Cell-Penetrating Chaperone Nuc1 for Small- and Large-Molecule Delivery Into Retinal Cells and Tissues.

Purpose: There are currently no means available for the efficient delivery of recombinant proteins into retinal cells in vivo. Although cell-penetrating peptides have been somewhat effective in protein delivery to the retina, they generally require conjugation chemistry with the payload, negatively impacting function of the therapeutic protein. In this study, we developed a novel peptide (Nuc1) that acts as a chaperone for delivery of small and large molecules, including steroids, peptides, antibodies, recombinant proteins, and viruses (adeno-associated viruses [AAVs]) across biological membranes in vivo without the need for conjugation. Methods: Nuc1 peptide was designed based on sequences known to bind heparan sulfate proteoglycans and nucleolin found on the surface of retinal cells. Nuc1 was injected into the vitreous of mice with a variety of molecules and retinas examined for uptake and function of these molecules. Results: Nuc1 engages the process of macropynocytosis for cell entry. The delivery of functional recombinant X-linked inhibitor of apoptosis protein to photoreceptors via the intravitreal route of injection inhibited retinal apoptosis. Nuc1 was found to enhance the delivery of anti-VEGF antibodies delivered intravitreally or topically in models of age-related macular degeneration (AMD). Nuc1 enhanced delivery of decorin, facilitating significant inhibition of neovascularization and fibrosis in a model of AMD. Finally, Nuc1 was found to enhance penetration of retinal cells and tissues by AAV via both the subretinal and intravitreal routes of injection. Conclusions: Nuc1 shows promise as a novel approach for the delivery of recombinant proteins into retinal cells in vivo.

Comparative Analysis of Surgically Retrieved Sperms in Cases of Obstructive and Nonobstructive Azoospermia Treated for Infertility.

BACKGROUND: Azoospermia is a highly evolving subject in the last few decades. In the past, use of donor sperm was the only option providing a realistic chance of conception for couples affected by azoospermia. Introduction of sperm retrieval techniques and assisted reproductive technologies, especially intracytoplasmic sperm injection (ICSI), has provided these men a chance to father their genetically own child and changed the management approach significantly. OBJECTIVE: The objective was to compare the sperm retrieval rate (SRR) and ICSI outcomes of surgically retrieved sperms in cases of obstructive and nonobstructive azoospermia (NOA) as well as to evaluate the efficacy of sperm retrieval techniques. MATERIALS AND METHODS: A total of sixty azoospermic patients were included in the study. The patients were divided between OA (16) and NOA groups (44). A retrospective outcome analysis was done on SRR and ICSI results among them. RESULTS: The overall SRR in patients with NOA and OA was 47.7% and 100%, respectively (P < 0.001). On subgroup analysis, higher serum follicle-stimulating hormone has shown significantly decreased sperm retrieval. The size of testes was not found to be related to sperm retrieval. Fertilization and embryo formation rate were found to be higher in OA cases in comparison to those of NOA cases. Clinical pregnancy rate showed no significant difference. CONCLUSION: Various sperm retrieval techniques can provide new dimensions for successful ICSI and managing azoospermia patients. Although SRRs as well as ICSI outcomes are lower in NOA patients than patients with OA, still they are potentially fertile. A systematic approach especially in patients with NOA is an important step. Microdissection testicular sperm extraction is an attractive option for NOA patients in order to increase the chances of successful sperm retrieval.

Occurrence of extended-spectrum ß-lactamase-producing bacteria in urban Clinton River habitat.

