Clinical and Immunologic Effects of Paraprobiotics in Long-COVID Patients: A Pilot Study.

BACKGROUND AND OBJECTIVES: After the enormous health burden during the acute stages of the COVID-19 pandemic, we are now facing another important challenge, that is, long-COVID, a clinical condition with often disabling signs and symptoms of the neuropsychiatric, gastrointestinal, respiratory, cardiovascular, and immune systems. While the pathogenesis of this syndrome is still poorly understood, alterations of immune function and the gut microbiota seem to play important roles. Because affected individuals are frequently unable to work for prolonged periods and suffer numerous health compromises, effective treatments represent a major unmet medical need. Multiple potential therapies have been tried, but none is approved yet. Approaches that are able to influence the immune system and gut microbiota such as probiotics and paraprobiotics, i.e., nonviable probiotics, seem promising candidates. We, therefore, evaluated the clinical and immunologic effects of paraprobiotics in a small pilot study. METHODS: A total of 6 patients with long-COVID were followed systematically for more than 12 months after disease onset using standardized validated questionnaires, a smartphone app, and wearable sensors to assess neurocognitive function, fatigue, depressiveness, autonomic nervous system alterations, and quality of life. We then offered patients defined paraprobiotics for 4 weeks and evaluated them at the end of the treatment period using the same questionnaires, smartphone app, and wearable sensors. In addition, a comprehensive immunophenotyping and gut microbiota analysis was performed before and after treatment. RESULTS: Improvements in several of the neurologic symptoms such as dysautonomia, fatigue, and depression were documented using both patient-reported outcomes and data from the smartphone app and wearable sensors. Of interest, the expression of activation markers on some immune cell populations such as B cells and nonclassical monocytes and the expression of toll-like receptor 2 (TLR2) on T cells were reduced after paraprobiotics treatment. DISCUSSION: Our results suggest that paraprobiotics might exert positive effects in patients with long-COVID most likely by modulating immune cell activation and expression of TLR2 on T cells. Further studies with paraprobiotics should confirm the promising observations of this small pilot study and hopefully not only improve the outcome of long-COVID but also unravel the pathomechanisms of this condition. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that paraprobiotics increase the probability of favorable changes of clinical and immunologic markers in patients with long-COVID.

HLA Associations of Intrathecal IgG Production against Specific Viruses in Multiple Sclerosis.

OBJECTIVE: Specific human leucocyte antigen (HLA) alleles are not only associated with higher risk to develop multiple sclerosis (MS) and other autoimmune diseases, but also with the severity of various viral and bacterial infections. Here, we analyzed the most specific biomarker for MS, that is, the polyspecific intrathecal IgG antibody production against measles, rubella, and varicella zoster virus (MRZ reaction), for possible HLA associations in MS. METHODS: We assessed MRZ reaction from 184 Swiss patients with MS and clinically isolated syndrome (CIS) and 89 Swiss non-MS/non-CIS control patients, and performed HLA sequence-based typing, to check for associations of positive MRZ reaction with the most prevalent HLA alleles. We used a cohort of 176 Swedish MS/CIS patients to replicate significant findings. RESULTS: Whereas positive MRZ reaction showed a prevalence of 38.0% in MS/CIS patients, it was highly specific (97.7%) for MS/CIS. We identified HLA-DRB1*15:01 and other tightly linked alleles of the HLA-DR15 haplotype as the strongest HLA-encoded risk factors for a positive MRZ reaction in Swiss MS/CIS (odds ratio [OR], 3.90, 95% confidence interval [CI] 2.05-7.46, padjusted = 0.0004) and replicated these findings in Swedish MS/CIS patients (OR 2.18, 95%-CI 1.16-4.02, padjusted = 0.028). In addition, female MS/CIS patients had a significantly higher probability for a positive MRZ reaction than male patients in both cohorts combined (padjusted <0.005). INTERPRETATION: HLA-DRB1*15:01, the strongest genetic risk factor for MS, and female sex, 1 of the most prominent demographic risk factors for developing MS, predispose in MS/CIS patients for a positive MRZ reaction, the most specific CSF biomarker for MS. ANN NEUROL 2024;95:1112-1126.

