Outcomes and characteristics of nonmelanoma skin cancers in patients with myeloproliferative neoplasms on ruxolitinib.

ABSTRACT: Nonmelanoma skin cancers (NMSCs) in ruxolitinib-treated patients with myeloproliferative neoplasms behave aggressively, with adverse features and high recurrence. In our cohort, mortality from metastatic NMSC exceeded that from myelofibrosis. Vigilant skin assessment, counseling on NMSC risks, and prospective ruxolitinib-NMSC studies are crucial.

Supplementary orthopaedic screening for children and adolescents to prevent permanent skeletal deformities - protocol for the "OrthoKids" study.

BACKGROUND: Skeletal deformities (SD) in children and adolescents can lead to arthritic conditions, impairment of quality of life, and high treatment costs in the long term. However, comprehensive data on the prevalence of SDs in children and adolescents are limited and it remains therefore unclear whether there is a healthcare gap. "OrthoKids" is a project that addresses this evidence gap by implementing an orthopaedic screening for children and adolescents that supplements existing detection examinations within statutory standard care in Germany. OBJECTIVE: To detect SDs so that they can be treated as needed at an early stage. METHODS: The implementation of the supplementary orthopaedic screening will be evaluated through an exploratory cohort study that is set up in the German state Baden-Wuerttemberg. 20,000 children and adolescents aged 10 to 14 years will be recruited as a prospective cohort. A retrospective control cohort will be formed based on claims data provided by two cooperating statutory health insurances (SHIs). Participating children and adolescents receive a one-time orthopaedic screening. If at least one SD is diagnosed, treatment will be provided as part of the statutory standard care. Within the scope of the project, a follow-up examination will be performed after one year. An IT-platform will complement the study. The primary outcome measure is the point prevalence of scoliosis, genu varum/valgum, hip dysplasia, and flat feet. Secondary outcome measures are (i) the point prevalence of further less common SDs, (ii) health-related quality of life (HRQoL), (iii) sports ability based on activity (physical/athletic), physical constraints, and (sports) injuries, as well as (iv) monetary consequences of the orthopaedic screenings' implementation. Implementation determinants will be evaluated, too. DISCUSSION: If the supplementary orthopaedic screening proves to be viable, it could be considered as a supplementary examination for children and adolescents within the frame of SHI in Germany. This could relieve the burden of disease among children and adolescents with SDs. In addition, it could disburden SHIs in the medium to long term. TRIAL REGISTRATION: The OrthoKids study was registered in the German Clinical Trials Registry (Deutsches Register Klinischer Studien (DRKS)) on 26th July 2022 under the number 00029057.

Effect of apical root resection, orthodontic extrusion, and surgical crown lengthening on load capability.

OBJECTIVES: This study aims to investigate the load-to-fracture of procedures changing crown-to-root ratio (RCRR) aimed to restore severely damaged upper central incisors to avoid tooth extraction compared to implant placement. There is no evidence on load capability after apical root resection (AR), orthodontic extrusion (OE), and surgical crown lengthening (SCL) in respect to RCRR, respectively. MATERIAL AND METHODS: Human maxillary central incisors were endodontically treated, decoronated, and divided into 4 groups (n = 48). The following specimen preparation was performed: (I) adhesive core-and-post build-up (control), (II) as (I) and 2 mm apical root resection (AR), (III) before adhesive core-and-post build-up teeth were shortened 2 mm coronally (OE) (IV) as (I), but specimens were embedded 4 mm instead of 2 mm below the CEJ (SCL), group (V) implant-borne restoration with individual all-ceramic abutments (n = 12; ∅4.1/l = 12 mm) (IBR). All specimens received all-ceramic crowns, thermo-mechanical (TML), and subsequent linear loading (LL) until failure. RCRR were calculated and log-rank, Kruskal-Wallis, Mann-Whitney U, ANOVA, and chi-square tests applied (p = 0.05). RESULTS: Fracture loads after subsequent LL differed significantly (p = 0.001) between groups, while implants showed the highest values. Fmax median (min/max) were as follows: (I) 252 (204/542), (II) 293 (243/443), (III) 253 (183/371), (IV) 195 (140/274), and (V) 446 (370/539). Pair-wise comparison showed significant differences (p = 0.001) between group I/IV and group V, I, and IV (p = 0.045), II and IV (p = 0.001), and III compared to IV (p = 0.033), respectively. RCRR below 1 significantly increased load capability compared to RCRR = 1. CONCLUSIONS: OE appears to preferably ensure biomechanical stability of teeth that are endodontically treated and receive core-and-post and crown placement compared to SCL. AR has no adverse biomechanical impact. RCRR < 1 is biomechanically beneficial. CLINICAL RELEVANCE: For endodontically treated and restored teeth, orthodontic extrusion should be preferred compared to surgical crown lengthening prior single-crown restoration. As orthodontic extrusion, apical root resection has no adverse effect on load capability. Single-crown implant-borne restorations are most load capable.

