Cellular and humoral immunogenicity of the COVID-19 vaccine and COVID-19 disease severity in individuals with immunodeficiency.

Background: A well-coordinated adaptive immune response is crucial for limiting COVID-19 disease. Some individuals with immunodeficiency are at a high risk of developing severe COVID-19. Therefore, the development of standardized methods for measuring different arms of the vaccine response in the setting of immunodeficiency is of particular interest. In this study, we compared the vaccine response of individuals living with immunodeficiency with healthy controls in terms of interferon gamma (IFN-γ) production and spike protein-specific antibody level post primary COVID-19 vaccination and booster vaccines. Additionally, the disease severity of those individuals who contracted COVID-19 was assessed. Methods: Whole blood was stimulated overnight from 71 participants and 99 healthy controls. Commercially available PepTivator® peptide pool and trimeric spike protein stimulation were used. ELISA was used to analyze IFN-γ levels. The total SARS-CoV-2 spike protein antibody titre was measured using a Roche Elecsys® S total antibody assay. Patient characteristics, COVID-19 infection status and IDDA 2.1 'Kaleidoscope' scores were recorded. Vaccine responses were scored from zero to three. Results: 99% of healthy controls, 89% of individuals with IEI and 76% with secondary immunodeficiency (SID) had an IFN-γ level above the validated reference range after peptide mix stimulation following primary vaccination. There was an increase in IFN-γ levels in patients with inborn errors of immunity (IEI) following the booster vaccine (p = 0.0156). 100% of healthy controls, 70% of individuals living with IEI and 64% of individuals living with SID had detectable spike protein-specific antibody levels following the primary vaccination. 55% of immunodeficiency patients who had mild COVID-19 and 10% with moderate/severe COVID-19 had detectable antibody and IFN-γ levels post vaccine. The mean pre-infection IDDA 2.1 scores were higher in individuals who developed moderate/severe COVID-19 (25.2 compared to 9.41). Conclusions: Covid whole-blood IGRA is a highly accurate, straightforward and robust assay and can be easily adapted to measure cellular response to COVID-19. A complete evaluation of the vaccine response may be particularly important for individuals living with immunodeficiency. A clinical immunodeficiency score and a validated vaccine response score may be valuable tools in estimating COVID-19 disease risk and identifying individuals living with immunodeficiency who may benefit from enhanced vaccination schedules.

High-resolution three-dimensional structural microscopy by single-angle Bragg ptychography.

Coherent X-ray microscopy by phase retrieval of Bragg diffraction intensities enables lattice distortions within a crystal to be imaged at nanometre-scale spatial resolutions in three dimensions. While this capability can be used to resolve structure-property relationships at the nanoscale under working conditions, strict data measurement requirements can limit the application of current approaches. Here, we introduce an efficient method of imaging three-dimensional (3D) nanoscale lattice behaviour and strain fields in crystalline materials with a methodology that we call 3D Bragg projection ptychography (3DBPP). This method enables 3D image reconstruction of a crystal volume from a series of two-dimensional X-ray Bragg coherent intensity diffraction patterns measured at a single incident beam angle. Structural information about the sample is encoded along two reciprocal-space directions normal to the Bragg diffracted exit beam, and along the third dimension in real space by the scanning beam. We present our approach with an analytical derivation, a numerical demonstration, and an experimental reconstruction of lattice distortions in a component of a nanoelectronic prototype device.

Efficient modeling of Bragg coherent x-ray nanobeam diffraction.

X-ray Bragg diffraction experiments that utilize tightly focused coherent beams produce complicated Bragg diffraction patterns that depend on scattering geometry, characteristics of the sample, and properties of the x-ray focusing optic. Here, we use a Fourier-transform-based method of modeling the 2D intensity distribution of a Bragg peak and apply it to the case of thin films illuminated with a Fresnel zone plate in three different Bragg scattering geometries. The calculations agree well with experimental coherent diffraction patterns, demonstrating that nanodiffraction patterns can be modeled at nonsymmetric Bragg conditions with this approach--a capability critical for advancing nanofocused x-ray diffraction microscopy.