OBJECTIVE: The aim of this study was to determine whether Clinton River water is contaminated with antibiotics and is a reservoir of antimicrobial-resistant bacteria. METHODS: Water samples were taken from two sites of Clinton River. Antimicrobial-resistant bacteria were enumerated on agar plates supplemented with six commonly used antibiotics. Extended-spectrum ß-lactamase (ESBL)-producing bacteria were identified using a BD Phoenix™ System and by 16S rRNA gene sequencing. Antimicrobial resistance gene transfer was performed by conjugation studies and the location of genes was determined by Southern hybridisation. Virulence properties of ESBL-producing isolates were determined by assessing their biofilm-forming ability, cellular toxicity, and induction of an inflammatory response in intestinal epithelial (Caco-2) cells. RESULTS: 16S rRNA analysis of water samples showed the presence of potentially pathogenic bacteria (e.g. Shigella flexneri, Klebsiella pneumoniae, Aeromonas punctata and Pseudomonas aeruginosa). Among 64 biochemically identified bacterial isolates tested, 42% were resistant to cefotaxime, 34% to chloramphenicol, 9% to tetracycline, 11% to ciprofloxacin and 9% to gentamicin. Of 27 cefotaxime-resistant isolates, 11 (41%) were ESBL-positive and possessed either blaCTX-M (n=9), blaTEM (n=1) or blaKPC (n=1). Comparative analysis of ESBL gene sequences from Clinton River water bacteria showed 98-100% identity with clinical isolates. ESBL-producing isolates from Clinton River water were found to form biofilms, induced inflammatory cytokines and caused toxicity to epithelial cells. CONCLUSIONS: Clinton River water contains isolates with ESBL genes identical to clinical isolates and possessing virulence properties, thus it could be a potential reservoir in causing human infections.

Successful management of ureteric endometriosis by laparoscopic ureterolysis - A review and report of three further cases.

OBJECTIVE: To review articles highlighting the effectiveness of conservative laparoscopic ureterolysis as a primary treatment option in patients with ureteric endometriosis and to report on a further three cases. PATIENTS AND METHODS: PubMed, EMBASE, Cochrane database were searched to identify articles reporting cases of laparoscopic management of ureteric endometriosis and, in particular management by ureterolysis. We further described three new cases of ureteric endometriosis managed at our institute. RESULTS: The present study illustrates the significance of laparoscopic ureterolysis in the management of patients with ureteric endometriosis. In our cases, a systematic surgical approach was followed in order to perform complete but careful excision of the all visible endometriotic implants. During follow-up successful treatment was established by relief of hydroureteronephrosis by ultrasonographic evaluation. CONCLUSION: Considering the risk of loss of renal function and due to the nonspecific symptoms, a prompt clinical suspicion and thorough preoperative assessment can potentially help in the diagnosis. We conclude that laparoscopic ureterolysis is a minimally invasive technique with low complication and recurrence rates. It is a suitable option as a primary approach for selected patients with ureteric endometriosis, if done in a systematic step-by-step approach.

G-quartet oligonucleotide mediated delivery of functional X-linked inhibitor of apoptosis protein into retinal cells following intravitreal injection.

There is currently no efficient method available for the delivery of full length functional proteins into the cytoplasm of retinal cells in vivo. Historically, the most successful approach for the treatment of retinal diseases has been intravitreal injection of antibodies or recombinant proteins, but this approach is not yet utilized for the delivery of proteins that require intracellular access for a therapeutic effect. Here we describe a platform for the delivery of functional proteins into ganglion cells, photoreceptors and retinal pigment epithelium via intravitreal injection. A nucleolin binding aptamer, AS1411, was biotinylated and complexed with traptavidin and utilized as a platform for the delivery of GFP or X-linked inhibitor of apoptosis (XIAP) proteins by intravitreal injection in BALB/c mice. Retinal sections were analyzed for uptake of proteins in the retina. Apoptosis was induced by intravitreal injection of N-methyl-D-aspartate (NMDA). Retinas were harvested for analysis of TUNEL and caspase 3/7 activity. Intravitreal injection of AS1411-directed GFP or XIAP complexes enabled delivery of these proteins into ganglion cells, photoreceptors and retinal pigment epithelium in vivo. AS1411-XIAP complexes conferred significant protection to cells in the outer and inner nuclear layers following NMDA induced apoptosis. A concomitant decrease in activity of Caspase 3/7 was observed in eyes injected with the AS1411-XIAP complex. In conclusion, AS1411 can be used as a platform for the delivery of therapeutic proteins into retinal cells. This approach can potentially be utilized to introduce a large variety of therapeutically relevant proteins that are previously well characterized to maintain the structural integrity and function of retina, thus, preventing vision loss due to ocular trauma or inherited retinal degeneration.

Microscopic varicocelectomy as a treatment option for patients with severe oligospermia.

