Effectiveness of PD-(L)1 inhibitors alone or in combination with platinum-doublet chemotherapy in first-line (1L) non-squamous non-small-cell lung cancer (Nsq-NSCLC) with PD-L1-high expression using real-world data.

BACKGROUND: Anti-programmed cell death protein (death-ligand) 1 [PD-(L)1] therapy alone [cancer immunotherapy (CIT)-mono] or combined with platinum-based chemotherapy (CIT-chemo) is used as the first-line treatment for patients with metastatic non-small-cell lung cancer (NSCLC). Our study compared clinical outcomes with CIT-mono versus CIT-chemo in the specific clinical scenario of non-squamous (Nsq)-NSCLC with a high PD-L1 expression of ≥50% [tumor proportion score (TPS) or tumor cells (TC)]. METHODS: This was a retrospective cohort study using a real-world de-identified database. Patients with metastatic Nsq-NSCLC with high PD-L1 expression initiating first-line CIT-mono or CIT-chemo between 24 October 2016 and 28 February 2019 were followed up until 28 February 2020. We compared overall survival (OS) and real-world progression-free survival (rwPFS) using the Kaplan-Meier methodology. Hazard ratios (HRs) were adjusted (aHR) for differences in baseline key prognostic characteristics using the inverse probability of treatment weighting methodology. RESULTS: Patients with PD-L1-high Nsq-NSCLC treated with CIT-mono (n = 351) were older and less often presented with de novo stage IV disease than patients treated with CIT-chemo (n = 169). With a median follow-up of 19.9 months for CIT-chemo versus 23.5 months for CIT-mono, median OS and rwPFS did not differ between the two groups [median OS: CIT-chemo, 21.0 months versus CIT-mono, 22.1 months, aHR = 1.03, 95% confidence interval (CI) 0.77-1.39, P = 0.83; median rwPFS: CIT-chemo, 10.8 months versus CIT-mono, 11.5 months, aHR = 1.04, 95% CI 0.78-1.37, P = 0.81]. CIT-chemo showed significant and meaningful improvement in OS and rwPFS versus CIT-mono only in the never-smoker subgroup, albeit among a small sample of patients (n = 50; OS HR = 0.25, 95% CI 0.07-0.83, interaction P = 0.02; rwPFS HR = 0.40, 95% CI 0.17-0.95, interaction P = 0.04). CONCLUSION: Except in the subgroup of never-smoker patients, sparing the chemotherapy in first-line CIT treatment does not appear to impact survival outcomes in Nsq-NSCLC patients with high PD-L1 expression.

Influence of surface coating on the intracellular behaviour of gold nanoparticles: a fluorescence correlation spectroscopy study.

In the biomedical applications of nanoparticles (NPs), the proper choice of surface chemistry is a crucial aspect in their design. The nature of the coating can heavily impact the interaction of NPs with biomolecules, affect the state of aggregation, and ultimately determine their biological fate. As such, protein corona formation and the aggregation behaviour of gold NPs (Au NPs) are studied here. Au NPs are prepared with four distinct surface functionalisations, namely mercaptosuccinic acid (MSA), N-4-thiobutyroil glucosamine, HS-PEG5000 and HS-alkyl-PEG600. Corona formation, aggregation, and the intracellular behaviour of the Au NPs are then investigated by means of Fluorescence Correlation Spectroscopy (FCS) in cell culture media and in live cells. To evaluate the state of aggregation and the formation of a protein corona, the Au NPs are incubated in cell media and the diffusion coefficient is determined via FCS. The in vitro behaviour is compared with the level of aggregation of the NPs in cells. Diffusion times of the NPs are estimated at different positions in the cell after a one hour incubation period. It is found that the majority of MSA and glucose-Au NPs are present inside the cell as slowly diffusing species with diffusion times (τD) greater than 6000 µs (hydrodynamic diameter >250 nm). PEGylated Au NPs adsorb a small amount of protein and manifest low agglomeration both in media and in living cells. In particular, the HS-alkyl-PEG600 coating shows an excellent correlation between lower protein adsorption, 4-fold lower compared to the MSA coated NPs, and limited intracellular aggregation. In the case of single HS-alkyl-PEG600 coated NPs, it is found that typical intracellular τD values range from 500 to 1500 µs, indicating that these particles display reduced aggregation in the intracellular environment.

