Selection, characterization and in vivo evaluation of novel CD44v6-targeting antibodies for targeted molecular radiotherapy.

Molecular radiotherapy combines the advantages of systemic administration of highly specific antibodies or peptides and the localized potency of ionizing radiation. A potential target for molecular radiotherapy is the cell surface antigen CD44v6, which is overexpressed in numerous cancers, with limited expression in normal tissues. The aim of the present study was to generate and characterize a panel of human anti-CD44v6 antibodies and identify a suitable candidate for future use in molecular radiotherapy of CD44v6-expressing cancers. Binders were first isolated from large synthetic phage display libraries containing human scFv and Fab antibody fragments. The antibodies were extensively analyzed through in vitro investigations of binding kinetics, affinity, off-target binding, and cell binding. Lead candidates were further subjected to in vivo biodistribution studies in mice bearing anaplastic thyroid cancer xenografts that express high levels of CD44v6. Additionally, antigen-dependent tumor uptake of the lead candidate was verified in additional xenograft models with varying levels of target expression. Interestingly, although only small differences were observed among the top antibody candidates in vitro, significant differences in tumor uptake and retention were uncovered in in vivo experiments. A high-affinity anti-CD44v6 lead drug candidate was identified, mAb UU-40, which exhibited favorable target binding properties and in vivo distribution. In conclusion, a panel of human anti-CD44v6 antibodies was successfully generated and characterized in this study. Through comprehensive evaluation, mAb UU-40 was identified as a promising lead candidate for future molecular radiotherapy of CD44v6-expressing cancers due to its high affinity, excellent target binding properties, and desirable in vivo distribution characteristics.

Diagnostic criteria for temporomandibular disorders: Diagnostic accuracy for general dentistry procedure without mandatory commands regarding myalgia, arthralgia and headache attributed to temporomandibular disorder.

The clinical examination in diagnostic criteria for temporomandibular disorders (DC/TMD) is a strict procedure and comprises mandatory commands. However, learning and using these mandatory commands in general practice have proven to be difficult and their use of DC/TMD is minimal. To investigate whether reliability on a diagnostic level for DC/TMD diagnoses differs between examiners using the mandatory commands or not. Six examiners were divided into two groups: one using the mandatory commands in DC/TMD for the clinical examination and one who did not use the mandatory commands. A reliability assessment was performed twice, one occasion for each group of examiners. The assessment was performed according to the guidelines from the International Network for Orofacial Pain and Related Disorders Methodology. Each group of examiners thereby examined 16 subjects (11 TMD patients and 5 healthy individuals) each, and the diagnostic agreement (reliability) as compared to diagnoses derived by a reference standard examiner was calculated with Cohen' s kappa coefficient. The DC/TMD diagnoses myalgia, arthralgia and headache attributed to TMD were included in the reliability assessment. There was no significant difference regarding diagnostic agreement reliability between the examiners using or not using the mandatory DC/TMD commands. This study indicates that not using the mandatory commands in DC/TMD in general practice does not impair the diagnostic reliability regarding the diagnoses myalgia, arthralgia and headache attributed to TMD compared to including the commands.

Chronic stress is linked to 5-HT(1A) receptor changes and functional disintegration of the limbic networks.

