Prospective study evaluating dynamic changes of cell-free HPV DNA in locoregional viral-associated oropharyngeal cancer treated with induction chemotherapy and response-adaptive treatment.

BACKGROUND: Human papillomavirus (HPV)-associated oropharyngeal cancer (OPC) has a favorable prognosis which has led to efforts to de-intensify treatment. Response-adaptive de-escalated treatment is promising, however improved biomarkers are needed. Quantitative cell-free HPV-DNA (cfHPV-DNA) in plasma represents an attractive non-invasive biomarker for grading treatment response and post-treatment surveillance. This prospective study evaluates dynamic changes in cfHPV-DNA during induction therapy, definitive (chemo)radiotherapy, and post-treatment surveillance in the context of risk and response-adaptive treatment for HPV + OPC. METHODS: Patients with locoregional HPV + OPC are stratified into two cohorts: High risk (HR) (T4, N3, [Formula: see text] 20 pack-year smoking history (PYH), or non-HPV16 subtype); Low risk (LR) (all other patients). All patients receive induction chemotherapy with three cycles of carboplatin and paclitaxel. LR with ≥ 50% response receive treatment on the single-modality arm (minimally-invasive surgery or radiation alone to 50 Gy). HR with ≥ 50% response or LR with ≥ 30% and < 50% response receive treatment on the intermediate de-escalation arm (chemoradiation to 50 Gy with cisplatin). All other patients receive treatment on the regular dose arm with chemoradiation to 70 Gy with concurrent cisplatin. Plasma cfHPV-DNA is assessed during induction, (chemo)radiation, and post-treatment surveillance. The primary endpoint is correlation of quantitative cfHPV-DNA with radiographic response. DISCUSSION: A de-escalation treatment paradigm that reduces toxicity without compromising survival outcomes is urgently needed for HPV + OPC. Response to induction chemotherapy is predictive and prognostic and can select candidates for de-escalated definitive therapy. Assessment of quantitative cfHPV-DNA in the context of response-adaptive treatment of represents a promising reliable and convenient biomarker-driven strategy to guide personalized treatment in HPV + OPC. TRIAL REGISTRATION: This trial is registered with ClinicalTrials.gov on October 1st, 2020 with Identifier: NCT04572100 .

Salivary gland macrophages and tissue-resident CD8+ T cells cooperate for homeostatic organ surveillance.

It is well established that tissue macrophages and tissue-resident memory CD8+ T cells (TRM) play important roles for pathogen sensing and rapid protection of barrier tissues. In contrast, the mechanisms by which these two cell types cooperate for homeostatic organ surveillance after clearance of infections is poorly understood. Here, we used intravital imaging to show that TRM dynamically followed tissue macrophage topology in noninflamed murine submandibular salivary glands (SMGs). Depletion of tissue macrophages interfered with SMG TRM motility and caused a reduction of interepithelial T cell crossing. In the absence of macrophages, SMG TRM failed to cluster in response to local inflammatory chemokines. A detailed analysis of the SMG microarchitecture uncovered discontinuous attachment of tissue macrophages to neighboring epithelial cells, with occasional macrophage protrusions bridging adjacent acini and ducts. When dissecting the molecular mechanisms that drive homeostatic SMG TRM motility, we found that these cells exhibit a wide range of migration modes: In addition to chemokine- and adhesion receptor-driven motility, resting SMG TRM displayed a remarkable capacity for autonomous motility in the absence of chemoattractants and adhesive ligands. Autonomous SMG TRM motility was mediated by friction and insertion of protrusions into gaps offered by the surrounding microenvironment. In sum, SMG TRM display a unique continuum of migration modes, which are supported in vivo by tissue macrophages to allow homeostatic patrolling of the complex SMG architecture.

Medium-chain glycerides affect gut morphology, immune- and goblet cells in post-weaning piglets: In vitro fatty acid screening with Escherichia coli and in vivo consolidation with LPS challenge.