Endocytosis and intracellular localisation of type 1 ribosome-inactivating protein saporin-s6.

Saporin-S6 is a single-chain ribosome-inactivating protein (RIP) that has low toxicity in cells and animals. When the protein is bound to a carrier that facilitates cellular uptake, the protein becomes highly and selectively toxic to the cellular target of the carrier. Thus, saporin-S6 is one of the most widely used RIPs in the preparation of immunoconjugates for anti-cancer therapy. The endocytosis of saporin-S6 by the neoplastic HeLa cells and the subsequent intracellular trafficking were investigated by confocal microscopy that utilises indirect immunofluorescence analysis and transmission electron microscopy that utilises a direct assay with gold-conjugated saporin-S6 and an indirect immunoelectron microscopy assay. Our results indicate that saporin-S6 was taken up by cells mainly through receptor-independent endocytosis. Confocal microscopy analysis showed around 30% co-localisation of saporin-S6 with the endosomal compartment and less than 10% co-localisation with the Golgi apparatus. The pathway identified by the immunofluorescence assay and transmission electron microscopy displayed a progressive accumulation of saporin-S6 in perinuclear vesicular structures. The main findings of this work are the following: i) the nuclear localisation of saporin-S6 and ii) the presence of DNA gaps resulting from abasic sites in HeLa nuclei after intoxication with saporin-S6.

The transformer gene of Ceratitis capitata: a paradigm for a conserved epigenetic master regulator of sex determination in insects.

The transformer gene in Ceratitis capitata (Cctra(ep)) is the founding member of a family of related SR genes that appear to act as the master epigenetic switch in sex determination in insects. A functional protein seems to be produced only in individuals with a female XX karyotype where it is required to maintain the productive mode of expression through a positive feedback loop and to direct female development by instructing the downstream target genes accordingly. When zygotic activation of this loop is prevented, male development follows. Recently, tra(ep) orthologues were isolated in more distantly related dipteran species including Musca domestica, Glossina morsitans and Lucilia cuprina and in the Hymenopterans Apis mellifera and Nasonia vitripennis. All of these tra(ep) orthologues seem to act as binary switches that govern all aspects of sexual development. Transient silencing leads to complete masculinization of individuals with a female karyotype. Reciprocally, in some systems it has been shown that transient expression of the functional TRA product is sufficient to transactivate the endogenous gene and implement female development in individuals with a male karyotype. Hence, a mechanism based on tra(ep) epigenetic autoregulation seems to represent a common and presumably ancestral single principle of sex determination in Insecta. The results of these studies will not only be important for understanding divergent evolution of basic developmental processes but also for designing new strategies to improve genetic sexing in different insect species of economical or medical importance.

Expressional and functional analysis of the male-specific cluster mst36F during Drosophila spermatogenesis.

mst36Fa and mst36Fb are two male-specific genes that are part of a novel gene family recently characterized in Drosophila melanogaster. The genes are strictly clustered and show an identical tissue and temporal expression pattern limited to the male germline. Here we demonstrate that the transcription of these two genes, which is triggered by different cis regulatory elements, responds to the same testisspecific factors encoded by the aly and can class meiotic arrest genes. RNA interference was used to decrease expression of these two genes. We obtained a reduction of fertility in the transgenic adult males compared to the wild type. These data suggest that the Mst36Fa and Mst36Fb proteins may have an important role in the production of functional sperm.

Refinement of a transplantation project in the non-human primate by the use of a humane endpoint.

