Additional booster doses in patients with chronic lymphocytic leukemia induce humoral and cellular immune responses to SARS-CoV-2 similar to natural infection regardless ongoing treatments: A study by ERIC, the European Research Initiative on CLL.

Profound immune dysregulation and impaired response to the SARS-CoV-2 vaccine put patients with chronic lymphocytic leukemia (CLL) at risk of severe COVID-19. We compared humoral memory and T-cell responses after booster dose vaccination or breakthrough infection. (Green) Quantitative determination of anti-Spike specific antibodies. Booster doses increased seroconversion rate and antibody titers in all patient categories, ultimately generating humoral responses similar to those observed in the postinfection cohort. In detail, humoral response with overscale median antibody titers arose in >80% of patients in watch and wait, off-therapy in remission, or under treatment with venetoclax single-agent. Anti-CD20 antibodies and active treatment with BTK inhibitors (BTKi) represent limiting factors of humoral response, still memory mounted in ~40% of cases following booster doses or infection. (Blue) Evaluation of SARS-CoV-2-specific T-cell responses. Number of T-cell functional activation markers documented in each patient. The vast majority of patients, including those seronegative, developed T-cell responses, qualitatively similar between treatment groups or between vaccination alone and infection cases. These data highlight the efficacy of booster doses in eliciting T-cell immunity independently of treatment status and support the use of additional vaccination boosters to stimulate humoral immunity in patients on active CLL-directed treatments.

Vitamin D receptor expression and acid sphingomyelinase activity in prefrontal region of a learning animal model.

Contextual fear conditioning (CFC) paradigm is routinely used to study fear-based learning in animals and it provides a useful model for understanding fear and anxiety in human. In the present study, such model was used following the previously established CFC protocol, and immunohistochemistry, enzymatic activity and western blotting analysis approaches were used to identify the expression of acid sphingomyelinase (aSMase) and vitamin D receptor (VDR) in prefrontal region brain of rat. Results revealed an increase of aSMase activity in conditioned rats, suggesting an apoptotic condition in such animals. In addition, an increase of density and organization of axonal neurofilaments and of VDR expression has been observed in brain of conditioned rats, supporting an induction of growth and organization of new neurons in prefrontal regions, whose contribution to various aspects of contextual fear learning is still largely unknown.

The thyroid lobes: the different twins.

Although differences in size of the right and left thyroid lobes are well defined, differences in morphology, follicles structure, cAMP production, thyrotropin receptor, and protein involved in cell signalling have not previously been reported. This study provides morpho-functional data of right and left thyroid lobes by biochemical, immunohistochemistry, immunoblotting and immunofluorescence analysis. We demonstrate that, in comparison with the left lobe, the right lobe has a higher activation index, is more sensitive to thyrotropin treatment, is rich in thyrotropin receptor and caveolin 1 involved in thyroid hormone synthesis as well as in epithelial thyroid cell homeostasis, is characterised by a high content of molecules involved in cell signalling such as stat3, raf1, sphingomyelinase and sphingomyelin-synthase whose activity ratio is necessary for epithelial cell activity and finally has more areas calcitonin-dependent. The relation between structure/function of right lobe and its susceptibility to the higher risk of pathological modifications with respect the left lobe is discussed.

The nuclear ceramide/diacylglycerol balance depends on the physiological state of thyroid cells and changes during UV-C radiation-induced apoptosis.

Intranuclear lipid metabolism modifications in relation to cell proliferation and/or apoptosis were demonstrated in hepatocytes. The aim of this study was to establish whether nuclear lipid metabolites influence cell function in different experimental models using a rat thyroid cell line (FRTL-5) treated with UV-C radiation. After UV-C irradiation cells proliferate and undergo apoptosis in the presence of thyrotropin, are quiescent and resistant to radiation-induced apoptosis in its absence and finally are proapoptotic for nutrition withdrawal. In nuclei purified from proliferating cells, irradiation stimulates neutral-sphingomyelinase activity and inhibits sphingomyelin-synthase, phosphatidylcholine-specific phospholipase C and phosphatidylinositol-specific phospholipase C activity with a consequent increase in the ceramide/diacylglycerol ratio. This effect is marked in proapoptotic cell nuclei and low in quiescent cell nuclei. In conclusion, UV-C radiation induces apoptosis, modifying nuclear lipid metabolism in relation to the physiological state of cells.

Antiphospholipid antibodies in patients with cancer.

