Potential antiviral effects of pantethine against SARS-CoV-2.

SARS-CoV-2 interacts with cellular cholesterol during many stages of its replication cycle. Pantethine was reported to reduce total cholesterol levels and fatty acid synthesis and potentially alter different processes that might be involved in the SARS-CoV-2 replication cycle. Here, we explored the potential antiviral effects of pantethine in two in vitro experimental models of SARS-CoV-2 infection, in Vero E6 cells and in Calu-3a cells. Pantethine reduced the infection of cells by SARS-CoV-2 in both preinfection and postinfection treatment regimens. Accordingly, cellular expression of the viral spike and nucleocapsid proteins was substantially reduced, and we observed a significant reduction in viral copy numbers in the supernatant of cells treated with pantethine. In addition, pantethine inhibited the infection-induced increase in TMPRSS2 and HECT E3 ligase expression in infected cells as well as the increase in antiviral interferon-beta response and inflammatory gene expression in Calu-3a cells. Our results demonstrate that pantethine, which is well tolerated in humans, was very effective in controlling SARS-CoV-2 infection and might represent a new therapeutic drug that can be repurposed for the prevention or treatment of COVID-19 and long COVID syndrome.

Protein-X, Pancreatic Stone-, Pancreatic thread-, reg-protein, P19, lithostathine, and now what? Characterization, structural analysis and putative function(s) of the major non-enzymatic protein of pancreatic secretions.

Reg protein was first found in pancreatic stones. It was named Pancreatic Stone Protein and later renamed lithostathine, as it was assumed to prevent stone formation. The 144 amino acid protein is O-glycosylated on Thr-5. The glycan chain is variable in length and in charge. Lithostathine 3-D organization is of the C-lectin type, even though it is unlikely to have any functional calcium-binding site. The Arg11-Ile12 bond is readily cleaved by trypsin; the resulting C-terminal polypeptide precipitates at physiological pH and tends to form fibrils. The protein was more recently found in the regenerating endocrine pancreas and it was named Reg (for regenerating) protein. Numerous proteins related to Reg have been identified successively in several mammalian species. They constitute the Reg superfamily. Reg genes show the same organization and are located in the same chromosome region. These genes are therefore likely to derive from a common ancestor gene by duplication. In the course of evolution, they may have diverged in tissue-related expression and function. In the endocrine pancreas, Reg protein stimulates islet beta-cell growth and reduces experimental diabetes via the activation of a high affinity receptor. The role of the protein produced by the exocrine pancreas, however, is controversial. Not only is Reg/lithostathine unlikely to be a physiologically relevant pancreatic stone inhibitor, but it may contribute to stone formation. We suggest that it might help prevent the harmful activation of protease precursors in the pancreatic juice. The protein provides a useful model for examining the conformational changes associated with globular to fibril transformation.

Ecdysone and 20 hydroxyecdysone: new hormones for the human parasite schistosoma mansoni.

The insect moulting hormones, ecdysone and 20 hydroxyecdysone, were detected by the combined use of radioimmunoassay and high performance liquid chromatography in the human parasite Schistosoma mansoni. On day 11 after infection only the ecdysone form is present, but, on day 40 after infection the ratio between ecdysone and 20 hydroxyecdysone changes with anatomic localization of the adult worms in mammalian host. In the eggs, the ratio of these two hormones is identical to the ratio found in sexually mature worms located in mesenteric veins. These data demonstrate for the first time that S. mansoni synthesizes the steroid hormones ecdysone and 20 OH ecdysone which are potent molecules in stimulating growth and vitello-genesis of this gonochoric trematode.

Excretion of ecdysteroids by schistosomes as a marker of parasite infection.

