Aqueous humor concentrations of topically administered caspofungin in rabbits.

BACKGROUND: The echinocandin caspofungin (CAS) is a novel antifungal drug with fungicidal in vitro activity against all Candida spp., which are the most frequent cause of fungal keratitis. Penetration of CAS through the cornea into the aqueous humor after topical administration was investigated. METHODS: A CAS solution with a concentration of 7 mg/ml was applied onto each rabbit's cornea. Drug application after corneal epithelium abrasion was processed in different time intervals: single application with aqueous humor sampling after 1 and 2 h. In addition, after continuous application of CAS every 30 min, aqueous humor concentrations of CAS after 1, 2 and 5 h were analyzed by liquid-chromatography tandem mass spectrometry. RESULTS: Topical administration of CAS without corneal epithelium abrasion resulted in no detectable amounts of the drug in the aqueous humor. However, with corneal abrasion, after a single application, levels of 2.16 +/- 1.57 microg/ml (n = 6) were reached after 1 h and then decreased to 1.76 +/- 0.88 microg/ml (n = 2) after 2 h. After serial application every 30 min, the following intracameral levels of CAS were detected: after 1 h, 2.11 +/- 1.09 microg/ml (n = 6); after 2 h, 4.94 +/- 1.80 microg/ml (n = 5), and after 5 h, 3.45 +/- 2.11 microg/ml (n = 6). CONCLUSION: In the aqueous humor, therapeutic drug levels can be reached that cover the MICs of most fungi after epithelial abrasion. To achieve a sustained high level of CAS as an effective antifungal therapy for corneal keratitis, CAS should be administered topically every 30 min after removal of the corneal epithelium.

Aqueous humor concentration of voriconazole after topical administration in rabbits.

OBJECTIVES: Voriconazol is a triazole antifungal drug with in vitro fungicidal activity against all Candida spp., Fusarium spp. and Aspergillus spp. which are frequent causes of fungal keratitis depending on geographic location. We investigated the penetration of voriconazole through the cornea into the aqueous humor (AH) after topical administration. METHODS: A 1% voriconazole solution was applied onto each rabbit's cornea. Topical drug application was processed at different time intervals: single drug application with AH sampling after 30 min, 1 h, 2 h, 3 h and 6 h. In addition, we evaluated AH samples after repeated topical application of voriconazole every 30 min after 1, 2, 4 and 6 h. Furthermore, after repeated drug application every hour, we analyzed voriconazole concentration after 2, 3, 4 and 6 h. All samples were analyzed by high-performance liquid chromatography (HPLC)-UV. RESULTS: A single application showed a maximum peak in AH of 3.58 microg/ml (N = 9) after 30 min. Within 3 h the concentration decreased to 0.04 microg/ml (N = 11). Application of voriconazole every half an hour revealed a peak value of 6.73 microg/ml (N = 10) after 2 h; after 4 h the value decreased to 6.19 microg/ml (N = 10) and was constant after 6 h (6.12 microg/ml, N = 6). When administrated every hour, only lower AH concentrations of voriconazole were reached with a maximum level of 2,06 microg/ml (N = 8) after four hours. CONCLUSION: In AH, therapeutic drug levels that cover the minimum inhibitory concentrations (MIC) of most fungi can be reached. To achieve a sustained high level of voriconazole as an effective antifungal therapy for corneal keratitis, voriconazole should be topically administered every 30 min.

Analysis of a single nucleotide polymorphism in codon 388 of the FGFR4 gene in malignant gliomas.

The FGFR4 codon 388 polymorphism (Arg(388), Arg/Gly(388) or Gly(388)) was determined in glioblastoma multiforme (GBM), anaplastic astrocytomas (AA), diffuse astrocytomas (DA), and control muscles. Arg(388) was rare in AA, GBM, muscles, and was absent in DA. The Arg/Gly(388) and the Gly(388) frequency was equal among GBM and controls. FGFR4 expression was not related to codon 388 in GBM, and no survival differences between Arg/Gly(388) and Gly(388) tumors were found. U87 cells (Arg/Gly(388)) did not show higher invasion than U138 cells (Gly(388)). This suggests that the FGFR4 codon 388 status does not play a major role in malignant gliomas.