Coherent Bragg nanodiffraction at the hard X-ray Nanoprobe beamline.

Bragg coherent diffraction with nanofocused hard X-ray beams provides unique opportunities for quantitative in situ studies of crystalline structure in nanoscale regions of complex materials and devices by a variety of diffraction-based techniques. In the case of coherent diffraction imaging, a major experimental challenge in using nanoscale coherent beams is maintaining a constant scattering volume such that coherent fringe visibility is maximized and maintained over the course of an exposure lasting several seconds. Here, we present coherent Bragg diffraction patterns measured from different nanostructured thin films at the Sector 26 Nanoprobe beamline at the Advanced Photon Source and demonstrate that with nanoscale positional control, coherent diffraction patterns can be measured with source-limited fringe visibilities more than 50% suitable for imaging by coherent Bragg ptychography techniques.

Quantitative nanoscale imaging of lattice distortions in epitaxial semiconductor heterostructures using nanofocused X-ray Bragg projection ptychography.

We imaged nanoscale lattice strain in a multilayer semiconductor device prototype with a new X-ray technique, nanofocused Bragg projection ptychography. Applying this technique to the epitaxial stressor layer of a SiGe-on-SOI structure, we measured the internal lattice behavior in a targeted region of a single device and demonstrated that its internal strain profile consisted of two competing lattice distortions. These results provide the strongest nondestructive test to date of continuum modeling predictions of nanoscale strain distributions.

Implications of eosinophilia in the normal duodenal biopsy - an association with allergy and functional dyspepsia.

BACKGROUND: Allergy and functional gastrointestinal disorders have been associated with eosinophilia in duodenal mucosa. AIM: To assess the prevalence of eosinophilia in duodenal biopsies of patients attending for oesophogastroduodenoscopy and delineate associated clinical conditions. METHODS: A total of 155 patients (mean age 55 years, 59% women) with normal duodenal biopsies were randomly selected for audit from histopathology files. Eosinophil counts in five high power fields (HPFs) were assessed. Records were analysed for symptoms, diagnosis and medications; patients were divided into five groups based on upper gastrointestinal (UGI) symptom profiles, including a control group of those without predominant UGI symptoms. The prevalence of duodenal eosinophilia (defined as >22/5HPFs a priori) was calculated. RESULTS: In the control group, the mean duodenal eosinophil count was 15/5HPFs; prevalence of duodenal eosinophilia was 22.5%. In postprandial distress syndrome (PDS), both mean eosinophil counts (20.2/5HPF, P < 0.04) and prevalence of duodenal eosinophilia (47.3%, P < 0.04) were significantly higher. Duodenal eosinophilia was significantly associated with allergy (OR 5.04, 95% CI 2.12-11.95, P < 0.001). There was no association with irritable bowel syndrome or medications. CONCLUSIONS: Subtle duodenal eosinophilia is relatively common in routine oesophogastroduodenoscopy and previously overlooked; it is associated with allergy and may indicate a hypersensitivity mechanism in some patients with PDS including early satiety.

A technique for the orientation of endoscopically resected laryngeal lesions.

Endoscopic resection is a widely accepted method of treating early laryngeal malignancies. Accurate histological assessment of resected laryngeal specimens can be difficult due to their small size and potential damage and distortion caused by standard orientation and processing techniques. A new technique is described which employs dehydrated cucumber to mount laryngeal specimens for orientation and processing. This technique is quick, easy, cheap and reliable, allowing accurate histological assessment of potentially malignant lesions. Better orientating and processing of laryngeal specimens can optimise treatment decisions based on histological results.

Use of backscattered electron detector arrays for forming backscattered electron images in the scanning electron microscope.

The backscattered electron (BSE) signal in the scanning electron microscope (SEM) can be used in two different ways. The first is to give a BSE image from an area that is defined by the scanning of the electron beam (EB) over the surface of the specimen. The second is to use an array of small BSE detectors to give an electron backscattering pattern (EBSP) with crystallographic information from a single point. It is also possible to utilize the EBSP detector and computer-control system to give an image from an area on the specimen--for example, to show the orientations of the grains in a polycrystalline sample ("grain orientation imaging"). Some further possibilities based on some other ways for analyzing the output from an EBSP detector array, are described.

