18F-fluoro-ethyl-tyrosine PET co-registered with MRI in patients with persisting acromegaly.

OBJECTIVE: To report our experience with 18F-fluoro-ethyl-tyrosine (FET) positron emission tomography-computed tomography (PET-CT) co-registered with magnetic resonance imaging (MRI) (FET-PET/MRICR) in the care trajectory for persistent acromegaly. DESIGN: Prospective case series. PATIENTS: Ten patients with insufficiently controlled acromegaly referred to our team to evaluate surgical options. MEASUREMENTS: FET-PET/MRICR was used to support decision-making if MRI alone and multidisciplinary team evaluation did not provide sufficient clarity to proceed to surgery. RESULTS: FET-PET/MRICR showed suspicious (para)sellar tracer uptake in all patients. In five patients FET-PET/MRICR was fully concordant with conventional MRI, and in one patient partially concordant. FET-PET/MRICR identified suggestive new foci in four other patients. Surgical re-exploration was performed in nine patients (aimed at total resection (6), debulking (2), diagnosis (1)), and one patient underwent radiation therapy. In 7 of 9 (78%) operated patients FET-PET/MRICR findings were confirmed intraoperatively, and in six (67%) also histologically. IGF-1 decreased significantly in eight patients (89%). All patients showed clinical improvement. Complete biochemical remission was achieved in three patients (50% of procedures in which total resection was anticipated feasible). Biochemistry improved in five and was unchanged in one patient. No permanent complications occurred. At six months, optimal outcome (preoperative intended goal achieved without permanent complications) was achieved in six (67%) patients and an intermediate outcome (goal not achieved, but no complications) in the other three patients. CONCLUSIONS: In patients with persisting acromegaly without a clear surgical target on MRI, FET-PET/MRICR is a new tracer to provide additional information to aid decision-making by the multidisciplinary pituitary team.

Periodontitis: A Plausible Modifiable Risk Factor for Neurodegenerative Diseases? A Comprehensive Review.

The aim of this comprehensive review is to summarize recent literature on associations between periodontitis and neurodegenerative diseases, explore the bidirectionality and provide insights into the plausible pathogenesis. For this purpose, systematic reviews and meta-analyses from PubMed, Medline and EMBASE were considered. Out of 33 retrieved papers, 6 articles complying with the inclusion criteria were selected and discussed. Additional relevant papers for bidirectionality and pathogenesis were included. Results show an association between periodontitis and Alzheimer's disease, with odds ratios of 3 to 5. A bidirectional relationship is suspected. For Parkinson's disease (PD), current evidence for an association appears to be weak, although poor oral health and PD seem to be correlated. A huge knowledge gap was identified. The plausible mechanistic link for the association between periodontitis and neurodegenerative diseases is the interplay between periodontal inflammation and neuroinflammation. Three pathways are hypothesized in the literature, i.e., humoral, neuronal and cellular, with a clear role of periodontal pathogens, such as Porphyromonas gingivalis. Age, gender, race, smoking, alcohol intake, nutrition, physical activity, socioeconomic status, stress, medical comorbidities and genetics were identified as common risk factors for periodontitis and neurodegenerative diseases. Future research with main emphasis on the collaboration between neurologists and dentists is encouraged.

The differences in the anatomy of the thoracolumbar and sacral autonomic outflow are quantitative.