The influence of medium-chain glycerides on performance and gastrointestinal well-being in weaning piglets was assessed. First, caproic (C6), caprylic (C8) and capric (C10) acid activity against Escherichia coli was screened in vitro. Pig flora of the whole small intestine was used as inoculum. Seven in vitro incubations were done in duplicate at pH = 3 and 5: C10 (15 mM), C8 (12 mM), C6 (15, 12, 10 mM), a non-incubated-negative control and incubated negative control. Culture suspensions were plated on E. coli-selective agar. Controls showed bacterial growth. C6 and C8 showed no growth at both pH-values, where C10 showed growth at pH = 5. Secondly, an in vivo study was done with 80 weaned piglets over 42 days, housed in pens of eight animals (five pens/treatment), fed a basal diet containing broken rice/soya bean meal/fish meal and supplemented with C6 and C8 in medium-chain glyceride form (MCT6/8, 0.175%) or antibiotic growth promoter (AGP, 0.020%) (Kasetsart University, Thailand) serving as control. Feed intake, daily gain and feed-to-gain ratio did not differ between MCT6/8 and AGP. Per replicate, two random selected piglets were challenged intravenously with E. coli-lipopolysaccharide (LPS) or saline solution (S) at Days 21 and 28. All challenged animals were sacrificed; blood and digestive tract samples (jejunum/ileum) were collected at Day 35. LPS challenge consistently reduced villus height and crypt depth for MCT6/8 and AGP. However, LPS-challenged piglets supplemented with MCT6/8 restored villus height, where AGP did not. MCT6/8 piglets had higher serum IgA, more jejunal IgA-positive plasma cells and goblet cells than AGP. At the ileal level, results were similar, though less pronounced. The present study offers new insight in the benefits of MCT6/8 over AGP in the post-weaning period. There is in vitro anti-microbial action of C6 and C8 on E. coli. In vivo, MCT6/8 also has protective effects in the small intestine that may result in growth promotion.

Control of primary mouse cytomegalovirus infection in lung nodular inflammatory foci by cooperation of interferon-gamma expressing CD4 and CD8 T cells.

Human cytomegalovirus (CMV) and mouse cytomegalovirus (MCMV) infection share many characteristics. Therefore infection of mice with MCMV is an important tool to understand immune responses and to design vaccines and therapies for patients at the risk of severe CMV disease. In this study, we investigated the immune response in the lungs following acute infection with MCMV. We used multi-color fluorescence microscopy to visualize single infected and immune cells in nodular inflammatory foci (NIFs) that formed around infected cells in the lungs. These NIFs consisted mainly of myeloid cells, T cells, and some NK cells. We found that the formation of NIFs was essential to reduce the number of infected cells in the lung tissue, showing that NIFs were sites of infection as well as sites of immune response. Comparing mice deficient for several leukocyte subsets, we identified T cells to be of prime importance for restricting MCMV infection in the lung. Moreover, T cells had to be present in NIFs in high numbers, and CD4 as well as CD8 T cells supported each other to efficiently control virus spread. Additionally, we investigated the effects of perforin and interferon-gamma (IFNγ) on the virus infection and found important roles for both mechanisms. NK cells and T cells were the major source for IFNγ in the lung and in in vitro assays we found that IFNγ had the potential to reduce plaque growth on primary lung stromal cells. Notably, the T cell-mediated control was shown to be perforin-independent but IFNγ-dependent. In total, this study systematically identifies crucial antiviral factors present in lung NIFs for early containment of a local MCMV infection at the single cell level.

The M25 gene products are critical for the cytopathic effect of mouse cytomegalovirus.

Cell rounding is a hallmark of the cytopathic effect induced by cytomegaloviruses. By screening a panel of deletion mutants of mouse cytomegalovirus (MCMV) a mutant was identified that did not elicit cell rounding and lacked the ability to form typical plaques. Altered cell morphology was assigned to the viral M25 gene. We detected an early 2.8 kb M25 mRNA directing the synthesis of a 105 kDa M25 protein, and confirmed that a late 3.1 kb mRNA encodes a 130 kDa M25 tegument protein. Virions lacking the M25 tegument protein were of smaller size because the tegument layer between capsid and viral envelope was reduced. The ΔM25 mutant did not provoke the rearrangement of the actin cytoskeleton observed after wild-type MCMV infection, and isolated expression of the M25 proteins led to cell size reduction, confirming that they contribute to the morphological changes. Yields of progeny virus and cell-to-cell spread of the ΔM25 mutant in vitro were diminished and replication in vivo was impaired. The identification of an MCMV gene involved in cell rounding provides the basis for investigating the role of this cytopathic effect in CMV pathogenesis.

Systemic Virus Infections Differentially Modulate Cell Cycle State and Functionality of Long-Term Hematopoietic Stem Cells In Vivo.

