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Robust biomarkers to predict response to immune checkpoint blockade (ICB) in metastatic urothelial carcinoma (mUC) are still in demand. Recently, early C-reactive protein (CRP) kinetics and especially the novel CRP flare-response phenomenon has been associated with immunotherapy response.We conducted a multicentre observational study comprising 154 patients with mUC treated with ICB to evaluate the predictive value of a previously described on-treatment CRP kinetics: CRP flare responders (at least doubling of baseline CRP within the first month after initiation of ICB followed by a decline below baseline within three months), CRP responders (decline in baseline CRP by ≥ 30% within three months without a prior flare) and the remaining patients as CRP non-responders. CRP kinetics groups were correlated with baseline parameters, PD-L1 status, progression-free survival (PFS) and overall survival (OS).Objective response was observed in 57.1% of CRP responders, 45.8% of CRP flare responders and 17.9% of CRP non-responders (P 0.001). CRP flare response was associated with prolonged PFS and OS (P 0.001). In multivariable Cox regression analysis, CRP flare responders showed a risk reduction of ∼70% for tumour progression and death compared to CRP non-responders. Subgroup analysis of CRP flare responders revealed that patients with a long-flare response (completed flare-response kinetics ≥6 weeks on-treatment) showed even more favourable outcomes following ICB (HR = 0.18, 95%-CI: 0.07-0.48, P 0.001).CRP (flare)response robustly predicts immunotherapy response and outcomes in mUC independent of PD-L1 status. Thus, early on-treatment CRP kinetics is a promising low-cost and easy-to-implement biomarker to optimise therapy monitoring in patients with mUC treated with ICB.

Supplementary Table 2. Baseline characteristics of the multicenter EV cohort.

Purpose:The antibody–drug conjugate enfortumab vedotin (EV) releases a cytotoxic agent into tumor cells via binding to the membrane receptor NECTIN-4. EV was recently approved for patients with metastatic urothelial carcinoma (mUC) without prior assessment of the tumor receptor status as ubiquitous NECTIN-4 expression is assumed. Our objective was to determine the prevalence of membranous NECTIN-4 protein expression in primary tumors (PRIM) and patient-matched distant metastases (MET).Experimental Design:Membranous NECTIN-4 protein expression was measured (H-score) by IHC in PRIM and corresponding MET (N = 137) and in a multicenter EV-treated cohort (N = 47). Progression-free survival (PFS) after initiation of EV treatment was assessed for the NECTIN-4–negative/weak (H-score 0–99) versus moderate/strong (H-score 100–300) subgroup. The specificity of the NECTIN-4 IHC staining protocol was validated by establishing CRISPR-Cas9–induced polyclonal NECTIN-4 knockouts.Results:In our cohort, membranous NECTIN-4 expression significantly decreased during metastatic spread (Wilcoxon matched pairs P < 0.001; median H-score = 40; interquartile range, 0–140), with 39.4% of MET lacking membranous NECTIN-4 expression. In our multicenter EV cohort, absence or weak membranous NECTIN-4 expression (34.0% of the cohort) was associated with a significantly shortened PFS on EV (log-rank P < 0.001).Conclusions:Membranous NECTIN-4 expression is frequently decreased or absent in mUC tissue. Of note, the clinical benefit of EV strongly depends on membranous NECTIN-4 expression. Thus, our results are of highest clinical relevance and argue for a critical reconsideration of the current practice and suggest that the NECTIN-4 receptor status should be determined (ideally in a metastatic/progressive lesion) before initiation of EV.See related commentary by Aggen et al., p. 1377

Background Ionotropic glutamate receptors AMPAR and NMDAR modulate proliferation, invasion and radioresistance in glioblastoma (GB). Pharmacological targeting is difficult as many in vitro-effective agents are not suitable for in patient applications. We aimed to develop a method to test the well tolerated AMPAR- and NMDAR-antagonist xenon gas as radiosensitizer in GB. Methods We designed a diffusion-based system to perform the colony formation assay (CFA), the radiobiological gold standard, under xenon exposure. Stable and reproducible gas atmosphere was validated with oxygen and carbon dioxide as tracer gases. After checking for AMPAR and NMDAR expression via immunofluorescence staining we performed the CFA with the glioblastoma cell lines U87 and U251 as well as the non-glioblastoma derived cell line HeLa. Xenon was applied after irradiation and additionally tested in combination with NMDAR antagonist memantine. Results The gas exposure system proved compatible with the CFA and resulted in a stable atmosphere of 50% xenon. Glutamate receptor subunits were present in glioblastoma-derived and to a lesser extent in HeLa cells. Significant radiosensitization by xenon was shown in U251 and U87 at irradiation doses of 4–8 Gy and 2–8 Gy, respectively (p < .05). The effect was further enhanced by the addition of memantine, showing significant reduction in the surviving fraction already at 2 Gy for both glioblastoma cell lines (p < .05). Xenon did not increase the radiosensitivity of HeLa cells until a radiation dose of 8 Gy. Conclusion The developed system allows for testing of gaseous agents with CFA. As a proof of concept, we have, for the first time, unveiled radiosensitizing properties of xenon gas in glioblastoma.

