Impact of tozinameran (BNT162b2) mRNA vaccine on kidney transplant and chronic dialysis patients: 3-5 months follow-up.

BACKGROUND: Determining the humoral immunogenicity of tozinameran (BNT162b2) in patients requiring chronic renal replacement therapy, and its impact on COVID-19 morbidity several months after vaccination, may guide risk assessment and changes in vaccination policy. METHODS: In a prospective post-vaccination cohort study with up to 5 months follow-up we studied outpatient dialysis and kidney transplant patients and respective healthcare teams. Outcomes were anti S1/S2 antibody responses to vaccine or infection, and infection rate during follow-up. RESULTS: One hundred seventy-five dialysis patients (40% women, 65 ± 15 years), 252 kidney transplant patients (33% women, 54 ± 14 years) and 71 controls (65% women, 44 ± 14 years) were followed. Three months or longer after vaccination we detected anti S1/S2 IgG antibodies in 79% of dialysis patients, 42% of transplant recipients and 100% of controls, whereas respective rates after infection were 94%, 69% and 100%. Predictors of non-response were older age, diabetes, history of cancer, lower lymphocyte count and lower vitamin-D levels. Factors associated with lower antibody levels in dialysis patients were modality (hemodialysis vs peritoneal) and high serum ferritin levels. In transplant patients, hypertension and higher calcineurin or mTOR inhibitor drug levels were linked with lower antibody response. Vaccination was associated with fewer subsequent infections (HR 0.23, p < 0.05). Moreover, higher antibody levels (particularly above 59 AU/ml) were associated with fewer events, with a HR 0.41 for each unit increased in log10titer (p < 0.05). CONCLUSIONS: Dialysis patients, and more strikingly kidney transplant recipients, mounted reduced antibody response to COVID-19 mRNA vaccination. Lesser humoral response was associated with more infections. Measures to identify and protect non-responsive patients are required.

Toxicology and Pharmacokinetic Studies in Mice and Nonhuman Primates of the Nontoxic, Efficient, Targeted Hexameric FasL: CTLA4-FasL.

Cytotoxic T-lymphocyte antigen 4 (CTLA4)-FasL, a homo-hexameric signal converter protein, is capable of inducing robust apoptosis in malignant cells of the B-cell lineage expressing its cognate B7 and Fas targets, while sparing nonmalignant ones. This fusion protein's striking proapoptotic efficacy stems from its complementary abilities to coordinately activate apoptotic signals and abrogate antiapoptotic ones. A limiting factor in translating FasL or Fas receptor agonists into the clinic has been lethal hepatotoxicity. Here, we establish CTLA4-FasL's in vivo efficacy in multiple murine and xenograft models, for both systemic and subcutaneous tumors. Significantly, good laboratory practice (GLP) toxicology studies in mice indicate that CTLA4-FasL given repeatedly at doses up to five times the effective dose was well-tolerated and resulted in no significant adverse events. An equivalent single dose of CTLA4-FasL administered to nonhuman primates was also well-tolerated, albeit with a moderate dose-dependent leukopenia that was completely reversible. Interestingly, monkey peripheral blood mononuclear cells were more sensitive to CTLA4-FasL-induced apoptosis when tested in vitro. In both species, there was short-term elevation in serum levels of IL6, IL2, and IFNγ, although this was not associated with clinical signs of proinflammatory cytokine release, and further, this cytokine elevation could be completely prevented by dexamethasone premedication. Liver toxicity was not observed in either species, as confirmed by serum liver enzyme levels and histopathologic assessment. In conclusion, CTLA4-FasL emerges from animal model studies as an effective and safe agent for targeted FasL-mediated treatment of B7-expressing aggressive B-cell lymphomas.

Ambulatory monitoring unmasks hypertension among kidney transplant patients: single center experience and review of the literature.

