Search

Background: Multiple myeloma (MM) cancers originate from plasma cells that have passed through the germinal center reaction where somatic hypermutation of Ig V regions takes place. Myeloma protein V regions often express many mutations and are thus a rich source of neoantigens (traditionally called idiotopes (Id)). Therefore, these are highly tumor-specific and excellent targets for immunotherapy., Methods: We have developed a DNA Id vaccine which as translated protein targets conventional dendritic cells (cDC) for CCL3-mediated delivery of myeloma protein V regions in a single-chain fragment variable (scFv) format. Vaccine efficacy was studied in the mouse MM model, mineral oil-induced plasmacytoma 315.BM., Results: The Id vaccine protected mice against a challenge with MM cells. Moreover, the vaccine had a therapeutic effect. However, in some of the vaccinated mice, MM cells not producing H chains escaped rejection, resulting in free light chain (FLC) MM. Depletion of CD8 + T cells abrogated vaccine efficacy, and protection was observed to be dependent on cDC1s, using Batf3 -/- mice. Modifications of scFv in the vaccine demonstrated that CD8 + T cells were specific for two mutated V H sequences., Conclusions: V H neoantigen-specific CD8 + T cells elicited by CCL3-containing Id vaccines had a therapeutic effect against MM in a mouse model. MM cells could escape rejection by losing expression of the H chain, thus giving rise to FLC MM., Competing Interests: Competing interests: BB is an inventor of patents related to Vaccibody vaccines. He is leader of the Scientific Advisory Board in the Nykode Therapeutics (former Vaccibody AS) company in which he holds shares., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Targeting antigens to dendritic cells represent a promising method for enhancing immune responses against specific antigens. However, many studies have focused on systemic delivery (intravenous or intraperitoneally) of targeted antigen, approaches that are not easily transferable to humans. Here we evaluate the efficacy of an influenza vaccine targeting Xcr1 + cDC1 administered by intranasal immunization. Intranasal delivery of antigen fused to the chemokine Xcl1, the ligand of Xcr1, resulted in specific uptake by lung CD103 + cDC1. Interestingly, intranasal immunization with influenza A/PR/8/34 haemagglutinin (HA) fused to Xcl1, formulated with poly(I:C), resulted in enhanced induction of antigen-specific IFNγ + CD4 + and IFNγ + CD8 + T cell responses in lung compared non-targeted anti-NIP-HA (αNIP-HA). Induction of antibody responses was, however, similar in Xcl1-HA and αNIP-HA immunized mice, but significantly higher than in mice immunized with monomeric HA. Both Xcl1-HA and αNIP-HA vaccines induced full protection when mice were challenged with a lethal dose of influenza PR8 virus, reflecting the strong induction of HA-specific antibodies. Our results demonstrate that i.n. delivery of Xcl1-HA is a promising vaccine strategy for enhancing T cell responses in addition to inducing strong antibody responses., (© 2021 The Authors. Scandinavian Journal of Immunology published by John Wiley & Sons Ltd on behalf of The Scandinavian Foundation for Immunology.)

Targeting antigen to conventional dendritic cells (cDCs) can improve antigen-specific immune responses and additionally be used to influence the polarization of the immune responses. However, the mechanisms by which this is achieved are less clear. To improve our understanding, we here evaluate molecular and cellular requirements for CD4 + T cell and antibody polarization after immunization with Xcl1-fusion vaccines that specifically target cDC1s. Xcl1-fusion vaccines induced an IgG2a/IgG2b-dominated antibody response and rapid polarization of Th1 cells both in vitro and in vivo . For comparison, we included fliC-fusion vaccines that almost exclusively induced IgG1, despite inducing a more mixed polarization of T cells. Th1 polarization and IgG2a induction with Xcl1-fusion vaccines required IL-12 secretion but were nevertheless maintained in BATF3 -/- mice which lack IL-12-secreting migratory DCs. Interestingly, induction of IgG2a-dominated responses was highly dependent on the early kinetics of Th1 induction and was important for optimal protection in an influenza infection model. Early Th1 induction was dominant, since a combined Xcl1- and fliC-fusion vaccine induced IgG2a/IgG2b polarized antibody responses similar to Xcl1-fusion vaccines alone. In summary, our results demonstrate that targeting antigen to Xcr1 + cDC1s is an efficient strategy for enhancing IgG2a antibody responses through rapid Th1 induction, which can be utilized for improved vaccine design., Competing Interests: BB is the inventor on patents on the vaccine molecules described herein (Vaccibodies). He is the leader of the scientific panel of Vaccibody AS and has shares in the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tesfaye, Bobic, Lysén, Huszthy, Gudjonsson, Braathen, Bogen and Fossum.)