There are increasing reports about stress related cognitive and psychic declines in subjects who have no psychiatric premorbidity, depression, or major life trauma. Yet, little is known about the underlying neurobiology. Based on the typical symptomatology, fMRI data suggesting that stress activates the limbic circuits, and animal data showing a major involvement of the 5-HT(1A) receptor in stress regulation, we hypothesized that enduring daily stress causes widespread limbic dysfunctions, and specific changes of the 5-HT(1A) receptor. To test these hypotheses combined PET studies were carried out in 16 chronically stressed, and 16 non-stressed subjects. Limbic function was tested by measuring cerebral blood flow during rest, and when using an odor activation paradigm. 5-HT(1A) receptor binding potential (BP) was assessed with [(11)C]WAY100635. All subjects went through a battery of neuropsychological tests. Stressed subjects showed a functional disconnection between the amygdala and ACC/medial prefrontal cortex (mPFC), and an impaired odor activation of the ACC. They also displayed a reduced 5-HT(1A) receptor BP in the anterior cingulate (ACC), the insular-cortex, and the hippocampus. Their performance in attention-, odor discrimination-, and semantic memory tasks was impaired, and correlated with the BP-values in the respective region. The degree of reported stress was inversely correlated with activation of ACC, and the 5-HT(1A) receptor BP in the amygdala and hippocampus. Enduring every day psychosocial stress seems to be associated with a limbic reduction of 5-HT(1A) receptor binding and functional disintegration of ACC/mPFC. These changes support the notion of an impaired top-down regulation of stress stimuli, and identify potential targets for early treatment.

Male-to-female transsexuals show sex-atypical hypothalamus activation when smelling odorous steroids.

One working hypothesis behind transsexuality is that the normal sex differentiation of certain hypothalamic networks is altered. We tested this hypothesis by investigating the pattern of cerebral activation in 12 nonhomosexual male-to-female transsexuals (MFTRs) when smelling 4,16-androstadien-3-one (AND) and estra-1,3,5(10),16-tetraen-3-ol (EST). These steroids are reported to activate the hypothalamic networks in a sex-differentiated way. Like in female controls the hypothalamus in MFTRs activated with AND, whereas smelling of EST engaged the amygdala and piriform cortex. Male controls, on the other hand, activated the hypothalamus with EST. However, when restricting the volume of interest to the hypothalamus activation was detected in MFTR also with EST, and explorative conjunctional analysis revealed that MFTR shared a hypothalamic cluster with women when smelling AND, and with men when smelling EST. Because the EST effect was limited, MFTR differed significantly only from male controls, and only for EST-AIR and EST-AND. These data suggest a pattern of activation away from the biological sex, occupying an intermediate position with predominantly female-like features. Because our MFTRs were nonhomosexual, the results are unlikely to be an effect of sexual practice. Instead, the data implicate that transsexuality may be associated with sex-atypical physiological responses in specific hypothalamic circuits, possibly as a consequence of a variant neuronal differentiation.

Implementation of semi-automated cloning and prokaryotic expression screening: the impact of SPINE.

The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.

Smelling of odorous sex hormone-like compounds causes sex-differentiated hypothalamic activations in humans.

The anatomical pathways for processing of odorous stimuli include the olfactory nerve projection to the olfactory bulb, the trigeminal nerve projection to somatosensory and insular cortex, and the projection from the accessory olfactory bulb to the hypothalamus. In the majority of tetrapods, the sex-specific effects of pheromones on reproductive behavior is mediated via the hypothalamic projection. However, the existence of this projection in humans has been regarded as improbable because humans lack a discernable accessory olfactory bulb. Here, we show that women smelling an androgen-like compound activate the hypothalamus, with the center of gravity in the preoptic and ventromedial nuclei. Men, in contrast, activate the hypothalamus (center of gravity in paraventricular and dorsomedial nuclei) when smelling an estrogen-like substance. This sex-dissociated hypothalamic activation suggests a potential physiological substrate for a sex-differentiated behavioral response in humans.

Elevations in antidiuretic hormone and aldosterone as possible causes of fluid retention in the Maze procedure.

