Impaired humoral and cellular immunity after SARS-CoV-2 BNT162b2 (tozinameran) prime-boost vaccination in kidney transplant recipients

Novel mRNA-based vaccines have been proven to be powerful tools in combating the global pandemic caused by SARS-CoV-2, with BNT162b2 (trade name: Comirnaty) efficiently protecting individuals from COVID-19 across a broad age range. Still, it remains largely unknown how renal insufficiency and immunosuppressive medication affect development of vaccine-induced immunity. We therefore comprehensively analyzed humoral and cellular responses in kidney transplant recipients after the standard second vaccination dose. As opposed to all healthy vaccinees and the majority of hemodialysis patients, only 4 of 39 and 1 of 39 transplanted individuals showed IgA and IgG seroconversion at day 8 ± 1 after booster immunization, with minor changes until day 23 ± 5, respectively. Although most transplanted patients mounted spike-specific T helper cell responses, frequencies were significantly reduced compared with those in controls and dialysis patients and this was accompanied by a broad impairment in effector cytokine production, memory differentiation, and activation-related signatures. Spike-specific CD8⁺ T cell responses were less abundant than their CD4⁺ counterparts in healthy controls and hemodialysis patients and almost undetectable in transplant patients. Promotion of anti-HLA antibodies or acute rejection was not detected after vaccination. In summary, our data strongly suggest revised vaccination approaches in immunosuppressed patients, including individual immune monitoring for protection of this vulnerable group at risk of developing severe COVID-19.     

Role of endothelins as mediators of injury-induced alterations of glial glutamate turnover

Astroglia terminate glutamatergic neurotransmission and prevent excitotoxic extracellular glutamate concentration by clearing synaptically released glutamate through the high-affinity, sodium-dependent glutamate transporters GLT-1 and GLAST. Many brain injures are associated with the disturbed expression of glial glutamate transporters and a subsequent increase of extracellular glutamate to neurotoxic levels. We have now followed up initial hints pointing to endothelins, a family of injury-regulated peptides, as mediators of this injury-induced loss of glial glutamate transporter expression. We observed that, in line with such a role, endothelins not only act as potent inhibitors of basal and exogenously (dbcAMP)-induced expression of GLT-1 in cortical astrocytes as shown before, but likewise inhibit expression of GLT-1 or GLAST in astrocytes cultured from the diencephalon, mesencephalon, cerebellum, and spinal cord. We further demonstrate that endothelins equally inhibit GLT-1 expression in cortical slice cultures, a culture system closely resembling the in vivo situation. Although brain injuries are usually associated with an increase in the expression of the glutamate-converting enzyme glutamine synthetase, cultured cortical astrocytes maintained with endothelins showed an almost complete loss of glutamine synthetase. Interestingly, the inhibitory effects of endothelins on the expression of glutamine synthetase, but not of glutamate transporters, was overridden by high extracellular glutamate, indicating that the primarily inhibitory action of endothelins on the various components of glial glutamate turnover dissociates in the injured brain.     

A perivascular niche in the bone marrow hosts quiescent and proliferating tumorigenic colorectal cancer cells

Disseminated tumor cells (dTCs) can frequently be detected in the bone marrow (BM) of colorectal cancer (CRC) patients, raising the possibility that the BM serves as a reservoir for metastatic tumor cells. Identification of dTCs in BM aspirates harbors the potential of assessing therapeutic outcome and directing therapy intensity with limited risk and effort. Still, the functional and prognostic relevance of dTCs is not fully established. We have previously shown that CRC cell clones can be traced to the BM of mice carrying patient-derived xenografts. However, cellular interactions, proliferative state and tumorigenicity of dTCs remain largely unknown. Here, we applied a coculture system modeling the microvascular niche and used immunofluorescence imaging of the murine BM to show that primary CRC cells migrate toward endothelial tubes. dTCs in the BM were rare, but detectable in mice with xenografts from most patient samples (8/10) predominantly at perivascular sites. Comparable to primary tumors, a substantial fraction of proliferating dTCs was detected in the BM. However, most dTCs were found as isolated cells, indicating that dividing dTCs rather separate than aggregate to metastatic clones—a phenomenon frequently observed in the microvascular niche model. Clonal tracking identified subsets of self-renewing tumor-initiating cells in the BM that formed tumors out of BM transplants, including one subset that did not drive primary tumor growth. Our results indicate an important role of the perivascular BM niche for CRC cell dissemination and show that dTCs can be a potential source for tumor relapse and tumor heterogeneity.     