Targeting Ag to surface receptors on conventional type 1 dendritic cells can enhance induction of Ab and T cell responses. However, it is unclear to what extent the targeted receptor influences the resulting responses. In this study, we target Ag to Xcr1, Clec9A, or DEC-205, surface receptors that are expressed on conventional type 1 dendritic cells, and compare immune responses in BALB/c and C57BL/6 mice in vitro and in vivo after intradermal DNA vaccination. Targeting hemagglutinin from influenza A to Clec9A induced Ab responses with higher avidity that more efficiently neutralized influenza virus compared with Xcr1 and DEC-205 targeting. In contrast, targeting Xcr1 resulted in higher IFN-γ + CD8 + T cell responses in spleen and lung and stronger cytotoxicity. Both Clec9A and Xcr1 targeting induced Th1-polarized Ab responses, although the Th1 polarization of CD4 + T cells was more pronounced after Xcr1 targeting. Targeting DEC-205 resulted in poor Ab responses in BALB/c mice and a more mixed Th response. In an influenza challenge model, targeting either Xcr1 or Clec9A induced full and long-term protection against influenza infection, whereas only partial short-term protection was obtained when targeting DEC-205. In summary, the choice of targeting receptor, even on the same dendritic cell subpopulation, may strongly influence the resulting immune response, suggesting that different targeting strategies should be considered depending on the pathogen., (Copyright © 2020 by The American Association of Immunologists, Inc.)

Introduction: Ogilvie syndrome, or acute colonic pseudo-obstruction (ACPO), represents a pathological entity, potentially with a severe outcome, due to the acute important dilation of the large bowel, in the absence of a mechanical luminal obstruction. Usually, it occurs in patients admitted in intensive care unit, that associate severe surgical or medical pathologies. The mechanism of the ACPO has not been completely explained, but it is assumed that the motor function of the colon may be affected, as a result of autonomic regulation disturbance. Early diagnosis and treatment help reduce the risk of severe outcome, such as ischemia or perforation. Material and Method: In addition to our experience, a literature search was elaborated in order to evaluate the incidence, the etiology, the clinical presentation and the diagnosis of the ACPO. Results and Conclusions: The present study may be of help in the process of guiding the optimal management of a critically ill patient is at high risk of developing colonic pseudo-obstruction., (Celsius.)

Immunogenicity of DNA vaccines can be increased by constructing the DNA in such a way that it encodes secreted homodimeric fusion proteins that target antigen-presenting cells (APCs). In this study, we have developed novel APC-targeting vaccine molecules with an increased flexibility due to introduction of a heterodimerization motif. The heterodimeric proteins permit four different fusions within a single molecule, thus allowing expression of two different APC-targeting moieties and two different antigens. Two types of heterodimeric fusion proteins were developed that employed either the ACID/BASE or the Barnase/Barstar motifs, respectively. The ACID/BASE heterodimeric vaccines conferred protection against challenges with either influenza virus or tumor cells in separate preclinical models. The ACID/BASE motif was flexible since a large number of different targeting moieties and antigens could be introduced with maintenance of specificity, antigenicity, and secretion. APC-targeting ACID/BASE vaccines expressing two different antigens induced antibody and T cell responses against either of the two antigens. Heterodimeric ACID/BASE DNA vaccines were of approximately the same potency as previously reported homodimeric DNA vaccines. The flexibility and potency of the ACID/BASE format suggest that it could be a useful platform for DNA vaccines that encode APC-targeting fusion proteins., (© 2020 The Author(s).)