Quiescent long-term hematopoietic stem cells (LT-HSCs) are efficiently activated by type I interferon (IFN-I). However, this effect remains poorly investigated in the context of IFN-I-inducing virus infections. Here we report that both vesicular stomatitis virus (VSV) and murine cytomegalovirus (MCMV) infection induce LT-HSC activation that substantially differs from the effects triggered upon injection of synthetic IFN-I-inducing agents. In both infections, inflammatory responses had to exceed local thresholds within the bone marrow to confer LT-HSC cell cycle entry, and IFN-I receptor triggering was not critical for this activation. After resolution of acute MCMV infection, LT-HSCs returned to phenotypic quiescence. However, non-acute MCMV infection induced a sustained inflammatory milieu within the bone marrow that was associated with long-lasting impairment of LT-HSC function. In conclusion, our results show that systemic virus infections fundamentally affect LT-HSCs and that also non-acute inflammatory stimuli in bone marrow donors can affect the reconstitution potential of bone marrow transplants.

The Mouse Cytomegalovirus Gene m42 Targets Surface Expression of the Protein Tyrosine Phosphatase CD45 in Infected Macrophages.

The receptor-like protein tyrosine phosphatase CD45 is expressed on the surface of cells of hematopoietic origin and has a pivotal role for the function of these cells in the immune response. Here we report that following infection of macrophages with mouse cytomegalovirus (MCMV) the cell surface expression of CD45 is drastically diminished. Screening of a set of MCMV deletion mutants allowed us to identify the viral gene m42 of being responsible for CD45 down-modulation. Moreover, expression of m42 independent of viral infection upon retroviral transduction of the RAW264.7 macrophage cell line led to comparable regulation of CD45 expression. In immunocompetent mice infected with an m42 deletion mutant lower viral titers were observed in all tissues examined when compared to wildtype MCMV, indicating an important role of m42 for viral replication in vivo. The m42 gene product was identified as an 18 kDa protein expressed with early kinetics and is predicted to be a tail-anchored membrane protein. Tracking of surface-resident CD45 molecules revealed that m42 induces internalization and degradation of CD45. The observation that the amounts of the E3 ubiquitin ligases Itch and Nedd4 were diminished in cells expressing m42 and that disruption of a PY motif in the N-terminal part of m42 resulted in loss of function, suggest that m42 acts as an activator or adaptor for these Nedd4-like ubiquitin ligases, which mark CD45 for lysosomal degradation. In conclusion, the down-modulation of CD45 expression in MCMV-infected myeloid cells represents a novel pathway of virus-host interaction.

In Vivo Killing Capacity of Cytotoxic T Cells Is Limited and Involves Dynamic Interactions and T Cell Cooperativity.

According to in vitro assays, T cells are thought to kill rapidly and efficiently, but the efficacy and dynamics of cytotoxic T lymphocyte (CTL)-mediated killing of virus-infected cells in vivo remains elusive. We used two-photon microscopy to quantify CTL-mediated killing in mice infected with herpesviruses or poxviruses. On average, one CTL killed 2-16 virus-infected cells per day as determined by real-time imaging and by mathematical modeling. In contrast, upon virus-induced MHC class I downmodulation, CTLs failed to destroy their targets. During killing, CTLs remained migratory and formed motile kinapses rather than static synapses with targets. Viruses encoding the calcium sensor GCaMP6s revealed strong heterogeneity in individual CTL functional capacity. Furthermore, the probability of death of infected cells increased for those contacted by more than two CTLs, indicative of CTL cooperation. Thus, direct visualization of CTLs during killing of virus-infected cells reveals crucial parameters of CD8(+) T cell immunity.

Nodular inflammatory foci are sites of T cell priming and control of murine cytomegalovirus infection in the neonatal lung.

Neonates, including mice and humans, are highly susceptible to cytomegalovirus (CMV) infection. However, many aspects of neonatal CMV infections such as viral cell tropism, spatio-temporal distribution of the pathogen as well as genesis of antiviral immunity are unknown. With the use of reporter mutants of the murine cytomegalovirus (MCMV) we identified the lung as a primary target of mucosal infection in neonatal mice. Comparative analysis of neonatal and adult mice revealed a delayed control of virus replication in the neonatal lung mucosa explaining the pronounced systemic infection and disease in neonates. This phenomenon was supplemented by a delayed expansion of CD8(+) T cell clones recognizing the viral protein M45 in neonates. We detected viral infection at the single-cell level and observed myeloid cells forming "nodular inflammatory foci" (NIF) in the neonatal lung. Co-localization of infected cells within NIFs was associated with their disruption and clearance of the infection. By 2-photon microscopy, we characterized how neonatal antigen-presenting cells (APC) interacted with T cells and induced mature adaptive immune responses within such NIFs. We thus define NIFs of the neonatal lung as niches for prolonged MCMV replication and T cell priming but also as sites of infection control.

