Virus-like particle vaccine displaying an external, membrane adjacent MUC16 epitope elicits ovarian cancer-reactive antibodies.

BACKGROUND: MUC16 is a heavily glycosylated cell surface mucin cleaved in the tumor microenvironment to shed CA125. CA125 is a serum biomarker expressed by > 95% of non-mucinous advanced stage epithelial ovarian cancers. MUC16/CA125 contributes to the evasion of anti-tumor immunity, peritoneal spread and promotes carcinogenesis; consequently, it has been targeted with antibody-based passive and active immunotherapy. However, vaccination against this self-antigen likely requires breaking B cell tolerance and may trigger autoimmune disease. Display of self-antigens on virus-like particles (VLPs), including those produced with human papillomavirus (HPV) L1, can efficiently break B cell tolerance. RESULTS: A 20 aa juxta-membrane peptide of the murine MUC16 (mMUC16) or human MUC16 (hMUC16) ectodomain was displayed either via genetic insertion into an immunodominant loop of HPV16 L1-VLPs between residues 136/137, or by chemical coupling using malemide to cysteine sulfhydryl groups on their surface. Female mice were vaccinated intramuscularly three times with either DNA expressing L1-MUC16 fusions via electroporation, or with alum-formulated VLP chemically-coupled to MUC16 peptides. Both regimens were well tolerated, and elicited MUC16-specific serum IgG, although titers were higher in mice vaccinated with MUC16-coupled VLP on alum as compared to L1-MUC16 DNA vaccination. Antibody responses to mMUC16-targeted vaccination cross-reacted with hMUC16 peptide, and vice versa; both were reactive with the surface of CA125+ OVCAR3 cells, but not SKOV3 that lack detectable CA125 expression. Interestingly, vaccination of mice with mMUC16 peptide mixed with VLP and alum elicited mMUC16-specific IgG, implying VLPs provide robust T help and that coupling may not be required to break tolerance to this epitope. CONCLUSION: Vaccination with VLP displaying the 20 aa juxta-membrane MUC16 ectodomain, which includes the membrane proximal cleavage site, is likely to be well tolerated and induce IgG targeting ovarian cancer cells, even after CA125 is shed.

Mus musculus papillomavirus 1 E8

High-risk human papillomaviruses (PV) account for approximately 600,000 new cancers per year. The early protein E8^E2 is a conserved repressor of PV replication, whereas E4 is a late protein that arrests cells in G2 and collapses keratin filaments to facilitate virion release. While inactivation of the Mus musculus PV1 (MmuPV1) E8 start codon (E8-) increases viral gene expression, surprisingly, it prevents wart formation in FoxN1nu/nu mice. To understand this surprising phenotype, the impact of additional E8^E2 mutations was characterized in tissue culture and mice. MmuPV1 and HPV E8^E2 similarly interact with cellular NCoR/SMRT-HDAC3 co-repressor complexes. Disruption of the splice donor sequence used to generate the E8^E2 transcript or E8^E2 mutants (mt) with impaired binding to NCoR/SMRT-HDAC3 activates MmuPV1 transcription in murine keratinocytes. These MmuPV1 E8^E2 mt genomes also fail to induce warts in mice. The phenotype of E8^E2 mt genomes in undifferentiated cells resembles productive PV replication in differentiated keratinocytes. Consistent with this, E8^E2 mt genomes induced aberrant E4 expression in undifferentiated keratinocytes. In line with observations for HPV, MmuPV1 E4-positive cells displayed a shift to the G2 phase of the cell cycle. In summary, we propose that in order to enable both expansion of infected cells and wart formation in vivo, MmuPV1 E8^E2 inhibits E4 protein expression in the basal keratinocytes that would otherwise undergo E4-mediated cell cycle arrest. IMPORTANCE Human papillomaviruses (PVs) initiate productive replication, which is characterized by genome amplification and expression of E4 protein strictly within suprabasal, differentiated keratinocytes. Mus musculus PV1 mutants that disrupt splicing of the E8^E2 transcript or abolish the interaction of E8^E2 with cellular NCoR/SMRT-HDAC3 co-repressor complexes display increased gene expression in tissue culture but are unable to form warts in vivo. This confirms that the repressor activity of E8^E2 is required for tumor formation and genetically defines a conserved E8 interaction domain. E8^E2 prevents expression of E4 protein in basal-like, undifferentiated keratinocytes and thereby their arrest in G2 phase. Since binding of E8^E2 to NCoR/SMRT-HDAC3 co-repressor is required to enable expansion of infected cells in the basal layer and wart formation in vivo, this interaction represents a novel, conserved, and potentially druggable target.