PURPOSE: We have re-evaluated the anatomical arguments that underlie the division of the spinal visceral outflow into sympathetic and parasympathetic divisions. METHODOLOGY: Using a systematic literature search, we mapped the location of catecholaminergic neurons throughout the mammalian peripheral nervous system. Subsequently, a narrative method was employed to characterize segment-dependent differences in the location of preganglionic cell bodies and the composition of white and gray rami communicantes. RESULTS AND CONCLUSION: One hundred seventy studies were included in the systematic review, providing information on 389 anatomical structures. Catecholaminergic nerve fibers are present in most spinal and all cranial nerves and ganglia, including those that are known for their parasympathetic function. Along the entire spinal autonomic outflow pathways, proximal and distal catecholaminergic cell bodies are common in the head, thoracic, and abdominal and pelvic region, which invalidates the "short-versus-long preganglionic neuron" argument. Contrary to the classically confined outflow levels T1-L2 and S2-S4, preganglionic neurons have been found in the resulting lumbar gap. Preganglionic cell bodies that are located in the intermediolateral zone of the thoracolumbar spinal cord gradually nest more ventrally within the ventral motor nuclei at the lumbar and sacral levels, and their fibers bypass the white ramus communicans and sympathetic trunk to emerge directly from the spinal roots. Bypassing the sympathetic trunk, therefore, is not exclusive for the sacral outflow. We conclude that the autonomic outflow displays a conserved architecture along the entire spinal axis, and that the perceived differences in the anatomy of the autonomic thoracolumbar and sacral outflow are quantitative.

Does social-norm messaging influence expected satiety and ideal portion-size selection?

A person's perception of how long a food will stave off hunger (expected satiety) and the ideal amount to consume (ideal portion size) are both influenced by food-to-mealtime norms. Here, we examine whether social norms can modulate this effect, in three experimental studies. In study 1 (n = 235) participants were exposed to a social norm suggesting most people enjoyed consuming pasta for breakfast. There was a main effect of food-to-mealtime congruence for expected satiety and ideal portion size (p < 0.001) - participants selected a smaller portion of pasta for breakfast (vs. lunch) - but there were no other main effects/interactions (p ≥ 0.15). Study 2 (n = 200) followed the same approach as study 1, but sought to examine whether the typical volume of food consumed at breakfast and lunch needed to be controlled. Again, there was a main effect of congruence (the same pattern) (p ≤ 0.02) but no other main effects/interactions (p ≥ 0.73). Study 3 (n = 208) followed the same approach as study 2, but the social-norm message was changed to suggest that most people who eat pasta for breakfast found it effectively reduced their hunger. Again, there was a main effect of congruence (the same pattern) (p < 0.001) but no other main effects/interaction (p ≥ 0.26). These studies provide further evidence for the food-to-mealtime effect, but do not provide any evidence that a single, simple social-norm statement can modulate expected satiety or ideal portion size, or interact with the food-to-mealtime effect.

Trans-cyclosulfamidate mannose-configured cyclitol allows isoform-dependent inhibition of GH47 α-d-mannosidases through a bump-hole strategy.

Class I inverting exo-acting α-1,2-mannosidases (CAZY family GH47) display an unusual catalytic itinerary featuring ring-flipped mannosides, 3S1 → 3H4‡ → 1C4. Conformationally locked 1C4 compounds, such as kifunensine, display nanomolar inhibition but large multigene GH47 mannosidase families render specific "isoform-dependent" inhibition impossible. Here we develop a bump-and-hole strategy in which a new mannose-configured 1,6-trans-cyclic sulfamidate inhibits α-d-mannosidases by virtue of its 1C4 conformation. This compound does not inhibit the wild-type GH47 model enzyme by virtue of a steric clash, a "bump", in the active site. An L310S (a conserved residue amongst human GH47 enzymes) mutant of the model Caulobacter GH47 awoke 574 nM inhibition of the previously dormant inhibitor, confirmed by structural analysis of a 0.97 Å structure. Considering that L310 is a conserved residue amongst human GH47 enzymes, this work provides a unique framework for future biotechnological studies on N-glycan maturation and ER associated degradation by isoform-specific GH47 α-d-mannosidase inhibition through a bump-and-hole approach.

The development of a broad-spectrum retaining ß-exo-galactosidase activity-based probe.