Priming of CD8+ T cells against cytomegalovirus-encoded antigens is dominated by cross-presentation.

CMV can infect dendritic cells (DCs), and direct Ag presentation could, therefore, lead to the priming of CMV-specific CD8(+) T cells. However, CMV-encoded immune evasins severely impair Ag presentation in the MHC class I pathway; thus, it is widely assumed that cross-presentation drives the priming of antiviral T cells. We assessed the contribution of direct versus cross priming in mouse CMV (MCMV) infection using recombinant viruses. DCs infected with an MCMV strain encoding the gB498 epitope from HSV-1 were unable to stimulate in vitro naive gB498-specific CD8(+) T cells from TCR transgenic mice. Infection of C57BL/6 mice with this recombinant virus led, however, to the generation of abundant numbers of gB498-specific T cells in vivo. Of the DC subsets isolated from infected mice, only CD8α(+) DCs were able to stimulate naive T cells, suggesting that this DC subset cross-presents MCMV-encoded Ag in vivo. Upon infection of mice with MCMV mutants encoding Ag that can either be well or hardly cross-presented, mainly CD8(+) T cells specific for cross-presented epitopes were generated. Moreover, even in the absence of immune evasion genes interfering with MHC class I-mediated Ag presentation, priming of T cells to Ag that can only be presented directly was not observed. We conclude that the host uses mainly DCs capable of cross-presentation to induce the CMV-specific CD8(+) T cell response during primary, acute infection and discuss the implications for the development of a CMV vaccine.

Renal tubular Fas ligand mediates fratricide in cisplatin-induced acute kidney failure.

Cisplatin, a standard chemotherapeutic agent for many tumors, has an unfortunately common toxicity where almost a third of patients develop renal dysfunction after a single dose. Acute kidney injury caused by cisplatin depends on Fas-mediated apoptosis driven by Fas ligand (FasL) expressed on tubular epithelial and infiltrating immune cells. Since the role of FasL in T cells is known, we investigated whether its presence in primary kidney cells is needed for its toxic effect. We found that all cisplatin-treated wild-type (wt) mice died within 6 days; however, severe combined immunodeficiency (SCID)/beige mice (B-, T-, and natural killer-cell-deficient) displayed a significant survival benefit, with only 55% mortality while exhibiting significant renal failure. Treating SCID/beige mice with MFL3, a FasL-blocking monoclonal antibody, completely restored survival after an otherwise lethal cisplatin dose, suggesting another source of FasL besides immune cells. Freshly isolated primary tubule segments from wt mice were co-incubated with thick ascending limb (TAL) segments freshly isolated from mice expressing the green fluorescent protein (GFP) transgene (same genetic background) to determine whether FasL-mediated killing of tubular cells is an autocrine or paracrine mechanism. Cisplatin-stimulated primary segments induced apoptosis in the GFP-tagged TAL cells, an effect blocked by MFL3. Thus, our study shows that cisplatin-induced nephropathy is mediated through FasL, functionally expressed on tubular cells that are capable of inducing death of cells of adjacent tubules.

Effective blockage of both the extrinsic and intrinsic pathways of apoptosis in mice by TAT-crmA.

Evidence accumulates that in clinically relevant cell death, both the intrinsic and extrinsic apoptotic pathway synergistically contribute to organ failure. In search for an inhibitor of apoptosis that provides effective blockage of these pathways, we analyzed viral proteins that evolved to protect the infected host cells. In particular, the cowpox virus protein crmA has been demonstrated to be capable of blocking key caspases of both pro-apoptotic pathways. To deliver crmA into eukaryotic cells, we fused the TAT protein transduction domain of HIV to the N terminus of crmA. In vitro, the TAT-crmA fusion protein was efficiently translocated into target cells and inhibited apoptosis mediated through caspase-8, caspase-9, and caspase-3 after stimulation with alpha-Fas, etoposide, doxorubicin, or staurosporine. The extrinsic apoptotic pathway was investigated following alpha-Fas stimulation. In vivo 90% of TAT-crmA-treated animals survived an otherwise lethal dose of alpha-Fas and showed protection from Fas-induced organ failure. To examine the intrinsic apoptotic pathway, we investigated the survival of mice treated with an otherwise lethal dose of doxorubicin. Whereas all control mice died within 31 days, 40% of mice that concomitantly received intraperitoneal injections of TAT-crmA survived. To test the ability to comprehensively block both the intrinsic and extrinsic apoptotic pathway in a clinically relevant setting, we employed a murine cardiac ischemia-reperfusion model. TAT-crmA reduced infarction size by 40% and preserved left ventricular function. In summary, these results provide a proof of principle for the inhibition of apoptosis with TAT-crmA, which might provide a new treatment option for ischemia-reperfusion injuries.

