Plasma of Acute Lymphoblastic Leukemia Patients React to the Culture of a Mycovirus Containing Aspergillus flavus.

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and is also seen in adults. Currently, no plasma-based test for the detection of ALL is available. We have cultured the home of a patient with ALL and isolated a mycovirus containing Aspergillus flavus. This culture was subjected to electron microscopy, purification, and mass spectrometry. Using enzyme-linked immunosorbent assay technique, plasma of patients with ALL and long-term survivors of this disease were tested for antibodies, utilizing supernatant of the culture of this organism. The results were compared with 3 groups of controls, including healthy individuals, patients with sickle cell disease, and solid tumors. Using electron microscopy, the isolated A. flavus contained mycovirus particles. In chemical analysis, this organism did not produce any aflatoxin. Using an enzyme-linked immunosorbent assay technique, the supernatant of the culture of the mycovirus containing A. flavus could differentiate ALL patients from each group of controls (P<0.001). These studies provide a new technique for the detection of ALL and may add information for future research regarding leukemogenesis.

Exploration of molecular factors impairing superoxide dismutase isoforms activity in human senile cataractous lenses.

PURPOSE: To explore different molecular factors impairing the activities of superoxide dismutase (SOD) isoforms in senile cataractous lenses. METHODS: Enzyme activity of SOD isoforms, levels of their corresponding cofactors copper (Cu), manganese (Mn), zinc (Zn), and expression of mRNA transcripts and proteins were determined in the lenses of human subjects with and without cataract. DNA from lens epithelium (LE) and peripheral blood was isolated. Polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) followed by sequencing was carried out to screen somatic mutations. The impact of intronic insertion/deletion (INDEL) variations on the splicing process and on the resultant transcript was evaluated. Genotyping of IVS4+42delG polymorphism of SOD1 gene was done by PCR-restriction fragment length polymorphism (RFLP). RESULTS: A significant decrease in Cu/Zn- and Mn-SOD activity (P < 0.001) and in Cu/Zn-SOD transcript (P < 0.001) and its protein (P < 0.05) were found in cataractous lenses. No significant change in the level of copper (P = 0.36) and an increase in the level of manganese (P = 0.01) and zinc (P = 0.02) were observed in cataractous lenses. A significant positive correlation between the level of Cu/Zn-SOD activity and the levels of Cu (P = 0.003) and Zn (P = 0.005) was found in the cataractous lenses. DNA sequencing revealed three intronic INDEL variations in exon4 of SOD1 gene. Splice-junction analysis showed the potential of IVS4+42delG in creating a new cryptic acceptor site. If it is involved in alternate splicing, it could result in generation of SOD1 mRNA transcripts lacking exon4 region. Transcript analysis revealed the presence of complete SOD1 mRNA transcripts. Genotyping revealed the presence of IVS4+42delG polymorphism in all subjects. CONCLUSIONS: The decrease in the activity of SOD1 isoform in cataractous lenses was associated with the decreased level of mRNA transcripts and their protein expression and was not associated with either modulation in the level of enzyme cofactors or with INDEL variations.

Estrogen mediated protection of cytoskeleton against oxidative stress.

BACKGROUND & OBJECTIVES: Cytoskeletal proteins are deregulated during oxidative stress and cataract formation. However, estrogen which protects against cataract formation and harmful effects of oxidative stress has not been tested on the cytoskeleton of lens epithelial cells (LECs). The current study was undertaken to assess if the protection rendered to LECs by estrogen was mediated by preserving the cytoskeletal proteins. METHODS: Oxidative stress was induced by 50 µM of H 2 O 2 in cultured goat LECs (gLECs) and effect of 1 µM 17ß-estradiol (E 2 ) was tested. After treatment, morphological analysis of cells was carried out using haematoxylin-eosin staining and cell density was also quantified. Cell viability was determined using Hoechst (Ho), YO-Pro (YP) and propidium iodide (PI). F-actin and vimentin were localized using phalloidin and anti-vimentin antibody, respectively, and viewed under fluorescence microscopy. Vimentin was further analysed at protein level by Western blotting. RESULTS: H 2 O 2 led to increased condensation of nucleus, cell death and apoptosis but these were prevented with pre- and co-treatment of E 2 with increase in cell viability (P<0.001). E 2 also prevented H 2 O 2 mediated depolymerization of cytoskeleton but was not able to reverse the changes when given after induction of oxidative stress. INTERPRETATION & CONCLUSIONS: Our findings showed that E 2 helped in preventing deteriorating effect of H 2 O 2 , inhibited cell death, apoptosis and depolymerisation of cytoskeletal proteins in LECs. However, the exact mechanism by which estrogen renders this protection to cytoskeleton of lens epithelial cells remains to be determined.

Histomorphological and immunofluorescence evaluation of clear corneal incisions after microcoaxial phacoemulsification with 2.2 mm and 1.8 mm systems.