Aß42 oligomer-specific antibody ALZ-201 reduces the neurotoxicity of Alzheimer's disease brain extracts.

BACKGROUND: In Alzheimer's disease (AD), amyloid-ß 1-42 (Aß42) neurotoxicity stems mostly from its soluble oligomeric aggregates. Studies of such aggregates have been hampered by the lack of oligomer-specific research tools and their intrinsic instability and heterogeneity. Here, we developed a monoclonal antibody with a unique oligomer-specific binding profile (ALZ-201) using oligomer-stabilising technology. Subsequently, we assessed the etiological relevance of the Aß targeted by ALZ-201 on physiologically derived, toxic Aß using extracts from post-mortem brains of AD patients and controls in primary mouse neuron cultures. METHODS: Mice were immunised with stable oligomers derived from the Aß42 peptide with A21C/A30C mutations (AßCC), and ALZ-201 was developed using hybridoma technology. Specificity for the oligomeric form of the Aß42CC antigen and Aß42 was confirmed using ELISA, and non-reactivity against plaques by immunohistochemistry (IHC). The antibody's potential for cross-protective activity against pathological Aß was evaluated in brain tissue samples from 10 individuals confirmed as AD (n=7) and non-AD (n=3) with IHC staining for Aß and phosphorylated tau (p-Tau) aggregates. Brain extracts were prepared and immunodepleted using the positive control 4G8 antibody, ALZ-201 or an isotype control to ALZ-201. Fractions were biochemically characterised, and toxicity assays were performed in primary mouse neuronal cultures using automated high-content microscopy. RESULTS: AD brain extracts proved to be more toxic than controls as demonstrated by neuronal loss and morphological determinants (e.g. synapse density and measures of neurite complexity). Immunodepletion using 4G8 reduced Aß levels in both AD and control samples compared to ALZ-201 or the isotype control, which showed no significant difference. Importantly, despite the differential effect on the total Aß content, the neuroprotective effects of 4G8 and ALZ-201 immunodepletion were similar, whereas the isotype control showed no effect. CONCLUSIONS: ALZ-201 depletes a toxic species in post-mortem AD brain extracts causing a positive physiological and protective impact on the integrity and morphology of mouse neurons. Its unique specificity indicates that a low-abundant, soluble Aß42 oligomer may account for much of the neurotoxicity in AD. This critical attribute identifies the potential of ALZ-201 as a novel drug candidate for achieving a true, clinical therapeutic effect in AD.

Dynamics of Inflammatory and Neurodegenerative Biomarkers after Autologous Hematopoietic Stem Cell Transplantation in Multiple Sclerosis.

Autologous hematopoietic stem cell transplantation (aHSCT) is a highly efficient treatment of multiple sclerosis (MS), and hence it likely normalizes pathological and/or enhances beneficial processes in MS. The disease pathomechanisms include neuroinflammation, glial cell activation and neuronal damage. We studied biomarkers that in part reflect these, like markers for neuroinflammation (C-X-C motif chemokine ligand (CXCL) 9, CXCL10, CXCL13, and chitinase 3-like 1 (CHI3L1)), glial perturbations (glial fibrillary acidic protein (GFAP) and in part CHI3L1), and neurodegeneration (neurofilament light chain (NfL)) by enzyme-linked immunosorbent assays (ELISA) and single-molecule array assay (SIMOA) in the serum and cerebrospinal fluid (CSF) of 32 MS patients that underwent aHSCT. We sampled before and at 1, 3, 6, 12, 24 and 36 months after aHSCT for serum, as well as before and 24 months after aHSCT for CSF. We found a strong increase of serum CXCL10, NfL and GFAP one month after the transplantation, which normalized one and two years post-aHSCT. CXCL10 was particularly increased in patients that experienced reactivation of cytomegalovirus (CMV) infection, but not those with Epstein-Barr virus (EBV) reactivation. Furthermore, patients with CMV reactivation showed increased Th1 phenotype in effector memory CD4+ T cells. Changes of the other serum markers were more subtle with a trend for an increase in serum CXCL9 early post-aHSCT. In CSF, GFAP levels were increased 24 months after aHSCT, which may indicate sustained astroglia activation 24 months post-aHSCT. Other CSF markers remained largely stable. We conclude that MS-related biomarkers indicate neurotoxicity early after aHSCT that normalizes after one year while astrocyte activation appears increased beyond that, and increased serum CXCL10 likely does not reflect inflammation within the central nervous system (CNS) but rather occurs in the context of CMV reactivation or other infections post-aHSCT.

