Immunotherapy targeting B cells and long-lived plasma cells effectively eliminates pre-existing donor-specific allo-antibodies.

Pre-existing anti-human leukocyte antigen (HLA) allo-antibodies constitute a major barrier to transplantation. Current desensitization approaches fail due to ineffective depletion of allo-specific memory B cells (Bmems) and long-lived plasma cells (LLPCs). We evaluate the efficacy of chimeric antigen receptor (CAR) T cells targeting CD19 and B cell maturation antigen (BCMA) to eliminate allo-antibodies in a skin pre-sensitized murine model of islet allo-transplantation. We find that treatment of allo-sensitized hosts with CAR T cells targeting Bmems and LLPCs eliminates donor-specific allo-antibodies (DSAs) and mitigates hyperacute rejection of subsequent islet allografts. We then assess the clinical efficacy of the CAR T therapy for desensitization in patients with multiple myeloma (MM) with pre-existing HLA allo-antibodies who were treated with the combination of CART-BCMA and CART-19 (ClinicalTrials.gov: NCT03549442) and observe clinically meaningful allo-antibody reduction. These findings provide logical rationale for clinical evaluation of CAR T-based immunotherapy in highly sensitized candidates to promote successful transplantation.

Modulation of the gut microbiota engages antigen cross-presentation to enhance antitumor effects of CAR T cell immunotherapy.

Several studies have shown the influence of commensal microbes on T cell function, specifically in the setting of checkpoint immunotherapy for cancer. In this study, we investigated how vancomycin-induced gut microbiota dysbiosis affects chimeric antigen receptor (CAR) T immunotherapy using multiple preclinical models as well as clinical correlates. In two murine tumor models, hematopoietic CD19+-A20 lymphoma and CD19+-B16 melanoma, mice receiving vancomycin in combination with CD19-directed CAR T cell (CART-19) therapy displayed increased tumor control and tumor-associated antigens (TAAs) cross-presentation compared with CART-19 alone. Fecal microbiota transplant from human healthy donors to pre-conditioned mice recapitulated the results obtained in naive gut microbiota mice. Last, B cell acute lymphoblastic leukemia patients treated with CART-19 and exposed to oral vancomycin showed higher CART-19 peak expansion compared with unexposed patients. These results substantiate the role of the gut microbiota on CAR T cell therapy and suggest that modulation of the gut microbiota using vancomycin may improve outcomes after CAR T cell therapy across tumor types.

Germinal center responses to SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals.

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. Herein, through a fine-needle aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant recipients (KTXs). We found that, unlike healthy subjects, KTXs presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cell, SARS-CoV-2 receptor binding domain-specific memory B cell, and neutralizing antibody responses. KTXs also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals and suggest a GC origin for certain humoral and memory B cell responses following mRNA vaccination.

Germinal center responses to SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals.

Vaccine-mediated immunity often relies on the generation of protective antibodies and memory B cells, which commonly stem from germinal center (GC) reactions. An in-depth comparison of the GC responses elicited by SARS-CoV-2 mRNA vaccines in healthy and immunocompromised individuals has not yet been performed due to the challenge of directly probing human lymph nodes. In this study, through a fine-needle-aspiration-based approach, we profiled the immune responses to SARS-CoV-2 mRNA vaccines in lymph nodes of healthy individuals and kidney transplant (KTX) recipients. We found that, unlike healthy subjects, KTX recipients presented deeply blunted SARS-CoV-2-specific GC B cell responses coupled with severely hindered T follicular helper cells, SARS-CoV-2 receptor-binding-domain-specific memory B cells and neutralizing antibodies. KTX recipients also displayed reduced SARS-CoV-2-specific CD4 and CD8 T cell frequencies. Broadly, these data indicate impaired GC-derived immunity in immunocompromised individuals, and suggest a GC-origin for certain humoral and memory B cell responses following mRNA vaccination.

Adoptive T cell immunotherapy for medullary thyroid carcinoma targeting GDNF family receptor alpha 4.

Metastatic medullary thyroid cancer (MTC) is a rare but often aggressive thyroid malignancy with a 5-year survival rate of less than 40% and few effective therapeutic options. Adoptive T cell immunotherapy using chimeric antigen receptor (CAR)-modified T cells (CAR Ts) is showing encouraging results in the treatment of cancer, but development is challenged by the availability of suitable target antigens. We identified glial-derived neurotrophic factor (GDNF) family receptor alpha 4 (GFRα4) as a putative antigen target for CAR-based therapy of MTC. We show that GFRα4 is highly expressed in MTC, in parafollicular cells within the thyroid from which MTC originates, and in normal thymus. We isolated two single-chain variable fragments (scFvs) targeting GFRα4 isoforms a and b by antibody phage display. CARs bearing the CD3ζ and the CD137 costimulatory domains were constructed using these GFRα4-specific scFvs. GFRα4-specific CAR Ts trigger antigen-dependent cytotoxicity and cytokine production in vitro, and they are able to eliminate tumors derived from the MTC TT cell line in an immunodeficient mouse xenograft model of MTC. These data demonstrate the feasibility of targeting GFRα4 by CAR T and support this antigen as a promising target for adoptive T cell immunotherapy and other antibody-based therapies for MTC.