Aim Less than a dozen cases of psoas abscesses in pregnancy have been described in the literature. We reviewed the literature when treating a patient with a psoas abscess after ipsilateral double J-ureteral stent placement (in the following: “double J-stent”) due to infected hydronephrosis. Methods In January 2022, this review was searched using the Pubmed/MEDLINE database and the mesh terms “Psoas Abscess” AND “Pregnancy”. Studies were included in any language and of all years, describing a psoas abscess during pregnancy. When patients did not have a psoas abscess, the abscess occurred after pregnancy, or when there was no full text available, the article was excluded. Main results Ten case reports about patients with psoas abscesses during pregnancy were included. The classical symptomatic triad of psoas muscle abscess included lower back pain, limping and persistent fever with daily spikes. However, in most cases, not all three symptoms can be found. Especially, fever is absent in more than half of the patients. Psoas abscesses are described between 13 and 39 weeks of gestation. Primary psoas abscesses with haematogenous spread are more common during pregnancy than secondary with spread per continuitatem. In the literature, the main reasons for psoas abscess are spinal tuberculosis, drug abuse or underlying diseases such as Crohn’s disease. It is not uncommon for the definite cause to be unclear. Regarding the patient's symptoms, pyelonephritis is often considered a possible aetiology. In general, the main treatment options include antibiotic treatment and abscess drainage. There is no higher caesarean section rate, and no negative outcome for the foetus has been described. Case presentation In our patient, a 38-year-old obese Caucasian woman, who had received a left double J-stent for infected hydronephrosis at 15 weeks of gestation, we successfully treated a psoas abscess of 20 × 10 cm with a sonographically assisted abscess drainage and antibiotics. The further course of pregnancy and the elective repeat caesarean section at 38 + 0 weeks of gestation were without any problems. Double J-stent placement and laser stone lithotripsy during puerperium were performed because of recurrent urolithiasis. Conclusions Although rare, psoas abscesses can occur during pregnancy, and it has often been treated surgically in the past. A psoas abscess as a complication after infected hydronephrosis and intervention during pregnancy has never been reported in the literature. Even for obese patients, minimally invasive therapy may be a treatment option that has rarely been reported in the literature.

Purpose: The antibody–drug conjugate enfortumab vedotin (EV) releases a cytotoxic agent into tumor cells via binding to the membrane receptor NECTIN-4. EV was recently approved for patients with metastatic urothelial carcinoma (mUC) without prior assessment of the tumor receptor status as ubiquitous NECTIN-4 expression is assumed. Our objective was to determine the prevalence of membranous NECTIN-4 protein expression in primary tumors (PRIM) and patient-matched distant metastases (MET). Experimental Design: Membranous NECTIN-4 protein expression was measured (H-score) by IHC in PRIM and corresponding MET (N = 137) and in a multicenter EV-treated cohort (N = 47). Progression-free survival (PFS) after initiation of EV treatment was assessed for the NECTIN-4–negative/weak (H-score 0–99) versus moderate/strong (H-score 100–300) subgroup. The specificity of the NECTIN-4 IHC staining protocol was validated by establishing CRISPR-Cas9–induced polyclonal NECTIN-4 knockouts. Results: In our cohort, membranous NECTIN-4 expression significantly decreased during metastatic spread (Wilcoxon matched pairs P < 0.001; median H-score = 40; interquartile range, 0–140), with 39.4% of MET lacking membranous NECTIN-4 expression. In our multicenter EV cohort, absence or weak membranous NECTIN-4 expression (34.0% of the cohort) was associated with a significantly shortened PFS on EV (log-rank P < 0.001). Conclusions: Membranous NECTIN-4 expression is frequently decreased or absent in mUC tissue. Of note, the clinical benefit of EV strongly depends on membranous NECTIN-4 expression. Thus, our results are of highest clinical relevance and argue for a critical reconsideration of the current practice and suggest that the NECTIN-4 receptor status should be determined (ideally in a metastatic/progressive lesion) before initiation of EV. See related commentary by Aggen et al., p. 1377

