Effect of Extended Release Steroid Implants on the Contralateral Eye.

PURPOSE: To investigate the contralateral effect of extended release steroid implants on cystoid macular edema (CME). METHODS: Retrospective study of patients with bilateral CME receiving intravitreal injections of long-acting intravitreal corticosteroid implants in one eye. Changes in CME and central subfield thickness (CST) in the contralateral eye on optical coherence tomography (OCT) were compared to an untreated control group. The main outcome measures were the change in central subfield thickness (CST) and the change in the macular volume. RESULTS: Thirteen study patients and 14 controls were included in the study. There was no difference in the baseline LogMAR visual acuity (0.32 ± 0.35 vs 0.43 ± 0.26, p = 0.37) or the baseline central subfield thickness (341.4 ± 76.6 vs 296.5 ± 65.0 µm, p = 0.12) between groups. In the treatment group CST remained stable in 92.3% of the patients. Of the controls, CST worsened in 21.4% and remained stable in 78.6%. The mean change in CST (6.3 ± 30.3 vs. 27.5 ± 66.1 µm, p = 0.2) and the mean change in macular volume (0.08 ± 0.34 vs. -0.05 ± 0.21 mm3, P = 0.8) were not statistically different between the treatment group and control group. In the post-hoc analysis restricting the treatment group to patients who had not received intravitreal injections in the study eye within 6 months, CST decrement was not statistically significant (p = 0.11). CONCLUSION: In this study there was no statistically significant effect on CME of contralateral intravitreal corticosteroid implants.

Long-Term Follow-Up of Outer Retinal Layers in Patients with Epiretinal Membranes.

PURPOSE: This study aimed to investigate the long-term effect of observed epiretinal membranes on the outer retinal layers and visual acuity. METHODS: It is a retrospective observational study. Subjects with an epiretinal membrane and consecutive optical coherence tomography scans were followed for changes in visual acuity, central macular thickness, ellipsoid zone loss, and outer foveal thickness (OFT). RESULTS: The study consisted of 24 eyes of 22 patients, with a mean follow-up of 5 ± 1.6 years. The mean visual acuity was slightly worse at the last follow-up (0.22 ± 0.36 LogMAR [20/33] vs. 0.27 ± 0.36 LogMAR [20/36], p = 0.05). Ellipsoid zone loss was found in 37.5% of eyes. Vision loss was associated with initial size of ellipsoid disruption (p = 0.048) and age (p = 0.027). A decrease in OFT was associated with an initially larger zone of ellipsoid disruption (p = 0.006) and an initially thicker OFT (p = 0.011). An epiretinal membrane associated with vitreomacular adhesion within 1,000 µm of the foveal center at baseline was associated with ellipsoid zone loss (p = 0.012) but not with a change in visual acuity. CONCLUSIONS: Ellipsoid zone changes were common in this study and tended to enlarge over time. Epiretinal membranes associated with vitreomacular adhesion within 1,000 µm of the foveal center may be a risk factor for ellipsoid zone loss.

UK consensus statement on the use of plerixafor to facilitate autologous peripheral blood stem cell collection to support high-dose chemoradiotherapy for patients with malignancy.

Plerixafor is a CXC chemokine receptor (CXCR4) antagonist that mobilizes stem cells in the peripheral blood. It is indicated (in combination with granulocyte-colony stimulating factor [G-CSF]) to enhance the harvest of adequate quantities of cluster differentiation (CD) 34+ cells for autologous transplantation in patients with lymphoma or multiple myeloma whose cells mobilize poorly. Strategies for use include delayed re-mobilization after a failed mobilization attempt with G-CSF, and rescue or pre-emptive mobilization in patients in whom mobilization with G-CSF is likely to fail. Pre-emptive use has the advantage that it avoids the need to re-schedule the transplant procedure, with its attendant inconvenience, quality-of-life issues for the patient and cost of additional admissions to the transplant unit. UK experience from 2 major centers suggests that pre-emptive plerixafor is associated with an incremental drug cost of less than £2000 when averaged over all patients undergoing peripheral blood stem cell (PBSC) transplant. A CD34+ cell count of <15 µl-1 at the time of recovery after chemomobilization or after four days of G-CSF treatment, or an apheresis yield of <1 × 106 CD34+ cells/kg on the first day of apheresis, could be used to predict the need for pre-emptive plerixafor.