BACKGROUND: Disagreements between clinic and ambulatory blood pressure (BP) measurements are well-described in the general population. Though hypertension is frequent in renal transplant recipients, only a few studies address the clinic-ambulatory discordance in this population. We aimed to describe the difference between clinic and ambulatory BP in kidney transplant patients at our institution. METHODS: We compared the clinic and ambulatory BP of 76 adult recipients of a kidney allograft followed at our transplant center and investigated the difference between these methods, considering confounding by demographic and clinical variables. RESULTS: Clinic systolic BP (SBP) and diastolic BP (DBP) were 128 ± 13/79 ± 9 mmHg. Awake SBP and DBP were 147 ± 18/85 ± 10 mmHg. The clinic-minus-awake SBP and DBP differences were - 18 and - 6 mmHg, respectively. The negative clinic-awake ΔSBP was more pronounced at age > 60 years (p = 0.026) and with tacrolimus use compared to cyclosporine (p = 0.046). Sleep SBP and DBP were 139 ± 21/78 ± 11 mmHg. A non-dipping sleep BP pattern was noted in 73% of patients and was associated with tacrolimus use (p = 0.020). CONCLUSIONS: Our findings suggest pervasive underestimation of BP when measured in the kidney transplant clinic, emphasizes the high frequency of a non-dipping pattern in this population and calls for liberal use of ambulatory BP monitoring to detect and manage hypertension.

Fn14·TRAIL fusion protein is oligomerized by TWEAK into a superefficient TRAIL analog.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) demonstrates specific anti-cancer activity, but insufficient efficacy in patients. A fusion protein Fn14·TRAIL, that combines soluble TRAIL molecule with a specific TWEAK receptor Fn14, is a better apoptosis-inducer for hepatocellular carcinomas than soluble TRAIL. However, Fn14·TRAIL does not effectively induce apoptosis in tumors of the lymphoid origin. As malignant cell apoptosis is strongly enhanced by secondary oligomerization of TRAIL, we tested the hypothesis that soluble Fn14·TRAIL can be oligomerized and become more active by adding TWEAK, a cytokine secreted in the tumor environment. We revealed that TWEAK and Fn14·TRAIL spontaneously formed a stable complex that induced apoptosis of malignant lymphoblasts earlier and more efficiently than TRAIL. The TWEAK-modified Fn14·TRAIL oligomer bound to target cells and delivered apoptotic signaling via TRAIL receptors. The oligomer induced faster and stronger cleavage of procaspase-8, -9, and -3; BID; poly-ADP ribose polymerase; and RIP compared to TRAIL. The oligomer also reduced expression of the anti-apoptotic proteins c-FLIP short and cIAP-1. Our data indicate that Fn14·TRAIL can be converted into a highly effective TRAIL oligomer upon binding to TWEAK.

Heart rate response to blood pressure variations: sympathetic activation versus baroreflex response in patients with end-stage renal disease.

BACKGROUND: Continuous systolic blood pressure (SBP) and interbeat intervals (IBI) recordings reveal sequences of consecutive beats in which SBP and heart rate change in opposite direction, representing negative feedback baroreflex mechanisms, as well as sequences in which SBP and heart rate change in the same direction (non-baroreflex), believed to represent feedforward control mechanisms. The present study was undertaken to assess the relationship between baroreflex and non-baroreflex sequences in end stage renal insufficiency. METHODOLOGY/PRINCIPAL FINDINGS: Continuous beat-to-beat SBP and IBI monitoring was performed in patients on chronic hemodialysis (HD, n=72), in age-matched patients after renal transplantation (TX, n=41) and healthy (control) individuals (C, n=34). The proportion of baroreflex and nonbaroreflex episodes and the b coefficients (the regression line slope of SBP-IBI correlation) were determined using a newly developed 1 minute sliding window method, the classical sequence technique and the "Z" coefficient method. Analysis using the 1 minute sliding window showed an increased proportion of baroreflex episodes in controls and HD, and predominance of nonbaroreflex episodes in TX. An increased proportion of nonbaroreflex episodes in TX patients relative to HD was also revealed by the "Z" method. Baroreflex and nonbaroreflex b coefficients obtained by all methods were markedly decreased in HD. This alteration was reversed at least partly in TX. In HD, both baroreflex and nonbaroreflex b coefficients were inversely correlated to age and CRP levels; in TX, the nonbaroreflex b coefficient was influenced by the type of calcineurin inhibitor. CONCLUSION/SIGNIFICANCE: Renal status affects the contribution of baroreflex and nonbaroreflex mechanisms and the strength of SBP-IBI relationship. The predominant contribution of nonbaroreflex mechanisms in TX may be suggestive of enhanced central sympathetic control. Our data may be relevant for understanding of the pathogenesis and selection of appropriate treatment of post-transplant hypertension.