Engineered FVIII-expressing cytotoxic T cells target and kill FVIII-specific B cells in vitro and in vivo.

Hemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII (FVIII) gene (F8). Treatment with recombinant or plasma-derived FVIII replacement therapy is standard therapy. A major problem in treating hemophilia A patients with therapeutic FVIII is that 20% to 30% of these patients produce neutralizing anti-FVIII antibodies (inhibitors) because they are not immunologically tolerant to this human protein. Hence, there is a need to establish tolerogenic protocols to FVIII epitopes. To specifically target FVIII-specific B cells, we engineered immunodominant FVIII domains (A2 and C2) as a chimeric antigen receptor expressed by both human and murine cytotoxic T cells. This FVIII domain engineered B-cell antibody receptor (BAR) that expresses T cells was capable of killing FVIII-reactive B-cell hybridomas in vitro and in vivo. Moreover, FVIII BAR CD8 T cells blocked the development of specific antibody from unimmunized spleen cells stimulated polyclonally with lipopolysaccharide in vitro. In addition, adoptive transfer of FVIII A2- and C2-BAR CD8 T cells significantly reduced the anti-FVIII antibody formation in hemophilic mice. These data suggest that BAR-engineered T cells are a promising approach for future prophylactic treatment for patients with severe hemophilia A who are at high risk of developing inhibitors.

Hemophilia A inhibitor treatment: the promise of engineered T-cell therapy.

Hemophilia A is a bleeding disorder caused by mutations in the gene encoding factor VIII (FVIII), a cofactor protein that is essential for normal blood clotting. Approximately, 1 in 3 patients with severe hemophilia A produce neutralizing antibodies (inhibitors) that block its biologic function in the clotting cascade. Current efforts to eliminate inhibitors consist of repeated FVIII injections under what is termed an "ITI" protocol (Immune Tolerance Induction). However, this method is extremely costly and approximately 30% of patients undergoing ITI do not achieve peripheral tolerance. Human T regulatory cells (Tregs) have been proposed as a new strategy to treat this antidrug antibody response, as well as other diseases. Polyclonal Tregs are nonspecific and could potentially cause general immunosuppression. Novel approaches to induce tolerance to FVIII include the use of engineered human and mouse antigen-specific Tregs, or alternatively antigen-specific cytotoxic cells, to delete, anergize, or kill FVIII-specific lymphocytes. In this review, we discuss the current state of engineered T-cell therapies, and we describe the recent progress in applying these therapies to induce FVIII-specific tolerance.

BIGH3 protein and macrophages in retinal endothelial cell apoptosis.

Diabetes is a pandemic disease with a higher occurrence in minority populations. The molecular mechanism to initiate diabetes-associated retinal angiogenesis remains largely unknown. We propose an inflammatory pathway of diabetic retinopathy in which macrophages in the diabetic eye provide TGFß to retinal endothelial cells (REC) in the retinal microvasculature. In response to TGFß, REC synthesize and secrete a pro-apoptotic BIGH3 (TGFß-Induced Gene Human Clone 3) protein, which acts in an autocrine loop to induce REC apoptosis. Rhesus monkey retinal endothelial cells (RhREC) were treated with dMCM (cell media of macrophages treated with high glucose and LDL) and assayed for apoptosis (TUNEL), BIGH3 mRNA (qPCR), and protein (Western blots) expressions. Cells were also treated with ΤGFß1 and 2 for BIGH3 mRNA and protein expression. Inhibition assays were carried out using antibodies for TGFß1 and for BIGH3 to block apoptosis and mRNA expression. BIGH3 in cultured RhREC cells were identified by immunohistochemistry (IHC). Distribution of BIGH3 and macrophages in the diabetic mouse retina was examined with IHC. RhRECs treated with dMCM or TGFß showed a significant increase in apoptosis and BIGH3 protein expression. Recombinant BIGH3 added to RhREC culture medium led to a dose-dependent increase in apoptosis. Antibodies (Ab) directed against BIGH3 and TGFß, as well as TGFß receptor blocker resulted in a significant reduction in apoptosis induced by either dMCM, TGFß or BIGH3. IHC showed that cultured RhREC constitutively expressed BIGH3. Macrophage and BIGH3 protein were co-localized to the inner retina of the diabetic mouse eye. Our results support a novel inflammatory pathway for diabetic retinopathy. This pathway is initiated by TGFß released from macrophages, which promotes synthesis and release of BIGH3 protein by REC and REC apoptosis.