Antiphospholipid antibodies are generally associated with Antiphospholipid Syndrome, which can occur as a primary disorder or may be secondary to connective tissue disease or tumour. The presence of antiphospholipid antibodies in patients with tumour disease is responsible for thrombotic complications. In a population of 53 tumor patients with positive carcinoembryonic antigen CEA, carbohydrate antigen CA19.9, CA125 and CA15.3 markers, IgM and IgG anticardiolipin and antiphosphatidylinositol were detected by solid-phase immunoassays. Our results show that moderate or high levels of antiphospholipid antibodies are present in a great number of patients with CEA and CA19.9 markers, suggesting a specific association with gastroenteric tumors. By testing for antiphosphatidylinositol antibodies, many patients not evidenced by the standard anticardiolipin assay were found to be antiphospholipid-positive. The analysis of antiphosphatidylinositol antibodies as a diagnostic tool in gastroenteric cancer to highlight patients with the risk of thromboembolic complications is discussed.

Low levels of serum cholesterol/phospholipids are associated with the antiphospholipid antibodies in monoclonal gammopathy.

A decrease in cholesterol blood level, not due to a decrease synthesis by the liver, has been observed in patients suffering from tumors. In this work cholesterol blood was evaluated in patients affected by monoclonal gammopathy who were not subjected to any treatment. The blood of 25 patients were analyzed for protein and lipid content. Patients were divided according to the gamma protein content into three groups, and it was demonstrated that the group with high levels of gamma proteins presented a strong decrease in blood cholesterol and phospholipids. In these patients the presence of antibodies against phospholipids by using cardiolipin and phosphatidylinositol as antigens has also been demonstrated. The antibodies were rare in patients with a low content of gamma proteins and normal level of lipids, but the frequency was more than 80% in patients with low blood lipid levels.

In Vitro Protective Effects of Lycium barbarum Berries Cultivated in Umbria (Italy) on Human Hepatocellular Carcinoma Cells.

Lycium barbarum is a famous plant in the traditional Chinese medicine. The plant is known to have health-promoting bioactive components. The properties of Lycium barbarum berries cultivated in Umbria (Italy) and their effect on human hepatocellular carcinoma cells (HepG2) have been investigated in this work. The obtained results demonstrated that the Lycium barbarum berries from Umbria region display high antioxidant properties evaluated by total phenolic content and ORAC method, on hydrophilic and lipophilic fractions. Moreover, on HepG2 cell line Lycium barbarum berries extract did not change cell viability analyzed by MTT and Trypan blue exclusion assay and did not induce genotoxic effect analyzed by comet assay. Furthermore, it was demonstrated, for the first time, that the berries extract showed a protective effect on DNA damage, expressed as antigenotoxic activity in vitro. Finally, Lycium barbarum berries extract was able to modulate the expression of genes involved in oxidative stress, proliferation, apoptosis, and cancer. In particular, downexpression of genes involved in tumor migration and invasion (CCL5), in increased risk of metastasis and antiapoptotic signal (DUSP1), and in carcinogenesis (GPx-3 and PTGS1), together with overexpression of tumor suppressor gene (MT3), suggested that Umbrian Lycium barbarum berries could play a protective role against hepatocellular carcinoma.

Sphingomyelin synthase in rat liver nuclear membrane and chromatin.

The presence of phospholipids in chromatin has been demonstrated, as well as the difference in composition and turnover compared to those present in the nuclear membrane. Recently, some enzymes were also evidenced in chromatin: the base exchange protein complex and neutral sphingomyelinase. The latter has a particular relevance, since sphingomyelin is one of the phospholipids more represented in chromatin. We therefore decided to study the synthesis of sphingomyelin in chromatin and in nuclear membrane isolated from liver nuclei. The evaluation of the enzyme was made (i) using [(3)H]phosphatidylcholine as donor of radioactive phosphorylcholine and (ii) by identifying the product isolated by thin layer chromatography. In both fractions the enzyme phosphatidylcholine:ceramide phosphocholine transferase or sphingomyelin synthase was present, although with higher activity in nuclear membrane. The enzyme present in the chromatin differs in pH optimum and K(m), showing a higher affinity for the substrates than that of nuclear membrane. The results presented show that sphingomyelin synthase is present not only in the cytoplasm at the level of the Golgi apparatus, but also in the nuclei, at the level of either the nuclear membrane or the chromatin.

Nuclear sphingomyelin protects RNA from RNase action.