Fn14•TRAIL effectively inhibits hepatocellular carcinoma growth.

BACKGROUND: New strategies for the treatment of hepatocellular carcinoma (HCC) are needed, given that currently available chemotherapeutics are inefficient. Since tumor growth reflects the net balance between pro-proliferative and death signaling, agents shifting the equilibrium toward the latter are of considerable interest. The TWEAK:Fn14 signaling axis promotes tumor cell proliferation and tumor angiogenesis, while TRAIL:TRAIL-receptor (TRAIL-R) interactions selectively induce apoptosis in malignant cells. Fn14•TRAIL, a fusion protein bridging these two pathways, has the potential to inhibit tumor growth, by interfering with TWEAK:Fn14 signaling, while at the same time enforcing TRAIL:TRAIL-R-mediated apoptosis. Consequently, Fn14•TRAIL's capacity to inhibit HCC growth was tested. RESULTS: Fn14•TRAIL induced robust apoptosis of multiple HCC cell lines, while sparing non-malignant hepatocyte cell lines. Differential susceptibility to this agent did not correlate with expression levels of TRAIL, TRAIL-R, TWEAK and Fn14 by these lines. Fn14•TRAIL was more potent than soluble TRAIL, soluble Fn14, or a combination of the two. The requirement of both of Fn14•TRAIL's molecular domains for function was established using blocking antibodies directed against each of them. Subcutaneous injection of Fn14•TRAIL abrogated HCC growth in a xenograft model, and was well tolerated by the mice. CONCLUSIONS: In this study, Fn14•TRAIL, a multifunctional fusion protein originally designed to treat autoimmunity, was shown to inhibit the growth of HCC, both in vitro and in vivo. The demonstration of this fusion protein's potent anti-tumor activity suggests that simultaneous targeting of two signaling axes by a single fusion can serve as a basis for highly effective anti-cancer therapies.

Amelioration of autoimmune neuroinflammation by the fusion molecule Fn14·TRAIL.

BACKGROUND: Multiple sclerosis (MS) is a, T cell-mediated autoimmune disease, the management of which remains challenging. The recently described fusion protein, Fn14·TRAIL, combining the extracellular domain of Fn14 (capable of blocking the pro-inflammatory TWEAK ligand) fused to the extracellular domain of the TRAIL ligand (capable of sending apoptotic signals through its receptors on activated inflammatory cells) was designed to modulate the immune system as an anti-inflammatory agent. The present study explores the efficacy of this purified protein as an anti-inflammatory agent, using the animal model of MS - experimental autoimmune encephalomyelitis (EAE). METHODS: EAE was induced by myelin oligodendrocyte glycoprotein (MOG). Fn14·TRAIL or vehicle were injected daily for 4 to 16 days, at different time points after disease induction. Animals were examined daily and evaluated for EAE clinical signs. Lymphocytes were analyzed for ex vivo re-stimulation, cytokine secretion, transcription factor expression and subtype cell analysis. Spinal cords were checked for inflammatory foci. The Mann- Whitney rank sum test, Student's t-test or ANOVA were used for statistical analysis. RESULTS: Significant improvement of EAE in the group treated with Fn14·TRAIL was noted from day 6 of disease onset and lasted until the end of follow-up (day 40 from disease induction), even in animals treated for 4 days only. Clinical improvement was linked to decreased lymphocyte infiltrates in the central nervous system (CNS) and to decreased Th1 and Th17 responses and to increased number of T- regulatory in the treated mice. No liver or kidney toxicity was evident. In vitro assays established the ability of Fn14·TRAIL to induce apoptosis of T cell lines expressing TRAIL receptors and TWEAK. CONCLUSIONS: In this study we established the potency of Fn14·TRAIL, a unique fusion protein combining two potentially functional domains, in inhibiting the clinical course of EAE, even when given for a short time, without apparent toxicity. These findings make Fn14·TRAIL a highly promising agent to be used for targeted amelioration of neuro-inflammatory processes, as well as other autoimmune pathologies.