Aberrant DNA and RNA Methylation Occur in Spinal Cord and Skeletal Muscle of Human SOD1 Mouse Models of ALS and in Human ALS: Targeting DNA Methylation Is Therapeutic.

Amyotrophic lateral sclerosis (ALS) is a fatal disease. Skeletal muscles and motor neurons (MNs) degenerate. ALS is a complex disease involving many genes in multiple tissues, the environment, cellular metabolism, and lifestyles. We hypothesized that epigenetic anomalies in DNA and RNA occur in ALS and examined this idea in: (1) mouse models of ALS, (2) human ALS, and (3) mouse ALS with therapeutic targeting of DNA methylation. Human superoxide dismutase-1 (hSOD1) transgenic (tg) mice were used. They expressed nonconditionally wildtype (WT) and the G93A and G37R mutant variants or skeletal muscle-restricted WT and G93A and G37R mutated forms. Age-matched non-tg mice were controls. hSOD1 mutant mice had increased DNA methyltransferase enzyme activity in spinal cord and skeletal muscle and increased 5-methylcytosine (5mC) levels. Genome-wide promoter CpG DNA methylation profiling in skeletal muscle of ALS mice identified hypermethylation notably in cytoskeletal genes. 5mC accumulated in spinal cord MNs and skeletal muscle satellite cells in mice. Significant increases in DNA methyltransferase-1 (DNMT1) and DNA methyltransferase-3A (DNMT3A) levels occurred in spinal cord nuclear and chromatin bound extracts of the different hSOD1 mouse lines. Mutant hSOD1 interacted with DNMT3A in skeletal muscle. 6-methyladenosine (6mA) RNA methylation was markedly increased or decreased in mouse spinal cord depending on hSOD1-G93A model, while fat mass and obesity associated protein was depleted and methyltransferase-like protein 3 was increased in spinal cord and skeletal muscle. Human ALS spinal cord had increased numbers of MNs and interneurons with nuclear 5mC, motor cortex had increased 5mC-positive neurons, while 6mA was severely depleted. Treatment of hSOD1-G93A mice with DNMT inhibitor improved motor function and extended lifespan by 25%. We conclude that DNA and RNA epigenetic anomalies are prominent in mouse and human ALS and are potentially targetable for disease-modifying therapeutics.

Vaccination with human alphapapillomavirus-derived L2 multimer protects against human betapapillomavirus challenge, including in epidermodysplasia verruciformis model mice.

Human alphapapillomaviruses (αHPV) infect genital mucosa, and a high-risk subset is a necessary cause of cervical cancer. Licensed L1 virus-like particle (VLP) vaccines offer immunity against the nine most common αHPV associated with cervical cancer and genital warts. However, vaccination with an αHPV L2-based multimer vaccine, α11-88x5, protected mice and rabbits from vaginal and skin challenge with diverse αHPV types. While generally clinically inapparent, human betapapillomaviruses (ßHPV) are possibly associated with cutaneous squamous cell carcinoma (CSCC) in epidermodysplasia verruciformis (EV) and immunocompromised patients. Here we show that α11-88x5 vaccination protected wild type and EV model mice against HPV5 challenge. Passive transfer of antiserum conferred protection independently of Fc receptors (FcR) or Gr-1+ phagocytes. Antisera demonstrated robust antibody titers against ten ßHPV by L1/L2 VLP ELISA and neutralized and protected against challenge by 3 additional ßHPV (HPV49/76/96). Thus, unlike the licensed vaccines, α11-88x5 vaccination elicits broad immunity against αHPV and ßHPV.

RG2-VLP: a Vaccine Designed to Broadly Protect against Anogenital and Skin Human Papillomaviruses Causing Human Cancer.

