Mechanical Properties and Nanomotion of BT-20 and ZR-75 Breast Cancer Cells Studied by Atomic Force Microscopy and Optical Nanomotion Detection Method.

Cells of two molecular genetic types of breast cancer-hormone-dependent breast cancer (ZR-75 cell line) and triple-negative breast cancer (BT-20 cell line)-were studied using atomic force microscopy and an optical nanomotion detection method. Using the Peak Force QNM and Force Volume AFM modes, we revealed the unique patterns of the dependence of Young's modulus on the indentation depth for two cancer cell lines that correlate with the features of the spatial organization of the actin cytoskeleton. Within a 200-300 nm layer just under the cell membrane, BT-20 cells are stiffer than ZR-75 cells, whereas in deeper cell regions, Young's modulus of ZR-75 cells exceeds that of BT-20 cells. Two cancer cell lines also displayed a difference in cell nanomotion dynamics upon exposure to cytochalasin D, a potent actin polymerization inhibitor. The drug strongly modified the nanomotion pattern of BT-20 cells, whereas it had almost no effect on the ZR-75 cells. We are confident that nanomotion monitoring and measurement of the stiffness of cancer cells at various indentation depths deserve further studies to obtain effective predictive parameters for use in clinical practice.

Second Primary Cancer Among Patients With Papillary Thyroid Carcinoma Following the Chernobyl Disaster.

Importance: To our knowledge, there are no complete population-based studies of the risks of developing second malignant tumors after papillary thyroid carcinoma (PTC) in patients following the Chernobyl nuclear accident. Objective: To study the risk of second primary cancers in patients with PTC after the Chernobyl disaster. Design, Setting, and Participants: This was a retrospective cohort study conducted in the Republic of Belarus over a 31-year time frame evaluating patients with primary PTC and second malignant tumors. Personal data from the Belarussian Cancer Registry were used in the investigation, and only second primary cancers were included in the analysis. Patients were observed from January 1, 1990, to December 31, 2021, for the establishment of second primary malignant tumors. Main Outcomes and Measures: For analysis, synchronous and metachronous tumors were grouped into 1 group (second primary cancer group). If the patient had more than 2 cancers, they were observed until development of a second tumor and, subsequently, the development of a third tumor. The starting point for calculating the number of person-years was the date of thyroid cancer diagnosis. The end point for calculating the number of person-years was the date of diagnosis of the second primary malignant tumor, the date of death, the date of the last visit of the patient, or December 31, 2021 (the end the of study period). The incidence of a second primary malignant tumor with PTC was calculated for the study groups using standardized incidence ratios. Results: Of the 30 568 patients with a primary PTC included in this study, 2820 (9.2%) developed a second malignant tumor (2204 women and 616 men); the mean (SD) age of all patients at time of the primary cancer was 53.9 (12.6) years and at time of the secondary cancer was 61.5 (11.8) years. Overall, the standardized incidence ratio was statistically significant for all types of cancer (1.25; 95% CI, 1.21-1.30), including solid malignant tumors (1.20; 95% CI, 1.15-1.25) and all leukemias (1.61; 95% CI, 2.17-2.13). Cancers of the digestive system (466 cases [21.1%]), genital organs (376 cases [17.1%]), and breasts (603 cases [27.4%]) were the most prevalent second primary tumors in women following PTC. Second primary tumors of the gastrointestinal tract (146 cases [27.7%]), genitourinary system (139 cases [22.6%]), and urinary tract (139 cases [22.6%]) were the most prevalent in men. Urinary tract cancers (307 cases [10.9%]) and gastrointestinal tumors (612 cases [21.4%]) were the most prevalent second primary tumors overall. Conclusions and Relevance: This cohort study reports the increased incidence of solid secondary tumors in men and women over a 31-year time frame after the Chernobyl disaster. Moreover, there was a statistically significant increased risk of second tumors of the breast, colon, rectum, mesothelium, eye, adnexa, meninges, and adrenal glands as well as Kaposi sarcoma. These data might have an effect on the follow-up of this cohort of patients to detect secondary malignant tumors at an early stage.

CD109-regulated mechanical properties of endothelial cells.

