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.

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.

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.

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.

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 5. 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 5. 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.

Cellular response to Schistosoma mansoni infection in Biomphalaria alexandrina strains selected for susceptibility and resistance.

Exposure of juvenile and adult Biomphalaria alexandrina to Schistosoma mansoni miracidia resulted, typically, in three susceptibility patterns: a) non-infected snails; b) normal infections, and c) retarded infections. Under laboratory conditions, a vigorous resistant-type cellular response to invading miracidia was seen in the histological sections of non-susceptible snails. Accordingly, they were classified as resistant snails. Data pertaining to the influence of host size on suceptibility to S. mansoni indicates that adult snails (i.e. 10-20 mm shell diameter) were significantly less likely to harbour sporocysts than juvenile ones (i.e. 5-10 mm shell diameter). Cellular reaction to the infection varied with sporocysts location and length of infection. At 2 days post-exposure (DPE), most sporocysts were viable. Approximately 8-12% of the sporocysts had elongated shaped transverse constriction and were categorized "normal", while those showing no elongation were categorized "retarded". All remaining sporocysts at 4 DPE were categorized "dead", while at 30 DPE, most sporocysts were "amorphous" with eosinophilic masses. Although encapsulation of sporocysts never occurred in susceptible snails, haemocyte aggregations could sometimes be observed in the proximity of well developed sporocysts. In resistant snails, this cellular response continued to increase and resulted in the encapsulation of the sporocysts. In susceptible snails, the nucleus of secretory cells of the albumen cytoplasm were different in susceptible and resistant snails.

Possible role of Schistosoma mansoni infection in male hypogonadism.

Schistosomiasis has been suggested to decrease the reproductive potential or castrate both invertebrate and vertebrate hosts. Furthermore, schistosomiasis may cause anatomic anomalies of the reproductive organs responsible for permanent or reversible infertility. To specify the effect of schistosomiasis on gonadal functions, production of testosterone (TS), leutinizing hormone (LH) and estradiol (E2) in Egyptian men infected with schistosomiasis were studied. All participants were tested for the following parameters: Clinical examination and diagnostic, semen, haematological and liver function tests and blood level of IL-2. The mean TS levels were at the lowest limit of normal range for liver cirrhotic patients. Mean E2 levels were increased in all patients, but patients with liver cirrhosis-related schistosomiasis had higher E2 levels. Linear regression analysis showed that the sex hormone levels correlated best with the patient's liver function parameters. The present data suggest that a sex hormone imbalance plays a role in patients with liver cirrhosis due to the inhibitory effects of schistosomiasis on gonadal functions.

Association of BCL-2 with membrane hyperpolarization and radioresistance.

The resting membrane potential of parental, neomycin control, and Bcl-2 transfected cells was measured, and the effect of membrane hyperpolarization or depolarization on radiosensitivity was studied. Bcl-2 transfected cells were significantly more radioresistant than control cells and were significantly hyperpolarized compared to parental and neomycin control transfected PW and HL60 cells. Hyperpolarization of the parental and neomycin control transfected cells by valinomycin significantly increased the radioresistance of these cells to such an extent that there was no longer a significant difference in the survival of the valinomycin treated and irradiated control cells compared to similarly irradiated Bcl-2 transfected cells. In contrast, depolarization of the Bcl-2 transfected PW and HL60 cells decreased the radioresistance of the Bcl-2 transfectants to a level similar to that of the control cells. The data presented here suggest that overexpression of Bcl-2 affects membrane potential and that this hyperpolarization is associated with increased radioresistance of cells that overexpress Bcl-2. Furthermore, Bcl-2 transfected cells were also less susceptible to the specific Na+/K(+)-ATPase inhibitor ouabain, suggesting that Bcl-2 may act at the level of the Na+/K(+)-ATPase pump.

Structural characterization of an Lyt-2/3 homolog expressed on Bufo regularis lymphocytes.