Introduction: The abdominal wall hernias in patients with liver cirrhosis have a significant higher prevalence than those in non- cirrhotic patients. The best surgical treatment option and the optimal operative time still represent a subject of debate. Material and method: We have retrospectively analyzed the cases of abdominal wall hernias in patients with liver cirrhosis admitted in the Surgical Department of the Sfantul Pantelimon Clinical Emergency Hospital from Bucharest, undergoing surgical treatment between January 2012 and December 2016. Data regarding the laboratory results (the serum albumin and bilirubin levels, the Prothrombin Time) and the clinical aspects (the presence of ascites or encephalopathy) that helped establish the grade of the disease according to Child classification system, and, also, the information regarding the type of hernia, the character of the surgical indication (emergency or elective), the surgical technique and the postoperative evolution have been collected from the medical documents of the patients included in the present study. The statistical analysis has been made using the chi- square test. Results: 32 cases out of the total 65 patients included in the study, that underwent surgical intervention for the treatment of the abdominal wall defects, had umbilical hernia (49,23%), 18 cases presented with inguinal hernia (27.69%), 11 with incisional hernia (16.92%) and 4 with epigastric hernia (6.15%). 29 patients were subjected to elective surgery (44.6%) and 36 to emergency surgery (55,4%). Regarding the Child classification system used in the present study, 24 patients presented with Child A grade of cirrhosis (36.92%), 30 with Child B (46.15%) and 11 with Child C (16.92%). The postoperative morbidity rate was 45,83% in Child A group, 56,66% in Child B group and 81,8% in Child C group. The mortality rate was 4,16% in the Child A group (one death), 13.33% in the Child B group (4 deaths) and 72.72% in Child C group (8 cases). The highest mortality rates have been registered in patients that underwent emergency surgical intervention. Conclusions: The parietal defects in patients with liver cirrhosis can be surgically treated with satisfactory outcomes. The best results have been registered in patients with compensated form of the cirrhosis or in cases undergoing elective surgery. The methods used for the surgical treatment of the abdominal wall defects vary from herniorrhaphy to alloplastic techniques. For patients with ascites, a good control of this complication represents an important factor for the favourable postoperative evolution. The emergency surgical interventions are associated with greater risks of morbidity and mortality., (Celsius.)

B cell activating factor (BAFF), also called the B lymphocyte stimulator, has been known to show increased expression in primary Sjögren's syndrome (pSS) which could explain increased B cell activation characteristic of this disease. Belimumab, a fully human IgG1λ recombinant monoclonal antibody directed against B lymphocyte stimulator (Blys), has been reported to be efficacious in systemic lupus erythematosus (SLE) through its B cell-mediated action. Randomized controlled trials of belimumab in a selected target population of pSS patients are further warranted.

Traumatic abdominal wall hernias (TAWHs) are rare. Their diagnosis is mostly clinical and can be overlooked in the setting of trauma and distracting injuries or they can be misinterpreted as parietal hematomas. Associated lesions can influence decision making regarding time of operation and surgical technique. Our case highlights the management of a high-energy TAWH that associates a small bowel traumatic lesion. Surgical repair of TAWHs should follow general hernia repair principles. Further exploration of surgical options is necessary for a consensus to be reached.

Background: The naso-ocular interaction in allergic rhinoconjunctivitis is well recognized from epidemiological, clinical, and experimental observations. The precise mechanisms remain incompletely understood. A new mouse model of allergic rhinoconjunctivitis was used to investigate the contribution of mast cells and trigeminal ganglia activation to conjunctival (conj.) inflammation after nasal allergen provocation., Methods: Sensitized mice were exposed to ovalbumin (OVA) via the nose and/or conjunctiva, and conj. homogenates were analyzed for histamine and substance P (using ELISA) and by eosinophil peroxidase (EPO) and beta-hexosaminidase assays. The conj. effects of nasal allergen deposition were compared with those induced by the mast cell activator C48/80 and with pretreatment of the mast cell stabilizer ketotifen or the transient receptor potential channel receptor (TRP) agonist capsaicin. Protachykinin 1 (TAC1) expression was quantified in the trigeminal ganglia using real time polymerase chain reaction., Results: At 1 hour after nasal application of OVA, increased conj. levels of beta-hexosaminidase (0.68 ± 0.03 nm versus 0.56 ± 0.02 nm; p = 0.02), histamine (751.1 ± 52.17 ng/mL versus 546.3 ± 76.91 ng/mL; p = 0.05), and EPO (0.66 ± 0.09 nm versus 0.37 ± 0.03 nm; p = 0.02) were detected compared with saline. Higher levels of TAC1 expression were found in the trigeminal ganglia at 24 hours after OVA application (1326 ± 255 versus 687.5 ± 90.77 TAC1/beta-actin; p = 0.04). Nasal challenge with C48/80 increased substance P and beta-hexosaminidase levels in the conjunctiva, as well as TAC1 expression. Pretreatment with ketotifen resulted in lower levels of substance P as well as TAC1 expression. Destruction of sensory nerves in the nose by capsaicin reduced the OVA-induced conj. levels of substance P, histamine, and beta-hexosaminidase., Conclusion: Nasal allergen deposition in sensitized mice induced trigeminal TAC1 expression and conj. mast cell degranulation. These data represent a significant step forward in understanding the close interaction between nasal and conj. inflammation in allergy.