The role of extracorporeal photopheresis in the management of cutaneous T-cell lymphoma, graft-versus-host disease and organ transplant rejection: a consensus statement update from the UK Photopheresis Society.

Extracorporeal photopheresis (ECP) has been used for over 35 years in the treatment of erythrodermic cutaneous T-cell lymphoma (CTCL) and over 20 years for chronic and acute graft-versus-host disease (GvHD) and solid organ transplant rejection. ECP for CTCL and GvHD is available at specialised centres across the UK. The lack of prospective randomised trials in ECP led to the development of UK Consensus Statements for patient selection, treatment schedules, monitoring protocols and patient assessment criteria for ECP. The recent literature has been reviewed and considered when writing this update. Most notably, the national transition from the UVAR XTS® machine to the new CELLEX machine for ECP with dual access and a shorter treatment time has led to relevant changes in these schedules. This consensus statement updates the previous statement from 2007 on the treatment of CTCL and GvHD with ECP using evidence based medicine and best medical practise and includes guidelines for both children and adults.

Efficacy and safety of eculizumab in atypical hemolytic uremic syndrome from 2-year extensions of phase 2 studies.

Atypical hemolytic uremic syndrome (aHUS) is a rare, possibly life-threatening disease characterized by platelet activation, hemolysis and thrombotic microangiopathy (TMA) leading to renal and other end-organ damage. We originally conducted two phase 2 studies (26 weeks and 1 year) evaluating eculizumab, a terminal complement inhibitor, in patients with progressing TMA (trial 1) and those with long duration of aHUS and chronic kidney disease (trial 2). The current analysis assessed outcomes after 2 years (median eculizumab exposure 100 and 114 weeks, respectively). At all scheduled time points, eculizumab inhibited terminal complement activity. In trial 1 with 17 patients, the platelet count was significantly improved from baseline, and hematologic normalization was achieved in 13 patients at week 26, and in 15 patients at both 1 and 2 years. The estimated glomerular filtration rate (eGFR) was significantly improved compared with baseline and year 1. In trial 2 with 20 patients, TMA event-free status was achieved by 16 patients at week 26, 17 patients at year 1, and 19 patients at year 2. Criteria for hematologic normalization were met by 18 patients at each time point. Improvement of 15 ml/min per 1.73 m(2) or more in eGFR was achieved by 1 patient at week 26, 3 patients at 1 year, and 8 patients at 2 years. The mean change in eGFR was not significant compared with baseline, week 26, or year 1. Eculizumab was well tolerated, with no new safety concerns or meningococcal infections. Thus, a 2-year analysis found that the earlier clinical benefits achieved by eculizumab treatment of aHUS were maintained at 2 years of follow-up.

DNA-mounted self-assembly: new approaches for genomic analysis and SNP detection.

This article presents an overview of new emerging approaches for nucleic acid detection via hybridization techniques that can potentially be applied to genomic analysis and SNP identification in clinical diagnostics. Despite the availability of a diverse variety of SNP genotyping technologies on the diagnostic market, none has truly succeeded in dominating its competitors thus far. Having been designed for specific diagnostic purposes or clinical applications, each of the existing bio-assay systems (briefly outlined here) is usually limited to a relatively narrow aspect or format of nucleic acid detection, and thus cannot entirely satisfy all the varieties of commercial requirements and clinical demands. This drives the diagnostic sector to pursue novel, cost-effective approaches to ensure rapid and reliable identification of pathogenic or hereditary human diseases. Hence, the purpose of this review is to highlight some new strategic directions in DNA detection technologies in order to inspire development of novel molecular diagnostic tools and bio-assay systems with superior reliability, reproducibility, robustness, accuracy and sensitivity at lower assay cost. One approach to improving the sensitivity of an assay to confidently discriminate between single point mutations is based on the use of target assembled, split-probe systems, which constitutes the main focus of this review.

