Humanin G (HNG) protects age-related macular degeneration (AMD) transmitochondrial ARPE-19 cybrids from mitochondrial and cellular damage.

Age-related macular degeneration (AMD) ranks third among the leading causes of visual impairment with a blindness prevalence rate of 8.7%. Despite several treatment regimens, such as anti-angiogenic drugs, laser therapy, and vitamin supplementation, being available for wet AMD, to date there are no FDA-approved therapies for dry AMD. Substantial evidence implicates mitochondrial damage and retinal pigment epithelium (RPE) cell death in the pathogenesis of AMD. However, the effects of AMD mitochondria and Humanin G (HNG), a more potent variant of the mitochondrial-derived peptide (MDP) Humanin, on retinal cell survival have not been elucidated. In this study, we characterized mitochondrial and cellular damage in transmitochondrial cybrid cell lines that contain identical nuclei but possess mitochondria from either AMD or age-matched normal (Older-normal (NL)) subjects. AMD cybrids showed (1) reduced levels of cell viability, lower mtDNA copy numbers, and downregulation of mitochondrial replication/transcription genes and antioxidant enzyme genes; and (2) elevated levels of genes related to apoptosis, autophagy and ER-stress along with increased mtDNA fragmentation and higher susceptibility to amyloid-β-induced toxicity compared to NL cybrids. In AMD cybrids, HNG protected the AMD mitochondria, reduced pro-apoptosis gene and protein levels, upregulated gp130 (a component of the HN receptor complex), and increased the protection against amyloid-β-induced damage. In summary, in cybrids, damaged AMD mitochondria mediate cell death that can be reversed by HNG treatment. Our results also provide evidence of Humanin playing a pivotal role in protecting cells with AMD mitochondria. In the future, it may be possible that AMD patient's blood samples containing damaged mitochondria may be useful as biomarkers for this condition. In conclusion, HNG may be a potential therapeutic target for treatment of dry AMD, a debilitating eye disease that currently has no available treatment. Further studies are needed to establish HNG as a viable mitochondria-targeting therapy for dry AMD

HSV Latency-Associated Transcript and Neuronal Apoptosis

Thompson and Sawtell report that the Promega Anti-PARP p85 antibody did not recognize cleaved PARP in mouse or rabbit cells in their experiments, and conclude that the results reported with this antibody by Perng et al. (1) are an artifact. The Promega antibody was generated against a peptide based on the sequence of human p85. Although the corresponding bovine sequence differs by two amino acids, the antibody reacts with both human and bovine p85 (2). The mouse and rat sequences for this region of p85 differ from the human sequence by a single amino acid that corresponds to one of the bovine amino acid differences. External testers have successfully stained mouse and rat p85 using Promega Anti-PARP p85 (2). Thus, the negative mouse results reported by Thompson and Sawtell are surprising, and call into question the validity of their negative rabbit results. Extracts that we prepared (Fig. 1) from rabbit skin cells induced to undergo apoptosis by staurosporin (lane RS-S) contained a band of approximately 85 kD that was recognized by Anti-PARP p85, and that comigrated with the p85 band induced in human Jurkat cells by staurosporin (lane Jurkat-S) or anti-Fas antibody (lane Jurkat-F). Clearly, then, the Promega antibody recognizes the rabbit cleaved PARP p85 protein, and the argument to the contrary by Thompson and Sawtell has no merit. Their negative mouse and rabbit results apparently stemmed from technical problems, a bad batch of antibody, or some other unknown factor

Characterizing the protective effects of SHLP2, a mitochondrial-derived peptide, in macular degeneration

Mitochondrial-derived peptides (MDPs) are rapidly emerging therapeutic targets to combat development of neurodegenerative diseases. SHLP2 (small humanin-like peptide 2) is a newly discovered MDP that is coded from the MT-RNR2 (Mitochondrially encoded 16S rRNA) gene in mitochondrial DNA (mtDNA). In the current study, we examined the biological consequences of treatment with exogenously-added SHLP2 in an in vitro human transmitochondrial age-related macular degeneration (AMD) ARPE-19 cell model. In AMD cells, we observed significant down-regulation of the MDP-coding MT-RNR2 gene, and remarkably reduced levels of all five oxidative phosphorylation (OXPHOS) complex I-V protein subunits that are involved in the electron transport chain; these results suggested mitochondrial toxicity and abnormal OXPHOS complex protein subunits' levels in AMD cells. However, treatment of AMD cells with SHLP2: (1) restored the normal levels of OXPHOS complex protein subunits, (2) prevented loss of viable cells and mitochondria, (3) increased the number of mtDNA copies, (4) induced anti-apoptotic effects, and (5) attenuated amyloid-β-induced cellular and mitochondrial toxicity. Cumulatively, our findings established the protective role of SHLP2 in AMD cells in vitro. In conclusion, this novel study supports the merit of SHLP2 in the treatment of AMD, a primary retinal disease that is a leading cause of blindness among the elderly population in the United States as well as worldwide