An assessment scheme was developed to establish a humane endpoint in a pig-to-primate renal xenotransplantation project, with a view to minimizing and controlling any pain or suffering conditions in the animals involved while still achieving the scientific objective. In particular, the assessment criteria for identifying the earliest endpoint are described, bearing in mind both the researcher's need to obtain top-quality data and the ethical need to safeguard the animals. The scheme should also be applicable to other experiments involving non-human primates (e.g. allotransplantation, survival after major surgery, pharmacological safety tests) because it considers reproducible general parameters together with aspects specific to each experimental model.

Associations of the plasma interleukin 6 (IL-6) levels with disability and mortality in the elderly in the Treviso Longeva (Trelong) study.

IL-6 expression is regulated by the interplay of several transcriptional and hormonal factors, including sex steroids and glucocorticoids. In late life IL-6 expression increases as a result from loss of the normally inhibiting sex steroids. IL-6 is one of several proinflammatory cytokines. It has been proposed that many chronic inflammatory diseases are the result of a dysregulation of IL-6 expression. In this work we demonstrate that increased IL-6 levels in elderly are associated with higher disability and mortality, also independently of age and comorbidity.

Occupational sensitization to ribosome-inactivating proteins in researchers.

BACKGROUND: Ribosome-inactivating proteins (RIPs) are expressed in many plants. Because of their anti-infectious and anti-proliferative effects, intensive research is going on for applying these toxins in therapy against viral infections or malignancies. Recently, we demonstrated that type I allergy against RIPs from elderberry can occur. OBJECTIVE: Stimulated by our study, a group of RIP researchers reported that some of the employees had suspected allergy to RIPs. METHODS AND RESULTS: We tested their sera in ELISA on natural RIPs. Specific IgE in four subjects were found against dianthin30, gelonin, momordin, PAP-S, saporin, ricin and volkensin. In contrast, asparin and lychnin did not show any IgE binding. When separating extracts of plants containing the toxins in SDS-PAGE, RIPs appeared to be the predominant constituents. Interestingly, among the other plant proteins, they were exclusively recognized by IgE in immunoblot. RIPs derived from close botanical families share high sequence homologies. Nevertheless, in IgE inhibition experiments with human sera, cross-reactivity between RIPs also derived from non-related plants could be demonstrated. CONCLUSION: We conclude that sensitization and IgE induction to RIPs may occur upon exposure. This has to be considered when applying them in therapy against malignancies or viral infections.

Toxicity of xanthine oxidoreductase to malignant B lymphocytes.

Reactive oxygen species (ROS) generated by xanthine oxidoreductase (XOR) were toxic to B lymphoma-derived Raji cells (positive for 8A monoclonal antibody, mAb). The sensitivity of these malignant cells to the hypoxanthine/XOR system was higher than that observed in peripheral human lymphocytes. The understanding of the mechanisms of cytotoxicity induced by XOR-produced ROS is essential in view of a possible clinical application. Cell death mostly had the feature of apoptosis and post-apoptotic necrosis and depended on the activity of XOR. Catalase, but not superoxide dismutase, protected cells from the toxicity of XOR, thus indicating that cell damage depended on the production of hydrogen peroxide. The toxicity of ROS was selectively targeted to malignant Raji cells by antibody-XOR conjugation, either directly, with an 8A-XOR conjugate, or indirectly, with an 8A mAb plus an anti-mouse IgG-XOR. Both direct and indirect immunotoxins induced apoptotic death to target cells in a dose-dependent manner. These conjugates showed no aspecific cytotoxicity in conditions very similar to the ex vivo treatment of cell suspension for bone marrow transplantation. Moreover, the prevalence of apoptotic death over necrosis may reduce the in vivo inflammatory response and its local and systemic consequences, thus becoming relevant in the construction of immunotoxins with therapeutic potential.

CD38 as a target of IB4 mAb carrying saporin-S6: design of an immunotoxin for ex vivo depletion of hematological CD38+ neoplasia.

