Fair governance of biotechnology : patents, private governance, and procedural justice

Published online: 14 Dec 201

Unexpected finding of uniparental disomy mosaicism in term placentas: Is it a common feature in trisomic placentas?

Objective: Non-invasive prenatal testing (NIPT) detects placental chromosome aberrations. When amniocentesis reveals a normal karyotype, confined placental mosaicism (CPM) may be assumed. In order to confirm this, placental cytogenetic studies were performed. Method: NIPT was conducted in the course of the Dutch TRIDENT study. Placentas of 10 cases with NIPT results indicating an autosomal trisomy and showing a normal (N = 9) or low mosaic karyotype (N = 1) in amniotic fluid (AF) were investigated. The cytotrophoblast as well as the mesenchymal core of two to four placental chorionic villi biopsies were studied with single nucleotide polymorphism (SNP) array. Clinical outcome data were collected. Results: In 10/10 cases, CPM was proven. In 3/10 cases trisomy/uniparental disomy (UPD)/biparental disomy (BPD) mosaicism was discovered. In 2/3 cases, all three cell lines were present in the placenta, whereas BPD was found in AF. In 1/3 cases trisomy 22/UPD22 was present in AF while trisomy 22/BPD22 mosaicism was found in the placenta. Five of 10 pregnancies were affected with pre-eclampsia, low birth weight, preterm delivery, and/or congenital malformations. Conclusion: The presence of trisomy/UPD/BPD mosaicism in 3/10 cases that we investigated proves that trisomic zygote rescue may involve multiple rescue events during early embryogenesis. UPD mosaicism, when present in crucial fetal tissues, may explain the abnormal phenotype in undiagnosed cases

Complement Component C1q as Serum Biomarker to Detect Active Tuberculosis.

Background: Tuberculosis (TB) remains a major threat to global health. Currently, diagnosis of active TB is hampered by the lack of specific biomarkers that discriminate active TB disease from other (lung) diseases or latent TB infection (LTBI). Integrated human gene expression results have shown that genes encoding complement components, in particular different C1q chains, were expressed at higher levels in active TB compared to LTBI. Methods: C1q protein levels were determined using ELISA in sera from patients, from geographically distinct populations, with active TB, LTBI as well as disease controls. Results: Serum levels of C1q were increased in active TB compared to LTBI in four independent cohorts with an AUC of 0.77 [0.70; 0.83]. After 6 months of TB treatment, levels of C1q were similar to those of endemic controls, indicating an association with disease rather than individual genetic predisposition. Importantly, C1q levels in sera of TB patients were significantly higher as compared to patients with sarcoidosis or pneumonia, clinically important differential diagnoses. Moreover, exposure to other mycobacteria, such as Mycobacterium leprae (leprosy patients) or BCG (vaccinees) did not result in elevated levels of serum C1q. In agreement with the human data, in non-human primates challenged with Mycobacterium tuberculosis, increased serum C1q levels were detected in animals that developed progressive disease, not in those that controlled the infection. Conclusions: In summary, C1q levels are elevated in patients with active TB compared to LTBI in four independent cohorts. Furthermore, C1q levels from patients with TB were also elevated compared to patients with sarcoidosis, leprosy and pneumonia. Additionally, also in NHP we observed increased C1q levels in animals with active progressive TB, both in serum and in broncho-alveolar lavage. Therefore, we propose that the addition of C1q to current biomarker panels may provide added value in the diagnosis of active TB

Exploring protective and pathogenic immune responses in the non-human primate model of tuberculosis

