unchanged in fellow eyes. Average RNFL thickness demonstrated a small but significant reduction between 12 and 36 months for both ON and fellow eyes. Change in RNFL thickness between 12 and 36 months, however, did not correlate with the degree of mfVEP latency delay. CONCLUSION: The results, therefore, show no association between the degree of permanent optic nerve demyelination (as measured by latency delay) and progressive axonal degeneration, at least in the early stages of the disease. The fact that fellow eyes demonstrated a similar degree of progressive axonal loss supports this suggestion.

Inhibition of farnesyltransferase induces regression of mammary and salivary carcinomas in ras transgenic mice.

For Ras oncoproteins to transform mammalian cells, they must be post-translationally modified with a farnesyl group in a reaction catalysed by the enzyme farnesyl-protein transferase (FPTase). Inhibitors of FPTase have therefore been proposed as anti-cancer agents. We show that L-744,832, which mimics the CaaX motif to which the farnesyl group is added, is a potent and selective inhibitor of FPTase. In MMTV-v-Ha-ras mice bearing palpable tumours, daily administration of L-744,832 caused tumour regression. Following cessation of treatment, tumours reappeared, the majority of which regressed upon retreatment. No systemic toxicity was found upon necropsy of L-744,832-treated mice. This first demonstration of anti-FPTase-mediated tumour regression suggests that FPTase inhibitors may be safe and effective anti-tumour agents in some cancers.

Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor.

To acquire transforming potential, the precursor of the Ras oncoprotein must undergo farnesylation of the cysteine residue located in a carboxyl-terminal tetrapeptide. Inhibitors of the enzyme that catalyzes this modification, farnesyl protein transferase (FPTase), have therefore been suggested as anticancer agents for tumors in which Ras contributes to transformation. The tetrapeptide analog L-731,735 is a potent and selective inhibitor of FPTase in vitro. A prodrug of this compound, L-731,734, inhibited Ras processing in cells transformed with v-ras. L-731,734 decreased the ability of v-ras-transformed cells to form colonies in soft agar but had no effect on the efficiency of colony formation of cells transformed by either the v-raf or v-mos oncogenes. The results demonstrate selective inhibition of ras-dependent cell transformation with a synthetic organic inhibitor of FPTase.

Monitoring of optic nerve function in Neurofibromatosis 2 children with optic nerve sheath meningiomas using multifocal visual evoked potentials.

Monitoring optic nerve sheath meningiomas (ONSM) in Neurofibromatosis type 2 (NF2) patients remains difficult. Other ocular manifestations of NF2 may obscure ophthalmic assessment of optic nerve function in these patients. Serial magnetic resonance imaging (MRI) used to assess the optic nerve is not without limitations, being expensive and often requiring general anaesthetic in children, with associated risks. This study was undertaken to describe the use of multifocal visual evoked potentials (multifocal VEP, mfVEP) in the regular monitoring of NF2 patients with ONSM. This study involved three NF2 patients with ONSM who undertook mfVEP testing at an academic ophthalmic centre. Same day mfVEP and routine ophthalmic testing were undertaken. Topographical function of the optic nerve was assessed, utilising tools such as asymmetry deviation and accumap severity index. Results were assessed alongside MRI and visual acuity (VA). From the three patients, five eyes had ONSMs, of which two caused unilateral blindness. The remaining three affected eyes had initial VAs 6/6, 6/24, and 6/18. Over follow up, ranging from 5 to 12 years, all tumours progressed, and VA declined for all patients. Multifocal VEP detected optic nerve functional loss corresponding with visual decline. This case series suggests mfVEP is effective in the objective topographic monitoring of optic nerve function in NF2 patients with ONSM. Due also to its safety in a paediatric population, the test may be considered in the routine monitoring of these patients, to be used to assist regular ophthalmic review and MRI scans.

Farnesyltransferase inhibition causes morphological reversion of ras-transformed cells by a complex mechanism that involves regulation of the actin cytoskeleton.