Background: Sinonasal inflammation and symptoms are often underdiagnosed in chronic obstructive pulmonary disease (COPD) patients. So far, it is not known to what extent anti-inflammatory nasal treatment may reduce sinonasal symptoms in COPD patients. This study was designed to examine the effects of nasal anti-inflammatory treatment on sinonasal symptoms and cough in COPD patients., Methods: Thirty-three COPD patients on stable bronchial therapy (salmeterol/fluticasone propionate 50/500 mg b.i.d. for >6 weeks) were randomized to receive fluticasone furoate (FF) or placebo nasal spray at 110 μg once daily for 12 weeks. Sinonasal symptoms and cough were monitored at baseline, at 6 and12 weeks of treatment, and at 4 weeks after cessation of the treatment using a visual analog scale. Levels of cytokines were measured in nasal secretions., Results: In contrast to the placebo group (n = 13), FF patients (n = 14) reported less nasal blockage (10.62 ± 4.21 mm versus 36.57 ± 8.01 mm; p = 0.0026), postnasal drip (1.46 ± 0.29 score versus 2.83 ± 0.38 score; p = 0.03), and nasal discharge (0.23 ± 0.12 score versus 1.77 ± 0.43 score; p = 0.01) after 6 weeks of treatment compared with baseline, which was still present at 12 weeks. FF patients reported less cough compared with baseline (25.54 ± 4.46 mm versus 36.79 ± 5.75 mm; p = 0.04), which was not the case in the placebo group (49.58 ± 10.44 mm versus 42.00 ± 8.05 mm; p = 0.38). Nine of 14 patients in the FF group (64%) reported slight to total relief of nasal symptoms, and this subgroup had a significant decrease in IL-8 levels in nasal secretions after 6 weeks of treatment (850.7 ± 207.2 pg/mL versus 1608 ± 696.5 pg/mL; p = 0.03) compared with baseline., Conclusion: Nasal FF treatment in COPD patients significantly reduced sinonasal symptoms, in parallel with reduced IL-8 in nasal secretion levels and cough.

Rationale: Airway hyperreactivity (AHR) is a key feature of bronchial asthma, and inhalation of irritants may facilitate development of nonallergic AHR. Swimmers exposed to hypochlorite (ClO(-))-containing water show a higher risk of developing AHR. We developed a mouse model in which instillation of ClO(-) before ovalbumin (OVA) induces AHR without bronchial inflammatory cells., Objectives: To investigate the mechanisms of ClO(-)-OVA-induced nonallergic AHR., Methods: The involvement of the transient receptor potential ankyrin (TRPA)1 channel was checked in vivo by the use of TRPA1(-/-) mice and in vitro by Ca(2+) imaging experiments. The role of substance P (SP) was investigated by pretreating animals with the receptor antagonist RP67580, by replacing ClO(-) with SP in vivo, and by immunofluorescent staining of large airways of exposed mice. The role of mast cells was evaluated by exposing mast cell-deficient Kit(Wh)/Kit(Wsh) mice to ClO(-)-OVA with or without mast cell reconstitution., Measurements and Main Results: ClO(-)-OVA did not induce AHR in TRPA1(-/-) mice, and ClO(-) generates a Ca(2+) influx in TRPA1-transfected cells. Pretreatment with RP67580 reduces ClO(-)-OVA-induced AHR, although no increased SP expression was shown in the airways. SP-OVA exposure resulted in the same AHR as induced by ClO(-)-OVA. Kit(Wsh)/Kit(Wsh) mice did not develop AHR in response to ClO(-)-OVA unless they were reconstituted with bone marrow-derived mast cells., Conclusions: Induction of AHR by exposure to ClO(-)-OVA depends on a neuroimmune interaction that involves TRPA1-dependent stimulation of sensory neurons and mast cell activation.