Background: The methylation status of Septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) in circulating cell-free DNA (ccfDNA) are validated pan-cancer biomarkers. The present proof-of-concept study aimed to investigate the potential and dynamics of quantitative SEPT9 and SHOX2 methylation in prostate cancer (PCa) patient tissue and ccfDNA during prostate biopsy as a diagnostic tool. Methods: The methylation patterns of SEPT9 and SHOX2 in prostate tissue were analyzed using The Cancer Genome Atlas data set (n = 498 PCa and n = 50 normal adjacent prostate tissue (NAT)). Next, dynamic changes of ccfDNA methylation were quantified in prospectively enrolled patients undergoing prostate biopsy (n = 72), local treatment for PCa (n = 7; radical prostatectomy and radiotherapy) as well as systemic treatment for PCa (n = 6; chemotherapy and 177-Lu-PSMA-therapy). Biomarker levels were correlated with clinicopathological parameters. Results: SEPT9 and SHOX2 were hypermethylated in PCa tissue (p < 0.001) and allowed discrimination of PCa and non-tumor prostate tissue (mSEPT9: AUC 0.87, 95%CI [0.82–0.92]; mSHOX2: AUC 0.89, 95%CI 0.84–0.94). SHOX2 methylation and mRNA levels were significantly higher in PCa tissue and increased with tumor stage and grade, as well as in patients suffering from biochemical recurrence following radical prostatectomy. SEPT9 and SHOX2 ccfDNA methylation allowed distinguishing patients with localized and metastatic disease (p < 0.001 for both). In addition, methylation levels increased shortly after prostate biopsy only in patients with PCa (ΔmSEPT9: p < 0.001 and ΔmSHOX2: p = 0.001). Conclusions: The early dynamics of methylated SEPT9 and SHOX2 in ccfDNA allow differentiation between PCa patients and patients without PCa and is a promising marker for tumor monitoring in the metastatic stage to determine tumor burden under systemic therapy.

Simple Summary With the introduction of immune checkpoint inhibitors (ICI) in recent years, the treatment landscape of metastatic urothelial cancer has undergone a substantial transformation. Nevertheless, disease progression after prior platinum-based chemotherapy and ICI pretreatment remains a challenging clinical situation with little evidence for following therapeutic options. The aim of this multicenter analysis was to examine the efficacy of the vinca alkaloid vinflunine after previous ICI therapy. In our cohort, post-ICI patients showed an overall response rate (ORR) of 22.4% compared to 15.6% within ICI-naïve patients (p = 0.451), and the clinical benefit rate (CBR) was 51.0% vs. 25.0% (p = 0.020), respectively. Post-ICI patients showed longer OS (8.78 vs. 5.72 months; p = 0.467) and longer PFS (3.09 vs. 2.14 months; p = 0.105). Our analysis demonstrates the clinical activity of vinflunine in a third- or later-line post-ICI setting, and the therapeutic benefit may be considerably higher than demonstrated in previous studies. Abstract Background: Immune checkpoint inhibitors (ICI) are standard of care in patients with metastatic urothelial carcinoma (mUC) ineligible for cisplatin, and as second-line therapy after platinum-based chemotherapy. To date, few data exist about the efficacy of the former second-line chemotherapeutic agent vinflunine after the failure of sequential platinum-based chemotherapy and ICI treatment. The aim of this analysis was to examine the efficacy of vinflunine in a post-ICI third- or later-line setting. Methods: In this retrospective German multicenter study, data of mUC patients treated with vinflunine were reviewed in six centers between February 2010 and December 2021. All of the 105 included patients had radiologic progression after first-line platinum-based chemotherapy. The objective was to describe the efficacy of vinflunine in terms of overall response rate (ORR), clinical benefit rate (CBR), overall survival (OS), and progression-free survival (PFS) for post-ICI and ICI-naïve patients, respectively. Results: In our cohort, 61 patients (58.1%) had preceding immunotherapy before vinflunine administration, and 44 patients (41.9%) were ICI-naïve. Patients with ICI pretreatment showed an ORR of 22.4% compared to 15.6% within ICI-naïve patients (p = 0.451), and CBR was 51.0% vs. 25.0% (p = 0.020), respectively. Post-ICI patients showed longer OS (8.78 vs. 5.72 months; p = 0.467) and longer PFS (3.09 vs. 2.14 months; p = 0.105). Conclusion: This analysis supports the sequential use of vinflunine in post-ICI patients since the vinca-alkaloid retains a measurable clinical activity in these heavily pretreated patients. The therapeutic benefit may be higher than demonstrated in previous studies.

Background: Impaired kidney function increases the risk of vascular events in stroke patients, when assessed by single measurements of serum creatinine and estimated glomerular filtration rate (eGFR). Whether repeated measurements improve risk prediction in acute stroke patients is unknown. Methods: The investigator-initiated, prospective, multicenter Systematic Monitoring for Detection of Atrial Fibrillation in Patients with Acute Ischemic Stroke study randomized 3,465 acute ischemic stroke patients without known AF to usual diagnostic procedures for detecting AF or an additive Holter-ECG. eGFR was estimated by the CKD-Epi formula. Dynamics of eGFR during hospitalization was categorized as “stable ≥60ml/min”, “rising” (by at least 15% from any baseline value), “falling” (by at least 15% and baseline eGFR≥60ml/min) and “stable Results: The dynamics of eGFR were available in 1,623 patients. In 1,001 (61.7%) eGFR was “stable≥60ml/min”, in 231 (6.7%) “rising”, in 74 (4.6%) “falling”, and in 317 (19.5%) “stable Conclusions: Hospitalized ischemic stroke patients with a falling eGFR (of at least 15%, baseline ≥60ml/min) are at similar risk for recurrent vascular events or death and at higher risk for death as stroke patients with constantly reduced kidney function. Repeated estimates of eGFR may therefore improve risk prediction.

