Activation of innate anti-viral immune response genes in symptomatic benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) is the most common urologic disease in men over age 50. Symptoms include acute urinary retention, urgency to urinate and nocturia. For patients with severe symptoms, surgical treatment is used to remove the affected tissue. Interestingly, the presence of histologic BPH does not always correlate with symptoms. The molecular basis of symptomatic BPH and how it differs from asymptomatic BPH is unknown. Investigation into the molecular players involved in symptomatic BPH will likely give insight into novel therapeutic, and potentially preventative, targets. We determined the expression of genes involved in the innate anti-viral immune response in tissues from patients undergoing surgery to alleviate the symptoms of BPH, and compared the results with prostate tissue with histologic BPH, but from patients with few urinary issues (asymptomatic BPH). We found that expression of complement factor I, apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like protein 3G, oligoadenylate synthetase 2 and interferon-induced tetratricopeptide 1, four genes whose protein products are involved in the innate anti-viral immune response, was significantly transcriptionally upregulated in symptomatic BPH. Additionally, we observe hypomethylation and concomitant expression of ancient retroviral-like sequences, the long interspersed nuclear element 1 retrotransposons, in symptomatic BPH when compared with normal prostate tissue. These findings merit further investigation into the anti-viral immune response in symptomatic BPH.

Oxidative damage in age-related macular degeneration.

Epidemiologic studies have suggested that elderly patients who consumed diets rich in antioxidants throughout their lives are less likely to be afflicted with age-related macular degeneration (AMD). This led to the Age-Related Eye Disease Study, which showed that supplements containing antioxidant vitamins and zinc reduce the risk of progression to severe stages of AMD. Despite these data that indirectly implicate oxidative damage in the pathogenesis of AMD, there has not been any direct demonstration of increased oxidative damage in the retinas of patients with AMD. In this study, we used biomarkers of oxidative damage in postmortem eyes from patients with AMD and comparably aged patients without AMD to directly assess for oxidative damage. Sections from 4 eyes with no pathologic features of AMD showed no immunofluorescent staining for markers of oxidative damage, while sections from 8 of 12 eyes with advanced geographic atrophy showed evidence of widespread oxidative damage in both posterior and anterior retina. Only 2 of 8 eyes with choroidal neovascularization and 2 of 16 eyes with diffuse drusen and no other signs of AMD showed evidence of oxidative damage. These data suggest that widespread oxidative damage occurs in the retina of some patients with AMD and is more likely to be seen in patients with advanced geographic atrophy. This does not rule out oxidative damage as a pathogenic mechanism in patients with CNV, but suggests that a subpopulation of patients with geographic atrophy may have a major deficiency in the oxidative defense system that puts the majority of cells in the retina at risk for oxidative damage.

Detection of CX3CR1 single nucleotide polymorphism and expression on archived eyes with age-related macular degeneration.

There is a significant genetic component in age-related macular degeneration (AMD). CX3CR1, which encodes the fractalkine (chemokine, CX3CL1) receptor, has two single nucleotide polymorphisms (SNPs): V249I and T280M. These SNPs are correlated with other aged-related diseases such as atherosclerosis. We have reported an association of CX3CR1 SNP and AMD. In this study we examined CX3CR1 SNP frequencies and protein expression on archived sections of AMD and normal eyes. We microdissected non-retinal, peripheral retinal and macular cells from archived slides of eyes of AMD patients and normal subjects. CX3CR1 SNP typing was conducted by PCR and restriction fragment length polymorphism analysis. CX3CR1 transcripts from retinal cells were also measured using RT-PCR. CX3CR1 protein expression was evaluated using avidin-biotin complex immunohistochemistry. We successfully extracted DNA from 32/40 AMD cases and 2/2 normal eyes. Among the 32 AMD cases, 18 had neovascular AMD and 14 had non-neovascular AMD. The M280 allele was detected in 19/64 (32 cases x2) with a frequency of 29.7%, which was significantly higher as compared to the frequency in the normal population (11.2%). We detected CX3CR1 expression in the various retinal cells. CX3CR1 transcript and protein levels were diminished in the macular lesions. This study successfully analyzed CX3CR1 SNP and transcript expression in microdissected cells from archived paraffin fixed slides. Our data suggest that the M280 allele, a SNP resulting in aberrant CX3CR1 and CX3CL1 interaction, as well as lowered expression of macular CX3CR1, may contribute to the development of AMD.