BACKGROUND: Reduced levels of atrial natriuretic peptide (ANP) has been suggested as a cause of fluid retention after combined Maze and valvular surgery. This study aimed to assess hormonal activation in the perioperative setting of isolated Maze procedures. METHODS: Changes in ANP, brain natriuretic peptide (BNP), antidiuretic hormone (ADH), aldosterone, and angiotensin II were measured in 16 patients (mean age 53+/-9 years) without concomitant heart disease undergoing the Maze (III) procedure. Ten matched patients (mean age 56+/-9 years) undergoing multivessel coronary artery bypass grafting served as controls. Measurements with hemodynamic correlates were obtained at baseline and after ventricular pacing (100 stimulations/minute), directly preoperatively, postoperatively and the first postoperative day. Weight gain and diuretic requirements were recorded. RESULTS: The major differences in hormonal response were significantly higher plasma levels of ADH (Maze preoperative 1.1+/-0.4, postoperative 24.9+/-16.7 pmol/L; controls preoperative 1.1+/-0.1, postoperative 3.7+/-3.5 pmol/L) and aldosterone (Maze preoperative 106+/-94, postoperative 678+/-343 pmol/L; controls preoperative 124+/-79, postoperative 171+/-93 pmol/L) in the Maze group on the first postoperative day (p < 0.001). Preoperative baseline plasma levels of ANP and pulmonary capillary wedge pressures (PCWP) were higher in the Maze group but this difference was abolished by pacing, and postoperatively, ANP levels changed in parallel to the PCWP in both groups. Diuretic requirements were significantly higher in the Maze group. CONCLUSIONS: Substantial increases in ADH and aldosterone were observed after the Maze procedure, indicating these hormones as important determinants in postoperative fluid retention. The role for ANP in this setting may be a less prominent than previously reported.

Brain activation during odor perception in males and females.

Several studies indicate that women outperform men in olfactory identification tasks. The psychophysical data are more divergent when it comes to gender differences at levels of odor processing which are cognitively less demanding. We therefore compared cerebral activation with H2(15)O PET in 12 females and 11 males during birhinal passive smelling of odors and odorless air. The odorous compounds (odorants) were pure olfactory, or mixed olfactory and weakly trigeminal. Using odorless air as the baseline condition, activations were found bilaterally in the amygdala, piriform and insular cortices in both sexes, irrespective of the odor. No gender difference was detected in the pattern of cerebral activation (random effect analysis SPM99, corrected p < 0.05) or in the subjective perception of odors. Males and females seem to use similar cerebral circuits during the passive perception of odors. The reported female superiority in assessing olfactory information including odor identification is probably an effect of a difference at a cognitive, rather than perceptive level of olfactory processing.

Influence of ejection fraction and valvular regurgitation on the accuracy of aortic valve area determination.

OBJECTIVE: To examine the influence of left ventricular dysfunction, aortic regurgitation, and mitral regurgitation on commonly used methods for aortic valve area (AVA) determination. BACKGROUND: Each method for AVA determination has its inherent limitations. METHODS: AVA determinations by transesophageal echocardiography (TEE) using planimetry, transthoracic echocardiography (TTE) with application of the continuity equation, and cardiac catheterization applying the Gorlin formula were performed in 74 patients with aortic stenosis. The severity of the aortic stenosis was defined by consensus of at least two methods. Over- or underestimation of AVA associated with ejection fraction, aortic regurgitation, mitral regurgitation, or severity of the aortic stenosis for each method in relation to the other two methods was assessed. RESULTS: Mean AVAs were 1.05 +/- 0.51 by TEE, 1.06 +/- 0.51 by TTE, and 1.08 +/- 0.53 by cardiac catheterization. An overestimation of the severity of the aortic stenosis by the Gorlin formula in patients with moderate-to-severe aortic regurgitation as compared to TEE-derived data was found (P = 0.014). A similar trend of overestimation by catheterization in comparison with the TTE data was found. In the context of moderate-to-severe mitral regurgitation, AVA determination by TTE overestimated the degree of aortic stenosis as compared to TEE (P = 0.011) and cardiac catheterization (P = 0.023). CONCLUSIONS: Overall mean AVA did not differ between methods, suggesting that these three methods are equally accurate in a nonselected clinical patient group. However, in the presence of significant aortic regurgitation, the two echocardiographic methods appear more accurate. Our observation of an overestimation of the severity of aortic stenosis by TTE in the presence of moderate-to-severe mitral regurgitation indicates that this possibility should be accounted for in clinical decisions based on TTE determinations of AVA.