An anti-CD38 mAb (IB4) coupled to saporin-S6, a type 1 ribosome-inactivating protein (RIP), was designed for ex vivo or loco-regional therapeutical applications in myeloma and lymphoma. The ability of this immunotoxin to eliminate CD38+ cells was studied in vitro on selected CD38+ human cell lines (Raji, HBL6, L540 and CEM) and on CD38+ neoplastic cells from a Non Hodgkin Lymphoma (NHL) patient. HBL6, Raji and L540 cells resulted very sensitive to the IB4/saporin-S6 conjugate, concentrations as low as 100 pM of the immunotoxin completely inhibited protein synthesis. CD38+ neoplastic cells from the NHL patient were completely eliminated after treatment with immunotoxin at 10 nM concentration. CFU-c rescue by bone marrow precursors was maintained after exposure to the immunotoxin. These results indicate that IB4/saporin-S6 is endowed with strong and specific cytotoxic effects on selected CD38+ tumor cells lineages. Consequently, it is reasonable to propose a clinical use of the IB4/saporin-S6 for ex vivo purging of unwanted cells (e.g. depletion of contaminating neoplastic cells in aphereses obtained from G-CSF-treated patients) or for loco-regional therapies of CD38+ tumors.

Immunotoxins and other conjugates: pre-clinical studies.

A wide variety of conjugates containing RIPs, of either chemical or recombinant type, have been made and tested against dangerous cells in vitro and in animal models. Many of these pre-clinical studies will be reviewed here dividing them on the basis of the target cell and the surface molecule specifically recognized.

The conjugate Rituximab/saporin-S6 completely inhibits clonogenic growth of CD20-expressing cells and produces a synergistic toxic effect with Fludarabine.

Immunotoxins are chimeric proteins consisting of a toxin coupled to an antibody. To date, several clinical trials have been conducted, and some are still ongoing, to evaluate their anti-tumor efficacy. In this view, we chemically constructed an anti-CD20 immunotoxin with the mAb Rituximab and the type 1 ribosome-inactivating protein (RIP) saporin-S6, designed for B cells non-Hodgkin's lymphoma (NHL) therapy. This immunotoxin showed a specific cytotoxicity for the CD20+ cell lines Raji and D430B, evidenced by inhibition of protein synthesis, evaluation of apoptosis and clonogenic assay. Upon conjugation, saporin-S6 increased its toxicity on target cells by at least 2 logs, with IC(50) values of 0.1-0.3 nM. The percentage of AnnexinV+ cells was over 95% in both cell lines treated with 10 nM immunotoxin. A complete elimination of Raji clones was reached with the 10 nM immunotoxin, whereas a mixture of free RIP and mAb gave about 90% of clonogenic growth. Rituximab/saporin-S6, at 10 nM concentration, also induced apoptosis in 80% of lymphoma cells from NHL patients. Moreover, sensitivity of Raji to Rituximab/saporin-S6 was augmented when cells were coincubated with Fludarabine. The synergistic toxic effect of the two drugs led to a total elimination of the neoplastic population.

Expression of CTLA-4 in nonhuman primate lymphocytes and its use as a potential target for specific immunotoxin-mediated apoptosis: results of in vitro studies.

T-cell-mediated immunoregulation is one of the main mechanisms implicated in induction and maintenance of transplantation tolerance. In this regard, deletion or modulation of xeno/alloantigen-specific T cells, as well as blocking of their interactions with other cell populations, are currently being pursued for tolerance induction in humans as well as nonhuman primates. In order to investigate whether cytotoxic T-lymphocyte antigen-4 (CTLA-4) may represent a suitable target for a T cell depletion approach in nonhuman primate models, we analysed CTLA-4 expression in peripheral blood mononuclear cells (PBMCs) from nonhuman primates and the potential role of two anti-CTLA-4 saporin-conjugated immunotoxins. The analysis was performed in PBMCs from 8 cynomolgus monkeys from Philippines and from Mauritius both at protein level by flow cytometry and at transcriptional level by RT-PCR. In addition, the apoptotic role of the immunotoxins was investigated. The results showed that CTLA-4 was expressed at variable levels depending on the origin of the cynomolgus monkeys and the resting or activated cell condition. CTLA-4 was not expressed on resting Mauritius PBMCs and showed a lower up-regulation upon PMA/PHA activation compared to the Philippines PBMCs that expressed CTLA-4 also before activation. Two CTLA-4 RNA transcripts (672 and 550 bp) were detected with levels variations after cell stimulation. Two anti-CTLA-4 immunotoxins induced in vitro apoptosis of activated PBMCs from both sources of cynomolgus monkeys. This is the first report that documents CTLA-4 expression both at protein and transcriptional level by nonhuman primate PBMCs and provides novel perspectives of xeno/allograft rejection immunotherapy based on CTLA-4 targeting.