Plerixafor for autologous peripheral blood stem cell mobilization in patients previously treated with fludarabine or lenalidomide.

Fludarabine and lenalidomide are essential drugs in the front-line treatment of non-Hodgkin lymphoma (NHL) and multiple myeloma (MM), respectively. Data suggests that fludarabine and lenalidomide therapy may have a deleterious effect on stem cell mobilization. In the European compassionate use program, 48 patients (median age 57 years) previously treated with fludarabine (median 5 cycles; range: 1-7 cycles) were given plerixafor plus granulocyte colony-stimulating factor (G-CSF) for remobilization following a primary mobilization attempt. The overall median number of CD34+ cells collected was 2.3 × 10(6)/kg (range: 0.3-13.4). The minimum required number of CD34+ cells (≥2.0 × 10(6)/kg) was collected from 58% of patients in a median of 2 days. Thirty-five patients (median age = 57 years) previously treated with lenalidomide (median 5 cycles; range: 1-10 cycles) were given plerixafor plus G-CSF for remobilization. The overall median number of CD34+ cells collected was 3.4 × 10(6)/kg (range: 1.1-14.8). The minimum required number of CD34+ cells (≥2.0 × 10(6) per kg) was collected from 69% of patients in a median of 2 days. In conclusion, salvage mobilization with plerixafor plus G-CSF is successful in the majority of patients with MM previously treated with lenalidomide. In fludarabine-exposed patients, only 58% of patients will achieve successful salvage mobilization with plerixafor plus G-CSF, suggesting the need for novel mobilization regimens algorithms in this subgroup of patients.

European data on stem cell mobilization with plerixafor in patients with nonhematologic diseases: an analysis of the European consortium of stem cell mobilization.

BACKGROUND: Plerixafor with granulocyte-colony-stimulating factor (G-CSF) has been shown to enhance stem cell mobilization in patients with multiple myeloma and lymphoma with previous mobilization failure. In this European named patient program we report the experience in insufficiently mobilizing patients diagnosed with nonhematologic diseases. STUDY DESIGN AND METHODS: Thirty-three patients with germ cell tumor (n=11), Ewing sarcoma (n=6), Wiscott-Aldrich disease (n=5), neuroblastoma (n=4), and other nonhematologic diseases (n=7) were included in the study. Plerixafor was limited to patients with previous or current stem cell mobilization failure and given after 4 days of G-CSF (n=21) or after chemotherapy and G-CSF (n=12) in patients who mobilized poorly. RESULTS: Overall, 28 (85%) patients succeeded in collecting at least 2×10(6)/kg body weight (b.w.) CD34+ cells (median, 5.0×10(6)/kg b.w. CD34+ cells; range, 2.0×10(6)-29.5×10(6)/kg b.w. CD34+ cells), and five (15%) patients collected a median of 1.5×10(6)/kg b.w. CD34+ cells (range, 0.9×10(6)-1.8×10(6)/kg b.w. CD34+ cells). Nineteen patients proceeded to transplantation. The median dose of CD34+ cells infused was 3.3×10(6)/kg b.w. (range, 2.3×10(6)-6.7×10(6)/kg b.w. CD34+ cells). The median numbers of days to neutrophil and platelet engraftment were 11 (range, 9-12) and 15 (range, 10-25) days, respectively. CONCLUSION: These data emphasize the role of plerixafor in combination with G-CSF or chemotherapy and G-CSF as an effective mobilization regimen with the potential of successful stem cell collection. Accordingly, plerixafor seems to be safe and effective in patients with nonhematologic diseases. Larger prospective studies are warranted to further assess its use in these patients.