Right-nostril dominance in discrimination of unfamiliar, but not familiar, odours.

In a recent PET study on processing of unfamiliar odours we observed that odour discrimination performance was superior during right compared with left nostril presentations, and that mainly the right cerebral hemisphere was activated. In the present study we investigated whether the asymmetric performance is present also during the processing of familiar odours. Seventy-one right-handed healthy subjects (age 21-49 years, 40 females) with normal nasal anatomy and olfactory thresholds participated. Forty pairs of odours (20 familiar and 20 unfamiliar) were presented in the same/different paradigm, alternating nostrils and balancing the order. The number of errors during the discrimination task was compared with respect to nostril and odour familiarity. The overall odour discrimination performance was superior on the right side. However, this difference was valid only for unfamiliar odours, whereas the performance for familiar odours was symmetrical. Familiar odours were easier to discriminate than unfamiliar ones. The present data are congruent with the idea of a semantic influence on odour processing. Odours seem to be processed with a right sided preponderance when not clearly familiar, and symmetrically when language becomes involved. Future studies on odour processing should therefore take into account odour familiarity and side of presentation.

The three-dimensional solution structure and dynamic properties of the human FADD death domain.

FADD (also known as MORT-1) is an essential adapter protein that couples the transmembrane receptors Fas (CD95) and tumor necrosis factor receptor-1 (TNF-R1) to intracellular cysteine proteases known as caspases, which propagate and execute the programmed cell death-inducing signal triggered by Fas ligand (FasL, CD95L) and TNF. FADD contains 208 amino acid residues, and comprises two functionally and structurally distinct domains: an N-terminal death effector domain (DED) that promotes activation of the downstream proteolytic cascade through binding of the DED domains of procaspase-8; and a C-terminal death domain (DD). FADD-DD provides the site of FADD recruitment to death receptor complexes at the plasma membrane by, for example, interaction with the Fas receptor cytoplasmic death domain (Fas-DD), or binding of the TNF-R1 adapter molecule TRADD. We have determined the three-dimensional solution structure and characterised the internal polypeptide dynamics of human FADD-DD using heteronuclear NMR spectroscopy of (15)N and (13)C,(15)N-labelled samples. The structure comprises six alpha-helices joined by short loops and displays overall similarity to the death domain of the Fas receptor. The analysis of the dynamic properties reveals no evidence of contiguous stretches of polypeptide chain with increased internal motion, except at the extreme chain termini. A pattern of increased rates of amide proton solvent exchange in the alpha3 helix correlates with a higher degree of solvent exposure for this secondary structure element. The properties of the FADD-DD structure are discussed with respect to previously reported mutagenesis data and emerging models for FasL-induced FADD recruitment to Fas and caspase-8 activation.

Clinical validation of intravascular ultrasound imaging for assessment of coronary stenosis severity: comparison with stress myocardial perfusion imaging.

OBJECTIVES: To validate intravascular ultrasound (IVUS) measurements for differentiating functionally significant from nonsignificant coronary stenosis. BACKGROUND: To date, there are no validated criteria for the definition of a flow-limiting coronary artery stenosis by IVUS. METHODS: Preinterventional IVUS imaging (30-MHz imaging catheter) of 70 de novo coronary lesions was performed. The lesion lumen area and three IVUS-derived stenosis indixes comparing lesion lumen area with the lesion external elastic lamina (EEL) area, the mean reference lumen area and the mean reference EEL area were compared with the results of stress myocardial perfusion imaging. RESULTS: The lesion lumen area and three IVUS-derived stenosis indexes showed sensitivities and specificities ranging between 80% and 90% using stress myocardial perfusion imaging as the gold standard. The lesion lumen area < or =4 mm2 is a simple and highly accurate criterion for significant coronary narrowing. CONCLUSIONS: Quantitative IVUS indices can be reliably used for identifying significant epicardial coronary artery stenoses.

