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Osteoclasts (OCs) and much less dendritic cells (DCs) induce significant expansion and functional activation of NK cells, and furthermore, the OC-expanded NK cells preferentially increase the expansion and activation of CD8+ T cells by targeting CD4+ T cells. When autologous OCs were used to expand patient NK cells much lower percentages of expanded CD8+ T cells, decreased numbers of expanded NK cells and decreased functions of NK cells could be observed, and the addition of allogeneic healthy OCs increased the patients’ NK function. Mechanistically, OC-expanded NK cells were found to lyse CD4+ T cells but not CD8+ T cells suggesting potential selection of CD8+ T cells before their expansion by OC activated NK cells. In agreement, Increased IFN-γ secretion, and NK cell-mediated cytotoxicity and higher percentages of CD8+ T cells, in various tissue compartments of oral tumor-bearing hu-BLT mice in response to immunotherapy by OC-expanded NK cells were observed. Thus, our results indicate an important relationship between NK and CD8+ T cells.

Background The cholinesterase inhibitor therapeutics (CI) approved for use in Alzheimer's disease (AD) are palliative for a limited time. Objective To examine the outcome of AD patients with add-on therapy of the omega-3 fatty acid drink Smartfish. Methods We performed a prospective study using Mini-Mental State Examination, amyloid-β (Aβ) phagocytosis blood assay, and RNA-seq of peripheral blood mononuclear cells in 28 neurodegenerative patients who had failed their therapies, including 8 subjective cognitive impairment (SCI), 8 mild cognitive impairment (MCI), 2 AD dementia, 1 frontotemporal dementia (FTD), 2 vascular cognitive impairment, and 3 dementia with Lewy bodies (DLB) patients. Results MCI, FTD, and DLB patients patients volunteered for the addition of a ω-3 fatty acid drink Smartfish protected by anti-oxidants to failing CI therapy. On this therapy, all MCI patients improved in the first year energy transcripts, Aβ phagocytosis, cognition, and activities of daily living; in the long term, they remained in MCI status two to 4.5 years. All FTD and DLB patients rapidly progressed to dementia. On in vivo or in vitroω-3 treatments, peripheral blood mononuclear cells of MCI patients upregulated energy enzymes for glycolysis and citric acid cycle, as well as the anti-inflammatory circadian genes CLOCK and ARNTL2. Conclusion Add-on ω-3 therapy to CI may delay dementia in certain patients who had failed single CI therapy.

Natural killer (NK) cells are the key immune effectors with the ability to mediate selection and differentiation of a number of different cancer stem cells/undifferentiated tumors via lysis, and secreted or membrane-bound interferon (IFN)-γ and tumor necrosis factor (TNF)-α, respectively, leading to curtailment of tumor growth and metastasis. In this review, we present an overview of our recent findings on the biology and significance of NK cells in selection and differentiation of stem-like tumors using in vitro and in vivo studies conducted in humanized-BLT mice and in cancer patients. In addition, we present current advances in NK cell expansion and therapeutic delivery, and discuss the utility of allogeneic supercharged NK cells in the treatment of cancer patients. Moreover, we discuss the potential loss of NK cell numbers and function at the neoplastic and pre-neoplastic stages of tumorigenesis in induction and progression of pancreatic cancer. Therefore, because of their indispensable role in targeting cancer stem-like/undifferentiated tumors, NK cells should be placed high in the armamentarium of tumor immunotherapy. A combination of allogeneic supercharged NK cells with other immunotherapeutic strategies such as oncolytic viruses, antibody-dependent cellular cytotoxicity (ADCC)-inducing antibodies, checkpoint inhibitors, chimeric antigen receptor (CAR) T cells, CAR NK cells, and chemotherapeutic and radiotherapeutic strategies can be used for the ultimate goal of tumor eradication. Keywords: NK cells, cancer stem cells, CSCs, differentiation, cancer immunotherapy, osteoclasts, supercharged NK cells, humanized-BLT mice, pre-neoplastic, KRAS