CD40·FasL and CTLA-4·FasL fusion proteins induce apoptosis in malignant cell lines by dual signaling.

Evolution of apoptosis resistance in both lymphoma and leukemia cells is well documented, and induction of apoptosis in malignant cells is a major goal of cancer therapy. Up-regulation of anti-apoptotic signals is one of the mechanisms whereby resistance to apoptosis emerges. We have previously described the fusion proteins CD40·FasL and CTLA-4·FasL, which are formed from two functional membrane proteins and induce apoptosis of activated T cells. The present study explores the potential use of CD40·FasL and CTLA-4·FasL for the killing of malignant cells of lymphatic origin. Using malignant B and T cell lines that differ in surface expression of costimulatory molecules, we found that CTLA-4·FasL induces effective apoptosis of cells expressing CD95 and activates caspases 3, 8, and 9. Only B7-expressing B cells responded to CTLA-4·FasL with rapid abrogation of cFLIP expression. CD40·FasL effectively killed only the T cells that express high levels of CD40L in addition to CD95. In these cells, CD40·FasL significantly diminished cFLIP expression. Importantly, each of the fusion proteins is more potent than its respective components parts, alone or in combination. Thus, the proteins with their two functional ends deliver a pro-apoptotic signal and, in parallel, inhibit an anti-apoptotic signal, thus optimizing the wanted, death-inducing effect. Therefore, these proteins emerge as promising agents to be used for targeted and specific tumor cell killing.

Human erythropoietin gene therapy for patients with chronic renal failure.

Gene therapy holds a major promise. However, until now, this promise was fulfilled only in few cases, in rare genetic diseases. One very common clinical condition is anemia. Patients with anemia of chronic renal failure are treated with erythropoietin. The objective of this study was to develop a therapeutic platform for serum-secreted proteins like erythropoietin. We developed a tissue protein factory based on dermal cores (Biopump) harvested and implanted autologously. In this study, an adenovector was designed to express the human erythropoietin under the control of the cytomegalovirus (CMV) promoter. This vector transduced the harvested dermal cores ex vivo. The transduced cores were implanted, and erythropoietin and reticulocyte counts were measured. Dermal cores were harvested from 13 patients with chronic renal failure, and implantation was performed in 10. There were no significant drug-related side effects to this procedure. Erythropoietin serum levels increased significantly to therapeutic levels from day 1 after implantation reaching a peak during the first week of follow-up. The expression period was transient for up to 14 days. The rise of erythropoietin was followed by a transient significant increase in reticulocyte counts. The decrease of erythropoietin expression coincided with a significant dermal infiltrate of CD8 cytotoxic T cells. Antierythropoietin antibodies were not detected until day 90 following implantation. Implantation of dermal cores ex vivo transduced with human genes could eventually be used in the clinical setting to express therapeutic serum proteins. However, nonimmunogenic delivery system should be tested as gene vehicles.

Sensing phosphate across the kingdoms.

PURPOSE OF REVIEW: The present review summarizes recent findings that may help in understanding how the cell senses changes in serum phosphate. RECENT FINDINGS: The sensing of phosphate determines the organism's response to change in supply of this essential nutrient. Phosphate depletion or surfeit results in homeostatic responses that involve changes in transcription, transcript stability, transporter recruitment or breakdown, and cell replication. These responses are shared across the biological kingdoms, and lessons from unicellular organisms may be relevant to multicellular mammals. An understanding of nutrient sensing in general may help in determining how the cell senses changes in phosphate concentration. SUMMARY: Research has yielded important advances in unravelling phosphate sensing and the response to nutrient phosphate supply. However, the actual sensing event for phosphate and most other nutrients must still be defined. Lessons may be learned from those examples in which the sensing event is known, and these are summarized here.