Potential benefits of using hydrogen sulfide, vitamin E and necrostatin-1 to counteract acetaminophen­induced hepatotoxicity in rats.

BACKGROUND: The use of acetaminophen (APAP) is increasing recently, especially with COVID-19 outbreaks. APAP is safe at therapeutic levels, however, an overdose can cause severe liver injury. This study aims to explore possible mechanisms involved in APAP­induced hepatotoxicity and compare different hepatoprotective agents, namely vitamin E, hydrogen sulfide (H2S) and necrostatin-1 (NEC-1). METHODS: Adult male albino rats were divided into groups: Control group, APAP­induced hepatotoxicity group, Vitamin E­treated group, H2S­treated group and NEC-1­treated group. Serum levels for aspartate aminotransferase (AST), alanine aminotransferase (ALT), interleukin-33 (IL-33), tumor necrosis factor alpha (TNF-α), reduced glutathione (GSH) and lipid profile were measured. Histopathological examinations of liver tissue with H(et)E stain and immunohistochemistry for activated caspase-3 were also done. RESULTS: APAP­treated group showed elevated liver transaminases, hyperlipidemia, and deficient liver anti-oxidative response together with disturbed hepatic architecture and increased immune-expression of activated caspase-3 in hepatic tissue. Pretreatment with vitamin E, H2S or NEC-1 reversed the affected parameters. Vitamin E and H2S showed greater improvement when compared to NEC-1. CONCLUSION: Vitamin E, H2S and NEC-1 showed protective effects against APAP-induced hepatotoxicity, thus they may be used as an adjuvant therapy when APAP is indicated for long periods as is the case in COVID-19 patients (Tab. 2, Fig. 2, Ref. 45). Text in PDF www.elis.sk Keywords: acetaminophen, hepatotoxicity, apoptosis, necrostatin-1, vitamin E, H2S.

Cross-reaction of antigen preparations from adult and larval stages of the parasite Setaria equina with sera from infected humans with Wuchereria bancrofti.

Crude antigenic preparations from Setaria equina were used in ELISA and Western blotting to examine cross-reaction with human sera from areas endemic for bancroftian filariasis. Sera from normal subjects from non-endemic areas were included as negative controls. Cross-reaction was found between S. equina antigens and antibodies in the sera of Wuchereria bancrofti-infected patients, with the highest levels observed between sera of chronic infected patients and Setaria spp. crude female worm surface antigen (CFSWA). In the absence of active transmission of Setaria spp. infection, CFWSA is useful to detect chronic W. bancrofti infection before patients become symptomatic, particularly when chronic patients are known to be amicrofilaraemic. In the presence of active S. equina infection, antigens from the adult and microfilaraemic stages showed the highest degree of cross-reaction with human sera.

Setaria equina: in vivo effect of diethylcarbamazine citrate on microfilariae in albino rats.

Although diethylcarbamazine citrate (DEC) is successful drug in eliminating human filariasis, yet, its mode of action is still debatable. Herein, the effect of DEC to treat albino rats infected with the animal filarial parasite Setaria equina was tested. Microfilarial (mf) counts and sections from liver, lung, kidney as well as spleen were investigated at different time points after treatment by light microscopy. After 45 and 300min of treatment, a significant decrease in blood mf was observed accompanied by adherence of degenerated mf to both kupffer cells and leukocyte in liver sections. In lung sections, loss of sheath was observed at 45min, while degeneration was observed at later time points. In kidney sections, more mf counts and less matrix were observed in the glomeruli at all time points after treatment. Degenerated mf were observed in spleen sections only at, late time point, 480min after treatment. In conclusion, one of the possible mechanisms by which DEC reduces blood microfilarial count is trapping larvae in organs and killing them through cellular adherence.

Ultrasound-guided adductor canal block after arthroscopic anterior cruciate ligament reconstruction: Effect of adding dexamethasone to bupivacaine, a randomized controlled trial.