Coadministration of swainsonine and doxorubicin attenuates doxorubicin-induced lethality in mice.

This study in mice concerns the protective effectiveness and mechanisms of action by which a coadministered regimen of an immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) protects against lethality induced by a single bolus intraperitoneal injection of LD50/14 doxorubicin. This swainsonine coadministration treatment regimen has been identified previously in our laboratory as the superior of the two optimal conditions for diminishing lethality in mice due to LD50/14 doxorubicin. The anthracycline, doxorubicin is a potent and widely used cancer chemotherapeutic agent whose clinical usefulness is limited by both a dose- and time-dependent cardiomyopathy. Specifically, mice were given simultaneous injections of swainsonine or its diluent buffer, phosphate buffered saline and LD50/14 doxorubicin on day 0, followed by twice daily injections of swainsonine or phosphate buffered saline up to day +9. The survival and well being of mice were monitored daily for 70 days, which may be considered equivalent to a period of 4 to 5 years in humans. This duration has a clinical implication with respect to the late manifestation of cardiotoxicity after doxorubicin treatment. We quantified the bone marrow cellularity of mice and performed in vitro progenitor cell assays to determine the effects of swainsonine coadministration treatment regimen on bone marrow competence after doxorubicin treatment. The effects of this regimen on doxorubicin-induced changes in heart morphology and on hematologic toxicities caused by doxorubicin were determined. This swainsonine coadministration treatment regimen significantly diminished doxorubicin-induced lethality and prolonged survival and well being of mice by preventing bone marrow pancytopenia from the start of therapy. It decreased bone marrow toxicity and facilitated its restoration. It accelerated restoration of blood hematocrit and total leukocyte levels. Also it facilitated the proliferation and differentiation of bone marrow pluripotent stem cells along the different paths to progenitor lineages, and significantly preserved the mouse heart morphology. These underlying mechanisms of action for the protection by swainsonine coadministration strongly suggest a potential role for swainsonine in high dose chemotherapy with doxorubicin.

Mice primed with swainsonine are protected against doxorubicin-induced lethality.

The anthracycline, doxorubicin is a potent cancer chemotherapeutic agent whose therapeutic usefulness is limited by both a dose- and time-dependent cardiomyopathy. We tested the ability of an immunomodulatory alkaloid swainsonine (8alphabeta-indolizidine-1alpha,2alpha,8beta-triol) to protect C57BL/6 mice against lethality within 70 days following a single bolus intraperitoneal injection of LD50/14 doxorubicin. Also, we sought the potential mechanisms responsible for this protection. This extended 70-day study in mice, which may be considered equivalent to a period of 4 to 5 years in humans, has clinical implication for delayed cardiotoxic sequela of therapy with high dose doxorubicin. Mice were pretreated with swainsonine or its diluent buffer, phosphate buffered saline for ten consecutive days prior to a single bolus intraperitoneal injection of a LD50/14 doxorubicin. We have previously defined this swainsonine pretreatment regimen as one of the two optimal conditions for swainsonine rescue of mice from death induced by LD50/14 doxorubicin. The survival and well being of groups of mice pretreated with swainsonine and phosphate buffered saline prior to LD50/14 doxorubicin, sham-treated and untreated were monitored daily for up to 70 days. The bone marrow cellularity of the mice were quantified, and in vitro progenitor cell assays were used to determine the effects of these treatment regimens on bone marrow competence following doxorubicin treatment. The effects of these treatment regimens on heart morphology and hematologic toxicities were also determined. This swainsonine pretreatment regimen significantly abrogated doxorubicin-induced lethality and prolonged survival of mice by facilitating restoration of bone marrow cellularity, accelerating restoration of blood hematocrit and total leukocyte levels, enhancing the proliferation and differentiation of bone marrow pluripotent stem cells along the different paths to progenitor lineages, and preserving the heart morphology. This study strongly suggests a potential role for swainsonine with doxorubicin in cancer chemotherapy.

Morphometric analysis of the macula in eyes with geographic atrophy due to age-related macular degeneration.