Repression of breast cancer cell growth by proteasome inhibitors in vitro: impact of mitogen-activated protein kinase phosphatase 1

Mitogen activated protein kinase phosphatase-1 (MKP-1) has emerged as an important protein mediating breast cancer oncogenesis and chemoresistance to cancer chemotherapies, especially proteasome inhibitors. In this in vitro study, we utilized the breast cancer epithelial cell lines MCF-7 and MDA-MB-231, in comparison to MCF-10A control cells, to examine the impact of MKP-1 on breast cancer cell growth and repression by proteasome inhibitors. We confirm that proteasome inhibitors MG-132 and bortezomib induce MKP-1 protein upregulation and we show that one of the ways in which bortezomib increases MKP-1 in breast cancer cells, in addition to inhibition of ubiquitin-proteasome system, is via upregulation of MKP-1 mRNA expression in p38 MAPK-mediated manner. Notably, these effects are specific to cancer cells, as bortezomib activated p38 MAPK and induced MKP-1 in MCF-7 and MDA-MB-231 breast cancer cells, but not in control cells (MCF-10A). We took a dual approach toward targeting MKP-1 to show that bortezomib-induced effects are enhanced. Firstly, treatment with the non-specific MKP-1 inhibitor triptolide reduces breast cancer cell growth and augments proteasome inhibitor-induced effects. Secondly, specific knock-down of MKP-1 with siRNA significantly repressed cell viability by reduced cyclin D1 expression, and enhanced repression of cancer cell growth by proteasome inhibitors. Taken together, these results indicate that removing the unwanted (MKP-1-inducing) effects of bortezomib significantly improves the efficacy of proteasome inhibition in breast cancer cells. Thus, future development of drugs targeting MKP-1 offer promise of combination therapies with reduced toxicity and enhanced cell death in breast cancer.     

X-Nuclei NMR Self-Diffusion Studies in Mesoporous Silica Foam and Microporous MOF CuBTC

A standard X-observe NMR probe was equipped with a z-gradient coil to enable high-sensitivity pulsed field gradient NMR diffusion studies of Li⁺ and Cs⁺ cations of aqueous salt solutions in a high-porosity mesocellular silica foam (MCF) and of CO₂ adsorbed in metal-organic frameworks (MOF). The coil design and the necessary probe modifications, which yield pulsed field gradients of up to ±16.2 Tm^-1, are introduced. The system was calibrated at ²H resonance frequency and successfully applied for diffusion studies at ⁷Li, ²³Na, ¹³C and ¹³³Cs frequencies. Significant reductions of the diffusivities of the cations in LiCl_ac and CsCl_ac solution introduced into MCFs are observed. By comparison of the diffusion behavior with the bulk solutions, a tortuosity of the silica foam of 4.5 ± 0.6 was derived. Single component self-diffusion of CO₂ and CH₄ (measured by ¹H NMR) as well as self-diffusion of the individual components in CO₂/CH₄ mixtures was studied in the MOF CuBTC. The experimental results confirm high mobilities of the adsorbed gases and trends for diffusion separation factors predicted by MD simulations.     