No evidence for cell-to-cell transmission of the unfolded protein response in cell culture.

The unfolded protein response (UPR) is one of the major cell-autonomous proteostatic stress responses. The UPR has been implicated in the pathogenesis of neurodegenerative diseases and is therefore actively investigated as therapeutic target. In this respect, cell non-autonomous effects of the UPR including the reported cell-to-cell transmission of UPR activity may be highly important. A pharmaca-based UPR induction was employed to generate conditioned media (CM) from CM-donating neuronal ('donor') cells (SK-N-SH and primary mouse neurons). As previously reported, upon subsequent transfer of CM to naive neuronal 'acceptor' cells, we confirmed UPR target mRNA and protein expression by qPCR and automated microscopy. However, UPR target gene expression was also induced in the absence of donor cells, indicating carry-over of pharmaca. Genetic induction of single pathways of the UPR in donor cells did not result in UPR transmission to acceptor cells. Moreover, no transmission was detected upon full UPR activation by nutrient deprivation or inducible expression of the heavy chain of immunoglobulin M in donor HeLa cells. In addition, in direct co-culture of donor cells expressing the immunoglobulin M heavy chain and fluorescent UPR reporter acceptor HeLa cells, UPR transmission was not observed. In conclusion, carry-over of pharmaca is a major confounding factor in pharmaca-based UPR transmission protocols that are therefore unsuitable to study cell-to-cell UPR transmission. In addition, the absence of UPR transmission in non-pharmaca-based models of UPR activation indicates that cell-to-cell UPR transmission does not occur in cell culture.

Granulovacuolar degeneration bodies are neuron-selective lysosomal structures induced by intracellular tau pathology.

Granulovacuolar degeneration bodies (GVBs) are membrane-bound vacuolar structures harboring a dense core that accumulate in the brains of patients with neurodegenerative disorders, including Alzheimer's disease and other tauopathies. Insight into the origin of GVBs and their connection to tau pathology has been limited by the lack of suitable experimental models for GVB formation. Here, we used confocal, automated, super-resolution and electron microscopy to demonstrate that the seeding of tau pathology triggers the formation of GVBs in different mouse models in vivo and in primary mouse neurons in vitro. Seeding-induced intracellular tau aggregation, but not seed exposure alone, causes GVB formation in cultured neurons, but not in astrocytes. The extent of tau pathology strongly correlates with the GVB load. Tau-induced GVBs are immunoreactive for the established GVB markers CK1δ, CK1ɛ, CHMP2B, pPERK, peIF2α and pIRE1α and contain a LAMP1- and LIMP2-positive single membrane that surrounds the dense core and vacuole. The proteolysis reporter DQ-BSA is detected in the majority of GVBs, demonstrating that GVBs contain degraded endocytic cargo. GFP-tagged CK1δ accumulates in the GVB core, whereas GFP-tagged tau or GFP alone does not, indicating selective targeting of cytosolic proteins to GVBs. Taken together, we established the first in vitro model for GVB formation by seeding tau pathology in primary neurons. The tau-induced GVBs have the marker signature and morphological characteristics of GVBs in the human brain. We show that GVBs are lysosomal structures distinguished by the accumulation of a characteristic subset of proteins in a dense core.