PURPOSE: To evaluate the extent of neural cell death in eyes with geographic atrophy (GA). METHODS: Ten eyes with GA and five age-matched control eyes were selected for morphometric analysis. The nuclei of the ganglion cell, inner nuclear, and outer nuclear layers were counted in contiguous 100-microm segments from 1,500 microm nasal to 1,500 microm temporal to the fovea. RESULTS: The outer nuclear layer was most severely attenuated in eyes with GA, demonstrating a 76.9% reduction relative to control eyes (P < 0.0001). A significant loss of ganglion cells (by 30.7%) was also observed (P = 0.0008). There was no significant difference in the inner nuclear layer cells (P = 0.30). Among the GA eyes, the nuclei in all three layers were significantly reduced in segments in which the retinal pigment epithelium was completely absent (P

Morphometric analysis of the macula in eyes with disciform age-related macular degeneration.

PURPOSE: To evaluate the extent of neural cell death in eyes with disciform age-related macular degeneration. METHODS: Six eyes with disciform degeneration at various stages and five age-matched control eyes were selected for morphometric analysis using digitized light microscopic images. Disciform scars were classified as subneurosensory retinal, subretinal pigment epithelial, or combined lesions. The nuclei of the ganglion cell, inner nuclear, and outer nuclear layers were counted in contiguous 100 microm segments spanning a distance from 1,500 microm nasal to 1,500 microm temporal to the fovea. RESULTS: The outer nuclear layer was most severely attenuated in eyes with disciform scars, demonstrating a 69.4% reduction in cell number relative to control eyes. A loss in retinal ganglion cells (by 7.3%) and an increase in inner nuclear layer cells (by 10%) were observed, but these changes were not significant. Photoreceptor loss was most pronounced when the disciform scar was not covered by the retinal pigment epithelium. CONCLUSION: The nuclei of the outer nuclear layer are significantly attenuated in eyes with disciform age-related macular degeneration, while the ganglion cell and inner nuclear layers are relatively preserved. These findings suggest that replacement of outer nuclear function, by either retinal transplantation or implantation of the intraocular retinal prosthesis, might be a feasible therapeutic option for patients with this condition.

Pathology and pathogenesis of retinal detachment.

Retinal detachment, separation of the neurosensory retina from the underlying retinal pigment epithelium, is a sight threatening condition that is considered one of the few ocular emergencies. The literature is enormously rich in studies that focused on different aspects of this disease process. Yet certain aspects remain largely unanswered. We briefly review major aspects of retinal detachment and discuss various important contributions in this field, focussing mainly on the pathogenesis of and predisposing factors to retinal detachment, and on the pathologic changes that occur following its development and following various surgical procedures currently used in its management.

Endogenous Serratia marcescens endophthalmitis with dark hypopyon: case report and review.

A case of endogenous Serratia marcescens endophthalmitis in a patient with diabetes, end-stage renal disease, and an indwelling venous catheter is reported. The patient presented with a tan hypopyon and elevated intraocular pressure. Diagnosis was established by positive blood, vitreous, conjunctival, and catheter tip cultures. After a deteriorating course the eye was enucleated. Gross and histopathologic examination revealed the presence of a dark hypopyon with iris necrosis and pigment dispersion and possible spontaneous globe perforation. This is the eleventh reported case of endogenous Serratia endophthalmitis. Previous association of a pink hypopyon and of pigmented vitreous fluid and Serratia endophthalmitis has been reported. This is the first case of dark hypopyon in endogenous Serratia marcescens endophthalmitis reported in the medical literature. Previous entities associated with dark hypopyon have been limited to intraocular melanoma and Listeria monocytogenes endophthalmitis. Dark hypopyon in the appropriate clinical setting may be useful in aiding diagnostic and therapeutic decisions.

The role of apoptosis in the pathogenesis of Fuchs endothelial dystrophy of the cornea.

