Assessing spirometry competence through certification in community-based healthcare settings in Australia and New Zealand: A position paper of the Australian and New Zealand Society of Respiratory Science.

Spirometry has been established as an essential test for diagnosing and monitoring respiratory disease, particularly asthma and COPD, as well as in occupational health surveillance. In Australia and New Zealand, there is currently no pathway for spirometry operators in community-based healthcare settings to demonstrate spirometry competence. The Australia and New Zealand Society of Respiratory Science (ANZSRS) has identified a need for developing a pathway for operators working in community-based practices in Australia and New Zealand to demonstrate spirometry competence and certification. Spirometry certification provides evidence to patients, clients, employers and organizations that an individual has participated in an assessment process that qualifies them to perform spirometry to current international spirometry standards set out by the American Thoracic Society and the European Respiratory Society (ATS/ERS). This document describes a competence assessment pathway that incorporates a portfolio and practical assessment. The completion of this pathway and the award of certification confer an individual is competent to perform spirometry for 3 years, after which re-certification is required. The adoption of this competency assessment and certification process by specialist organizations, and the commitment of operators performing spirometry to undergo this process, will enhance spirometry quality and practice in community-based healthcare settings.

Designed Ankyrin Repeat Protein (DARPin) to target chimeric antigen receptor (CAR)-redirected T cells towards CD4+ T cells to reduce the latent HIV+ cell reservoir.

Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4+ target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir.

Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

Receptor-targeted lentiviral vectors (LVs) can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV) glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance). Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV) glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4) was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

Adults with Philadelphia chromosome-like acute lymphoblastic leukemia frequently have IGH-CRLF2 and JAK2 mutations, persistence of minimal residual disease and poor prognosis.

Philadelphia-like B-cell precursor acute lymphoblastic leukemia (Ph-like ALL) is characterized by distinct genetic alterations and inferior prognosis in children and younger adults. The purpose of this study was a genetic and clinical characterization of Ph-like ALL in adults. Twenty-six (13%) of 207 adult patients (median age: 42 years) with B-cell precursor ALL (BCP-ALL) were classified as having Ph-like ALL using gene expression profiling. The frequency of Ph-like ALL was 27% among 95 BCP-ALL patients negative for BCR-ABL1 and KMT2A-rearrangements. IGH-CRLF2 rearrangements (6/16; P=0.002) and mutations in JAK2 (7/16; P<0.001) were found exclusively in the Ph-like ALL subgroup. Clinical and outcome analyses were restricted to patients treated in German Multicenter Study Group for Adult ALL (GMALL) trials 06/99 and 07/03 (n=107). The complete remission rate was 100% among both Ph-like ALL patients (n=19) and the "remaining BCP-ALL" cases (n=40), i.e. patients negative for BCR-ABL1 and KMT2A-rearrangements and the Ph-like subtype. Significantly fewer Ph-like ALL patients reached molecular complete remission (33% versus 79%; P=0.02) and had a lower probability of continuous complete remission (26% versus 60%; P=0.03) and overall survival (22% versus 64%; P=0.006) at 5 years compared to the remaining BCP-ALL patients. The profile of genetic lesions in adults with Ph-like ALL, including older adults, resembles that of pediatric Ph-like ALL and differs from the profile in the remaining BCP-ALL. Our study is the first to demonstrate that Ph-like ALL is associated with inferior outcomes in intensively treated older adult patients. Ph-like adult ALL should be recognized as a distinct, high-risk entity and further research on improved diagnostic and therapeutic approaches is needed. (NCT00199056, NCT00198991).

Specific gene delivery to liver sinusoidal and artery endothelial cells.

Different types of endothelial cells (EC) fulfill distinct tasks depending on their microenvironment. ECs are therefore difficult to genetically manipulate ex vivo for functional studies or gene therapy. We assessed lentiviral vectors (LVs) targeted to the EC surface marker CD105 for in vivo gene delivery. The mouse CD105-specific vector, mCD105-LV, transduced only CD105-positive cells in primary liver cell cultures. Upon systemic injection, strong reporter gene expression was detected in liver where mCD105-LV specifically transduced liver sinusoidal ECs (LSECs) but not Kupffer cells, which were mainly transduced by nontargeted LVs. Tumor ECs were specifically targeted upon intratumoral vector injection. Delivery of the erythropoietin gene with mCD105-LV resulted in substantially increased erythropoietin and hematocrit levels. The human CD105-specific vector (huCD105-LV) transduced exclusively human LSECs in mice transplanted with human liver ECs. Interestingly, when applied at higher dose and in absence of target cells in the liver, huCD105-LV transduced ECs of a human artery transplanted into the descending mouse aorta. The data demonstrate for the first time targeted gene delivery to specialized ECs upon systemic vector administration. This strategy offers novel options to better understand the physiological functions of ECs and to treat genetic diseases such as those affecting blood factors.

