Immunological memory diversity in the human upper airway.

The upper airway is an important site of infection, but immune memory in the human upper airway is poorly understood, with implications for COVID-19 and many other human diseases1-4. Here we demonstrate that nasal and nasopharyngeal swabs can be used to obtain insights into these challenging problems, and define distinct immune cell populations, including antigen-specific memory B cells and T cells, in two adjacent anatomical sites in the upper airway. Upper airway immune cell populations seemed stable over time in healthy adults undergoing monthly swabs for more than 1 year, and prominent tissue resident memory T (TRM) cell and B (BRM) cell populations were defined. Unexpectedly, germinal centre cells were identified consistently in many nasopharyngeal swabs. In subjects with SARS-CoV-2 breakthrough infections, local virus-specific BRM cells, plasma cells and germinal centre B cells were identified, with evidence of local priming and an enrichment of IgA+ memory B cells in upper airway compartments compared with blood. Local plasma cell populations were identified with transcriptional profiles of longevity. Local virus-specific memory CD4+ TRM cells and CD8+ TRM cells were identified, with diverse additional virus-specific T cells. Age-dependent upper airway immunological shifts were observed. These findings provide new understanding of immune memory at a principal mucosal barrier tissue in humans.

Single-neuron sequencing analysis of L1 retrotransposition and somatic mutation in the human brain.

A major unanswered question in neuroscience is whether there exists genomic variability between individual neurons of the brain, contributing to functional diversity or to an unexplained burden of neurological disease. To address this question, we developed a method to amplify genomes of single neurons from human brains. Because recent reports suggest frequent LINE-1 (L1) retrotransposition in human brains, we performed genome-wide L1 insertion profiling of 300 single neurons from cerebral cortex and caudate nucleus of three normal individuals, recovering >80% of germline insertions from single neurons. While we find somatic L1 insertions, we estimate <0.6 unique somatic insertions per neuron, and most neurons lack detectable somatic insertions, suggesting that L1 is not a major generator of neuronal diversity in cortex and caudate. We then genotyped single cortical cells to characterize the mosaicism of a somatic AKT3 mutation identified in a child with hemimegalencephaly. Single-neuron sequencing allows systematic assessment of genomic diversity in the human brain.

Late Pleistocene horse and camel hunting at the southern margin of the ice-free corridor: reassessing the age of Wally's Beach, Canada.

The only certain evidence for prehistoric human hunting of horse and camel in North America occurs at the Wally's Beach site, Canada. Here, the butchered remains of seven horses and one camel are associated with 29 nondiagnostic lithic artifacts. Twenty-seven new radiocarbon ages on the bones of these animals revise the age of these kill and butchering localities to 13,300 calibrated y B.P. The tight chronological clustering of the eight kill localities at Wally's Beach indicates these animals were killed over a short period. Human hunting of horse and camel in Canada, coupled with mammoth, mastodon, sloth, and gomphothere hunting documented at other sites from 14,800-12,700 calibrated y B.P., show that 6 of the 36 genera of megafauna that went extinct by approximately 12,700 calibrated y B.P. were hunted by humans. This study shows the importance of accurate geochronology, without which significant discoveries will go unrecognized and the empirical data used to build models explaining the peopling of the Americas and Pleistocene extinctions will be in error.

New evidence for, and challenges in, linking small CGG repeat expansion FMR1 alleles with Parkinson's disease.

We recently reported a significant increase in the frequency of carriers of grey zone (GZ) alleles of FMR1 gene in Australian males with Parkinson's disease (PD) from Victoria and Tasmania. Here, we report data comparing an independent sample of 817 PD patients from Queensland to 1078 consecutive Australian male newborns from Victoria. We confirmed the earlier finding by observing a significant excess of GZ alleles in PD (4.8%) compared to controls (1.5%). Although both studies provided evidence in support of an association between GZ-carrier status and increased risk for parkinsonism, the existing evidence in the literature from screening studies remains equivocal and we discuss the need for alternative approaches to resolve the issue.

Pathogenic aberrations revealed exclusively by single nucleotide polymorphism (SNP) genotyping data in 5000 samples tested by molecular karyotyping.