1H NMR spectroscopic identification of protonable sites in cryptolepines with C-11 substituents containing two amino functionalities.

Knowledge of protonable sites and acid dissociation constants of cryptolepine derivatives having C-11 substituents containing two amino functionalities is of great importance to the understanding of the mechanism of their antimalarial action, which may contribute to their further development as drug candidates. In this work, we applied (1)H NMR titration to investigate the acid-base characteristics of these polyprotic compounds in the pH range 3-13. We identified three acid-base equilibria with most acid dissociation constants (pK(a)*) being greater than 10.5, which prevented us from using the potentiometric method. Overall, (1)H NMR titration was sensitive and suitable for the determination of pK(a) values for these drug leads.

Bilateral neuroretinits and exudative retinal detachment with multifocal subretinal deposits secondary to Bartonella henselae infection.

PURPOSE: to describe a case of bilateral neuroretinitis with bullous retinal detachment and multiple subretinal lesions, in a 10-year-old immunocompetent girl. OBSERVATIONS: A broad workup for infectious, inflammatory and masquerade etiologies was done for the patient, resulting in positive IgM and IgG for Bartonella henselae. The patient demonstrated improvement in the visual acuity, and rapid resolution of the retinal detachment and subretinal lesions in both eyes in response to systemic rifampin, doxycycline and corticosteroids. CONCLUSIONS AND IMPORTANCE: Bartonella henselae neuroretinitis may present as an acute form of bullous retinal detachment with multiple subretinal lesions and markedly reduced vision. Significant visual improvement may occur with prompt treatment with a combination of systemic antibiotics and corticosteroids.

Biophysical and X-ray crystallographic analysis of Mps1 kinase inhibitor complexes.

The dual-specificity protein kinase monopolar spindle 1 (Mps1) is a central component of the mitotic spindle assembly checkpoint (SAC), a sensing mechanism that prevents anaphase until all chromosomes are bioriented on the metaphase plate. Partial depletion of Mps1 protein levels sensitizes transformed, but not untransformed, human cells to therapeutic doses of the anticancer agent Taxol, making it an attractive novel therapeutic cancer target. We have previously determined the X-ray structure of the catalytic domain of human Mps1 in complex with the anthrapyrazolone kinase inhibitor SP600125. In order to validate distinct inhibitors that target this enzyme and improve our understanding of nucleotide binding site architecture, we now report a biophysical and structural evaluation of the Mps1 catalytic domain in the presence of ATP and the aspecific model kinase inhibitor staurosporine. Collective in silico, enzymatic, and fluorescent screens also identified several new lead quinazoline Mps1 inhibitors, including a low-affinity compound termed Compound 4 (Cpd 4), whose interaction with the Mps1 kinase domain was further characterized by X-ray crystallography. A novel biophysical analysis demonstrated that the intrinsic fluorescence of SP600125 changed markedly upon Mps1 binding, allowing spectrophotometric displacement analysis and determination of dissociation constants for ATP-competitive Mps1 inhibitors. By illuminating the structure of the Mps1 ATP-binding site our results provide novel biophysical insights into Mps1-ligand interactions that will be useful for the development of specific Mps1 inhibitors, including those employing a therapeutically validated quinazoline template.

Infection frequently triggers thrombotic microangiopathy in patients with preexisting risk factors: a single-institution experience.