Towards a Rational Design of an Asymptomatic Clinical Herpes Vaccine: The Old, the New, and the Unknown

The best hope of controlling the herpes simplex virus type 1 and type 2 (HSV-1 and HSV-2) pandemic is the development of an effective vaccine. However, in spite of several clinical trials, starting as early as 1920s, no vaccine has been proven sufficiently safe and efficient to warrant commercial development. In recent years, great strides in cellular and molecular immunology have stimulated creative efforts in controlling herpes infection and disease. However, before moving towards new vaccine strategy, it is necessary to answer two fundamental questions: (i) why past herpes vaccines have failed? (ii) Why the majority of HSV seropositive individuals (i.e., asymptomatic individuals) are naturally “protected” exhibiting few or no recurrent clinical disease, while other HSV seropositive individuals (i.e., symptomatic individuals) have frequent ocular, orofacial, and/or genital herpes clinical episodes? We recently discovered several discrete sets of HSV-1 symptomatic and asymptomatic epitopes recognized by CD4+ and CD8+ T cells from seropositive symptomatic versus asymptomatic individuals. These asymptomatic epitopes will provide a solid foundation for the development of novel herpes epitope-based vaccine strategy. Here we provide a brief overview of past clinical vaccine trials, outline current progress towards developing a new generation “asymptomatic” clinical herpes vaccines, and discuss future mucosal “asymptomatic” prime-boost vaccines that could optimize local protective immunity

The Gene That Encodes the Herpes Simplex Virus Type 1 Latency-Associated Transcript Influences the Accumulation of Transcripts (Bcl-x\u3csub\u3eL\u3c/sub\u3e and Bcl-x\u3csub\u3es\u3c/sub\u3e) That Encode Apoptotic Regulatory Proteins

The herpes simplex virus type 1 latency-associated transcript (LAT) inhibits apoptosis. We demonstrate here that LAT influences the accumulation of the Bcl-xL transcript versus the Bcl-xS transcript in Neuro-2A cells. Bcl-xL encodes an antiapoptotic protein, whereas Bcl-xS encodes a proapoptotic protein. Promoting the accumulation of Bcl-xL in neurons may inhibit apoptosis, thus enhancing the latency-reactivation cycle

The Gene That Encodes the Herpes Simplex Virus Type 1 Latency-Associated Transcript Influences the Accumulation of Transcripts (Bcl-x\u3csub\u3eL\u3c/sub\u3e and Bcl-x\u3csub\u3es\u3c/sub\u3e) That Encode Apoptotic Regulatory Proteins

The herpes simplex virus type 1 latency-associated transcript (LAT) inhibits apoptosis. We demonstrate here that LAT influences the accumulation of the Bcl-xL transcript versus the Bcl-xS transcript in Neuro-2A cells. Bcl-xL encodes an antiapoptotic protein, whereas Bcl-xS encodes a proapoptotic protein. Promoting the accumulation of Bcl-xL in neurons may inhibit apoptosis, thus enhancing the latency-reactivation cycle

Linear and Branched Glyco-Lipopeptide Vaccines Follow Distinct Cross-Presentation Pathways and Generate Different Magnitudes of Antitumor Immunity