Chromatin phospholipidic fraction, as previously demonstrated, shows the same localization as RNA inside the nuclei. DNase and RNase treatment of nuclei removed almost totally the DNA, 63% of RNA and caused a 50% loss of phospholipids. The aim of the present investigation is to study the fraction of RNase undigested nuclear RNA and its relationship with the phospholipids still present in the nuclei. Isolated hepatocyte nuclei were treated with Triton X-100 and digested with RNase and DNase. The undigested nuclear material contained proteins (98%) and a small amount of RNA (1.7%), DNA (0.4%) and phospholipids (0.18%). The analysis of phospholipids showed the presence of two components only, namely phosphatidylcholine and sphingomyelin. In the same complex, the activity of sphingomyelin synthase, phosphatidylcholine-dependent phospholipase C and neutral sphingomyelinase has been detected. Treatment of isolated RNA with neutral sphingomyelinase modified the RNA in RNase sensitive RNA, thus suggesting that the SM may represent a bridge between two RNA strands possibly regulating transcription.

Antiphosphatidylinositol antibody in deep venous thrombosis patients.

Antiphospholipid antibodies are a heterogeneous group of immunoglobulins with specificity for a number of phospholipids, phospholipid-binding proteins and phospholipid-protein complexes. The association between antiphospholipid antibodies and a variety of pathologic disorders, such as arterial and venous thrombosis and recurrent pregnancy loss is recognized as Antiphospholipid Syndrome. The immunoassay currently used to detect antiphospholipid antibodies is the anticardiolipin test. Anticardiolipin antibodies are believed to be polyspecific antibodies that cross-react with all the anionic phospholipids. Therefore, testing only for anticardiolipin antibodies does not always permit detection of all antiphospholipid antibodies, specially when only IgG are evaluated. In a selected population of 74 idiopathic and secondary deep venous thrombosis patients, IgG anticardiolipin, antiphosphatidylinositol and antiphosphatidylserine antibodies were detected by solid-phase immunoassays. Our results show that by testing for each antiphospholipid family, many patients, not evidenced by the standard anticardiolipin assay, were found to be antiphospholipid-positive. The anticardiolipin positive patients have always low, moderate or high levels of antiphospholipid antibodies, suggesting that the antiphospholipid positivity is predictive of anticardiolipin positivity. It should be noted that the patients with only antiphosphatidylinositol positive antibody have a story of nervous system pathology. The meaning of these results is at present under discussion.

Rat liver chromatin phospholipids.

To shed light on the question whether the phospholipids present in chromatin are native or are due to contamination from nuclear membranes, we labeled the phospholipids of isolated nuclei and determined the amount of phospholipids (PL) and PL fatty acid composition in nuclei and chromatin. The hepatocyte nuclei were isolated and radioiodinated by the lactoperoxidase method under saturating and nonsaturating conditions, and the radioactivity associated with chromatin extracted from these nuclei was monitored. Whereas 97% the label was recovered in the nuclear membranes, only 0.08-0.6% was found in chromatin. The PL present in chromatin were relative to the amounts present in the entire nuclei and calculated as percentage of total, phosphatidylethanolamine (10%), phosphatidylserine (22%), phosphatidylinositol (19%) phosphatidylcholine (14%), and sphingomyelin (35%). In sphingomyelin of chromatin-associated PL an enrichment in polyunsaturated fatty acids was seen. The data indicated that the PL found in isolated chromatin do not seem to be due to contamination from the nuclear membrane.

Reinterpretation of mouse thyroid changes under space conditions: the contribution of confinement to damage.

During space missions, astronauts work in a state of separation from their daily social environment and in physical confinement. It has been shown that confinement influences mood and brain cortical activity, but no data has been obtained with regard to its effect on the thyroid gland, the structure and function of which change during spaceflights. Here, we report the results of a study on the effects of confinement on mouse thyroid, which was implemented with the Mice Drawer System Facility maintained on the ground, a system used for spaceflight experiments. The results show that confinement changes the microscopic structure of the thyroid gland and that it exhibits symptoms similar to those that result from physiological and/or pathological hyperfunction. What is left unchanged, however, is the sphingomyelinase-thyrotropin receptor relationship, which is important for thyrotropin response with a consequential production of hormones that act on the metabolism of almost all tissues and reduces the production of calcitonin, a hormone involved in bone metabolism. During space missions, the overexpression of pleiotrophin, a widespread cytokine up-regulated after tissue injury that acts on bone remodeling, attenuates changes to the thyroid that are spaceflight-dependent; therefore we studied the thyroids of pleiotrophin-transgenic mice in the Mice Drawer System Facility. In confinement, pleiotrophin overexpression does not protect from the loss of calcitonin. The contribution of confinement to thyroid damage during spaceflights is discussed.