Success and survival of post-restorations: six-year results of a prospective observational practice-based clinical study.

AIM: This prospective, noninterventional, multi-centre, practice-based study aimed to evaluate the longevity of endodontically treated teeth (ETT) restored with posts and to analyse factors influencing the success and survival of endodontic posts. METHODOLOGY: Eight general dental practitioners each placed up to 27 endodontic posts without any restriction to size and material. Teeth were restricted to incisors, canines and premolars. Multi-level Cox proportional hazards models were used to evaluate the association between clinical factors and time until failure. RESULTS: A total of 195 endodontic posts were followed-up for up to 6.5 years in 195 patients. Of these, 140 posts were judged as successful [mean success time: 59 (55-63) months]; the mean annual failure rate was 8.6%. This decreased to 4.4% when excluding recementations. 152 posts survived [mean survival time: 64 (60-67) months]. Recemented restorations had an eight times higher failure rate compared with new restorations. Furthermore, restorations with glass fibre post had a significantly lower success rate compared with titanium posts. CONCLUSION: Relatively low success and survival rates occurred for restorations with posts after root canal treatment in a private practice setting after a follow-up of up to 6.5 years. Recemented crowns had a high risk of failure.

Longevity of composite build-ups without posts-10-year results of a practice-based study.

AIM: The aim of this prospective, non-interventional, multi-center, practice-based study was, firstly, to evaluate the longevity of composite build-ups in endodontically treated teeth (ETT) without post placement and, secondly, to analyze factors influencing the success of these composite build-ups. METHODOLOGY: Each of seven general dental practitioners placed up to 50 composite build-ups without additional posts in ETT. Teeth were restricted to incisors, canines, and premolars. Several clinical data were recorded for 192 coronal restorations on ETT in 192 patients. Cox proportional hazard models were applied to analyze associations between clinical factors and time until failure. RESULTS: Within a follow-up period of 10 years, 167 restorations were judged as successful [mean success time, 110 (105-115) months] and 180 teeth survived [mean survival time, 114 (110-119) months]. The main failure type was fracture of the restoration (n = 15). The annual failure rate was 2.4%. In bivariate Cox regression, both factors such as number of restored tooth surfaces and adhesive were significantly associated with the failure rate. In multivariate Cox proportional hazards regression, none of the investigated factors were significantly associated with the failure rate. CONCLUSION: For composite build-ups in ETT without post placement, high success rates could be found after up to 10 years of observation time. Within the limitations of the present study, none of the analyzed factors such as "tooth type" or "number of restored tooth surfaces" was a significant predictor for the failure rate. CLINICAL RELEVANCE: Endodontically treated teeth can be successfully directly restored with composite build-ups even when no additional post is inserted. TRIAL REGISTRATION: The study was registered in the German Clinical Trials Register (DRKS-ID: DRKS00012882).

Direct restoration of endodontically treated maxillary central incisors: post or no post at all?

OBJECTIVES: The aim of this ex-vivo study was to evaluate the impact of cavity size and glass-fiber post (GFP) placement on the load capability of endodontically treated maxillary incisors directly restored with resin composite. MATERIALS AND METHODS: Ninety-six extracted human maxillary central incisors were endodontically treated and distributed to four groups (n = 24): access cavity (A), access cavity and uni-proximal class III cavity (U), access cavity and bi-proximal class III cavity (B), and decoronated tooth (D). Specimens were restored with resin composite, and 12 specimen of each group received an adhesively placed glass-fiber post (P). Prior to linear loading, specimens were exposed to thermo-mechanical loading (TCML). Statistical analysis was performed using log-rank test after TCML, Kruskall-Wallis and Mann-Whitney U test to compare load capabilities (Fmax). RESULTS: Significantly more failures occurred in group D for specimens without GFP during TCML (p = 0.001). Fmax (mean (SD) in N was (A) 513 (124), (AP) 554 (201), (U) 438 (171), (UP) 537 (232) (B) 483 (219), (BP) 536 (281), D 143 (181), and DP 500 (331), and differed significantly among groups (p = 0.003). Pair-wise comparison revealed lower Fmax values for group D compared to all other groups (p < 0.034) except group DP. CONCLUSIONS: Endodontically treated maxillary central incisors with cavity sizes up to bi-proximal class III may be successfully directly restored with resin composite. Post placement shows no additional effect except for decoronated endodontically treated incisors. CLINICAL RELEVANCE: Endodontically treated incisors with access cavities to class III cavities can be successfully restored with resin composite. Post placement for decoronated ETT is recommended.

