COVID-19 Induces Neuroinflammation and Suppresses Peroxisomes in the Brain.

OBJECTIVE: Peroxisome injury occurs in the central nervous system (CNS) during multiple virus infections that result in neurological disabilities. We investigated host neuroimmune responses and peroxisome biogenesis factors during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using a multiplatform strategy. METHODS: Brain tissues from coronavirus disease 2019 (COVID-19) (n = 12) and other disease control (ODC) (n = 12) patients, as well as primary human neural cells and Syrian hamsters, infected with a clinical variant of SARS-CoV-2, were investigated by droplet digital polymerase chain reaction (ddPCR), quantitative reverse transcriptase PCR (RT-qPCR), and immunodetection methods. RESULTS: SARS-CoV-2 RNA was detected in the CNS of 4 patients with COVID-19 with viral protein (NSP3 and spike) immunodetection in the brainstem. Olfactory bulb, brainstem, and cerebrum from patients with COVID-19 showed induction of pro-inflammatory transcripts (IL8, IL18, CXCL10, NOD2) and cytokines (GM-CSF and IL-18) compared to CNS tissues from ODC patients (p < 0.05). Peroxisome biogenesis factor transcripts (PEX3, PEX5L, PEX11ß, and PEX14) and proteins (PEX3, PEX14, PMP70) were suppressed in the CNS of COVID-19 compared to ODC patients (p < 0.05). SARS-CoV-2 infection of hamsters revealed viral RNA detection in the olfactory bulb at days 4 and 7 post-infection while inflammatory gene expression was upregulated in the cerebrum of infected animals by day 14 post-infection (p < 0.05). Pex3 transcript levels together with catalase and PMP70 immunoreactivity were suppressed in the cerebrum of SARS-CoV-2 infected animals (p < 0.05). INTERPRETATION: COVID-19 induced sustained neuroinflammatory responses with peroxisome biogenesis factor suppression despite limited brainstem SARS-CoV-2 neurotropism in humans. These observations offer insights into developing biomarkers and therapies, while also implicating persistent peroxisome dysfunction as a contributor to the neurological post-acute sequelae of COVID-19. ANN NEUROL 2023;94:531-546.

Genetic characterization of measles virus genotype D6 subacute sclerosing panencephalitis case, Alberta, Canada.

Subacute sclerosing panencephalitis (SSPE) is a progressive and eventually fatal neurological disease arising from a persistent infection with measles virus (MV) acquired at a young age. SSPE measles virus strains are defective and unable to produce progeny virions, due to multiple and extensive mutations in a number of key genes. We sequenced the full MV genome from our recently reported SSPE case, which typed as genotype D6, and compared it with other genotype D6 wild type and SSPE sequences. The Alberta D6 strain was significantly different from other reported SSPE D6 sequences. Mutations were observed in all the genes of the Alberta strain, with the greatest sequence divergence noted in the M gene with 17.6% nucleotide and 31% amino acid variation. The L gene showed the least variation with 1.3% nucleotide and 0.7% amino acid differences respectively. The nucleotide variability for 15,672 bases of the complete genome compared to the wild type and other SSPE D6 strains was around 3%.

Evaluation of kidney function and risk factors of retinopathy in Type 2 diabetes mellitus people in South Africa.

AIM: Diabetic retinopathy (DR) is a disease of the retina and an important microvascular complication of diabetes, frequently causing blindness in adults. The study aims to evaluate kidney function and risk factors for the development of retinopathy in Type 2 diabetes mellitus (DM) patients. METHODS: A total of 140 participants (54 Type 2DM without retinopathy, 44 with DR and 42 normal, healthy controls) consented to participate in the study. Fasting plasma glucose (FPG), glycosylated haemoglobin (HbA1c), lipid profile and renal function tests were measured using routine laboratory methods. RESULTS: A significant increase in FPG, HbA1c, serum triglycerides (TG), serum urea, urine albumin, the urine albumin/creatinine ratio (UACR) and, a significant decrease in high-density lipoprotein cholesterol (HDL-C) was observed in DR patients, compared to controls. In multivariate logistic analysis, FPG, insulin treatment and UACR were the risk factors found to be significantly associated with the presence of retinopathy. According to the Receiver Operating Characteristic (ROC) curve analysis, the diagnostic performance of UACR≥1.45mg/mmol-1l-1 was the optimal cut-off point. CONCLUSION: A mild alteration in renal function was observed in DR patients and UACR is found to be an important risk factor and can be a valuable marker for predicting DR.

