Molecular profiling in bee venom allergy: clinical and therapeutic characterization in a Portuguese cohort.

Summary: Background. Bee venom allergy (BVA) can trigger local and systemic allergic reactions, including anaphylaxis. Recently, the molecular sensitization profile has gained importance in the reaction's stratification and venom immunotherapy (VIT). Methods. Retrospective analysis of patients with hypersensitivity to BVA, confirmed by specific sIgE to Apis mellifera ≥0.35 kU/L and/or positive skin tests to bee venom commercial extract, evaluated in specialized consultation. Demographic, clinical, and laboratory data (including molecular Api m 1, 4, and 10) were analyzed, looking for risk factors associated with the severity of the index reaction and reactions during VIT. Results. 93 patients were included (55.9% male; median age of 46 years), 57.3% with atopic comorbidities, and 23.4% with cardiovascular comorbidities. The median specific IgE to Apis mellifera was 6.7 kU/L (IQR 1.0-20.3) kU/L. Regarding the molecular profile, the median IgE to Api m 1 was 0.5 kU/L (57.5% positive out of all measurements); Api m 4 - 0.01 kU/L (11.9% positive), and Api m 10 - 0.3 kU/L (50.0% positive). No patient was monosensitized to Api m 4. The median age of the most severe sting reaction was 36 (IQR 26-48) years, with a median severity (Müeller scale) of 3 (IQR 2-3). Forty-seven patients (50.5%) underwent VIT, with 35.6% of reactions recorded. Allergic reactions during VIT were recorded in 35.6% of cases. The severity of the index reaction correlated positively with older ages (p=0.040; r=0.249), in contrast to monosensitization to Api m 1, which was an independent predictor of milder reactions (p=0.015). Sensitization to Api m 10 was associated with a higher likelihood of reactions during VIT (p=0.038) but potentially less systemic reactions at re-stings (p=0.097). Conclusions. Molecular sensitization profile appears to be relevant not only to the severity of index reactions but also during VIT. Studies of a large cohort of patients with molecular profiles are essential to validate these results and improve the clinical and therapeutic approach to BVA.

Expression of IL-17RA in Innate Cells of Patients with Common Variable Immunodeficiency (CVID) and its Clinical Implications.

Summary: Background. Common Variable Immunodeficiency (CVID) is a primary immunodeficiency disorder characterized by B-cell dysfunction and immunoglobulin production deficiency. Dysregulation of interleukin-17 (IL-17) and its receptor IL-17RA have been reported in various immune disorders. This study aimed to investigate the expression of IL-17RA in innate immune cells of CVID patients and its correlation with clinical manifestations. Methods. A cross-sectional study included 22 CVID patients and 14 age- and sex-matched healthy controls. IL-17RA expression was assessed in various immune cell subsets using flow cytometry. Demographic and clinical data were collected, and statistical analysis was performed. Results. CVID patients had elevated IL-17RA expression in neutrophils, non-classical monocytes, and dendritic cells compared to healthy controls. Patients with a history of intestinal microbial colonization, particularly with Campylobacter jejuni and Giardia intestinalis, showed significantly higher IL-17RA expression in innate cells. Elevated IL-17RA expression in monocytes and dendritic cells also correlated with higher fecal calprotectin levels in CVID patients, regardless of microbial colonization. Conclusions. The study suggests that despite previous reports of reduced circulating Th17 cells and IL-17 levels in CVID patients, IL-17RA expression in innate cells may be elevated, potentially indicating altered IL-17 signaling. This heightened IL-17RA expression could contribute to a persistent pro-inflammatory state, possibly due to microbial translocation or other inflammatory factors. The association of IL-17RA expression with gastrointestinal microbial colonization and its correlation with fecal calprotectin underscores the complexity of IL-17RA's role in CVID pathophysiology. Further research in larger cohorts could elucidate the implications of IL-17RA expression in both infectious and non-infectious inflammatory aspects of CVID.

Allergic emergencies in the prehospital setting: a 5 year retrospective study.

