Efficacy and Safety of Tinzaparin Thromboprophylaxis in Lung Cancer Patients with High Thromboembolic Risk: A Prospective, Observational, Single-Center Cohort Study.

BACKGROUND: The aim of this study was to record and assess the efficacy and safety ofthromboprophylaxis with an intermediate dose of Tinzaparin in lung cancer patients with high thrombotic risk. METHODS: This was a non-interventional, single-arm, prospective cohort study of lung cancer patients who received thromboprophylaxis with Tinzaparin 10.000 Anti-Xa IU in 0.5 mL, OD, used in current clinical practice. Enrolled ambulatory patients signed informed consent. Anti-Xa levels were tested. RESULTS: In total, 140 patients were included in the study, of which 81.4% were males. The histology of the tumor was mainly adenocarcinoma. Lung cancer patients with high thrombotic risk based on tumor, patient, treatment, and laboratory-related factors were enrolled. Only one patient experienced a thrombotic event (0.7%), and 10 patients had bleeding events (7.1%), including only one major event. Anti-Xa levels measured at 10 days and 3 months did not differ significantly between patients who developed hemorrhagic events and those who did not (p = 0.26 and p = 0.32, respectively). CONCLUSION: Thromboprophylaxis with an intermediate Tinzaparin dose in high thrombotic-risk lung cancer patients is a safe and effective choice for the prevention of VTE.

Digging into the NGS Information from a Large-Scale South European Population with Metastatic/Unresectable Pancreatic Ductal Adenocarcinoma: A Real-World Genomic Depiction.

Despite ongoing oncological advances, pancreatic ductal adenocarcinoma (PDAC) continues to have an extremely poor prognosis with limited targeted and immunotherapeutic options. Its genomic background has not been fully characterized yet in large-scale populations all over the world. Methods: Replicating a recent study from China, we collected tissue samples from consecutive Greek patients with pathologically-confirmed metastatic/unresectable PDAC and retrospectively investigated their genomic landscape using next generation sequencing (NGS). Findings: From a cohort of 409 patients, NGS analysis was successfully achieved in 400 cases (56.50% males, median age: 61.8 years). Consistent with a previous study, KRAS was the most frequently mutated gene in 81.50% of tested samples, followed by TP53 (50.75%), CDKN2 (8%), and SMAD4 (7.50%). BRCA1/2 variants with on-label indications were detected in 2%, and 87.50% carried a variant associated with off-label treatment (KRAS, ERBB2, STK11, or HRR-genes), while 3.5% of the alterations had unknown/preliminary-studied actionability (TP53/CDKN2A). Most of HRR-alterations were in intermediate- and low-risk genes (CHEK2, RAD50, RAD51, ATM, FANCA, FANCL, FANCC, BAP1), with controversial actionability: 8% harbored a somatic non-BRCA1/2 alteration, 6 cases had a high-risk alteration (PALB2, RAD51C), and one co-presented a PALB2/BRCA2 alteration. Elevated LOH was associated with HRR-mutated status and TP53 mutations while lowered LOH was associated with KRAS alterations. Including TMB/MSI data, the potential benefit from an NGS-oriented treatment was increased from 1.91% to 13.74% (high-MSI: 0.3%, TMB > 10 muts/MB: 12.78%). TMB was slightly increased in females (4.75 vs. 4.46 muts/MB) and in individuals with age > 60 (4.77 vs. 4.40 muts/MB). About 28.41% showed PD-L1 > 1% either in tumor or immune cells, 15.75% expressed PD-L1 ≥ 10%, and only 1.18% had PD-L1 ≥ 50%. This is the largest depiction of real-world genomic characteristics of European patients with PDAC, which offers some useful clinical and research insights.

Delineating the SARS-CoV-2 Induced Interplay between the Host Immune System and the DNA Damage Response Network.