Decreased total antioxidant levels and increased oxidative stress in South African type 2 diabetes mellitus patients

Background: Chronic hyperglycaemia in diabetes mellitus leads to increased lipid peroxidation in the body, followed by the development of chronic complications due to oxidative stress.Objective: The aim of this study was to compare total antioxidant (TAO) levels and oxidative stress in type 2 diabetes mellitus (T2DM) patients with that of healthy controls without diabetes.Methods: A total of 98 participants (57 T2DM and 41 healthy people) gave their consent and participated in the study. Routine biochemical methods were used for fasting plasma glucose (FPG), glycosylated haemoglobin (HbA1c) and lipid profile measurements. Serum TAO levels, malondialdehyde (MDA), oxidised low-density lipoprotein (ox-LDL) levels and superoxide dismutase (SOD) activity were analysed using standard commercial reagent kits.Results: A significant rise in FPG, HbA1c, triglycerides, MDA and ox-LDL, and a significant reduction in TAO and high-density lipoprotein cholesterol (HDL-C) was observed in T2DM patients compared with controls. A significant negative relationship was observed between TAO levels and MDA levels in the T2DM group. Increased lipid peroxidation and reduced antioxidant levels were observed in T2DM patients.Conclusion: Early management through an antioxidant-rich diet and lifestyle changes in T2DM patients would help to avert the debilitating complications of diabetes

Cotranslational protein folding within the ribosome tunnel influences trigger-factor recruitment.

In recent years, various folding zones within the ribosome tunnel have been identified and explored through x-ray, cryo-electron microscopy (cryo-EM), and molecular biology studies. Here, we generated ribosome-bound nascent polypeptide complexes (RNCs) with different polyalanine (poly-A) inserts or signal peptides from membrane/secretory proteins to explore the influence of nascent chain compaction in the Escherichia coli ribosome tunnel on chaperone recruitment. By employing time-resolved fluorescence resonance energy transfer and immunoblotting, we were able to show that the poly-A inserts embedded in the passage tunnel can form a compacted structure (presumably helix) and reduce the recruitment of Trigger Factor (TF) when the helical motif is located in the region near the tunnel exit. Similar experiments on nascent chains containing signal sequences that may form compacted structural motifs within the ribosome tunnel and lure the signal recognition particle (SRP) to the ribosome, provided additional evidence that short, compacted nascent chains interfere with TF binding. These findings shed light on the possible controlling mechanism of nascent chains within the tunnel that leads to chaperone recruitment, as well as the function of L23, the ribosomal protein that serves as docking sites for both TF and SRP, in cotranslational protein targeting.

Induction of amyloid fibrils by the C-terminal fragments of TDP-43 in amyotrophic lateral sclerosis.

TAR DNA-binding protein 43 (TDP-43) has been identified as the major ubiquitinated aggregates in the inclusion bodies in the patients of amyotrophic lateral sclerosis (ALS) since 2006 and become a crucial culprit for ALS and related motor neuron diseases. Recent literature has further indicated that the major components of these aggregates are hyper-phosphorylated TDP-43 C-terminus. In an effort to clarify the conformational and physical properties of its disordered C-terminal domain, we have synthesized several peptide fragments and shown that only D1 within D1-4 can form twisted fibrils with a cross section of approximately 11 nm in width under the incubation of phosphate buffer. In contrast, the D2-4 peptides all formed amorphous aggregates, showing different aggregation propensities. In addition to D1, two pathological mutant peptides, A315T and G294A, can also form fibrils that share similar shape and morphology with neuronal cytoplasmic inclusions. We propose that the residues with this region (287-322), which contains myriads of glycine repeats, may contribute significantly to the fiber formation as well as aggregation propensity. Moreover, from the conformational characterizations of D1, A315T, and G294A with EM, CD, fluorescence, and Raman spectroscopy, we found that all three peptides formed an amyloid structure, providing insights into the nature of its aggregation vis a vis the other fragments in the C-terminus of TDP-43.