Lactic acid fermentation within a cascading approach for biowaste treatment.

Limited availability of resources and increased amounts of waste coupled with an ever-increasing demand for raw materials are typical characteristics of our times. As such, there is an urgent need to accordingly update waste treatment technology. The aim of this study was to determine whether a separate treatment of the liquid and the solid fraction of biowaste could enhance overall efficiency. Liquid fractions obtained from two different separation procedures were fermented at a pH of 5 and uncontrolled pH conditions for 72 h. The fermentation conditions leading to highest lactic acid productivity and yield were evaluated. The substrates gained by both separation procedures showed efficient lactic acid production up to <25 g L(-1). The pH control increased lactic acid concentration by about 27 %. Furthermore, quantitative real-time PCR analyses revealed stronger Lactobacilli growth in these fermentations. As identified via Illumina sequencing Lactobacillus delbrueckii and its closest relatives seemed to drive the fermentation independent of the substrate. These results could help to improve today's resourcing concept by providing a separate treatment of the liquid and solid biowaste fraction.

T-cell receptor gene transfer exclusively to human CD8(+) cells enhances tumor cell killing.

Transfer of tumor-specific T-cell receptor (TCR) genes into patient T cells is a promising strategy in cancer immunotherapy. We describe here a novel vector (CD8-LV) derived from lentivirus, which delivers genes exclusively and specifically to CD8(+) cells. CD8-LV mediated stable in vitro and in vivo reporter gene transfer as well as efficient transfer of genes encoding TCRs recognizing the melanoma antigen tyrosinase. Strikingly, T cells genetically modified with CD8-LV killed melanoma cells reproducibly more efficiently than CD8(+) cells transduced with a conventional lentiviral vector. Neither TCR expression levels, nor the rate of activation-induced death of transduced cells differed between both vector types. Instead, CD8-LV transduced cells showed increased granzyme B and perforin levels as well as an up-regulation of CD8 surface expression in a small subpopulation of cells. Thus, a possible mechanism for CD8-LV enhanced tumor cell killing may be based on activation of the effector functions of CD8(+) T cells by the vector particle displaying OKT8-derived CD8-scFv and an increase of the surface density of CD8, which functions as coreceptor for tumor-cell recognition. CD8-LV represents a powerful novel vector for TCR gene therapy and other applications in immunotherapy and basic research requiring CD8(+) cell-specific gene delivery.

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors.

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.

An optimal antitumor response by a novel CEA/CD3 bispecific antibody for colorectal cancers.

BACKGROUND: CD3-based bispecific T cell engagers (bsTCEs) are one of the most promising bispecific antibodies for effective cancer treatments. To elicit target-specific T cell-mediated cytotoxicity, these bsTCEs contain at least one binding unit directed against a tumor antigen and another binding unit targeting CD3 in T cell receptor complex. Development of CD3-based bsTCEs, however, has been severely hampered by dose-limiting toxicities due to cytokine release syndrome. To address this limitation, we developed a novel functionally trivalent T cell engager (t-TCE) antibody containing affinity-reduced CD3 binding unit positioned to ensure monovalent CD3 engagement, in combination with bivalent tumor antigen binding of carcinoembryonic antigen (CEA). METHODS: We modeled the variable region of anti-CD3 in the complementarity-determining regions of the heavy chain and obtained CD3 binders with reduced binding affinity. Two optimized versions CEA/CD3-v1 and CEA/CD3-v2 were identified and generated in tetravalent format, characterized and compared in vitro and in vivo for functional activity. RESULTS: Our lead candidate, CEA/CD3-v2, demonstrated subnanomolar binding and picomolar potency against a panel of CEA-expressing cancer cell lines. In addition, we detected reduced T cell cytokine release with potent cytotoxic activity. Our t-TCE CEA/CD3-v2 molecule demonstrated strong antitumor effect in a dose-dependent manner in human peripheral blood mononuclear cell (PBMC) xenograft model. Furthermore, combination of CEA/CD3-v2 with atezolizumab provided synergistic antitumor effect. CONCLUSIONS: Because of its effective tumor cell killing in vitro and in vivo with reduced cytokine release, CEA/CD3 bsTCE may greatly benefit in CEA-positive cancer immunotherapy.