Expression and function of somatostatin receptors in peripheral nerve sheath tumors.

Although somatostatin receptors have been detected in many normal and neoplastic tissues, little is known of their expression and function in peripheral nerve tumors. In the present study, we examined the expression of all 5 somatostatin receptor subtypes (sst1-5) in 3 normal peripheral nerves, 3 traumatic neuromas, 27 schwannomas, 18 neurofibromas, and 177 malignant peripheral nerve sheath tumors (MPNSTs) by immunohistochemistry as well as by Western blot and reverse transcriptase-polymerase chain reaction investigations in 2 normal peripheral nerves, one neurofibroma, 5 schwannomas, and 5 MPNSTs. Immunoreactive somatostatin receptors were not detectable in normal peripheral nerve and in nonneoplastic Schwann cell proliferations. In contrast, sst2A mRNA and protein was present in 89% of schwannomas. This receptor subtype was less frequently detected in neurofibromas (22%) and MPNSTs (15%). Interestingly, sst4 was seen in 32% of MPNSTs and was almost exclusively expressed in this malignant tumor type. In support of a role in Schwann cell tumor growth control by somatostatin was the observation of induced internalization of sst2A and inhibition of cell proliferation in an NF1-associated MPNST cell line. Moreover, administration of an sst2A-selective agonist resulted in induction of MPNST cell apoptosis. We conclude that peripheral nerve sheath tumors often express at least one functional somatostatin receptor. Furthermore, our findings suggest a potential clinical role for somatostatin receptor agonists in tumor imaging and/or treatment of schwannomas and MPNSTs.

Alterations of cell cycle regulators in gliomatosis cerebri.

Gliomatosis cerebri (GC) is regarded as a rare glial neoplasm of unknown origin, and a detailed analysis of molecular alterations underlying this disease has started only recently. However, because GC characteristically affects large parts of the brain and spinal cord, the distribution of genetic alterations may be highly variable between different tumor areas. Additionally, tumor areas with varying degrees of differentiation may be present, raising the possibility to model the genetic events associated with astrocytoma progression. Here we analyzed various tumor regions with features of low-grade and high-grade astrocytomas from 9 autopsy-proven GC cases for the immunoexpression of the cell cycle-controlling proteins mdm2, p21, p27/kip1, p16, and Rb. The samples were also screened for EGFR expression, and for amplification of the EGFR and MDM2 genes. Furthermore, allelic losses of the CDKN2A gene and of a PTEN flanking region of chromosome 10 were determined. We detected tumor regions with immunoexpression of p21 only rarely in our series, without association to the tumor grade. No MDM2 gene amplification was detected. In contrast, three cases demonstrated maintained Rb expression. The expression of p27(kip1) showed a clear reduction with increasing astrocytoma malignancy in 7 cases. Allelic loss of the CDKN2A gene occurred in 5 patients but was not related to the tumor grading, nor to the intensity of p16 immunoexpression. No homozygous CDKN2Adeletions were detected. EGFR amplification was also absent in our series, but one case demonstrated EGFR expression only in the high-grade tumor area. Allelic losses on chromosome 10 were found in one out of six informative cases. However, marked differences in the immunoexpression, as well as in the distribution of genetic aberrations were seen between different tumor samples within a given case. The distribution of the alterations suggests that these molecular genetic changes represent secondary events, which may develop within tumor clones derived from a common founder tumor clone characterized by extraordinary spreading through the brain. Moreover, the detected aberrations in gliomatosis cerebri can reflect the tumor progression associated with secondary malignant astrocytoma formation even within a single case.

Different activation of mitogen-activated protein kinase and Akt signaling is associated with aggressive phenotype of human meningiomas.