BackgroundPersistent antiphospholipid antibodies (aPL) constitute the serological hallmark of the antiphospholipid syndrome (APS). Recently, various new assay technologies for the detection of aPL better suited to multiplex reaction environments than ELISAs emerged. We evaluated the diagnostic performance of such a novel line immunoassay (LIA) for the simultaneous detection of 10 different aPL.MethodsFifty-three APS patients and 34 healthy controls were investigated for criteria (antibodies against cardiolipin [aCL], β2-glycoprotein I [aβ2-GPI]) and non-criteria aPL (antibodies against phosphatidic acid [aPA], phosphatidyl-choline [aPC], -ethanolamine [aPE], -glycerol [aPG], -inositol [aPI], -serine [aPS], annexin V [aAnnV], prothrombin [aPT]) IgG and IgM by LIA. Criteria aPL were additionally determined with the established Alegria (ALE), AcuStar (ACU), UniCap (UNI), and AESKULISA (AES) systems and non-criteria aPL with the AES system. Diagnostic performance was evaluated with a gold standard for criteria aPL derived from the results of the four established assays via latent class analysis and with the clinical diagnosis as gold standard for non-criteria aPL.ResultsAssay performance of the LIA for criteria aPL was comparable to that of ALE, ACU, UNI, and AES. For non-criteria aPL, sensitivities of the LIA for aPA-, aPI-, aPS-IgG and aPA-IgM were significantly higher and for aPC-, aPE-, aAnnV-IgG and aPC- and aPE-IgM significantly lower than AES. Specificities did not differ significantly.ConclusionsThe LIA constitutes a valuable diagnostic tool for aPL profiling. It offers increased sensitivity for the detection of aPL against anionic phospholipids. In contrast, ELISAs exhibit strengths for the sensitive detection of aPL against neutral phospholipids.

Purpose Anti-phospholipid antibodies (aPL) analyzed by line immunoassay (LIA) can recognize beta2-glycoprotein I (β2GPI) domain 1 (D1) epitopes depending on β2GPI binding to distinct phospholipids. The aPL LIA was compared with consensus ELISA to investigate whether both techniques can discriminate anti-phospholipid syndrome (APS) patients from aPL-positive, systemic autoimmune rheumatic diseases (SARD) patients without clinical symptoms of APS and controls. Methods Thirty-four APS patients (14 arterial/venous thrombosis, 16 pregnancy morbidity, and 4 both), 41 patients with SARD lacking clinical APS criteria but demonstrating positivity for anti-β2GPI (aβ2GPI) IgG, and 20 healthy subjects (HS) were tested for aPL to cardiolipin (aCL), phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol (aPG), phosphatidylinositol, phosphatidylserine, β2GPI, prothrombin, and annexin V by LIA. Samples were also tested for aCL, aβ2GPI, aβ2GPI-domain 1 (aD1), and aβ2GPI-domains 4–5 (aD4–5) by ELISA and for lupus anti-coagulant. Results Comparison of LIA with ELISA revealed a good agreement for the consensus criteria aPL aβ2GPI and aCL IgG (kappa = 0.69, 0.68, respectively) and a moderate agreement for IgM (kappa = 0.52, 0.49, respectively). Regarding ELISA, aD1/aD4–5 demonstrated the best performance of differentiating APS from asymptomatic SARD [area under the curve (AUC): 0.76]. aPG IgG had the best performance by LIA (AUC: 0.72) not significantly different from aD1/aD4–5. There was a good agreement for aPG IgG with aD1/aD4–5 (kappa = 0.71). Conclusions aD1/aD4–5 (ELISA) and aPG IgG (LIA) differentiate APS from SARD patients. PG appears to interact with β2GPI of APS patients and exposes D1 thereof for disease-specific aPL binding in LIA.

