Nasal delivery of an IgM offers broad protection from SARS-CoV-2 variants.

Resistance represents a major challenge for antibody-based therapy for COVID-191-4. Here we engineered an immunoglobulin M (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulin G (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern-B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)-and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044 mg per kg body weight confers prophylactic efficacy and a single dose at 0.4 mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19.

Host-microbiome interactions in human type 2 diabetes following prebiotic fibre (galacto-oligosaccharide) intake.

Aberrant microbiota composition and function have been linked to several pathologies, including type 2 diabetes. In animal models, prebiotics induce favourable changes in the intestinal microbiota, intestinal permeability (IP) and endotoxaemia, which are linked to concurrent improvement in glucose tolerance. This is the first study to investigate the link between IP, glucose tolerance and intestinal bacteria in human type 2 diabetes. In all, twenty-nine men with well-controlled type 2 diabetes were randomised to a prebiotic (galacto-oligosaccharide mixture) or placebo (maltodextrin) supplement (5·5 g/d for 12 weeks). Intestinal microbial community structure, IP, endotoxaemia, inflammatory markers and glucose tolerance were assessed at baseline and post intervention. IP was estimated by the urinary recovery of oral 51Cr-EDTA and glucose tolerance by insulin-modified intravenous glucose tolerance test. Intestinal microbial community analysis was performed by high-throughput next-generation sequencing of 16S rRNA amplicons and quantitative PCR. Prebiotic fibre supplementation had no significant effects on clinical outcomes or bacterial abundances compared with placebo; however, changes in the bacterial family Veillonellaceae correlated inversely with changes in glucose response and IL-6 levels (r -0·90, P=0·042 for both) following prebiotic intake. The absence of significant changes to the microbial community structure at a prebiotic dosage/length of supplementation shown to be effective in healthy individuals is an important finding. We propose that concurrent metformin treatment and the high heterogeneity of human type 2 diabetes may have played a significant role. The current study does not provide evidence for the role of prebiotics in the treatment of type 2 diabetes.

An ACE2 decamer viral trap as a durable intervention solution for current and future SARS-CoV.

The capacity of SARS-CoV-2 to evolve poses challenges to conventional prevention and treatment options such as vaccination and monoclonal antibodies, as they rely on viral receptor binding domain (RBD) sequences from previous strains. Additionally, animal CoVs, especially those of the SARS family, are now appreciated as a constant pandemic threat. We present here a new antiviral approach featuring inhalation delivery of a recombinant viral trap composed of ten copies of angiotensin-converting enzyme 2 (ACE2) fused to the IgM Fc. This ACE2 decamer viral trap is designed to inhibit SARS-CoV-2 entry function, regardless of viral RBD sequence variations as shown by its high neutralization potency against all known SARS-CoV-2 variants, including Omicron BQ.1, BQ.1.1, XBB.1 and XBB.1.5. In addition, it demonstrates potency against SARS-CoV-1, human NL63, as well as bat and pangolin CoVs. The multivalent trap is effective in both prophylactic and therapeutic settings since a single intranasal dosing confers protection in human ACE2 transgenic mice against viral challenges. Lastly, this molecule is stable at ambient temperature for more than twelve weeks and can sustain physical stress from aerosolization. These results demonstrate the potential of a decameric ACE2 viral trap as an inhalation solution for ACE2-dependent coronaviruses of current and future pandemic concerns.

Single scan SeHCAT studies: a model for the prediction of the 3-h counts.

OBJECTIVES: As part of the 75-Selenium homocholic acid taurine (SeHCAT) study, counts are acquired as a baseline to allow the calculation of the retention at 7 days. In this work, we evaluated whether it was possible to replace the baseline image with a predictive model based on the patient's height and weight. METHOD: Height and weight data from 723 patients scanned at three hospitals using seven gamma cameras were compiled. A number of different models were trialled, with fitting parameters determined by regression. A predictive model based on height and logarithm of weight was found to have the best correlation with the measured counts in the 3-h study. RESULTS: There was a strong correlation (R2 = 0.91) between the measured counts and the predicted counts using a model based on height and logarithm of weight. Treating the standard SeHCAT test result as the gold standard, the test result when predicted baseline counts were used had a sensitivity and specificity of 97.5 and 98.0%, respectively, at a threshold of 15%. In total 694/723 (96.0%) of patients had no change to their severity grading when using the predicted baseline counts. CONCLUSION: This work presents a model that was able to predict the counts in the 3 h SeHCAT study for patients on seven gamma cameras. This can enable a single scan study, giving significant savings to patient and staff time and imaging resources.