We investigated the use of a system with an ingestible sensor (Proteus Digital Health Feedback system) coencapsulated with antiretrovirals (ARVs) to measure real-time adherence. To assess the safety and impact, if any, coencapsulation might have on ARV concentrations, we evaluated the pharmacokinetics of ARVs coencapsulated with an ingestible sensor for eight commonly used fixed-dose combination ARVs: emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF); FTC/tenofovir alafenamide (TAF); efavirenz (EFV)/FTC/TDF; abacavir (ABC)/lamivudine (3TC); dolutegravir (DTG)/ABC/3TC; rilpivirine (RPV)/TAF/FTC; elvitegravir (EVG)/cobicistat (COBI)/FTC/TAF; and bictegravir (BIC)/FTC/TAF. The steady-state apparent peak plasma concentration (Cmax) and area under the concentration-time curve (AUC) were determined from plasma concentrations measured at predose, 1, 2, 4, and 6 h postdose, and compared with literature values. A total of 49 unique patients on stable regimens for at least 12 weeks with undetectable viral loads were recruited. Cmax and AUC values were not statistically significantly different from literature values for all of the formulations except the Cmax of FTC/TDF, Cmax of BIC, and the Cmax of RPV. In a subsequent evaluation of FTC/TDF and BIC/FTC/TAF using a crossover design, the geometric mean ratio (GMR) between the coencapsulated and the unencapsulated formulations for FTC/TDF were the following: FTC, 84.6% (90% confidence interval [CI] 66.6–107.4) for AUC and 77.5% (60.1–99.9) for Cmax. For tenofovir (TFV), the GMR was 96.2% (90% CI 89.2–103.8) for AUC and 87.3% (64.2–118.7) for Cmax. The GMR for BIC (from the BIC/FTC/TAF formulation) was 98.0% (90% CI 84.5–113.5) for AUC and 89.9% (84.5–95.7) for Cmax. The observed deviation in FTC/TDF (Truvada) may be due to participant characteristics, fasted/fed conditions, and/or random variation and may warrant further investigations with a larger sample size. These findings provide assurance for use of coencapsulated ARVs for future HIV treatment-adherence research.

We have recently shown that natural killer (NK) cells select and differentiate cancer stem cells (CSCs)/undifferentiated tumors via secreted and membrane bound IFN-gamma (IFN-γ) and TNF-alpha (TNF-α), preventing tumor growth and inducing remodeling of the tumor microenvironment. Since many conventional therapeutic strategies, including chemotherapy and radiotherapy remain fairly unsuccessful in treating CSCs/poorly differentiated tumors, there has been an increasing interest in NK cell-targeted immunotherapy for the treatment of aggressive tumors. In our recent studies, we used humanized-BLT (hu-BLT) mouse model with transplanted human bone marrow, liver and thymus to demonstrate the efficacy of adoptive transfer of ex vivo expanded, super-charged NK cells in selection and differentiation of stem-like tumors within the context of a fully reconstituted human immune system. Furthermore, we have demonstrated that CSCs differentiated with split-anergized NK cells prior to implantation in hu-BLT mice were not able to grow or metastasize. However, when NK cell-mediated tumor differentiation was blocked by the addition of antibodies to IFN-γ and TNF-α, tumors grew and metastasized. In this review, we present current advances in NK cell expansion and therapeutic delivery, and discuss the utility of allogeneic super-charged NK cells in treatment of cancer patients. In addition, NK suppression occurs not only at the stage of overt cancer, but also at the pre-neoplastic stage. Therefore, due to the indispensable role of NK cells in targeting CSCs/undifferentiated tumors and their role in differentiation of the tumors, NK cells should be placed high in the armamentarium of tumor immunotherapy.