Purpose: Many studies have shown improved semen parameters after varicocele surgery; however, the benefit in terms of improved pregnancy rates and live births is still disputed in cases of severe oligoasthenozoospermia (OAS). The present study evaluated the outcome of microscopic subinguinal varicocelectomy in terms of the spontaneous pregnancy rate in patients with severe OAS. Materials and Methods: This was a retrospective, observational, analytic study of 56 men with OAS who underwent microscopic varicocelectomy at our center between 2008 and 2015. The subjects were followed for a mean period of 12.4 months. Outcome was compared among groups of men with mild (sperm concentration, 10.2-19 million/mL), moderate (5.7-9.5 million/mL), and severe (<5 million/mL) OAS who were operated on during the same period. Results: A total of 13 of 35 men (37.1%) with severe OAS achieved spontaneous pregnancy. Mean sperm density increased from 2.29 million/mL preoperatively to 14.09 million/mL postoperatively. The mean time to pregnancy from the date of surgery was 8.5 months. The spontaneous pregnancy rate in men with mild and moderate OAS was 62.5% and 46.2%, respectively. Conclusions: Although pregnancy rates after varicocele surgery are lower preoperatively in men with severe OAS than in men with mild or moderate OAS, the spontaneous pregnancy rate of 37.1% still compares very favorably with outcomes after a single attempt at in vitro fertilization.

Occurrence of Multidrug Resistant Extended Spectrum Beta-Lactamase-Producing Bacteria on Iceberg Lettuce Retailed for Human Consumption.

Antibiotic resistance in bacteria is a global problem exacerbated by the dissemination of resistant bacteria via uncooked food, such as green leafy vegetables. New strains of bacteria are emerging on a daily basis with novel expanded antibiotic resistance profiles. In this pilot study, we examined the occurrence of antibiotic resistant bacteria against five classes of antibiotics on iceberg lettuce retailed in local convenience stores in Rochester, Michigan. In this study, 138 morphologically distinct bacterial colonies from 9 iceberg lettuce samples were randomly picked and tested for antibiotic resistance. Among these isolates, the vast majority (86%) demonstrated resistance to cefotaxime, and among the resistant bacteria, the majority showed multiple drug resistance, particularly against cefotaxime, chloramphenicol, and tetracycline. Three bacterial isolates (2.17%) out of 138 were extended spectrum beta-lactamase (ESBL) producers. Two ESBL producers (T1 and T5) were identified as Klebsiella pneumoniae, an opportunistic pathogen with transferable sulfhydryl variable- (SHV-) and TEM-type ESBLs, respectively. The DNA sequence analysis of the bla SHV detected in K. pneumoniae isolate T1 revealed 99% relatedness to bla SHV genes found in clinical isolates. This implies that iceberg lettuce is a potential reservoir of newly emerging and evolving antibiotic resistant bacteria and its consumption poses serious threat to human health.

Catalase therapy corrects oxidative stress-induced pathophysiology in incipient diabetic retinopathy.

PURPOSE: Preclinical studies have highlighted retinal oxidative stress in the pathogenesis of diabetic retinopathy. We evaluated whether a treatment designed to enhance cellular catalase reduces oxidative stress in retinal cells cultured in high glucose and in diabetic mice corrects an imaging biomarker responsive to antioxidant therapy (manganese-enhanced magnetic resonance imaging [MEMRI]). METHODS: Human retinal Müller and pigment epithelial cells were chronically exposed to normal or high glucose levels and treated with a cell-penetrating derivative of the peroxisomal enzyme catalase (called CAT-SKL). Hydrogen peroxide (H2O2) levels were measured using a quantitative fluorescence-based assay. For in vivo studies, streptozotocin (STZ)-induced diabetic C57Bl/6 mice were treated subcutaneously once a week for 3 to 4 months with CAT-SKL; untreated age-matched nondiabetic controls and untreated diabetic mice also were studied. MEMRI was used to analytically assess the efficacy of CAT-SKL treatment on diabetes-evoked oxidative stress-related pathophysiology in vivo. Similar analyses were performed with difluoromethylornithine (DFMO), an irreversible inhibitor of ornithine decarboxylase. RESULTS: After catalase transduction, high glucose-induced peroxide production was significantly lowered in both human retinal cell lines. In diabetic mice in vivo, subnormal intraretinal uptake of manganese was significantly improved by catalase supplementation. In addition, in the peroxisome-rich liver of treated mice catalase enzyme activity increased and oxidative damage (as measured by lipid peroxidation) declined. On the other hand, DFMO was largely without effect in these in vitro or in vivo assays. CONCLUSIONS: This proof-of-concept study raises the possibility that augmentation of catalase is a therapy for treating the retinal oxidative stress associated with diabetic retinopathy.