BACKGROUND: Several recent studies have demonstrated the use of single nucleotide polymorphism (SNP) arrays for the investigation of intellectual disability, developmental delay, autism or congenital abnormalities. In addition to LogR 'copy number' data, these arrays provide SNP genotyping data for gene level autozygosity mapping, estimating low levels of mosaicism, assessing long continuous stretches of homozygosity (LCSH), detection of uniparental disomy, and 'autozygous' regions. However, there remains little specific information on the clinical utility of this genotyping data. METHODS: Molecular karyotyping, using SNP array, was performed on 5000 clinical samples. RESULTS: Clinically significant 'LogR neutral' genotyping abnormalities were detected in 0.5% of cases. Among these were a single case of chimerism, 12 cases with low level chromosome mosaicism, and 11 cases with an LCSH associated with uniparental disomy. In addition, the genotyping data revealed several LCSH associated with clinically relevant 'recessive type' genetic defects. CONCLUSIONS: These results demonstrate the utility of SNP genotyping data for detection of clinically significant abnormalities, including chimerism/mosaicism and recessive Mendelian disorders associated with autozygosity. The incidence of clinically significant low level mosaicism inferred from these cases suggests that this has hitherto been underestimated and chromosome mosaicism frequently occurs in the absence of indicative clinical features. The growing appreciation among clinicians and demand for SNP genotyping data poses significant challenges for the interpretation of LCSH, especially where there is no detailed phenotypic description to direct laboratory analysis. Finally, reporting of unexpected or hidden consanguinity revealed by SNP array analysis raises potential ethical and legal issues.

Diverse Roles of Akt in T cells.

Akt kinases translate various external cues into intracellular signals that control cell survival, proliferation, metabolism and differentiation. This review discusses the requirement for Akt and its targets in determining the fate and function of T cells. We discuss the importance of Akt at various stages of T cell development including ß-selection during which Akt fulfills the energy requirements of highly proliferative DN3 cells. Akt also plays an integral role in CD8 T cell biology where its regulation of Foxo transcription factors and mTORC1 metabolic activity controls effector versus memory CD8 T cell differentiation. Finally, Akt promotes the differentiation of naïve CD4 T cells into Th1, Th17 and Tfh cells but inhibits the development of Treg cells. We also highlight how modulating Akt in T cells is a promising avenue for enhancing cell-based cancer immunotherapy.

MiR-338-3p regulates neuronal maturation and suppresses glioblastoma proliferation.

Neurogenesis is a highly-regulated process occurring in the dentate gyrus that has been linked to learning, memory, and antidepressant efficacy. MicroRNAs (miRNAs) have been previously shown to play an important role in the regulation of neuronal development and neurogenesis in the dentate gyrus via modulation of gene expression. However, this mode of regulation is both incompletely described in the literature thus far and highly multifactorial. In this study, we designed sensors and detected relative levels of expression of 10 different miRNAs and found miR-338-3p was most highly expressed in the dentate gyrus. Comparison of miR-338-3p expression with neuronal markers of maturity indicates miR-338-3p is expressed most highly in the mature neuron. We also designed a viral "sponge" to knock down in vivo expression of miR-338-3p. When miR-338-3p is knocked down, neurons sprout multiple primary dendrites that branch off of the soma in a disorganized manner, cellular proliferation is upregulated, and neoplasms form spontaneously in vivo. Additionally, miR-338-3p overexpression in glioblastoma cell lines slows their proliferation in vitro. Further, low miR-338-3p expression is associated with increased mortality and disease progression in patients with glioblastoma. These data identify miR-338-3p as a clinically relevant tumor suppressor in glioblastoma.

Protective effect of enprostil against aspirin-induced gastroduodenal mucosal injury in man. Comparison with cimetidine and sucralfate.

A single-blind endoscopic study was undertaken to test the relative efficacy of enprostil, a synthetic analogue of prostaglandin E2, cimetidine, and sucralfate in the prevention of aspirin-induced gastroduodenal mucosal injury. Fifty healthy, non-smoking male volunteers completed the study after having been randomly assigned to receive two weeks of therapy with one of the following regimens: enprostil 35 micrograms twice daily; enprostil 35 micrograms in the morning; cimetidine 200 mg three times daily and 400 mg at night; sucralfate 1 g four times daily; or placebo. In the second week, aspirin (900 mg three times daily) was also administered. Endoscopies were performed before and after the aspirin phase of the study, and lesions (mucosal erosions plus submucosal hemorrhages) were counted in the stomach and duodenal bulb. All treatments were superior to placebo (p less than 0.05). The mean number of lesions in the 70-micrograms enprostil group (8.5) was significantly less than in the 35-micrograms enprostil group, (11.1), the sucralfate group (12.4), or the placebo group (16.0); the benefit over cimetidine (10.1), however, was not statistically significant. The protective effect of enprostil was greatest in the antrum, the site of maximal mucosal injury. Gastrointestinal side effects were reported in all groups, though abdominal pain and dyspepsia were noted more frequently in those taking enprostil.

