Improvement in inner retinal function in glaucoma with nicotinamide (vitamin B3) supplementation: A crossover randomized clinical trial

IMPORTANCE: Retinal ganglion cells endure significant metabolic stress in glaucoma but maintain capacity to recover function. Nicotinamide, a precursor of NAD+ , is low in serum of glaucoma patients and its supplementation provides robust protection of retinal ganglion cells in preclinical models. However, the potential of nicotinamide in human glaucoma is unknown. BACKGROUND: To examine the effects of nicotinamide on inner retinal function in glaucoma, in participants receiving concurrent glaucoma therapy. DESIGN: Crossover, double-masked, randomized clinical trial. Participants recruited from two tertiary care centres. PARTICIPANTS: Fifty-seven participants, diagnosed and treated for glaucoma. METHODS: Participants received oral placebo or nicotinamide and reviewed six-weekly. Participants commenced 6 weeks of 1.5 g/day then 6 weeks of 3.0 g/day followed by crossover without washout. Visual function measured using electroretinography and perimetry. MAIN OUTCOME MEASURES: Change in inner retinal function, determined by photopic negative response (PhNR) parameters: saturated PhNR amplitude (Vmax), ratio of PhNR/b-wave amplitude (Vmax ratio). RESULTS: PhNR Vmax improved beyond 95% coefficient of repeatability in 23% of participants following nicotinamide vs 9% on placebo. Overall, Vmax improved by 14.8% [95% CI: 2.8%, 26.9%], (P = .02) on nicotinamide and 5.2% [-4.2%, 14.6%], (P = .27) on placebo. Vmax ratio improved by 12.6% [5.0%, 20.2%], (P = .002) following nicotinamide, 3.6% [-3.4%, 10.5%], (P = .30) on placebo. A trend for improved visual field mean deviation was observed with 27% improving ≥1 dB on nicotinamide and fewer deteriorating (4%) compared to placebo (P = .02). CONCLUSIONS: Nicotinamide supplementation can improve inner retinal function in glaucoma. Further studies underway to elucidate the effects of long-term nicotinamide supplementation.Flora Hui, Jessica Tang, Pete A. Williams, Myra B. McGuinness, Xavier Hadoux, Robert J. Casson, Michael Coote, Ian A. Trounce, Keith R. Martin, Peter van Wijngaarden, Jonathan G. Crowsto

Haemolytic and cytotoxic activities of the Tween 80-extracted putative haemolysin of Pasteurella multocida B:2

The objective of this study was to investigate the haemolytic and cytotoxic activity of Pasteurella multocida B:2 strains, originally from cases of haemorrhagic septicaemia in cattle. All six P. multocida B:2 strains were non-haemolytic on sheep blood agar (SBA) and horse blood agar (HBA) when grown aerobically and on SBA anaerobically but they were haemolytic on HBA when grown anaerobically. No haemolytic activity against horse red blood cells was detected in culture supernates from aerobically or anaerobically grown cultures and only very weak haemolytic activity was obtained in supernates or pellet fractions from sonicated cells. However, after repeated extraction of sonicated cells with Tween 80, haemolytic activity was found in various cell fractions, both Tween-soluble and -insoluble. The Tween-extracted putative haemolysin and other bacterial fractions were also cytotoxic for mouse macrophage-like J774.2 cells. Further characterisation of the putative haemolysin revealed it to be a heat-labile, non-pore-forming protein of molecular weight >10ákDa whose activity was completely destroyed by trypsin and greatly reduced with protease and proteinase K treatment. Congo red also reduced the haemolytic activity. Non-denaturing gel-electrophoresis and RBC agar overlay revealed clear haemolytic zones but suggested that Tween was bound to some component of the P. multocida B:2 fractions and was responsible, to some extent, for the haemolytic activity observed. However, the effect of heat and other reagents on the Tween-extracted fractions and the lack of haemolytic activity in different Tween-extracted cell fractions of organisms other than P. multocida suggested that some proteinaceous component of the organism could indeed act as a haemolysin. This putative haemolysin may be one of the virulence attributes of P. multocida, but its characterisation and role in pathogenesis require further stud