Impact of simulated reduced alveolar bone support, increased tooth mobility, and distal post-supported, root-treated abutment tooth on load capability of all-ceramic zirconia-supported cantilever FDP.

OBJECTIVES: The aim of this in vitro study was an analysis of the impact of simulated reduced alveolar bone support and post-restored, endodontically treated distal abutment tooth on load capability of all-ceramic zirconia-based cantilever-fixed dental prosthesis (CFDP). MATERIAL AND METHODS: The roots of human lower sound premolars (n = 80) were divided into five experimental groups to be restored with all-ceramic zirconia-supported three-unit CFDP regarding bone loss (BL) relative to the cement-enamel junction (CEJ): 2 mm below CEJ = 0% BL (control group), group 25% distal BL, group 50% distal BL, group 50% mesial and distal BL, and group 50% distal BL and adhesive post-supported restoration. Specimens were exposed to simulated clinical function by thermo-mechanical loading (6.000 cycles 5°-55°; 1.2 × 106 cycles 0-50 N) and subsequent linear loading until failure. RESULTS: Tooth mobility increased significantly for groups with simulated bone loss (p < 0.001). Four specimens failed during thermal cycling and mechanical loading (TCML). The maximum load capability ranged from 350 to 569 N, and did not differ significantly between experimental groups (p = 0.095). Groups with simulated bone loss revealed more tooth fractures at distal abutment teeth, whereas technical failures were more frequent in the control group (p = 0.024). CONCLUSIONS: Differences of alveolar bone support and respectively increased tooth mobility between mesial and distal abutments did not influence load capability. A distal adhesively post-and-core-supported, root-treated abutment tooth did not increase risk of three-unit CFDP failure. CLINICAL RELEVANCE: CFDPs are a treatment option used with caution when reduced alveolar bone support, increased tooth mobility, and distal post-supported, root-treated abutment teeth are involved.

Measuring changes in the optic nerve sheath diameter in patients with idiopathic normal-pressure hydrocephalus: a useful diagnostic supplement to spinal tap tests.

BACKGROUND AND PURPOSE: Cerebrospinal fluid (CSF) removal improves clinical symptoms of many patients with idiopathic normal-pressure hydrocephalus (iNPH). The aim of this study was to investigate the correlation of changes in the optic nerve sheath diameter (ONSD) with patient responses to CSF removal. METHODS: Transorbital ultrasonography was performed to obtain ONSD measurements in 31 patients with iNPH before and after lumbar puncture. Measurements were obtained while patients were supine and upright. Changes in the ONSD between supine and upright positions [ONSD variability (ONSD-V)] were assessed and compared with those in 60 healthy volunteers. ONSD-V was correlated with relative changes in a validated iNPH severity (Boon) score. RESULTS: Mean pre-puncture ONSD-V was significantly lower in healthy volunteers and patients with no response to CSF removal (Fisher test) [0.05 ± 0.14 mm (SD)] than in responsive patients [0.37 ± 0.20 mm (SD), P < 0.001]. ONSD-V predicted response to the spinal tap test (odds ratio, 0.30; 95% confidence interval, 0.12-0.75 mm, P = 0.011). The higher the ONSD-V, the better the therapeutic effect (χ2 = 14.980, P < 0.001). The post-spinal tap test ONSD-V correlated significantly with clinical severity in the motor portion of the Boon score [0.16 ± 0.23 mm (SD), P = 0.003]. CONCLUSIONS: The ONSD-V before and after spinal tap test correlated well with the clinical effects of CSF removal. Transorbital ultrasonography seems to be a reliable, safe add-on to the Fisher test and may support selection of patients for shunt intervention.

Anti-contactin-associated protein-2 encephalitis: relevance of antibody titres, presentation and outcome.