Sympathetic nervous system activity in adipose tissues during pregnancy and lactation of the rat.

The concentration and turnover of norepinephrine in white adipose and liver tissues were determined in pregnant, lactating, and age-matched virgin rats to elucidate the adaptations in sympathetic nervous system activity. In study 1, at d 18 of pregnancy and d 7 and 21 of lactation, animals were killed, and liver and cardiac perimetrial and retroperitoneal adipose depots were quick-frozen and then assayed for norepinephrine as a gross estimate of sympathetic innervation. In study 2, the same design was used to measure the turnover of norepinephrine as a measure of sympathetic activity. Animals were treated with alpha-methylparatyrosine, an inhibitor of norepinephrine synthesis, and killed at 0, 1.5, and 3 h after injection. In pregnant animals, basal norepinephrine concentrations were decreased compared with unbred controls in perimetrial and retroperitoneal depots. By d 21 of lactation, all adipose depots from lactating animals had more norepinephrine than did controls. The turnover of norepinephrine decreased in noncardiac adipose depots of pregnant animals. By d 21 of lactation, norepinephrine concentration was greater in all of the adipose depots than in controls. The turnover rate was faster in all adipose tissue depots but only significantly different in the cardiac depot. Sympathetic nervous activity in adipose tissue is diminished in pregnant rats, presumably to save energy for fetal growth and maternal fat storage. In late lactation, activity is increased, presumably to direct fatty acids away from adipose tissue towards milk production. The data from this study are consistent with the hypothesis that the sympathetic nervous system plays a role in the regulation of adipose metabolism in lactation.

An algorithm for evaluating human cytotoxic T lymphocyte responses to candidate AIDS vaccines.

Development of an effective vaccine against HIV-1 will likely require the induction of a broad array of immune responses, including virus-specific CTLs and neutralizing antibodies. One promising vaccine approach involves live recombinant canarypox (CP)-based vectors (ALVAC) containing multiple HIV-1 genes. In phase I clinical trials in HIV-1-seronegative volunteers, the cumulative rate of detection of HIV-1-specific CTLs has been as high as 60-70%. In the present study, the factors associated with CTL responsiveness were evaluated in a subset of vaccinees immunized with a CP vector expressing portions of the gag, pro, and env genes of HIV-1 (ALVAC-HIV). CTL responses were detected in one of seven examined. While the responding individual had both CD4+ and CD8+ CTLs directed at multiple HIV-1 antigens, this response was not detectable 1 year after the last vaccination. In-depth characterization of "CTL nonresponders" showed that nonresponsiveness was not associated with defects in antigen processing or presentation. A generalized defect in CTL responsiveness was ruled out by parallel assays to detect CMV-specific CTLs from these same volunteers. Furthermore, HIV-1-specific memory CTLs were not detectable by peptide stimulation or by a novel technique for flow cytometric visualization of Gag epitope-specific T lymphocytes while HIV-1-seropositive donors frequently had 0.1-3% of CD8+ cells stain positively for this epitope (SLYNTVATL). Taken together, these results suggest that the lack of detectable HIV-1 CTLs in these volunteers was not due to classic MHC-linked nonresponsiveness.

Developing the role of the CNS in osteoporosis.

Nurses can have a major impact on the prevention and treatment of osteoporosis. The role of the osteoporosis nurse specialist is being developed. As nurse specialist posts are created it is essential that UKCC criteria are met. Comparability of grades and areas of expertise are required.

Regional differences in chronic neuroleptic effects on extracellular dopamine activity.