Stable marking and transgene expression without progression to monoclonality in canine long-term hematopoietic repopulating cells transduced with lentiviral vectors.

Lentiviral gene transfer vectors have a number of potential advantages over gammaretroviral vectors including more efficient transduction of nondividing cells, a more favorable integration site profile, and the ability to accommodate large transgenes. Here, we present long-term follow-up data of animals that received lentivirus-transduced CD34-enriched cells. Six long-term surviving dogs were available for analysis. Transgene expression was analyzed from at least 12 months to more than 5 years after transplantation in peripheral blood cells and multiple cell lineages. All animals demonstrated long-term stable transgene expression in peripheral blood myeloid, lymphoid, and red blood cells as well as in platelets. Vector integration sites were analyzed by linear amplification-mediated polymerase chain reaction and showed a polyclonal repopulation pattern in all animals. There was no evidence of any development of monoclonality or leukemia in the animals. The stable long-term multilineage transgene expression, together with detection of the same integration site in myeloid and lymphoid cells, strongly suggests the transduction of long-term repopulating stem cells. Our data demonstrate safe and efficient transduction of multilineage long-term repopulating cells with lentiviral vectors and support the use of such vectors for gene therapy studies in patients.

Long-term polyclonal and multilineage engraftment of methylguanine methyltransferase P140K gene-modified dog hematopoietic cells in primary and secondary recipients.

Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies, and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n = 157) and after (n = 129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogene TSS after chemotherapy. We also wanted to determine whether in vivo selected cells retained fundamental characteristics of hematopoietic stem cells. To that end, we performed secondary transplantation of MGMTP140K gene-modified cells after in vivo selection in dog leukocyte antigen (DLA)-matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs more than 800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.

Comparison of mesenchymal stem cells from different tissues to suppress T-cell activation.

Graft-versus-host disease (GvHD) and graft rejection have remained significant complications of allogeneic stem cell transplantation. Mesenchymal stem cells (MSCs) from the bone marrow have been shown to suppress T-cell activation in vitro and in vivo, and may be used to reduce GvHD in the recipient or to facilitate engraftment across MHC barriers. MSCs can be derived from a variety of tissues. Thus, we asked whether MSCs from different tissues might have differential effects on T-cell responses. We were particularly interested in MSCs derived from adipose tissue because of its abundance and accessibility. We investigated and compared the immunosuppressive potential of murine MSCs derived from muscle tissue, adipose tissue, omentum, and bone. Cells from the different tissues were enriched for MSCs and cultured for 2-3 weeks to deplete hematopoietic cells. Mixed lymphocyte reactions (MLRs) including MSCs were performed using concanavalin A or allogeneic T cells as inducers of T-cell activation. MSCs from all tissues differentiated into multiple lineages. Mitogen-induced T-cell activation, as well as allogeneic T-cell responses, was reduced in MLRs mediated by the addition of MSCs. Reduction of T-cell activation was most pronounced for muscle tissue in the mitogen-induced MLR and fat tissue during the allogeneic MLR. These data demonstrate that MSCs from multiple tissues efficiently reduce T-cell activation. The results suggest that MSCs from adipose tissue can serve as an alternative source for MSCs to bone or bone marrow for the modulation of GvHD after allogeneic stem cell transplantation or to enhance engraftment across MHC barriers.

Unique integration profiles in a canine model of long-term repopulating cells transduced with gammaretrovirus, lentivirus, or foamy virus.