Summary: Background. Patients with severe allergic conditions often request support from the prehospital emergency services given the rapid, unexpected and potentially life-threatening nature of the reactions, such as anaphylaxis. Studies regarding prehospital incidents for allergic conditions are scarce. This study aimed to characterize prehospitalar medical requesting assistance due to suspected hypersensitivity reactions (HSR). Methods. Retrospective study of allergic-related requesting assistances between 2017-2022 of a Portuguese emergency dispatch centre - Emergency and Resuscitation Medical Vehicle (VMER), in Coimbra University Hospital. Demographic and clinical variables were analysed, including clinical manifestations, anaphylaxis severity grading, therapeutic interventions, and post-incident allergic work-up. Regarding anaphylactic events, three diagnosis timings were compared: on-site, hospital emergency department and Investigator-diagnosis based on data reviewed. Results. Out of 12689 VMER requesting assistances, 210 (1.7%) were classified as suspected HSR reactions. After on-site medical evaluation, 127 (60.5%) cases maintained the HSR classification (median age 53 years; 56% males) and the main diagnoses included HSR to Hymenoptera venom (29.9%), food allergy (29.1%), and pharmaceutical drugs (25.5%). Anaphylaxis was assumed on-site in 44 (34.7%) cases, in the hospital emergency department in 53 cases (41.7%) and by investigators in 76 (59.8%) cases. Regarding management, epinephrine was administered on-site in 50 cases (39.4%). Conclusions. The main reason for prehospital requesting assistance was HSR to Hymenoptera venom. A high proportion of incidents met the criteria for anaphylaxis and despite the inherent difficulties of the prehospital setting, many of the on-site diagnoses agreed with the criteria. Regarding management, epinephrine was underused in this setting. Referral to specialized consultation is crucial for the management of prehospital incidents.

Quantifying chaos in stock markets before and during COVID-19 pandemic from the phase space reconstruction.

From a methodology in the reconstruction scheme, applicable to chaotic time series of economic indices, this paper presents an analysis of the underlying dynamics of stock markets of North America, Europe and Asia. The same global fit model and reconstruction parameters-employed to study the time evolution of S&P 500, NASDAQ Composite, IBEX 35, EURONEXT 100, Nikkei 225 and SSE Composite Index-led a convenient simplification in the analysis. The tools chosen to analyse the time dependence of the level of chaos concerning weeks of economic activity were scatter plots, histograms and sample Spearman correlation coefficients. The results permit to evaluate the impact of the pandemic in the underlying dynamics of different stock markets and to compare them to one another.

The diverse roles of T cell subsets in asthma.

SUMMARY: T cells are coordinators of the immune response and have been shown to play a central role in the pathophysiology of asthma. A good understanding of the T cells functions in asthma is important for therapeutic reasons, in particular for the choice of biological treatments in severe asthma. Although classically considered a Th2 disease, it is now clear that other types of T cells contribute for the pathophysiology and the heterogeneity of asthma. We here review how the different subsets of T cells are involved in the different phenotypes/endotypes of asthma and how this may influence the treatment of the disease.

Co-occurrence of Eutrombicula alfreddugesi and Oswaldofilaria chabaudi in Tropidurus torquatus and first report of microfilariae in the chigger mite: possible evidence of a lifecycle pathway?

While much attention has been paid to vector-borne filariasis, diseases that threaten millions of people in tropical and subtropical countries, the literature on host-parasite associations and transmission strategies of filarial nematodes in wildlife is scarce. Here, we report the co-occurrence of chigger mites (Eutrombicula alfreddugesi) and onchocercid nematodes (Oswaldofilaria chabaudi) parasitizing the lizard Tropidurus torquatus in the State of Minas Gerais, Brazil. Examination of chiggers established, for the first time, the occurrence of microfilariae in trombiculid mites (Trombiculidae). These larvae were morphologically similar to those recovered from adult females of O. chabaudi. The current evidence suggests that chiggers do not play a role in the transmission of filarioid nematodes, but rather act as accidental or dead-end hosts. Nevertheless, considering the polyphagous nature of trombiculid mites, similar to blood-sucking insects involved in the transmission of several infectious diseases, further studies may shed light on the potential role of chiggers as vectors of filarioids.