COVID-19 is an infectious disease caused by the SARS-CoV-2 coronavirus and characterized by an extremely variable disease course, ranging from asymptomatic cases to severe illness. Although all individuals may be infected by SARS-CoV-2, some people, including those of older age and/or with certain health conditions, including cardiovascular disease, diabetes, cancer, and chronic respiratory disease, are at higher risk of getting seriously ill. For cancer patients, there are both direct consequences of the COVID-19 pandemic, including that they are more likely to be infected by SARS-CoV-2 and more prone to develop severe complications, as well as indirect effects, such as delayed cancer diagnosis or treatment and deferred tests. Accumulating data suggest that aberrant SARS-CoV-2 immune response can be attributed to impaired interferon signaling, hyper-inflammation, and delayed adaptive immune responses. Interestingly, the SARS-CoV-2-induced immunological abnormalities, DNA damage induction, generation of micronuclei, and the virus-induced telomere shortening can abnormally activate the DNA damage response (DDR) network that plays a critical role in genome diversity and stability. We present a review of the current literature regarding the molecular mechanisms that are implicated in the abnormal interplay of the immune system and the DDR network, possibly contributing to some of the COVID-19 complications.

Oxidative Stress and Deregulated DNA Damage Response Network in Lung Cancer Patients.

The deregulated DNA damage response (DDR) network is associated with the onset and progression of cancer. Herein, we searched for DDR defects in peripheral blood mononuclear cells (PBMCs) from lung cancer patients, and we evaluated factors leading to the augmented formation of DNA damage and/or its delayed/decreased removal. In PBMCs from 20 lung cancer patients at diagnosis and 20 healthy controls (HC), we analyzed oxidative stress and DDR-related parameters, including critical DNA repair mechanisms and apoptosis rates. Cancer patients showed higher levels of endogenous DNA damage than HC (p < 0.001), indicating accumulation of DNA damage in the absence of known exogenous genotoxic insults. Higher levels of oxidative stress and apurinic/apyrimidinic sites were observed in patients rather than HC (all p < 0.001), suggesting that increased endogenous DNA damage may emerge, at least in part, from these intracellular factors. Lower nucleotide excision repair and double-strand break repair capacities were found in patients rather than HC (all p < 0.001), suggesting that the accumulation of DNA damage can also be mediated by defective DNA repair mechanisms. Interestingly, reduced apoptosis rates were obtained in cancer patients compared with HC (p < 0.001). Consequently, the expression of critical DDR-associated genes was found deregulated in cancer patients. Together, oxidative stress and DDR-related aberrations contribute to the accumulation of endogenous DNA damage in PBMCs from lung cancer patients and can potentially be exploited as novel therapeutic targets and non-invasive biomarkers.

Latest evidence on immune checkpoint inhibitors in metastatic colorectal cancer: A 2022 update.

The long-term remissions induced by immune-checkpoint inhibitors (ICIs) in many types of cancers have opened up the possibility of a broader use of immunotherapy in less immunogenic but genetically heterogeneous tumours. Regarding metastatic colorectal cancer (mCRC), in first-line setting, pembrolizumab has been approved as preferred option and nivolumab, alone or in combination with ipilimumab as alternative option for patients with mismatch-repair-deficient and microsatellite instability-high (dMMR/MSI-H) disease, independently of their eligibility for intensive chemotherapy. In subsequent lines, both these immunotherapeutic regimens (e.g., pembrolizumab and nivolumab+/-ipilimumab) as well as dostarlimab-gxly are currently recommended for patients with dMMR/MSI-H chemo-resistant mCRC who have not previously received an ICI. Beginning from the rationale behind the immune-mediated interplay in the dMMR/MSI-H bowel microenvironment, we provide here an update on the evolution status of all available, approved or not, ICIs in mCRC, describing their efficacy and toxicity profile with an emphasis on the pivotal trials supporting current colorectal indications. For each ICI agent, the results from combinations under investigation, particularly for those being upgraded in clinical phasing, the perspectives but also the limitations of main ongoing trials are thoroughly discussed. In the close future, upcoming data are expected to confirm the clinical benefit of ICIs and to further expand their role in mCRC.

Front-Line Bevacizumab plus Chemotherapy with or without Maintenance Therapy for Metastatic Breast Cancer: An Observational Study by the Hellenic Oncology Research Group.