To this day, tuberculosis remains a complex global health problem, the elimination of which is hindered by knowledge gaps related to protective immunity and diagnosis. In an effort to address these knowledge gaps, this thesis utilized the non-human primate model of tuberculosis to investigate protective and pathogenic immune responses after vaccination and Mycobaterium tuberculosis (Mtb) infection, as well as the dynamics of a potential biomarker of active tuberculosis (TB) disease. First, by applying a dose-escalation study design, we showed that rhesus and cynomolgus macaques are not dissimilar in their susceptibility to infection. However, also at low challenge doses, cynomolgus macaques remained more resistant to the development of TB pathology than rhesus macaques. We used this differential disease susceptibility to characterize early and local immune responses that may underlie tuberculosis disease development and resistance. An early, local proinflammatory innate immune response was observed in cynomolgus macaques, while rhesus macaques presented with increased expression of markers associated with immune suppression. We next built upon of the observation that administration of 1 CFU of Mtb resulted in infection in a subset of exposed rhesus macaques. Intradermal or pulmonary BCG vaccinated rhesus macaques were subjected eight times to this limiting infectious dose to investigate whether we could assess vaccine-mediated prevention of infection, in addition to (partial) prevention of disease. We observed that pulmonary BCG vaccination was superior to intradermal BCG in preventing both infection and disease. This enhanced protection correlated with the induction of local, polyfunctional, IL17A producing T-cells and IL-10 production. Subsequently, we evaluated whether these correlates of protection would also be elicited by mucosal administration of MTBVAC, a novel, live attenuated, Mtb-derived TB vaccine. Like BCG, MTBVAC was found to induce local IL10 production and IL17A producing T-cells. These antigen-specific IL17A-producing T-cells were profiled more in depth, and we showed that cytokine-producing T-cells induced by vaccination display a distinct tissue-residency phenotype and express more mucosal homing markers. Cytokine producing T-cells induced by Mtb infection lacked this phenotype. Antibodies induced by both mucosal vaccination with BCG and MTBVAC were able to bind live Mtb and enhance pathogen uptake by phagocytes. Finally, we utilized biosamples of the studies described in this thesis as well as prior studies performed to assess the association of complement component C1q with TB disease severity. As observed in patients, C1q levels in serum were increased in animals with more TB pathology. In macaques, C1q levels correlated with the amount of pathology assessed post-mortem or by PET-CT imaging. Elevated C1q levels were detected from 6 weeks post-infection onward. An increase in C1q levels in BAL fluid and C1q production by BAL cells could be observed in Mtb infected, but also pulmonary BCG vaccinated animals. Taken together, the NHP studies described in this thesis have added to the model and contributed to our knowledge of protective TB immunity, thereby furthering the efforts towards elimination of TB

Exploring protective and pathogenic immune responses in the non-human primate model of tuberculosis

To this day, tuberculosis remains a complex global health problem, the elimination of which is hindered by knowledge gaps related to protective immunity and diagnosis. In an effort to address these knowledge gaps, this thesis utilized the non-human primate model of tuberculosis to investigate protective and pathogenic immune responses after vaccination and Mycobaterium tuberculosis (Mtb) infection, as well as the dynamics of a potential biomarker of active tuberculosis (TB) disease. First, by applying a dose-escalation study design, we showed that rhesus and cynomolgus macaques are not dissimilar in their susceptibility to infection. However, also at low challenge doses, cynomolgus macaques remained more resistant to the development of TB pathology than rhesus macaques. We used this differential disease susceptibility to characterize early and local immune responses that may underlie tuberculosis disease development and resistance. An early, local proinflammatory innate immune response was observed in cynomolgus macaques, while rhesus macaques presented with increased expression of markers associated with immune suppression. We next built upon of the observation that administration of 1 CFU of Mtb resulted in infection in a subset of exposed rhesus macaques. Intradermal or pulmonary BCG vaccinated rhesus macaques were subjected eight times to this limiting infectious dose to investigate whether we could assess vaccine-mediated prevention of infection, in addition to (partial) prevention of disease. We observed that pulmonary BCG vaccination was superior to intradermal BCG in preventing both infection and disease. This enhanced protection correlated with the induction of local, polyfunctional, IL17A producing T-cells and IL-10 production. Subsequently, we evaluated whether these correlates of protection would also be elicited by mucosal administration of MTBVAC, a novel, live attenuated, Mtb-derived TB vaccine. Like BCG, MTBVAC was found to induce local IL10 production and IL17A producing T-cells. These antigen-specific IL17A-producing T-cells were profiled more in depth, and we showed that cytokine-producing T-cells induced by vaccination display a distinct tissue-residency phenotype and express more mucosal homing markers. Cytokine producing T-cells induced by Mtb infection lacked this phenotype. Antibodies induced by both mucosal vaccination with BCG and MTBVAC were able to bind live Mtb and enhance pathogen uptake by phagocytes. Finally, we utilized biosamples of the studies described in this thesis as well as prior studies performed to assess the association of complement component C1q with TB disease severity. As observed in patients, C1q levels in serum were increased in animals with more TB pathology. In macaques, C1q levels correlated with the amount of pathology assessed post-mortem or by PET-CT imaging. Elevated C1q levels were detected from 6 weeks post-infection onward. An increase in C1q levels in BAL fluid and C1q production by BAL cells could be observed in Mtb infected, but also pulmonary BCG vaccinated animals. Taken together, the NHP studies described in this thesis have added to the model and contributed to our knowledge of protective TB immunity, thereby furthering the efforts towards elimination of TB

Fair Governance of Biotechnology : Patents, Private Governance, and Procedural Justice

Published online: 14 Dec 201

Abnormal non-invasive prenatal test results concordant with karyotype of cytotrophoblast but not reflecting abnormal fetal karyotype