PURPOSE: Activation of intracellular signaling cascades has been implicated in the growth control of benign meningiomas, but their role for meningioma progression and outcome is unknown. Here we determined the expression and function of proteins involved in mitogen-activated protein kinase (MAPK) and phosphinositol-3 kinase (PI3K)/Akt signaling in benign, atypical, and malignant meningiomas and studied their association with clinicopathologic data including meningioma recurrence. EXPERIMENTAL DESIGN: Expression of various MAPK and PI3K signaling proteins was determined in 70 primary meningiomas and, if present, in recurrent tumors by immunohistochemistry and Western blotting. The expression patterns in primary and recurrent tumors were related to clinical data. The effect of MAPK and PI3K pathway inhibition on cell proliferation and apoptosis was determined using a primary malignant meningioma cell culture. RESULTS: Atypical and malignant meningiomas showed higher levels of phospho-Akt compared with benign tumors, and their proliferation could be inhibited by PI3K blocking using wortmannin. PI3K inhibition did not induce apoptosis in malignant meningioma cells. In contrast, expression of phospho-Raf and phospho-MAPK was decreased in aggressive meningiomas compared with benign tumors, but MAPK inhibition by PD98059 resulted in tumor cell apoptosis and decreased proliferation. Reduced MAPK activation was associated with meningioma recurrence, and PI3K activation was associated with poor preclinical condition and brain invasion of malignant meningiomas. CONCLUSIONS: Both MAPK and PI3K/Akt pathways are activated at different levels in benign and malignant meningiomas. Activation of PI3K/Akt signaling contributes to the aggressive behavior of malignant meningiomas, whereas MAPK activation is involved in both proliferation and apoptosis of malignant meningiomas.

Hereditary lattice corneal dystrophy is associated with corneal amyloid deposits enclosing C-terminal fragments of keratoepithelin.

PURPOSE: To investigate the molecular basis of hereditary lattice corneal dystrophy (LCD) type IIIA associated with corneal amyloid deposits afflicting several members of a four-generation family. METHODS: Histologic, immunohistochemical and biochemical studies were performed on corneal tissue samples obtained after perforating keratoplasty. DNA was extracted from peripheral blood leukocytes. All exons of the keratoepithelin-encoding TGFBI gene were amplified and sequenced. The presence of a mutation was confirmed by digestion of the isolated PCR product with the restriction enzyme AlwNI. RESULTS: The cornea of the index patient (II-1) contained large patchy deposits of amyloid, which were immunoreactive for the C terminus of keratoepithelin. Western blot analysis of the polypeptide chains extracted from the amyloid deposits of paraffin-embedded tissue revealed that these represented mainly fragments of the full-length protein. The smallest fragments were 6.5 and 6.9 kDa. DNA analyses of the TGFBI gene revealed a heterozygous T-->C transition at the second position of codon 540 in exon 12, indicating that replacement of phenylalanine by serine (Phe540Ser) leads to dominant disease. The mutation creates a new restriction site for the enzyme AlwNI. Five of the examined family members carried this mutation. Three of them (aged >/=41 years) had the disease, two family members (aged <20 years) do not yet show any clinical symptoms. An additional inconsequential single-nucleotide polymorphism (T1667C) was found at the third position of the same codon (Phe540Phe) in three unaffected family members. CONCLUSIONS: This is the first report of a single-nucleotide mutation at codon 540 of TGFBI leading to LCD, and the first to demonstrate that the amyloid deposits in LCD contain proteolytic fragments of keratoepithelin.

Single-cell analysis of mtDNA deletion levels in sporadic amyotrophic lateral sclerosis.

One possible cause for the neuronal loss in sporadic amyotrophic lateral sclerosis (S-ALS) is an increase of free radicals, which may produce oxidative damage to susceptible biomolecules, which, in turn, can damage the mitochondrial DNA (mtDNA). Following laser microdissection of single motor neurons from paraffin-embedded autopsy tissue, we analyzed the presence of a common mtDNA deletion, the 5 kb common deletion (CD). Spinal cord neurons showed slightly higher CD detection rate in patients than controls (94% vs 75%). No significant differences were found between patients and controls for neurons derived from other motor or non-motor regions. A PCR assay of serial DNA dilutions (10-fold) showed no CD level differences between motor neurons in S-ALS and controls. These data suggest that neuronal death in S-ALS is not associated with significant accumulation of mtDNA deletions.

Effects of axonal injury on ganglion cell survival and glutamate homeostasis.