Previous work has demonstrated that cerebral echo contrast enhancement can be assessed by means of transcranial ultrasound using transient response second harmonic imaging (HI). The current study was designed to explore possible advantages of two new contrast agent specific imaging modes, contrast burst imaging (CBI) and time variance imaging (TVI), that are based on the detection of destruction or splitting of microbubbles caused by ultrasound in comparison with contrast harmonic imaging (CHI), which is a broadband phase-inversion—based implementation of HI. Nine healthy individuals with adequate acoustic temporal bone windows were included in the study. Contrast harmonic imaging, CBI, and TVI examinations were performed in an axial diencephalic plane of section after an intravenous bolus injection of 4 g galactose-based microbubble suspension in a concentration of 400 mg/mL. Using time-intensity curves, peak intensities and times-to peak-intensity (TPIs) were calculated off-line in anterior and posterior parts of the thalamus, in the region of the lentiform nucleus, and in the white matter. The potential of the different techniques to visualize cerebral contrast enhancement in different brain areas was compared. All techniques produced accurate cerebral contrast enhancement in the majority of investigated brain areas. Contrast harmonic imaging visualized signal increase in 28 of 36 regions of interest (ROIs). In comparison, TVI and CBI examinations were successful in 32 and 35 investigations, respectively. In CHI examinations, contrast enhancement was most difficult to visualize in posterior parts of the thalamus (6 of 9) and the lentiform nucleus (6 of 9). In TVI examinations, anterior parts of the thalamus showed signal increase in only 6 of 9 examinations. For all investigated imaging modes, PIs and TPIs in different ROIs did not differ significantly, except that TVI demonstrated significantly higher PIs in the lentiform nucleus as compared with the thalamus and the white matter ( P < 0.05). The current study demonstrates for the first time that CBI and TVI represent new ultrasonic tools that allow noninvasive assessment of focal cerebral contrast enhancement and that CBI and TVI improve diagnostic sensitivity as compared with CHI.

Background —Second harmonic imaging (SHI) is a new ultrasound technique that is able to detect microbubbles in the tissue vascular space. The aim of this pilot study was to prove that this technique may detect focal abnormalities of cerebral echo-contrast enhancement in acute hemispheric stroke. Case Descriptions —Two male patients (aged 72 and 64 years) were included who presented with acute onset of severe hemiparesis and no established demarcation of the ischemic area in CT scans. After bolus application of galactose-based microbubbles, axial SHI examinations in a diencephalic plane of sections were performed using the transtemporal approach. Ultrasound investigations were recorded and evaluated offline. In both individuals demarcated focal abnormalities of cerebral contrast enhancement were detectable: in patient 1 the region of the lentiform nucleus and the adjacent parts of the temporoparietal lobe was affected, and in patient 2 a large region including the lentiform nucleus and cortical white matter was involved for at least 24 hours. Follow-up CT scans demonstrated a striatocapsular infarct in patient 1 and complete MCA infarction in patient 2, correlating with the presumed ischemic area in acute ultrasound examinations. The patient with complete MCA infarction showed missing contrast enhancement in the entire hemisphere of the affected side in follow-up SHI examinations. He died of malignant space-occupying brain edema. In the patient with the striatocapsular infarction, reappearance of echo-contrast enhancement in the ischemic area was assessable after 1 week. Conclusions —SHI may identify focal abnormalities of cerebral echo-contrast enhancement in acute hemispheric stroke. Furthermore, this technique helps to determine size, localization, and prognosis of the ischemic region and could be useful for bedside assessment of echo-contrast agent distribution related to brain tissue perfusion.

To the Editor: Baumgartner et al1 recently reported on the diagnostic value of contrast-enhanced transcranial color-coded duplex sonography (CE-TCCS) in ischemic cerebrovascular disease. In this study, 33 patients with insufficient temporal insonation conditions (21 patients had ischemic stroke and 12 suffered from transient ischemic attack) were investigated after application of a galactose-based echo contrast agent. The presence of an insufficient temporal bone window was indicated when two sonographers estimated that they were unable to evaluate the basal cerebral arteries by means of color and spectral Doppler imaging in unenhanced examinations. After application of a galactose-based echo contrast agent, 66% of the CE-TCCS examinations were considered conclusive. Cross-flow through the anterior and posterior communicating arteries due to extracranial occlusive disease could be demonstrated in 3 and 2 patients, respectively. No stenoses or occlusions of intracranial arteries could be visualized. We would like to add our CE-TCCS experiences in severely affected stroke individuals with insufficient acoustic bone windows (IABW). 30 patients (17 women and 13 men; mean age, 75.2 [range, 59 to 86] years) with IABW and severe cerebrovascular event (European Stroke Scale score of

Background and Purpose Elevations of protein S-100 (S-100) in cerebrospinal fluid and serum have been reported after cerebral infarctions. The aim of our study was to evaluate the time course of serum S-100 concentrations after territorial middle cerebral artery (MCA) infarctions in correlation with clinical data and prognosis. Methods S-100 serum levels were serially determined in 26 patients with an acute infarction in the territory of the MCA at day 0 (within 12 hours after onset of symptoms), day 1 (24 hours after stroke onset), and days 2, 3, 4, 5, 7 or 8, and 10 after stroke and in 26 age- and sex-matched control subjects. S-100 assays were performed using a two-site radioimmunoassay technique. The clinical status was documented using the Scandinavian Stroke Scale. The functional deficit 4 weeks after stroke onset was scored by use of the modified Rankin scale. A cranial computed tomography (CCT) was performed initially and at day 4 or 5. Results Elevated concentrations of S-100 (>0.2 μg/L) were observed in 21 of 26 patients with MCA infarction but in none of the control subjects. S-100 levels peaked at days 2 and 3 after stroke. The S-100 concentrations in serum were significantly higher in patients with severe neurological deficits at admission, with extensive infarctions and a space-occupying effect of ischemic edema as compared with the rest of the population. S-100 values were not significantly correlated with the functional prognosis. Conclusions Presence of S-100 in serum after ischemic stroke may be due to combined leakage out of necrotic glial cells and passage through an impaired brain-blood barrier, indicating severe ischemic cell injury. Therefore, S-100 in serum can be used as a peripheral marker of ischemic focal brain damage and may be helpful for therapeutic decisions in acute ischemic stroke.