PURPOSE: To compare changes in the incision's histomorphology and denaturation of collagen I in rabbit eyes having microcoaxial phacoemulsification through 2.2 mm and 1.8 mm incision-compatible systems. DESIGN: Randomized experimental trial. SETTING: Iladevi Cataract & IOL Research Centre, Ahmedabad, India. METHODS: Thirty rabbit eyes were randomized into Group 1 (microcoaxial phacoemulsification through 2.2 mm incisions using Infiniti system [torsional ultrasound]) and Group 2 (microcoaxial phacoemulsification through 1.8 mm incisions using Stellaris system [longitudinal ultrasound]). Each group was then divided into 3 subgroups of 5 eyes each based on 1 of the 3 intervention options: phacoemulsification only, intraocular lens (IOL) insertion only, and phacoemulsification with IOL insertion. Left eyes were randomized for microcoaxial phacoemulsification, and right eyes were treated as controls. RESULTS: After phacoemulsification, eyes in Group 1 showed loss of epithelium at the roof of the incisions and Descemet membrane detachment at the floor of the incisions. These findings did not change after IOL insertion. After phacoemulsification, eyes in Group 2 showed loss of epithelium, but Descemet membrane remained attached. There was a longitudinal split in the incision's stroma in the direction of internal entry. The stromal damage increased after IOL implantation. Immunofluorescence studies showed no obvious irregularities in the arrangement of collagen I in either group. A dot blot analysis showed significant denaturation of collagen I in Group 2. CONCLUSION: The histomorphology of the 2.2 mm system incision showed localized Descemet membrane detachment and endothelial cell loss. The 1.8 mm system incision showed exaggerated stromal damage after IOL insertion.

Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells.

Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (n=52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (n=11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student's t-test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.

Comparison of the effect of torsional and microburst longitudinal ultrasound on clear corneal incisions during phacoemulsification.

PURPOSE: To compare incision integrity after clear corneal microcoaxial phacoemulsification using longitudinal and torsional ultrasound (US). SETTING: Iladevi Cataract & IOL Research Centre, Ahmedabad, India. DESIGN: Prospective randomized experimental clinical trial. METHODS: Part 1 comprised an experimental study of rabbit eyes. Group 1 received longitudinal US. Group 2 received torsional US. The right eye of each rabbit served as a control. Samples were processed for histomorphology and collagen I denaturation by immunofluorescence. Part 2 comprised a clinical trial of patients. Group 1 received torsional US. Group 2 received longitudinal US. At the end of surgery, trypan blue 0.0125% was instilled. After 2 minutes, 0.1 mL of aqueous was aspirated and its optical density measured. RESULTS: In part 1, incision histomorphology was comparable in both modalities. Collagen denaturation tests (immunofluorescence, dot blot analysis) showed no irregularity in collagen arrangement in either group. In Group 2, Descemet membrane was detached and endothelial cells were minimal at the roof of the incision. In part 2, trypan blue ingress into the anterior chamber was significantly greater in Group 1 than in Group 2 (mean 3.40 + 0.6 log units versus and 3.77 + 0.82 log units) (P<.007). CONCLUSIONS: Incision histomorphology in the torsional group showed minimal Descemet membrane detachment and minimal endothelial cell loss at the roof of the incision. Minimal ingress of trypan blue into the anterior chamber was observed with torsional US, indicating better wound integrity than with longitudinal US. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Matrix metalloproteinase-2 and -9 activities in the human lens epithelial cells and serum of steroid induced posterior subcapsular cataracts.

PURPOSE: To evaluate the level of matrix metalloproteinase (MMP)-2 and MMP-9 activities in patients with steroid induced posterior subcapsular cataract (PSC). METHODS: This prospective, observational study comprised of 156 patients having either steroid induced PSC (n=50) or non-steroidal PSC (n=106) were performed to evaluate the level of MMP-2 and MMP-9 activities in the lens epithelial cells (LECs) and the serum. Anterior lens capsules harboring LECs were obtained during phacoemulsification and peripheral blood was collected from patients before administration of anesthesia. Serum was separated by centrifugation at 10,000× g for 15 min at 4 °C. The LECs and serum samples were processed to analyze MMP-2 and MMP-9 activities using succinylated gelatin assay. Quantitative real time-PCR (qRT-PCR) was performed to determine the mRNA levels of MMP-2 and MMP-9 in LECs. The mRNA levels were expressed as a ratio, using the delta-delta method for comparing the relative expression results between cases with steroid induced PSC and cases with non-steroidal PSC. MMP-2 and MMP-9 levels were also compared in the two groups using immunolocalization. RESULTS: The level of MMP-2 and MMP-9 activity was found to be high in LECs and serum of cases with steroid induced PSC. Further in all steroid induced cases, a 1.4 fold increase was observed in MMP-2 activity in LECs and a 1.4 fold increase in MMP-9 activity in the serum. Both qRT-PCR and immunolocalization showed increased expression of MMP-2 and MMP-9 activity. CONCLUSIONS: MMP-2 and MMP-9 activity in both LECs and serum was significantly higher in cases with steroid induced PSC. The possible use of MMP-9 as a non-invasive biomarker in ascertaining the presence of steroid induced PSC should be evaluated using a larger sample size.

Fungal scleral keratitis caused by Phomopsis phoenicicola.

We report a case of scleral keratitis caused by Phomopsis phoenicicola. Pterygium surgery was a predisposing factor, and the patient was treated with natamycin and fluconazole eye drops and oral fluconazole. The fungus was identified by sequencing of the internal transcribed spacer (ITS) region of the fungal ribosomal DNA (rDNA) locus and confirmed on the basis of its typical pycnidia and conidia.