Spinal cord lesions of progressive multifocal leukoencephalopathy in an acquired immunodeficiency syndrome patient.

Progressive multifocal leukoencephalopathy (PML) is a deadly demyelinating disease of the central nervous system, which occurs in immunosuppressed individuals. This disease is caused by a reactivation of the polyomavirus JC (JCV). Clinical presentation can be variable from patient to patient as lesions can occur anywhere in the CNS white matter; however, they appear to spare the optic nerves and the spinal cord. The authors present a case of PML in the setting of acquired immunodeficiency syndrome (AIDS) who developed PML lesions in the spinal cord, discovered during the postmortem examination. This finding is significant because PML has recently been diagnosed in patients with multiple sclerosis (MS) treated with the novel immunomodulatory medication natalizumab. Indeed, spinal cord lesions are frequent in MS. Therefore clinicians should be aware that in addition to the brain, PML may also affect the spinal cord white matter.

Effects of turn stability on the kinetics of refolding of a hairpin in a beta-sheet.

As part of our continuing study of the effects of the turn sequence on the conformational stability as well as the mechanism of folding of a beta-sheet structure, we have undertaken a parallel investigation of the solution structure, conformational stability, and kinetics of refolding of the beta-sheet VFIVDGOTYTEV(D)PGOKILQ. The latter peptide is an analogue of the original Gellman beta-sheet VFITS(D)PGKTYTEV(D)PGOKILQ, wherein the TS(D)PGK turn sequence in the first hairpin has been replaced by VDGO. Thermodynamics studies revealed comparable conformational stability of the two peptides. However, unlike the Gellman peptide, which showed extremely rapid refolding of the first hairpin, early kinetic events associated with the refolding of the corresponding hairpin in the VDGO mutant were found to be significantly slower. A detailed study of the conformation of the modified peptide suggested that hydrophobic interactions might be contributing to its stability. Accordingly, we surmise that the early kinetic events are sensitive to whether the formation of the hairpin is nucleated at the turn or by sequestering of the hydrophobic residues across the strand, before structural rearrangements to produce the nativelike topology. Nucleation of the hairpin at the turn is expected to be intrinsically rapid for a strong turn. However, if the process must involve collapse of hydrophobic side chains, the nucleation should be slower as solvent molecules must be displaced to sequester the hydrophobic residues. These findings reflect the contribution of different forces toward nucleation of hairpins in the mechanism of folding of beta-sheets.

Measuring the refolding of beta-sheets with different turn sequences on a nanosecond time scale.

Whether turns play an active or passive role in protein folding remains a controversial issue at this juncture. Here we use a photolabile cage strategy in combination with laser-flash photolysis and photoacoustic calorimetry to study the effects of different turns on the kinetics of beta-hairpin refolding on a nanosecond time scale. This strategy opens up a temporal window to allow the observation of early kinetic events in the protein refolding process at ambient temperature and pH without interference from any denaturants. Our results provide direct evidence demonstrating that even a one-residue difference in the turn region can change the refolding kinetics of a peptide. This observation suggests an active role for turn formation in directing protein folding.

Noncompliance after kidney transplantation: a systematic review.

We performed a systematic review of the literature on medical noncompliance after kidney transplantation in the cyclosporine era. We wished to define commonalities that may help the clinician identify patients for early intervention. We found that patients who were at a higher risk of noncompliance after kidney transplants were younger, female, unmarried, and non-Caucasians. Patients who were recipients of living donor transplants and had been transplanted for a longer time with a history of a previous transplant were also at risk of noncompliance. We also found that patients displaying emotional problems, such as anxiety, hostility, depression, distress, lack of coping, and avoidant behaviors, were also at risk for noncompliance after kidney transplantation.