Spinocerebellar ataxia type 6 (SCA6), episodic ataxia type 2 (EA2) and familial hemiplegic migraine type 1 (FHM1) are allelic disorders of the gene CACNA1A encoding the P/Q subunit of a voltage gated calcium channel. While SCA6 is related to repeat expansions affecting the C-terminal part of the protein, EA2 and FHM phenotypes are usually associated with nonsense and missense mutations leading to impaired channel properties. In three unrelated families with dominant cerebellar ataxia, symptoms cosegregated with CACNA1A missense mutations of evolutionary highly conserved amino acids (exchanges p.E668K, p.R583Q and p.D302N). To evaluate pathogenic effects, in silico, protein modeling analyses were performed which indicate structural alterations of the novel mutation p.E668K within the homologous domain 2 affecting CACNA1A protein function. The phenotype is characterised by a very slowly progressive ataxia, while ataxic episodes or migraine are uncommon. These findings enlarge the phenotypic spectrum of CACNA1A mutations.

"The occupational skill structure depends on the business cycle if employers respond to shortages of applicants during upturns by lowering their hiring standards. The notion and relevance of hiring standards adjustment was advanced by Reder (1955) and investigated formally in a search-theoretic framework by Mortensen (1970). Devereux (2002) implements empirical tests for these theories and finds affirmative evidence for the U.S labour market. We replicate his analysis using German employment register data. Regarding the occupational skill composition we obtain somewhat lower but qualitatively similar responses to the business cycle despite of well known institutional differences between the U.S. and German labour market. The responsiveness of occupational composition wages to the business cycle is considerably lower in Germany." (Author's abstract, IAB-Doku) ((en))

I will discuss my experience with Stata's ml method d2 when coding and estimator for a multi-state Markov transition model with unobserved heterogeneity. When analytical derivatives are available, programming a "d2" estimator is in principle straightforward and offers potentially huge rewards in terms of convergence and speed of convergence: When the likelihood is flat, method "d0" may fail to converge (after a many iterations) as numerical derivatives cannot be computed, whereas convergence is often achieved quickly with method "d2". However, when the likelihood function is non-standard, programming a "d2" estimator may be complicated by Stata's limited range of matrix commands. In these cases, the researcher has to be inventive and may have to take a significant "diversion" to compute blocks of the Hessian that should have been straightforward with enhanced matrix capabilities. These "diversions" may be difficult to code and increase evaluation time significantly. With large datasets, this may also push the memory requirements beyond the available limit.

People with brain injuries involving the amygdala are often poor at recognizing facial expressions of fear, but the extent to which this impairment compromises other signals of the emotion of fear has not been clearly established. We investigated N.M., a person with bilateral amygdala damage and a left thalamic lesion, who was impaired at recognizing fear from facial expressions. N.M. showed an equivalent deficit affecting fear recognition from body postures and emotional sounds. His deficit of fear recognition was not linked to evidence of any problem in recognizing anger (a common feature in other reports), but for his everyday experience of emotion N.M. reported reduced anger and fear compared with neurologically normal controls. These findings show a specific deficit compromising the recognition of the emotion of fear from a wide range of social signals, and suggest a possible relationship of this type of impairment with alterations of emotional experience.