BACKGROUND: Dexamethasone improves the quality and duration of peripheral nerve block when used as an adjuvant to local anaesthetic. We evaluated the effect of adding dexamethasone to bupivacaine on the duration of postoperative analgesia in patients undergoing knee arthroscopy using ultrasound-guided adductor canal block. METHODS: The study was a randomized, double-blinded trial. Sixty patients scheduled for arthroscopic anterior cruciate ligament reconstruction were randomly allocated into two groups to receive adductor canal block. The control group received 20 mL bupivacaine 0.5% + 2 mL normal saline, and the dexamethasone group received 20 mL bupivacaine 0.5% + 2 mL dexamethasone (8 mg). Measurements included onset and duration of sensory blockade, visual analog score, time to first analgesic requirement, analgesic consumption, satisfaction score and assessment of quadriceps strength. RESULTS: Duration of sensory block was significantly longer in the dexamethasone group (17.42 ± 5.24 h) than the control group (12.52 ± 1.16 h), p < 0.001. The visual analog score was significantly lower (p < 0.05) in the dexamethasone group. Time to first analgesic requirement was significantly longer in the dexamethasone group (13.37 ± 3.68 h) compared with the control group (10.57 ± 0.93 h), p < 0.001. Ketorolac dose as a rescue analgesic was significantly higher in the control group (p < 0.001), whereas patients' satisfaction score was significantly higher in the dexamethasone group (p < 0.001). CONCLUSION: The addition of dexamethasone to bupivacaine in adductor canal block provides prolonged postoperative analgesia and less postoperative analgesic consumption than bupivacaine alone in anterior cruciate ligament arthroscopic surgery. SIGNIFICANCE: Adding dexamethasone to bupivacaine in adductor canal block significantly increases the duration of sensory block, time to first analgesic requirement and patients' satisfaction score in anterior cruciate ligament arthroscopic surgery.

Ultrasound enhanced drug toxicity on Chinese hamster ovary cells in vitro.

Chinese hamster ovary cells (HA1) were exposed to therapeutic ultrasound (F = 2.025 MHz) in the presence of various drugs at temperatures of 37-43 degrees C. The space averaged intensities used were 0.5-2 W/cm2. The survival of these cells was subsequently tested using the clonogenic assay. Marked enhancement by ultrasound of the cytotoxicity of Adriamycin and amphotericin B was observed. For Adriamycin, the potentiation was dependent upon the intensity of sonication (exposure duration being 30 min). At 0.5 W/cm2, there was enhancement of cytotoxicity above 41 degrees C. At 1 W/cm2, there was a 3-order increase in cytotoxicity at 37 degrees C. Thus an increase in intensity resulted in a decrease in "threshold" temperature. The effect with Adriamycin could be explained in part by an increase in net uptake of drug into the cells. Further, ultrasound was observed to increase the sensitivity of cells to Adriamycin. For amphotericin B, the enhancement was observed only at exposure durations greater than 30 min and at 43 degrees C. There was no enhancement observed for cisplatin and etoposide. From these results, it appears that ultrasound potentiates the cytotoxicity of drugs the mode of action of which (at least in part) involves the plasma membrane.

Effect of unilateral adrenalectomy on acute immobilization stress response in rats.

OBJECTIVES: The aim of the present study was to investigate the differential effect of unilateral adrenalectomy, right vs. left, in response to acute immobilization stress (IS) in rats. METHODS: Adult male rats were subjected to unilateral right or left adrenalectomy or sham operation (control). Two weeks later, the rats were sacrificed either immediately or 3 hours after IS exposure. Plasma samples were used for determination of catecholamines (CAs), adrenocorticotrophic hormone (ACTH), corticosterone (CORT), sodium, potassium, and glucose levels. After terminating the experiment, both or remaining adrenals were removed, weighed, and used for estimation of CAs and nitric oxide (NO) levels. RESULTS: Under basal conditions, either right or left adrenal kept all the tested parameters near to the control levels, except the adrenal weight and CAs content. These were significantly higher in the remaining right than left adrenal. However, the remaining right adrenal responded better to IS exposure than the remaining left one in the term of compensatory adrenal growth and plasma parameters which were all kept insignificantly different from those of IS intact group. CONCLUSION: Our data indicate that the adrenal glands may substitute each other under basal conditions. However, the right adrenal seems to be dominant during exposure to acute immobilization stress.