Influence of anterograde flow in the infarct artery on the incidence of late potentials after acute myocardial infarction.

In patients after myocardial infarction, survival is influenced by the presence or absence of anterograde flow in the infarct artery, and late potentials on signal-averaged electrocardiography identify those at risk for tachyarrhythmias and sudden death. To assess the frequency of late potentials in survivors of first infarction, coronary arteriography and signal-averaged electrocardiography were performed in 109 subjects (64 men, 45 women, aged 30 to 77 years), 49 with (group I) and 60 without (group II) anterograde flow in the infarct artery. The groups were similar in age, sex, infarct artery, severity of coronary artery disease and left ventricular function. However, only 4 (8%) of group I had late potentials, whereas 24 (40%) of group II had late potentials (p less than 0.001). Thus, anterograde flow in the infarct artery after myocardial infarction is associated with a low incidence of late potentials on signal-averaged electrocardiography, whereas the absence of anterograde flow is more often associated with late potentials.

X chromosome inactivation in fibroblasts of mentally retarded female carriers of the fragile site Xq27.3: application of the probe M27 beta to evaluate X inactivation status.

Over 30% of female carriers of the fragile X [fra(X)] syndrome are clinically affected. A nonrandom X chromosome inactivation in these cases could be a plausible explanation. A review of previous studies addressing this question showed inconclusive results; thus, we analysed the X inactivation pattern in fibroblasts of 4 unrelated, mentally retarded fra(X) carriers with a high expression of the fragile site Xq27.3. Using Southern analysis with a highly polymorphic probe M27 beta that recognizes methylation differences between the active and inactive X chromosome we found a 50/50 inactivation pattern in 2 cases and skewed patterns in the other 2. As biased patterns were also observed in control females we conclude that at present no evidence exists for a nonrandom X chromosome inactivation in the fra(X) syndrome in females.

Risk factors for recurrence of hemorrhagic endovasculitis of the placenta.

OBJECTIVE: To assess the potential for recurrence of placental hemorrhagic endovasculitis and to identify clinical or pathologic cofactors that might influence recurrence of this lesion or subsequent pregnancy outcome. METHODS: Ninety-seven women with a placenta affected by hemorrhagic endovasculitis, who also had at least one placenta referred to the Michigan Placental Tissue Registry from a subsequent pregnancy, were identified from 10,531 referrals between 1978 and 1988. Histologic slides from 209 placentas and clinical data from 211 infants (two sets of twins) from initial (first) and subsequent referrals were analyzed. Placentas were graded for the presence, extent, and severity of hemorrhagic endovasculitis and chronic villitis of unknown etiology; for placental lesions indicative of hypertensive maternal vessel disease; and for intravascular nucleated erythrocytes and chorionic thrombi. Maternal data included age, gravidity, number of previous losses, and history of toxemia or hypertension. All data were analyzed for significance using chi2 and t tests. Outcome assessment was based on recurrence of hemorrhagic endovasculitis and infant viability with the second referral. RESULTS: With first referrals, 80 of 98 infants (81.6%) were stillborn. Among second referrals, 26 of 98 infants (26.5%) were stillborn. Hemorrhagic endovasculitis recurred in 28 second placentas (28.9%); of these, 18 infants (64.3%) were stillborn. Higher rates of recurrence were found with progressively higher first-referral chronic villitis severity scores (P < .02), higher hypertensive placental lesion scores (P < .001), and first referrals with a history of toxemia or hypertension (P < .02). Recurrence of hemorrhagic endovasculitis was higher in patients with two or more of these factors in first referrals (P < .001). Subsequent stillbirth was more frequent with progressively higher first-referral hypertensive placental lesion scores (P < .01) and in first placentas with two or more risk factors (P = .064). Hemorrhagic endovasculitis severity scores, intravascular nucleated erythrocytes, and chorionic thrombi were associated with stillbirth in index pregnancies only. Maternal age, gravidity, or history of prior losses were not predictive. CONCLUSIONS: Placental hemorrhagic endovasculitis is associated with pregnancy loss and can recur in some patients. Interrelations among placental hemorrhagic endovasculitis, chronic villitis, maternal hypertension, and adverse outcomes in subsequent pregnancies are apparent. This information may be useful in patient counseling.

Automated fibronectin assay using light-scattering optics on a centrifugal analyzer.