CD109 antigen on the endothelial cell surface plays an important role in vascular pathology. The aim of the work was to investigate the effect of the immobilization of CD109 antigen with specific antibodies on nanomechanical properties of human umbilical endothelial cells (HUVECs) using atomic force microscopy in quantitative nanomechanical property mapping mode (PeakForce QNM). Anti-CD109 antibodies induced significant stiffening of the cell surface Me(LQ; UQ): in 1.45(1.07;2.29) times with respect to control cells for fixed cells and in 4.9(3.6;5.9) times with respect to control cells for living cells, and changes in the spatial distribution of cell surface mechanical properties. The changes in the HUVEC's mechanical properties were accompanied by the activation of the TGF-/Smad2/3 signaling pathway and reorganization of the vimentin and actin cytoskeletal elements. Our findings show that blocking CD109 antigen using anti-CD109 antibodies leads in HUVECs to the processes similar to that occur after cell TGF-ß-signaling activation. Therefore, we suggest that CD109 antigen may be involved in regulating the mechanical behavior of endothelial cells.

Prognostic Value of Immunohistochemical Markers for Locally Advanced Rectal Cancer.

The aim of this study is to reveal the potential roles of apoptosis markers (Bcl2 and p53), proliferation markers (Ki-67 and CyclD1), and the neuroendocrine marker Chromogranin A as markers for the radioresistance of rectal cancer. Statistically significant differences were found in the expression of p53, Ki-67, and Chromogranin A in groups of patients with and without a favorable prognosis after radiotherapy. The survival analysis revealed that the marker of neuroendocrine differentiation, Chromogranin A, also demonstrated a high prognostic significance, indicating a poor prognosis. Markers of proliferation and apoptosis had no prognostic value for patients who received preoperative radiotherapy. Higher Chromogranin A values were predictors of poor prognosis. The results obtained from studying the Chromogranin A expression suggest that the secretion of biologically active substances by neuroendocrine cells causes an increase in tumor aggressiveness.

Potential role of tumor-infiltrating T-, B-lymphocytes, tumor-associated macrophages and IgA-secreting plasma cells in long-term survival in the rectal adenocarcinoma patients.

AIMS: Many studies investigated the associations between the role of immune cells of rectal cancer microenvironment and survival during the first 5 years post-surgery. This is problematic as this disease has the potential to progress even after 5 years after relapse and infiltrating immune cells could play key roles. Therefore, this retrospective study investigates expression and roles of tumor-infiltrating T-lymphocytes (TIL-T), tumor-infiltrating B-lymphocytes (TILB), IgA+ plasma cells (IgA+ PC) and tumor-associated macrophages (TAM) in patients with or without progression over 5 years survival with rectal adenocarcinoma. MAIN METHODS: Here we used immunohistochemical staining of CD3, CD20, IgA, CD68 positive cells and its detection in rectal cancer stroma. Data was analyzed using Mann Whitney U test, ROC, survival and Cox's regression analysis. KEY FINDINGS: The number of TIL-T (p = 0.0276), TIL-B (p < 0.0001) and IgA+ PC (p = 0.015) immune cells was significantly higher in rectal cancer stroma of patients with favorable outcome. Univariate Cox's regression analysis revealed a predictive role of TIL-T (HR = 0.482; 95% CI, 0.303 to 0.704; p < 0.0001), TIL-B (HR = 0.301; 95% CI, 0.198 to 0.481; p < 0.0001) and IgA+-PC (HR = 0.488; 95% CI, 0.322 to 0.741; p < 0.0001). Multivariate Cox's regression analysis showed prognostic role of TIL-B (HR = 0.940; 95% CI, 0.914 to 0.968; p < 0.0001) and IgA+-PC (HR = 0.985; 95% CI, 0.975 to 0.996; p = 0.006) play role in long time survival. SIGNIFICANCE: CD20+ TIL-B and IgA+ cells have significant associations with long -term survival of patients with rectal cancer, with potential therapeutic intervention in cancer immunotherapy.

Heterogeneity of nanomechanical properties of the human umbilical vein endothelial cell surface.