Biological variations in adrenal gland response to immobilization and glucoprivation stressors in rats.

OBJECTIVES: The aim of the present study was to investigate the potential variation in adrenal gland response to two different types of acute stressors, immobilization and glucoprivation. METHODS: Twenty-four adult male albino rats were randomly divided into three main groups (8 rats/group): a) control, i.e. non-stressed group, b) immobilized group (IS), and c) glucoprivated (GS) group. Plasma catecholamines (CAs), including epinephrine (E), norepinephrine (NE), dopamine (DA), adrenocorticotrophic hormone (ACTH), corticosterone (CORT), sodium, potassium, and glucose were measured. Adrenals weight, CAs levels, and nitric oxide (NO) content were also determined. RESULTS: Immobilized group of rats showed significantly higher plasma NE and DA levels along with a significantly lower adrenal NE content than GS group. On the other hand, GS group was associated with significantly higher plasma E, ACTH, CORT, glucose, and Na+ levels as well as higher adrenal DA and NO levels along with significantly lower plasma K+ levels and adrenal E content in comparison with IS group. CONCLUSION: Stress response is unique according to the nature of the stressor. Adrenal glands play a key role in this stress-induced differentiated response probably via modulation of its adreno-medullary and/or adrenocortical hormone levels in order to assign the body cope with different types of stress challenges during the life.

Computer simulations and the use of radiolabelled sulphur colloid to measure the efficiency of the mononuclear phagocyte system.

Techniques are described whereby the clearance of the radiolabelled blood borne colloid can be continuously and reproducibly measured non-invasively from the same animal in vivo or from the isolated perfused intact liver in vitro. Using these techniques, the rate of removal of radiolabelled sulphur colloid by the mononuclear phagocytes in vivo and in vitro was shown to be biexponential. The pattern of clearance of colloid and the factors contributing to this were analysed with the aid of a computer program which mimicked the in vitro liver perfusion.

Characterization of Biomphalaria alexandrina-derived lectins recognizing a fucosyllactose-related determinant on schistosomes.

Two novel lectins that bind selectively to a schistosome-associated fucosyllactose-related determinant have been characterized and purified from the hemolymph of Biomphalaria alexandrina, the snail vector of Schistosoma mansoni. Both lectins were purified by affinity chromatography on a column of equimolar mixture of D- and L-glucose coupled to epoxy-activated Sepharose 6B and sequential elution by D-glucose (designated BaSI) and L-fucose (designated BaSII). Assessment of the structural characteristics, by one- and two-dimensional polyacrylamide gel electrophoresis, indicated that BaSI and BaSII were structurally distinct, and exist in their native forms as multimers of non-covalently associated subunits, that were of different sizes in BaSI and of equal size in BaSII. Removal of N-linked glycans by Endo-beta-N-acetylglucosaminidase F resolved the heterodisperse pattern of BaSI subunits into two spots of 13.2 kDa (pI 7.2) and 10.1 kDa (pI 5.8), and collapsed the acidic charge microheterogeneity of the BaSII subunit into a single spot that corresponded in terms of molecular weight and pI to the basic 13.2-kDa subunit of BaSI. In miracidial binding and inhibition assays with different sugars, both lectins exhibited selectivity towards a fucosyllactose sequence, but BaSII had a higher binding preference to fucose moieties. BaSII-Sepharose 4B affinity chromatography and analysis on two-dimensional gels indicated that multiple copies of the fucosyllactose-related determinant were expressed by heterogeneous, acidic glycoproteins in the miracidial stage of S. mansoni.

Sex-associated differences in the mitogenic responsiveness of snake blood lymphocytes.

