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In the present study, the effects of high temperatures on immune responses in the clam Chamelea gallina (Bivalvia: Veneridae) were evaluated.

In order to investigate the influence of anoxic stress on haemocyte immune response, specimens of Chamelea gallina were exposed to 24 and 48 h anoxia. To evaluate recovery capacity clams were maintained, at the end of the anoxic phase, for 24 h in reoxygenated seawater. In this paper activity and expression of the antioxidant enzyme superoxide dismutase (SOD) were studied on haemocyte lysate and haemolymph. Reported results have shown that the anoxic stress changed strongly the response of Chamelea gallina blood cells. Indeed, at the end of the anoxic phase in both experiments (24 and 48 h of anoxia exposure) SOD activity in haemocyte lysate decreased significantly with respect to the control, likely because of a decreasing superoxide anion generation in anoxia. Expression analyses were coherent with activity values. In the first experiment (24 h anoxia) reoxygenation determined an increase in activity of both Cu/Zn-SOD and Mn-SOD, but with values that remained significantly lower than those of the controls. It seems that after the applied anoxic stress, 24 h of recovery is not sufficient to restore pre-anoxic conditions. In the second experiment (48 h anoxia), SOD isoforms showed a different response during the recovery of animals. Cu/Zn-SOD activity dropped below the values showed by haemocytes of anoxic bivalves, while Mn-SOD activity values exceeded significantly those of controls . The different haemocyte response could be probably due to a further stress suffered by the clams because of a massive spawning during the reoxygenation phase. Therefore the high values of activity shown by Mn-SOD during the recovery is likely to be due to the high inducibility of this isoform . In Cu/Zn-SOD expression analyses two immunoreactive bands were highlighted in both experiments. The former (apparent molecular weight of 16 kDa), corresponds to the expression of SOD1 and the latter (apparent molecular weight of 28-30 kDa) could be attributed to EC-SOD (SOD3), a Cu/Zn-SOD isoform located in extracellular ambient and identified both in vertebrates and invertebrates. The strong SOD3 expression during anoxia exposure and the further spawning stress (second experiment) testified its inducibility in C. gallina haemocytes and haemolymph in response to stressful conditions.

The effects of anoxia on the immune responses of a commercially important bivalve species, Chamelea gallina, were studied in two different experiments (24-h and 48-h exposure to deoxygenated seawater). After anoxic stress, the capability of clams to recover was also evaluated by maintaining animals for 24 h in aerated seawater. Total haemocyte count (THC), phagocytosis and lysozyme activity (in both haemocyte lysate and cell-free haemolymph) were chosen as cellular biomarkers of exposure to anoxic conditions. Anoxia significantly reduced THC with respect to controls in both experiments. However, clams which recovered from the 24-h anoxia test showed THC values similar to those of controls, whereas in the 48-h test recovery clam THC was similar to that of stressed clams. Significantly decreased phagocytic activity with respect to controls was also observed in both experiments. In the first experiment, haemocytes from recovery clams showed slightly higher phagocytic activity with respect to controls, whereas after 48 h of exposure haemocytes from recovery animals were unable to recover their phagocytic activity. After both 24-h and 48-h anoxia, lysozyme activity was significantly reduced in both haemocyte lysate and cell-free haemolymph. In recovery clams from 24-h anoxia, the lysozyme activity of lysate was significantly lower than in control and stressed clams, whereas in haemolymph it was significantly higher. Conversely, in recovery clams from 48-h anoxia, the enzyme activity of both media decreased significantly with respect to controls. Interestingly, an unexpected additional stress during the 48-h anoxia test was recorded: recovery clams spawned during the recovery phase. To validate the hypothesis that spawning acts as an additional type of stress, the survival-in-air test was also performed. Significant decreases in resistance to air exposure were observed after 48 h anoxia in both stressed and recovery animals. Results show that anoxia strongly affects the functional responses of haemocytes, reducing immunosurveillance in stressed clams. The present study also highlights that additional stress, such as spawning, may further decrease immunosurveillance and reduce the resistance of clams to exposure to air.