Erratum: A Library-Based Screening Strategy for the Identification of DARPins as Ligands for Receptor-Targeted AAV and Lentiviral Vectors.

[This corrects the article DOI: 10.1016/j.omtm.2018.07.001.].

Combining T-cell-specific activation and in vivo gene delivery through CD3-targeted lentiviral vectors.

Genetic modification of T lymphocytes is a key issue in research and therapy. Conventional lentiviral vectors (LVs) are neither selective for T cells nor do they modify resting or minimally stimulated cells, which is crucial for applications, such as efficient in vivo modification of T lymphocytes. Here, we introduce novel CD3-targeted LVs (CD3-LVs) capable of genetically modifying human T lymphocytes without prior activation. For CD3 attachment, agonistic CD3-specific single-chain variable fragments were chosen. Activation, proliferation, and expansion mediated by CD3-LVs were less rapid compared with conventional antibody-mediated activation owing to lack of T-cell receptor costimulation. CD3-LVs delivered genes not only selectively into T cells but also under nonactivating conditions, clearly outperforming the benchmark vector vesicular stomatitis-LV glycoproteins under these conditions. Remarkably, CD3-LVs were properly active in gene delivery even when added to whole human blood in absence of any further stimuli. Upon administration of CD3-LV into NSG mice transplanted with human peripheral blood mononuclear cells, efficient and exclusive transduction of CD3+ T cells in all analyzed organs was achieved. Finally, the most promising CD3-LV successfully delivered a CD19-specific chimeric antigen receptor (CAR) into T lymphocytes in vivo in humanized NSG mice. Generation of CAR T cells was accompanied by elimination of human CD19+ cells from blood. Taken together, the data strongly support implementation of T-cell-activating properties within T-cell-targeted vector particles. These particles may be ideally suited for T-cell-specific in vivo gene delivery.

Retroviral display and high throughput screening.

Retroviruses distinguish themselves from all other mammalian viruses by their abilities to infect and propagate in mammalian cells without causing a cytopathic effect and to stably integrate their genetic information into the genome of the host cell. These unique properties make them an ideal platform for the display and directed evolution of proteins in a mammalian cell environment. This review will describe the essentials about retrovirus biology and then discuss in detail display and screening strategies that have been developed during the past 15 years of retroviral display technology.

Displaying Tetra-Membrane Spanning Claudins on Enveloped Virus-Like Particles for Cancer Immunotherapy.

Virus-like particles (VLPs) displaying foreign antigens have become an important tool in vaccination including the induction of immune responses against self-antigens. Claudin 6 (CLDN6) has been identified as tumor-associated antigen and is therefore a potential target for tumor vaccination strategies. However, as tetra-membrane spanning protein its incorporation into VLPs while preserving a native fold is challenging. Here, we attempted the incorporation of a panel of engineered CLDN6 variants into the membrane of retrovirus-derived VLPs. Interestingly, wild-type CLDN6 revealed the most efficient display. VLPs presenting CLDN6 or CLDN9 derived from different donor species were produced and preservation of their native confirmation was demonstrated by antibody binding assays. VLPs displaying murine CLDN6 were used to immunize mice. Antibodies recognizing native CLDN6 as displayed on cell surfaces and mediating complement-dependent cytotoxicity were elicited in vaccinated animals. The data suggest applications of CLDN6 displaying VLPs in cancer immunotherapy.

CD30-targeted oncolytic viruses as novel therapeutic approach against classical Hodgkin lymphoma.