Thrombotic microangiopathies are rare conditions characterized by microangiopathic hemolytic anemia, microthrombi, and multiorgan insult. The disorders, which include hemolytic uremic syndrome and thrombotic thrombocytopenic purpura, are often acute and life threatening. We report a retrospective analysis of 65 patients presenting to our institution from 1997 to 2008 with all forms of thrombotic microangiopathy. Therapeutic plasma exchange was a requirement for analysis and 65 patients were referred to our institution; 66% of patients were female and median age at presentation was 52 years. Bacterial infection was the most commonly identified etiologic factor and in the multivariate model was the only significant variable associated with survival outcome (odds ratio 5.1, 95% confidence interval, 1.2-21.7). As infection can be considered a common trigger event for thrombotic microangiopathy, patients with hepatobiliary sepsis may benefit from elective cholecystectomy. We conclude that bacterial infection frequently triggers TTP and other thrombotic microangiopathies in patients with preexisting risk factors and propose a model for the development of these syndromes.

Molecular dynamics study of chemically engineered green fluorescent protein mutants: comparison of intramolecular fluorescence resonance energy transfer rate.

Because of its unusual spectroscopic properties, green fluorescent protein (GFP) has become a useful tool in molecular genetics, biochemistry and cell biology. Here, we computationally characterize the behavior of two GFP constructs, designed as bioprobes for enzymatic triggering using intramolecular fluorescence resonance energy transfer (FRET). These constructs differ in the location of an intramolecular FRET partner, an attached chemical chromophore (either near an N-terminal or C-terminal site). We apply the temperature replica exchange molecular dynamics method to the two flexible constructs in conjunction with a generalized Born implicit solvent model. The calculated rate of FRET was derived from the interchromophore distance, R, and orientational factor, kappa(2). In agreement with experiment, the construct with the C-terminally attached dye was predicted to have higher energy transfer rate than observed for the N-terminal construct. The molecular basis for this observation is discussed. In addition, we find that the orientational factor, kappa(2), deviates from the commonly assumed value, the implications of which are also considered.

Discovery of novel inhibitors of Trypanosoma cruzi trans-sialidase from in silico screening.

trans-Sialidase from Trypanosoma cruzi (TcTS) has emerged as a potential drug target for treatment of Chagas disease. Here, we report the results of virtual screening for the discovery of novel TcTS inhibitors, which targeted both the sialic acid and sialic acid acceptor sites of this enzyme. A library prepared from the Evotec database of commercially available compounds was screened using the molecular docking program GOLD, following the application of drug-likeness filters. Twenty-three compounds selected from the top-scoring ligands were purchased and assayed using a fluorimetric assay. Novel inhibitor scaffolds, with IC(50) values in the submillimolar range were discovered. The 3-benzothiazol-2-yl-4-phenyl-but-3-enoic acid scaffold was studied in more detail, and TcTS inhibition was confirmed by an alternative sialic acid transfer assay. Attempts to obtain crystal structures of these compounds with TcTS proved unsuccessful but provided evidence of ligand binding at the active site.

Rational drug design in parasitology: trans-sialidase as a case study for Chagas disease.

Trypanosoma cruzi trans-sialidase is a potential target for Chagas disease chemotherapy. From the specific need of T. cruzi to obtain sialic acid through trans-sialidase-mediated transfers from host sources and the lack of alternative to this for the parasite, a good case can be made for T. cruzi trans-sialidase to serve as a potential drug target against Chagas disease. This review deals with both the particular aspects relevant to T. cruzi trans-sialidase as a target and generalises the situation for drug design in its broader aspects on the basis of some special problems in terms of rational drug design that T. cruzi trans-sialidase raises, particularly those of multiple gene copies and active site plasticity.

Quantum dot FRET biosensors that respond to pH, to proteolytic or nucleolytic cleavage, to DNA synthesis, or to a multiplexing combination.

Fluorescent acceptors have been immobilized on nanoparticulate quantum dots (QDs), which serve in turn as their FRET donors. The broad excitation and narrow emission bands of QDs mark them as having excellent potential as donors for FRET and, in principle, differently colored QDs could be excited simultaneously. The present work describes the preparation and operation of FRET-based QD bioprobes individually able to detect the actions of protease, deoxyribonuclease, DNA polymerase, or changes in pH. In addition, two such QD-mounted biosensors were excited at a single wavelength, and shown to operate simultaneously and independently of each other in the same sample solution, allowing multiplex detection of the action of a protease, trypsin, in the presence of deoxyribonuclease.