Conformational dynamics and molecular recognition: backbone dynamics of the estrogen receptor DNA-binding domain.

We examined the internal mobility of the estrogen receptor DNA-binding domain (ER DBD) using NMR15N relaxation measurements and compared it to that of the glucocorticoid receptor DNA-binding domain (GR DBD). The studied protein fragments consist of residues Arg183-His267 of the human ER and residues Lys438-Gln520 of the rat GR. The15N longitudinal (R1) and transverse (R2) relaxation rates and steady state {1H}-15N nuclear Overhauser enhancements (NOEs) were measured at 30 degrees C at1H NMR frequencies of 500 and 600 MHz. The NOE versus sequence profile and calculated order parameters for ER DBD backbone motions indicate enhanced internal dynamics on pico- to nanosecond time-scales in two regions of the core DBD. These are the extended strand which links the DNA recognition helix to the second zinc domain and the larger loop region of the second zinc domain. The mobility of the corresponding regions of the GR DBD, in particular that of the second zinc domain, is more limited. In addition, we find large differences between the ER and GR DBDs in the extent of conformational exchange mobility on micro- to millisecond time-scales. Based on measurements of R2as a function of the15N refocusing (CPMG) delay and quantitative (Lipari-Szabo-type) analysis, we conclude that conformational exchange occurs in the loop of the first zinc domain and throughout most of the second zinc domain of the ER DBD. The conformational exchange dynamics in GR DBD is less extensive and localized to two sites in the second zinc domain. The different dynamical features seen in the two proteins is consistent with previous studies of the free state structures in which the second zinc domain in the ER DBD was concluded to be disordered whereas the corresponding region of the GR DBD adopts a stable fold. Moreover, the regions of the ER DBD that undergo conformational dynamics on the micro- to millisecond time-scales in the free state are involved in intermolecular protein-DNA and protein-protein interactions in the dimeric bound state. Based on the present data and the previously published dynamical and DNA binding properties of a GR DBD triple mutant which recognize an ER binding site on DNA, we argue that the free state dynamical properties of the nuclear receptor DBDs is an important element in molecular recognition upon DNA binding.

Effect of externally applied focused acoustic energy on clot disruption in vitro.

Application of low-frequency ultrasound for clot disruption has been suggested as a potential therapy to enhance thrombus dissolution, but the optimal mode for delivery of ultrasound with clot-disruptive properties has not yet been extensively explored. Target-specific effects are desirable and may be accomplished by focusing the ultrasound. Adequate focusing, however, requires a short wavelength. The aim of this study was to compare the clot-disruptive effects of different modalities of focused acoustic energy. An in vitro model (10 blood clots for each modality) was used to test the clot-disruptive capacity of (i) shock waves generated in an electrohydraulic lithotriptor; (ii) focused continuous ultrasound of frequency 1.1 MHz, delivered from a specially constructed piezoelectric transducer; and (iii) focused pulse-modulated ultrasound of frequency 1.1 MHz delivered from the same transducer. Exposure to 30 s of focused pulse-modulated ultrasound caused a marked reduction (99+/-2%) in clot weight compared with 30 shock waves (11+/-5%) or 30 s exposure to focused continuous wave ultrasound (11+/-6%) (P<0.0001). The observed marked and rapid disruptive effect on blood clots of focused high-frequency ultrasound indicates an alternative approach for external ultrasound-mediated thrombus destruction in vivo. The focused pulse-modulated technique has potential to exhibit the desired effect in a well-defined target volume and provides the means for control of the average power.

13C-urea breath test results in pigs challenged with Helicobacter pylori or other urease producing bacteria.