Estrogen-induced retinal endothelial cell proliferation: possible involvement of pigment epithelium-derived factor and phosphoinositide 3-kinase/mitogen-activated protein kinase pathways.

PURPOSE: Diabetic retinopathy is a leading cause of blindness due to a progressive damage of the retina by neovascularization and other related ocular complications. However, the molecular mechanism underlying the development of diabetic retinopathy is not well understood. An increase in estrogen levels during puberty is associated with an accelerated development of diabetic retinopathy. Previously, we have introduced 17ß-estradiol (E2) to rhesus retinal capillary endothelial cells (RhRECs) in culture and observed a dose- and time-dependent increase in the number of viable cells. The purpose of this present study was to investigate the molecular signaling pathway associated with this estrogen-induced proliferation of RhRECs. METHODS: Estrogen receptor (ER) ER(α) and ER(ß) mRNA expression, and protein synthesis were measured at 0, 3, 6, and 12 h using nested polymerase chain reaction and Western blots. Phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathway inhibitors were introduced into culture media to study their effects on E2-induced cell proliferation and pigment epithelium-derived factor (PEDF) synthesis. The levels of PEDF in the conditioned media were measured by enzyme-linked immunosorbent assay. RESULTS: Exogenous E2 induced a significant increase in the expression of ER(ß) along with an increase in the number of viable RhRECs. Cotreatment of E2 with PI3K and MAPK inhibitors significantly reduced the E2-induced effect on cell proliferation and PEDF production in a dose-dependent manner. CONCLUSION: Results from the present study suggest that an E2-induced increase in the proliferation of RhRECs may be mediated by the action of ER(ß.) Both PI3K and MAPK signaling pathways are involved in this E2-induced cell proliferation, which may follow changes in PEDF levels controlled by these pathways. Further studies will provide additional details on the interaction between these pathways to control changes in PEDF levels and cell proliferation.

Effects of tamoxifen versus raloxifene on retinal capillary endothelial cell proliferation.

PURPOSE: Endothelial cell proliferation in angiogenesis is active in conditions such as cancers and diabetic retinopathy. Tamoxifen (T) and raloxifene (R) have been compared in numerous studies as a prophylaxis for breast cancer, and T is used to treat breast cancer. T, unlike R, has been linked to an increase in uterine cancers, thrombo-embolic events, and cataract. The purpose of our study was to evaluate the efficacies of T and R in reducing estrogen-induced retinal capillary endothelial cell proliferation. METHODS: Rhesus monkey retinal capillary endothelial cells (ATCC RF/6A) were used to assay cell proliferation when treated with 0.0, 0.1, 1.0, and 10.0 nM 17 ß estradiol (E2) for 24 and 48 h. Viable cells were counted using a Neubauer hemocytometer with a trypan blue exclusion method to determine the number of viable cells. Cell counts were also performed using 1.0 nM E2 with 0.01, 0.1, 1.0, and 10.0 nM concentrations of either T or R. Cell medium, collected at 24 h, was evaluated for vascular endothelial growth factor and pigment epithelium-derived factor. RESULTS: Viable cells were significantly greater in cultures treated with 1.0 or 10.0 nM E2, compared to cells treated with 0.0 or 0.1 nM E2 both at 24 and 48 h. Viable cell counts were reduced significantly in cultures treated with 0.1, 1.0, or 10.0 nM T or R in addition to the 1.0 nM E2. Cell counts were not significantly different when comparing equal concentrations of T and R, that is, 1.0 nM E2+1 nM T or R. Vascular endothelial growth factor and pigment epithelium-derived factor protein/10,000 cells was reduced by 1.0 nM E2, but returned to higher levels with the introduction of T and R to growth media. CONCLUSIONS: T and R showed similar potency in inhibiting estrogen-induced retinal capillary endothelial cell proliferation. Considering drug safety profiles, our results, when extended to animals and humans, suggest that R is preferable to T in treating angiogenic retinal diseases. Further studies on the signaling mechanism of estrogen-induced endothelial cell proliferation may lead to new treatment strategies in the treatment of ocular angiogenic diseases.

Mitochondrion-specific antioxidants as drug treatments for Alzheimer disease.