Endothelial cells, due to heterogeneity in the cell structure, can potentially form an inhomogeneous on structural and mechanical properties of the inner layer of the capillaries. Using quantitative nanomechanical mapping mode of atomic force microscopy, the parameters of the structural, elastic, and adhesive properties of the cell surface for living and glutaraldehyde-fixed human umbilical vein endothelial cells were studied. A significant difference in the studied parameters for three cell surface zones (peripheral, perinuclear, and nuclear zones) was established. The perinuclear zone appeared to be the softest zone of the endothelial cell surface. The heterogeneity of the endothelial cell mechanical properties at the nanoscale level can be an important mechanism in regulating the endothelium functions in blood vessels.

Cathepsin L-induced galectin-1 may act as a proangiogenic factor in the metastasis of high-grade serous carcinoma.

BACKGROUND: New treatment options for metastasised high-grade serous carcinoma (HGSC) are urgently needed. HGSC frequently metastasises to the omentum, inducing angiogenesis in the local omental microvasculature to facilitate tumour growth. We previously showed that HGSC-secreted cathepsin L (CathL) induces pro-angiogenic changes in disease relevant human omental microvascular endothelial cells (HOMECs), suggesting a role in tumour angiogenesis. Here we investigate whether CathL acts by inducing local production of the carbohydrate-binding protein galectin-1 (Gal1), which has been reported to be involved in tumourigenesis in other tumours. METHODS: HOMECs were used for all experiments. Gal1 mRNA and protein levels were measured by RT-PCR and ELISA respectively. Gal1-induced cell proliferation was assessed using WST-1 assay, migration using a transwell assay and in vivo Gal1 expression by immunohistochemistry. RESULTS: CathL transcriptionally regulated HOMEC production and secretion of Gal1 via activation of NFκB (significantly inhibited by sulfasalazine). Gal1 significantly enhanced HOMEC migration (p < 0.001) and proliferation (p < 0.001), suggesting an autocrine action. The latter was significantly reduced by the MEK/ERK1/2 inhibitors U0126 and PD98059 suggesting downstream activation of this pathway. Immunohistochemical analysis of omenta from HGSC patients with or without metastatic disease demonstrated a positive correlation between Gal1 expression and number of microvessels (r = 0.8702, p < 0.001), and area of vessels (r = 0.7283, p < 0.001), supporting a proangiogenic role for Gal1 in omental metastases. CONCLUSION: HOMEC Gal1 transcription and release in response to CathL secreted from metastasising HGSC acts in an autocrine manner on the local microvasculature to induce pro-angiogenic changes, highlighting a potential new therapeutic target.

Risk of Thyroid Nodules in Residents of Belarus Exposed to Chernobyl Fallout as Children and Adolescents.

Context: Although radiation exposure is an important predictor of thyroid cancer on diagnosis of a thyroid nodule, the relationship between childhood radiation exposure and thyroid nodules has not been comprehensively evaluated. Objective: To examine the association between internal I-131 thyroid dose and thyroid nodules in young adults exposed during childhood. Design, setting, and participants: In this cross-sectional study, we screened residents of Belarus aged ≤18 years at the time of the Chernobyl nuclear accident for thyroid disease (median age, 21 years) with thyroid palpation, ultrasonography, blood/urine analysis, and medical follow-up when appropriate. Eligible participants (N = 11,421) had intact thyroid glands and doses based on direct individual thyroid activity measurements. Main outcome measures: Excess odds ratios per Gray (EOR/Gy, scaled at age 5 years at exposure) for any thyroid nodule and for nodules grouped by cytology/histology, diameter size, and singularity. Results: Risk of any thyroid nodule increased significantly with I-131 dose and, for a given dose, with younger age at exposure. The EOR/Gy (95% confidence intervals) for neoplastic nodules (3.82; 0.87 to 15.52) was significantly higher than for nonneoplastic nodules (0.32; <0.03 to 0.70) and did not vary by size; whereas the EOR/Gy for nonneoplastic nodules did vary by size (P = 0.02) and was 1.55 (0.36 to 5.46) for nodules ≥10 mm and 0.02 (<-0.02 to 0.70) for nodules <10 mm. EORs/Gy for single and multiple nodules were comparable. Conclusions: Childhood exposure to internal I-131 is associated with increased risk of neoplastic thyroid nodules of any size and nonneoplastic nodules ≥10 mm.