Acid ß-galactosidase (GLB1) and galactocerebrosidase (GALC) are retaining exo-ß-galactosidases involved in lysosomal glycoconjugate metabolism. Deficiency of GLB1 may result in the lysosomal storage disorders GM1 gangliosidosis, Morquio B syndrome, and galactosialidosis, and deficiency of GALC may result in Krabbe disease. Activity-based protein profiling (ABPP) is a powerful technique to assess the activity of retaining glycosidases in relation to health and disease. This work describes the use of fluorescent and biotin-carrying activity-based probes (ABPs) to assess the activity of both GLB1 and GALC in cell lysates, culture media, and tissue extracts. The reported ABPs, which complement the growing list of retaining glycosidase ABPs based on configurational isomers of cyclophellitol, should assist in fundamental and clinical research on various ß-galactosidases, whose inherited deficiencies cause debilitating lysosomal storage disorders.

Enhanced HPV16 E6/E7+ tumor eradication via induction of tumor-specific T cells by therapeutic vaccination with virosomes presenting synthetic long peptides.

Therapeutic cancer vaccines trigger CD4 + and CD8 + T cell responses capable of established tumor eradication. Current platforms include DNA, mRNA and synthetic long peptide (SLP) vaccines, all aiming at robust T cell responses. SLPs linked to the Amplivant® adjuvant (Amplivant-SLP) have shown effective delivery to dendritic cells, resulting in improved immunogenicity in mice. We have now tested virosomes as a delivery vehicle for SLPs. Virosomes are nanoparticles made from influenza virus membranes and have been used as vaccines for a variety of antigens. Amplivant-SLP virosomes induced the expansion of more antigen-specific CD8 + T memory cells in ex vivo experiments with human PBMCs than Amplivant-SLP conjugates alone. The immune response could be further improved by including the adjuvants QS-21 and 3D-PHAD in the virosomal membrane. In these experiments, the SLPs were anchored in the membrane through the hydrophobic Amplivant adjuvant. In a therapeutic mouse model of HPV16 E6/E7+ cancer, mice were vaccinated with virosomes loaded with either Amplivant-conjugated SLPs or lipid-coupled SLPs. Vaccination with both types of virosomes significantly improved the control of tumor outgrowth, leading to elimination of the tumors in about half the animals for the best combinations of adjuvants and to their survival beyond 100 days.

Online prediction tools for melanoma survival: A comparison.

BACKGROUND: Breslow thickness, patient age and ulceration are the three most valuable clinical and pathological predictors of melanoma survival. A readily available reliable online tool that accurately considers these and other predictors could be valuable for clinicians managing melanoma patients. OBJECTIVE: To compare online melanoma survival prediction tools that request user input on clinical and pathological features. METHODS: Search engines were used to identify available predictive nomograms. For each, clinical and pathological predictors were compared. RESULTS: Three tools were identified. The American Joint Committee on Cancer tool inappropriately rated thin tumours as higher risk than intermediate tumours. The University of Louisville tool was found to have six shortcomings: a requirement for sentinel node biopsy, unavailable input of thin melanoma or patients over 70 years of age and less reliable hazard ratio calculations for age, ulceration and tumour thickness. The LifeMath.net tool was found to appropriately consider tumour thickness, ulceration, age, sex, site and tumour subtype in predicting survival. LIMITATIONS: The authors did not have access to the base data used to compile various prediction tools. CONCLUSION: The LifeMath.net prediction tool is the most reliable for clinicians in counselling patients with newly diagnosed primary cutaneous melanoma regarding their survival prospects.

Implications of disparate uterine and ovarian development observed among heifers evaluated during the peripubertal period.

The incidence and implications of disparate ovarian and uterine development during the peripubertal period were evaluated in two experiments. In Experiment 1, two consecutive pre-breeding evaluations were performed on 469 heifers. In Experiment 2, data from 22,174 heifers were retrospectively analyzed. For heifers in both experiments, ovarian and uterine maturity were independently assessed via transrectal evaluation, and a two-digit reproductive tract score (RTS: first digit = ovarian; second digit = uterine) was assigned. Measures of the physical maturity of heifers were recorded at the time of pre-breeding evaluation. Heifers were subjected to 14-day progestin-based protocols for synchronization of estrus, and artificial insemination (AI) was performed. Pregnancy diagnosis was performed via transrectal ultrasonography. Incidence of disparate ovarian and uterine score was 33.7 % (158/469) in Experiment 1 % and 16.3 % (3622/22,174) in Experiment 2. Observations of disparate ovarian and uterine maturity were correlated with physical maturity. Heifers with RTS < 3-3 demonstrated poor reproductive performance, as lesser proportions of these animals conceived to the first AI service in Experiment 2 (P < 0.01) or throughout the breeding season in Experiment 1 (P = 0.03). Conception did not differ between heifers assigned congruent or disparate scores of greater than RTS = 3-3. Disparities in ovarian and uterine development are likely observed as the result of rapid, yet asynchronous growth of reproductive tissues during the peripubertal period and are not indicative of inherently reduced potential for fertility. Independent assessment of ovarian and uterine maturity may increase precision in characterizing physiologic maturity of mixed groups of prepubertal, peripubertal, and pubertal heifers.

