Journal of Cytology & Histology

Cell-mediated immune responses play a central role in the control of infections and tumor growth. In particular, cytolytic T lymphocytes (CTL) and natural killer (NK) cells are fundamental effectors against virus-infected, tumor and leukemia cells. Both T and NK cells are particularly efficient also in allogeneic settings such as the allogeneic haemopoietic stem cell transplantation (HSCT) to cure hematologic malignancies. Another particularly promising approach of cellular therapy is the use of genetically-engineered autologous T cells with chimeric antigen receptors (CAR) conferring specificity for antigens expressed by tumor cells. Also NK cells can be genetically engineered with CAR. Different from CAR-T, NK cells, equipped with an array of receptors involved in tumor cell recognition and killing, retain their ability to target neoplastic cells through such receptors, possibly making tumor escape mechanisms less effective. In addition, they may be complementary to CAR-T cells. However, NK cell transfection resulted quite challenging. Thus, viral transduction display to have variable levels of transgene expression and may

compromise NK cell viability. Moreover, viral transduction requires dedicated facilities, high costs and lengthy preparation. Recently, electroporation of mRNA has been proposed as alternative of viral methods. Although the mRNA electroporation has a very low effect on the vitality and good efficacy, a relevant drawback are represented by the short-time expression of the transgene. Here we show a new procedure for NK cells transfection with plasmid DNA. With an efficiency of up to 50% and viability up to 65% it is the most efficient, non-viral, methodology existing so far to deliver exogenous DNA into NK cells. By applying this method, we transfected exogenous CCR7 chemokine receptor conferring to the NK cells the ability to efficiently migrate in response to the chemokines. Moreover, the introduction of an anti-CD19 CAR confer to transfected NK cells a specific and powerful cytotoxicity against CD19+ leukemic cells. These results illustrate some of potential important applications of this novel transfection approach. Notably, the electroporation of DNA may allow to a non-integrating gene transfer with episomal vectors.

The Crigler Najjar syndrome type I (CNSI) is a rare recessive disorder caused by mutations in the Ugt1a1 gene. There is no permanent cure except for liver transplantation, and current therapies present several shortcomings. Since stem cell-based therapy offers a promising alternative for the treatment of this disorder, we evaluated the therapeutic potential of a population of stem cells isolated from cryopreserved hepatocytes known as human liver stem cells (HLSC) in immune-compromised NOD SCID Gamma (NSG)/Ugt1-/- mice, which closely mimic the pathological manifestations in CNSI patients. In order to assess whether HLSC expressed UGT1A1, decellularised mouse liver scaffolds were repopulated with these cells. After 15 days' culture in this 3D setting ex vivo, HLSC differentiated into hepatocyte-like cells expressing markers such as albumin and cytochrome 1a1. For the in vivo human cell engraftment and recovery experiments in the Crigler-Najjar mouse model, NSG/Ugt1-/- mice were generated. A single dose of HLSC was injected in the liver parenchyma of 5 days old phototherapy-treated NSG/Ugt1-/- pups and HLSC functionality and phenotype rescue were assessed in vivo at post-natal Day 21. HLSC expressed UGT1A1 in vivo, induced a decrease in serum unconjugated bilirubin, and improved phenotype and survival compared to untreated controls. A significant reduction in eosinophilic neurons was also observed in HLSC-injected mutant mice hippocampus and cerebellum reflecting recovery from brain damage versus controls. Our results show that HLSC express UGT1A1 in vivo and improve the phenotype and survival of NSG/Ugt1-/- mice, and show promises for the treatment of CNSI.

Crigler-Najjar syndrome is a rare autosomal recessive inherited disorder characterized by the absence or decreased activity of UDP-glucuronosyl transferase, an enzyme required for glucuronidation of unconjugated bilirubin in the liver. It is one of the major causes of congenital non-hemolytic jaundice. The increased concentration of unconjugated bilirubin is the sole cause of disease manifestation. The disease severity depends upon the number of enzymes produced required for the glucuronidation of bilirubin. Newborns may present with hyperbilirubinemia, but other signs progressively develop later in life.