The blastogenic response of male and female snake (Psammophis sibilans) peripheral blood lymphocytes to concanavalin A, phytohaemagglutinin, and E. coli lipopolysaccharide was tested for putative sex-associated differences. Data obtained suggest that normal female blood lymphocytes respond relatively vigorous in comparison to male cells or cells taken from gravid female snakes. The data are discussed in terms of possible correlations with the normal status of the animal.

Ontogeny of con A responsiveness and mixed leucocyte reactivity in the lizard, Chalcides ocellatus.

The immune system of phylogenetically key animals will contribute significantly to our understanding of the evolution of immune response in higher vertebrates. Reptiles, being evolutionary precursors of both birds and mammals, represent a pivotal group and thus a study of their immune system is of special significance. Here we described the emergence of T-cell immune capability in the viviparous lizard (Chalcides ocellatus) throughout embryonic development (stages 36-41 of Zada and El Deeb, 1984) and in newborns. The response of embryonic thymocytes (5 X 10(5) cells/ml) to Con A (5 micrograms/ml in culture) was first detected at stages 36-37, increased gradually during successive stages and then declined at birth to yield low responses in newborn lizards. In addition, embryonic thymocytes cultured in two-way MLR, using several combination sets, were significantly responsive at all stages. Our results reveal a degree of immunological T-cell maturation during reptitilian embryonic life which is similar to results in amphibians and mammals, but not clear with respect to fish and birds where comparative information still somewhat limited.

Effect of hydrocortisone on immune system of the lizard, Chalcides ocellatus. I. Response of lymphoid tissues and cells to in vivo and in vitro hydrocortisone.

A single dose of 1 mg/g body weight of hydrocortisone acetate (HC) administered intraperitoneally to adult lizards, Chalcides ocellatus induced rapidly a reduction of about 85% of thymic lymphocytes. Histological evidence indicated that cortical, as well as, medullary thymocytes are sensitive to HC exposure. Around 40-50% of lymphocytes in peripheral blood (PB) and spleen were depleted at 3-7 days post-HC injection; such depletion durated about 4 weeks for PB but was rather temporary in spleen. Increase in number of bone marrow (BM) lymphocytes was negligible and transient and could by no way account for the dramatic cell losses in the different lymphoid tissues. The findings thus suggested that HC-mediated lymphocyte depletion in lizards is not attributable to redistribution between the different lymphoid compartments but rather to destruction. In direct conformation, lymphocytes were readily lysed in vitro by 10(-3)M HC, thymocytes being more vulnerable greater than PB greater than spleen greater than BM lymphocytes.

Effect of hydrocortisone on immune system of the lizard, Chalcides ocellatus. II. Differential action on T and B lymphocytes.

Lymphocytes of thymus, spleen, peripheral blood (PB) and bone marrow (BM) collected from adult lizards, Chalcides ocellatus were cultured for 24 hr in the presence of 10(-3)M hydrocortisone acetate (HC) in order to assess the effect of in vitro HC on lizard T and B cell viability. The results indicated that HC induced stepwise, time-dependent mortality of the majority of thymocytes carrying T cell specific antigen(s) (TSA), 30-50% of T cells of spleen, PB and BM, and of a proportion of splenic B lymphocytes. Administration of 1 mg/g body weight HC to adult Ch. ocellatus lead to depletion of all TSA+ thymocytes. In contrast, T lymphocytes in the peripheral lymphoid compartments revealed both sensitivity and resistance to HC; similarly, B lymphocytes constituted susceptible and resistant subpopulations.

Effect of hydrocortisone on immune system of the lizard, Chalcides ocellatus. III. Effect on cellular and humoral immune responses.