BACKGROUND AND PURPOSE: To clarify the relevance of titres of IgG antibodies against contactin-associated protein-2 (CASPR2) in diagnosing anti-CASPR2 encephalitis and to describe features and outcomes. METHODS: This was a retrospective analysis of 64 patients with CASPR2 antibodies, categorized independently as 'autoimmune encephalitis' or 'other disease'. Logistic regression methods were performed to identify potential predictors of 'autoimmune encephalitis' in addition to CASPR2 antibodies. RESULTS: An upfront CASPR2 antibody serum titre cut-off at ≥1:200 had a diagnostic sensitivity of 85% and a specificity of 81%. Logistic regression analyses indicated that, in addition to titre, encephalitic magnetic resonance imaging (MRI) was a significant predictor of 'autoimmune encephalitis' (Nagelkerke's R2 = 0.81, P < 0.001) with high sensitivity (84%) and very high specificity (100%). Patients with CASPR2 antibodies and an estimated probability of >70% of having anti-CASPR2 encephalitis (n = 22) had limbic encephalitis (n = 18, one patient plus ataxia), Morvan syndrome (n = 2) or a hyperkinetic movement disorder (n = 2). Median modified Rankin score (mRS) at diagnosis was 3 (range 1-4). Twenty patients were male; median age was 64 (range 54-75) years; 5/15 patients with cerebrospinal fluid data had intrathecal CASPR2 antibody synthesis, and 12/19 with follow-ups >3 months (median 12 months, range 4-43 months) improved by ≥1 mRS point resulting in a median mRS of 2 (range 0-6; one death; all but one having received immunotherapy); and 2/15 patients with follow-up MRI developed hippocampal atrophy. CONCLUSIONS: Only higher CASPR2 serum antibody titres indicate anti-CASPR2 encephalitis, and diagnostic accuracy increases if MRI findings are considered. Anti-CASPR2 encephalitis has characteristic features and a favourable outcome with immunotherapy.

Chiral Weyl Pockets and Fermi Surface Topology of the Weyl Semimetal TaAs.

Tantalum arsenide is a member of the noncentrosymmetric monopnictides, which are putative Weyl semimetals. In these materials, three-dimensional chiral massless quasiparticles, the so-called Weyl fermions, are predicted to induce novel quantum mechanical phenomena, such as the chiral anomaly and topological surface states. However, their chirality is only well defined if the Fermi level is close enough to the Weyl points that separate Fermi surface pockets of opposite chirality exist. In this Letter, we present the bulk Fermi surface topology of high quality single crystals of TaAs, as determined by angle-dependent Shubnikov-de Haas and de Haas-van Alphen measurements combined with ab initio band-structure calculations. Quantum oscillations originating from three different types of Fermi surface pockets were found in magnetization, magnetic torque, and magnetoresistance measurements performed in magnetic fields up to 14 T and temperatures down to 1.8 K. Of these Fermi pockets, two are pairs of topologically nontrivial electron pockets around the Weyl points and one is a trivial hole pocket. Unlike the other members of the noncentrosymmetric monopnictides, TaAs is the first Weyl semimetal candidate with the Fermi energy sufficiently close to both types of Weyl points to generate chiral quasiparticles at the Fermi surface.

Damage of lithium-disilicate all-ceramic restorations by an experimental self-adhesive resin cement used as core build-ups.

OBJECTIVES: This in vitro study aimed to predict the potential of fracture initiation after long-term incubation (LTI) of lithium-disilicate restorations due to a hygroscopic expansion of self-adhesive resin cement (SARC) used as core build-up material. METHODS: Human maxillary central incisors were divided into four groups (n = 10). Teeth were endodontically treated and decoronated. Specimens were restored in a one-stage post-and-core procedure using experimental dual-curing SARC. Three application protocols to build up the core were compared as follows: I, auto-polymerisation; II, dual curing including 40 s light-initiated polymerisation; and III, an open matrix technique in a dual-curing mode. In group IV, a chemical-curing composite core build-up material served as control. For all specimens, a 2-mm ferrule design was ensured. Full anatomic lithium-disilicate crowns were adhesively luted. One-year LTI in 0.5 % chloramine solution at 37 °C was performed. Restorations were examined after 3, 6, 9 and 12 month of storage. Survival rates were calculated using log-rank statistics (p = 0.05). RESULTS: Fifty per cent of lithium-disilicate crowns of groups I and II showed visible crack propagation after 9 months of incubation, while one crown failed in group III. No failure was observed in group IV. The survival rates differed significantly (p = 0.017). CONCLUSION: SARC used to build up the core of severely damaged endodontically treated teeth does have the potential to cause fracture of lithium-disilicate crown restorations. CLINICAL RELEVANCE: Hygroscopic expansion of self-adhesive resin cements used as a core build-up material might have an adverse impact on longevity of glass-ceramic crowns.