Design, creation and in vitro testing of a reduced immunogenicity humanized anti-CD25 monoclonal antibody that retains functional activity.

Humanized and fully human sequence-derived therapeutic antibodies retain the capacity to induce anti-drug antibodies. Daclizumab (humanized version of the murine anti-Tac antibody; E.HAT) was selected for a proof of concept application of engineering approaches to reduce potential immunogenicity due to its demonstrated immunogenicity in the clinic. Reduced immunogenicity variants of E.HAT were created by identifying and modifying a CD4+ T cell epitope region in the VH region. Variant epitope region peptides were selected for their reduced capacity to induce CD4+ T cell proliferative responses in vitro. Variant antibody molecules were created, and CD25 affinity and potency were similar to the unmodified parent antibody. Fab fragments from the variant antibodies induced a lower frequency and magnitude of responses in human peripheral blood mononuclear cells proliferation tests. By the empirical selection of two amino acid mutations, fully functional humanized E.HAT antibodies with reduced potential to induce immune responses in vitro were created.

Fecal Enterobacteriales enrichment is associated with increased in vivo intestinal permeability in humans.

Type 2 diabetes (T2D) has been linked with increased intestinal permeability, but the clinical significance of this phenomenon remains unknown. The objective of this study was to investigate the potential link between glucose control, intestinal permeability, diet and intestinal microbiota in patients with T2D. Thirty-two males with well-controlled T2D and 30 age-matched male controls without diabetes were enrolled in a case-control study. Metabolic parameters, inflammatory markers, endotoxemia, and intestinal microbiota in individuals subdivided into high (HP) and normal (LP) colonic permeability groups, were the main outcomes. In T2D, the HP group had significantly higher fasting glucose (P = 0.034) and plasma nonesterified fatty acid levels (P = 0.049) compared with the LP group. Increased colonic permeability was also linked with altered abundances of selected microbial taxa. The microbiota of both T2D and control HP groups was enriched with Enterobacteriales. In conclusion, high intestinal permeability was associated with poorer fasting glucose control in T2D patients and changes in some microbial taxa in both T2D patients and nondiabetic controls. Therefore, enrichment in the gram-negative order Enterobacteriales may characterize impaired colonic permeability prior to/independently from a disruption in glucose tolerance.

The CD25-binding antibody Daclizumab High-Yield Process has a distinct glycosylation pattern and reduced antibody-dependent cell-mediated cytotoxicity in comparison to Zenapax®.

The CD25-binding antibody daclizumab high-yield process (DAC HYP) is an interleukin (IL)-2 signal modulating antibody that shares primary amino acid sequence and CD25 binding affinity with Zenapax®, a distinct form of daclizumab, which was approved for the prevention of acute organ rejection in patients receiving renal transplants as part of an immunosuppressive regimen that includes cyclosporine and corticosteroids. Comparison of the physicochemical properties of the two antibody forms revealed the glycosylation profile of DAC HYP differs from Zenapax in both glycan distribution and the types of oligosaccharides, most notably high-mannose, galactosylated and galactose-α-1,3-galactose (α-Gal) oligosaccharides, resulting in a DAC HYP antibody material that is structurally distinct from Zenapax. Although neither antibody elicited complement-dependent cytotoxicity in vitro, DAC HYP antibody had significantly reduced levels of antibody-dependent cell-mediated cytotoxicity (ADCC). The ADCC activity required natural killer (NK) cells, but not monocytes, suggesting the effects were mediated through binding to Fc-gamma RIII (CD16). Incubation of each antibody with peripheral blood mononuclear cells also caused the down-modulation of CD16 expression on NK cells and the CD16 down-modulation was greater for Zenapax in comparison to that observed for DAC HYP. The substantive glycosylation differences between the two antibody forms and corresponding greater Fc-mediated effector activities by Zenapax, including cell killing activity, manifest as a difference in the biological function and pharmacology between DAC HYP and Zenapax.

Variation of DaTSCAN quantification between different gamma camera types.

OBJECTIVE: To acquire data from a 123I filled Alderson phantom on different gamma cameras types and compare the relative uptake results from processing using the QuantiSPECT program (GE Healthcare). METHODS: A DaTSCAN phantom was filled using the standard protocol and imaged on seven different gamma camera types and on two identical cameras of the same type. The standard GE Healthcare protocols for the given cameras were used. Aliquots of the striatum and brain background were counted in a gamma counter to determine variations in filling concentration. All the raw DaTSCAN SPECT data was imported into QuantiSPECT and processed by the three different algorithms (two box, three box and crescent) to determine the relative uptake in the striatum. Inter-operater and intra-operator variation was also determined. RESULTS: The 10% variation in filling concentration found across the sites was compensated for in the final results. There was a 5-15% variation between cameras depending on the processing algorithm used. There was an intra-operator variation of between 5 and 12% which reflected the proportion of operator intervention within the processing method. There was no statistical variation between operators. CONCLUSIONS: The transfer of a DaTSCAN database between camera types is feasible, but ideally all data would be acquired on a single camera type and phantom data used to normalize the database accordingly.