Syncytial Hepatitis of Tilapia ( Oreochromis niloticus L.) is Associated With Orthomyxovirus-Like Virions in Hepatocytes.

Using transmission electron microscopy (TEM), the presented work expands on the ultrastructural findings of an earlier report on "syncytial hepatitis," a novel disease of tilapia (SHT). Briefly, TEM confirmed the presence of an orthomyxovirus-like virus within the diseased hepatocytes but not within the endothelium. This was supported by observing extracellular and intracellular (mostly intraendosomal), 60-100 nm round virions with a trilaminar capsid containing up to 7 electron-dense aggregates. Other patterns noted included enveloped or filamentous virions and virion-containing cytoplasmic membrane folds, suggestive of endocytosis. Patterns atypical for orthymyxovirus included the formation of syncytia and the presence of virions within the perinuclear cisternae (suspected to be the Golgi apparatus). The ultrastructural morphology of SHT-associated virions is similar to that previously reported for tilapia lake virus (TiLV). A genetic homology was investigated using the available reverse transcriptase polymerase chain reaction (RT-PCR) probes for TiLV and comparing clinically sick with clinically normal fish and negative controls. By RT-PCR analysis, viral nucleic acid was detected only in diseased fish. Taken together, these findings strongly suggest that a virus is causally associated with SHT, that this virus shares ultrastructural features with orthomyxoviruses, and it presents with partial genetic homology with TiLV (190 nucleotides).

Risk of thyroid follicular adenoma among children and adolescents in Belarus exposed to iodine-131 after the Chornobyl accident.

Several studies reported an increased risk of thyroid cancer in children and adolescents exposed to radioactive iodines, chiefly iodine-131 ((131)I), after the 1986 Chornobyl (Ukrainian spelling) nuclear power plant accident. The risk of benign thyroid tumors following such radiation exposure is much less well known. We have previously reported a novel finding of significantly increased risk of thyroid follicular adenoma in a screening study of children and adolescents exposed to the Chornobyl fallout in Ukraine. To verify this finding, we analyzed baseline screening data from a cohort of 11,613 individuals aged ≤18 years at the time of the accident in Belarus (mean age at screening = 21 years). All participants had individual (131)I doses estimated from thyroid radioactivity measurements and were screened according to a standardized protocol. We found a significant linear dose response for 38 pathologically confirmed follicular adenoma cases. The excess odds ratio per gray of 2.22 (95% confidence interval: 0.41, 13.1) was similar in males and females but decreased significantly with increasing age at exposure (P < 0.01), with the highest radiation risks estimated for those exposed at <2 years of age. Follicular adenoma radiation risks were not significantly modified by most indicators of past and current iodine deficiency. The present study confirms the (131)I-associated increases in risk of follicular adenoma in the Ukrainian population and adds new evidence on the risk increasing with decreasing age at exposure.

A mechano-chemiosmotic model for the coupling of electron and proton transfer to ATP synthesis in energy-transforming membranes: a personal perspective.

ATP is synthesized using ATP synthase by utilizing energy either from the oxidation of organic compounds, or from light, via redox reactions (oxidative- or photo phosphorylation), in energy-transforming membranes of mitochondria, chloroplasts, and bacteria. ATP synthase undergoes several changes during its functioning. The generally accepted model for ATP synthesis is the well-known rotatory model (see e.g., Junge et al., Nature 459:364-370, 2009; Junge and Müller, Science 333:704-705, 2011). Here, we present an alternative modified model for the coupling of electron and proton transfer to ATP synthesis, which was initially developed by Albert Lester Lehninger (1917-1986). Details of the molecular mechanism of ATP synthesis are described here that involves cyclic low-amplitude shrinkage and swelling of mitochondria. A comparison of the well-known current model and the mechano-chemiosmotic model is also presented. Based on structural, and other data, we suggest that ATP synthase is a Ca(2+)/H(+)-K(+) Cl(-)-pump-pore-enzyme complex, in which γ-subunit rotates 360° in steps of 30°, and 90° due to the binding of phosphate ions to positively charged amino acid residues in the N-terminal γ-subunit, while in the electric field. The coiled coil b 2-subunits are suggested to act as ropes that are shortened by binding of phosphate ions to positively charged lysines or arginines; this process is suggested to pull the α 3 ß 3-hexamer to the membrane during the energization process. ATP is then synthesized during the reverse rotation of the γ-subunit by destabilizing the phosphated N-terminal γ-subunit and b 2-subunits under the influence of Ca(2+) ions, which are pumped over from storage-intermembrane space into the matrix, during swelling of intermembrane space. In the process of ATP synthesis, energy is first, predominantly, used in the delivery of phosphate ions and protons to the α 3 ß 3-hexamer against the energy barrier with the help of C-terminal alpha-helix of γ-subunit that acts as a lift; then, in the formation of phosphoryl group; and lastly, in the release of ATP molecules from the active center of the enzyme and the loading of ADP. We are aware that our model is not an accepted model for ATP synthesis, but it is presented here for further examination and test.