The family of human papillomaviruses (HPV) includes over 400 genotypes. Genus α genotypes generally infect the anogenital mucosa, and a subset of these HPV are a necessary, but not sufficient, cause of cervical cancer. Of the 13 high-risk (HR) and 11 intermediate-risk (IR) HPV associated with cervical cancer, genotypes 16 and 18 cause 50% and 20% of cases, respectively, whereas HPV16 dominates in other anogenital and oropharyngeal cancers. A plethora of ßHPVs are associated with cutaneous squamous cell carcinoma (CSCC), especially in sun-exposed skin sites of epidermodysplasia verruciformis (EV), AIDS, and immunosuppressed patients. Licensed L1 virus-like particle (VLP) vaccines, such as Gardasil 9, target a subset of αHPV but no ßHPV. To comprehensively target both α- and ßHPVs, we developed a two-component VLP vaccine, RG2-VLP, in which L2 protective epitopes derived from a conserved αHPV epitope (amino acids 17 to 36 of HPV16 L2) and a consensus ßHPV sequence in the same region are displayed within the DE loop of HPV16 and HPV18 L1 VLP, respectively. Unlike vaccination with Gardasil 9, vaccination of wild-type and EV model mice (Tmc6Δ/Δ or Tmc8Δ/Δ) with RG2-VLP induced robust L2-specific antibody titers and protected against ß-type HPV5. RG2-VLP protected rabbits against 17 αHPV, including those not covered by Gardasil 9. HPV16- and HPV18-specific neutralizing antibody responses were similar between RG2-VLP- and Gardasil 9-vaccinated animals. However, only transfer of RG2-VLP antiserum effectively protected naive mice from challenge with all ßHPVs tested. Taken together, these observations suggest RG2-VLP's potential as a broad-spectrum vaccine to prevent αHPV-driven anogenital, oropharyngeal, and ßHPV-associated cutaneous cancers. IMPORTANCE Licensed preventive HPV vaccines are composed of VLPs derived by expression of major capsid protein L1. They confer protection generally restricted to infection by the αHPVs targeted by the up-to-9-valent vaccine, and their associated anogenital cancers and genital warts, but do not target ßHPV that are associated with CSCC in EV and immunocompromised patients. We describe the development of a two-antigen vaccine protective in animal models against known oncogenic αHPVs as well as diverse ßHPVs by incorporation into HPV16 and HPV18 L1 VLP of 20-amino-acid conserved protective epitopes derived from minor capsid protein L2.

The impact of antenatal massage practice on intrapartum massage application and their associations with the use of analgesics during labour : Sub-analysis of a randomised control trial.

BACKGROUND: Massage during labour is one form of intrapartum non-pharmacological pain relief but it is not known whether the frequency of practicing these massage techniques among couples during the antenatal period could enhance the effectiveness of intrapartum massage. This study was to evaluate the association between compliance of antenatal massage practice with intrapartum application and their impact on the use of analgesics during labour. METHODS: This was a sub-analysis of a childbirth massage programme which was carried out in two public hospitals with total births of around 8000 per year. Data from women who were randomized to the massage group were further analysed. After attending the pre-birth training class on massage at 36 weeks gestation, couples would be encouraged to practice at home. Their compliance with massage at home was classified as good if they had practiced for at least 15 minutes for three or more days in a week, or as poor if the three-day threshold had not been reached. Application of intrapartum massage was quantified by the duration of practice divided by the total duration of the first stage of labour. Women's application of intrapartum massage were then divided into above and below median levels according to percentage of practice. Logistic regression was used to assess the use of epidural analgesia or pethidine, adjusted for duration of labour and gestational age when attending the massage class. RESULTS: Among the 212 women included, 103 women (48.6%) achieved good home massage compliance. No significant difference in the maternal characteristics or birth outcomes was observed between the good and poor compliance groups. The intrapartum massage application (median 21.1%) was inversely associated with duration of first stage of labour and positively associated with better home massage practice compliance (p = 0.04). Lower use of pethidine or epidural analgesia (OR 0.33 95% CI 0.12, 0.90) was associated with above median intrapartum massage application but not antenatal massage compliance, adjusted for duration of first stage of labour. CONCLUSIONS: More frequent practice of massage techniques among couples during antenatal period could enhance the intrapartum massage application, which may reduce the use of pethidine and epidural analgesia. TRIAL REGISTRATION: (CCRBCTR) Unique Trial Number CUHK_ CCRB00525 .

Non-small cell lung cancer with iris metastasis controlled with osimertinib and monthly intravitreal bevacizumab.

PURPOSE: Iris metastases from lung cancer occur rarely. Current treatment options such as surgical iridectomy or radiotherapy are invasive and can potentially lead to negative side effects. Other less invasive alternatives include chemotherapy and intracameral bevacizumab. OBSERVATIONS: An 81 year-old female with metastatic non-small cell lung adenocarcinoma to the iris in the right eye was treated with daily oral osimertinib 80mg, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), for her systemic lung cancer. In addition, 8 monthly intravitreal bevacizumab (1.25mg/0.05 cc) injections were given. The iris tumor demonstrated complete regression by the third cycle of osimertinib. Following 21 months of osimertinib and 8 bevacizumab injections, the tumor remained regressed. Subsequent iris biopsy confirmed complete tumor regression. CONCLUSIONS AND IMPORTANCE: In this case, osimertinib and monthly intravitreal bevacizumab controlled iris metastasis due to non-small cell lung cancer. Osimertinib can be beneficial for patients with EGFR-positive lung cancer for both ocular and systemic control.