OBJECTIVE: To investigate the potential role of apoptosis in the pathogenesis of Fuchs endothelial dystrophy of the cornea. METHODS: Twenty-one corneal buttons from patients with Fuchs dystrophy and 15 control corneas were studied. Apoptosis was assessed by the in situ end-labeling of double-stranded DNA breaks, and by immunohistochemical characterization of cellular markers associated with apoptosis (Fas, FasL, Bcl-2, and Bax). Expression of Bcl-2 and Bax mRNA in the corneal stroma and endothelium was separately analyzed by a semiquantitative reverse transcriptase polymerase chain reaction. Furthermore, cultivated keratocytes generated from diseased corneal buttons and donor rims were exposed to camptothecin, an apoptotic inducer, for 6 and 24 hours. They were then examined for protein and messenger RNA (mRNA) expression of apoptotic regulatory molecules. RESULTS: DNA fragmentation was seen in the epithelium, stroma, and endothelium in 6 of 7 corneas with Fuchs dystrophy. A statistically significant difference was identified in the expression of Bax and its mRNA in the stroma, but not in the endothelium of Fuchs dystrophy corneas. Following exposure to camptothecin, keratocytes from patients with Fuchs dystrophy responded with an increased level of Bax and a low level of Bcl-2. This trend was distinctively different from the response of normal keratocytes. CONCLUSIONS: The evidence in this study points to a disease-related disturbance in the regulation of apoptosis in Fuchs dystrophy. Our findings suggest that excessive apoptosis may be an important mechanism in the pathogenesis of Fuchs dystrophy.

Clinicopathologic studies of age-related macular degeneration with classic subfoveal choroidal neovascularization treated with photodynamic therapy.

BACKGROUND: Photodynamic therapy (PDT) is a relatively new modality that is currently under clinical and experimental evaluation for treatment of subfoveal choroidal neovascularization (CNV). The authors report the case of an 82-year-old woman who underwent verteporfin-mediated PDT for classic subfoveal CNV. Fluorescein angiography performed 2 weeks after treatment disclosed reduction of the initial area of neovascularization and leakage by approximately 60%. Three weeks after PDT, however, the area of leakage was almost the same size as that before treatment. The patient underwent submacular membranectomy almost 4 weeks after treatment. The authors describe the ultrastructural vascular changes after PDT and a clinicopathologic study of classic CNV. METHODS: The submacular membrane was studied by light and electron microscopy and immunohistochemical techniques. RESULTS: Ultrastructural examination of the peripheral vessels showed evidence of endothelial cell degeneration with platelet aggregation and thrombus formation. Occasional occluded vessels were surrounded by macrophages, a phenomenon previously reported to describe the process of resorption of such blood vessels. The vessels in the center of the membrane were unremarkable. CONCLUSION: Photodynamic therapy causes endothelial cell damage, thrombus formation, and vascular occlusion of classic CNV in age-related macular degeneration.

Vitrectomy for retained lens material after cataract extraction: the relationship between histopathologic findings and the time of vitreous surgery.

PURPOSE: To report the histopathologic features of vitreous specimens obtained after vitrectomy for retained lens material and to correlate these findings with the time after cataract surgery at which vitreous surgery was performed. DESIGN: Observational case series. PARTICIPANTS: All 135 patients who had vitreous specimens submitted to the pathology laboratory after vitreous surgery for retained lens material after elective senile cataract surgery. METHODS: A review of histopathologic reports of 135 consecutive vitreous specimens that were obtained during vitreous surgery for retained lens material after surgery for senile cataract. MAIN OUTCOME MEASURES: Identification of lens material, macrophages, phacolytic cells, and giant cells in the vitrectomy specimens. RESULTS: Lens material was observed in 100% of cases. Macrophages without ingested lens material were present in 48% (65 of 135), phacolytic cells in 24% (32 of 135), and a mixture of neutrophils and multinucleated giant cells in 8% (11 of 135). No cells were discovered in eyes in which vitrectomy was performed within 3 days of cataract extraction. The percentages of eyes with macrophages increased from 35% when vitrectomy was performed 4 to 7 days after cataract surgery to 80% when this time interval was 61 to 90 days. CONCLUSION: Lens-induced inflammatory reaction seems to increase with time that retained lens material remains in the eye.

Interphase fluorescence in situ hybridisation of the X and Y chromosomes in the human eye.