Recent advances have allowed for improved retrovirus-mediated gene transfer, and therapeutic benefits have been described in patients. These successes have shown the potential of hematopoietic stem cell (HSC) gene therapy, but treatment-related leukemia and benign expansion of gene-modified clones have shifted the attention toward safety. The delayed onset of adverse events in gene therapy clinical trials emphasizes the importance of long-term integration site studies in large animal models. We have addressed safety by characterizing the genomic location of 555 integration sites of the three most commonly used integrating retroviral vectors, that is, gammaretrovirus, lentivirus, and foamy virus, in long-term repopulating cells from dogs. Gammaretroviral integrants showed the most significant frequency of occurrence very close (<2.5 kb) to transcription start sites, but a substantial portion of all three retroviral integrants were within 50 kb. Importantly, gammaretroviral integrants were found more frequently in and near proto-oncogenes, suggesting this retroviral system may be the most prone to adverse gene activation. These data suggest that gammaretroviral vectors may have the highest intrinsic risk, but also emphasize that no vector system can be defined as "safe" based solely on integration profile.

Foamy-virus-mediated gene transfer to canine repopulating cells.

Foamy virus (FV) vectors are particularly attractive gene-transfer vectors for stem-cell gene therapy because they form a stable transduction intermediate in quiescent cells and can efficiently transduce hematopoietic stem cells. Here, we studied the use of FV vectors to transduce long-term hematopoietic repopulating cells in the dog, a clinically relevant large animal model. Mobilized canine peripheral blood (PB) CD34+ cells were transduced with an enhanced green fluorescent protein (EGFP)-expressing FV vector in an 18-hour transduction protocol. All 3 dogs studied had rapid neutrophil engraftment to greater than 500/microL with a median of 10 days. Transgene expression was detected in all cell lineages (B cells, T cells, granulocytes, red blood cells, and platelets), indicating multilineage engraftment of transduced cells. Up to 19% of blood cells were EGFP+, and this was confirmed at the DNA level by real-time polymerase chain reaction (PCR) and Southern blot analysis. These transduction rates were higher than the best results we obtained previously with lentiviral vectors in a similar transduction protocol. Integration site analysis also demonstrated polyclonal repopulation and the transduction of multipotential hematopoietic repopulating cells. These data suggest that FV vectors should be useful for stem-cell gene therapy, particularly for applications in which short transduction protocols are critical.

Tylosin detection in animal feed by liquid chromatography-tandem mass spectrometry with enzymatic hydrolysis of the tylosin urea adduct.

When the use of tylosin as a feed additive was forbidden by Council Regulation 2821/98, the necessity of a chemical confirmation method for the monitoring of the ban was created. Recently a method was developed for the detection of tylosin in animal feed by means of LC-MS/MS. During the validation high deviating values for the decision limit, detection capability, and repeatability for tylosin in cattle feed were observed, and the presence of urea and the formation of a tylosin urea adduct (TUA) were suggested as possible explanations. In this study two hydrolysis approaches for the TUA adduct were compared, namely, a chemical hydrolysis and an enzymatic hydrolysis with urease. The latter yielded a more complete hydrolysis of urea and was used for further validation. The recovery increased by approximately 15-25% depending on the amount of urea present in the feed (0.5-2%). The decision limit and detection capability were hardly influenced by the enzymatic hydrolysis.

Efficient lentiviral gene transfer to canine repopulating cells using an overnight transduction protocol.

The use of lentiviral vectors for the transduction of hematopoietic stem cells has evoked much interest owing to their ability to stably integrate into the genome of nondividing cells. However, published large animal studies have reported highly variable gene transfer rates of typically less than 1%. Here we report the use of lentiviral vectors for the transduction of canine CD34(+) hematopoietic repopulating cells using a very short, 18-hour transduction protocol. We compared lentiviral transduction of hematopoietic repopulating cells from either stem cell factor (SCF)- and granulocyte-colony stimulating factor (G-CSF)-primed marrow or mobilized peripheral blood in a competitive repopulation assay in 3 dogs. All dogs engrafted rapidly within 9 days. Transgene expression was detected in all lineages (B cells, T cells, granulocytes, and red blood cells as well as platelets) indicating multilineage engraftment of transduced cells, with overall long-term marking levels of up to 12%. Gene transfer levels in mobilized peripheral blood cells were slightly higher than in primed marrow cells. In conclusion, we show efficient lentiviral transduction of canine repopulating cells using an overnight transduction protocol. These results have important implications for the design of stem cell gene therapy protocols, especially for those diseases in which the maintenance of stem cells in culture is a major limitation.