New targets of nascent lymphatic vessels in ocular diseases.

Recent advancements in the field of endothelial markers of lymphatic vessels and lymphangiogenic factors have shed light on the association between several ocular diseases and ocular nascent lymphatic vessels. The immune privilege of corneal tissue typically limits the formation of lymphatic vessels in a healthy eye. However, vessels in the eyes can potentially undergo lymphangiogenesis and be conditionally activated. It is evident that nascent lymphatic vessels in the eyes contribute to various ocular pathologies. Conversely, lymphatic vessels are present in the corneal limbus, ciliary body, lacrimal glands, optic nerve sheaths, and extraocular muscles, while a lymphatic vasculature-like system exists in the choroid, that can potentially cause several ocular pathologies. Moreover, numerous studies indicate that many ocular diseases can influence or activate nascent lymphatic vessels, ultimately affecting patient prognosis. By understanding the mechanisms underlying the onset, development, and regression of ocular nascent lymphatic vessels, as well as exploring related research on ocular diseases, this article aims to offer novel perspectives for the treatment of such conditions.

Preparation of highly activated natural killer cells for advanced lung cancer therapy.

Background: Natural killer (NK) cells can be used as an adoptive immunotherapy to treat cancer patients. Purpose: In this study, we evaluated the efficacy of highly activated NK (HANK) cell immunotherapy in patients with advanced lung cancer. Patients and methods: Between March 2016 and September 2017, we enrolled 13 patients who met the enrollment criteria. Donor peripheral blood monocytes were isolated from patients and the NK cells were expanded. After 12 days of culture, the cells were collected and infused intravenously on days 13 to 15. The enrolled patients received at least one course including three times of infusions. The lymphocyte subsets, cytokine production, and the expression of carcinoembryonic antigen (CEA) and thymidine kinase 1 (TK1) were measured before treatment and after the last infusion. Results: No side effects were observed. After a three-month follow-up, the percentage of patients who achieved stable disease and progressive disease was 84.6% and 15.4%. Moreover, the level of IFN-γ was significantly higher after treatment and the level of CEA decreased substantially. The overall immune function of the patients who received the NK cell therapy remained stable. Conclusion: This is the first study to describe the efficacy of NK cell therapy of patients with advanced lung cancer. These clinical observations demonstrated that NK cell is safe and efficient for advanced lung cancer therapy.

Chemical induction of endogenous retrovirus particles from the vero cell line of African green monkeys.

Endogenous retroviral sequences are present in high copy numbers in the genomes of all species and may be expressed as RNAs; however, the majority are defective for virus production. Although virus has been isolated from various Old World monkey and New World monkey species, there has been no report of endogenous retroviruses produced from African green monkey (AGM) tissues or cell lines. We have recently developed a stepwise approach for evaluating the presence of latent viruses by chemical induction (Khan et al., Biologicals 37:196-201, 2009). Based upon this strategy, optimum conditions were determined for investigating the presence of inducible, endogenous retroviruses in the AGM-derived Vero cell line. Low-level reverse transcriptase activity was produced with 5-azacytidine (AzaC) and with 5'-iodo-2'-deoxyuridine (IUdR); none was detected with sodium butyrate. Nucleotide sequence analysis of PCR-amplified fragments from the gag, pol, and env regions of RNAs, prepared from ultracentrifuged pellets of filtered supernatants, indicated that endogenous retrovirus particles related to simian endogenous type D betaretrovirus (SERV) sequences and baboon endogenous virus type C gammaretrovirus (BaEV) sequences were induced by AzaC, whereas SERV sequences were also induced by IUdR. Additionally, sequence heterogeneity was seen in the RNAs of SERV- and BaEV-related particles. Infectivity analysis of drug-treated AGM Vero cells showed no virus replication in cell lines known to be susceptible to type D simian retroviruses (SRVs) and to BaEV. The results indicated that multiple, inducible endogenous retrovirus loci are present in the AGM genome that can encode noninfectious, viruslike particles.

Optimization of chemical induction conditions for human herpesvirus 8 (HHV-8) reactivation with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from latently-infected BC-3 cells.

Human herpesvirus 8 (HHV-8) persists as episomal DNA in latently-infected cells and can establish two alternative life cycles, latent or lytic. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is a known inducer of HHV-8 in several human primary effusion lymphoma cell lines and has been widely used for HHV-8 reactivation; however, induction conditions have differed, resulting in varying levels of virus expression. We have used HHV-8 latently-infected BC-3 cells as a model to determine critical parameters for optimizing virus reactivation by TPA. We found that cell growth properties and drug treatment conditions were important for maximum reactivation of HHV-8. Addition of TPA to cells in the early log phase of a sigmoidal growth curve, which was tightly associated with high percentage of the cells in early S phase and with lower histone deacetylase activity in the cells, provided the optimum cell conditions for latent virus to switch to lytic replication. Furthermore, increasing TPA concentration (up to 320 ng per ml) at 48 h exposure time resulted in increased virus production. The results demonstrate the use of a step-wise strategy with chemical induction that may facilitate broad detection of latent DNA viruses and novel virus discovery.

Proposed algorithm to investigate latent and occult viruses in vaccine cell substrates by chemical induction.

The recent urgency to develop new vaccines for emerging and re-emerging diseases, such as pandemic influenza, has necessitated the use of cell substrates not previously used in the manufacture of licensed vaccines. A major safety concern in the use of novel cell substrates is the presence of potential adventitious agents, such as latent and occult viruses, that may not be detected by currently used conventional assays. In cases where the novel cell substrate is known to be tumorigenic, there are additional safety issues related to tumorigenicity of intact cells and oncogenicity of residual cellular DNA. We have developed a strategy for evaluating vaccine cell substrates for the presence of latent/occult viruses, including endogenous retroviruses, latent RNA viruses and oncogenic DNA viruses, by optimizing conditions for chemical induction of viruses and using a combination of broad and specific assays to enable detection of known and novel viruses.