AIM: To determine the sex of individual cells in paraffin sections of the human eye by fluorescence in situ hybridisation (FISH) of the X and Y chromosomes. METHODS: The authors developed a protocol for FISH of the X and Y chromosomes in paraffin sections of human eyes. RESULTS: In all the specimens that had been fixed in 10% formalin and with a fixation time of up to 3 days sex determination of individual cells was achieved. The percentage of cells with clearly identifiable signals was up to 98% for corneal epithelium, keratocytes, corneal endothelium, trabecular meshwork, lens epithelium, retina, and optic nerve. CONCLUSIONS: FISH allows the determination of the sex of single cells in paraffin sections of human eyes without destruction of the tissue structure. Its main application is the histological analysis of sex mismatched corneal, RPE, or neuroretinal transplants to distinguish host and donor cells.

Retinal pigment epithelial cell transplantation after subfoveal membranectomy in age-related macular degeneration: clinicopathologic correlation.

PURPOSE: To report the histopathology after retinal pigment epithelial cell transplantation and subfoveal membranectomy in age-related macular degeneration. METHODS: An 85-year-old white woman with bilateral choroidal neovascularization underwent subfoveal membranectomy combined with transplantation of a sheet of human adult retinal pigment epithelium (retinal pigment epithelium) under the foveal center in the right eye. The patient was immunosuppressed postoperatively with prednisone, cyclosporine, and azathioprine. The patient died from congestive heart failure 114 days after surgery. RESULTS: A patch of hyperpigmentation was visible at the transplant site under the foveola after surgery. Mound-like clusters of individual round, large densely pigmented cells were present in the subretinal space and outer retina in this area. There was loss of the photoreceptor outer segments and native retinal pigment epithelium in the center of the transplant bed, with disruption of the outer nuclear layer predominantly over regions of multilayered pigmented cells. Cystic spaces were present in the inner and outer retina. A residual intra-Bruchs membrane component of the original choroidal neovascular complex was present under the transplant site. CONCLUSIONS: The transplant site contained clusters of round, pigmented cells that did not form a uniform monolayer in most areas. The morphology at the transplant site is consistent with the lack of visual improvement seen after surgery in this patient.

Sympathetic ophthalmia associated with pars plana vitrectomy without antecedent penetrating trauma.

PURPOSE: To evaluate, describe, and categorize the clinical presentation, clinical course, histopathology, and response to therapy in patients without a history of penetrating ocular trauma who developed sympathetic ophthalmia following pars plana vitrectomy. METHODS: The records of patients without a history of trauma who underwent pars plana vitrectomy and developed sympathetic ophthalmia were retrospectively reviewed. Cases were analyzed with respect to clinical presentation, fluorescein angiographic findings, anatomic and visual outcomes, histopathology, and response to therapy. RESULTS: Eight eyes were identified. The median age at presentation was 55 years, with a range of 14 to 62 years. The time from vitrectomy to diagnosis of sympathetic ophthalmia ranged from 2 months to greater than 2 years, with a median of 7 months. Six of eight patients (75%) presented with anterior chamber reaction. All eight patients presented with a vitreous inflammatory response. The optic nerve was inflamed clinically or angiographically in four of eight cases (50%). Small yellow-white sub-retinal pigment epithelial deposits were present in four of eight cases (50%). Two eyes had lesions characterized as multifocal choroiditis. One eye had larger yellow placoid-like lesions. One eye presented with vitritis but no retinal lesions. Subretinal choroidal neovascularization was noted in the inciting eye of one patient. Vision improved in the sympathizing eye with immunosuppressive therapy in five of eight cases (62.5%). CONCLUSIONS: Sympathetic ophthalmia can be seen following pars plana vitrectomy in patients without penetrating injuries or a history of trauma. Indeed, it may be seen after successful vitrectomy for retinal detachment. Diverse clinical presentations are possible, and persistent or atypical uveitis following vitrectomy should alert the surgeon to the development of sympathetic ophthalmia.

Characterization of the CD154-positive and CD40-positive cellular subsets required for pathogenesis in retrovirus-induced murine immunodeficiency.