Synthesis of broad-specificity activity-based probes for exo-ß-mannosidases.

Exo-ß-mannosidases are a broad class of stereochemically retaining hydrolases that are essential for the breakdown of complex carbohydrate substrates found in all kingdoms of life. Yet the detection of exo-ß-mannosidases in complex biological samples remains challenging, necessitating the development of new methodologies. Cyclophellitol and its analogues selectively label the catalytic nucleophiles of retaining glycoside hydrolases, making them valuable tool compounds. Furthermore, cyclophellitol can be readily redesigned to enable the incorporation of a detection tag, generating activity-based probes (ABPs) that can be used to detect and identify specific glycosidases in complex biological samples. Towards the development of ABPs for exo-ß-mannosidases, we present a concise synthesis of ß-manno-configured cyclophellitol, cyclophellitol aziridine, and N-alkyl cyclophellitol aziridines. We show that these probes covalently label exo-ß-mannosidases from GH families 2, 5, and 164. Structural studies of the resulting complexes support a canonical mechanism-based mode of action in which the active site nucleophile attacks the pseudoanomeric centre to form a stable ester linkage, mimicking the glycosyl enzyme intermediate. Furthermore, we demonstrate activity-based protein profiling using an N-alkyl aziridine derivative by specifically labelling MANBA in mouse kidney tissue. Together, these results show that synthetic manno-configured cyclophellitol analogues hold promise for detecting exo-ß-mannosidases in biological and biomedical research.

Evaluation of the 7 & 7 Synch and 7-day CO-Synch + CIDR treatment regimens for control of the estrous cycle among beef cows prior to fixed-time artificial insemination with conventional or sex-sorted semen.

An experiment was conducted to compare the 7 & 7 Synch and 7-day CO-Synch + controlled internal drug release (CIDR®) treatment regimens before fixed-time artificial insemination (FTAI) of beef cows with conventional or sex-sorted semen. Cows (n = 1538) were blocked based on age and days postpartum (DPP) and randomly assigned to treatment regimen and semen type. Cows assigned to the 7-day CO-Synch + CIDR treatment regimen (n = 769) were administered gonadotropin-releasing hormone (GnRH) and an intravaginal progesterone-releasing insert (CIDR) on Day - 10, and administration of prostaglandin F2α (PG) coincident with CIDR removal on Day - 3. Cows assigned to 7 & 7 Synch (n = 769) were administered PG and a CIDR device on Day - 17, GnRH on Day - 10, and PG coincident with CIDR removal on Day - 3. Cows were administered GnRH coincident with FTAI, which was performed 66 h after CIDR removal with conventional (20 × 106 cells) or sex-sorted (4 × 106 cells) semen. Expression of estrus was affected by treatment regimen (P = 0.01) and by treatment regimen × DPP (P = 0.0004), as a result of imposing the 7 & 7 Synch regimen; therefore, a greater percentage of cows expressed estrus (82% compared with 64%), particularly among cows with greater DPP. Pregnancy percentages resulting from FTAI were less (P < 0.0001) when using sex-sorted semen but greater among cows treated with 7 & 7 Synch (conventional semen: 72%; sex-sorted semen: 52%) compared with 7-day CO-Synch + CIDR (conventional semen: 61%; sex-sorted semen: 44%).

pUL21 regulation of pUs3 kinase activity influences the nature of nuclear envelope deformation by the HSV-2 nuclear egress complex.

