Erratum to: Phagocytic Activity of Rat Primary Astrocytes Is Regulated by Insulin and Ganglioside GM1

Journal of Evolutionary Biochemistry and Physiology -     

Dimeric transferrin inhibits phagocytosis of residual bodies by testicular rat Sertoli cells

Transferrin is well known as an iron transport glycoprotein. Dimeric or tetrameric transferrin forms have recently been reported to modulate phagocytosis by human leukocytes. It is mainly synthesized by the liver, and also by other sources, such as Sertoli cells of the testis. Sertoli cells show a strong phagocytic activity toward apoptotic germ cells and residual bodies. Here, we provide evidence that purified human dimeric transferrin from commercial sources decreased residual body phagocytosis, unlike monomeric transferrin. The presence of iron appeared essential for dimeric transferrin inhibitory activity. Importantly, dimeric transferrin could be visualized by immunoblotting in Sertoli cell lysates as well as in culture media, indicating that dimeric transferrin could be physiologically secreted by Sertoli cells. By siRNA-mediated knockdown, we show that endogenous transferrin significantly inhibited residual body ingestion by Sertoli cells. These results are the first to identify dimeric transferrin in Sertoli cells and to demonstrate its implication as a physiological modulator of residual body phagocytosis by Sertoli cells.     

Recombinant MHC tetramers for isolation of virus-specific CD8<Superscript>+</Superscript> cells from healthy donors: Potential approach for cell therapy of posttransplant cytomegalovirus infection

Patients undergoing allogeneic hematopoietic stem cell transplantation have a high risk of cytomegalovirus reactivation, which in the absence of T-cell immunity can result in the development of an acute inflammatory reaction and damage of internal organs. Transfusion of the virus-specific donor T-lymphocytes represents an alternative to a highly toxic and often ineffective antiviral therapy. Potentially promising cell therapy approach comprises transfusion of cytotoxic T-lymphocytes, specific to the viral antigens, immediately after their isolation from the donor’s blood circulation without any in vitro expansion. Specific T-cells could be separated from potentially alloreactive lymphocytes using recombinant major histocompatibility complex (MHC) multimers, carrying synthetic viral peptides. Rapid transfusion of virus-specific T-cells to patients has several crucial advantages in comparison with methods based on the in vitro expansion of the cells. About 30% of hematopoietic stem cell donors and 46% of transplant recipients at the National Research Center for Hematology were carriers of the HLA-A*02 allele. Moreover, 94% of Russian donors have an immune response against the cytomegalovirus (CMV). Using recombinant HLA-A*02 multimers carrying an immunodominant cytomegalovirus peptide (NLV), we have shown that the majority of healthy donors have pronounced T-cell immunity against this antigen, whereas shortly after the transplantation the patients do not have specific T-lymphocytes. The donor cells have the immune phenotype of memory cells and can be activated and proliferate after stimulation with the specific antigen. Donor lymphocytes can be substantially enriched to significant purity by magnetic separation with recombinant MHC multimers and are not activated upon cocultivation with the antigen-presenting cells from HLA-incompatible donors without addition of the specific antigen. This study demonstrated that strong immune response to CMV of healthy donors and prevalence of HLA-A*02 allele in the Russian population make it possible to isolate a significant number of virus-specific cells using HLA-A*02–NLV multimers. After the transfusion, these cells should protect patients from CMV without development of allogeneic immune response.     

Evaluation of the genome stability and mitochondrial potential of tumor cells in vivo under the action of nanoferromagnetics with different sizes of nanoparticles

The transmembrane potential and genome stability of tumor cells under nanoferromagnetic action with different sizes of nanoparticles were studied in vivo. An evaluation of the genotoxic influence of nanoeferromagnetics on tumor cells and changes in their mitochondrial potential was carried out with the application of a micronuclei test and mitochondrial fluorescent probe JC-1. Nanoferromagnetics with nanoparticle diameters of 40 and 100 nm at a concentration of 3 mg/kg were not found to deminish the trasmembrane mitochondrial potential and the genome of tumor cells and increase the micronucleus frequency of tumor cells to only a small extent. Iron nanoparticles at a concentration of 6 mg/kg were noted to demonstrate an antidromonous character of the changing mitochondrial potential and micronuclei frequency depending on their size. Iron nanoparticles with a smaller diameter were found to provoke an essential rise in the transmembrane potential of tumor cell metachondria on the background of a constant number of micronuclei, and the opposite was true for those with a greater diameter. The data obtained can serve as an important criterion for further studies in experimental oncology to create an optimal system for delivering anticancer preparations to tissue targets.     