Analysis of thyroid malignant pathologic findings identified during 3 rounds of screening (1997-2008) of a cohort of children and adolescents from belarus exposed to radioiodines after the Chernobyl accident.

BACKGROUND: Recent studies of children and adolescents who were exposed to radioactive iodine-131 (I-131) after the 1986 Chernobyl nuclear accident in Ukraine exhibited a significant dose-related increase in the risk of thyroid cancer, but the association of radiation doses with tumor histologic and morphologic features is not clear. METHODS: A cohort of 11,664 individuals in Belarus who were aged ≤18 years at the time of the accident underwent 3 cycles of thyroid screening during 1997 to 2008. I-131 thyroid doses were estimated from individual thyroid activity measurements taken within 2 months after the accident and from dosimetric questionnaire data. Demographic, clinical, and tumor pathologic characteristics of the patients with thyroid cancer were analyzed using 1-way analysis of variance, chi-square tests or Fisher exact tests, and logistic regression. RESULTS: In total, 158 thyroid cancers were identified as a result of screening. The majority of patients had T1a and T1b tumors (93.7%), with many positive regional lymph nodes (N1; 60.6%) but few distant metastases (M1; <1%). Higher I-131 doses were associated with higher frequency of solid and diffuse sclerosing variants of thyroid cancer (P < .01) and histologic features of cancer aggressiveness, such as lymphatic vessel invasion, intrathyroidal infiltration, and multifocality (all P < .03). Latency was not correlated with radiation dose. Fifty-two patients with self-reported thyroid cancers which were diagnosed before 1997 were younger at the time of the accident and had a higher percentage of solid variant cancers compared with patients who had screening-detected thyroid cancers (all P < .0001). CONCLUSIONS: I-131 thyroid radiation doses were associated with a significantly greater frequency of solid and diffuse sclerosing variants of thyroid cancer and various features of tumor aggressiveness.

Influence of the dietary protein:lipid ratio and fish oil substitution on fatty acid composition and metabolism of Atlantic salmon (Salmo salar) reared at high water temperatures.

A factorial, two-way, experimental design was used for this 10-week nutritional trial, aiming to elucidate the interactive effects of decreasing dietary protein:lipid level and substitution of fish oil (FO) with rapeseed oil (RO) on tissue fatty acid (FA) composition and metabolism of large Atlantic salmon (Salmo salar L.) reared at high water temperatures (sub-optimal, summer temperatures: 11·6°C). The six experimental diets were isoenergetic and formulated to include either FO or RO (60 % of the added oil) at three dietary protein:lipid levels, specifically (1) 350 g/kg protein and 350 g/kg lipid, (2) 330 g/kg protein and 360 g/kg lipid, (3) 290 g/kg protein and 380 g/kg lipid. Final weight, specific growth rate and thermal growth coefficient were positively affected by the dietary RO inclusion at the expense of FO, while no significant effects were seen on growth due to the decreasing protein level. The oil source had a significant effect on muscle and liver FA composition. However, the changes in muscle and liver FA indicate selective utilisation or retention of individual FA and moderate reductions in tissue EPA and DHA. Pyloric caeca phospholipid FA composition was significantly affected by the two factors and, in some cases, significant interactions were also revealed. Liver and red muscle ß-oxidation capacities were significantly increased due to RO inclusion, while an interactive effect of protein level and oil source was shown for white muscle ß-oxidation capacity. The results could explain, at least partially, the better performance that was shown for the RO groups and the enhanced protein-sparing effect.