It is well established that the herpesvirus nuclear egress complex (NEC) has an intrinsic ability to deform membranes. During viral infection, the membrane-deformation activity of the NEC must be precisely regulated to ensure efficient nuclear egress of capsids. One viral protein known to regulate herpes simplex virus type 2 (HSV-2) NEC activity is the tegument protein pUL21. Cells infected with an HSV-2 mutant lacking pUL21 (ΔUL21) produced a slower migrating species of the viral serine/threonine kinase pUs3 that was shown to be a hyperphosphorylated form of the enzyme. Investigation of the pUs3 substrate profile in ΔUL21-infected cells revealed a prominent band with a molecular weight consistent with that of the NEC components pUL31 and pUL34. Phosphatase sensitivity and retarded mobility in phos-tag SDS-PAGE confirmed that both pUL31 and pUL34 were hyperphosphorylated by pUs3 in the absence of pUL21. To gain insight into the consequences of increased phosphorylation of NEC components, the architecture of the nuclear envelope in cells producing the HSV-2 NEC in the presence or absence of pUs3 was examined. In cells with robust NEC production, invaginations of the inner nuclear membrane were observed that contained budded vesicles of uniform size. By contrast, nuclear envelope deformations protruding outwards from the nucleus, were observed when pUs3 was included in transfections with the HSV-2 NEC. Finally, when pUL21 was included in transfections with the HSV-2 NEC and pUs3, decreased phosphorylation of NEC components was observed in comparison to transfections lacking pUL21. These results demonstrate that pUL21 influences the phosphorylation status of pUs3 and the HSV-2 NEC and that this has consequences for the architecture of the nuclear envelope.

Common Reference-Based Tandem Mass Tag Multiplexing for the Relative Quantification of Peptides: Design and Application to Degradome Analysis of Diphtheria Toxoid.

Currently, animal tests are being used to confirm the potency and lack of toxicity of toxoid vaccines. In a consistency approach, animal tests could be replaced if production consistency (compared to known good products) can be proven in a panel of in vitro assays. By mimicking the in vivo antigen processing in a simplified in vitro approach, it may be possible to distinguish aberrant products from good products. To demonstrate this, heat-exposed diphtheria toxoid was subjected to partial digestion by cathepsin S (an endoprotease involved in antigen processing), and the peptide formation/degradation kinetics were mapped for various heated toxoids. To overcome the limitations associated with the very large number of samples, we used common reference-based tandem mass tag (TMT) labeling. Instead of using one label per condition with direct comparison between the set of labels, we compared multiple labeled samples to a common reference (a pooled sample containing an aliquot of each condition). In this method, the number of samples is not limited by the number of unique TMT labels. This TMT multiplexing strategy allows for a 15-fold reduction of analysis time while retaining the reliability advantage of TMT labeling over label-free quantification. The formation of the most important peptides could be followed over time and compared among several conditions. The changes in enzymatic degradation kinetics of diphtheria toxoid revealed several suitable candidate peptides for use in a quality control assay that can distinguish structurally aberrant diphtheria toxoid from compliant toxoids.

Complications and survival after hybrid and fully minimally invasive oesophagectomy.