A potent and specific small molecule inhibitor of farnesyl-protein transferase, L-739,749, caused rapid morphological reversion and growth inhibition of ras-transformed fibroblasts (Rat1/ras cells). Morphological reversion occurred within 18 h of L-739,749 addition. The reverted phenotype was stable for several days in the absence of inhibitor before the transformed phenotype reappeared. Cell enlargement and actin stress fiber formation accompanied treatment of both Rat1/ras and normal Rat1 cells. Significantly, inhibition of Ras processing did not correlate with the initiation or maintenance of the reverted phenotype. While a single treatment with L-739,749 was sufficient to morphologically revert Rat1/ras cells, repetitive inhibitor treatment was required to significantly reduce cell growth rate. Thus, the effects of L-739,749 on transformed cell morphology and cytoskeletal actin organization could be separated from effects on cell growth, depending on whether exposure to a farnesyl-protein transferase inhibitor was transient or repetitive. In contrast, L-739,749 had no effect on the growth, morphology, or actin organization of v-raf-transformed cells. Taken together, the results suggest that the mechanism of morphological reversion is complex and may involve farnesylated proteins that control the organization of cytoskeletal actin.

Comparison of objective diagnostic tests in glaucoma: Heidelberg retinal tomography and multifocal visual evoked potentials.

PURPOSE: To compare sensitivity and specificity of functional and structural changes in glaucoma using two objective tests: the multifocal visual evoked potential (m-VEP) and Heidelberg retinal tomography II (HRT). PATIENTS AND METHODS: 41 glaucoma patients and 25 normal individuals participated in the study. One eye per individual was included in the study. Individuals were evaluated with Humphrey visual field (HVF) perimetry, m-VEP, and HRT. Moorfield regression analysis findings of HRT were compared with presence of scotoma on m-VEP. Linear regression analysis of quantitative variables, such as HVF mean deviation (MD), m-VEP discriminant score (Accumap Severity Index) and, global HRT parameters was also performed. RESULTS: m-VEP sensitivity and specificity were 93% and 96% respectively. HRT sensitivity and specificity were 79% and 92% respectively. The area under the receiver operating characteristic curve (ROC) for m-VEP was 0.96 and for HRT varied from 0.79 to 0.86 depending on the parameters used. Linear correlation between MD and Accumap Severity Index score was -77%, while that between HRT global parameters, Accumap Severity Index and MD were at best around 50%. Topographic comparison of the presence of scotoma on HVF and m-VEP in different areas of the visual field showed good agreement. Comparison of optic nerve head structural abnormality with corresponding areas of field defects on HVF and m-VEP showed poor to moderate agreement. CONCLUSION: The objective test of optic nerve function (m-VEP) and structure (HRT) can detect glaucomatous damage, with limited correlation. The 2 functional tests, HVF and m-VEP correlate better with each other than with HRT. It remains important to look for both functional and structural changes in order to detect all glaucoma cases.

Evaluation of farnesyl:protein transferase and geranylgeranyl:protein transferase inhibitor combinations in preclinical models.