Revisiting the metallothionein genes polymorphisms and the risk of oral squamous cell carcinoma in a Brazilian population.

BACKGROUND: Metallothioneins (MTs) gene polymorphisms have been associated with the ability of free radical scavenging and detoxification of heavy metals leading to cancer development. Our aim was to revisit, in a Brazilian population, single-nucleotide polymorphisms (SNPs) of the MT gene family previously associated with oral squamous cell carcinoma (OSCC). MATERIAL AND METHODS: A case-control investigation with 28 OSCC patients and 45 controls was conducted, using conventional risk factors (tobacco use and alcohol consumption) as covariates. SNPs genotyping for rs8052334 (MT1B), rs964372 (MT1B), and rs1610216 (MT2A) was performed by PCR-RFLP, and SNPs for rs11076161 (MT1A) were analyzed by TaqMan assay. RESULTS: The only SNP associated with increased risk for OSCC was the MT-1A AA genotype (OR = 4.7; p = 0.01). We have also evidenced for the first time a significant linkage disequilibrium between the SNPs of MT-2A and MT-1A in this population with the highest frequency (30%) of the unfavorable haplotype G/A/C/T (rs1610216 / rs11076161 / rs964372 / rs8052334) of MT gene polymorphisms (OR = 6.2; p = 0.04). Interestingly, after removing the effects of conventional risk factors, we have uncovered the significance of the AA genotype of the rs11076161 with increased odds of 19-fold higher towards OSCC development. CONCLUSIONS: This is the first demonstration that a significant linkage disequilibrium among gene polymorphisms of the MT family may affect susceptibility to oral cancer, which is conditioned by the G/A/C/T haplotype (rs1610216/rs11076161/rs964372/ rs8052334) and the MT-1A gene polymorphism has a potential clinical utility for the OSCC risk assessment.

Towards an international language for incontinence-associated dermatitis (IAD): design and evaluation of psychometric properties of the Ghent Global IAD Categorization Tool (GLOBIAD) in 30 countries.

BACKGROUND: Incontinence-associated dermatitis (IAD) is a specific type of irritant contact dermatitis with different severity levels. An internationally accepted instrument to assess the severity of IAD in adults, with established diagnostic accuracy, agreement and reliability, is needed to support clinical practice and research. OBJECTIVES: To design the Ghent Global IAD Categorization Tool (GLOBIAD) and evaluate its psychometric properties. METHODS: The design was based on expert consultation using a three-round Delphi procedure with 34 experts from 13 countries. The instrument was tested using IAD photographs, which reflected different severity levels, in a sample of 823 healthcare professionals from 30 countries. Measures for diagnostic accuracy (sensitivity and specificity), agreement, interrater reliability (multirater Fleiss kappa) and intrarater reliability (Cohen's kappa) were assessed. RESULTS: The GLOBIAD consists of two categories based on the presence of persistent redness (category 1) and skin loss (category 2), both of which are subdivided based on the presence of clinical signs of infection. The agreement for differentiating between category 1 and category 2 was 0·86 [95% confidence interval (CI) 0·86-0·87], with a sensitivity of 90% and a specificity of 84%. The overall agreement was 0·55 (95% CI 0·55-0·56). The Fleiss kappa for differentiating between category 1 and category 2 was 0·65 (95% CI 0·65-0·65). The overall Fleiss kappa was 0·41 (95% CI 0·41-0·41). The Cohen's kappa for differentiating between category 1 and category 2 was 0·76 (95% CI 0·75-0·77). The overall Cohen's kappa was 0·61 (95% CI 0·59-0·62). CONCLUSIONS: The development of the GLOBIAD is a major step towards a better systematic assessment of IAD in clinical practice and research worldwide. However, further validation is needed.

Impact of the anatomical location, alcoholism and smoking on the prevalence of advanced oral cancer in Brazil.