We present a unique case in which non-invasive and invasive prenatal diagnoses showed abnormal, but discordant, results. A patient with abnormal non-invasive prenatal test (NIPT) results, indicating a 99% risk for monosomy X, was referred to our center for genetic counseling and confirmatory studies. Cytogenetic analysis of uncultured mesenchymal core of chorionic villi (CV) revealed a mosaic male karyotype consisting of two abnormal cell lines: one with monosomy X and the other with an isodicentric chromosome Y. Array analysis of the trophoblast confirmed the NIPT results. Based on the CV results, the patient opted for termination of pregnancy. After extensive counseling by a clinical geneticist about the possible outcomes and by a gynecologist about the risk of a second-trimester abortion procedure, the patient agreed to undergo early amniocentesis. Amniocentesis confirmed that the fetus had a male karyotype with an isodicentric chromosome Y, and the single nucleotide polymorphism (SNP) array profile suggested absence of the monosomy X cell line. The male infant was expected to be infertile. The patient finally decided to continue the pregnancy. Our case confirms that NIPT results are comparable with those of short-term cultured CV investigating the cytotrophoblast. Our patient was not aware that the NIPT results reveal the placental karyotype, which sometimes may be different from the fetal karyotype. Pretest counseling and providing the risk figures for false-positive and false-negative NIPT results are of great importance in order to discourage women from terminating pregnancies based on NIPT results alone. Copyright (C) 2014 ISUOG. Published by John Wiley & Sons Ltd

A protective, single-visit TB vaccination regimen by co-administration of a subunit vaccine with BCG

Abstract The only licensed tuberculosis (TB) vaccine, Bacillus Calmette Guerin (BCG), fails to reliably protect adolescents and adults from pulmonary TB, resulting in ~1.6 million deaths annually. Protein subunit vaccines have shown promise against TB in clinical studies. Unfortunately, most subunit vaccines require multiple administrations, which increases the risk of loss to follow-up and necessitates more complex and costly logistics. Given the well-documented adjuvant effect of BCG, we hypothesized that BCG co-administration could compensate for a reduced number of subunit vaccinations. To explore this, we developed an expression-optimized version of our H107 vaccine candidate (H107e), which does not cross-react with BCG. In the CAF®01 adjuvant, a single dose of H107e induced inferior protection compared to three H107e/CAF®01 administrations. However, co-administering a single dose of H107e/CAF®01 with BCG significantly improved protection, which was equal to BCG co-administered with three H107e/CAF®01 doses. Importantly, combining BCG with a single H107e/CAF®01 dose also increased protection in previously BCG-primed animals. Overall, a single dose of H107e/CAF®01 with BCG induced long-lived immunity and triggered BCG-specific Th17 responses. These data support co-administration of BCG and subunit vaccines in both BCG naïve and BCG-primed individuals as an improved TB vaccine strategy with reduced number of vaccination visits

Correction: selective blockade of CD28-mediated T cell costimulation protects rhesus monkeys against acute fatal experimental autoimmune encephalomyelitis

Costimulatory and coinhibitory receptor-ligand pairs on T cells and APC control the immune response. We have investigated whether selective blockade of CD28-CD80/86 costimulatory interactions, which preserves the coinhibitory CTLA4-CD80/86 interactions and the function of regulatory T (Treg) cells, abrogates the induction of experimental autoimmune encephalomyelitis (EAE) in rhesus monkeys. EAE was induced by intracutaneous immunization with recombinant human myelin oligodendrocyte glycoprotein (rhMOG) in CFA on day 0. FR104 is a monovalent, PEGylated-humanized Fab' Ab fragment against human CD28, cross-reactive with rhesus monkey CD28. FR104 or placebo was administered on days 0, 7, 14, and 21. FR104 levels remained high until the end of the study (day 42). Placebo-treated animals all developed clinical EAE between days 12 and 27. FR104-treated animals did not develop clinical EAE and were sacrificed at the end of the study resulting in a significantly prolonged survival. FR104 treatment diminished T and B cell responses against rhMOG, significantly reduced CNS inflammation and prevented demyelination. The inflammatory profile in the cerebrospinal fluid and brain material was also strongly reduced. Recrudescence of latent virus was investigated in blood, spleen, and brain. No differences between groups were observed for the beta-herpesvirus CMV and the polyomaviruses SV40 and SA12. Cross-sectional measurement of lymphocryptovirus, the rhesus monkey EBV, demonstrated elevated levels in the blood of FR104-treated animals. Blocking rhesus monkey CD28 with FR104 mitigated autoreactive T and B cell activation and prevented CNS pathology in the rhMOG/CFA EAE model in rhesus monkeys