We describe an automated, kinetic nephelometric method for fibronectin on the Multistat III F/LS Centrifugal Analyzer (Instrumentation Laboratory Inc., Lexington, MA 02173). Antiserum is diluted with polyethylene glycol. Calibrators and samples are prediluted with potassium phosphate buffer (10 mmol/L, pH 7.0) containing 8.5 g of NaCl per litre. Intensity (I) is read at 5 and 180 s and the resulting delta I plotted against concentration. A non-linear least squares curve fitting and interpolation of results is carried out automatically. Three controls, with values of between 190-370 gave coefficients of variation between 4 and 7 percent, and the sensitivity of the method is to 25 mg/L.

Fluorometric measurement of serum magnesium with a centrifugal analyzer.

We developed a rapid fluorometric assay for measurement of serum magnesium using the ligand, 8-hydroxy-quinoline-5-sulfonate and adapted the procedure to the Multistat Fluorescence Light Scattering Centrifugal Analyzer. The standard curve extends from 0.26 to 4.11 mmol/l. There was no interference from calcium or inorganic phosphorus at concentrations of 4.95 and 5.0 mmol/l, lead, iron, zinc or copper at twice the normal levels found in serum, bilirubin at concentrations of 10 mg/dl, or lipemic samples with triglyceride concentrations of 2400 mg/dl. Citrate and EDTA lowered magnesium concentrations in serum. Analytical recovery of magnesium added to four serum specimens averaged 97%. Between-run and within-run precision of the assay gave CVs which ranged from 2.9 to 7.6%. Magnesium concentrations, measured by our fluorometric procedure, were compared with those obtained by atomic absorption and colorimetric procedures. Correlation coefficients of 0.91 and 0.88 were obtained.

Deletions in GRID2 lead to a recessive syndrome of cerebellar ataxia and tonic upgaze in humans.

OBJECTIVE: To identify the genetic cause of a syndrome causing cerebellar ataxia and eye movement abnormalities. METHODS: We identified 2 families with cerebellar ataxia, eye movement abnormalities, and global developmental delay. We performed genetic analyses including single nucleotide polymorphism genotyping, linkage analysis, array comparative genomic hybridization, quantitative PCR, and Sanger sequencing. We obtained eye movement recordings of mutant mice deficient for the ortholog of the identified candidate gene, and performed immunohistochemistry using human and mouse brain specimens. RESULTS: All affected individuals had ataxia, eye movement abnormalities, most notably tonic upgaze, and delayed speech and cognitive development. Homozygosity mapping identified the disease locus on chromosome 4q. Within this region, a homozygous deletion of GRID2 exon 4 in the index family and compound heterozygous deletions involving GRID2 exon 2 in the second family were identified. Grid2-deficient mice showed larger spontaneous and random eye movements compared to wild-type mice. In developing mouse and human cerebella, GRID2 localized to the Purkinje cell dendritic spines. Brain MRI in 2 affected children showed progressive cerebellar atrophy, which was more severe than that of Grid2-deficient mice. CONCLUSIONS: Biallelic deletions of GRID2 lead to a syndrome of cerebellar ataxia and tonic upgaze in humans. The phenotypic resemblance and similarity in protein expression pattern between humans and mice suggest a conserved role for GRID2 in the synapse organization between parallel fibers and Purkinje cells. However, the progressive and severe cerebellar atrophy seen in the affected individuals could indicate an evolutionarily unique role for GRID2 in the human cerebellum.

Somatic activation of AKT3 causes hemispheric developmental brain malformations.

Hemimegalencephaly (HMG) is a developmental brain disorder characterized by an enlarged, malformed cerebral hemisphere, typically causing epilepsy that requires surgical resection. We studied resected HMG tissue to test whether the condition might reflect somatic mutations affecting genes critical to brain development. We found that two out of eight HMG samples showed trisomy of chromosome 1q, which encompasses many genes, including AKT3, a gene known to regulate brain size. A third case showed a known activating mutation in AKT3 (c.49G→A, creating p.E17K) that was not present in the patient's blood cells. Remarkably, the E17K mutation in AKT3 is exactly paralogous to E17K mutations in AKT1 and AKT2 recently discovered in somatic overgrowth syndromes. We show that AKT3 is the most abundant AKT paralog in the brain during neurogenesis and that phosphorylated AKT is abundant in cortical progenitor cells. Our data suggest that somatic mutations limited to the brain could represent an important cause of complex neurogenetic disease.