Chronic Intermittent Mild Whole-Body Hypothermia Is Therapeutic in a Mouse Model of ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that causes motor neuron degeneration. There are no cures or effective treatments for ALS. Therapeutic hypothermia is effectively used clinically to mitigate mortality in patients with acute acquired brain injury and in surgical settings to minimize secondary brain injury. The efficacy of therapeutic hypothermia in chronic neurodegenerative disorders has not been examined. We tested the hypothesis that mild hypothermia/cold acclimation is therapeutic in a transgenic mouse model of ALS caused by expression of mutated human superoxide dismutase-1 gene. At presymptomatic stages of disease, body temperatures (oral and axial) of mutant male mice were persistently hyperthermic (38-38.5 °C) compared to littermate controls, but at end-stage disease mice were generally hypothermic (36-36.5 °C). Presymptomatic mutant mice (awake-freely moving) were acclimated to systemic mild hypothermia using an environmentally controlled chamber (12 h-on/12-off or 24 h-on/24 h-off) to lower body temperature (1-3 °C). Cooled ALS mice showed a significant delay in disease onset (103-112 days) compared to normothermia mice (80-90 days) and exhibited significant attenuation of functional decline in motor performance. Cooled mice examined at 80 days had reduced motor neuron loss, mitochondrial swelling, and spinal cord inflammation compared to non-cooled mice. Cooling attenuated the loss of heat-shock protein 70, mitochondrial uncoupling protein-3, and sumoylated-1 (SUMO1)-conjugated proteins in skeletal muscle and disengaged the mitochondrial permeability transition pore. Cooled ALS mice had a significant extension of lifespan (148 ± 7 days) compared to normothermic mice (135 ± 4 days). Thus, intermittent systemic mild hypothermia is therapeutic in mouse ALS with protective effects manifested within the CNS and skeletal muscle that target mitochondria.

EXPRESSION OF E8

Human papillomavirus (HPV) E1 and E2 proteins activate genome replication. E2 also modulates viral gene expression and is involved in the segregation of viral genomes. In addition to full length E2, almost all PV share the ability to encode an E8^E2 protein, that is a fusion of E8 with the C-terminal half of E2 which mediates specific DNA-binding and dimerization. HPV E8^E2 acts as a repressor of viral gene expression and genome replication. To analyze the function of E8^E2 in vivo, we used the Mus musculus PV1 (MmuPV1)-mouse model system. Characterization of the MmuPV1 E8^E2 protein revealed that it inhibits transcription from viral promoters in the absence and presence of E1 and E2 proteins and that this is partially dependent upon the E8 domain. MmuPV1 genomes, in which the E8 ATG start codon was disrupted (E8-), displayed a 10- to 25-fold increase in viral gene expression compared to wt genomes in cultured normal mouse tail keratinocytes in short-term experiments. This suggests that the function and mechanism of E8^E2 is conserved between MmuPV1 and HPVs. Surprisingly, challenge of athymic nude Foxn1nu/nu mice with MmuPV1 E8- genomes did not induce warts on the tail in contrast to wt MmuPV1. Furthermore, viral gene expression was completely absent at E8- MmuPV1 sites 20 - 22 weeks after DNA challenge on the tail or quasivirus challenge in the vaginal vault. This reveals that expression of E8^E2 is necessary to form tumors in vivo and that this is independent from the presence of T-cells.IMPORTANCE HPV encode an E8^E2 protein which acts as repressors of viral gene expression and genome replication. In cultured normal keratinocytes, E8^E2 is essential for long-term episomal maintenance of HPV31 genomes, but not for HPV16. To understand E8^E2's role in vivo, the Mus musculus PV1 (MmuPV1)-mouse model system was used. This revealed that E8^E2's function as a repressor of viral gene expression is conserved. Surprisingly, MmuPV1 E8^E2 knock out genomes did not induce warts in T-cell deficient mice. This shows for the first time that expression of E8^E2 is necessary for tumor formation in vivo independently of T cell immunity. This indicates that E8^E2 could be an interesting target for anti-viral therapy in vivo.

Skeletal Muscle-Restricted Expression of Human SOD1 in Transgenic Mice Causes a Fatal ALS-Like Syndrome.