BackgroundGlyco-lipopeptides, a form of lipid-tailed glyco-peptide, are currently under intense investigation as B- and T-cell based vaccine immunotherapy for many cancers. However, the cellular and molecular mechanisms of glyco-lipopeptides (GLPs) immunogenicity and the position of the lipid moiety on immunogenicity and protective efficacy of GLPs remain to be determined.Methods/Principal FindingsWe have constructed two structural analogues of HER-2 glyco-lipopeptide (HER-GLP) by synthesizing a chimeric peptide made of one universal CD4+ epitope (PADRE) and one HER-2 CD8+ T-cell epitope (HER420–429). The C-terminal end of the resulting CD4–CD8 chimeric peptide was coupled to a tumor carbohydrate B-cell epitope, based on a regioselectively addressable functionalized templates (RAFT), made of four α-GalNAc molecules. The resulting HER glyco-peptide (HER-GP) was then linked to a palmitic acid moiety, attached either at the N-terminal end (linear HER-GLP-1) or in the middle between the CD4+ and CD8+ T cell epitopes (branched HER-GLP-2). We have investigated the uptake, processing and cross-presentation pathways of the two HER-GLP vaccine constructs, and assessed whether the position of linkage of the lipid moiety would affect the B- and T-cell immunogenicity and protective efficacy. Immunization of mice revealed that the linear HER-GLP-1 induced a stronger and longer lasting HER420–429-specific IFN-γ producing CD8+ T cell response, while the branched HER-GLP-2 induced a stronger tumor-specific IgG response. The linear HER-GLP-1 was taken up easily by dendritic cells (DCs), induced stronger DCs maturation and produced a potent TLR- 2-dependent T-cell activation. The linear and branched HER-GLP molecules appeared to follow two different cross-presentation pathways. While regression of established tumors was induced by both linear HER-GLP-1 and branched HER-GLP-2, the inhibition of tumor growth was significantly higher in HER-GLP-1 immunized mice (p<0.005).SignificanceThese findings have important implications for the development of effective GLP based immunotherapeutic strategies against cancers

Identification of Herpes Simplex Virus Type 1 Latency-Associated Transcript Sequences That both Inhibit Apoptosis and Enhance the Spontaneous Reactivation Phenotype

The herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene is essential for the high spontaneous and induced reactivation phenotype of HSV-1 in the rabbit ocular model and for the high induced reactivation phenotype in the mouse ocular model. Recently we showed that LAT has an antiapoptosis function, and we hypothesized that LAT’s ability to inhibit apoptosis played an important role in LAT’s ability to enhance the reactivation phenotype. Expression of just the first 1.5 kb of the 8.3-kb LAT gene is sufficient for both inhibition of apoptosis in an in vitro transient-transfection assay and the high spontaneous reactivation phenotype in vivo. Here we show the results of more complex mapping studies in which inhibition of apoptosis and the enhanced spontaneous reactivation phenotype also appear to be linked. The HSV-1 mutant virus dLAT371 has a high spontaneous reactivation phenotype in rabbits, suggesting that the LAT region deleted in this mutant (LAT nucleotides 76 to 447) is not required for this phenotype. The LAT3.3A viral mutant (which expresses LAT nucleotides 1 to 1499) also has a high spontaneous reactivation phenotype, suggesting that the region of LAT not expressed by this mutant (LAT nucleotide 1500 to the end of LAT) is also not required for this phenotype. Surprisingly, LAT2.9A, which is a combination of dLAT371 and LAT3.3A (i.e., it expresses LAT nucleotides 1 to 76 and 447 to 1499), has a low spontaneous reactivation phenotype indistinguishable from that of LAT null mutants. We report here that consistent with the low spontaneous reactivation phenotype of LAT2.9A, a plasmid expressing the identical LAT RNA did not inhibit caspase 9-induced apoptosis. In contrast, plasmids containing the same deletion but able to transcribe up to or past LAT nucleotide 2850 (rather than just up to LAT nucleotide 1499) inhibited caspase 9-induced apoptosis, consistent with the high spontaneous reactivation phenotype of dLAT371. Thus, LAT2.9A may have a low spontaneous reactivation phenotype because the LAT RNA that is made cannot block apoptosis, and dLAT371 apparently has a high spontaneous reactivation phenotype because the LAT RNA made has significant antiapoptosis activity. Furthermore, LAT appeared to have at least two regions capable of interfering with caspase 9-induced apoptosis. One region partially overlaps LAT nucleotides 76 to 447. The second region is partially (or completely) downstream of LAT nucleotide 1499

Differential Epigenetic Status and Responses to Stressors between Retinal Cybrids Cells with African versus European Mitochondrial DNA: Insights into Disease Susceptibilities.