Front-line bevacizumab (BEV) in combination with taxanes offers benefit in progression-free survival (PFS) in metastatic breast cancer (mBC). The medical records of mBC patients, treated with front-line BEV-based chemotherapy, were retrospectively reviewed in order to generate real life safety and efficacy data. Patients with human epidermal growth factor receptor 2 (HER2)-negative mBC treated with front-line BEV in combination with chemotherapy were eligible. Maintenance therapy with BEV and/or hormonal agents was at the physicians' discretion. Among the 387 included patients, the most common adverse events were anemia (61.9%, mainly grade 1), grade 3/4 neutropenia (16.5%), grade 1/2 fatigue (22.3%), and grade 1/2 neuropathy (19.6%). Dose reductions were required in 164 cycles (7.1%) and toxicity led to treatment discontinuation in 21 patients (5.4%). The median PFS and the median overall survival (OS) were 13.3 (95% CI: 11.7-14.8) and 32.3 months (95% CI: 27.7-36.9), respectively. Maintenance therapy, with hormonal agents (ET) and/or BEV, was associated with longer OS versus no maintenance therapy (47.2 versus 23.6 months; p < 0.001) in patients with hormone receptor (HR)-positive disease and BEV maintenance offered longer OS versus no maintenance in patients with HR-negative disease (52.8 versus 23.3; p = 0.023). These real-life data show that front-line BEV-based chemotherapy in HER2-negative mBC patients is an effective treatment with an acceptable toxicity profile. The potential benefit of maintenance treatment, especially ET, is important and warrants further research.

Application of the International System for Reporting Serous Fluid Cytopathology with Cytohistological Correlation and Risk of Malignancy Assessment.

The International System for Reporting Serous Fluid Cytopathology (TIS) classifies serous effusions into five categories: non-diagnostic (ND), negative for malignancy (NFM), atypia of unknown significance (AUS), suspicious for malignancy (SFM) and malignant (MAL). The main objectives of this classification comprise the establishment of a universal code of communication between cytopathologists and clinicians and histopathologists, as well as between different laboratories worldwide, paving the way for the setting of clinical management guidelines based on the risk of malignancy assessment for each diagnostic category. We retrieved the total number of pleural and peritoneal effusion cases of our department for the three-year time period between 2018 and 2020, yielding a total of 528 and 500 cases, respectively. We then proceeded to reclassify each specimen according to TIS guidelines and calculate the risk of malignancy (ROM) for each category by searching each patients' histology records, medical history and clinical follow-up. For pleural effusions, 3 (0.57%) cases were classified as ND, 430 (81.44%) cases as NFM, 15 (2.84%) as AUS, 15 (2.84%) as SFM and 65 (12.31%) as MAL. ROM amounted to 0%, 5.3%, 33.33%, 93.33% and 100% for each category, respectively. As far as peritoneal effusions are concerned, 6 (1.2%) were categorized as ND with ROM estimated at 16.66%, 347 (69.4%) as NFM (ROM = 9%), 13 (2.6%) as AUS (ROM = 38.46%), 12 (2.4%) as SFM (ROM = 83.33%) and 122 (24.4%) as MAL (ROM = 100%). Our results underline the utility of the current classification, both as a means of communication between doctors of different specialties and as general guidelines for the further clinical management of patients.

Correction: Aberrant DNA Damage Response Pathways May Predict the Outcome of Platinum Chemotherapy in Ovarian Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0117654.].

DNA Damage Repair: Predictor of Platinum Efficacy in Ovarian Cancer?