Administration of a single injection of 1.0 mg/g body weight hydrocortisone acetate (HC) to adult lizards, Chalcides ocellatus in summer elicited a high and lasting rise in serum corticosterone and cortisol, peak levels being, however, in the physiologic ranges of 10 micrograms % (2 X 10(-7) M) and 40 micrograms % (1 X 10(-6) M), respectively. Elevation of serum corticosteroid (CS) concentrations by exogenous HC impaired the proliferative capacity of spleen cells in mixed leukocyte reaction (MLR) and significantly delayed skin allograft rejection. In vivo HC also abrogated the primary in vivo and in vitro immune responses of lizards to rat erythrocytes (RRBC). Reactivity to allogeneic cells in MLR, and antibody production against RRBC were, however, recovered when serum CS levels resumed normal, basal values i.e. at three weeks post-HC injection. These data indicate that rise in circulating CS induces severe immunosuppression in lizards.

Sexual dimorphism on the immune responses of the snake, Psammophis sibilans.

Recent trends in research have documented the influence of sex factors on the immune response of higher vertebrates. The present study is the first to indicate the existence of sex-dependent differences in the immune potentiality in reptiles. In optimal seasonal conditions, kinetics of the primary anti-rat erythrocytes (RRBC) antibody response were compared in individual male and female snakes, Psammophis sibilans. Antigen specific rosette forming cells (RFC) were enumerated in the spleen after in vivo primary immunization with 0.5 ml of 10% RRBC. A greater RFC activity as well as highest antibody titers were noted in females as compared to males. In addition, the peak of primary antibody response to RRBC in males was somewhat delayed in comparison with females. A similar pattern of sex-dependent differences was also observed regarding mitogen responsiveness to Con A, PHA and LPS with female lymphocytes being always the highest responders. In conclusion, female snakes demonstrated an elevated response in almost all comparative tests. Thereafter, at least under physiological conditions, our observations suggest a possible influence of sex hormones as immunoenhancers/immunosuppressors in reptiles.

Endogenous corticosteroids mediate seasonal cyclic changes in immunity of lizards.

Endogenous corticosteroid (CS) blood levels were radioimmunoassayed in fresh, field-collected lizards Chalcides ocellatus at two week-intervals throughout the four consecutive seasons. These animals were used in parallel to investigate the splenic T and B lymphocyte level, lymphoproliferative responsiveness to concanavalin A and primary antibody production in vitro against rat erythrocytes (RRBC). The recorded data indicated that fully developed splenic lymphoid tissue and powerful immune responsiveness are coincident with a continuously low CS level, and characterize the period from spring through early autumn. On the other hand, the dramatic lymphocytic destruction and impairment of immune reactivity observed in autumn and winter are associated with not only a high, but above all sustained, rise in endogenous CS levels. Apparently, exposure of lizard lymphocytes to comparatively high, yet physiologic, levels of endogenous CS for prolonged periods of time lead to impairment of their immune functions. In support, long-term administration of exogenous hydrocortisone acetate (HC) to "summer" lizards resulted in a high and lasting elevation in blood CS levels that was associated with a considerable depletion of lymphoid elements and abrogation of immune reactivity, exactly as in normal lizards collected from the field in autumn through winter. In addition, pharmacologic inhibition of CS synthesis by administration of metyrapone at the beginning of autumn greatly modulated the lizard lymphocyte response to the autumn-related immunodepression. The study thus strongly suggests that the autumn/winter-dependent immunosuppression in lizards is essentially due to a high and lasting rise in levels of endogenous CS. The results are discussed from the perspective of the role played by CS in mediating the seasonal rhythms that affect reptilian immunity.

Mixed leukocyte reaction, graft-versus-host reaction, and skin allograft rejection in the lizard, Chalcides ocellatus.

In optimal seasonal conditions, splenocytes derived from adult lizards, Chalcides ocellatus, responded to vigorous proliferation in two- and one-way mixed leukocyte reaction cultures (MLRC). Data based on a large number of reciprocal MLRC provided evidence for the presence of strong and diverse lymphocyte activating determinants. These findings were fully confirmed in studies of systemic graft-versus-host reaction as intraperitoneal injection of splenocytes into newborn allogeneic recipients consistently induced splenomegaly, retarded growth and mortality. In favourable ambient conditions, adult lizards were also able to reject skin allografts in a subacute manner (mean survival time +/- S.E. = 28.8 +/- 0.88). The results clearly indicate that the lizard, Chalcides ocellatus, is endowed with an advanced type of cell-mediated immunity, and possesses strong and polymorphic histocompatibility antigens. Cellular alloreactivity in MLRC and towards skin grafts was, however, abrogated in winter and significantly diminished during spring through mid-summer as compared to mid-summer till autumn. The present study, thus, suggests that immunological defects attributed to reptiles might be more apparent than real, and essentially ascribable to the fact that the immune capacity of these ectothermic vertebrates is profoundly modulated by environmental conditions.