Classical Hodgkin lymphoma (cHL) is a hematopoietic malignancy with a characteristic cellular composition. The tumor mass is made up of infiltrated lymphocytes and other cells of hematologic origin but only very few neoplastic cells that are mainly identified by the diagnostic marker CD30. While most patients with early stage cHL can be cured by standard therapy, treatment options for relapsed or refractory cHL are still not sufficient, although immunotherapy-based approaches for the treatment of cHL patients have gained ground in the last decade. Here, we suggest a novel therapeutic concept based on oncolytic viruses selectively destroying the CD30+-positive cHL tumor cells. Relying on a recently described CD30-specific scFv we have generated CD30-targeted measles virus (MV-CD30) and vesicular stomatitis virus (VSV-CD30). For VSV-CD30 the VSV glycoprotein G reading frame was replaced by those of the CD30-targeted MV glycoproteins. Both viruses were found to be highly selective for CD30-positive cells as demonstrated by infection of co-cultures of target and non-target cells as well as through blocking infection by soluble CD30. Notably, VSV-CD30 yielded much higher titers than MV-CD30 and resulted in a more rapid and efficient killing of cultivated cHL-derived cell lines. Mouse tumor models revealed that intratumorally, as well as systemically injected VSV-CD30, infected cHL xenografts and significantly slowed down tumor growth resulting in a substantially prolonged survival of tumor-bearing mice. Taken together, the data support further preclinical testing of VSV-CD30 as novel therapeutic agent for the treatment of cHL and other CD30+-positive malignancies.

A Library-Based Screening Strategy for the Identification of DARPins as Ligands for Receptor-Targeted AAV and Lentiviral Vectors.

Delivering genes selectively to the therapeutically relevant cell type is among the prime goals of vector development. Here, we present a high-throughput selection and screening process that identifies designed ankyrin repeat proteins (DARPins) optimally suited for receptor-targeted gene delivery using adeno-associated viral (AAV) and lentiviral (LV) vectors. In particular, the process includes expression, purification, and in situ biotinylation of the extracellular domains of target receptors as Fc fusion proteins in mammalian cells and the selection of high-affinity binders by ribosome display from DARPin libraries each covering more than 1012 variants. This way, DARPins specific for the glutamate receptor subunit GluA4, the endothelial surface marker CD105, and the natural killer cell marker NKp46 were generated. The identification of DARPins best suited for gene delivery was achieved by screening small-scale vector productions. Both LV and AAV particles displaying the selected DARPins transduced only cells expressing the corresponding target receptor. The data confirm that a straightforward process for the generation of receptor-targeted viral vectors has been established. Moreover, biochemical analysis of a panel of DARPins revealed that their functional cell-surface expression as fusion proteins is more relevant for efficient gene delivery by LV particles than functional binding affinity.

Covalent coupling of high-affinity ligands to the surface of viral vector particles by protein trans-splicing mediates cell type-specific gene transfer.

We have established a novel approach for the covalent coupling of large polypeptides to the surface of fully assembled adeno-associated viral gene transfer vector (AAV) particles via split-intein mediated protein-trans-splicing (PTS). This way, we achieved selective gene transfer to distinct cell types. Single-chain variable fragments (scFvs) or designed ankyrin repeat proteins (DARPins), exhibiting high-affinity binding to cell surface receptors selectively expressed on the surface of target cells, were coupled to AAV particles harboring mutations in the capsid proteins which ablate natural receptor usage. Both, the AAV capsid protein VP2 and multiple separately produced targeting ligands recognizing Her2/neu, EpCAM, CD133 or CD30 were genetically fused with complementary split-intein domains. Optimized coupling conditions led to an effective conjugation of each targeting ligand to the universal AAV capsid and translated into specific gene transfer into target receptor-positive cell types in vitro and in vivo. Interestingly, PTS-based AAVs exhibited significantly less gene transfer into target receptor-negative cells than AAVs displaying the same targeting ligand but coupled genetically. Another important consequence of the PTS technology is the possibility to now display scFvs or other antibody-derived domain formats harboring disulfide-bonds in a functionally active form on the surface of AAV particles. Hence, the custom combination of a universal AAV vector particle and targeting ligands of various formats allows for an unprecedented flexibility in the generation of gene transfer vectors targeted to distinct cell types.

Quality of vitamin K antagonist oral anticoagulation in 322 patients with atrial fibrillation - real-life data from a survey in Eastern Switzerland.