The extracellular levels of dopamine (DA) and DA metabolites in the caudate-putamen (CPu) and the nucleus accumbens (NA) of rats following administration of haloperidol (HAL) decanoate and fluphenazine (FLU) decanoate for 8 months were assessed using intracranial microdialysis 1 month after final injection. Both HAL- and FLU-treated animals showed persisting plasma neuroleptic levels at time of sacrifice. Extracellular basal levels of homovanillic acid (HVA) in the CPu were significantly elevated in the FLU-treated animals, while basal levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in the CPu were significantly elevated in the HAL-treated animals. Basal levels of DA and the serotonin metabolite, 5-hydroxyindoleacetic acid (5HIAA) in the CPu were not significantly different between groups. No significant between-group differences were found for basal levels of any of the analytes in the NA. Neuroleptic-treated animals showed an enhanced response to direct infusion through the dialysis probe of amphetamine (1 microM) and nomifensine (10 microM) in the CPu but not the NA. These results suggest that chronic neuroleptic treatment produces enhanced extracellular DA activity in nigrostriatal, but not mesolimbic DA pathways.

Changes in striatal dopamine release and metabolism during and after subchronic haloperidol administration in rats.

The release and metabolism of dopamine (DA) in the striatum of rats during and after subchronic haloperidol (HAL) administration (3 weeks) was assessed using in vivo microdialysis. Basal extracellular levels of DA, DA metabolites (homovanillic acid and 3,4-dihydroxyphenylacetic acid) and the serotonin metabolite (5-hydroxyindoleacetic acid) were not significantly different from control values at 3 weeks of HAL administration and 3 days after drug withdrawal. The specific DA D2 receptor antagonist, raclopride (0.5 mg/kg, i.p.), significantly increased DA release and metabolism in control animals, but decreased DA release in the HAL-treated groups at 3 weeks of drug treatment. This effect was not significant following drug withdrawal. These results contrast with our previous finding that chronic HAL treatment (32 weeks) increases basal DA metabolism and further support the possibility that changes in DA function differ following short term vs. long term neuroleptic exposure.

Use of aminopeptidase M as a hypotensive agent in spontaneously hypertensive rats.

The present investigation determined that a commercially available aminopeptidase M (AmM, Sigma Chemical) can be utilized to lower blood pressure in normotensive and hypertensive rats. In vitro analyses indicated that the predominant peptidase present in this preparation was AmM; however, it also contained some aminopeptidase A (AmA) and less DAP IV. Although no DAP IV-mediated metabolism of angiotensin II (AII) or angiotensin III (AIII) was measured, both AmM and AmA metabolized AII and AIII. Upon further examination, it appeared that AII could be converted to AIII by either AmM or AmA; however, Arg was cleaved from the N-Terminal of AIII predominantly by AmM. The aminopeptidase inhibitors actinonin (AC), amastatin (AM), and bestatin (BE) effectively blocked the AmM-induced hydrolysis of the Asp-Arg bond of AII, and the Arg-Val bond of AIII. The activity of AmA was inhibited by AM but was relatively resistant to inhibition by AC and BE. Next, exogenous aminopeptidase replacement was employed in the anesthetized spontaneously hypertensive rat (SHR) in an attempt to temporarily correct a hypothesized brain deficiency of receptor-associated peptidases and lower blood pressure. Third-ventricle infusion of AmM produced significant drops in blood pressure and heart rate in both SHRs and Wistar-Kyoto normotensive controls. Pretreatment with AC or BE was particularly effective at interfering with the subsequent AmM-induced hypotensive effect, while AM was less effective. The central mechanisms underlying these effects are in need of further investigation; however, they are at least partially dependent upon the brain angiotensin system.

Hypotensive effects of sarthran in normotensive and spontaneously hypertensive rat strains.

The specific angiotensin receptor antagonist [Sar1, Thr8]AII (sarthran) was intracerebroventricularly (ICV) infused in anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) normotensive controls. The results extend earlier findings by determining that: 1) the hypotensive effect of ICV-infused sarthran could be enhanced in anesthetized as compared with alert animals; 2) SHRs revealed a greater hypotensive response as compared with WKY rats; and 3) no sarthran-induced agonistic effects were observed in contrast with previous results using alert SHRs. These findings support the use of sarthran as a potent angiotensin receptor antagonist to investigate the role of the brain angiotensin system in the control of normal and dysfunctional blood pressure.

Aminopeptidase-induced elevations and reductions in blood pressure in the spontaneously hypertensive rat.