Two alloantisera and a monoclonal antibody (mAb 53-6.7) of proven specificities to the murine Lyt-2/3 macromolecule labeled, in indirect immunofluorescent assays, a distinct lymphocyte population in the toad, Bufo regularis. Lyt-2/3 antigenic activities expressed by B. regularis lymphocytes have been solubilized and purified by mAb 53-6.7 affinity chromatography and found to be associated with a single 67 kDa macromolecule in SDS-PAGE. Upon reduction, this macromolecule resolved into 38 kDa, 34 kDa and 28 kDa subunits corresponding to the alpha, alpha' and beta subunits of the murine Lyt-2/3 complex. Comparisons based on the S delta Q index of differences in amino acid compositions of HPLC-purified alpha- and alpha'-subunits of the amphibian Lyt-2/3 molecule indicated a significant structural relatedness to their murine counterpart as well as to the human CD8 polypeptide. Our observations point to an early phylogenetic emergence of Lyt-2/3 as an important component of the T cell cytolytic apparatus during vertebrate evolution.

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.

Ovarian activity during follicular-phase down regulation in in vitro fertilization is associated with advanced maternal age and a high recurrence rate in subsequent cycles.

PURPOSE: Previous reports have suggested that the ovarian response to leuprolide acetate is predictive of in vitro fertilization pregnancy rates. This study evaluated the outcome of in vitro fertilization cycles complicated by elevated estradiol levels during leuprolide acetate down regulation and the outcome of subsequent cycles in the same patients. METHODS: Two hundred fifty-two in vitro fertilization cycles were initiated utilizing leuprolide acetate down regulation beginning on cycle day 1. RESULTS: Seventy-four of these cycles had an elevated estradiol level at the time of the baseline scan (28%). This group of patients had a higher maternal age, a higher cycle cancellation rate (27.5 vs 16.3%), and a high rate of recurrence on subsequent cycles (63%). CONCLUSIONS: The pregnancy rate per retrieval was equivalent in the two groups. This suggests that patients with advanced maternal age or a history of failure to suppress in a previous cycle may benefit from alternate regimens of superovulation.

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.

Identification of peanut agglutinin-binding glycoproteins on lizard lymphocytes.

The expression of PNA-binding glycoproteins on lizard lymphocytes was investigated by studying the reactivity of FITC-PNA towards lizard lymphocytes obtained from the different lymphoid organs. Direct immunofluorescence assays have demonstrated that the majority of lizard thymocytes (70%) and only a fraction of lymphocytes in the spleen, peripheral blood and bone marrow were PNA-positive. This positivity was selectively inhibited by galactose as well as lactose, indicating the specificity of binding. Putative PNA receptors were purified from lizard thymocytes and splenocytes by affinity chromatography on a PNA-Sepharose 4B column and resulted in fractions enriched 1,792-fold and 3,141-fold for the PNA-binding component expressed on lizard thymocytes and splenocytes, respectively. Analysis on reducing and non-reducing SDS-PAGE revealed that both thymic and splenic PNA-binding glycoproteins migrated as a single component of 35 KDa, with no evidence for the association into higher multimers in both tissues. Analyses for amino acid and carbohydrate compositions indicated that the thymic and splenic glycoproteins have similar amino acid composition and differed in the content of neutral and amino-sugars as well as sialic acid. The content of the latter residue was relatively higher in the splenic form of the receptor compared to its thymic counterpart, and was inversely correlated with the content of galactosyl residues in both forms of the receptor. The functional significance of PNA-binding glycoproteins during vertebrate evolution is discussed.

Modulation of potassium channels by protein tyrosine kinase inhibitors.

Exposure of non-excitatory cells to the tyrosine kinase (PTK) inhibitors, genistein, herbimycin A, and tyrphostin, induced at least two families of K+ currents. The first, a TEA-insensitive slow-inactivating K+ current, is induced within 3 min following treatment with 140 mM genistein or 100 nM herbimycin A. The second current, a TEA-sensitive delayed rectifier, is induced within 30 min following treatment with 50 mM genistein or 10 nM herbimycin A. Currents with similar biophysical and pharmacological characteristics are induced in these cells following exposure to ionizing radiation. The radiation-induced currents are inhibited by pretreatment with the free radical scavenger, N-Acetyl L-Cysteine, or by pretreatment with the protein kinase C inhibitor, staurosporine; those induced by PTK inhibitors are not. The latter, therefore, do not appear to be mediated through free radicals or require serine/threonine phosphorylation for activation. Once the channels are activated by the PTK inhibitors, phosphorylation of the channel at serine/threonine residues results in slower inactivation of the induced current. We propose that protein tyrosine phosphorylation of the K+ channel protein itself or of a factor that interacts with it maintains the K+ channels of non-excitatory cells in a closed state. Following exposure to ionizing radiation, free radical-induced activation of serine/threonine kinase(s) results in phosphorylation of the channel and/or inactivation of a tyrosine kinase that in turn leads to activation of the K+ channels.

Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels.

In mammalian cells, little is known about the initial events whose ultimate consequence is mutagenesis or DNA repair. The role the plasma membrane may play as an initiator of such a pathway is not understood. We show, for the first time, that membrane voltage-dependent potassium (K+) currents, activated by ionizing radiation (Kuo et al., 1993), play a significant role in radiation mutagenesis. Specifically, we show that the frequency of mutation at the HGPRT locus is increased as expected to 37.6 +/- 4.0 mutations per 100,000 survivors by 800 cGy of ionizing radiation from a spontaneous frequency of 1.5 +/- 1.5. This increase, however, is abolished if either K+ channel blocker, CsCl or BaCl2, is present for 2 h following irradiation of the cells. RbCl, chemically similar to CsCl but known not to block K+ channels, is ineffective in reducing the mutation frequency. Treatment of cells with CsCl or BaCl2 had no effect on radiation-induced cell killing.

Thymus histogenesis in the lizard, Chalcides ocellatus.

Different stages of thymus morphogenesis and thymocyte differentiation have been studied at the ultrastructural level in the lizard, Chalcides ocellatus. On stage 36 of embryonic development, the thymus primordium was composed principally of undifferentiated epithelial cells and some lymphoid stem-cells. From stage 37 to 38, the lymphoid stem-cells differentiate into lymphoblasts and then transform into typical lymphocytes. A clasmotosis phenomenon seems to be involved in this transformation. In the developing cortical regions, lymphoblasts accumulated rapidly, stretching the epithelial cells which become stellate in shape. From stage 39 to 40, a phase of intense proliferation occurs and numerous lymphocytes die in the thymic tissue and are phagocytosed by macrophages. On stage 41, the presence of interdigitating cells in the medullary area completed cortico-medullary differentiation. On neonatal and juvenile lizards, small cortical thymocytes differentiated and the thymus possessed all characteristic of an adult thymus. Thus, at birth, the histogenesis of the lizard thymus was achieved and the only further modification consisted in a gain of weight.

Activation of potassium channels by hypoxia and reoxygenation in the human lung adenocarcinoma cell line A549.

Active oxygen species are generated in cells during pathophysiologic conditions such as inflammation and postischemic reperfusion. If oxygen radical scavengers are added before reperfusion, then the magnitude of injury is reduced. We investigated whether free radicals generated following exposure to hypoxia and reoxygenation activate voltage-dependent K+ ion channels in tumor cells in vitro. Using the technique of whole cell voltage clamping, we recorded currents from two families of potassium (K+) channels that were activated following reoxygenation. One of these groups possessed the electrophysical characteristics of a tetraethylammonium (TEA)-sensitive delayed rectifier channel and the other possessed characteristics of a Tea-insensitive slow inactivating channel. We present evidence which suggests that K+ channels are activated following reoxygenation but not during the hypoxia phase. The K+ currents decayed with time following reoxygenation. The decay characteristics of the K+ currents depended on the duration and level of hypoxia to which the cells were exposed. To determine whether activation of K+ channels by reoxygenation was initiated by free radicals, we pretreated cells with N-Acetyl L-Cysteine (NAC), a free radical scavenger, and found that this pretreatment abolished the currents induced by reoxygenation. We also present evidence that free radicals do not directly act on the channel itself, but activate a protein kinase which, in turn, activates the K+ channels. Taken together, these results indicate that one of the early responses to oxidative stress is the activation of K+ currents.