Development of a combined audiovisual and extremity dose monitoring software tool for use in nuclear medicine.

OBJECTIVES: Conventional extremity dose monitoring in nuclear medicine, using thermoluminescent dosimeters, provides a convenient method of determining integral doses from a series of procedures. Although semiconductor extremity probes are able to add time information and allow doses from individual procedures to be determined, it can be difficult to relate individual operations to the dose-time curve. Solutions to this problem have been identified and developed. METHODS: A novel software tool (Extremity Dose Information Package, EDIP) has been developed that uniquely combines and synchronizes two audiovisual and extremity probe data-streams. The value of this extra information was assessed by acquiring audiovisual and extremity dose information in nuclear medicine and radiopharmacy settings. RESULTS: The ability of the software tool to synchronize audiovisual and dose data-streams was verified. Preliminary studies of handling techniques in radiopharmacy and radioiodine administrations using this tool showed areas in which techniques could be adapted to reduce extremity doses, which would have been difficult or impossible to identify using the dose-time information alone. CONCLUSIONS: This low-cost multimedia extremity dose monitoring package can be used, for example, to aid staff training and pinpoint issues with current operating procedures within a clinical nuclear medicine department. Its unique ability to combine and synchronize audiovisual and dosimetry data is also likely to be of benefit to other industries handling unsealed radioactive materials.

Comparison of different methods of DatSCAN quantification.

BACKGROUND: The quantification of DaTSCAN images can be used as an adjunct to visual assessment to differentiate between Parkinson's syndrome and essential tremor. Many programs have been written to assess the relative uptake in the striatum. AIM: To compare two of the commercially available programs: QuantiSPECT, which analyses isolated data in two dimensions, and BRASS, which performs three-dimensional processing referencing a normal image template. METHOD: Twenty-two patients (11 with Parkinson's syndrome and 11 with essential tremor) were visually assessed by two nuclear medicine consultants. The patient data were then processed using two commercial programs to determine the relative uptake in the striatum. A comparison of the results from the programs was performed, together with a comparison with the visual assessment. The inter-operator and intra-operator variabilities were also ascertained. RESULTS: All programs and processing methods could distinguish between Parkinson's syndrome and essential tremor. There was also a good correlation between the results from the three- and two-dimensional methods. The intra-operator and inter-operator variabilities were dependent on the amount of operator intervention. CONCLUSION: Both programs allowed statistical differentiation between Parkinson's syndrome and essential tremor. Strict operator protocols are needed with QuantiSPECT to reduce inter- and intra-operator variation. The three-dimensional method (BRASS) gave greater concordance than the two-dimensional method (QuantiSPECT) with the visual assessment, but at a cost of increased operator time.

Delivery and collection of radioactive packages to and from UK hospital nuclear medicine departments.

Under radiation protection legislation in the UK, employers have a duty to maintain appropriate records to account for radioactive materials in their possession and to ensure security of these materials. This applies to radioactive packages, containing items such as technetium generators, which are regularly delivered to hospital nuclear medicine departments. It also applies to the collection of packages, such as those containing used generators for return to the supplier. This article has been written by the professional bodies representing nuclear medicine in the UK in order to provide guidance to hospitals on appropriate procedures that will comply with the legislation. General principles, which should be met by any acceptable protocol, are stated, and practical guidance on how these may be implemented is given. Some example scenarios are outlined.

Dissociation of Tc-99m DTPA in a nebulizer.

An unexpected biodistribution of nebulized Tc-99m diethylene triamine pentaacetic acid was detected after the ventilation of a patient referred for diagnosis of pulmonary embolism. Radiochemical purity testing of the stock vial showed more than 95% labelling. Further investigation indicated that the problem was possibly associated with a cleaning agent used for the nebulizer, leading to the dissociation of the Tc-99m diethylene triamine pentaacetic acid. Manufacturer's instructions for cleaning of the nebulizer specify the use of 70% denatured ethanol solution, but a chlorine-based agent had been used inadvertently.

The importance of scatter correction for the assessment of lung shunting prior to yttrium-90 radioembolization therapy.