Observing the mouse thyroid sphingomyelin under space conditions: a case study from the MDS mission in comparison with hypergravity conditions.

This is a case report of apparent thyroid structural and functional alteration in a single mouse subjected to low Earth orbit spaceflight for 91 days. Histological examination of the thyroid gland revealed an increase in the average follicle size compared to that of three control animals and three animals exposed to hypergravity (2g) conditions. Immunoblotting analysis detected an increase in two thyroid gland enzymes, sphingomyelinase and sphingomyelin-synthase1. In addition, sphingomyelinase, an enzyme confined to the cell nucleus in the control animals, was found in the mouse exposed to hypogravity to be homogeneously distributed throughout the cell bodies. It represents the first animal observation of the influence of weightlessness on sphingomyelin metabolism.

Thyrotropin receptor and membrane interactions in FRTL-5 thyroid cell strain in microgravity.

The aim of this work was to analyze the possible alteration of thyrotropin (TSH) receptors in microgravity, which could explain the absence of thyroid cell proliferation in the space environment. Several forms of the TSH receptor are localized on the plasma membrane associated with caveolae and lipid rafts. The TSH regulates the fluidity of the cell membrane and the presence of its receptors in microdomains that are rich in sphingomyelin and cholesterol. TSH also stimulates cyclic adenosine monophosphate (cAMP) accumulation and cell proliferation. Reported here are the results of an experiment in which the FRTL-5 thyroid cell line was exposed to microgravity during the Texus-44 mission (launched February 7, 2008, from Kiruna, Sweden). When the parabolic flight brought the sounding rocket to an altitude of 264 km, the culture media were injected with or without TSH in the different samples, and weightlessness prevailed on board for 6 minutes and 19 seconds. Control experiments were performed, in parallel, in an onboard 1g centrifuge and on the ground in Kiruna laboratory. Cell morphology and function were analyzed. Results show that in microgravity conditions the cells do not respond to TSH treatment and present an irregular shape with condensed chromatin, a modification of the cell membrane with shedding of the TSH receptor in the culture medium, and an increase of sphingomyelin-synthase and Bax proteins. It is possible that real microgravity induces a rearrangement of specific sections of the cell membrane, which act as platforms for molecular receptors, thus influencing thyroid cell function in astronauts during space missions.

The role of intranuclear lipids.

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCss1 having a role in the onset of DNA synthesis and PC-PLCgamma1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an enhanced rate of phosphorylation has been demonstrated in cells induced to differentiate. These molecules probably favour RNA transcription, counteracting the inhibition of H1 on RNA polymerase II. Plasmalogens were demonstrated in the nucleus and their increase favours the increased activity of phosphatidylcholine-dependent phospholipase C when DNA synthesis starts. Moreover, two forms of cholesterol has been described in chromatin: one, a less soluble sphingomyelin-linked form and a free fraction. Cholesterol increases during liver regeneration, first as a linked fraction and then, when DNA synthesis starts, as a free fraction. The changes of these components have been summarised in relation to cell function in order to give an overview of their possible roles in the different phases of cell duplication and their influence on cell differentiation and during apoptosis. Finally, the relevance of these molecules as intranuclear signals is discussed and future directions are indicated in clarifying pathological process such as tumour cell transformation and the possibility in finding new therapeutic tools.

Chromatin neutral sphingomyelinase and its role in hepatic regeneration.

Ceramide acts as a second messenger and modulates many cellular functions. This molecule can be produced by enzymatic digestion of sphingomyelin, a phospholipid which has been shown to be in high concentration in a chromatin phospholipidic fraction. In order to clarify whether chromatin sphingomyelin has a role in this signal transduction pathway, it is necessary to define the sphingomyelin cycle. Neutral sphingomyelinase represents the first step of the cycle. In this paper we demonstrate that sphingomyelinase activity can be detected also in the chromatin of rat hepatocyte nuclei and it increases in relation to the entrance in S phase of hepatocyte after hepatectomy. The enzyme can be distinguished from that present in the nuclear envelope on the basis of optimum pH and Km. The role of the spingomyelin pathway in relation to liver regeneration is discussed.

Choline base exchange activity in rat hepatocyte nuclei and nuclear membranes.