Simple Summary Natural Killer cells are known to eliminate tumors directly or via antibody dependent cellular cytotoxicity. The complete modes and mechanisms of such killings are yet to be delineated. It is also unclear at what stages of tumor differentiation NK cells are capable of mediating the two modes of tumor killing. In this report we provide evidence that NK cells mediate killing of both stem-like/poorly differentiated tumors and well-differentiated tumors via direct cytotoxicity and antibody dependent cellular cytotoxicity, respectively. By using antibodies to MICA/B, EGFR and PDL1 surface receptors expressed on well-differentiated but not on stem-like/poorly differentiated tumors we demonstrate significant NK cell mediated antibody dependent cellular cytotoxicity in the absence of direct killing. In addition, our results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and antibody dependent cellular cytotoxicity, resulting in the competitive use of these receptors in either direct killing or antibody dependent cellular cytotoxicity. Abstract Tumor cells are known to upregulate major histocompatibility complex-class I chain related proteins A and B (MICA/B) expression under stress conditions or due to radiation exposure. However, it is not clear whether there are specific stages of cellular maturation in which these ligands are upregulated or whether the natural killer (NK) cells differentially target these tumors in direct cytotoxicity or antibody-dependent cell cytotoxicity (ADCC). We used freshly isolated primary and osteoclast (OCs)-expanded NK cells to determine the degree of direct cytotoxicity or of ADCC using anti-MICA/B monoclonal antibodies (mAbs) against oral stem-like/poorly-differentiated oral squamous cancer stem cells (OSCSCs) and Mia PaCa-2 (MP2) pancreatic tumors as well as their well-differentiated counterparts: namely, oral squamous carcinoma cells (OSCCs) and pancreatic PL12 tumors. By using phenotypic and functional analysis, we demonstrated that OSCSCs and MP2 tumors were primary targets of direct cytotoxicity by freshly isolated NK cells and not by ADCC mediated by anti-MICA/B mAbs, which was likely due to the lower surface expression of MICA/B. However, the inverse was seen when their MICA/B-expressing differentiated counterparts, OSCCs and PL12 tumors, were used in direct cytotoxicity and ADCC, in which there was lower direct cytotoxicity but higher ADCC mediated by the NK cells. Differentiation of the OSCSCs and MP2 tumors by NK cell-supernatants abolished the direct killing of these tumors by the NK cells while enhancing NK cell-mediated ADCC due to the increased expression of MICA/B on the surface of these tumors. We further report that both direct killing and ADCC against MICA/B expressing tumors were significantly diminished by cancer patients’ NK cells. Surprisingly, OC-expanded NK cells, unlike primary interleukin-2 (IL-2) activated NK cells, were found to kill OSCCs and PL12 tumors, and under these conditions, we did not observe significant ADCC using anti-MICA/B mAbs, even though the tumors expressed a higher surface expression of MICA/B. In addition, differentiated tumor cells also expressed higher levels of surface epidermal growth factor receptor (EGFR) and programmed death-ligand 1(PDL1) and were more susceptible to NK cell-mediated ADCC in the presence of anti-EGFR and anti-PDL1 mAbs compared to their stem-like/poorly differentiated counterparts. Overall, these results suggested the possibility of CD16 receptors mediating both direct cytotoxicity and ADCC, resulting in the competitive use of these receptors in either direct killing or ADCC, depending on the differentiation status of tumor cells and the stage of maturation and activation of NK cells.

Chronic lymphocytic leukemia (CLL) accounts for 10% of hematologic malignancies. CLL is a malignancy of CD5+ B cells and it is characterized by the accumulation of small, mature-appearing neoplastic lymphocytes in the blood, bone marrow, and secondary lymphoid tissues. In the present case, a middle-aged female patient with poor prognosis unmutated IGHV CLL achieved cytogenetic and molecular remission with minimal adverse events following six cycles of low dose recombinant human IL-2 (rIL-2) in combination with low dose targeted venetoclax. Personalized low dose rIL-2 in combination with either lenalidomide or venetoclax mediates natural killer stimulation and is an effective non-toxic immunotherapy administered in the outpatient setting for poor prognosis CLL.

Natural killer (NK) cells are known to select and differentiate cancer stem-like cells/undifferentiated tumors via lysis, and secreted/membrane bound IFN-γ and TNF-α respectively, resulting in the control of tumor growth. Several in vivo mouse models including humanized-BLT mice have been used to study the biology and significance of NK cells in selection/differentiation of stem-like tumors within the context of a reconstituted human immune system. In addition, we discuss the evidence and significance of NK cell loss at the pre-neoplastic stage. Therefore, because of their indispensable role in targeting CSCs/undifferentiated tumors, NK-cells should be placed high in the armamentarium of tumor therapy.