Productive infection of cerebellar granule cell neurons by JC virus in an HIV+ individual.

BACKGROUND: In the setting of severe immunosuppression, the polyomavirus JC (JCV) can cause a lytic infection of oligodendrocytes. This demyelinating disease of the CNS white matter (WM) is called progressive multifocal leukoencephalopathy (PML). JCV has a very narrow host-cell range and productive infection of neurons has never been demonstrated. Patient, methods, and results: An HIV-1-infected patient presented with signs of pyramidal tract and cerebellar dysfunction. Brain MRI revealed T2 hyperintensities in the WM of both frontal lobes and cerebellar atrophy. His disease progressed despite therapy and he died 6 months later. In addition to classic PML findings in the frontal lobe WM, autopsy revealed scattered foci of tissue destruction in the internal granule cell layer (IGCL) of the cerebellum. In these foci, enlarged granule cell neurons identified by the neuronal markers MAP-2 and NeuN reacted with antibodies specific for the polyomavirus VP1 capsid protein. Electron microscopy showed 40 nm viral particles, consistent with polyomaviruses, in these granule cell neurons. In addition, JCV DNA was detected by PCR after laser capture microdissection of cells from the areas of focal cell loss. Finally, in situ hybridization studies demonstrated that many granule cell neurons were infected with JCV but did not contain viral proteins. Sequence analysis of the JCV regulatory region from cerebellar virions showed a tandem repeat pattern also found in PML lesions of the frontal lobe WM. CONCLUSION: JCV can productively infect granule cell neurons of the IGCL of the cerebellum. This suggests a role for JCV infection of neurons in cerebellar atrophy occurring in HIV-infected individuals.

Influence of dialysis on post-transplant events.

INTRODUCTION: We examined the effect of haemodialysis (HD) or peritoneal dialysis (PD) on acute rejection, delayed graft function (DGF), graft and patient survival after cadaveric renal transplantation. MATERIALS AND METHODS: We carried out a retrospective analysis of 325 patients (cyclosporin [CyA]-based therapy) who had their first cadaver renal transplant between January 1991 and December 1996 and followed up for a mean of 61 +/- 26 months. They were divided into three groups: HD, PD and CD (where both PD and HD was used for at least 3 months). Delayed graft function was diagnosed if the patient needed dialysis in the first week post-transplant while primary non-function (PNF) was diagnosed if the kidney never achieved function. Graft rejection was confirmed by biopsy; early acute rejection (EAR) was defined as acute rejection occurring before 90 days and late acute rejection (LAR) as one after 90 d. RESULTS: A total of 183 patients had PD, 117 HD and 25 CD. The mean time period in which the patients were on dialysis for PD was 24 months, HD 34.5 months and CD 50.6 months (p < 0.01). The recipients were matched for age and gender. The donor variables (age, gender and cold ischaemia time) did not differ between the groups. The mean time for the development of first acute rejection following renal transplant in each group was as follows: PD group: 68.8 d, HD group: 81.3 d and CD group: 105 d (p = 0.08). The number of patients who developed EAR was 90 (49.2%) in PD group, 51 (43.6%) in HD group and 11 (56%) in CD group (p = 0.6); the number who developed LAR was nine in PD group (4.9%), six in HD group (5.1%) and one in CD group (4%) (p = 0.9). Fifty-six patients with PD had DGF compared with 58 with HD (p = 0.01). There was no difference in the number and severity of rejection episodes or DGF based on the duration of dialysis. The 5-yr survival of patients was 79% for PD, 81% HD and 78% CD groups (p = n.s), while the graft survival for PD group was 61%, HD group 63% and CD group 74% (p = n.s). SUMMARY: We could find no difference in the patient or graft survival between patients who had pre-transplant HD, PD or CD. There was no difference in the incidence of acute rejection episodes between the three groups of patients as well. However, we found a significantly higher rate of DGF in the HD versus PD patients.