Farnesyl:protein transferase (FPTase) inhibitors (FTIs) were originally developed as potential anticancer agents targeting the ras oncogene and are currently in clinical trials. Whereas FTIs inhibit the farnesylation of Ha-Ras, they do not completely inhibit the prenylation of Ki-Ras, the allele most frequently mutated in human cancers. Whereas farnesylation of Ki-Ras is blocked by FTIs, Ki-Ras remains prenylated in FTI-treated cells because of its modification by the related prenyltransferase, geranylgeranyl:protein transferase type I (GGPTase-I). Hence, cells transformed with Ki-ras tend to be more resistant to FTIs than Ha-ras-transformed cells. To determine whether Ki-ras-transformed cells can be targeted by combining an FTI with a GGPTase-I inhibitor (GGTI), we evaluated potent, selective FTIs, GGTIs, and dual prenylation inhibitors (DPIs) that have both FTI and GGTI activity. We find that in human PSN-1 pancreatic tumor cells, which harbor oncogenic Ki-ras, and in other tumor lines having either wild-type or oncogenic Ki-ras, treatment with an FTI/GGTI combination or with a DPI blocks Ki-Ras prenylation and induces markedly higher levels of apoptosis relative to FTI or GGTI alone. We demonstrate that these compounds can inhibit their enzyme targets in mice by monitoring pancreatic and tumor tissues from treated animals for inhibition of prenylation of Ki-Ras, HDJ2, a substrate specific for FPTase, and Rap1A, a substrate specific for GGPTase-I. Continuous infusion (72 h) of varying doses of GGTI in conjunction with a high, fixed dose of FTI causes a dose-dependent inhibition of Ki-Ras prenylation. However, a 72-h infusion of a GGTI, at a dose sufficient to inhibit Ki-Ras prenylation in the presence of an FTI, causes death within 2 weeks of the infusion when administered either as monotherapy or in combination with an FTI. DPIs are also lethal after a 72-h infusion at doses that inhibit Ki-Ras prenylation. Because 24 h infusion of a high dose of DPI is tolerated and inhibits Ki-Ras prenylation, we compared the antitumor efficacy from a 24-h FTI infusion to that of a DPI in a nude mouse/PSN-1 tumor cell xenograft model and in Ki-ras transgenic mice with mammary tumors. The FTI and DPI were dosed at a level that provided comparable inhibition of FPTase. The FTI and the DPI displayed comparable efficacy, causing a decrease in growth rate of the PSN-1 xenograft tumors and tumor regression in the transgenic model, but neither treatment regimen induced a statistically significant increase in tumor cell apoptosis. Although FTI/GGTI combinations elicit a greater apoptotic response than either agent alone in vitro, the toxicity associated with GGTI treatment in vivo limits the duration of treatment and, thus, may limit the therapeutic benefit that might be gained by inhibiting oncogenic Ki-Ras through dual prenyltransferase inhibitor therapy.

Farnesyltransferase inhibitors versus Ras inhibitors.

Over the past few years, the idea that farnesyl-protein transferase (FPTase) inhibitors might be effective antiproliferative/antitumor agents has been realized in studies of cultured cells and in rodent models of cancer. Most of the studies with FPTase inhibitors have focused on inhibiting the growth of ras-transformed cells in vitro or the growth of ras-dependent tumors in mice. More recently, it has been recognized that the antiproliferative effect of FPTase inhibitors may extend beyond ras-driven tumors. It now seems likely that the ability of FPTase inhibitors to reverse the malignant phenotype results, at least in part, from inhibiting the farnesylation of proteins other than Ras.

Objective perimetry using the multifocal visual evoked potential in central visual pathway lesions.

AIMS: To examine the ability of the multifocal pattern visual evoked potential (mVEP) to detect field loss in neurological lesions affecting the visual pathway from the chiasm to the cortex. METHOD: The mVEPs recorded in the clinic were retrospectively reviewed for any cases involving central neurological lesions. Recordings had been performed with the AccuMap V1.3 objective perimeter, which used an array of four bipolar occipital electrodes to provide four differently oriented channels for simultaneous recording. 19 patients with hemianopias were identified. Of these there were 10 homonymous hemianopias with hemifield type loss, two bitemporal hemianopias, and seven homonymous hemianopias with quadrantanopic distribution. A comparison with subjective field results and CT/MRI findings was done to determine the relation between the two methods of visual field mapping and any relation with the anatomical location of the lesion and the mVEP results. RESULTS: In all hemianopic type cases (12) the defect was demonstrated on the mVEP and showed good correspondence in location of the scotoma (nine homonymous and two bitemporal). The topographic distribution was similar but not identical to subjective testing. Of the seven quadrantanopic type hemianopias, only four were found to have corresponding mVEP losses in the same areas. In the three cases where the mVEP was normal, the type of quadrantanopia had features consistent with an extra-striate lesion being very congruous, complete, and respecting the horizontal meridian. CONCLUSIONS: The mVEP can detect field loss from cortical lesions, but not in some cases of homonymous quadrantanopia, where the lesion may have been in the extra-striate cortex. This supports the concept that the mVEP is generated in V1 striate cortex and that it may be able to distinguish striate from extra-striate lesions. It implies caution should be used when interpreting "functional" loss using the mVEP if the visual field pattern is quadrantic.