Background and Aims: We have previously demonstrated that the stage of differentiation of tumors has profound effect on the function of NK cells, and that stem-like/poorly differentiated tumors were preferentially targeted by the NK cells. Therefore, in this study we determined the role of super-charged NK cells in immune mobilization, lysis, and differentiation of stem-like/undifferentiated tumors implanted in the pancreas of humanized-BLT (hu-BLT) mice fed with or without AJ2 probiotics. The phenotype, growth rate and metastatic potential of pancreatic tumors differentiated by the NK cells (NK-differentiated) or patient derived differentiated or stem-like/undifferentiated pancreatic tumors were investigated. Methods: Pancreatic tumor implantation was performed in NSG and hu-BLT mice. Stage of differentiation of tumors was determined using our published criteria for well-differentiated tumors exhibiting higher surface expression of MHC- class I, CD54, and PD-L1 (B7H1) and lower expression of CD44 receptors. The inverse was seen for poorly-differentiated tumors. Results: Stem-like/undifferentiated pancreatic tumors grew rapidly and formed large tumors and exhibited lower expression of above-mentioned differentiation antigens in the pancreas of NSG and hu-BLT mice. Unlike stem-like/undifferentiated tumors, NK-differentiated MP2 (MiaPaCa-2) tumors or patient-derived differentiated tumors were not able to grow or grew smaller tumors, and were unable to metastasize in NSG or hu-BLT mice, and they were susceptible to chemotherapeutic drugs. Stem-like/undifferentiated pancreatic tumors implanted in the pancreas of hu-BLT mice and injected with super-charged NK cells formed much smaller tumors, proliferated less, and exhibited differentiated phenotype. When differentiation of stem-like tumors by the NK cells was prevented by the addition of antibodies to IFN-&gamma, and TNF-&alpha, tumors grew rapidly and metastasized, and they remained resistant to chemotherapeutic drugs. Greater numbers of immune cells infiltrated the tumors of NK-injected and AJ2-probiotic bacteria-fed mice. Moreover, increased IFN-&gamma, secretion in the presence of decreased IL-6 was seen in tumors resected and cultured from NK-injected and AJ2 fed mice. Tumor-induced decreases in NK cytotoxicity and IFN-&gamma, secretion were restored/increased within PBMCs, spleen, and bone marrow when mice received NK cells and were fed with AJ2. Conclusion: NK cells prevent growth of pancreatic tumors through lysis and differentiation, thereby curtailing the growth and metastatic potential of stem-like/undifferentiated-tumors.

Therapeutic role of NK cells in solid tumors was challenged previously even though their role in hematological malignancies has clearly been established. Furthermore, functions and numbers of NK cells are greatly suppressed in oral cancer patients necessitating effective future NK immunotherapeutic strategies to aid in the control of disease. The humanized-BLT (hu-BLT) mice were used to implant stem-like/undifferentiated oral tumors to study the role of super-charged NK cells with and without feeding with AJ2 probiotic bacteria. Implanted CSC/undifferentiated tumors resected from NK-injected mice exhibited differentiated phenotype, grew slowly, and did not cause weight loss, whereas those from tumor-bearing mice without NK-injection remained relatively more stem-like/poorly-differentiated, grew faster, and caused significant weight loss. Moreover, in vitro NK-differentiated tumors were sensitive to chemotherapeutic drugs, and when implanted in the oral-cavity grew no or very small tumors in mice. When NK-mediated differentiation of tumors was blocked by IFN-γ and TNF-αantibodies before implantation, tumors grew rapidly, remained stem-like/poorly-differentiated and became resistant to chemotherapeutic drugs. Loss of NK cytotoxicity and decreased IFN-γ secretion in tumor-bearing mice in PBMCs, splenocytes, bone marrow derived immune cells and enriched NK cells was restored by the injection of super-charged NK cells with or without feeding with AJ2. Much greater infiltration of CD45+ and T cells were observed in tumors resected from the mice, along with the restored secretion of IFN-γ from purified T cells from splenocytes in NK-injected tumor-bearing mice fed with AJ2 probiotic bacteria. Thus, super-charged NK cells prevent tumor growth by restoring effector function resulting in differentiation of CSCs/undifferentiated-tumors in hu-BLT mice.