Background and Purpose —Gray-scale harmonic imaging is the first method to visualize blood perfusion and capillary blood flow with ultrasound after intravenous contrast agent application. The purpose of the present study was to evaluate the potential of transient response second harmonic imaging (TRsHI) to assess normal echo contrast characteristics in different brain areas by transcranial ultrasound. Methods —In 18 patients without cerebrovascular diseases, TRsHI examinations were performed bilaterally with the use of the transtemporal approach after application of 6.5 mL of a galactose-based microbubble suspension (400 mg/mL). The transmission rate was once every 4 cardiac cycles. Regional cerebral contrast was visually assessed and then quantified off-line with the use of time-intensity curves. In 4 different regions of interest (ROI) (posterior part of the thalamus [ROIa], anterior part of the thalamus [ROIb], lentiform nucleus [ROIc], and white matter [ROId]), the following parameters were evaluated: peak intensity, area under the curve (AUC), and time to peak intensity. AUC ratios for ROIc/a, d/a, c/b, and d/b were calculated. Results —In all patients parenchymal contrast enhancement was visually detectable. One hundred thirty-one characteristic time-intensity curves (baseline phase, peak contrast intensity, slow washout phase) were demonstrable in 144 ROIs. In ROIc and ROId, characteristic contrast curves could be observed most frequently (68/72 examinations), whereas time-intensity curves in ROIa and ROIb could not be evaluated because of inadequate contrast enhancement in 9 of 72 examinations. Time to peak intensity varied between 20 and 52 cardiac cycles; in 1 patient it was 88 cardiac cycles. In all individuals AUCs and in 16 of 18 subjects peak intensity in ROIc and ROId showed a 2- to 10-fold increase compared with ROIa and ROIb. In no examination did AUC ratios show a >2-fold side difference irrespective of the ROI. Conclusions —The present study demonstrates for the first time that TRsHI produces accurate contrast in different brain areas and represents an ultrasonic tool related to brain perfusion. Absolute values of quantitative parameters show high variations caused by different temporal bone thicknesses and a complex relationship between echo contrast concentrations and measurements of optic intensities. Ratios between different ROIs help to compare contrast enhancement in different brain areas. Furthermore, because of the fact that attenuation of contrast enhancement in TRsHI depends strictly on the insonation depth, harmonic imaging studies of brain perfusion cannot be compared directly with other imaging techniques such as positron emission tomography.

Introduction Indirect immunofluorescence (IIF) employing ethanol-fixed neutrophils (ethN) is still the method of choice for assessing antineutrophil cytoplasmic antibodies (ANCA) in ANCA-associated vasculitides (AAV). However, conventional fluorescence microscopy is subjective and prone to high variability. The objective of this study was to evaluate novel pattern recognition algorithms for the standardized automated interpretation of ANCA patterns. Methods Seventy ANCA-positive samples (20 antimyeloperoxidase ANCA, 50 antiproteinase3 ANCA) and 100 controls from healthy individuals analyzed on ethN and formalin-fixed neutrophils (formN) by IIF were used as a 'training set' for the development of pattern recognition algorithms. Sera from 342 patients ('test set') with AAV and other systemic rheumatic and infectious diseases were tested for ANCA patterns using the novel pattern recognition algorithms and conventional fluorescence microscopy. Results Interpretation software employing pattern recognition algorithms was developed enabling positive/negative discrimination and classification of cytoplasmic ANCA (C-ANCA) and perinuclear ANCA (P-ANCA). Comparison of visual reading of the 'test set' samples with automated interpretation revealed Cohen's kappa (κ) values of 0.955 on ethN and 0.929 on formN for positive/negative discrimination. Analysis of the 'test set' with regard to the discrimination between C-ANCA and P-ANCA patterns showed a high agreement for ethN (κ = 0.746) and formN (κ = 0.847). There was no significant difference between visual and automated interpretation regarding positive/negative discrimination on ethN and formN, as well as ANCA pattern recognition (P > 0.05, respectively). Conclusions Pattern recognition algorithms can assist in the automated interpretation of ANCA IIF. Automated reading of ethN and formN IIF patterns demonstrated high consistency with visual ANCA assessment.

Introduction Diagnosis of antiphospholipid syndrome (APS) still remains a laboratory challenge due to the great diversity of antiphospholipid antibodies (aPL) and their significance regarding APS-diagnostic criteria. Methods A multi-line dot assay (MLDA) employing phosphatidylserine (PS), phosphatidylinositol (PI), cardiolipin (CL), and beta2-glycoprotein I (β2 GPI) was used to detect aPL, immunoglobulin G (IgG) and immunoglobulin M (IgM) in 85 APS patients, 65 disease controls, and 79 blood donors. For comparison, anti-CL and anti-β2 GPI IgG and IgM were detected by enzyme-linked immunosorbent assay (ELISA). Results The level of agreement of both methods was good for anti-CL IgG, moderate for anti-CL IgM, very good for anti-β2 GPI IgG, and moderate for anti-β2 GPI IgM (kappa = 0.641, 0.507, 0.803 and 0.506, respectively). The frequency of observed discrepancies for anti-CL IgG (1.75%), anti-CL IgM (3.93%), anti-β2 GPI IgG (1.75%), and anti-β2 GPI IgM (0.87%) was low (McNemar test, P < 0.05, not-significant, respectively). Sensitivity, specificity, positive (+LR) and negative (-LR) likelihood ratios for at least one positive aPL antibody assessed by ELISA were 58.8%, 95.8%, 14.1, and 0.4, respectively, and for at least three positive aPl IgM and/or one positive aPL IgG by MLDA were 67.1%, 96.5%, 19.3, and 0.3, respectively. The frequency of IgM to PI, PS and CL, and combination of three or more aPL IgM detected by MLDA was significantly higher in APS patients with cerebral transient ischemia (P < 0.05, respectively). Conclusions The novel MLDA is a readily available, single-step, sensitive diagnostic tool for the multiplex detection of aPL antibodies in APS and a potential alternative for single aPL antibody testing by ELISA.