Are implants more reliable than severely compromised endodontically treated teeth as abutments for zirconia-based FPDs? : In vitro results of long-term preclinical load simulation.

OBJECTIVES: The aim was to study the impact of the defect size of endodontically treated incisors compared to dental implants as abutments on the survival of zirconia two-unit anterior cantilever-fixed partial dentures (2U-FPDs) during 10-year simulation. MATERIALS AND METHODS: Human maxillary central incisors were endodontically treated and divided into three groups (n = 24): I, access cavities rebuilt with composite core; II, teeth decoronated and restored with composite; and III as II supported by fiber posts. In group IV, implants with individual zirconia abutments were used. Specimens were restored with zirconia 2U-FPDs and exposed to two sequences of thermal cycling and mechanical loading. STATISTICS: Kaplan-Meier; log-rank tests. RESULTS: During TCML in group I two tooth fractures and two debondings with chipping were found. Solely chippings occurred in groups II (2×), IV (2×), and III (1×). No significant different survival was found for the different abutments (p = 0.085) or FPDs (p = 0.526). Load capability differed significantly between groups I (176 N) and III (670 N), and III and IV (324 N) (p < 0.024). CONCLUSION: Within the limitations of an in vitro study, it can be concluded that zirconia-framework 2U-FPDs on decoronated teeth with/without post showed comparable in vitro reliability as restorations on implants. The results indicated that restorations on teeth with only access cavity perform worse in survival and linear loading. CLINICAL RELEVANCE: Even severe defects do not justify per se a replacement of this particular tooth by a dental implant from load capability point of view.

Mutations in the gene encoding epsilon-sarcoglycan cause myoclonus-dystonia syndrome.

The dystonias are a common clinically and genetically heterogeneous group of movement disorders. More than ten loci for inherited forms of dystonia have been mapped, but only three mutated genes have been identified so far. These are DYT1, encoding torsin A and mutant in the early-onset generalized form, GCH1 (formerly known as DYT5), encoding GTP-cyclohydrolase I and mutant in dominant dopa-responsive dystonia, and TH, encoding tyrosine hydroxylase and mutant in the recessive form of the disease. Myoclonus-dystonia syndrome (MDS; DYT11) is an autosomal dominant disorder characterized by bilateral, alcohol-sensitive myoclonic jerks involving mainly the arms and axial muscles. Dystonia, usually torticollis and/or writer's cramp, occurs in most but not all affected patients and may occasionally be the only symptom of the disease. In addition, patients often show prominent psychiatric abnormalities, including panic attacks and obsessive-compulsive behavior. In most MDS families, the disease is linked to a locus on chromosome 7q21 (refs. 11-13). Using a positional cloning approach, we have identified five different heterozygous loss-of-function mutations in the gene for epsilon-sarcoglycan (SGCE), which we mapped to a refined critical region of about 3.2 Mb. SGCE is expressed in all brain regions examined. Pedigree analysis shows a marked difference in penetrance depending on the parental origin of the disease allele. This is indicative of a maternal imprinting mechanism, which has been demonstrated in the mouse epsilon-sarcoglycan gene.

Loading standardization of postendodontic restorations in vitro: impact of restorative stage, static loading, and dynamic loading.

OBJECTIVE: The load capability of post-restored endodontically treated teeth (ETT) can be determined at different restorative stages. It was the aim of this study to compare the load capability of ETT at these stages. MATERIALS AND METHODS: Maxillary central incisors were divided into 4 groups (n=10) and endodontically treated. Specimens were restored with: (I) only glass fiber posts (GFP); (II) GFP and composite build-up with 2 mm ferrule; (III and IV) with additional adhesively luted all-ceramic crowns. Group (I) to (III) were statically loaded, and group (IV) was exposed to thermomechanical loading (TML) and subsequent static loading. RESULTS: The lowest median load level of 73 N was determined for group (I). The maximum median load value of 331 N was found for group (III). The comparison of Fmax [N] of group (I), (II) and (III) revealed significant differences between the groups (p<0.001). The specimens of group (IV) failed at significantly lower load values (p<0.005) as similarly restored specimens of group (III) which were only statically loaded. The stage of restoration and TML loading prior to static loading had a significant impact on fracture patterns (p=0.006). CONCLUSION: Every additional restorative step torwards a final crown-restored ETT significantly increased the load capability. TML prior to load-to-fracture testing of the complete restorative complex, ie. post, core and crown, significantly decreased maximum load capability.