Crigler-Najjar syndrome is of two types based on the clinical criteria such as molecular and functional features, the severity of clinical presentation, and phenobarbitol response. Type I is the most severe form with an almost complete absence of UDP-glucuronosyltransferase enzyme activity, whereas type II is less severe with a reduced level of enzyme activity. Central nervous system involvement complicated by kernicterus is seen mainly in Crigler-Najjar type I.

Cellular based therapeutic approaches for cancer rely on careful consideration of finding the optimal cell to execute the cellular goal of cancer treatment. Cell lines and primary cell cultures have been used in some studies to compare the in vitro and in vivo efficacy of autologous vs. allogeneic tumour cell vaccines. This study examines the effect of -irradiation on a range of tumor cell lines in conjunction with suicide gene therapy of cancer. To determine the efficacy of this modality, a series of in vitro and in vivo experiments were conducted using genetically modified and unmodified tumor cell lines. Following co-culture of herpes simplex virus thymidine kinase (HSV-TK) modified tumor cells and unmodified tumor cells both in vitro and in vivo we observed that the PA-STK ovarian tumor cells were sensitive to -irradiation, completely abolishing their ability to induce bystander killing of unmodified tumor cells. In contrast, TK-modified human and mouse mesothelioma cells were found to retain their in vitro and in vivo bystander killing effect after -irradiation. Characterisation of tumor cell death showed that PA-STK cells underwent pyknosis (necrosis) after -irradiation. These results suggest that PA-STK cells are not suitable for clinical application of suicide gene therapy of cancer, as lethal -irradiation (100Gy) interferes with their bystander killing activity. However, the human mesothelioma cell line CRL-5830-TK retained its bystander killing potential after exposure to similarly lethal -irradiation (100Gy). CRL-5830 may therefore be a suitable vehicle for HSV-TK suicide gene therapy. This study highlights the diversity among tumor cell lines and the careful considerations needed to find the optimal tumor cell line for this type of suicide gene therapy of cancer.

The focal target in disease treatment is to slaughter the dangerous cells while making close to nothing or ideally no inadvertent blow-back solid cells. Self-destruction quality treatment, as applied to the therapy of malignancy, holds the possibility to accomplish only that. A model is the addition of the herpes simplex infection thymidine kinase (HSV-TK) quality into disease cells which are therefore initiated to "end it all" when within the sight of in any case non-poisonous portions of ganciclovir (GCV). This specific harmful impact of the purine simple ganciclovir is on the grounds that HSV-TK phosphorylates ganciclovir, changing over it at last to ganciclovir-triphosphate, a poisonous compound when embedded into the DNA of these transfected cells. Similarly as with some other quality treatment procedure, and any enemy of malignancy treatment approach, its principle constraint is the particular focusing on and transduction of all tumor cells in vivo. In any case, it may not be important to transduce each tumor cell in vivo to achieve a clinically-significant enemy of tumor impact. For sure, it has been exhibited that two kinds of "onlooker tumor cell murdering" components are interceded by this methodology: (a) a "immediate" spectator impact, because of the exchange of ganciclovir triphosphate from HSV-TK-positive tumor cells into untransfected adjoining cells, (b) a fundamental immunologically-intervened observer impact because of the in vivo safe introduction of tumor-explicit/related antigens following the slaughtering of HSV-TK—communicating cells.  

The hereditarily adjusted HSV-TK human ovarian disease cell line, PA1-STK, has been utilized for the therapy of strong tumors (directed intraperitoneally in patients with ovarian malignant growth). The in vitro culture of these cells within the sight of ganciclovir incited onlooker slaughtering, yet with restricted cytotoxic action in vivo. The reasoning for this technique was that PA-STK cells, infused in the region of the patient's tumor mass, could connect with, and seed themselves onto, the patient's tumor cells in vivo and, after treatment with ganciclovir, could end it all and slaughter the patient's tumor cells by a "immediate" spectator system (for example hole intersection interceded move of the phosphorylated ganciclovir from the HSV-TK positive cells to the TK negative cells. This immediate cytotoxicity could then initiate a more foundational immunological onlooker tumor-executing impact.