Amyotrophic lateral sclerosis (ALS) is a fatal heterogeneous neurodegenerative disease that causes motor neuron (MN) loss and skeletal muscle paralysis. It is uncertain whether this degeneration of MNs is triggered intrinsically and is autonomous, or if the disease initiating mechanisms are extrinsic to MNs. We hypothesized that skeletal muscle is a primary site of pathogenesis in ALS that triggers MN degeneration. Some inherited forms of ALS are caused by mutations in the superoxide dismutase-1 (SOD1) gene, that encodes an antioxidant protein, so we created transgenic (tg) mice expressing wild-type-, G37R-, and G93A-human SOD1 gene variants only in skeletal muscle. Presence of human SOD1 (hSOD1) protein in skeletal muscle was verified by western blotting, enzyme activity gels, and immunofluorescence in myofibers and satellite cells. These tg mice developed limb weakness and paresis with motor deficits, limb and chest muscle wasting, diaphragm atrophy, and age-related fatal disease with a lifespan shortening of 10-16%. Brown and white adipose tissue also became wasted. Myofibers of tg mice developed crystalline-like inclusions, individualized sarcomere destruction, mitochondriopathy with vesiculation, DNA damage, and activated p53. Satellite cells became apoptotic. The diaphragm developed severe loss of neuromuscular junction presynaptic and postsynaptic integrity, including decreased innervation, loss of synaptophysin, nitration of synaptophysin, and loss of nicotinic acetylcholine receptor and scaffold protein rapsyn. Co-immunoprecipitation identified hSOD1 interaction with rapsyn. Spinal cords of tg mice developed gross atrophy. Spinal MNs formed cytoplasmic and nuclear inclusions, axonopathy, mitochondriopathy, accumulated DNA damage, activated p53 and cleaved caspase-3, and died. Tg mice had a 40-50% loss of MNs. This work shows that hSOD1 in skeletal muscle is a driver of pathogenesis in ALS, that involves myofiber and satellite cell toxicity, and apparent muscle-adipose tissue disease relationships. It also identifies a non-autonomous mechanism for MN degeneration explaining their selective vulnerability as likely a form of target-deprivation retrograde neurodegeneration.

Insights into the Role of Innate Immunity in Cervicovaginal Papillomavirus Infection from Studies Using Gene-Deficient Mice.

We demonstrate that female C57BL/6J mice are susceptible to a transient lower genital tract infection with MmuPV1 mouse papillomavirus and display focal histopathological abnormalities resembling those of human papillomavirus (HPV) infection. We took advantage of strains of genetically deficient mice to study in vivo the role of innate immune signaling in the control of papillomavirus. At 4 months, we sacrificed MmuPV1-infected mice and measured viral 757/3139 spliced transcripts by TaqMan reverse transcription-PCR (RT-PCR), localization of infection by RNAscope in situ hybridization, and histopathological abnormities by hematoxylin and eosin (H&E) staining. Among mice deficient in receptors for pathogen-associated molecular patterns, MyD88-/- and STING-/- mice had 1,350 and 80 copies of spliced transcripts/µg RNA, respectively, while no viral expression was detected in MAVS-/- and Ripk2-/- mice. Mice deficient in an adaptor molecule, STAT1-/-, for interferon signaling had 46,000 copies/µg RNA. Among mice with targeted deficiencies in the inflammatory response, interleukin-1 receptor knockout (IL-1R-/-) and caspase-1-/- mice had 350 and 30 copies/µg RNA, respectively. Among mice deficient in chemokine receptors, CCR6-/- mice had 120 copies/µg RNA, while CXCR2-/- and CXCR3-/- mice were negative. RNAscope confirmed focal infection in MyD88-/-, STAT1-/-, and CCR6-/- mice but was negative for other gene-deficient mice. Histological abnormalities were seen only in the latter mice. Our findings and the literature support a working model of innate immunity to papillomaviruses involving the activation of a MyD88-dependent pathway and IL-1 receptor signaling, control of viral replication by interferon-stimulated genes, and clearance of virus-transformed dysplastic cells by the action of the CCR6/CCL20 axis.IMPORTANCE Papillomaviruses infect stratified squamous epithelia, and the viral life cycle is linked to epithelial differentiation. Additionally, changes occur in viral and host gene expression, and immune cells are activated to modulate the infectious process. In vitro studies with keratinocytes cannot fully model the complex viral and host responses and do not reflect the contribution of local and migrating immune cells. We show that female C57BL/6J mice are susceptible to a transient papillomavirus cervicovaginal infection, and mice deficient in select genes involved in innate immune responses are susceptible to persistent infection with variable manifestations of histopathological abnormalities. The results of our studies support a working model of innate immunity to papillomaviruses, and the model provides a framework for more in-depth studies. A better understanding of mechanisms of early viral clearance and the development of approaches to induce clearance will be important for cancer prevention and the treatment of HPV-related diseases.

Ileocolic vascular curvature: a new CT finding of cecal volvulus.