BapC autotransporter protein is a virulence determinant of Bordetella pertussis

A protein designated Bap-5 (GenBank accession no. AF081494) or BapC (GenBank accession no. AJ277634) has been identified as a member of the Bordetella pertussis autotransporter family and the present work suggests that this protein, like the previously characterised BrkA, is a Bvg-regulated serum resistance factor and virulence determinant. B. pertussis bapC and brkA, bapC mutants were created and, like a brkA mutant, showed greater sensitivity to killing by normal human serum than their parent strains but they were not as sensitive as a bvg mutant. Competition assays also showed an important role for BapC, like BrkA, in virulence of B. pertussis in mice after intranasal infection. Moreover, the bapC and brkA, bapC mutants, like the brkA mutant, were found to be more sensitive to the antimicrobial peptide cecropin P1 than the parent strains. In the genome sequence of B. pertussis strain Tohama, bapC is designated as a pseudogene due, in part, to a frameshift in a poly(C) tract near the 5' end of the gene which creates a truncated BapC protein. Sequence analyses of the bapC region spanning the poly(C) tract of a number of B. pertussis strains showed minor nucleotide and amino acid polymorphisms but it appeared that all had an ORF that would be able to produce BapC. (C) 2011 Elsevier Ltd. All rights reserve

Effect of different forms of adenylate cyclase toxin of Bordetella pertussis on protection afforded by an acellular pertussis vaccine in a murine model

Four recombinant forms of the cell-invasive adenylate cyclase toxin (CyaA) of Bordetella pertussis were compared for the ability to enhance protection against B. pertussis in mice when coadministered with an acellular pertussis vaccine (ACV). The four forms were as follows: fully functional CyaA, a CyaA form lacking adenylate cyclase enzymatic activity (CyaA*), and the nonacylated forms of these toxins, i.e., proCyaA and proCyaA*, respectively. None of these forms alone conferred significant (P > 0.05) protection against B. pertussis in a murine intranasal challenge model. Mice immunized with ACV alone showed significant (P < 0.05) reductions in bacterial numbers in the lungs after intranasal challenge compared with those for control mice. When administered with ACV, both CyaA and CyaA* further reduced bacterial numbers in the lungs of mice after intranasal challenge compared with those for ACV-immunized mice, but the enhanced protection was only significant (P < 0.05) with CyaA*. Coadministration of CyaA* with ACV caused a significant (P < 0.05) increase in immunoglobulin G2a antibody levels against pertactin compared with those in mice immunized with ACV alone. Spleen cells from mice immunized with ACV plus CyaA* secreted larger amounts of interleukin-5 (IL-5), IL-6, gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) than did cells from mice immunized with ACV plus CyaA or ACV alone after stimulation in vitro with a mixture of B. pertussis antigens. Spleen cells from mice immunized with ACV plus CyaA* also secreted larger amounts of IFN-γ and GM-CSF than did cells from mice immunized with CyaA* alone after stimulation in vitro with CyaA*. Macrophages from mice immunized with ACV plus CyaA* produced significantly (P < 0.05) higher levels of nitric oxide than did macrophages from mice immunized with CyaA* alone, ACV alone, or ACV plus CyaA after stimulation in vitro with a mixture of B. pertussis antigens or heat-killed B. pertussis cells. These data suggest that the enhancement of protection provided by CyaA* was due to an augmentation of both Th1 and Th2 immune responses to B. pertussis antigens

Virulence spectra of typed strains of Campylobacter jejuni from different sources: a blinded in vivo study

<i>Campylobacter jejuni</i> is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. The aims of the present blinded study were to measure and compare the <i>in vivo</i> properties of 40 serotyped, biotyped and genotyped <i>C. jejuni</i> isolates from different sources and genetic makeup. An 11-day-old chick embryo lethality assay, which measured embryo deaths and total viable bacteria over 72 h following inoculation of bacteria into the chorioallantoic membrane, revealed a spectrum of activity within the <i>C. jejuni</i> strains. Human and chicken isolates showed similar high virulence values for embryo deaths while the virulence of the bovine isolates was less pronounced. A one-way ANOVA comparison between the capacity of the strains to kill the chick embryos after 24 h with cytotoxicity towards cultured CaCo-2 cells was significant (<i>P</i>=0.025). After inoculation with a <i>Campylobacter</i> strain, mouse ligated ileal loops were examined histologically and revealed degrees of villous atrophy, abnormal mucosa, dilation of the lumen, congestion and blood in lumen, depending on the isolate examined. A total pathology score', derived for each <i>C. jejuni</i> strain after grading the pathology features for degree of severity, showed no apparent relationship with the source of isolation. Some relationship was found between amplified fragment length polymorphism groups and total ileal loop pathology scores, and a one-way ANOVA comparison of the mouse pathology scores against total chick embryo deaths after 72 h was significant (<i>P</i>=0.049)