In vitro results indicated that human placenta-derived aminopeptidase A (APA) was very effective at hydrolyzing aspartate from the angiotensin molecule, thus converting angiotensin II to angiotensin III, but was not active against angiotensin III. In vivo experiments revealed significant elevations in blood pressure when APA was intracerebroventricularly infused into anesthetized spontaneously hypertensive rats (SHR) and Wistar-Kyoto normotensive control rats (WKY), with maximum mean (+/- s.e.m.) increases of 30.0 +/- 2.5 and 32.5 +/- 3.7 mmHg, respectively. By contrast, in vitro incubation results utilizing leucine aminopeptidase M (LAP-M) indicated very active degradation of angiotensin III, with less rapid degradation of angiotensin II. The intracerebroventricular infusion of LAP-M significantly reduced blood pressure, particularly in the SHR, but also in WKY, -65.8 +/- 5.1 and -42.5 +/- 6.1 mmHg, respectively. Pretreatment with the specific angiotensin receptor antagonist, Sar1, Thr8 angiotensin II (sarthran) significantly diminished the subsequent APA-induced increase in blood pressure in members of both strains. Pretreatment with sarthran has previously been shown to significantly diminish LAP-M-induced decreases in blood pressure in SHR. Thus, the effects of these aminopeptidases appear to be primarily dependent upon the brain angiotensinergic system, and are consistent with the hypothesis that angiotensin III is the primary active form of central angiotensin.

Intracerebroventricularly infused [D-Arg1]angiotensin III, is superior to [D-Asp1]angiotensin II, as a pressor agent in rats.

Two D-amino acid substitution angiotensin analogues were compared against native angiotensin II (AII) and angiotensin III (AIII) for their resistance to brain tissue-induced degradation and for pressor potency when intracerebroventricularly (i.c.v.) infused in Sprague-Dawley rats. The in vitro results indicate that [D-Asp1]AII was very resistant to degradation, AII and [D-Arg1]AIII were degraded at similar rates, while AIII was the most rapidly degraded. In vivo results revealed that AII, AIII and [D-Arg1]AIII produced greater pressor responses than [D-Asp1]AII. Intracerebroventricular pretreatment with the aminopeptidase A inhibitor, amastatin, significantly reduced the subsequent pressor response to i.c.v. infused [D-Asp1]AII presumably by inhibiting its conversion to AIII. In contrast, pretreatment with the aminopeptidase B inhibitor, bestatin, potentiated the subsequent pressor response to i.c.v. infused [D-Arg1]AIII, presumably by inhibiting the conversion of [D-Arg1]AIII to the less active hexapeptide AII(3-8). Next, i.c.v. pretreatment with the specific angiotensin receptor antagonist, [Sar1, Thr8]AII (Sarthran) was found to greatly diminish the subsequent pressor responses to i.c.v. infused [D-Asp1]AII and [D-Arg1]AIII, suggesting that these analogues are having their effect at the same brain angiotensin receptor site. These results support the hypothesis that AIII, or AIII-like ligands, may serve as the active form of brain angiotensin.

Creatinine excretion as an index of myofibrillar protein mass in dystrophic mice.

1. Daily creatinine excretion in the urine of normal and dystrophic mice was determined and then the carcass proteins were quantitatively extracted into soluble, myofibrillar and collagenous fractions. 2. On a live body-weight basis, total carcass protein was 15% lower in dystrophic than in normal mice. Relative to carcass weight, however, the amount of protein was significantly lower only in male dystrophic mice. 3. The myofibrillar protein fraction comprised 36 . 3 and 32 . 5% of the total protein in male and female dystrophic mice and 48 . 8 and 45 . 0% respectively in normal mice. The decrease in myofibrillar protein in dystrophic mice was accompanied by an increase in the residual collagenous fraction of proteins. 4. The rate of excretion of creatinine was strongly correlated (r = +0 . 98) with the myofibrillar protein mass in each mouse. This relationship was the same for both normal and dystrophic mice, each gram of myofibrillar protein being associated with 3 . 6 mumol of creatinine excreted/day. 5. The creatinine excretion rate is a valid index of contractile muscle mass in murine dystrophy.