Effects of benzo(a)pyrene [B(a)P] (at a nominal concentration of 0.5 mg/L) on immune responses of the clam Chamelea gallina were investigated after 1, 7, and 12 days exposure. Total hemocyte count (THC), hemocyte volume, phagocytic activity, lysozyme-like activity in both hemocyte lysate (HL) and cell-free hemolymph (CFH) were measured. As unexpected alterations in hemocyte adhesion capability were observed in short-term hemocyte cultures for phagocytosis assays after a 1-day exposure, an adhesion test (not included in the original experimental setup) was performed after 7 and 12 days of exposure only. The survival-in-air test was carried out to evaluate general stress conditions in B(a)P-exposed clams. No alterations in THC was observed, whereas exposure for 7 and 12 days to B(a)P significantly decreased phagocytic activity and adhesion capability when compared with controls. Significant decreases in lysozyme activity were observed in CFH and HL, with respect to controls. B(a)P was also shown to alter the resistance to air exposure of clams. The LT(50) values fell from 9 days in control clams to 7 days in 1-day-exposed animals, and from 6 days in control clams to 5 days in 7-day-exposed bivalves. No significant variations in LT(50) values were recorded after 12 days of exposure. Results highlight a relationship between B(a)P exposure and alterations in hemocyte functionality and suggest that the contaminant induced irreversible immunosuppression in C. gallina, by altering phagocytic activity, adhesion capability, and enzymatic activity. Conversely, reduction in resistance to air exposure was reversible, suggesting that impairment of important physiological functions of clams occurred in the first phases of exposure only.

SUMMARY – The expression of HIF-1a and HSF1 was investigated in plasma samples (T0: at birth; T12: 12 hours after birth) of 9 neonates (4 healthy and 5 sick foals); plasma MDA concentration was also evaluated in 16 neonates (10 healthy and 6 sick foals). MDA concentration, HIF-1a and HSF1 expression decreased significantly at T12 if compared with T0 (p< 0.05). HIF-1a was significantly higher in sick foals at all sampling times (p< 0.05) and resulted correlated to pCO2 at T0 and to MDA at T12 (p< 0,05; R=0,83 e R=-0,72, respectively). HSF was significantly correlated to HCT and RBC at T0 (p< 0,05; R=0,68 e R=0,77, respectively). The parameters measured could be considered potential indexes of perinatal hypoxia.

Bidimensional electrophoresis (2-DE) protocols were adapted on Chamelea gallina digestive glands studies by the analysis of Heat Shock Proteins (HSP) compared with monodimensional electrophoresis (1-DE) results. Because polycyclic aromatic hydrocarbons (PAH) act on HSPs, C. gallina specimens were exposed to 0.5 mg/L of benzo[a]pyrene (B[a]P) for 24 h, 7 and 12 days. Immunoblotting after 1-DE showed a single band of 70 kDa significantly induced after 7 days of B[a]P exposure. After 2-DE, eight major high-resolved spots between 17 and 98 kDa were revealed. Three spots fell within the range of 62–98 kDa and of 5–6 pI, parameters which could include HSP70. Two spots of 77 and 72 kDa, obtained after 2-DE immunoblotting, could correspond to constitutive HSC70 and to inducible HSP70 forms respectively. Changes observed in inducible and in constitutive forms might be related to an adaptation to stress and to a normal protein synthesis capability, respectively. Employment of 2-DE and relationship between HSP70 and HSC70 may be useful to clarify their role in molluscs subjected to stress events.

The present study is the first to evaluate the expression and activity of MnSOD, Cu/ZnSOD and catalase in human gastric samples, since ROS play a significant role in the pathogenesis of different forms of malignancy inducing mutations and various diseases such as gastric cancer. Biopsies and surgical samples from 53 patients (male/female 22/31, mean age 56.5+/-15.8 years) consisted of 15 healthy, 12 autoimmune atrophic gastritis, 10 Helicobacter pylori (HP) infection, 8 HP-negative chronic gastritis (CG) and 8 adenocarcinoma cases. Enzyme activity and expression were evaluated by spectrophotometry and immunoblotting after specific extraction in phosphate buffer. We found that MnSOD activity was increased in adenocarcinoma, CG and HP tissues (p

HSP70, HSP27 and TNF-a were measured by immunoblotting in samples of pathological and healthy cutaneous and gland tissues (20 dogs of different ages and sex). HSP70 and HSP27 expressions are respectively characterized by a significant increase (p

The expression of inducible and constitutive forms of HSP70s, respectively HSP70 and HSC70, were investigated in skin samples of pathological and healthy tissues (28 dogs of different ages and sex). HSP70 increased in 54.3% (p

To prove CYP1A-immunopositive protein presence and to evaluate MnSOD behaviour in haemocytes and haemolymph, specimens of C. gallina were exposed to 0.5 mg/L of Benzo[a]pyrene for 12 days. CYP1A-immunopositive protein showed two bands of 53.5 kDa and 63.8 kDa not differently expressed, whereas MnSOD activity decreased after 7 and 12 days of exposure.