[Botulinum toxin in focal hyperhidrosis. An update]. / Botulinumtoxin bei fokaler Hyperhidrose. Ein aktueller Überblick.

Eight years after its approval, intralesional injections of botulinum toxin type A have become established as an easily performed, highly effective and almost complication-free therapeutic option in primary axillary hyperhidrosis. Sweat production is decreased to about a sixth of previous amounts, and the effect persists for 7 months on average. Restoration of the often significantly impaired quality of life has been convincingly documented in large studies. The effect of botulinum toxin is based on the inhibition of the release of acetylcholine into the synaptic cleft. In addition to this approved use, botulinum toxin is also successfully employed in other forms of focal hyperhidrosis, particularly in gustatory sweating. However, its use in palmoplantar hyperhidrosis, the second most common form of primary hyperhidrosis, is limited because of the pain from numerous injections, need for increased doses of the expensive toxin and the relatively short effective period of about 4 months. Botulinum toxin type B appears to be comparably effective as type A products but is more often associated with systemic adverse events.

Self-adhesive cements as core build-ups for one-stage post-endodontic restorations?

AIM: To investigate the load capability of root filled teeth restored with glass fibre posts when the same self-adhesive composite resin cement was used as post cement and core build-up material. METHODOLOGY: Human maxillary central incisors were divided into four groups (n=10). Teeth were root filled, decoronated and restored using glass fibre posts luted with different cements and composite resins for core build-up (i) RelyX Unicem/Clearfil Core (RXU/CC), (ii) RelyX Unicem/ RelyX Unicem (RXU/RXU), (iii) RelyX Unicem/LuxaCore-Dual (RXU/LCD) and (iv) LuxaCore-Dual/Clearfil (LCD/CC). A 2-mm ferrule crown preparation was always performed. All specimens were restored with adhesively luted all-ceramic crowns and were exposed to thermal cycling and mechanical loading (TCML) and subsequently statically loaded. For analysis of cycles-to-failure during TCML, log-rank statistics were calculated. The nonparametric Kruskal-Wallis test was applied to study group mean differences. Differences in the frequency of the failure modes between the groups were evaluated by Fisher's exact test. All tests were two-sided (α=0.05). RESULTS: Three specimens of RXU/LCD and two of RXU/RXU and LCD/CC, respectively, failed during TCML (P=0.379). For these specimens, the load capability value was set at 0 N. The median fracture load values (min/max) in (N) were RXU/CC=294 (209/445), RXU/RXU = 166 (0/726), RXU/LCD=241 (0/289) and LCD/CC=200 (0/371) (P=0.091). The RXU/CC had the highest (80%) and RXU/LCD the lowest (20%) percentage of restorable failures (P=0.028). CONCLUSIONS: These results imply that self-adhesive composite achieved similar load capabilities when used as core build-up materials in root filled teeth restored with glass fibre posts and all-ceramic crowns.

Anti-PF4 VITT antibodies are oligoclonal and variably inhibited by heparin.