Spatial distribution of antibodies to Salmonella enterica serovar Typhimurium O antigens in bulk milk from Texas dairy herds.

Environmental factors that enhance either the survivability or dispersion of Salmonella enterica serovar Typhimurium (S. Typhimurium) could result in a spatial pattern of disease risk. The objectives of this study were to: (1) describe herd status based on antibody response to Salmonella Typhimurium as estimated from bulk tank milk samples and (2) to describe the resulting geographical patterns found among Texas dairy herds. Eight hundred and fifty-two bulk milk samples were collected from georeferenced dairy farms and assayed by an indirect enzyme-linked immunosorbent assay (ELISA) using S. Typhimurium lipopolysaccharide (LPS). ELISA signal-to-noise ratios for each bulk tank milk sample were calculated and used for geostatistical analyses. Best-fit parameters for the exponential theoretical variogram included a range of 438.8 km, partial sill of 0.060 and nugget of 0.106. The partial sill is the classical geostatistical term for the variance that can be explained by the herd's location and the nugget is the spatially random component of the variance. We have identified a spatial process in bulk milk tank titers for S. Typhimurium in Texas dairy herds and present a map of the expected smoothed surface. Areas with higher expected titers should be targeted in further studies on controlling Salmonella infection with environmental modifications.

Atrial myxoma with multiple myeloma.

A male patient was observed for seven years, from the onset of neurologic symptoms to removal of a left atrial myxoma. Mild broad-based elevation of the serum gamma-globulin level with a normal bone marrow aspirate was seen early in the course of disease. The broad-based gamma-globulin level increased, and at the time of surgical removal of the tumor, the patient was found to have a monoclonal increase of K light chain IgG with 60% infiltration of the bone with immature plasma cells. The findings are consistent with the view that continuous immunologic stimulation by products of the atrial tumor resulted in malignant transformation of immunocytes with resulting IgG multiple myeloma.

Sulfonylmethanesulfonamide inhibitors of carbonic anhydrase.

A series of sulfonylmethanesulfonamide derivatives is described, which are inhibitors of carbonic anhydrase (CA). The most potent of these is the racemic fluoro sulfone 9, which inhibits carbon dioxide hydration catalyzed by human CA II (CA-II) with an IC50 of 3 nM. Binding competition studies versus dansylamide indicate that the enantiomers of 9 have different affinities for CA-II, with equilibrium dissociation constants of 3.6 and 0.6 nM. QSAR analysis suggests that the key factors involved in achieving high affinity in this series are sulfonamide acidity, hydrophobicity, and minimization of steric demands at the carbon atom adjacent to the sulfonamide group.

Topically active carbonic anhydrase inhibitors. 3. Benzofuran- and indole-2-sulfonamides.

Derivatives of benzofuran- and indole-2-sulfonamide were prepared for evaluation as topically active ocular hypotensive agents. These compounds were found to be excellent inhibitors of carbonic anhydrase and to lower intraocular pressure in a rabbit model of ocular hypertension. However, the development of these compounds for clinical use was precluded by the observation that they cause dermal sensitization in guinea pigs. A correlation between electrophilicity, as assessed by in vitro reactivity with reduced glutathione, and dermal sensitization potential was further documented.

HIV-1 protease inhibitors based on hydroxyethylene dipeptide isosteres: an investigation into the role of the P1' side chain on structure-activity.

A systematic investigation was undertaken to determine the role of the P1' sidechain in a series of hydroxyethylene isostere based inhibitors of HIV-1 protease. Substitution and homologation of the benzyl P1' side chain of the Phe-Phe isostere based pseudo peptides 1 (L-682,679) and 2 (L-685,434) with various heteroalkyl groups leads to a series of extremely potent inhibitors of the enzyme. Several examples of the most potent inhibitors were very effective in an ex vivo cell based viral spread assay using human H9 T-lymphocytes and the IIIb isolate of HIV-1. Compound 19 is 120 times more potent than 1 and 16 times more potent than 2 in inhibiting the spread of infection in this assay.