Ovarian cancer (OC) is the seventh most common type of cancer in women worldwide. Treatment for OC usually involves a combination of surgery and chemotherapy with carboplatin and paclitaxel. Platinum-based agents exert their cytotoxic action through development of DNA damage, including the formation of intra- and inter-strand cross-links, as well as single-nucleotide damage of guanine. Although these agents are highly efficient, intrinsic and acquired resistance during treatment are relatively common and remain a major challenge for platinum-based therapy. There is strong evidence to show that the functionality of various DNA repair pathways significantly impacts tumor response to treatment. Various DNA repair molecular components were found deregulated in ovarian cancer, including molecules involved in homologous recombination repair (HRR), nucleotide excision repair (NER), mismatch repair (MMR), non-homologous end-joining (NHEJ), and base excision repair (BER), which can be possibly exploited as novel therapeutic targets and sensitive/effective biomarkers. This review attempts to summarize published data on this subject and thus help in the design of new mechanistic studies to better understand the involvement of the DNA repair in the platinum drugs resistance, as well as to suggest new therapeutic perspectives and potential targets.

Subcutaneous Trastuzumab Combined with Pertuzumab and Docetaxel as First-line Treatment of Advanced HER2-positive Breast Cancer.

BACKGROUND/AIM: Subcutaneous (s.c.) trastuzumab was introduced in the (neo)adjuvant setting, based on the non-inferiority results and patient preference. In the advanced setting, preliminary safety data have only been reported. We conducted an observational study of s.c. trastuzumab in combination with i.v. pertuzumab and docetaxel in the first-line setting of human epidermal growth factor receptor 2 (HER2)-positive advanced breast cancer. PATIENTS AND METHODS: In this single-institution study, patients received 600 mg s.c. trastuzumab in combination with 840 mg pertuzumab for the first cycle and 420 mg for the following cycles, and 75-100 mg/m2 docetaxel, followed by maintenance with s.c. trastuzumab and pertuzumab until disease progression or unacceptable toxicity. Endpoints were efficacy and safety. RESULTS: Forty patients were enrolled. The median number of cycles with docetaxel was six, while the median number of maintenance cycles was 21. With a median follow-up of 37 months, median progression-free survival and overall survival were 24 and 35 months. CONCLUSION: Subcutaneous trastuzumab in combination with pertuzumab and docetaxel is well tolerated and effective in HER2-positive advanced breast cancer.

Biweekly Gemcitabine/Nab-Paclitaxel as First-line Treatment for Advanced Pancreatic Cancer.

BACKGROUND/AIM: During recent years, a survival advantage was reported for first-line treatment of advanced pancreatic cancer with two new regimens, FOLFIRINOX and gemcitabine/nab-paclitaxel, over gemcitabine monotherapy. Gemcitabine/nab-paclitaxel administration on days 1, 8 and 15 of a 4-week cycle is associated with some practical disadvantages. We adopted a biweekly regimen with the same dose density. PATIENTS AND METHODS: Patients with Eastern Cooperative Oncology Group performance status 0-2 diagnosed with advanced histologically or cytologically confirmed pancreatic cancer and no prior treatment were included in the study. Study combination included 1.5 g/m2 gemcitabine and 175 mg/m2 nab-paclitaxel given every 2 weeks. Survival analysis was performed using the Kaplan-Meier method. RESULTS: Forty-six patients were treated with this regimen. Adverse events were similar to those of the original regimen. Median progression-free and overall survival were 5 and 10 months, respectively. CONCLUSION: Biweekly gemcitabine/nab-paclitaxel seems to have a similar safety and efficacy profile as the original regimen.

Real-world experience of everolimus as second-line treatment in metastatic renal cell cancer after failure of pazopanib.