BACKGROUND: To evaluate the prevalence of oral cancer in Brazil according to the clinical stage, anatomical location, alcoholism and smoking. MATERIAL AND METHODS: Data referring to 31,217 cases of oral cancer, from 2000 to 2010, were obtained from the Integrator Module of the Hospital Registry of Cancer. Inconsistent data ("non-classified" cases) was eliminated and 21,160 cases were analyzed. The frequency distribution according to clinical stage, anatomical location, alcoholism and smoking was analyzed descriptively and through a binary logistic regression model (α<0.05). The clinical stage (dependent variable) was dichotomized in early stage (I and II) or advanced stage (III and IV). The year of diagnosis, anatomical location and deleterious habits (alcoholism and smoking) were considered independent variables. RESULTS: The most frequent characteristics were: oropharynx location (n=3856, 18.41%), clinical stage IV (n=11924, 56.09%) and combined use of alcohol and tobacco (n=19226; 61.59%). The year 2009 (p<0.01, PR = 1.162, CI-95%=1.053-1.283) and location at the base of tongue (p<0.01, PR = 2.485, CI-95% = 2.182-2.807) presented a higher prevalence ratio for advanced stage oral cancer. The combined use of alcohol and tobacco showed a higher prevalence rate for the advanced clinical stage of cancer (p<0.01, PR =1.449, CI-95%=1.382-1.520) if compared to individuals without habits, or just alcoholics. CONCLUSIONS: Higher prevalence of advanced stage of oral cancer is related to the localization at the base of the tongue and to the concomitant use of alcohol and tobacco. Therefore, it can be suggested that all these characteristics lead to a worse prognosis of oral cancer.

Safety and immunogenicity of neoadjuvant treatment using WT1-immunotherapeutic in combination with standard therapy in patients with WT1-positive Stage II/III breast cancer: a randomized Phase I study.

PURPOSE: This Phase I, multicenter, randomized study (ClinicalTrials.gov NCT01220128) evaluated the safety and immunogenicity of recombinant Wilms' tumor 1 (WT1) protein combined with the immunostimulant AS15 (WT1-immunotherapeutic) as neoadjuvant therapy administered concurrently with standard treatments in WT1-positive breast cancer patients. METHODS: Patients were treated in 4 cohorts according to neoadjuvant treatment (A: post-menopausal, hormone receptor [HR]-positive patients receiving aromatase inhibitors; B: patients receiving chemotherapy; C: HER2-overexpressing patients on trastuzumab-chemotherapy combination; D: HR-positive/HER2-negative patients on chemotherapy). Patients (cohorts A-C) were randomized (2:1) to receive 6 or 8 doses of WT1-immunotherapeutic or placebo together with standard neoadjuvant treatment in a double-blind manner; cohort D patients received WT1-immunotherapeutic in an open manner. Safety was assessed throughout the study. WT1-specific antibodies were assessed pre- and post-vaccination. RESULTS: Sixty-two patients were randomized; 60 received ≥ one dose of WT1-immunotherapeutic. Two severe toxicities were reported: diarrhea (cohort C; also reported as a grade 3 serious adverse event) and decreased left ventricular ejection fraction (cohort B; also reported as a grade 2 adverse event). Post-dose 4 of WT1-immunotherapeutic, 10/10 patients from cohort A, 0/8 patients from cohort B, 6/11 patients from cohort C, and 2/3 patients from cohort D were humoral responders. The sponsor elected to close the trial prematurely. CONCLUSIONS: Concurrent administration of WT1-immunotherapeutic and standard neoadjuvant therapy was well tolerated and induced WT1-specific antibodies in patients receiving neoadjuvant aromatase inhibitors. In patients on neoadjuvant chemotherapy or trastuzumab-chemotherapy combination, the humoral response was impaired or blunted, likely due to either co-administration of corticosteroids and/or the chemotherapies themselves.

Endemic species may have complex histories: within-refugium phylogeography of an endangered Iberian vole.