Introduction Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) is a basic tool for the serological diagnosis of systemic rheumatic disorders. Automation of autoantibody IIF reading including pattern recognition may improve intra- and inter-laboratory variability and meet the demand for cost-effective assessment of large numbers of samples. Comparing automated and visual interpretation, the usefulness for routine laboratory diagnostics was investigated. Methods Autoantibody detection by IIF on human epithelial-2 (HEp-2) cells was conducted in a total of 1222 consecutive sera of patients with suspected systemic rheumatic diseases from a university routine laboratory (n = 924) and a private referral laboratory (n = 298). IIF results from routine diagnostics were compared with a novel automated interpretation system. Results Both diagnostic procedures showed a very good agreement in detecting AAB (kappa = 0.828) and differentiating respective immunofluorescence patterns. Only 98 (8.0%) of 1222 sera demonstrated discrepant results in the differentiation of positive from negative samples. The contingency coefficients of chi-square statistics were 0.646 for the university laboratory cohort with an agreement of 93.0% and 0.695 for the private laboratory cohort with an agreement of 90.6%, P < 0.0001, respectively. Comparing immunofluorescence patterns, 111 (15.3%) sera yielded differing results. Conclusions Automated assessment of AAB by IIF on HEp-2 cells using an automated interpretation system is a reliable and robust method for positive/negative differentiation. Employing novel mathematical algorithms, automated interpretation provides reproducible detection of specific immunofluorescence patterns on HEp-2 cells. Automated interpretation can reduce drawbacks of IIF for AAB detection in routine diagnostics providing more reliable data for clinicians.

To the Editor: We read with great interest the recent article by Seidel et al1 on visualization of brain perfusion with harmonic imaging. The authors observed a homogeneous dose-dependent increase in the brightness of brain parenchyma in 12 healthy volunteers after intravenous bolus application of a perfluorpropane-containing echo contrast agent and concluded that it is possible to detect brain perfusion with ultrasound. We would like to add our experience with another ultrasound technique related to brain perfusion and in this way encourage further research on this new topic. In all echo-contrast–specific imaging techniques, interaction between ultrasound waves and microbubbles depends on acoustic pressures. Harmonic imaging exploits the fact that at lower transmit powers, echoes reflected from microbubbles exhibit harmonics of the transmit signal. Harmonic frequencies can be separated from the unwanted fundamental frequency by filter settings. At higher acoustic pressures, compared with harmonic imaging examinations, microbubbles undergo destruction, fusion, and splitting. This phenomenon has been termed stimulated acoustic emission . Signals resulting from bubble destruction are interpreted as flow signal and can be displayed color coded even from stationary or low-flow microbubbles. For this reason, ultrasound techniques that enable detection of these …

Background Antiphospholipid antibodies (aPL) can be detected in asymptomatic carriers and infectious patients. The aim was to investigate whether a novel line immunoassay (LIA) differentiates between antiphospholipid syndrome (APS) and asymptomatic aPL+ carriers or patients with infectious diseases (infectious diseases controls (IDC)). Methods Sixty-one patients with APS (56 primary, 22/56 with obstetric events only, and 5 secondary), 146 controls including 24 aPL+ asymptomatic carriers and 73 IDC were tested on a novel hydrophobic solid phase coated with cardiolipin (CL), phosphatic acid, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, beta2-glycoprotein I (β2GPI), prothrombin, and annexin V. Samples were also tested by anti-CL and anti-β2GPI ELISAs and for lupus anticoagulant activity. Human monoclonal antibodies (humoAbs) against human β2GPI or PL alone were tested on the same LIA substrates in the absence or presence of human serum, purified human β2GPI or after CL-micelle absorption. Results Comparison of LIA with the aPL-classification assays revealed good agreement for IgG/IgM aß2GPI and aCL. Anti-CL and anti-ß2GPI IgG/IgM reactivity assessed by LIA was significantly higher in patients with APS versus healthy controls and IDCs, as detected by ELISA. IgG binding to CL and ß2GPI in the LIA was significantly lower in aPL+ carriers and Venereal Disease Research Laboratory test (VDRL) + samples than in patients with APS. HumoAb against domain 1 recognized β2GPI bound to the LIA-matrix and in anionic phospholipid (PL) complexes. Absorption with CL micelles abolished the reactivity of a PL-specific humoAb but did not affect the binding of anti-β2GPI humoAbs. Conclusions The LIA and ELISA have good agreement in detecting aPL in APS, but the LIA differentiates patients with APS from infectious patients and asymptomatic carriers, likely through the exposure of domain 1. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-1018-x) contains supplementary material, which is available to authorized users.