Previous investigations have demonstrated the presence of phospholipids as a component of chromatin; however the mechanism of their synthesis, namely if they are synthesized in the nuclei or in the cytoplasm (microsomal fraction), from where they may eventually be transported to the nucleus, has not yet been clarified. The phosphatidylcholine, for example, can be formed, albeit in a limited amount, by an interconversion reaction between bases. The aim of the present research was to ascertain the presence of the enzyme complex responsible for this reaction in hepatocyte nuclei and in isolated nuclear membrane. The incorporation of [14C]-choline in phosphatidylcholine was assayed in microsomes, hepatocyte nuclei, liver nuclei and nuclear membranes of rat liver. The reaction was Ca(2+)-dependent and the specific activity was higher in microsomes but was present, albeit at a low level, also in nuclei and in nuclear membranes. Possible contaminations were excluded by specific microsomal markers and by the reaction time course. In fact, the nuclear reaction reached the maximum level slowly with respect to microsomes. Since the phosphatidylcholine extracted from the nuclei show an enrichment in unsaturated fatty acids of monoenoic fraction, such as oleic acid, the difference in reaction kinetics has been tentatively explained as due to the phosphatidylcholine fatty acid content. The presence of this base exchange enzyme complex may allow a fast change in chromatin phospholipid composition.

Chromatin-associated sphingomyelin: metabolism in relation to cell function.

After the first histochemical demonstration by Chayen and Gahan of the presence of phospholipids and especially of sphingomyelin in chromatin, this became the object of long debate and of contradictory results. The general conclusion was that the presence of phospholipids may due to contamination during the isolation of chromatin. More recently the existence of a phospholipid chromatin fraction was confirmed by demonstrating that isolated hepatocyte nuclei, labelled by saturated and unsaturated radioiodination method, showed the presence of radioactivity only in the membrane and not in the isolated chromatin. The phospholipid composition showed an enrichment in sphingomyelin which increased during hepatocyte maturation or erythroleukemic cell differentiation induced by DMSO. A decrease in sphingomyelin was observed at the beginning of the S-phase in regenerating liver or in cultured proliferating cells. These changes were due to the presence of sphingomyelinase and sphingomyelin synthase in the chromatin, the activity of which paralleled the variation in sphingomyelin content. The sphingomyelin was co-localized with RNA as shown by biochemical and electron microscopy methods. Using bromo-uridine it was demonstrated that labelled RNA and sphingomyelin were present in actively transcribing nuclear regions. Isolated nuclear complexes after DNase and RNase digestion contained not only protein, but also RNA and sphingomyelin. After hydrolysis of sphingomyelin the RNAse-resistant RNA becomes RNAse sensitive. It can therefore be concluded that sphingomyelin and the related enzymes are present in the chromatin; sphingomyelin may have a role in RNA transcription protecting RNA by RNAse digestion before its transfer to the cytoplasm.

Phosphatidylcholine-dependent phospholipase C in rat liver chromatin.

Phosphatidylcholine-dependent phospholipase C is an enzyme which hydrolyses phosphatidylcholine giving origin to diacylglicerol and phosphorylcholine. Diacylglicerol has many effect and activates also protein kinase C. Since the presence of protein kinase C in the hepatocyte nuclei and the existence of a phospholipidic fraction in the chromatin have been demonstrated, we investigated if phosphatidylcholine-dependent phospholipase C could be present in the nuclei. The results obtained have shown the presence of this enzyme in the chromatin fraction which differs with respect to that of nuclear membrane in pH and Km. The activity has been also evaluated during liver regeneration. In the chromatin an increase of activity has been shown 12 h and 30 h after hepatectomy, i.e. at the beginning of hepatocyte S-phase. No similar behaviour has been observed in the nuclear membrane. It has been suggested that diacylglicerol, produced by the hydrolysis of chromatin phosphatidylcholine, may have a role in initiating DNA synthesis through the prolonged activation of the nuclear form of protein kinase C.

Phospholipids and nuclear RNA.

It has been demonstrated that in hepatocyte nuclei the chromatin phospholipid fraction is localized near the RNA in decondensed chromatin. The aim of the present study was to see if there is any linkage between phospholipids and other nuclear components. Isolated hepatocyte nuclei and nuclear membranes were treated with deoxyribonuclease and ribonuclease. No loss of phospholipids was observed after DNA digestion, whereas 48% was lost following enzymatic RNA removal. This loss of phospholipids, localized either near the membrane or inside the nucleus, was not homogeneous for all phospholipids: phosphatidylserine and sphingomyelin being the most affected. It can be concluded that 48% of nuclear phospholipids, in particular sphingomyelin, is lost with RNA removal. This result is discussed in view of a possible role of phospholipids in DNA synthesis and RNA transcription.