Sex determination in flies, fruitflies and butterflies.

Sex determination mechanisms, differing in their modality, are widely represented in all the various animal taxa, even at the intraspecific level. Within the highly diversified Class Insecta, Drosophila has been used to unravel the mechanistic molecular and genetic interactions that are involved in sex determination. Indeed, the molecularly characterized genes of the Drosophila sex determination hierarchy X:A > Sxl > tra > dsx have been fruitful starting points in the cloning of homologous genes from other insect species. This cascade seems to control sex determination in all Drosophila species. However, no sex-specific regulatory Sxl homologues have been isolated from the Mediterranean fruitfly (medfly), Ceratitis capitata, the housefly, Musca domestica, Chrysomya rufifacies nor from the distantly related phorid fly Megaselia scalaris. Moreover, all these other species use primary signals different from the intricate X:A counting system of Drosophila. However, dsx homologues isolated from these and other dipteran species as well as from the silkmoth, Bombyx mori, share a conserved sex-specific regulation based on alternative splicing. An understanding of the sex determination mechanisms in insects that are of agricultural or public health importance may help in the development of improved methods for their control using the sterile insect technique.

The Mediet Project.

Preliminary evidence from a case control study of healthy postmenopausal women living in Palermo, Sicily, is presented to investigate the potential impact of a traditional Mediterranean diet on the risk of developing breast cancer. Of the 230 women who fulfilled specific eligibility criteria, 115 were enrolled in the study based on serum testosterone values equal to or greater than the median population value (0.14 microg/ml). Women were then individually randomized into a diet intervention (n = 58) and a control (n = 55) group. Women in the intervention group attended a weekly "cooking course" for 1 year, being trained by professional chefs in the correct use of the natural ingredients of the traditional Mediterranean diet, including whole cereals, legumes, seeds, fish, cruciferous vegetables, and many others. The intervention group was subsequently instructed to follow the learned diet at home, while the control group was only advised to increase the consumption of fruits and vegetables, as recommended by WHO. The following measures were taken at the beginning, middle, and end of the study: (a) fasting blood and 12-hour urine samples to assay defined hormonal endpoints; (b) height, weight, and circumference of the waist and hip; and (c) a food frequency and computerized 24-hour dietary recall questionnaire. After 1 year, both the control and the intervention groups showed satisfactory compliance rates (81 and 85%, respectively). In addition, preliminary results so far obtained reveal an unequivocal trend towards weight loss, a strong reduction in cholesterol levels, and a psychophysical feeling of well-being by women adopting the Mediterranean diet. The study is currently ongoing to verify the association of changes in serum and urine hormone levels and breast cancer risk in the intervention group.

Transferrin toxin but not transferrin receptor immunotoxin is influenced by free transferrin and iron saturation.