Topically active carbonic anhydrase inhibitors. 2. Benzo[b]thiophenesulfonamide derivatives with ocular hypotensive activity.

Derivatives of benzo[b]thiophene-2-sulfonamide were prepared to investigate their potential utility as topically active inhibitors of ocular carbonic anhydrase. Such an agent would be useful in the treatment of glaucoma. Among the compounds described are 6-hydroxybenzo[b]thiophene-2-sulfonamide (16) and its acetate ester (23), which are among the most potent ocular hypotensive agents in this class, as assessed in the alpha-chymotrypsinized rabbit. These compounds were selected for clinical evaluation.

Topically active carbonic anhydrase inhibitors. 1. O-acyl derivatives of 6-hydroxybenzothiazole-2-sulfonamide.

A series of O-acyl derivatives of 6-hydroxybenzothiazole-2-sulfonamide (4, L-643,799) was prepared and the potential utility of each series member as a topically active inhibitor of ocular carbonic anhydrase was determined. In vitro studies showed these esters to be substrates for ocular esterases which liberate 4 during corneal translocation. The most interesting series member, 2-sulfamoyl-6-benzothiazolyl 2,2-dimethylpropionate (22, L-645,151), acting as a prodrug form of 4, was found to enhance delivery through the isolated albino rabbit cornea by 40-fold when compared to the parent phenol 4. Studies in rabbits revealed that 22 is a potent topically active ocular hypotensive carbonic anhydrase inhibitor.

Design and synthesis of HIV protease inhibitors. Variations of the carboxy terminus of the HIV protease inhibitor L-682,679.

A series of tetrapeptide analogues of 1 (L-682,679), in which the carboxy terminus has been shortened and modified, was prepared and their inhibitory activity measured against the HIV protease in a peptide cleavage assay. Selected examples were tested as inhibitors of virus spread in cell culture. Compound 12 was a 10-fold more potent enzyme inhibitor than 1 in vitro and 30-fold more potent in inhibiting the viral spread in cells.

Topically active carbonic anhydrase inhibitors. 4. [(Hydroxyalkyl)sulfonyl]benzene and [(hydroxyalkyl)sulfonyl]thiophenesulfonamides.

For several decades a tantalizing goal for the treatment of primary open-angle glaucoma has been the development of a topically active carbonic anhydrase inhibitor. Recent results from several research groups indicate that considerable progress has been made toward this objective. In this report, we present the design and synthesis of (hydroxyalkyl)sulfonyl-substituted benzene- and thiophenesulfonamides. These compounds exhibit inhibition of carbonic anhydrase II in the nanomolar range and lower intraocular pressure in the alpha-chymotrypsinized rabbit model of ocular hypertension after topical instillation.

Pseudopeptide inhibitors of Ras farnesyl-protein transferase.

Inhibitors of Ras farnesyl-protein transferase are described. These are reduced pseudopeptides related to the C-terminal tetrapeptide of the Ras protein that signals farnesylation. Deletion of the carbonyl groups between the first two residues of the tetrapeptides either preserves or improves activity, depending on the peptide sequence. The most potent in vitro enzyme inhibitor described (IC50 = 5 nM) is Cys [psi CH2NH]Ile[psi CH2NH]Phe-Met (3). To obtain compounds able to suppress Ras farnesylation in cell culture, further structural modification to include a homoserine lactone prodrug was required. Compound 18 (Cys[psi CH2NH]Ile[psi CH2NH]Ile-homoserine lactone) reduced the extent of Ras farnesylation by 50% in NIH3T3 fibroblasts in culture at a concentration of 50 microM. Structure-activity studies also led to 12 (Cys[psi CH2NH]Val-Ile-Leu), a potent and selective inhibitor of a related enzyme, the type-I geranylgeranyl protein transferase.