A chimerical phagocytosis model reveals the recruitment by Sertoli cells of autophagy for the degradation of ingested illegitimate substrates

Phagocytosis and autophagy are typically dedicated to degradation of substrates of extrinsic and intrinsic origins respectively. Although overlaps between phagocytosis and autophagy were reported, the use of autophagy for ingested substrate degradation by nonprofessional phagocytes has not been described. Blood-separated tissues use their tissue-specific nonprofessional phagocytes for homeostatic phagocytosis. In the testis, Sertoli cells phagocytose spermatid residual bodies produced during germ cell differentiation. In the retina, pigmented epithelium phagocytoses shed photoreceptor tips produced during photoreceptor renewal. Spermatid residual bodies and shed photoreceptor tips are phosphatidylserine-exposing substrates. Activation of the tyrosine kinase receptor MERTK, which is implicated in phagocytosis of phosphatidylserine-exposing substrates, is a common feature of Sertoli and retinal pigmented epithelial cell phagocytosis. The major aim of our study was to investigate to what extent phagocytosis by Sertoli cells may be tissue specific. We analyzed in Sertoli cell cultures that were exposed to either spermatid residual bodies (legitimate substrates) or retina photoreceptor outer segments (illegitimate substrates) the course of the main phagocytosis stages. We show that whereas substrate binding and ingestion stages occur similarly for legitimate or illegitimate substrates, the degradation of illegitimate but not of legitimate substrates triggers autophagy as evidenced by the formation of double-membrane wrapping, MAP1LC3A-II/LC3-II clustering, SQSTM1/p62 degradation, and by marked changes in ATG5, ATG9 and BECN1/Beclin 1 protein expression profiles. The recruitment by nonprofessional phagocytes of autophagy for the degradation of ingested cell-derived substrates is a novel feature that may be of major importance for fundamentals of both apoptotic substrate clearance and tissue homeostasis.     

Distribution of transferrin and transferrin receptor of the eype 1 in the process of formation of the rat eye retina in early postnatal ontogenesis

By the method of indirect immunohistochemistry, distribution of transferrin and of transferrin receptor of the type 1 (TFR1) was studied in the formed rat eye retina at the period of early postnatal ontogenesis (from birth to opening of eyelids). It has been established that the character of distribution of these proteins and intensity of specific staining change dependent on the retina formation stage. Retina of the newborn rat is characterized by diffuse transferrin distribution in nuclear retina layer (in the neuroblast layer-NBL) and in the ganglionic cell layer (GCL) as well as in the eye pigment epithelium (PE); relative immunoreactivity to transferrin is not high. At the 5th postnatal day, immunoreactivity to transferrin is maximal and is revealed both in nuclear and in plexiform layers of retina and in the eye PE, the greatest signal being characteristic of NBL. At the 10th postnatal day the transferrin signal intensity in retina decreases, specific staining is revealed in GCL, PE, and in the area of formed outer segments of photoreceptors. At the 15th postnatal day, transferrin is revealed in GCL, in outer and inner photoreceptor segments and in the eye PE. TFR1 is present in all retina layers at all stages of the retina formation; the relative immunoreactivity to TFR1 sharply rises beginning from the 10th postnatal day; correlation between distribution of transferrin and TFR1 is detected in the entire retina of newborn rats as well as in the external retina area at subsequent stages of its development. A possible role of transferrin at various stages of formation of retina is discussed.     