Ecdysteroids produced by schistosomes are released in biological fluids of infected hosts. In the sera, the concentration of ecdysteroids correlates with the permissiveness of the host to schistosome infection and its detection is available in the absence of positive parasitological tests. In the urine, ecdysteroid concentration decreases markedly after chemotherapy. 20-Hydroxyecdysone and its epimer were identified in the urine of infected patients using mass spectrometry. These data demonstrate for the first time that ecdysteroids are released by organisms. Moreover, they are potent molecules of parasite infection and can be used for parasite diagnosis.

Ecdysteroid-like compounds in the serum and urine of African patients infected with Loa loa and Mansonella perstans microfilariae.

Ecdysteroids are compounds related to 20-hydroxyecdysone, the insect moulting hormone. Surprisingly, they have been found in serum and urine of patients infected with helminths. In these cases, the substances are assumed to be produced by the parasites and, therefore, might be used as a marker of parasitic infection. Thus, we need to know exactly which species, at which developmental stage, can release ecdysteroids in such large quantities that they could be detected in the biological fluids of the host. Large-scale investigations must, accordingly, be devoted to the major species of helminths. In the present study, we examined 100 African patients with Loa loa and/or Mansonella perstans microfilaraemia. About 70 of them had high levels of ecdysteroid-like materials in serum or urine. In contrast, uninfected patients and European controls had much lower concentrations. However, the ecdysteroid titres did not reflect the concentration of microfilariae actually present in the blood, and some heavily infected patients were even negative. Nevertheless, the most important point was that high ecdysteroid levels in man were always associated with a pathological condition. The precise significance of the phenomenon should be determined.

Hyaluronate levels and markers of oxidative stress in the serum of Sudanese subjects at risk of infection with Schistosoma mansoni.

We showed previously that infection by Schistosoma mansoni not only triggers the production of reactive oxygen species in mouse liver but also leads to the alteration of antioxidant defences. To determine whether such events occur in humans, we measured the serum markers of oxidative stress, i.e., lipid peroxides and protein carbonyl, as well as hyaluronate levels in subjects in the Managil area of the Sudan. Grades of fibrosis were determined by ultrasonographic examination. Two groups were used as controls, one Sudanese and the other European. We found that Sudanese subjects in the endemic area differed from the control groups, both Sudanese and European, insofar as they had higher levels of the serum metabolites measured. The latter increased with the grade of fibrosis. Moreover, protein carbonyl and hyaluronic acid levels correlated positively with lipid peroxide levels. These findings indicate that oxidative stress might contribute to S. mansoni-associated pathology in man. The serum markers considered in our study, obtained by relatively simple techniques, may provide a useful biochemical index for the identification of almost asymptomatic patients who, however, are at risk of developing severe schistosomiasis.

Ecdysteroid-like compounds in human urine: they can occur in the absence of any parasitic infection.

Ecdysteroids (compounds related to 20-hydroxyecdysone, the insect molting hormone) can appear in the blood and urine of man, as a result of an infection with helminths. It has been assumed that the products are released by parasites. However, we found that the phenomenon is not restricted to helminthiases, but is widely spread among patients suffering from various diseases or injuries: twenty percent of hospital in-patients had urine highly positive in our test. This was due to the appearance of immunoreactive compounds not found in healthy people. Among them, one was remarkable for being largely predominant in some patients. These findings indicate that the origin and significance of ecdysteroids in man should be reconsidered. Since they appear only in association with severe pathological conditions, they could be of potential interest as a clinical marker.

Ecdysteroids in urine of African patients infected with helminths.

The insect molting hormone and related compounds (ecdysteroids) have been found in patients infected with helminths. To investigate this phenomenon, we quantified and analyzed the urinary ecdysteroids in Malian subjects suffering from various helminthiases, as well as in Europeans. Very high titers (up to 100 nM) were found in some patients, whereas healthy persons had a basic level of 4 nM only. The high RIA activity was mainly due to two compounds. One of them was remarkable for being present in all the positive samples; it comigrated with the 20-hydroxyecdysone standard, both in HPLC and in TLC. The origin and the physiological significance of these compounds are questionable, since there was no clear relationship between their levels and the severity of the diseases. Numerous patients who were heavily infected had normal titers. Nevertheless, there is no doubt that high concentrations of ecdysteroid-like compounds in human urine indicate a pathological condition.