BACKGROUND: Minimally invasive oesophagectomy (MIO) is reported to produce fewer respiratory complications than open oesophagectomy. This study assessed differences in postoperative complications between MIO and hybrid MIO (HMIO) employing thoracoscopy and laparotomy, along with the influence of co-morbidities on postoperative outcomes. METHODS: Patients with oesophageal cancer undergoing three-stage MIO or three-stage HMIO between 1999 and 2018 were identified from a prospectively developed database, which included patient demographics, co-morbidities, preoperative therapies, and cancer stage. The primary outcome was postoperative complications in the two groups. Secondary outcomes included duration of operation, blood transfusion requirement, duration of hospital stay, and overall survival. RESULTS: There were 828 patients, of whom 722 had HMIO and 106 MIO, without significant baseline differences. Median duration of operation was longer for MIO (325 versus 289 min; P < 0.001), but with less blood loss (median 250 versus 300 ml; P < 0.001) and a shorter hospital stay (median 12 versus 13 days; P = 0.006). Respiratory complications were not associated with operative approach (31.1 versus 35.2 per cent for MIO and HMIO respectively; P = 0.426). Anastomotic leak rates (10.4 versus 10.2 per cent) and 90-day mortality (1.0 versus 1.7 per cent) did not differ. Cardiac co-morbidity was associated with more medical and surgical complications. Overall survival was associated with AJCC stage and co-morbidities, but not operative approach. CONCLUSION: MIO had a small benefit in terms of blood loss and hospital stay, but not in operating time. Oncological outcomes were similar in the two groups. Postoperative complications were associated with pre-existing cardiorespiratory co-morbidities rather than operative approach.

Delayed Extension Block Pinning in 27 Patients With Mallet Fracture.

Background: Untreated bony mallet fingers can cause an array of problems; therefore, adequate treatment is essential. The primary aim of this study was to determine the patient-reported functional outcome of delayed surgical intervention of bony mallet fingers. The secondary aim was to determine the complication rate of delayed surgical intervention. Methods: In this single-center retrospective cohort study, all consecutive patients treated between 2010 and 2016 at our level 2 regional teaching hospital were included. Inclusion criterion was a bony mallet finger injury (excluding the thumb), presenting >21 days after injury, treated with extension block pinning. Indications for surgery were >2 mm fragment displacement or volar subluxation of the distal interphalangeal joint. Six to 82 months postoperatively, patients completed the Patient-Rated Wrist and Hand Evaluation (PRWHE) by phone. Complications were extracted by chart review. Results: Twenty-seven patients were included, and all completed the PRWHE. Median time to surgery was 35 days (interquartile range [IQR] = 29-42; range = 22-61). Reasons for delay in surgical treatment were patient/physician delay in 24 cases and failed conservative treatment in 3 cases. The median PRWHE score was 0 (IQR = 0-5; range = 0-22.5). After retrospective assessment of the outpatient charts, no early symptoms of malunion or nonunion were found. One patient had a loss of Kirschner-wire fixation, which was corrected. Three patients had an infection that required antibiotic treatment. Conclusions: Delayed surgical management of bony mallet fingers demonstrated adequate functional outcome with minimal complications when compared with prior literature.

Evaluation of later timepoints for split-time artificial insemination when using sex-sorted semen among beef heifers following the 14-d CIDR®-PG protocol.

An experiment was designed to evaluate later timepoints for Split-Time AI (STAI), with the hypothesis that delaying AI may improve estrous response and pregnancy per AI when using sex-sorted semen. Timing of estrus was synchronized among 794 heifers using the 14-d CIDR®-PG protocol (1.38 g progesterone intravaginal insert from Day 0-14, followed by 25 mg dinoprost tromethamine on Day 30) with STAI performed based on estrous status. Heifers were blocked based on breed, source, sire, reproductive tract score (RTS), and BW and assigned within block to one of two approaches. In Approach 66, heifers that were estrual by 66 h after PG administration were inseminated at 66 h, and remaining heifers were inseminated 24 h later (90 h). In Approach 72, heifers that were estrual by 72 h were inseminated at 72 h, and remaining heifers were inseminated 24 h later (96 h). With both approaches, heifers that were non-estrual by the final timepoint were administered 100 µg gonadorelin acetate (GnRH). Within approach, heifers were pre-assigned to receive SexedULTRA 4M™ sex-sorted or conventional semen. The proportion of heifers estrual by the first timepoint was greater (P < 0.0001) with Approach 72 (76 %; 302/395) compared to Approach 66 (61 %; 242/399). The proportion of heifers pregnant as a result of AI differed (P = 0.0005) by semen type (59 % [240/404] for conventional compared with 48 % [187/390] for sex-sorted) but was not affected by approach or approach × semen type. In summary, pregnancy per AI of heifers receiving sex-sorted or conventional semen following the 14-d CIDR®-PG protocol did not differ when STAI was delayed 6 h. The proportion of estrual heifers prior to the first timepoint, however, was greater with later STAI.