PURPOSE: To evaluate the diagnostic performance of a proposed new CT observation in cecal volvulus, marked hook-like curvature of ileocolic vessels termed "ileocolic vascular curvature." METHODS: Contrast-enhanced CT scans of 14 patients with diagnoses of cecal volvulus on original CT reports were compared with scans of 40 control patients with dilated cecum but no cecal volvulus on original CT reports, accrued consecutively from January, 2006 through July, 2017. Two independent blinded readers retrospectively evaluated scans for cecal dilatation, ileocolic vascular curvature and seven previously reported CT features of cecal volvulus. Statistical methods included the exact binomial distribution to define 95% confidence intervals (95% CIs), logistic regression, receiver operating characteristic analysis, and the exact McNemar test. Pathology reports and clinical records served as reference standards. RESULTS: All patients had abdominal pain and cecal distension. All 14 patients with CT diagnoses of cecal volvulus, and no control patients, had cecal volvulus by reference standards. Ileocolic vascular curvature and ectopic cecal location were the only features independently and significantly associated with cecal volvulus in multivariable regression (odds ratio 178, p = 0.014, and 63, p = 0.013, respectively) and also the only features with both sensitivity (12/14 [85.7%, 95% CI 57.2-98.2%] and 13/14 [92.9%, 95% CI 66.1-99.8%], respectively) and specificity (40/40 [100.0%, 95% CI 91.2-100.0%] and 38/40 [95.0%, 95% CI 83.1-99.4%], respectively) that differed significantly from 50.0%. CONCLUSIONS: Ileocolic vascular curvature was independently and significantly associated with cecal volvulus and exhibited both substantial sensitivity and substantial specificity for cecal volvulus, and thus is potentially a valuable new CT finding of cecal volvulus.

DNA damage accumulates and responses are engaged in human ALS brain and spinal motor neurons and DNA repair is activatable in iPSC-derived motor neurons with SOD1 mutations.

DNA damage is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). However, relationships between DNA damage accumulation, DNA damage response (DDR), and upper and lower motor neuron vulnerability in human ALS are unclear; furthermore, it is unknown whether epigenetic silencing of DNA repair pathways contributes to ALS pathogenesis. We tested the hypotheses that DNA damage accumulates in ALS motor neurons along with diminished DDR, and that DNA repair genes undergo hypermethylation. Human postmortem CNS tissue was obtained from ALS cases (N = 34) and age-matched controls without neurologic disease (N = 15). Compared to age-matched controls, abasic sites accumulated in genomic DNA of ALS motor cortex and laser capture microdissection-acquired spinal motor neurons but not in motor neuron mitochondrial DNA. By immunohistochemistry, DNA damage accumulated significantly in upper and lower motor neurons in ALS cases as single-stranded DNA and 8-hydroxy-deoxyguanosine (OHdG) compared to age-matched controls. Significant DDR was engaged in ALS motor neurons as evidenced by accumulation of c-Abl, nuclear BRCA1, and ATM activation. DNA damage and DDR were present in motor neurons at pre-attritional stages and throughout the somatodendritic attritional stages of neurodegeneration. Motor neurons with DNA damage were also positive for activated p53 and cleaved caspase-3. Gene-specific promoter DNA methylation pyrosequencing identified the DNA repair genes Ogg1, Apex1, Pnkp and Aptx as hypomethylated in ALS. In human induced-pluripotent stem cell (iPSC)-derived motor neurons with familial ALS SOD1 mutations, DNA repair capacity was similar to isogenic control motor neurons. Our results show that vulnerable neurons in human ALS accumulate DNA damage, and contrary to our hypothesis, strongly activate and mobilize response effectors and DNA repair genes. This DDR in ALS motor neurons involves recruitment of c-Abl and BRCA1 to the nucleus in vivo, and repair of DNA double-strand breaks in human ALS motor neurons with SOD1 mutations in cell culture.

Neonatal Brain Injury and Genetic Causes of Adult-Onset Neurodegenerative Disease in Mice Interact With Effects on Acute and Late Outcomes.