Mitochondrial (mt) DNA can be classified into haplogroups, which represent populations with different geographic origins. Individuals of maternal African backgrounds (L haplogroup) are more prone to develop specific diseases compared those with maternal European-H haplogroups. Using a cybrid model, effects of amyloid-β (Amyβ), sub-lethal ultraviolet (UV) radiation, and 5-Aza-2'-deoxycytidine (5-aza-dC), a methylation inhibitor, were investigated. Amyβ treatment decreased cell metabolism and increased levels of reactive oxygen species in European-H and African-L cybrids, but lower mitochondrial membrane potential (ΔΨM) was found only in African-L cybrids. Sub-lethal UV radiation induced higher expression levels of CFH, EFEMP1, BBC3, and BCL2L13 in European-H cybrids compared to African-L cybrids. With respect to epigenetic status, the African-L cybrids had (a) 4.7-fold higher total global methylation levels (p = 0.005); (b) lower expression patterns for DNMT3B; and (c) elevated levels for HIST1H3F. The European-H and African-L cybrids showed different transcription levels for CFH, EFEMP1, CXCL1, CXCL8, USP25, and VEGF after treatment with 5-aza-dC. In conclusion, compared to European-H haplogroup cybrids, the African-L cybrids have different (i) responses to exogenous stressors (Amyβ and UV radiation), (ii) epigenetic status, and (iii) modulation profiles of methylation-mediated downstream complement, inflammation, and angiogenesis genes, commonly associated with various human diseases

A tissue-targeted prime/pull/keep therapeutic herpes simplex virus vaccine protects against recurrent ocular herpes infection and disease in HLA-A*0201 transgenic rabbits

Herpes simplex virus type 1 (HSV-1) continues to be one of the most prevalent viral infections globally, with approximately 3.72 billion individuals affected worldwide. A clinical herpes vaccine is still lacking. In the present study, a novel prime/pull/keep vaccine was tested in a human leukocyte antigen transgenic rabbit model of ocular herpes (HLA-A*0201 Tg rabbit). Ten asymptomatic (ASYMP) CD8+ T-cell peptide epitopes and 3 CD4+ T-cell epitopes were selected from the HSV-1 glycoproteins D and B (gD and gB), viral tegument proteins (VP11/12 and VP13/14), and the DNA replication-binding helicase (UL9), all preferentially recognized by CD8+ and CD4+ T cells from "naturally protected" HSV-1-seropositive healthy ASYMP individuals (who never had recurrent corneal herpetic disease). HLA Tg rabbits were ocularly infected with HSV-1, then during latency at day 30 post-infection, the rabbits were ocularly vaccinated with a recombinant neurotropic AAV8 vector (107GC/ eye) encoding for the 10 CD8+ T-cell peptide and 4 CD4+ T-cell peptide (prime), T-cell attracting CXCL-11 (pull), and T-cell keeping IL-2/IL-15 cytokines (keep). The rabbits were followed up for corneal disease and viral loads in tears for 28 days. The frequency, function, and protective efficacy of HSV-specific CD8+ T cells induced by the prime/pull/keep vaccine were assessed in the trigeminal ganglia (TG), cornea, spleen, and peripheral blood. Compared to the mock group (unvaccinated), the peptides/CXCL11/IL-2/IL-15 vaccine generated frequent resident CD8+ T cells that infiltrated the TG. In ocularly HSV-1-infected and prime/pull/keep vaccinated rabbits, CD8+ T cell mobilization and retention into TG were associated with a significant reduction in corneal herpes infection and disease. These findings draw attention to the novel prime/pull/keep therapeutic vaccine strategy to mobilize and retain antiviral T cells to tissues protecting them against herpetic infection and disease.ImportanceThere is an urgent need for a vaccine against widespread human herpes simplex virus infections. The present study demonstrates that immunization of humanized HLA-A*0201 transgenic rabbits with CD8+ and CD4+ T-cell epitope peptides (prime)/ CXCL11 (pull)/ IL-2/IL-15 (keep) AAV8-based vaccine triggered mobilization and retention of HSV-1-specific CD8+ T cells locally in the cornea and TG, the sites of acute and latent herpes infections. Mobilization and retention of antiviral CD8+ T cells into the cornea and TG of HSV-1-infected rabbits that received the prime/pull/keep vaccine was associated with protection against ocular herpes infection and disease. These results highlight the importance of the prime/pull/keep vaccine strategy to bolster the number and function of protective CD8+ T cells within infected tissues