BACKGROUND: Treatment of inoperable hepatocellular carcinoma or secondary metastases by radioembolization using yttrium-90 (90Y) microspheres is a promising method for the treatment of unresectable liver metastases. A pretreatment scintigraphy planar scan using 99mTc-labelled macroaggregated albumin (99mTc-MAA) injected directly into the hepatic artery is carried out to assess the degree of portal shunting of blood between the liver and the lungs. The quantitative results of this scan are used to modulate the activity of therapeutic 90Y microspheres injected into the patient to limit the radiation dose received by the lungs. The presence of scattered events in the MAA lung shunt scan leads to an overestimation of the true shunting ratio, which in turn leads to the administered therapeutic activity being lowered unnecessarily to comply with the protocols of radiation protection. Overall, this may impact the efficacy of treatment. MATERIALS AND METHODS: This study analyses the impact of a window-based analytical scatter-correction method on lung shunt analysis using an anthropomorphic torso phantom, and retrospectively analysed three patient case studies. RESULTS: Our results of scatter in the phantom show a marked decrease in the lung shunt percentage. Clinical analysis of patient data shows that the lung shunt percentage can be overestimated by up to 50% in clinical cases, and depending on the lung shunt percentage, the efficacy of treatment by therapeutic dose reduction may be compromised. CONCLUSION: Our results indicate that scatter correction should be used on 90Y pretreatment 99mTc-MAA scans in order to more accurately assess the lung shunting percentage before therapy.

Outcome measures and their everyday use in chiropractic practice.

OBJECTIVES: To describe the extent to which chiropractors utilize standardized outcome and various clinical measures to systematically document patients' baseline health status and responses to treatment, with particular consideration being given towards quantifiable outcome instruments. STUDY DESIGN: Cross-sectional mailed survey. PARTICIPANTS: Registered chiropractors in the province of Saskatchewan. METHODS: A survey was mailed to all registrants of the Chiropractors' Association of Saskatchewan. Respondents graded their frequency of using various standardized pencil-and-paper instruments and functional chiropractic, orthopaedic and neurological tests in the contexts of both the initial intake assessment ('always,' 'commonly,' 'occasionally,' or 'never') and the course of subsequent treatment (after 'each visit,' after '9-12 visits,' 'annually,' when patient 'not responding,' on 'dismissal/discharge,' 'never' or for some 'other' reason). Data were tabulated for all item and response category combinations as frequencies and percentages using the total sample size as the denominator. RESULTS: Of 164 registered chiropractors, 62 (38%) returned a completed questionnaire. A pain diagram was the most commonly used subjective outcome measure and was administered routinely (either "always" or "commonly") by 75% of respondents, at either the initial consultation or during a subsequent visit. Numerical rating and visual analogue scales were less popular (routinely used by 59% and 42% respectively). The majority of respondents (80%) seldom ("occasionally" or "never") used spine pain-specific disability indices such as the Low Back Revised Oswestry, Neck Disability Index or the Roland-Morris Questionnaire. As well, they did not use standardized psychosocial instruments such as the Beck Depression Index, or general health assessment measures such as the SF-36 or SF-12 questionnaire. Neurological testing was the most commonly used objective outcome measure. Most respondents (84% to 95%) indicated that they continually monitored neurological status through dermatomal, manual muscle strength and deep tendon reflex testing. Ranges of motion were routinely measured by 95% of respondents, usually visually (96%) rather than goniometrically or by some other specialized device (7%). CONCLUSIONS: Our findings suggest that the majority of chiropractors do not use psychosocial questionnaires or condition-specific disability indices to document baseline or subsequent changes in health status. Chiropractors are more likely to rely on medical history taking and pain drawings during an initial intake assessment, as well as neurological and visually estimated range of motion testing during both initial intake and subsequent treatment visits.

Measurement of functional capacity requirements of police officers to aid in development of an occupation-specific cardiac rehabilitation training program.

This study was designed to measure the functional capacity of healthy subjects during strenuous simulated police tasks, with the goal of developing occupation-specific training for cardiac rehabilitation of police officers. A calibrated metabolic instrument and an oxygen consumption data collection mask were used to measure the oxygen consumption and heart rates of 30 Dallas Police Academy officers and cadets as they completed an 8-event obstacle course that simulated chasing, subduing, and handcuffing a suspect. Standard target heart rates (85% of age-predicted maximum heart rate, or 0.85 x [220 - age]) and metabolic equivalents (METs) were calculated; a matched-sample t test based on differences between target and achieved heart rate and MET level was used for statistical analysis. Peak heart rates during the obstacle course simulation were significantly higher than the standard target heart rates (those at which treadmill stress tests in physicians' offices are typically stopped) (t(29) = 12.81, P < 0.001) and significantly higher than the suggested maximum of 150 beats/min during cardiac rehabilitation training (t(29) = 17.84, P < 0.001). Peak MET levels during the obstacle course simulation were also significantly higher than the goal level (8 METs) that patients typically achieve in a cardiac rehabilitation program (t(29) = 14.73, P < 0.001). We conclude that police work requires a functional capacity greater than that typically attained in traditional cardiac rehabilitation programs. Rehabilitation professionals should consider performing maximal stress tests and increasing the intensity of cardiac rehabilitation workouts to effectively train police officers who have had a cardiac event.