Placental growth factor (PlGF) and its receptor vascular endothelial growth factor receptor 1 (VEGFR1) play an important role in pathological conditions related to angiogenesis, vascular leakage, and inflammation. This study investigated their contributions to inflammation and the formation of edema in allergic asthma. The expression of PlGF and VEGFR1 was measured in induced sputum of patients with asthma (n = 11) and healthy subjects (n = 11), and in bronchial biopsies of house dust mite (HDM)-allergic patients stimulated with HDM allergens. The effects of the endonasal administration of human PlGF-2 and PlGF deficiency on inflammation and edema were evaluated in a murine model of allergic asthma. The migration of human neutrophils in response to hPlGF-2 was tested in vitro. The expression of PlGF and VEGFR1 was significantly higher in the sputum of patients with asthma, and in Der p 1-induced PlGF in biopsies from HDM-allergic patients. PlGF was increased in the bronchi of ovalbumin (OVA)-challenged mice compared with control mice (65 ± 17 pg/mg versus 18 ± 1 pg/mg, respectively; P < 0.01), and VEGFR1 was expressed in bronchial epithelium, endothelium (control mice), and inflammatory cells (OVA-challenged mice). The endonasal instillation of hPlGF-2 in wild-type, OVA-challenged mice led to an increase in bronchial neutrophils, lung tissue wet/dry ratio, and IL-17. PlGF-deficient mice showed lower numbers of BAL-infiltrating neutrophils, a reduced lung wet/dry ratio, and lower production of IL-17, macrophage inflammatory protein-2, and granulocyte chemotactic protein-2/LPS-induced chemokine compared with wild-type, OVA-challenged mice. hPlGF-2 induced the migration of human neutrophils in vitro in a VEGFR1-dependent way. PlGF and its receptor VEGFR1 are up-regulated in allergic asthma and play a proinflammatory role by inducing tissue edema, and increasing tissue neutrophilia and the production of IL-17.

Background: Some epidemiologic studies have indicated that attendance to chlorinated swimming pools is associated with airway hyperreactivity (AHR), allergies and asthma., Aim: To investigate the effects of sodium hypochlorite (NaClO), the main pool disinfectant, on allergic sensitization and airway inflammation in mice., Methods: In a first series of experiments, mice were sensitized to ovalbumin (OVA), followed by OVA aerosols with or without prior nasal instillation of NaClO (3ppm active chlorine). In a second series, naïve mice received 1-7 nasal instillations of OVA, 10min after instillations of NaClO or water. After 1, 3, 5 and 7 exposures airway reactivity to methacholine, cellular inflammation in bronchoalveolar lavage (BAL), serum OVA-specific IgEs and lung Th2 cytokines were measured., Results: In the first mouse model, airway allergy parameters were not significantly altered upon NaClO administration. However in the second model, NaClO exposure prior to OVA did induce AHR, already after 1 combined application. Combined NaClO+OVA exposure did not lead to an influx of inflammatory cells in BAL fluid or production of anti-OVA IgEs. No AHR developed when OVA was heat-denatured, pre-chlorinated, or replaced by bovine serum albumin or lipopolysaccharide., Conclusion: Nasal instillation of NaClO prior to OVA induces AHR without allergic sensitization. This response is OVA-specific., (Copyright © 2011. Published by Elsevier Ireland Ltd.)