Axonal trauma leads to a series of pathologic events that can culminate in neuronal death. Optic nerve crush can be used to explore histologic and molecular changes in traumatic central nervous system malfunction. Although the precise mechanisms of retinal ganglion cell death after optic nerve crush have not been elucidated, glutamate antagonists can protect retinal ganglion cells after axotomy. We, therefore, evaluated the effect of optic nerve crush on levels of extracellular glutamate. Ganglion cell survival and extracellular glutamate levels were assessed from 1 to 28 days after optic nerve crush in Long-Evans rats. Optic nerve crush led to a rise in extracellular glutamate; this rise was blocked by treatment with memantine, riluzole, and nimodipine. Partial optic nerve crush leads to an increase in vitreal glutamate, perhaps through release of intracellular contents. This released glutamate can contribute to additional ganglion cell loss. Future work will help to additionally unravel the steps by which axotomy induces excitotoxic damage to ganglion cells, and perhaps indicate protective interventions.

Region-specific analysis of mitochondrial DNA deletions in neurodegenerative disorders in humans.

Mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) aberrations has been implicated in the neuronal death in neurodegenerative disorders. Significant neuronal damage can occur if the percentage of mtDNA mutations may reach a critical threshold. mtDNA mutations also accumulate during normal aging. Here we quantified the 5 kB common mtDNA deletion (CD) using real-time PCR in various brain regions from neurodegenerative disorders and controls. We confirmed previous results that the CD levels increase with age, reaching highest levels in the basal ganglia. High CD levels were also found in affected regions in frontotemporal dementia, Parkinson's disease, and dementia with Lewy bodies, but not in Alzheimer's disease. This suggests that mtDNA damage may occur in a region-specific distribution in neurodegenerative disorders.

Prognostic relevance of MAPK expression in glioblastoma multiforme.

The objective of this study was to determine the immunoexpression pattern of the mitogen-activated protein kinase (MAPK), and related signalling proteins [protein kinase C (PKC), phospholipase Cgamma (PLCgamma)], in glioblastoma multi-forme, and to investigate their prognostic value. Paraffin-embedded biopsy samples from 26 patients [13 patients with long-term survival (LTS; N=13; median 28 months, range 13-76 months), and, for comparison, 13 patients with short-term survival (STS; N=13; median 7 months, range 1-12 months)] were investigated for the immunoexpression of MAPK, the activated pMAPK, PKC, PLCgamma, EGFR, and PTEN. Additionally, the MIB-1 proliferation index was determined. The immunoexpression pattern were related to clinical data, including analysis of their prognostic value using the Cox-proportional hazard model. No significant differences were found between STS and LTS in terms of age, Karnofsky performance status, and treatment. Whereas EGFR expression did not differ between STS and LTS and does not influence survival, expression of MAPK and activated pMAPK was significantly correlated with survival time. The percentage of pMAPK expressing cells correlated strongly with the percentage of MIB-1 positive cells. Furthermore, survival in patients with tumors expressing PKC or PLCgamma was significantly shorter. No differences were found for PTEN expression. Our findings indicate that the MAPK pathway is correlated with proliferation in gliomas, and that patient subgroups exist, in which expression of MAPK-related signalling proteins (PKC, PLCgamma) is associated with poorer prognosis. These patient subgroups may benefit from additional chemotherapeutic agents which specifically inhibit these signalling proteins.

Combined topical fluconazole and corticosteroid treatment for experimental Candida albicans keratomycosis.

PURPOSE: To determine the most efficient time point and concentration of topical corticosteroids in Candida albicans keratitis treated with fluconazole. METHODS: Corneas of 105 rabbits were infected with viable yeast cells of C. albicans (2.5 x 10(5)). After a 48-hour incubation period, seven groups of animals were treated for 21 days with fluconazole, with group I acting as a control, and groups II to VII receiving adjunct therapy with the corticosteroid prednisolone (5 or 10 times daily; 3, 9, or 15 days after infection). The degree of corneal infiltration, ulceration, corneal clouding, hypopyon, conjunctivitis, neovascularization, and corneal perforation was monitored over a 24-day period, as well as recultivation and resistance to fluconazole of the C. albicans pathogen. RESULTS: The control group showed the highest level of corneal clouding and neovascularization. In comparison, by day 24, the majority of groups also treated with prednisolone displayed significantly less corneal clouding and neovascularization. An immediate decrease in corneal clouding was observed in groups treated with additional low- or high-dose prednisolone from day 9 after inoculation. After additional prednisolone treatment from day 9 or 15 after inoculation, no significant difference was detected in the recultivation rate of C. albicans compared with the control. Early administration of prednisolone (day 3, low and high dose) resulted in the recultivation of significantly more C. albicans. CONCLUSIONS: Fluconazole plus adjunct high-dose prednisolone treatment was most effective when administered 9 days after infection. The delayed application of corticosteroids after treatment with antimycotic drugs in cases of fungal keratitis is therefore not contraindicated and may be beneficial in patients.