Effects of decontaminated fish oil or a fish and vegetable oil blend on persistent organic pollutant and fatty acid compositions in diet and flesh of Atlantic salmon ( Salmo salar).

The health benefits of seafood are well documented and based on the unique supply of n-3 highly unsaturated fatty acids (HUFA). Aquaculture now contributes about 50 % of food-grade seafood globally and Atlantic salmon (Salmo salar) is a rich source of n-3 HUFA. However, salmon and other oily fish can accumulate lipophilic persistent organic pollutants (POP), including dioxins (PCDD/F), polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE), derived largely from feed. In the present study, triplicate groups of salmon, of initial weight 0.78 kg, were fed one of three experimental diets for 11 weeks. The diets were coated with either a northern fish oil (FO) with a high POP content (cNFO), the same oil that had been decontaminated (deNFO) or a blend of southern fish oil, rapeseed and soyabean oils (SFO/RO/SO). Dietary PCDD/F+dioxin-like PCB (DL-PCB) concentrations were 17.36, 0.45 and 0.53 ng toxic equivalents (TEQ)/kg, respectively. After 11 weeks, the flesh concentrations in fish fed the cNFO, deNFO and SFO/RO/SO diets were 6.42, 0.34 and 0.41 ng TEQ/kg, respectively. There were no differences in flesh EPA and DHA between fish fed the cNFO or deNFO diets although EPA and DHA were reduced by 50 and 30 %, respectively, in fish fed the SFO/RO/SO diet. Thus, decontaminated FO can be used to produce salmon high in n-3 HUFA and low in POP. Salmon produced using deNFO would be of high nutritional value and very low in POP and would utilise valuable fish oils that would otherwise be destroyed due to their high pollutant concentrations.

An evidence for the equilibrium unfolding intermediates of ribonuclease A by tritium labeling method.

A formation of a molten globule in the unfolding of ribonuclease A could be considered as an evidence supporting a hypothesis on the existence of such intermediates on the pathway of a protein folding. Using a novel technique (tritium labeling method) we have showed that the ribonuclease A equilibrium unfolding in urea and guanidinium chloride (GuCl) solutions proceeds through a formation of intermediates whose properties (compactness, retention of the larger part hydrophobic core, secondary structure, and native-like folding pattern) correspond to the fundamental characteristics of the molten globule state. The both intermediates are the "wet" molten globules (the globule interior contains the water molecules). The results reveal the noticeable distinctions in intermediates structure, first of all, in the extent of their compactness. The urea intermediate is less compact than that in GuCl. It is shown that the refolding of the protein denatured by GuCl results in the formation of the intermediate which enzyme activity is virtually the same as the activity of the native protein.

Strontium-induced rat egg activation.

Parthenogenetic agents that evoke cytosolic calcium concentration ([Ca2+]i) oscillations similar to those evoked by sperm, mimic fertilization more faithfully than agents that trigger a single [Ca2+]i transient. Strontium chloride (SrCl2) binds to and activates the Ca2+-binding site on the inositol 1,4,5-trisphosphate receptor and evokes [Ca2+]i oscillations. Although SrCl2 has been reported to activate mouse eggs, little is known regarding the pattern of the [Ca2+]i oscillations it evokes in rat eggs and their effect on the early events of egg activation: cortical granule exocytosis (CGE) and completion of meiosis (CM). In the current study we investigated the effect of various concentrations of SrCl2 (2, 4 or 6 mM) on [Ca2+]i, by monitoring [Ca2+]i oscillations in fura-2-loaded rat eggs. Treatment with 2 mM SrCl2 was optimal for inducing the first [Ca2+]i transient, which was similar in duration to that triggered by sperm. However, the frequency and duration of the subsequent [Ca2+]i oscillations were lower and longer in SrCl2-activated than in sperm-activated eggs. The degree of CGE was identical in eggs activated by either sperm or SrCl2, as assessed by semi-quantitative immunohistochemistry combined with confocal microscopy. Evoking 1, 2 or 10 [Ca2+]i oscillations (8, 15 or 60 min in SrCl2 respectively) had no effect on the intensity of fluorescent CGE reporter dyes, while 60-min exposure to SrCl2 caused a delay in CM. Our results demonstrate that SrCl2 is an effective parthenogenetic agent that mimics rat egg activation by sperm, as judged by the generation of [Ca2+]i oscillations, CGE and CM.