BACKGROUND: COVID-19 vaccines have been associated with a rare thrombotic and thrombocytopenic reaction, Vaccine-induced immune thrombotic thrombocytopenia (VITT) characterized by platelet-activating anti-PF4 antibodies. This study sought to assess clonality of VITT antibodies and evaluate their characteristics in antigen-based and functional platelet studies. METHODS: Anti-PF4 antibodies were isolated from five patients with VITT secondary to ChAdOx1 nCoV-19 (n=1) or Ad26.COV2.S (n=4) vaccination. For comparative studies with heparin-induced thrombocytopenia (HIT), anti-PF4 antibodies were isolated from one patient with spontaneous HIT, another with "classical" HIT, and two patients with non-pathogenic (non-platelet activating) anti-PF4 antibodies. Isolated antibodies were subject to ELISA and functional testing, and mass spectrometric evaluation for clonality determination. RESULTS: All five VITT patients had oligoclonal anti-PF4 antibodies (3 monoclonal, one bi- and one tri-clonal antibodies), while HIT anti-PF4 antibodies were polyclonal. Notably, like VITT antibodies, anti-PF4 antibodies from a spontaneous HIT patient were monoclonal. The techniques employed did not detect non-pathogenic anti-PF4 antibodies. The ChAdOx1 nCoV-19-associated VITT patient made an excellent recovery with heparin treatment. In vitro studies demonstrated strong inhibition of VITT antibody-induced platelet activation with therapeutic concentrations of heparin in this and one Ad26.COV2.S-associated VITT patient. Oligoclonal VITT antibodies with persistent platelet-activating potential were detected at 6 and 10 weeks after acute presentation in two patients tested. Two of the 5 VITT patients had recurrence of thrombocytopenia and one patient had focal seizures several weeks after acute presentation. CONCLUSION: Oligoclonal anti-PF4 antibodies mediate VITT. Heparin use in VITT needs to be further studied.

Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors nuclear factor kappaB and activator protein 1 and the induction of inflammatory cytokines.

We have studied the effect of human bacterial pathogen Neisseria gonorrhoeae (Ngo) on the activation of nuclear factor (NF)-kappaB and the transcriptional activation of inflammatory cytokine genes upon infection of epithelial cells. During the course of infection, Ngo, the etiologic agent of gonorrhea, adheres to and penetrates mucosal epithelial cells. In vivo, localized gonococcal infections are often associated with a massive inflammatory response. We observed upregulation of several inflammatory cytokine messenger RNAs (mRNAs) and the release of the proteins in Ngo-infected epithelial cells. Moreover, infection with Ngo induced the formation of a NF-kappaB DNA-protein complex and, with a delay in time, the activation of activator protein 1, whereas basic leucine zipper transcription factors binding to the cAMP-responsive element or CAAT/enhancer-binding protein DNA-binding sites were not activated. In supershift assays using NF-kappaB-specific antibodies, we identified a NF-kappaB p50/p65 heterodimer. The NF-kappaB complex was formed within 10 min after infection and decreased 90 min after infection. Synthesis of tumor necrosis factor alpha and interluekin (IL)-1beta occurred at later times and therefore did not account for NF-kappaB activation. An analysis of transiently transfected IL-6 promoter deletion constructs suggests that NF-kappaB plays a crucial role for the transcriptional activation of the IL-6 promoter upon Ngo infection. Inactivation of NF-kappaB conferred by the protease inhibitor N-tosyl--phenylalanine chloromethyl ketone inhibited mRNA upregulation of most, but not all, studied cyctokine genes. Activation of NF-kappaB and cytokine mRNA upregulation also occur in Ngo-infected epithelial cells that were treated with cytochalasin D, indicating an extracellular signaling induced before invasion.

Coordinate activation of activator protein 1 and inflammatory cytokines in response to Neisseria gonorrhoeae epithelial cell contact involves stress response kinases.

Neisseria gonorrhoeae (Ngo), the etiologic agent of gonorrhea, induce a number of proinflammatory cytokines by contact to epithelial cells. Cytokine genes and a variety of other immune response genes are activated as a result of the regulatory function of immediate early response transcription factors including activator protein 1 (AP-1). Since it is established that phosphorylation of c-Jun, the central component of AP-1, by the stress-activated c-Jun NH2-terminal kinase (JNK) increases the transcriptional activity of AP-1, we studied whether Ngo could induce stress response pathways involving JNK. We found that virulent Ngo strains induce phosphorylation and activation of JNK but not of p38 kinase. Analysis of a nonpathogenic Ngo strain revealed only weak JNK activation. In respect to the molecular components upstream of the JNK signaling cascade, we show that a dominant negative mutant of MAP kinase kinase 4 (MKK4) represses transcription of an AP-1-dependent reporter gene. Regarding upstream stress response factors involved in Ngo-induced MKK4/JNK/AP-1 activation, we identified p21-activated kinase (PAK) but not MAPK/ERK kinase kinase (MEKK1). Inhibition of small GTPases including Rac1 and Cdc42 by Toxin B prevented JNK and AP-1 activation. Our results indicate that Ngo induce the activation of proinflammatory cytokines via a cascade of cellular stress response kinases involving PAK, which directs the signal from the Rho family of small GTPases to JNK/AP-1 activation.