The gastric bacterial flora and its influence on the 13C-urea breath test (UBT) for detection of Helicobacter pylori infection was studied in a pig model. Seven SPF minipigs were used. H. pylori or a mix of other urease positive bacteria were administered orally. UBT, serum and biopsies for histology and culture were collected. Our results show that UBT is not specific for H. pylori in pigs as the gastric bacterial flora is responsible for the high UBT values observed. Furthermore, the Ellegaard Göttingen SPF minipigs are not useful in an animal model for H. pylori studies.

Cooling System Permits Effective Transcutaneous Ultrasound Clot Lysis In Vivo Without Skin Damage.

Previous in vivo studies have shown that transcutaneous ultrasound enhances clot dissolution in the presence of either streptokinase or microbubbles. However, ultrasound-induced skin damage has been a major drawback. The objective was to evaluate the effect of a cooling system to prevent the skin damage that has heretofore been associated with transcutaneous low-frequency, high-intensity ultrasound clot dissolution. After thrombi were induced in both iliofemoral arteries in 15 rabbits, streptokinase (25,000 U/kg) was given intravenously and dodecafluoropentane was injected slowly (2 mL/15 min) through an infusion catheter into the abdominal aorta. One iliofemoral artery was randomized to receive ultrasound treatment, and the contralateral artery was treated as a control (receiving streptokinase and dodecafluoropentane alone). In six rabbits (group 1), the skin below the ultrasound transducer was protected by the use of a balloon cooling system, and in the other nine rabbits (group 2), ultrasound was used without a cooling system. Seven of nine (78%) arteries treated without the cooling system, and six of six (100%) arteries treated with the cooling system were angiographically recanalized after ultrasound + streptokinase + dodecafluoropentane treatment. Thermal damage was present in the skin and soft tissues of all nine rabbits treated without a cooling system. However, the skin and soft tissues were grossly and histologically normal in the six rabbits in which the transcutaneous ultrasound was used with the cooling system. Low-frequency, high-intensity ultrasound energy can be delivered transcutaneously for clot dissolution without concomitant tissue damage when coupled with the use of a cooling system to prevent thermal injury.

Preliminary NMR studies of Thermus thermophilus ribosomal protein S19 overproduced in Escherichia coli.

The gene for the ribosomal protein S19 from Thermus thermophilus was cloned, sequenced and overexpressed in Escherichia coli. A simple procedure for isolating the recombinant protein was developed. Preliminary NMR studies revealed a high content of alpha-helical secondary structure in the protein.

Highly localized arterial remodeling in patients with coronary atherosclerosis: an intravascular ultrasound study.

BACKGROUND: Preservation of luminal area and symmetry in the presence of irregular plaques necessitates local expansion of the artery wall. METHODS AND RESULTS: Cross-sectional dimensions of coronary arteries in 65 patients were measured with the use of intravascular ultrasound. A total of 104 arterial segments were studied, of which 88 had atherosclerosis; 16 served as nonatherosclerotic control segments. Three features of atherosclerotic arterial segments were classified: (1) plaque formation, (2) lumen shape, and (3) shape of arterial external elastic lamina. With our intravascular ultrasound-based three-level classification system, we identified three patterns that accounted for 89% of all atherosclerotic arterial segments: (1) concentric plaque with a circular lumen and a circular external elastic lamina (n= 17), (2) eccentric plaque with a circular lumen and an oval external elastic lamina (n=35), and (3) eccentric plaque with an oval lumen and a circular external elastic lamina (n=26). A circular lumen was preserved in 66% of all atherosclerotic arterial segments. Arterial segments with a circular lumen in the presence of an eccentric plaque had a significantly larger lumen area than the other two main groups (P<.05). CONCLUSIONS: With our intravascular ultrasound-based classification, we provided information regarding the local remodeling response in the coronary artery wall. In a majority of cases, a circular lumen is maintained. Failure of this highly localized response to be operative may contribute to the development of stenotic lesions at a specific site in the artery.