Neonatal brain damage and age-related neurodegenerative disease share many common mechanisms of injury involving mitochondriopathy, oxidative stress, excitotoxicity, inflammation, and neuronal cell death. We hypothesized that genes causing adult-onset neurodegeneration can influence acute outcome after CNS injury at immaturity and on the subsequent development of chronic disability after early-life brain injury. In two different transgenic (Tg) mouse models of adult-onset neurodegenerative disease, a human A53T-α-synuclein (hαSyn) model of Parkinson's disease (PD) and a human G93A-superoxide dismutase-1(hSOD1) model of amyotrophic lateral sclerosis (ALS), mortality and survivor morbidity were significantly greater than non-Tg mice and a Tg mouse model of Alzheimer's disease after neonatal traumatic brain injury (TBI). Acutely after brain injury, hαSyn neonatal mice showed a marked enhancement of protein oxidative damage in forebrain, brain regional mitochondrial oxidative metabolism, and mitochondriopathy. Extreme protein oxidative damage was also observed in neonatal mutant SOD1 mice after TBI. At 1 month of age, neuropathology in forebrain, midbrain, and brainstem of hαSyn mice with neonatal TBI was greater compared to sham hαSyn mice. Surviving hαSyn mice with TBI showed increased hαSyn aggregation and nitration and developed adult-onset disease months sooner and died earlier than non-injured hαSyn mice. Surviving hSOD1 mice with TBI also developed adult-onset disease and died sooner than non-injured hSOD1 mice. We conclude that mutant genes causing PD and ALS in humans have significant impact on mortality and morbidity after early-life brain injury and on age-related disease onset and proteinopathy in mice. This study provides novel insight into genetic determinants of poor outcomes after acute injury to the neonatal brain and how early-life brain injury can influence adult-onset neurodegenerative disease during aging.

Birth ball for pregnant women in labour research protocol: a multi-centre randomised controlled trial.

BACKGROUND: Birth ball is one of the non-pharmacologic pain relief methods to help mothers cope with the labouring process. A randomised controlled trial (RCT) is conducted to evaluate the effectiveness, safety and harm of birth ball use by pregnant women in labour compared to treatment as usual group. METHODS: A prospective multi-centre randomised controlled trial (RCT) will be conducted in Obstetrics and Gynaecological units of five public hospitals in Hong Kong, China. Data will be collected from March 2016 onward for 2 years. The target population is Chinese women with an uncomplicated singleton pregnancy at gestational age of 37 to 42 weeks. Participants are randomised based on parity (nulliparous and multiparous) and type of labour onset (spontaneous and induced). Women in the intervention group are actively offered and taught how to use a birth ball; those in the control group receive the usual midwifery care. The target sample size is 512. The primary outcome measures are maternal pain intensity, satisfaction with pain relief, sense of control in labour, assisted delivery and satisfaction with childbirth experience. Labour pain relief is measured by visual analogue scale (VAS). Other outcomes will be measured through four different validated questionnaires. To control for potential cluster effects, a linear mixed model will be used. An intention-to-treat analysis is adopted and performed by researchers unknown to subjects' group allocation. DISCUSSION: Results will provide rigorous scientific evidence for policy development and practice. We are using stratified randomisation according to potential confounders of parity and type of labour onset to give four possible combinations. If the results are favourable, it will facilitate systematic implementation to promote birth ball use for women in labour. TRIAL REGISTRATION: Chinese Clinical Trial Register (ChiCTR), Registration number: ChiCTR-IIC-16008275 , Date of registration 12 April 2016 (retrospectively registered), Date of enrolment of the first participant to the trial 1 March 2016.

Roles of Fc Domain and Exudation in L2 Antibody-Mediated Protection against Human Papillomavirus.

To address how L2-specific antibodies prevent human papillomavirus (HPV) infection of the genital tract, we generated neutralizing monoclonal antibodies (MAbs) WW1, a rat IgG2a that binds L2 residues 17 to 36 (like mouse MAb RG1), and JWW3, a mouse IgG2b derivative of Mab24 specific for L2 residues 58 to 64. By Western blotting, WW1 recognized L2 of 29/34 HPV genotypes tested, compared to only 13/34 for RG1 and 25/34 for JWW3. WW1 IgG and F(ab')2 bound HPV16 pseudovirions similarly; however, whole IgG provided better protection against HPV vaginal challenge. Passive transfer of WW1 IgG was similarly protective in wild-type and neonatal Fc receptor (FcRn)-deficient mice, suggesting that protection by WW1 IgG is not mediated by FcRn-dependent transcytosis. Rather, local epithelial disruption, required for genital infection and induced by either brushing or nonoxynol-9 treatment, released serum IgG in the genital tract, suggesting Fc-independent exudation. Depletion of neutrophils and macrophages reduced protection of mice upon passive transfer of whole WW1 or JWW3 IgGs. Similarly, IgG-mediated protection by L2 MAbs WW1, JWW3, and RG1 was reduced in Fc receptor knockout compared to wild-type mice. However, levels of in vitro neutralization by WW1 IgG were similar in TRIM21 knockout and wild-type cells, indicating that Fc does not contribute to antibody-dependent intracellular neutralization (ADIN). In conclusion, the Fc domain of L2-specific IgGs is not active for ADIN, but it opsonizes bound extracellular pseudovirions for phagocytes in protecting mice from intravaginal HPV challenge. Systemically administered neutralizing IgG can access the site of infection in an abrasion via exudation without the need for FcRn-mediated transcytosis.IMPORTANCE At least 15 alpha HPV types are causative agents for 5% of all cancers worldwide, and beta types have been implicated in nonmelanoma skin cancer, whereas others produce benign papillomas, such as genital warts, associated with considerable morbidity and health systems costs. Vaccines targeting the minor capsid protein L2 have the potential to provide broad-spectrum immunity against medically relevant HPVs of divergent genera via the induction of broadly cross-neutralizing serum IgG. Here we examine the mechanisms by which L2-specific serum IgG reaches the viral inoculum in the genital tract to effect protection. Abrasion of the vaginal epithelium allows the virus to access and infect basal keratinocytes, and our findings suggest that this also permits the local exudation of neutralizing IgG and vaccine-induced sterilizing immunity. We also demonstrate the importance of Fc-mediated phagocytosis of L2 antibody-virion complexes for humoral immunity, a protective mechanism that is not detected by current in vitro neutralization assays.