AIM: We aimed to provide real-life data on the outcomes of metastatic renal cell carcinoma (mRCC) patients treated with everolimus as second-line treatment after failure of first-line pazopanib. PATIENTS AND METHODS: Data from the medical charts of mRCC patients from 8 centers in Greece and Spain were reviewed. All patients had received or were continuing to receive second-line everolimus treatment after failure of first-line treatment with pazopanib. No other previous therapies were allowed. The primary end point was the determination of progression-free survival (PFS). RESULTS: In total, 31 patients were enrolled. Of these, 26% had performance status (PS) >0, 88% were of intermediate/poor Memorial Sloan-Kettering Cancer Center (MSKCC) risk group, and only 61% had undergone prior nephrectomy. Median PFS was 3.48 months (95% CI: 2.37-5.06 months). Median overall survival (OS) from everolimus initiation was 8.9 months (95% CI: 6.47-13.14 months). Median OS from pazopanib initiation was 14.78 months (95% CI: 10.54-19.08 months). Furthermore, 32% of patients temporarily discontinued everolimus due to adverse events (AEs), and 22% of patients discontinued everolimus permanently due to toxicity. Most common toxicities were anemia (29%), stomatitis (26%), pneumonitis (19%), and fatigue (10%). Moreover, 14 AEs (27%) were graded as 3 or 4 and were reported by 13 patients (42%). CONCLUSION: This study provides data exclusively on the sequence pazopanib-everolimus in mRCC. Everolimus has a favorable safety profile and is active. The short PFS and OS could be attributed to the fact that the pazopanib-everolimus sequence was mainly offered to patients with adverse prognostic features, resulting in a modest increase in the combined OS of our population.

Bevacizumab, pemetrexed and carboplatin in first-line treatment of non-small cell lung cancer patients: Focus on patients with brain metastases.

Data concerning bevacizumab plus pemetrexed plus carboplatin as first-line treatment for patients with non-squamous non-small cell lung cancer (NSCLC) with or without brain metastases (BM) are lacking. The present study analyzed the efficacy and safety of this combination as induction therapy, followed by maintenance therapy with bevacizumab plus pemetrexed in non-squamous NSCLC patients with or without BM. Treatment-naïve patients with advanced non-squamous NSCLC and an Eastern Cooperative Oncology Group performance status score of 0-2 were eligible. Treatment consisted of carboplatin (area under the curve of 5), pemetrexed (500 mg/m2) and bevacizumab (15 mg/kg) every 3 weeks for 6 cycles. Responders and patients with stable disease received maintenance therapy with bevacizumab plus pemetrexed until disease progression, which was evaluated every 3 cycles, or unacceptable toxicity. Kaplan-Meier median progression-free survival (PFS) and overall survival (OS) times were the primary endpoints, and safety was the secondary endpoint. In total, 39 patients, aged 44-78 years (median, 60 years), were treated; 11 (28.2%) of whom presented with BM. The majority of patients (56.4%) completed 6 cycles of induction therapy, and 26 patients continued on to maintenance therapy. The median PFS time was 8.2 months [95% confidence interval (CI), 7.05-9.35] and the median OS time was 14.0 months (95% CI, 8.46-19.54). Median PFS and OS times did not differ significantly between patients with or without BM (log rank (Mantel-Cox): PFS, P=0.748 and OS, P=0.447). The majority of patients (76.9%) did not experience adverse events during treatment. Overall, bevacizumab plus pemetrexed plus carboplatin as induction therapy, followed by bevacizumab plus pemetrexed as maintenance therapy was effective and well tolerated in advanced NSCLC, whether brain metastases were present or not.

Aberrant DNA damage response pathways may predict the outcome of platinum chemotherapy in ovarian cancer.

Ovarian carcinoma (OC) is the most lethal gynecological malignancy. Despite the advances in the treatment of OC with combinatorial regimens, including surgery and platinum-based chemotherapy, patients generally exhibit poor prognosis due to high chemotherapy resistance. Herein, we tested the hypothesis that DNA damage response (DDR) pathways are involved in resistance of OC patients to platinum chemotherapy. Selected DDR signals were evaluated in two human ovarian carcinoma cell lines, one sensitive (A2780) and one resistant (A2780/C30) to platinum treatment as well as in peripheral blood mononuclear cells (PBMCs) from OC patients, sensitive (n = 7) or resistant (n = 4) to subsequent chemotherapy. PBMCs from healthy volunteers (n = 9) were studied in parallel. DNA damage was evaluated by immunofluorescence γH2AX staining and comet assay. Higher levels of intrinsic DNA damage were found in A2780 than in A2780/C30 cells. Moreover, the intrinsic DNA damage levels were significantly higher in OC patients relative to healthy volunteers, as well as in platinum-sensitive patients relative to platinum-resistant ones (all P<0.05). Following carboplatin treatment, A2780 cells showed lower DNA repair efficiency than A2780/C30 cells. Also, following carboplatin treatment of PBMCs ex vivo, the DNA repair efficiency was significantly higher in healthy volunteers than in platinum-resistant patients and lowest in platinum-sensitive ones (t1/2 for loss of γH2AX foci: 2.7±0.5h, 8.8±1.9h and 15.4±3.2h, respectively; using comet assay, t1/2 of platinum-induced damage repair: 4.8±1.4h, 12.9±1.9h and 21.4±2.6h, respectively; all P<0.03). Additionally, the carboplatin-induced apoptosis rate was higher in A2780 than in A2780/C30 cells. In PBMCs, apoptosis rates were inversely correlated with DNA repair efficiencies of these cells, being significantly higher in platinum-sensitive than in platinum-resistant patients and lowest in healthy volunteers (all P<0.05). We conclude that perturbations of DNA repair pathways as measured in PBMCs from OC patients correlate with the drug sensitivity of these cells and reflect the individualized response to platinum-based chemotherapy.