Glacial refugia protected and promoted biodiversity during the Pleistocene, not only at a broader scale, but also for many endemics that contracted and expanded their ranges within refugial areas. Understanding the evolutionary history of refugial endemics is especially important in the case of endangered species to recognize the origins of their genetic structure and thus produce better informed conservation practices. The Iberian Peninsula is an important European glacial refugium, rich in endemics of conservation concern, including small mammals, such as the Cabrera vole (Microtus cabrerae). This near-threatened rodent is characterized by an unusual suite of genetic, life history and ecological traits, being restricted to isolated geographic nuclei in fast-disappearing Mediterranean subhumid herbaceous habitats. To reconstruct the evolutionary history of the Cabrera vole, we studied sequence variation at mitochondrial, autosomal and sex-linked loci, using invasive and noninvasive samples. Despite low overall mitochondrial and nuclear nucleotide diversities, we observed two main well-supported mitochondrial lineages, west and east. Phylogeographic modelling in the context of the Cabrera vole's detailed fossil record supports a demographic scenario of isolation of two populations during the Last Glacial Maximum from a single focus in the southern part of the Iberian Peninsula. In addition, our data suggest subsequent divergence within the east, and secondary contact and introgression of the expanding western population, during the late Holocene. This work emphasizes that refugial endemics may have a phylogeographic history as rich as that of more widespread species, and conservation of such endemics includes the preservation of that genetic legacy.

5-ALA-mediated photodynamic therapy reduces the parasite load in mice infected with Leishmania braziliensis.

Photodynamic therapy (PDT) has proven to be an effective alternative for the treatment of cutaneous leishmaniasis. Skin lesions consist of ulcers with well-defined raised edges, and granular floor. Th1 immune response is the protective profile in patients infected with Leishmania. In this study, the photodynamic therapy with 5-aminolevulinic acid, the parasitic load, and the modulation of the immune response was evaluated in mice infected with Leishmania braziliensis. Balb/c mice were infected with L. braziliensis and subsequently treated with three sections of PDT. The parasite load and mRNA expression of cytokines (IFN-γ, IL-4, IL-17, IL-22, IL-27, IL-10) and transcription factors (GATA-3, Foxp3 and T-bet) were analysed by quantitative PCR. The parasite load in the treated group was significantly lower than in the untreated group (P<.0001); in PDT treated animals, we observed an increase in IFN-γ and T-bet mRNA (P=.012 and P=.0071). There was a significant reduction in mRNA expression of IL-22 associated with an increased expression of IL-27 mRNA in the animals treated with light only (P=.0001). 5-ALA associated with photodynamic therapy promotes a reduction in parasite load and an increased expression of IFN-γ and T-bet mRNA.

Helminth parasites of South American fishes: current status and characterization as a model for studies of biodiversity.

The South American subcontinent supports one of the world's most diverse and commercially very important ichthyofauna. In this context, the study of South American fish parasites is of increased relevance in understanding their key roles in ecosystems, regulating the abundance or density of host populations, stabilizing food webs and structuring host communities. It is hard to estimate the number of fish parasites in South America. The number of fish species studied for parasites is still low (less than 10%), although the total number of host-parasite associations (HPAs) found in the present study was 3971. Monogeneans, with 835 species (1123 HPAs, 28.5%), and trematodes, with 662 species (1127 HPAs, 30.9%), are the more diverse groups. Data gathered from the literature are useful to roughly estimate species richness of helminths from South American fish, even though there are some associated problems: the reliability of information depends on accurate species identification; the lack of knowledge about life cycles; the increasing number of discoveries of cryptic species and the geographically biased number of studies. Therefore, the closest true estimations of species diversity and distribution will rely on further studies combining both molecular and morphological approaches with ecological data such as host specificity, geographical distribution and life-cycle data. Research on biodiversity of fish parasites in South America is influenced by problems such as funding, taxonomic impediments and dispersion of research groups. Increasing collaboration, interchange and research networks in the context of globalization will enable a promising future for fish parasitology in South America.

Evaluation of helper-dependent canine adenovirus vectors in a 3D human CNS model.

Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.

Increased titer and reduced lactate accumulation in recombinant retrovirus production through the down-regulation of HIF1 and PDK.