Streptococcus iniae type II infections in rainbow trout Oncorhynchus mykiss.

Clinical and pathological findings (anorexia, hemorrhage, lethargy, loss of orientation and exophthalmia) indicated that Streptococcus iniae type II is responsible for a fatal disease in rainbow trout. Histopathological findings revealed that S. iniae type II produces a systemic disease, including a diffuse necrotizing myositis. The distribution of viable bacteria in infected tissues substantiated the pathological findings, confirming that S. iniae type II is responsible for a generalized septic disease of rainbow trout.

Streptococcus iniae infections in Red Sea cage-cultured and wild fishes.

Streptococcus iniae was isolated from 2 moribund wild Red Sea fishes, Pomadasys stridens (Pomadasyidae) and Synodus variegatus (Synodontidae), both collected in shallow waters along the Israeli coast of the Gulf of Eilat. The site is approximately 2 km from a mariculture cage farm in which streptococcal infections were diagnosed in previous years in the red drum Sciaenops ocellatus. This is the first report of S. iniae in Red Sea fishes. Biochemical and molecular similarities between the isolates from cultured fishes and those from the wild specimens suggest that a single strain is involved, and that 'amplification' and dispersal of this pathogen from captive to feral fishes have occurred. At the molecular level, the pathogen is different from the S. iniae isolates that have been afflicting the Israeli freshwater aquaculture in recent years. Although S. iniae prevalence in the wild fish populations of the area remains to be determined, the northernmost region of the Gulf of Eilat, virtually landlocked and with generally calm seas and weak currents, seems to be particularly vulnerable to the impact of diseases that develop in this mariculture system.

Streptococcus iniae inhibition of apoptosis of nonspecific cytotoxic cells: a mechanism of activation of innate immunity in teleosts.

Nonspecific cytotoxic cells (NCC) may provide innate anti-bacterial resistance against Streptococcus iniae infections in tilapia. The mechanism of immunity would be elaboration and release of various cytokines, augmentation of inflammation and amplification of increased antigen processing. To investigate bacterial regulation of NCC function, 2 different processes of cellular pathology were examined: apoptosis and necrosis. Different isolates of S. iniae from diseased teleosts, a dolphin and a human were tested. All isolates were examined for their ability to produce apoptosis and/or necrosis on freshly purified tilapia NCC and on a tilapia continuous cell line (i.e. TMB-8 cells). Two different isolates (9033 and 173) inhibited the outer membrane expression of phosphatidylserine (PS) by NCC, an early sign of apoptosis. This occurred at 4 h post-treatment and lasted throughout the 24 h treatment period. All other isolates either did not differ from control levels or produced a small increase in PS expression by NCC. The early reduction in PS expression occurred concomitantly with increased necrosis associated with nonspecific DNA fragmentation. Two-color flow cytometry (Annexin-V vs propidium iodide staining) demonstrated the specificity of Annexin-V binding. Experiments were also done to determine the effects of S. iniae on TMB-8 cells. Treated TMB-8 cells did not produce appreciable Annexin-V binding. Compared to the ATCC strain, 9033 produced high levels of necrosis-associated DNA fragmentation of TMB-8 cells at 4 and 8 h post-treatment. These data indicated that different isolates of S. iniae may regulate NCC anti-bacterial resistance by causing reduced levels of programmed cell death (PCD), increased necrosis and associated enhancement of inflammatory responses. Understanding the relevance of these bacterial effects on NCC may be an important consideration in the evaluation of isolates used in vaccine/ bacterin production.

Recovery of Streptococcus iniae from diseased fish previously vaccinated with a streptococcus vaccine.

Streptococcus iniae was recovered from diseased rainbow trout (Oncorhynchus mykiss, Walbaum) previously vaccinated against streptococcosis. PCR and serological methods indicate the presence of a new serotype in the diseased fish.