Blood testosterone level: a season-dependent factor regulating immune reactivity in lizards.

An attempt to study the interaction between testosterone (Ts) and the immune system of the lizard Chalcides ocellatus led to three major findings: 1) Endogenous serum Ts levels in both males and females peak in spring and are minimal during summer; 2) Injection of Ts in either male or female lizards induces significant depletion of lymphoid elements, reduction in serum antibody titers to rat erythrocytes and increase in skin allograft survival; 3) A distinct inverse correlation between endogenous serum Ts levels and lizard immunocompetence is observed from March to September. The data obtained strongly suggested that concentration of circulating Ts is a season-related factor that is critical in defining the immune profile of lizards.

Seasonal distribution and hormonal modulation of reptilian T cells.

Using PNA and anti-Thy-1 fluorescent binding assays, T lymphocytes of the lizard, Chalcides ocellatus were phenotypically distinguishable into four subpopulations (PNA+ Thy-1-, PNA+ Thy-1+, PNA- Thy-1+ and PNA-Thy-1-), which seemed to be affected independently by endogenous steroid levels. Indeed, the size of PNA+ thymocytes is maximal and coincides with the low level of circulating cortisol during spring through summer and decreases gradually with the elevation of the cortisol level. On the other hand, as the endogenous testosterone (TS) level begins its physiological rise, lympholysis of Thy-1+ thymic cells begins in spring with gradual increase in size and with the decrease in TS levels. Among splenocytes and bone marrow lymphocytes, seasonal-dependent alterations in the size of both lymphocyte subpopulations seemed to correlate in part with the status of the thymus. Direct support of this observation was derived from subsequent in vitro studies with exogenous hydrocortisone (HC) and testosterone propionate (TP) treatments in spring and autumn. In all incidents, the data were indicative of the selective susceptibility of the PNA+ Thy-1- subpopulation to HC in the thymus and not in the periphery, and the susceptibility of the PNA- Thy-1+ subpopulation to TP in all three lymphoid organs tested. In vivo studies with a purified fraction of thymosin alpha 1 (T alpha 1) suggested that the PNA+ Thy-1- subpopulation in the different organs was the selective target for the action of T alpha 1. Finally, the dual treatment with T alpha 1 in vivo followed by TP or HC in vitro confirmed that TP-sensitivity was confined to the PNA- Thy-1+ and HC to PNA(+)-Thy-1- subpopulations in any of the three lymphoid organs.

Concanavalin A responsiveness and interleukin 2 production in the snake Spalerosophis diadema.

Thymocytes and splenocytes (SC) of adult snakes, Spalerosophis diadema, responded to concanavalin A (Con A) in vitro by strong proliferation during the spring and autumn seasons. Con A-mediated mitogenesis was, however, abrogated in summer and winter. Conditioned medium (CM) collected from snake SC cultures stimulated with Con A in spring or autumn could enhance the Con A summer and winter responses and support the proliferation of splenic lymphoblasts. Gel filtration of native CM on Sephadex G-100 revealed the presence of two biologically active peaks of molecular weight 39-42 and 15 KD. However, only one peak of activity corresponding to molecular weight (m.w.) of 14-15 KD was observed when CM was subjected to analysis by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE). The active molecular forms exhibited isoelectric points of 5.5-5.8 and 6.4-6.6. The findings suggest that Con A activation of snake lymphocytes in optimal seasonal conditions is associated with the secretion of a lymphokine analogous to the interleukin 2 (IL 2) of endothermic vertebrates.