Uterine myometrial ferritin and blood plasma transferrin as natural modulators of Ca-calmodulin-independent cyclic nucleotide phosphodiesterase from cow uterine myometrium

The iron storage protein ferritin was purified to electrophoretic homogeneity from cow uterine myometrium. Its Mr did not exceed 440, 000 and H-chains predominated in the subunit composition; the iron saturation was 43 iron ions per protein molecule. The uterine myometrial ferritin was a potent natural modulator of Ca-calmodulin-independent phosphodiesterase (Ca-CM-independent PDE, EC 3.1.4.17) isolated from the same tissue. Addition of iron-poor ferritin from uterine myometrium and iron-reach liver ferritin caused three- and two-fold inhibition of the enzyme activity, respectively. The iron transport protein transferrin in iron-saturated and iron-depleted forms can also inhibit Ca-CM-independent PDE activity by two-fold. In both cases, the degree of saturation with iron was not crucial for the inhibitory effects of these proteins on the enzyme activity. These data suggest that iron homeostasis proteins can modulate the cyclic nucleotide level in non-nervous tissue via interaction with enzymes involved in cyclic nucleotide hydrolysis.     

Early and delayed changes in potassium transport during the initiation of cell proliferation in CHO culture

Stimulation by serum of cell proliferation in G1-arrested culture of Chinese hamster ovary cells CHO-K1 was accompanied by an early (during the first minutes) and delayed (2-10 h) activation of Na+,K+-ATPase and an increase in cell K+ content from 0.5-0.6 to 0.7-0.8 mmol per gram protein. Isoproterenol acted synergistically with serum in eliciting both early and delayed changes in K+ transport and in stimulating G1----S transition. Isoproterenol alone (without serum) induced a transient increase in K+ influx via Na+,K+-ATPase without changing the cell K+ content or having any mitogenic effect. Theophylline enhanced the serum-induced early activation of Na+,K+-ATPase but inhibited both the delayed increase in cell K+ and the G1----S transition. Early serum-induced increase in K+ transport was not affected by cycloheximide, whereas net accumulation of cell K+ was abolished by the drug. It is concluded that the early and the delayed activation of Na+,K+-ATPase induced by mitogens can be dissociated; the early ionic response is related to the primary transduction of membrane signal, whereas the delayed modulation of ion transport via Na+,K+-ATPase has another function and is associated with cell growth.     

Morphological Study of the Encystment and Excystment of Vermamoeba vermiformis Revealed Original Traits

Free‐living amoebae are ubiquitous protozoa commonly found in water. Among them, Acanthamoeba and Vermamoeba (formerly Hartmannella) are the most represented genera. In case of stress, such as nutrient deprivation or osmotic stress, these amoebae initiate a differentiation process, named encystment. It leads to the cyst form, which is a resistant form enabling amoebae to survive in harsh conditions and resist disinfection treatments. Encystment has been thoroughly described in Acanthamoeba but poorly in Vermamoeba. Our study was aimed to follow the encystment/excystment processes by microscopic observations. We show that encystment is quite rapid, as mature cysts were obtained in 9 h, and that cyst wall is composed of two layers. A video shows that a locomotive form is likely involved in clustering cysts together during encystment. As for Acanthamoeba, autophagy is likely active during this process. Specific vesicles, possibly involved in ribophagy, were observed within the cytoplasm. Remarkably, mitochondria rearranged around the nucleus within the cyst, suggesting high needs in energy. Unlike Acanthamoeba and Naegleria, no ostioles were observed in the cyst wall suggesting that excystment is original. During excystment, large vesicles, likely filled with hydrolases, were found in close proximity to cyst wall and digest it. Trophozoite moves inside its cyst wall before exiting during excystment. In conclusion, Vermamoeba encystment/excystment displays original trends as compare to Acanthamoeba.     

Distribution of Proteins Providing Homeostasis of Iron Ions in Bovine Retina

Distribution of proteins providing homeostasis of iron ions in bovine retina was studied by methods of indirect immunohistochemistry, which allowed detection of localization of transferrin, ferritin, and transferrin receptor. In bovine retina, transferrin is revealed in the region of outer and inner segments of photoreceptors and in the external plexiform layer. Distributions of ferritin and transferrin receptor are identical; they are revealed in all layers of retina, the maximal immunoreactivity against these proteins is found in pigment epithelium, in the region of inner segments of photoreceptors, in the external plexiform and internal nuclear layers. The obtained results are discussed from the point of view of mechanisms providing with iron the cells of the outer and inner retina.