Mindfulness in Patients with Upper-Extremity Conditions: A Summary of Existing Literature.

Mindfulness implies entering a mental state of awareness which allows for the reframing of an experience, and functionality has shown to be influenced by mindset. The aim of this systematic review was to assess effects of mindfulness in patients with upper-extremity conditions. PubMed, Embase, Cochrane, and CINAHL databases were searched on June 19, 2019, for studies investigating mindfulness in patients with upper-extremity conditions. Two validated instruments for methodologic assessment were used to assess study quality. Studies that reported pain, psychological, or functional outcome measures were included. One randomized controlled trials and three observational studies were included, which together included 335 patients that completed final follow-up. The weighted average age was 52.4 years and 48% of the patients were male. Evaluation of the outcome measures used was immediately after the mindfulness intervention or assessment in all studies. Mindfulness appeared to be positively associated with less pain (though below the minimal clinically important difference), increased mood, and better function. Mindfulness is associated with increased mood and possibly better functionality in adults with a large range of upper-extremity conditions when measured or used as an intervention. Future researcher should expand the subject as only four studies were included in this review. This is a Level IV study.

Degradomics-Based Analysis of Tetanus Toxoids as a Quality Control Assay.

Currently, batch release of toxoid vaccines, such as diphtheria and tetanus toxoid, requires animal tests to confirm safety and immunogenicity. Efforts are being made to replace these tests with in vitro assays in a consistency approach. Limitations of current in vitro assays include the need for reference antigens and most are only applicable to drug substance, not to the aluminum adjuvant-containing and often multivalent drug product. To overcome these issues, a new assay was developed based on mimicking the proteolytic degradation processes in antigen-presenting cells with recombinant cathepsin S, followed by absolute quantification of the formed peptides by liquid chromatography-mass spectrometry. Temperature-exposed tetanus toxoids from several manufacturers were used as aberrant samples and could easily be distinguished from the untreated controls by using the newly developed degradomics assay. Consistency of various batches of a single manufacturer could also be determined. Moreover, the assay was shown to be applicable to Al(OH)3 and AlPO4-adsorbed tetanus toxoids. Overall, the assay shows potential for use in both stability studies and as an alternative for in vivo potency studies by showing batch-to-batch consistency of bulk toxoids as well as for aluminum-containing vaccines.

Novel Formaldehyde-Induced Modifications of Lysine Residue Pairs in Peptides and Proteins: Identification and Relevance to Vaccine Development.

Formaldehyde-inactivated toxoid vaccines have been in use for almost a century. Despite formaldehyde's deceptively simple structure, its reactions with proteins are complex. Treatment of immunogenic proteins with aqueous formaldehyde results in heterogenous mixtures due to a variety of adducts and cross-links. In this study, we aimed to further elucidate the reaction products of formaldehyde reaction with proteins and report unique modifications in formaldehyde-treated cytochrome c and corresponding synthetic peptides. Synthetic peptides (Ac-GDVEKGAK and Ac-GDVEKGKK) were treated with isotopically labeled formaldehyde (13CH2O or CD2O) followed by purification of the two main reaction products. This allowed for their structural elucidation by (2D)-nuclear magnetic resonance and nanoscale liquid chromatography-coupled mass spectrometry analysis. We observed modifications resulting from (i) formaldehyde-induced deamination and formation of α,ß-unsaturated aldehydes and methylation on two adjacent lysine residues and (ii) formaldehyde-induced methylation and formylation of two adjacent lysine residues. These products react further to form intramolecular cross-links between the two lysine residues. At higher peptide concentrations, these two main reaction products were also found to subsequently cross-link to lysine residues in other peptides, forming dimers and trimers. The accurate identification and quantification of formaldehyde-induced modifications improves our knowledge of formaldehyde-inactivated vaccine products, potentially aiding the development and registration of new vaccines.