High dose intracoronary N-acetylcysteine in a porcine model of ST-elevation myocardial infarction

We sought to evaluate the safety and efficacy of N-acetylcysteine (NAC) on ischemia and reperfusion in a pig model focusing on cardio-renal protection. High doses of NAC may provide protection from contrast induced nephropathy (CIN). NAC has also been demonstrated to reduce myocardial infarction size and improve left ventricular function after ischemia in both humans and animals studies. In this study we tested the safety and cardiorenal protective efficacy of intracoronary NAC delivered in the radiographic contrast agent in a pig model that simulates the catheter based reperfusion therapy of ST elevation myocardial infarctions. 27 pigs underwent 45 min of ischemia after surgical ligation of distal left descending coronary artery. With coronary reperfusion the animals received at total of 200 mL of the contrast agent Iopamidol with and without NAC to mimic radiographic contrast use during invasive reperfusion therapy. At 24 h the following endpoints were compared: LV function (MRI, echocardiography), myocardial injury (infarct size, area-at-risk, troponin, creatinine kinase) and CIN (creatinine, BUN and renal histology). The effects of NAC on platelet reactivity were also evaluated. Intracoronary administration of NAC administered in the contrast agent is safe. NAC reduces platelet reactivity and there was a trend towards a better cardiac function at 24 h. There was no significant difference in the size of the myocardial infarction. In this model of ischemia–reperfusion high dose NAC did not protect from CIN. High dose intracoronary NAC administered with the radiographic contrast is safe but does not provide significant cardio-renal protection.     

Impact of Bacillus thuringiensis toxin Cry1Ab on rumen epithelial cells (REC) – A new in vitro model for safety assessment of recombinant food compounds

The growing use of genetically modified crops necessitates viable screening methods for safety evaluation of recombinant feed, particularly for ruminants. A new sheep rumen epithelial cell culture is introduced as an in vitro cell system for safety evaluation especially focussing on feed and food compounds. We used lactate dehydrogenase (LDH) release, WST-1 conversion, ATP content and caspase 3/7 activity to evaluate cytotoxicity of Cry1Ab, one of the newly expressed Bt-proteins in transgene maize. The results were compared to the effects of valinomycin, a potassium ionophore known to induce cytotoxic effects on a wide range of cells. Whereas no toxicity of Cry1Ab was observed in short as well as in long term experiments, even at non-physiological high concentrations, exposure to valinomycin induced apoptosis and a significant response of all viability parameters after a number of hours. The ATP content and the WST-1 conversion reflecting the energy metabolism of the cells appear to be more sensitive indicators of valinomycin toxicity than the LDH release, a parameter which reflects the membrane integrity. This study presents an in vitro model system, that may be useful as a supplementary tool in toxicity screening before testing substances on animals in vivo.     

Pregnancy Does Not Increase Susceptibility to Bupivacaine in Spinal Root Axons

Background: The underlying mechanism of enhanced antinociceptive effects and increased susceptibility to local anesthetics during pregnancy is not known. Mechanical, hormonal, biochemical, and neural changes have been suggested. The authors measured the susceptibility of individual spinal root axons to bupivacaine during late pregnancy in rats and compared them with similar measurements in nonpregnant rats.

Methods: Lumbar dorsal and ventral roots were excised from anesthetized pregnant and nonpregnant rats. Single-fiber dissection and recording techniques were used to isolate activity in individual axons. Supramaximal constant voltage stimuli were delivered to the distal end of the root. During in vitro perfusion, each root was exposed to increasing concentrations of bupivacaine, and the minimum blocking concentration (Cm) and the concentration that increased conduction latency by 50% (EC50) were measured.

Results: Myelinated and unmyelinated dorsal and ventral root axons of pregnant rats appeared to be less sensitive to steady-state conduction block and to the latency-increasing effects of bupivacaine than were equivalent axons from nonpregnant rats. Although when comparing specific axon types, only the difference in C-fibers was significant (Cm = 29.8 micro Meter for pregnant and Cm = 22.1 micro Meter for nonpregnant rats, P < 0.05; EC50 = 19.9 micro Meter and 13.6 micro Meter, respectively).