Genetically susceptible C57BL/6 (B6) mice that are infected with the LP-BM5 isolate of murine retroviruses develop profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, terminal B-cell lymphomas, and an immunodeficiency state bearing many similarities to the pathologies seen in AIDS. Because of these similarities, this syndrome has been called murine AIDS (MAIDS). We have previously shown that CD154 (CD40 ligand)-CD40 molecular interactions are required both for the initiation and progression of MAIDS. Thus, in vivo anti-CD154 monoclonal antibody (MAb) treatment inhibited MAIDS symptoms in LP-BM5-infected wild-type mice when either a short course of anti-CD154 MAb treatment was started on the day of infection or a course was initiated 3 to 4 weeks after LP-BM5 administration, after disease was established. Here, we further characterize this required CD154-CD40 interaction by a series of adoptive transfer experiments designed to elucidate which cellular subsets must express CD154 or CD40 for LP-BM5 to induce MAIDS. Specifically with regard to CD154 expression, MAIDS-insusceptible B6 nude mice reconstituted with highly purified CD4+ T cells from wild-type, but not from CD154 knockout, B6 donors displayed clear MAIDS after LP-BM5 infection. In contrast, nude B6 recipients that received CD8+ T cells from wild-type B6 donors did not develop MAIDS after LP-BM5 infection. B6 CD40 knockout mice, which are also relatively resistant to LP-BM5-induced MAIDS, became susceptible to LP-BM5-induced disease after reconstitution with highly purified wild-type B cells but not after receiving purified wild-type dendritic cells (DC) or a combined CD40+ population composed of DC and macrophages obtained from B6 SCID mouse donors. Based on these and other experiments, we thus conclude that the cellular basis for the requirement for CD154-CD40 interactions for MAIDS induction and progression can be accounted for by CD154 expression on CD4+ T cells and CD40 expression on B cells.

Human immunodeficiency virus-specific and CD3-redirected cytotoxic T lymphocyte activity in the human female reproductive tract: lack of correlation between mucosa and peripheral blood.

CD8(+) T cell phenotype and function were assessed in the female reproductive tracts (FRTs) of 3 human immunodeficiency virus (HIV)-positive patients who had undergone hysterectomy. FRT cytotoxic T lymphocyte (CTL) lytic activity from 1 patient (patient 872) was detected by using CD3-dependent redirected-lysis assay and HIV-specific assay, concomitant with the presence of CD8(+) cells. In contrast, samples from the 2 other HIV-positive patients (patients 1356 and 1364), who also were asymptomatic for HIV-associated illnesses, demonstrated no CTL activity in any solid tissue tested by either assay, despite activity by autologous peripheral blood mononuclear cells (PBMC). This absence of CTL activity was correlated with a relative absence of CD8(+) cells in the FRT, whereas CD8(+) cells were present in PBMC. Thus, CTL activity in PBMC may fail to correlate with mucosal activity. The finding of CTL activity in the FRT of patient 872 represents the first description of CTL in upper and lower FRT tissues of an HIV-positive woman.

Clinicopathologic findings in eyes with retained perfluoro-n-octane liquid.

OBJECTIVE: To describe the clinical and histopathologic findings in five eyes with retained perfluoro-n-octane (PFO) liquid after retinal reattachment surgery. DESIGN: Retrospective, noncomparative, clinicopathologic case series. PARTICIPANTS: Surgical specimens from five eyes were studied. METHODS: Surgical specimens from eyes with prior intraoperative PFO use submitted to the W. Richard Green Eye Pathology Laboratory at the Wilmer Ophthalmological Institute were identified and reviewed. MAIN OUTCOME MEASURES: Histopathologic analysis and energy dispersive spectroscopy identified intracellular vacuoles containing PFO. RESULTS: Five cases were identified. Three specimens were obtained at the time of further surgery for recurrent retinal detachment; one at repeat penetrating keratoplasty, and one at removal of retained PFO. Each eye had macroscopic white flake-like material on intraocular structures noted before or during surgery. Histopathologic analysis disclosed an inflammatory response featuring macrophages with intracellular vacuoles containing PFO. Removal of the PFO in all five eyes combined with repeat retinal reattachment surgery in three eyes resulted in resolution of the inflammatory response. CONCLUSIONS: Retention of PFO after surgery elicits an inflammatory response. We suspect that young patients, those with considerable residual vitreous gel, and eyes with larger amounts of retained PFO may be at higher risk for this complication.

Pneumocystis carinii granuloma of the optic nerve: a histopathologic case report.

A 30-year-old man under treatment for acute leukemia died of Candida sepsis and Pneumocystis carinii pneumonia. A small granuloma containing Pneumocystis carinii was found in one optic nerve.