Ciliary body metastasis from renal cell carcinoma successfully treated with intravitreal bevacizumab.

PURPOSE: To report an innovative new treatment option for ciliary body tumor metastasis from renal cell carcinoma treated with intravitreal bevacizumab. OBSERVATIONS: A 70-year-old Caucasian man presented with a ciliary body tumor as a result from metastasis from renal cell carcinoma. It was preferable to treat the ciliary body tumor in the least invasive manner possible based on the patient's health, systemic metastasis, and the invasiveness of local resection. The tumor was treated with intravitreal bevacizumab for attempted tumor regression. Complete tumor resolution occurred following three injections. CONCLUSIONS AND IMPORTANCE: There is no consensus on treatment for ciliary body metastases from renal cell carcinoma. Based on the successful result of our patient, intravitreal bevacizumab could be an acceptable treatment option for this type of intraocular tumor. It affords a relatively non-invasive method of tumor regression without undergoing major intraocular surgery.

Traumatic reopening of macular hole following successful surgical repair.

A 75-year-old Caucasian female with a full-thickness macular hole underwent a successful surgical repair. Vision improved from 20/400 to 20/30 after surgery. Two years later, the patient presented with a decreased vision after a fall onto her face with a reopened full-thickness macular hole. Vision decreased to 20/400 and then she underwent a second surgery with an internal limiting membrane peeling, which resulted in a second successful closure of her macular hole. Vision following the second surgery was 20/100. Reopening of a surgically repaired macular hole commonly follows cataract surgery, epiretinal membrane formation, and development of cystoid macular edema. This case demonstrates that surgically repaired macular holes may reopen after trauma as well.

Enforced DNA repair enzymes rescue neurons from apoptosis induced by target deprivation and axotomy in mouse models of neurodegeneration.

It is unknown whether DNA damage accumulation is an upstream instigator or secondary effect of the cell death process in different populations of adult postmitotic neurons in the central nervous system. In two different mouse models of injury-induced neurodegeneration characterized by relatively synchronous accumulation of mitochondria, oxidative stress, and DNA damage prior to neuronal apoptosis, we enforced the expression of human 8-oxoguanine DNA glycosylase (hOGG1) and human apurinic-apyrimidinic endonuclease-1/Ref1 (hAPE) using recombinant adenoviruses (Ad). Thalamic lateral geniculate neurons and lumbar spinal cord motor neurons were transduced by Ad-hOGG1 and Ad-hAPE injections into the occipital cortex and skeletal muscle, respectively, prior to their target deprivation- and axotomy-induced retrograde apoptosis. Enforced expression of hOGG1 and hAPE in thalamus and spinal cord was confirmed by western blotting and immunohistochemistry. In injured populations of neurons in thalamus and spinal cord, a DNA damage response (DDR) was registered, as shown by localization of phospho-activated p53, Rad17, and replication protein A-32 immunoreactivities, and this DDR was attenuated more effectively by enforced hAPE expression than by hOGG1 expression. Enforced expression of hOGG1 and hAPE significantly protected thalamic neurons and motor neurons from retrograde apoptosis induced by target deprivation and axotomy. We conclude that a DDR response is engaged pre-apoptotically in different types of injured mature CNS neurons and that DNA repair enzymes can regulate the survival of retrogradely dying neurons, suggesting that DNA damage and activation of DDR are upstream mechanisms for this form of adult neurodegeneration in vivo, thus identifying DNA repair as a therapeutic target for neuroprotection.