AIM OF THE STUDY: To better appreciate the role of the non-vitamin K oral anticoagulants (NOACs) for patients with non-valvular atrial fibrillation in Switzerland we aimed to assess the quality of vitamin K antagonist (VKA) anticoagulation in daily practice. METHODS: In a cross-sectional study, clinically stable patients on VKA treatment for non-valvular atrial fibrillation for at least 6 months, documentation of international normalised ratio (INR) values for at least 3 months and with at least two INR values were included. The percentage of INR values within the therapeutic range of 2.0 to 3.0 and the time in therapeutic range (TTR; Rosendaal method) and predictors for these measures of VKA anticoagulation quality were assessed. RESULTS: We studied 332 patients (62% male, mean age 74 ± 9 years) with median (interquartile range) CHA2DS2Vasc and HAS-BLED scores of 4 (3-5) and 3 (2-4) points. The median number of INR values per patient was 8 (5-14), and the average interval between INR measurements was 20 (13-27) days. The percentage of INR values between 2.0 and 3.0 was 67% (50-83%). The median TTR was 69% (51-89%), and TTR ≥65% was found in 202 (61%) patients. Independent predictors of ≥80% INR values between 2.0 and 3.0 included a longer interval between INR measurements and the non-use of spironolactone. The non-use of amiodarone and spironolactone and a longer interval between INR measurements were the only independent predictors of a TTR ≥65%. CONCLUSIONS: The quality of VKA anticoagulation in Switzerland is highly variable. Importantly, only 60% of patients achieve a TTR ≥65%, which is currently considered to be the minimal acceptable TTR required for VKA therapy. There are few clinical predictors of a good VKA anticoagulation quality. These data may represent a novel basis for decision making regarding the choice of anticoagulation for atrial fibrillation in Switzerland.

CD30 Receptor-Targeted Lentiviral Vectors for Human Induced Pluripotent Stem Cell-Specific Gene Modification.

Cultures of induced pluripotent stem cells (iPSCs) often contain cells of varying grades of pluripotency. We present novel lentiviral vectors targeted to the surface receptor CD30 (CD30-LV) to transfer genes into iPSCs that are truly pluripotent as demonstrated by marker gene expression. We demonstrate that CD30 expression is restricted to SSEA4(high) cells of human iPSC cultures and a human embryonic stem cell line. When CD30-LV was added to iPSCs during routine cultivation, efficient and exclusive transduction of cells positive for the pluripotency marker Oct-4 was achieved, while retaining their pluripotency. When added during the reprogramming process, CD30-LV solely transduced cells that became fully reprogrammed iPSCs as confirmed by co-expression of endogenous Nanog and the reporter gene. Thus, CD30-LV may serve as novel tool for the selective gene transfer into PSCs with broad applications in basic and therapeutic research.

An ETV6-ABL1 fusion in a patient with chronic myeloproliferative neoplasm: Initial response to Imatinib followed by rapid transformation into ALL.

We report the case of a 26 year-old patient presenting with a persistent leukocytosis and CML-like marrow but no evidence of a BCR/ABL1 fusion. Molecular cytogenetics revealed that a portion of the ETV6 locus was inserted into the ABL1 locus. An ETV6/ABL1 fusion transcript could subsequently be confirmed. The patient was started on imatinib and went into complete cytomorphological remission. QRT-PCR measurements showed a 4 log reduction of the ETV6/ABL1 fusion. 15 months later, the disease transformed into ALL and the patient expired. Thus, an ETV6/ABL1 fusion positive MPN has the potential to transform very rapidly into ALL.

Waste Separation Press (WSP): a mechanical pretreatment option for organic waste from source separation.

An efficient biological treatment of source separated organic waste from household kitchens and gardens (biowaste) requires an adequate upfront mechanical preparation which possibly includes a hand sorting for the separation of contaminants. In this work untreated biowaste from households and gardens and the screen overflow >60mm of the same waste were mechanically treated by a Waste Separation Press (WSP). The WSP separates the waste into a wet fraction for biological treatment and a fraction of dry contaminants for incineration. The results show that it is possible to replace a hand sorting of contaminants, the milling and a screening of organic waste before the biological treatment by using the WSP. A special focus was put on the contaminants separation. The separation of plastic film from the untreated biowaste was 67% and the separation rate of glass was about 92%. About 90% of the organics were transferred to the fraction for further biological treatment. When treating the screen overflow >60mm with the WSP 86% of the plastic film and 88% of the glass were transferred to the contaminants fraction. 32% of the organic was transferred to the contaminants fraction and thereby lost for a further biological treatment. Additionally it was calculated that national standards for glass contaminants in compost can be met when using the WSP to mechanically treat the total biowaste. The loss of biogas by transferring biodegradable organics to the contaminants fraction was about 11% when preparing the untreated biowaste with the WSP.