In Vivo Role of TLR2 and MyD88 Signaling in Eliciting Innate Immune Responses in Staphylococcal Endophthalmitis.

PURPOSE: The purpose of this study was to investigate the protective mechanisms evoked by TLR2 and MyD88 signaling in bacterial endophthalmitis in vivo. METHODS: Endophthalmitis was induced in wild-type (WT), TLR2(-/-), MyD88(-/-), and Cnlp(-/-) mice by intravitreal injections of a laboratory strain (RN6390) and two endophthalmitis isolates of Staphylococcus aureus. Disease progression was monitored by assessing corneal and vitreous haze, bacterial burden, and retinal tissue damage. Levels of inflammatory cytokines/chemokines were determined using quantitative RT-PCR (qRT-PCR) and ELISA. Flow cytometry was used to assess neutrophil infiltration. Cathelicidin-related antimicrobial peptide (CRAMP) expression was determined by immunostaining and dot blot. RESULTS: Eyes infected with either laboratory or clinical isolates exhibited higher levels of inflammatory mediators at the early stages of infection (≤24 hours) in WT mice than in TLR2(-/-) or MyD88(-/-) mice. However, their levels surpassed that of WT mice at the later stages of infection (>48 hours), coinciding with increased bacterial burden and retinal damage. Both TLR2(-/-) and MyD88(-/-) retinas produced reduced levels of CRAMP, and its deficiency (Cnlp(-/-)) rendered the mice susceptible to increased bacterial burden and retinal tissue damage as early as 1 day post infection. Analyses of inflammatory mediators and neutrophil levels in WT versus Cnlp(-/-) mice showed a trend similar to that observed in TLR2 and MyD88 KO mice. Furthermore, we observed that even a 10-fold lower infective dose of S. aureus was sufficient to cause endophthalmitis in TLR2(-/-) and MyD88(-/-) mice. CONCLUSIONS: TLR2 and MyD88 signaling plays an important role in protecting the retina from staphylococcal endophthalmitis by production of the antimicrobial peptide CRAMP.

Pathogenicity of ocular isolates of Acinetobacter baumannii in a mouse model of bacterial endophthalmitis.

PURPOSE: To determine the virulence properties of ocular isolates of Acinetobacter baumannii in causing endophthalmitis in a mouse model. METHODS: Endophthalmitis was induced by intravitreal injections of the bacteria into C57BL/6 (B6) mouse eyes. The disease progression was monitored by ophthalmoscopic, electroretinography (ERG), histologic, cell death (TUNEL labeling), and microbiological parameters. The expression of cytokines/chemokines was checked by quantitative RT-PCR (qRT-PCR) and ELISA. Flow cytometry was used to determine cellular infiltration. The role of neutrophils was determined using neutropenic mice. The virulence traits (biofilm formation, adherence, and cytotoxicity) of the ocular isolates were tested using corneal epithelial cells. RESULTS: Among the three clinical isolates and a standard ATCC 19606 strain tested, a biofilm producing multidrug resistant (MDR) strain of A. baumannii AB12 caused severe endophthalmitis (100% destruction of the eyes) leading to the loss of retinal function as assessed by ERG analysis. Elevated levels of inflammatory mediators (TNF-α, IL-1ß, CXCL2, and IL-6) were detected in AB12-infected eyes. Histologic and TUNEL staining revealed increased retinal cell death and the flow cytometry data showed the presence of inflammatory cells, primarily neutrophils (CD45(+)/Ly6G(+)). Neutropenic mice showed an increased bacterial burden, reduced inflammatory response, and severe tissue destruction. CONCLUSIONS: These results indicate that A. baumannii causes severe intraocular inflammation and retinal damage. Furthermore, neutrophils play an important role in the pathogenesis of A. baumannii endophthalmitis.