Hypogonadism and ecdysteroid production in Loa loa and Mansonella perstans filariasis.

Possible endocrinological repercussions of infection with Loa loa and Mansonella perstans filariae were studied in Gabonese subjects. Microfilaremic males were compared with amicrofilaremic controls. In the infected group 13/105 subjects (12%) presented only abnormally low serum levels of testosterone (less than 4 ng/ml), 25/105 (24%) only abnormally high serum levels of gonadotrophins, FSH (greater than 15 mIU/ml) and LH (greater than 20 mIU/ml), and 22/105 (21%) presented anomalies in both testosterone and gonadotrophin levels. One out of 68 control subjects had 3.6 ng/ml seric testosterone and all had normal levels of gonadotrophins. Ecdysteroids were detected (greater than 0.025 ng/ml) in the serum of 87/97 (90%) microfilaremic subjects (GM 0.123 ng/ml) compared to 12/64 (19%) controls (GM 0.030 ng/ml). Ecdysteroids were detected in the urine of all subjects, infected (GM 8.468 ng/ml) as well as control (GM 1.245 ng/ml). The hormonal perturbations were correlated with the levels of Loa loa microfilaremia but not with those of serum and urinary ecdysteroids. These results demonstrate that microfilaremic subjects often show endocrinal signs of hypogonadism and present appreciable levels of ecdysteroids in serum and urine. A direct role for parasitic ecdysteroids in hypogonadism remains to be demonstrated.

Lithostathine, the presumed pancreatic stone inhibitor, does not interact specifically with calcium carbonate crystals.

Lithostathine (pancreatic stone protein, Reg protein) is, in addition to albumin, the major nonenzymatic protein of the pancreatic juice. It has been assumed to inhibit calcium carbonate precipitation and therefore to prevent stone formation in the pancreatic ducts. This function is, however, debatable. The assumption is based on the inhibition of in vitro crystal nucleation and growth by lithostathine. Considering that these phenomena occur only under certain critical conditions, we re-examined the question using a protein preparation where the purity and folding have been tested by mass spectroscopy and NMR in the absence of nonprotein contaminants. Under these conditions, we showed conclusively that lithostathine does not inhibit calcium carbonate nucleation and crystal growth. We demonstrated that previous findings on the alleged inhibition can be attributed to the uncontrolled presence of salts in the protein preparation used. Moreover, the affinity of lithostathine to calcite crystals, expressed as the half-life of bound iodinated protein in the presence of unlabeled competitor, was significantly lower than that of bovine serum albumin (8.8 and 11.2 h, respectively). Using glass microspheres instead of crystals did not significantly change the half-life of bound lithostathine (8.0 h). These findings are incompatible with the hypothesis of a specific interaction of lithostathine with calcium carbonate crystals. In conclusion, considering that components of pancreatic juice such as NaCl and phosphate ions are powerful inhibitors of calcium carbonate crystal growth, the mechanism of stone formation in pancreatic ducts must be reconsidered. The presence in normal pancreatic juice of small amounts of the 133-residue isoform of lithostathine (PSP-S1), which precipitates at physiological pH, should be noted, and the possibility should be considered that they form micro-precipitates that aggregate and are progressively calcified.

Changes in ecdysteroid and juvenile hormone titers in the hemolymph of Galleria mellonella larvae and pupae.