Purpose: Combinatorial bio/chemical approach was applied to investigate dioxin-like contamination of soil and sediment at the petrochemical and organochlorine plant in Pancevo, Serbia, after the destruction of manufacturing facilities that occurred in the spring of 1999 and subsequent remediation actions., Materials and Methods: Soil samples were analyzed for indicator polychlorinated biphenyls (PCBs) by gas chromatography/electron capture detection (GC/ECD). Prioritized soil sample and sediment samples from the waste water channel were analyzed for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) by high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). Microethoxyresorufin o-deethylase (Micro-EROD) and H4IIE-luciferase bioassays were used for monitoring of dioxin-like compounds (DLC) and for better characterization of dioxin-like activity of soil samples., Results: Bioanalytical results indicated high dioxin-like activity in one localized soil sample, while the chemical analysis confirmed the presence of large quantities of DLC: 3.0 × 10(5) ng/g d.w. of seven-key PCBs, 8.2 ng/g d.w. of PCDD/Fs, and 3.0 × 10(5) ng/g d.w. of planar and mono-ortho PCBs. In the sediment, contaminant concentrations were in the range 2-8 ng/g d.w. of PCDD/Fs and 9-20 ng/g d.w. of PCBs., Conclusions: This study demonstrates the utility of combined application of bioassays and instrumental analysis, especially for developing and transition country which do not have capacity of the expensive instrumental analysis. The results indicate the high contamination of soil in the area of petrochemical plant, and PCDD/Fs contamination of the sediment from the waste water channel originating from the ethylene dichloride production.

Pulmonary function analysis is an important tool in the evaluation of mouse respiratory disease models, but much controversy still exists on the validity of some tests. Most commonly used pulmonary function variables of humans are not routinely applied in mice, and the question of which pulmonary function is optimal for the monitoring of a particular disease model remains largely unanswered. Our study aimed to delineate the potential and restrictions of existing pulmonary function techniques in different respiratory disease models, and to determine some common variables between humans and mice. A noninvasive (unrestrained plethysmography) and two invasive pulmonary function devices (forced maneuvers system from Buxco Research Systems [Wilmington, NC] and forced oscillation technique from SCIREQ [Montreal, PQ, Canada]) were evaluated in well-established models of asthma (protein and chemical induced): a model of elastase-induced pulmonary emphysema, and a model of bleomycin-induced pulmonary fibrosis. In contrast to noninvasive tests, both invasive techniques were efficacious for the quantification of parenchymal disease via changes in functional residual capacity, total lung capacity, vital capacity, and compliance of the respiratory system. Airflow obstruction and airflow limitation at baseline were only present in emphysema, but could be significantly induced after methacholine challenge in mice with asthma, which correlated best with an increase of respiratory resistance. Invasive pulmonary functions allow distinction between respiratory diseases in mice by clinically relevant variables, and should become standard in the functional evaluation of pathological disease models.

Lectins as carbohydrate-binding proteins have been employed in various biological assays for the detection and characterization of glycan structures on glycoproteins, including clinical biomarkers in disease states. A mannose-specific banana lectin (BanLec) is unique in its specificity for internal alpha1,3 linkages as well as beta1,3 linkages at the reducing termini. The immunomodulatory potential of natural BanLec was recognized by a strong immunoglobulin G4 antibody response and T cell mitogen activity in humans. To explore its applicability in glycoproteomics and its modulatory potential, the gene of banana lectin was cloned, sequenced and a recombinant protein was produced in Escherichia coli. The obtained cDNA revealed a novel banana lectin isoform, with an open reading frame of 426 nucleotides, encoding a cytoplasmatic protein of 141 amino acids. The molecular mass of rBanLec determined by ESI FT-MS and N-terminal sequencing confirmed the cDNA at the protein level. The specificity of rBanLec for detection glycan structures was the same as for natural BanLec as examined with five protein extracts rich in glycoprotein content, as well as with horseradish peroxidase glycoprotein. Besides, the immunomodulatory potential of rBanLec and nBanLec were comparable as assessed by an inhibition assay and a human T cell proliferation assay where they induced a strong proliferation response in CD3+, CD4+, and CD8+ populations of human PBMCs. This recombinant BanLec is a useful reagent for glycoproteomics and lectin microarrays, with a potential for modulation of the immune response.

In this study level of soil contamination by polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) in two oil refineries in Vojvodina region of Serbia was assessed using combined bio/chemical approach. Toxicity of the samples, determined by microEROD analysis, could not be exclusively attributed to the content of measured PCBs and PAHs, but also to the presence of unknown dioxin-like compounds (DLC), and/or positive interactions among similarly acting chemicals. The results proved that biotests, when applied in ecotoxicological assessments, should be used either as a screening tool or initial step in effect-directed analyses.