Development and validation of a PCR-based assay for the selection of patients more likely to benefit from therapeutic treatment with alkylating drugs.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: Previous studies have indicated that the levels of DNA damage induced in peripheral blood mononuclear cells by the alkylating drugs melphalan, cisplatin and carboplatin can serve as useful biomarkers predictive of the therapeutic response of cancer patients to these drugs. WHAT THIS STUDY ADDS: In the present study we developed a quantitative PCR-based assay, for the measurement of DNA damage. The advantages of this methodology are based on: its far greater sensitivity (about 250 times) than the traditional Southern blot-based method (the detection limit is ~10-20 lesions/10(6) nucleotides from the equivalent DNA of ~8000 cells); its simplicity and speed (results obtained within ~8h); its excellent reproducibility, with a coefficient of variance of 10-15% for different DNA preparations from similarly treated cells; its requirement for only minute amounts of material, and; the avoidance of radioisotope labeling. Moreover, emphasis was given to translate basic research findings into clinical practice through the validation of this assay for prediction of clinical outcome in multiple myeloma patients. AIM: In order to develop and validate a simple, sensitive and rapid method for the quantitation of alkylating drug-induced DNA damage. METHODS: HepG2 cells and blood samples were treated with alkylating drugs (melphalan, cisplatin, carboplatin). Gene-specific damage was examined using Southern blot and a multiplex long quantitative PCR (QPCR) carried out in a 7 kb fragment (part of the p53 gene) and a 0.5 kb fragment (part of the IFN-ß1 sequence; internal standard). RESULTS: The extent of PCR amplification of a p53 fragment was inversely proportional to the treatment concentrations of all anticancer drugs examined, indicating a dose-related inhibition by the DNA adducts formed. Parallel analysis of the same samples using both Southern blot and QPCR showed that the DNA adducts measured by QPCR corresponded to the interstrand cross-links in the case of melphalan, and to total drug-induced lesions in the case of the platinum drugs. The detection limit was ~10-20 lesions/10(6) nucleotides using DNA from ~8000 cells. The method is about 250 times more sensitive than the Southern blot-based method and the reproducibility is excellent, with an intraday coefficient of variance (CV) of 5-9% and an interday CV of 4-12%. Application of the QPCR assay to ex vivo melphalan-treated peripheral blood mononuclear cells from multiple myeloma patients, showed that the positive predictive value of this assay for clinical response to melphalan therapy was 92.9%. CONCLUSION: The PCR-based assay developed in this study can be used for the selection of cancer patients more likely to benefit from therapeutic treatment with alkylating drugs.

Assessment of species specificity of moulting accelerating compounds in Lepidoptera: comparison of activity between Bombyx mori and Spodoptera littoralis by in vitro reporter and in vivo toxicity assays.