Changes of retinal glutamate transporter GLT-1 mRNA levels following optic nerve damage.

PURPOSE: Under physiological conditions, levels of the excitatory neurotransmitter glutamate within the retina are regulated by retinal glutamate transporters to prevent toxic accumulation. Alterations in this glutamate buffering have been implicated in retinal ganglion cell (RGC) death. We quantified the changes in the level of glutamate transporter mRNA in a model of acute rat optic nerve injury. METHODS: Optic nerve damage was induced in one eye of 25 adult Wistar rats by partial optic nerve crush (ONC). Total mRNA levels of the retinal glutamate transporter GLT-1 (EAAT-2) were determined by quantitative real-time PCR. GLT-1 mRNA levels were measured 1, 3, 7, 14, and 28 days following optic nerve injury. Additionally, control values were obtained from the retinas of five control rats (sham-crush). RESULTS: In the very early phase (1 day post-ONC), a 3.9 fold increase in levels of GLT-1 mRNA was observed in the ONC retinae compared with control eyes. This was followed by a rapid decrease towards control levels at day 3 post-ONC. GLT-1 mRNA levels remained up to 14 days post-crush. However, in the late phase post-ONC (day 28), the level of GLT-1 mRNA increased again, but still remained not significant to control levels. CONCLUSIONS: Changes in GLT-1 mRNA expression following axonal trauma of RGCs can lead to an imbalance of glutamate homeostasis. This may cause local accumulation of toxic concentrations of the neurotransmitter glutamate and further irreversible excitotoxic damage of RGCs.

Traumatic axonal injury results in biphasic calpain activation and retrograde transport impairment in mice.

Traumatic axonal injury (TAI) is one of the most important pathologies associated with closed head injury, and contributes to ensuing morbidity. The authors evaluated the potential role of calpains in TAI using a new model of optic nerve stretch injury in mice. Male C57BL/6 mice were anesthetized, surgically prepared, and subjected to a 2.0-mm optic nerve stretch injury (n = 34) or sham injury (n = 18). At various intervals up to 2 weeks after injury, optic nerves were examined for neurofilament proteins and calpain-mediated spectrin breakdown products using immunohistochemistry. In addition, fluorescent tracer was injected into the superior colliculi of mice 1 day before they were killed, to investigate the integrity of retrograde axonal transport to the retina. Optic nerve stretch injury resulted in persistent disruption of retrograde axonal transport by day 1, progressive accumulation and dephosphorylation of neurofilament protein in swollen and disconnected axons, and subsequent loss of neurofilament protein in degenerating axons at day 14. Calpains were transiently activated in intact axons in the first minutes to hours after stretch injury. A second stage of calpain-mediated proteolysis was observed at 4 days in axonal swellings, bulbs, and fragments. These data suggest that early calpain activation may contribute to progressive intraaxonal structural damage, whereas delayed calpain activation may be associated with axonal degeneration.

GM1 ganglioside administration protects spinal neurons after glutamate excitotoxicity.

Injury of the nervous system initiates a cascade of signal transduction including a rise in extracellular glutamate which leads to subsequent secondary neuronal loss. Excitotoxicity is known to play a crucial role in cell death spinal cord trauma. Gangliosides, particularly monosialogangliosides (GM1), are protective against various neurological insults, including excitotoxicity. Gangliosides may improve outcome following human spinal cord injury in humans. To further explore the relationship between excitotoxicity and GM1, dissociated murine spinal cord preparations were exposed to glutamate (0.5 mM) with the subsequent administration of GM1 (80 micro M) at 8 and 13 days in culture. The addition of GM1 30 minutes after exposure to glumatate significantly reduced neuronal damage and preserved membrane structure and integrity. These results demonstrate that post-treatment with GM1 gangliosides after glutamate-induced excitotoxicity is effective in protecting spinal cord neurons.