BACKGROUND: Cytotoxic agents can be targeted successfully to cancer cells. The efficacy of such novel and potent anticancer strategies may be influenced by variables of iron metabolism. METHODS: The in vitro cytotoxicity against glioma cells of transferrin (Tf)-based targeted toxins was compared with that of alpha-transferrin receptor (TfR)-immunotoxin. RESULTS: Of four Tf-based targeted toxins, Tf-gelonin, Tf-pokeweed antiviral protein, Tf-momordin and Tf-saporin, inhibitory concentration 50% values against glioma-derived cell lines HS683 and U251, ranged from [4.8 +/- 1.5] x 10(-10) m for Tf-saporin to [26.9 +/- 15.3] x 10(-10) m for Tf-gelonin in [(3)H]-leucine incorporation assays. Tf-saporin and alpha-TfR-saporin-immunotoxin had similar efficacy, even in the more quantitative clonogenic assay (4-5 log kill with 1 x 10(-9) m) using the myeloma cell line RPMI 8226 and glioma cell line U251. However, on RPMI 8226, the efficacy of Tf-saporin 1 x 10(-9) m was reduced by 90% in the presence of 150 microg mL(-1)(=20% of normal plasma value) competing diferric transferrin, whereas the efficacy of the corresponding immunotoxin was affected only marginally. In addition, the efficacy of Tf-based conjugates will depend on their iron saturation state. Iron desaturation of Tf-saporin was demonstrated by [(59)Fe]-labelling, subsequent CM-Sepharose chromatography and SDS-PAGE. Desaturation led to virtually complete loss of affinity for the transferrin receptor, as determined by flow cytometry, which could be largely restored upon resaturation. CONCLUSION: Transferrin-based toxin conjugates are strongly influenced by the presence of free transferrin and the iron saturation state. The corresponding alpha-transferrin receptor-immunotoxin does not show these disadvantages, has similar efficacy and should be preferred for further experiments.

Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model.

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies' growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.

Genomic organization and characterization of the white locus of the Mediterranean fruitfly, Ceratitis capitata.

An approximately 14-kb region of genomic DNA encoding the wild-type white eye (w+) color gene from the medfly, Ceratitis capitata has been cloned and characterized at the molecular level. Comparison of the intron-exon organization of this locus among several dipteran insects reveals distinct organizational patterns that are consistent with the phylogenetic relationships of these flies and the dendrogram of the predicted primary amino acid sequence of the white loci. An examination of w+ expression during medfly development has been carried out, displaying overall similarity to corresponding studies for white gene homologues in Drosophila melanogaster and other insects. Interestingly, we have detected two phenotypically neutral allelic forms of the locus that have arisen as the result of an apparently novel insertion or deletion event located in the large first intron of the medfly white locus. Cloning and sequencing of two mutant white alleles, w1 and w2, from the we,wp and M245 strains, respectively, indicate that the mutant conditions in these strains are the result of independent events--a frameshift mutation in exon 6 for w1 and a deletion including a large part of exon 2 in the case of w2.

Reliable sequence determination of ribosome- inactivating proteins by combining electrospray mass spectrometry and Edman degradation.

The primary structure of saporin-S9 and MAP-S, two type-1 ribosome-inactivating proteins isolated from the seeds of Saponaria officinalis L. and Mirabilis jalapa, respectively, was determined using a combined approach based on Edman degradation and electrospray ionization mass spectrometry (ESMS). Saporin-S9 has 253 amino acids with a calculated molecular mass of 28,492.99, which is in good agreement with that determined by ESMS (28 495 +/- 2 Da). Unlike other saporins with known primary structure, saporin-S9 contains four histidinyl residues (positions 111, 121, 216 and 248). By comparing the amino acid sequence of saporin-S9 with that of saporin-S6, we found 22 amino acid substitutions (8.7%), 13 of which are conservative and nine non-conservative. The residues known to be involved in the definition of the active site and with RNA base recognition are conserved. The four histidinyl residues and especially Lys for Gln203 contribute to the higher calculated pI value (10.17) of saporin-S9 compared with saporin-S6 (9.98). MAP-S contains 250 amino acid residues with a calculated molecular mass of 27,789.49, in good agreement with that determined by ESMS (27,789 +/- 2). Cys36 and Cys220 form a disulphide bridge and only four amino acid residues are different from the amino acid sequence of MAP, isolated from the roots of the same plant, i.e. Leu34 (Glu), Ile161 (Leu), Asp185 (Glu) and Asp191 (Glu) (in parentheses, the residues present in MAP). The reported approach can provide rapid and reliable sequence screening in the analysis of homologous proteins, including the presence of disulphide bridges.