Age-related dementias such as Alzheimer disease (AD) have been linked to vascular disorders like hypertension, diabetes and atherosclerosis. These risk factors cause ischemia, inflammation, oxidative damage and consequently reperfusion, which is largely due to reactive oxygen species (ROS) that are believed to induce mitochondrial damage. At higher concentrations, ROS can cause cell injury and death which occurs during the aging process, where oxidative stress is incremented due to an accelerated generation of ROS and a gradual decline in cellular antioxidant defense mechanisms. Neuronal mitochondria are especially vulnerable to oxidative stress due to their role in energy supply and use, causing a cascade of debilitating factors such as the production of giant and/or vulnerable young mitochondrion who's DNA has been compromised. Therefore, mitochondria specific antioxidants such as acetyl-L-carnitine and R-alphalipoic acid seem to be potential treatments for AD. They target the factors that damage mitochondria and reverse its effect, thus eliminating the imbalance seen in energy production and amyloid beta oxidation and making these antioxidants very powerful alternate strategies for the treatment of AD.

Flavones from root of Scutellaria baicalensis Georgi: drugs of the future in neurodegeneration?

Flavonoids are natural, plant-derived compounds which exert diverse biological activities, also valuable neuroprotective actions within the brain and currently are intensively studied as agents able to modulate neuronal function and to prevent age-related neurodegeneration. Among them, flavones isolated from Scutellaria baicalensis root exhibit strong neuroprotective effects on the brain and are not toxic in the broad range of tested doses. Their neuroprotective potential has been shown in both oxidative stress-induced and amyloid-beta and alpha-synuclein-induced neuronal death models. Baicalein, the main flavone present in Scutellaria baicalensis root, strongly inhibited aggregation of neuronal amyloidogenic proteins in vitro and induces dissolution of amyloid deposits. It exerts strong antioxidative and anti-inflammatory activities and also exhibits anti-convulsive, anxiolytic, and mild sedative actions. Importantly, baicalein, and also another flavone: oroxylin A, markedly enhanced cognitive and mnestic functions in animal models of aging brains and neurodegeneration. In the preliminary study, wogonin, another flavone from Scutellaria baicalensis root, has been shown to stimulate brain tissue regeneration, inducing differentiation of neuronal precursor cells. This concise review provides the main examples of neuroprotective activities of the flavones and reveals their potential in prevention and therapyof neurodegenerative diseases.

Implication of oncogenic signaling pathways as a treatment strategy for neurodegenerative disorders - contemporary approaches.

Recent evidence has associated the aberrant, proximal re-expression of various cell cycle control elements with neuronal cell vulnerability in Alzheimer's and Parkinson's diseases, as a common chronic neurodegeneration. This phenomenon associated with oncogenic transduction pathway activation has attracted the interest of scientists all over the world for a few years now. The purpose of this paper is to outline areas of research related to oncogenic factors or medicines in the context of potential applications for future treatment of the above mentioned chronic and, largely, incurable diseases.

Antioxidants in health, disease and aging.

There is growing scientific agreement that antioxidants, particularly the polyphenolic forms, may help lower the incidence of disease, such as certain cancers, cardiovascular and neurodegenerative diseases, DNA damage, or even have anti-aging properties. On the other hand, questions remain as to whether some antioxidants or phytochemicals potentially could do more harm than good, as an increase in glycation-mediated protein damage (carbonyl stress) and some risk has been reported. Nevertheless, the quest for healthy aging has led to the use of antioxidants as a means to disrupt age-associated deterioration in physiological function, dysregulated metabolic processes or prevention of many age-related diseases. Although a diet rich in polyphenolic forms of antioxidants does seem to offer hope in delaying the onset of age-related disorders, it is still too early to define their exact clinical benefit for treating age-related disease. Regardless of where the debate will end, it is clear that any deficiency in antioxidant vitamins or adequate enzymatic antioxidant defenses can manifest in many disease states and shift the redox balance in some diseases. This updated review critically examines general antioxidant compounds in health, disease and aging with hope that a better understanding of the many mechanisms involved with these diverse compounds may lead to better health and novel treatment approaches for age-related diseases.

Ginsenoside-Rb1 Induces ARPE-19 Proliferation and Reduces VEGF Release.

Rb1, a ginsenoside from ginseng root extract, possesses antiangiogenic effects, but its role on ocular cells has not been studied. We hypothesize that Rb1 inhibits the production of the angiogenic cytokine VEGF from ARPE-19 cells, leading to a significant reduction in the proliferation of ocular vasculatures. Data from our experiments show that Rb1 induced an increase in the number of ARPE cells in culture, while VEGF release (pg/10,000 viable cells) was significantly reduced. Treatment with VEGF and cotreatment with Rb1 and VEGF showed that this Rb1-induced cell proliferation was mediated by VEGF. Because VEGF from RPE plays a major role in promoting angiogenesis in ocular vasculatures. Our finding that Rb1 inhibits the release of VEGF from RPE cells suggests that Rb1 has a significant role in the eye to protect against angiogenic diseases such as age-related macular degeneration.