Functional and structural studies on different forms of the adenylate cyclase toxin of Bordetella pertussis

A comparison was made of the cytotoxic activity and secondary structural features of four recombinant forms of adenylate cyclase toxin (CyaA). These forms were fully functional CyaA, CyaA lacking adenylate cyclase enzymatic activity (CyaA*), and non-acylated forms of these toxins, proCyaA and proCyaA*. At a toxin concentration >1 μg/ml, CyaA* was as cytotoxic towards J774.2 cells as CyaA and mediated cell killing at a faster rate than CyaA. At concentrations <0.5 μg/ml, CyaA* was less cytotoxic than CyaA and, at <0.1 μg/ml of CyaA*, no activity was detected. CyaA, but not CyaA*, was able to induce caspase 3/7 activity, a measure of apoptosis. ProCyaA and proCyaA* had no detectable cytotoxic or apoptotic activity. CyaA caused 50% inhibition of the zymosan-stimulated oxidative burst at 0.003 μg/ml, whereas a not, vert, similar500-fold greater toxin concentration of CyaA* or proCyaA was needed for 50% inhibition. ProCyaA* was inactive. CyaA is a calcium-binding protein and far UV circular dichroism (CD), near UV CD and fluorescence spectra analyses showed that all the forms of CyaA had similar overall structures at different calcium concentrations up to 5.0 mM. At 7.5 mM CaCl2, the far UV spectrum of CyaA altered significantly, indicating a change in secondary structure associated with high β-sheet content or a β-aggregated state, whereas the spectrum of CyaA* showed only a slight alteration at this calcium concentration. Near UV CD and fluorescence studies were consistent with a rearrangement of secondary structural elements in the presence of CaCl2 for all CyaA forms. There was a marked dependence on protein concentration of the far UV spectra of these CyaA forms, implying an interaction between individual molecules at higher protein concentrations

A model to measure fluid outflow in rabbit capsules post glaucoma implant surgery

Purpose.: Prior models of glaucoma filtration surgery assess bleb morphology, which does not always reflect function. Our aim is to establish a model that directly measures tissue hydraulic conductivity of postsurgical outflow in rabbit bleb capsules following experimental glaucoma filtration surgery. Methods.: Nine rabbits underwent insertion of a single-plate pediatric Molteno implant into the anterior chamber of their left eye. Right eyes were used as controls. The rabbits were then allocated to one of two groups. Group one had outflow measurements performed at 1 week after surgery (n = 5), and group two had measurements performed at 4 weeks (n = 4). Measurements were performed by cannulating the drainage tube ostium in situ with a needle attached to a pressure transducer and a fluid column at 15 mm Hg. The drop in the fluid column was measured every minute for 5 minutes. For the control eyes (n = 6), the anterior chamber of the unoperated fellow eye was cannulated. Animals were euthanized with the implant and its surrounding capsule dissected and fixed in 4% paraformaldehyde, and embedded in paraffin before 6-μm sections were cut for histologic staining. Results.: By 7 days after surgery, tube outflow was 0.117 ± 0.036 μL/min/mm Hg at 15 mm Hg (mean ± SEM), whereas at 28 days, it was 0.009 ± 0.003 μL/min/mm Hg. Control eyes had an outflow of 0.136 ± 0.007 μL/min/mm Hg (P = 0.004, one-way ANOVA). Hematoxylin and eosin staining demonstrated a thinner and looser arrangement of collagenous tissue in the capsules at 1 week compared with that at 4 weeks, which had thicker and more densely arranged collagen. Conclusions.: We describe a new model to directly measure hydraulic conductivity in a rabbit glaucoma surgery implant model. The principal physiologic endpoint of glaucoma surgery can be reliably quantified and consistently measured with this model. At 28 days post glaucoma filtration surgery, a rabbit bleb capsule has significantly reduced tissue hydraulic conductivity, in line with loss of implant outflow facility, and increased thickness and density of fibrous encapsulation

Improvement in inner retinal function in glaucoma in response to nicotinamide (Vitamin B3) supplementation: a crossover randomized clinical trial

Abstract #3493Flora Hui, Jessica Tang, Pete Williams, Myra McGuinness, Xavier Hadoux, Robert James Casson, Michael Coote, Ian Trounce, Keith R Martin, Peter van Wijngaarden, Jonathan Guy Crowsto