The impact of late acute rejection after cadaveric kidney transplantation.

BACKGROUND: Acute graft rejection (AR) following renal transplantation results in reduced graft survival. However, there is uncertainty regarding the definition, aetiology and long-term graft and patient outcome of AR occurring late in the post-transplant period. AIM: To determine if rejection episodes can be classified by time from transplantation by their impact on graft survival into early acute rejection (EAR) and late acute rejection (LAR). MATERIALS AND METHODS: 687 consecutive adult renal transplant recipients who received their first cadaveric renal transplant at a single centre. All received cyclosporine (CyA)-based immunosuppression, from 1984 to 1996, with a median follow-up of 6.9 yr. Details were abstracted from clinical records, with emphasis on age, sex, co-morbid conditions, HLA matching, rejection episodes, patient and graft survival. ANALYSIS: Patients were classified by the presence and time to AR from the date of transplantation. Using those patients who had no AR (NAR) as a baseline, we determined the relative risk of graft failure by time to rejection. The characteristics of patients who had no rejection, EAR and LAR were compared. RESULTS: Compared with NAR, the risk of graft failure was higher for those patients who suffered a rejection episode. A much higher risk of graft failure was seen when the first rejection episode occurred after 90 d. Thus, a period of 90 d was taken to separate EAR and LAR (relative risk of 3.06 and 5.27 compared with NAR as baseline, p<0.001). Seventy-eight patients (11.4%) had LAR, 271 (39.4%) had EAR and 338 (49.2%) had NAR. The mean age for each of these groups differed (LAR 39.6 yr, EAR 40.8 yr compared with NAR 44 yr, p<0.003). The 5-yr graft survival for those who had LAR was 45% and 10-yr survival was 28%. HLA mismatches were more frequent in those with EAR vs. NAR (zero mismatches in HLA-A: 36 vs. 24%, HLA-B: 35 vs. 23% and HLA-DR: 63 vs. 41%, p<0.003). There was no difference in mismatching frequency between NAR and LAR. CONCLUSIONS: AR had a deleterious impact on graft survival, particularly if occurring after 90 d. AR episodes should therefore be divided into early and late phases. In view of the very poor graft survival associated with LAR, it is important to gain further insight into the main aetiological factors. Those such as suboptimal CyA blood levels and non-compliance with medication should be further investigated with the aim of developing more effective immunosuppressive regimens in order to reduce the incidence of LAR.

JCV-specific cellular immune response correlates with a favorable clinical outcome in HIV-infected individuals with progressive multifocal leukoencephalopathy.

Most immunosuppressed individuals who develop progressive multifocal leukoencephalopathy (PML) have a rapid fatal outcome, whereas some become long-term survivors. We explored the impact of the cellular immune response against JC virus (JCV) on the clinical outcome of 7 HIV+ and 3 HIV- individuals with PML. Of the 4 HIV+/PML survivors, all had detectable cytotoxic T lymphocytes (CTL) specific for JCV T or VP 1 proteins compared to none of the 3 HIV+/PML progressors tested. Of the 3 HIV-/PML patients, 1 was recently diagnosed with PML and showed evidence of neurologic improvement without any treatment. This patient had CTL specific for the VP1 protein of JCV. The other 2 HIV-/PML survivors were stable 3-8 years after the diagnosis of PML. They did not have any detectable CTL against JCV. These findings suggest that JCV-specific immune response is associated with favorable outcome in HIV+ individuals with PML. The lack of detectable JCV-specific CTL in 2 HIV-/PML survivors might indicate a burnt-out disease without sufficient antigenic stimulation to maintain the cellular immune response. The detection of JCV-specific CTL in an HIV- patient recently diagnosed with PML, who was showing evidence of neurological improvement without any treatment, indicates that this finding may be used as a favorable prognostic marker of disease evolution in the clinical management of patients with PML. As the quest for an effective treatment of PML continues, JCV-specific cellular immune response deserves further attention because it appears to play a crucial role in the prevention of disease progression.