BACKGROUND: Dibenzoylhydrazine analogues have been developed successfully as a new group of insect growth regulators, called ecdysone agonists or moulting accelerating compounds. A notable feature is their high activity against lepidopteran insects, raising the question as to whether species-specific analogues can be isolated. In this study, the specificity of ecdysone agonists was addressed through a comparative analysis in two important lepidopterans, the silkworm Bombyx mori L. and the cotton leafworm Spodoptera littoralis (Boisd.). RESULTS: When collections of non-steroidal ecdysone agonists containing different mother structures (dibenzoylhydrazine, acylaminoketone, tetrahydroquinoline) were tested, in vitro reporter assays showed minor differences using cell lines derived from both species. However, when compounds with high ecdysone agonist activity were examined in toxicity assays, larvicidal activity differed considerably. Of note was the identification of three dibenzoylhydrazine analogues with > 100-fold higher activity against Bombyx than against Spodoptera larvae. CONCLUSION: The present study demonstrated that species-specific ecdysone-agonist-based insecticides can be developed, but their species specificity is not based on differences in the activation of the ecdysone receptor but rather on unidentified in vivo parameters such as permeability of the cuticle, uptake/excretion by the gut or metabolic detoxification.

Endoparasitoid wasp bracovirus-mediated inhibition of hemolin function and lepidopteran host immunosuppression.

Successful embryonic development of parasitoid wasps in lepidopteran hosts is achieved through co-injection of polydna viruses whose gene products are thought to target the immune responses of the host. One gene product of the endosymbiont bracovirus of the parasitic wasp Cotesia rubecula, CrV1, has been reported to inhibit the immune responses of its endoparasitized lepidopteran host through interference with the haematocyte cytoskeletal structure. Here we establish that CcV1, the Cotesia congregata bracovirus orthologue of CrV1, is also uptaken by lepidopteran haemocytes and haemocyte-like established cell lines, but we also report on a different function of CcV1, which is highly relevant to the inhibition of the host immune responses and is based on its direct interaction with the pattern recognition molecule hemolin. Recombinant CcV1 inhibits hemolin functions, such as lipopolysaccharide binding and bacterial agglutination as well as bacterial phagocytosis by haemocytes and haemocyte-like cell lines, producing functional phenotypes equivalent to those observed to arise from RNAi-based inhibition of hemolin gene expression. Finally, we show that CcV1 and hemolin colocalize on the membrane surface of hemolin-expressing cells, a finding suggesting that CcV1 may be uptaken by haemocytes and inhibit haemocyte function as a result of its interaction with membrane-anchored hemolin.

High-throughput screening of ecdysone agonists using a reporter gene assay followed by 3-D QSAR analysis of the molting hormonal activity.

In this study, 172 diacylhydrazine analogs were examined for their ability to activate an ecdysone (molting hormone)-dependent reporter gene in a silkworm (Bombyx mori) cell-based high-throughput screening assay. The measured EC(50) values (concentration required to cause an effect in 50% of the cells) were used to construct a 3-D QSAR model that describes the ecdysone agonist activities of the diacylhydrazine analogs. Of these compounds, 14 exhibited no activity and were excluded from the 3-D QSAR analysis. The resulting equation described approximately 74% of the activity for 158 compounds. The final equation consisted of 42% electrostatic and 58% steric effects (r(2) = 0.74 and q(2) = 0.45). Comparative molecular field analysis (CoMFA) was used to visualize the steric and electrostatic potential fields that were favorable and unfavorable for biological activity. Of particular interest was the observation that the hydrophobic parameter (logP) was not necessary for describing the observed activities, although previous studies have cited the importance of hydrophobic parameters in both classical and 3-D QSAR analyses of these compounds. Modeling studies of the B. mori ecdysone receptor supported the observed physicochemical parameters required for activity reported by the CoMFA models. Comparison of the present analysis with those performed using other lepidopteran assay systems evidenced a high degree of correlation (r(2) = 0.81 for a Sf-9 cell-based assay and r(2) = 0.89 for a Chilo suppressalis integument-based assay), indicating that it is valid to compare the results generated with the B. mori cell-based system to those generated with previous lepidopteran assays. This novel assay system is amendable to a high-throughput screening format and should greatly increase our ability to discover novel agonists of molting hormone (ecdysone) activity.