Many mammalian cell lines used in the manufacturing of biopharmaceuticals exhibit high glycolytic flux predominantly channeled to the production of lactate. The accumulation of lactate in culture reduces cell viability and may also decrease product quality. In this work, we engineered a HEK 293 derived cell line producing a recombinant gene therapy retroviral vector, by down-regulating hypoxia inducible factor 1 (HIF1) and pyruvate dehydrogenase kinase (PDK). Specific productivity of infectious viral titers could be increased more than 20-fold for single gene knock-down (HIF1 or PDK) and more than 30-fold under combined down-regulation. Lactate production was reduced up to 4-fold. However, the reduction in lactate production, alone, was not sufficient to enhance the titer: high-titer clones also showed significant enrollment of metabolic routes not related to lactate production. Transcriptome analysis indicated activation of biological amines metabolism, detoxification routes, including glutathione metabolism, pentose phosphate pathway, glycogen biosynthesis and amino acid catabolism. The latter were validated by enzyme activity assays and metabolite profiling, respectively. High-titer clones also presented substantially increased transcript levels of the viral genes expression cassettes. The results herein presented demonstrate the impact of HIF1 and PDK down-regulation on the production performance of a mammalian cell line, reporting one of the highest fold-increase in specific productivity of infectious virus titers achieved by metabolic engineering. They additionally highlight the contribution of secondary pathways, beyond those related to lactate production, that can be also explored to pursue improved metabolic status favoring a high-producing phenotype.

Plant growth-promoting Burkholderia species isolated from annual ryegrass in Portuguese soils.

AIMS: To search for culturable Burkholderia species associated with annual ryegrass in soils from natural pastures in Portugal, with plant growth-promoting effects. METHODS AND RESULTS: Annual ryegrass seedlings were used to trap Burkholderia from two different soils in laboratory conditions. A combined approach using genomic fingerprinting and sequencing of 16S rRNA and recA genes resulted in the identification of Burkholderia strains belonging to the species Burkholderia graminis, Burkholderia fungorum and the Burkholderia cepacia complex. Most strains were able to solubilize mineral phosphate and to synthesize indole acetic acid; some of them could produce siderophores and antagonize the phytopathogenic oomycete, Phytophthora cinnamomi. A strain (G2Bd5) of B. graminis was selected for gnotobiotic plant inoculation experiments. The main effects were the stimulation of root growth and enhancement of leaf lipid synthesis and turnover. Fluorescence in situ hybridization and confocal laser microscopy evidenced that strain G2Bd5 is a rhizospheric and endophytic colonizer of annual ryegrass. CONCLUSIONS: This work revealed that annual ryegrass can naturally associate with members of the genus Burkholderia. A novel plant growth promoting strain of B. graminis was obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel strain belongs to the plant-associated Burkholderia cluster and is a promising candidate for exploitation as plant inoculant in field conditions.

Impact of adenovirus life cycle progression on the generation of canine helper-dependent vectors.

Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus-cell interaction.

Single-step cloning-screening method: a new tool for developing and studying high-titer viral vector producer cells.

This article describes a novel method merging the cloning of viral vector producer cells with vector titer screening, allowing for screening 200-500 clones in 2 weeks. It makes use of a GFP separated into two fragments, S10 and S11 (Split GFP), fluorescing only upon transcomplementation. Producer cells carrying a S11 viral transgene are cloned in 96-well plates and co-cultured with target cells stably expressing S10. During the period of clone expansion, S11 viruses infect S10 target cells reconstituting the GFP signal. Transcomplemented fluorescence data provide direct estimation of the clone's productivity and can be analyzed in terms of density distribution, offering valuable information on the average productivity of the cell population and allowing the identification of high-producing clones. The method was validated by establishing a retrovirus producer from a nude cell line, in <3 months, inserting three vector constructs without clone selection or screening in between. Clones producing up to 10(8) infectious particles per ml were obtained, delivering optimal ratios of infectious-to-total particles (1 to 5). The method was additionally used to evaluate the production performance of HEK 293 and HEK 293T cell lines demonstrating that the latter sustains increased titers. Finally, it was used to study genetic manipulation of glutathione metabolism in retrovirus production showing that changing cell metabolism steers higher vector expression with titer increases of more than one order of magnitude.This method is a valuable tool not only for cell line development but also for genetic manipulation of viral vector and/or producer cells contributing to advancing the field of viral gene therapy.