Virulence properties of multidrug resistant ocular isolates of Acinetobacter baumannii.

PURPOSE: Acinetobacter (A.) baumannii is an opportunistic pathogen and has been reported as a causative agent of ocular infections. The aim of this study is to identify virulence properties (biofilm formation, adhesion, invasion and cytotoxicity) and antibiotic resistance among A. baumannii isolates recovered from the eye. MATERIALS AND METHODS: The Microscan Walk-Away®, an automated bacterial identification and susceptibility testing system was used to determine antibiotic resistance. Clonal relatedness was assessed by Pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis. Conjugation experiments were carried out to determine the transfer of antibiotic resistance genes and PCR was used to confirm gene transfer. Virulence properties of the isolates were determined by biofilm formation using crystal violet and immunofluorescence staining, adherence and internalization using cultured corneal epithelial cells, and cytotoxicity by TUNEL-staining and LDH release assays. RESULTS: All ocular isolates (n = 12) exhibited multidrug resistant (MDR) phenotype and one of the isolate (AB12) was resistant to 18 antibiotics (ß-lactam, aminoglycosides, tetracycline, chloramphenicol and quinolones). The plasmid profile analysis showed the presence of multiple plasmids in each isolate and a total of 10 different profiles were observed. However, PFGE analysis was more discriminatory which revealed 12 distinct genotypes. Antibiotic resistance (tetracycline and quinolone) was transferable from the isolate AB12 to a recipient Escherichia coli J53. Ten isolates were strong biofilm producers and the remaining two (AB5 and AB7) were moderate producers. All isolates demonstrated adherence and invasive properties towards HCECs. A similar trend was observed in their ability to cause cell death and toxicity. CONCLUSIONS: Our results indicate that ocular isolates of A. baumannii are biofilm producers and adherent and invasive to corneal epithelium, a first step in the pathogenesis of ocular infection. In addition, they demonstrated plasmid-mediated transfer of MDR traits making them a reservoir of resistance genes at ocular surface.

FK506 binding protein 12 deficiency in endothelial and hematopoietic cells decreases regulatory T cells and causes hypertension.

Patients treated with the immunosuppressive drug tacrolimus (FK506), which binds FK506 binding protein 12 (FKBP12) and then inhibits the calcium-dependent phosphatase calcineurin, exhibit decreased regulatory T cells, endothelial dysfunction, and hypertension; however, the mechanisms and whether altered T-cell polarization play a role are unknown. Tacrolimus treatment of mice for 1 week dose-dependently decreased splenic CD4(+)/FoxP3(+) (regulatory T cells), increased splenic CD4(+)/IL-17(+) (T-helper 17) cells, and caused endothelial dysfunction and hypertension. To determine the mechanisms, we crossed floxed FKBP12 mice with Tie2-Cre mice to generate offspring lacking FKBP12 in endothelial and hematopoietic cells only (FKBP12EC knockout [KO]). Given the role of FKBP12 in inhibiting transforming growth factor-ß receptor activation, Tie2-Cre-mediated deletion of FKBP12 increased transforming growth factor-ß receptor activation and SMAD2/3 signaling. FKBP12EC KO mice exhibited increased vascular expression of genes and proteins related to endothelial cell activation and inflammation. Serum levels of the proinflammatory cytokines IL-2, IL-6, interferon-γ, IL-17a, IL-21, and IL-23 were increased significantly, suggesting a T-helper 17 cell-mediated inflammatory state. Flow cytometry studies confirmed this, because splenic levels of CD4(+)/IL-17(+) cells were increased significantly, whereas CD4(+)/FoxP3(+) cells were decreased in FKBP12EC KO mice. Furthermore, spleens from FKBP12EC KO mice showed increased signal transducer and activator of transcription 3 activation, involved in T-helper 17 cell induction, and decreased signal transducer and activator of transcription 5 activation, involved in regulatory T-cell induction. FKBP12EC KO mice also exhibited endothelial dysfunction and hypertension. These data suggest that tacrolimus, through its activation of transforming growth factor-ß receptors in endothelial and hematopoietic cells, may cause endothelial dysfunction and hypertension by activating endothelial cells, reducing regulatory T cells, and increasing T-helper 17 cell polarization and inflammation.