An engineered human IgG1 antibody with longer serum half-life.

The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). By binding to FcRn in endosomes, IgG Abs are salvaged from lysosomal degradation and recycled to the circulation. Several studies have demonstrated a correlation between the binding affinity of IgG Abs to FcRn and their serum half-lives in mice, including engineered Ab fragments with longer serum half-lives. Our recent study extended this correlation to human IgG2 Ab variants in primates. In the current study, several human IgG1 mutants with increased binding affinity to human FcRn at pH 6.0 were generated that retained pH-dependent release. A pharmacokinetics study in rhesus monkeys of one of the IgG1 variants indicated that its serum half-life was approximately 2.5-fold longer than the wild-type Ab. Ag binding was unaffected by the Fc mutations, while several effector functions appeared to be minimally altered. These properties suggest that engineered Abs with longer serum half-lives may prove to be effective therapeutics in humans.

Design of humanized antibodies: from anti-Tac to Zenapax.

Since the introduction of hybridoma technology, monoclonal antibodies have become one of the most important tools in the biosciences, finding diverse applications including their use in the therapy of human disease. Initial attempts to use monoclonal antibodies as therapeutics were hampered, however, by the potent immunogenicity of mouse (and other rodent) antibodies in humans. Humanization technology has made it possible to remove the immunogenicity associated with the use of rodent antibodies, or at least to reduce it to an acceptable level for clinical use in humans, thus facilitating the application of monoclonal antibodies to the treatment of human disease. To date, nine humanized monoclonal antibodies have been approved for use as human therapeutics in the United States. In this paper, we describe procedures for antibody humanization with an emphasis on strategies for designing humanized antibodies with the aid of computer-guided modeling of antibody variable domains, using as an example the humanized anti-CD25 monoclonal antibody, Zenapax.

Amphiregulin and epidermal hyperplasia: amphiregulin is required to maintain the psoriatic phenotype of human skin grafts on severe combined immunodeficient mice.

Overexpression of amphiregulin has been shown to induce psoriasiform changes in the skin of transgenic mice shortly after birth. Therefore, amphiregulin has been suggested as a target for anti-psoriatic therapy. To test this theory, a humanized monoclonal antibody capable of neutralizing human amphiregulin was examined for anti-proliferative effects in the human skin-severe combined immunodeficient (SCID) mouse transplant model. The anti-amphiregulin antibody reduced epidermal thickness of transplanted psoriatic skin and also inhibited the hyperplastic response that developed in nonpsoriatic skin after transplantation. The same antibody also suppressed keratinocyte proliferation in monolayer culture in a dose-dependent manner. Under the same conditions in which keratinocyte proliferation was inhibited, the antibody had little effect on proliferation of human dermal fibroblasts and no effect on type I procollagen production by these cells. Taken together, these data indicate an important role for amphiregulin in psoriatic hyperplasia and suggest that inhibition of amphiregulin activity could be an efficacious therapeutic strategy for psoriasis. These data also suggest that the hyperplastic response occurring in nonpsoriatic human skin on transplantation to the SCID mouse is mediated, in large part, by amphiregulin.

Engineered human IgG antibodies with longer serum half-lives in primates.

The neonatal Fc receptor (FcRn) plays an important role in regulating the serum half-lives of IgG antibodies. A correlation has been established between the pH-dependent binding affinity of IgG antibodies to FcRn and their serum half-lives in mice. In this study, molecular modeling was used to identify Fc positions near the FcRn binding site in a human IgG antibody that, when mutated, might alter the binding affinity of IgG to FcRn. Following mutagenesis, several IgG2 mutants with increased binding affinity to human FcRn at pH 6.0 were identified at Fc positions 250 and 428. These mutants do not bind to human FcRn at pH 7.5. A pharmacokinetics study of two mutant IgG2 antibodies with increased FcRn binding affinity indicated that they had serum half-lives in rhesus monkeys approximately 2-fold longer than the wild-type antibody.