Chloramphenicol (CA) is a largely used antibiotic and it is an inhibitor of protein synthesis that also induces ROS production. In this work there were investigated activities and expressions in the Adriatic bivalve Chamelea gallina of some antioxidant and detoxification proteins like superoxide dismutase (Mn–SOD, Cu/Zn–SOD), catalase (CAT) and Cytochrome P450 (CYP1A). Clams exposed to 5 mg l −1 of chloramphenicol were sampled 2, 4 and 8 days after treatment (CA2, CA4 and CA8). SODs, CAT, and CYP1A activity and/or expression were detected in pooled digestive glands by Western blotting and by spectrophotometrical analysis. Enzymes activities increase during the entire antibiotic exposure. With respect to the control Cu/Zn–SOD expression increases, while Mn–SOD expression decreases significantly after 4 days. Two CYP1A immunopositive-proteins (57.7 and 59.8 kDa) were detected. The lower band significantly decreases in CA8, the upper one also in CA4 condition. High levels of Mn–SOD, CAT activity and Cu/Zn–SOD expression, indicate intense ROS production while Mn–SOD expression inhibition might be ascribable to mitochondrial alterations due to CA and indirectly to ROS. CYP1A1 action determines H 2 O 2 production that would contribute to a CYP1A1 gene promoter down regulation, a response to oxidative stress with the antioxidant enzymes activation as a final result. This study highlights the close association, in C. gallina , in presence of chloramphenicol, between SOD/CAT and CYP system, and it appear particularly interesting to the lack of similar researches on mollusc species.

Environmental stresses are responsible of HSPs induction, that guarantee tolerance of otherwise lethal conditions. This study was performed to evaluate HSP70 expression in Chamelea gallina (Bivalvia) digestive gland in response to oxygen, temperature, salinity changes and B[a]P exposure. The results obtained confirm general induction of HSP70 in all the stress conditions tested.

The effects of different salinity levels (28, 34 and 40‰) on functional responses of Chamelea gallina haemocytes were evaluated in a two part study dealing with modulations of immune parameters. This part (Part II) of the study was focused on the superoxide dismutase (SODs) activity and expression in haemocyte lysate and cell-free haemolymph. Results of this study established that the exposure of C. gallina specimens at 40‰ salinity provoked a decrease in Mn-SOD and Cu/Zn-SOD activities in haemocyte lysate suggesting a declining superoxide anion generation at the highest salinity tested. Expression of MnSOD was coherent with activity values, while Cu/ZnSOD showed two immunoreactive bands. The former corresponds to the cytosolic Cu/Zn-SOD (16 kDa) was not coherent with the enzyme activity and the second (28–30 kDa) probably attributed to EC-SOD. In cell-free haemolymph, Mn-SOD activity decrease and Cu/Zn-SOD activity increase at 40‰ were observed, likely due to EC-SOD contribution, strongly induced at the same salinity. After EC-SOD detection with two different antibody, we postulate that the EC-SOD like-protein band (29 kDa) may be constitute partly by EC-SOD and probably by Cu/Zn-SOD dimeric form not completely dissociated under reducing condition or a Cu/Zn-SOD degenerated but still recognized by antibody. The cell-free haemolymph increase of EC-SOD at high salinity values plays an important role in immune defence of C. gallina. According to the conclusion of Part I of this study, our data confirmed the destabilizing effect of 40‰ salinity on haemocyte functionality, while to 28‰ exposure, data don’t confirm its stressful action as instead stated by results of Part I. Further studies are necessary to clear up this discrepancy.

SOD activity and expression were evaluated in haemocytes of Chamelea gallina exposed for seven days to three different temperatures. The values recorded for the acitivity of Total SOD, Mn-SOD and CuZn-SOD were significantly lower in specimens kept at 30° C. Even the results of immumological analysis (Western Blotting) revealed a minor expression at the end of the exposure at the same temperature for Mn-SOD, CuZn-SOD and EC-SOD. The high mortality recorded at 30° C is probably related to a lower phagocytosis activity and to a lower production of ROS, a useful defence against foreign elements.

I ROS rivestono un importante ruolo microbicida e allo stesso tempo sono responsabili di molteplici danni a diverse macromolecole biologiche (stress ossidativi). Per contrastare lo stress ossidativo gli organismi aerobi dispongono di sistemi di difesa. In particolare la superossido dismutasi (SOD) è il principale enzima coinvolto nella dismutazione dello ione superossido (O2-•). Dal momento che le variazioni della tensione di ossigeno e della temperatura sono responsabili di potenziali rischi immunologici, con la presente ricerca ci proponiamo di valutare gli effetti della anossia/riossigenazione e della temperatura sull'attività ed espressione della SOD negli emociti del bivalve Chamelea gallina.