The variations in circulating ecdysteroids and juvenile hormones (JH) in Galleria, from the end of the antepenultimate larval stage until emergence of adults, have been determined. The two hormonal families were extracted separately from the same hemolymph sample and quantified by two radioimmunoassays. Juvenile hormone RIA activity was about 35 nM in larvae of the antepenultimate and penultimate stages. It dropped before each molt and increased thereafter. Moreover, it gradually decreased during the last larval instar. In pupae, it was generally low, but it rose drastically during the late pupal development and in young adults. This rise was very much higher in females than in males. Three different RIA-active compounds were found; they were assumed to be JH-I, JH-II, and JH-III according to their retention times in HPLC. The three compounds were present in almost equal concentration in larvae of the penultimate stage: JH-I predominated, however, during the last larval instar. In late pupae, the main hormone was JH-III both in males and in females. There is no clear relationship between ecdysteroid and juvenile hormone changes, except for a female-specific ecdysteroid rise which coincides with the juvenile hormone release in late pupae. This double hormonal stimulation can be involved in the regulation of vitellogenin synthesis and deposition in oocytes.

Changes in ecdysteroid and juvenile hormone titers correlated to the initiation of vitellogenesis in two Aedes species (Diptera, Culicidae).

Ecdysteroid and juvenile hormone titers were determined in the whole body of females of Aedes detritus and A. caspius. Since both hormones were assayed from the same extract, this method allowed determination of their simultaneous variations during egg formation, i.e., from the time the females emerged until the onset of oviposition. A drastic hormonal increase was observed at the beginning of vitellogenesis. This increase occurred as two high and sharp peaks, the first of ecdysteroids and the second, which took place 8 hr later, of juvenile hormones. The two peaks together lasted less than 12 hr, with the highest level at about 3 X 10(-7) mumol/mg fresh tissue. After the juvenile hormone peak, the oocytes entered into stage III/b, the time at which the intensive phase of vitellin accumulation in the eggs begins.

Fundamental aspects and potential roles of ecdysteroids in schistosomes an update overview.

The human digenic trematodeSchistosoma mansoni produces ecdysteroid hormones. Both ecdysone (22R)-2ß,3ß,14,22,25-pentahydroxy-cholest-7-en-6-one and 20-hydroxyecdysone (22R)-2ß,3ß, 14,20,22,25-hexahydroxycholest-7-en-6-one were detected during the critical development stages of this bisexual parasite: during the exponential growth phase to adult stage (day 11 postinfection) and during the sexual maturation of adult and egg laying (day 40 postinfection). The parasites released their ecdysteroid hormones in the biological fluids of the infected vertebrates as soon as six days postinfection. In addition, the time course of the ecdysteroid titer was correlated with the susceptibility or the innate resistance of the host to schistosome infection. Moreover, we demonstrated that after a schistomicide therapy, ecdysteroids from urine of infected children decreased markedly four days after drug administration. We also have demonstrated that immunization of rodents with an ecdysone-BSA complex led to the reduction of the worm burden. By in vitro studies, we have shown that the ecdysterone antibodies were able to kill the juvenile worms within 24 hr. In addition, we have demonstrated that the enzymes Superoxide dismutase (SOD, EC 1.15.1.1) were present in schistosomes and that the total Superoxide dismutase activities in both males and females could be correlated with the 20-hydroxyecdysone within parasites.

What function for human lithostathine?: structural investigations by three-dimensional structure modeling and high-resolution NMR spectroscopy.

Human lithostathine is a 144-residue protein, expressed in various organs and pathologies. Several biological functions have been proposed for this protein. Among others, inhibition of nucleation and growth of CaCO3 crystals in the pancreas and bacterial aggregation has retained attention, because lithostathine presents high sequence similarities with calcium-dependent (or C-type) lectins. To study its structure-function relationship and compare it with that of C-type lectins, we have built a model for lithostathine. This model is derived from the only two C-type lectins of known structures: rat mannose binding protein and human E-selectin. An original strategy, inspired by that proposed by Havel and Snow, was designed for model building. We have undertaken NMR studies on the natural protein. Although complete structure determination has not yet been achieved, the NMR studies did confirm the main characteristics of the model. From analysis of the proposed model, we concluded that lithostathine is not expected to present sugar- or calcium-binding properties. Therefore, the mechanisms of bacterial aggregation and inhibition of CaCO3 nucleation and growth have not yet been elucidated.