SNP detection for cytochrome P450 alleles by target-assembled tandem oligonucleotide systems based on exciplexes.

We report the first use of exciplex-based split-probes for detection of the wild type and *3 mutant alleles of human cytochrome P450 2C9. A tandem 8-mer split DNA oligonucleotide probe system was designed that allows detection of the complementary target DNA sequence. This exciplex-based fluorescence detector system operates by means of a contiguous hybridization of two oligonucleotide exciplex split-probes to a complementary target nucleic acid target. Each probe oligonucleotide is chemically modified at one of its termini by a potential exciplex-forming partner, each of which is fluorescently silent at the wavelength of detection. Under conditions that ensure correct three-dimensional assembly, the chemical moieties on suitable photoexcitation form an exciplex that fluoresces with a large Stokes shift (in this case 130 nm). Preliminary proof-of-concept studies used two 8-mer probe oligonucleotides, but in order to give better specificity for genomic applications, probe length was extended to give coverage of 24 bases. Eight pairs of tandem 12-mer oligonucleotide probes spanning the 2C9*3 region were designed and tested to find the best set of probes. Target sequences tested were in the form of (i) synthetic oligonucleotides, (ii) embedded in short PCR products (150 bp), or (iii) inserted into plasmid DNA (approximately 3 Kbp). The exciplex system was able to differentiate wild type and human cytochrome P450 2C9 *3 SNP (1075 A-->C) alleles, based on fluorescence emission spectra and DNA melting curves, indicating promise for future applications in genetic testing and molecular diagnostics.

Target-assembled exciplexes based on Scorpion oligonucleotides.

Scorpion probes, specific DNA probe sequences maintained in a hairpin-loop, can be modified to carry the components of an exciplex for use as a novel fluorescence-based method for specific detection of DNA. The exciplex partners (5'-pyrenyl and 3'-naphthalenyl) were attached to oligonucleotides via phosphoramidate links to terminal phosphate groups. Hybridization of the probe to a complementary target in a buffer containing trifluoroethanol produced an obvious fluorescence change from blue (pyrene locally excited state emission) to green (exciplex emission).

Extracorporeal photopheresis improves nephrogenic fibrosing dermopathy/nephrogenic systemic fibrosis: three case reports and review of literature.

Nephrogenic fibrosing dermopathy/nephrogenic systemic fibrosis (NFD/NSF) is a recently recognized systemic fibrosing disorder that develops in the context of renal insufficiency. Its predominant manifestation is severe cutaneous fibrosis, often causing disabling contractures of neighboring joints. No therapy is consistently effective, and etiology and pathogenesis remain obscure. However, gadolinium-containing contrast agents used in magnetic resonance imaging or angiography have recently been strongly linked to the development of fibrosis in NSF. We report a clinical response to treatment with extracorporeal photopheresis (ECP) using the Therakos UVAR XTS system in three cases of NFD/NSF associated with flexion contractures affecting all four limbs. All three patients were receiving renal replacement therapy for longstanding end-stage renal failure. All three showed clinical response with softening of skin plaques at the end of four cycles, and improved range of movement in all four limbs on completion of treatment. The first patient, with NFD/NSF of 4.5 year's duration, was able to resume most activities of daily living although still chairbound, while a second patient with more recent onset of NSF was able to walk short distances with the help of Zimmer frames by the end of 16-18 cycles of treatment, having been chairbound pretreatment. A third patient with milder initial symptoms also experienced significant improvement after a shorter course of ECP treatment. Our experience with these three patients confirms previous case reports, suggesting that ECP may be effective in NFD/NSF.