Functional-State Dependence of Picosecond Protein Dynamics.

We examine temperature-dependent picosecond dynamics of two benchmarking proteins lysozyme and cytochrome c using temperature-dependent terahertz permittivity measurements. We find that a double Arrhenius temperature dependence with activation energies E1 ∼ 0.1 kJ/mol and E2 ∼ 10 kJ/mol fits the folded and ligand-free state response. The higher activation energy is consistent with the so-called protein dynamical transition associated with beta relaxations at the solvent-protein interface. The lower activation energy is consistent with correlated structural motions. When the structure is removed by denaturing, the lower-activation-energy process is no longer present. Additionally, the lower-activation-energy process is diminished with ligand binding but not for changes in the internal oxidation state. We suggest that the lower-energy activation process is associated with collective structural motions that are no longer accessible with denaturing or binding.

Terahertz near-field spectroscopy of filled subwavelength sized apertures in thin metal films.

We have measured terahertz near-field spectra of cesium iodide crystals as small as ~10 µm in diameter, which were deposited on single, sub-wavelength-sized apertures created in thin gold films on a substrate. The advantage of using small apertures for terahertz microspectroscopy is that only terahertz light that has interacted with the cesium iodide is observed. We find that around the transverse optical phonon frequency of cesium iodide, the amplitude transmission is as much influenced by the refractive index as by the absorption. We show that the ability to measure in the near-field of the apertures, where signals are relatively strong, allows us to measure on sample volumes as small as ~5×10(-16) m(3).

Evidence of protein collective motions on the picosecond timescale.

We investigate the presence of structural collective motions on a picosecond timescale for the heme protein, cytochrome c, as a function of oxidation and hydration, using terahertz (THz) time domain spectroscopy and molecular dynamics simulations. The THz response dramatically increases with oxidation, with the largest increase for lowest hydrations, and highest frequencies. For both oxidation states the THz response rapidly increases with hydration saturating above ∼25% (g H(2)O/g protein). Quasiharmonic vibrational modes and dipole-dipole correlation functions were calculated from molecular dynamics trajectories. The collective mode density of states alone reproduces the measured hydration dependence, providing strong evidence of the existence of these motions. The large oxidation dependence is reproduced only by the dipole-dipole correlation function, indicating the contrast arises from diffusive motions consistent with structural changes occurring in the vicinity of buried internal water molecules. This source for the observed oxidation dependence is consistent with the lack of an oxidation dependence in nuclear resonant vibrational spectroscopy measurements.

Influence of the dielectric substrate on the terahertz electric near-field of a hole in a metal.

We have studied theoretically and experimentally the influence of a dielectric substrate on the frequency-dependent terahertz electric near-field of a small hole in a metal layer. We find that the near-field transmission spectrum and the two-dimensional field distribution of an empty hole in a thin metal layer on a substrate are almost identical to that of a hole which is also filled with the same dielectric material as the substrate. For thicker metal layers, however, the near-field spectra of filled and unfilled holes become very different. In addition, for thick metal layers, the two-dimensional field distributions are more strongly affected by the substrate, especially if we allow for an air gap between the metal and the substrate. Our results validate the -somewhat unusual- two-dimensional field distribution measured beneath a hole in a thick metal foil and highlight the effect that a substrate can have on the measurement of the near-field of an object.

Terahertz near-field vectorial imaging of subwavelength apertures and aperture arrays.

We present measurements of the complete terahertz (THz) electric near-field distribution, E(x), E(y) and E(z), in both the time- and frequency-domains, for subwavelength apertures and subsections of subwavelength aperture arrays. Measuring the individual components of the THz near-field with subwavelength spatial resolution, as they emerge from these structures, illustrates how the field interacts with the apertures. We observe the small but measurable y- and z-components of the electric field for both single apertures and arrays. Resonant contributions, attributed to Bloch modes, are detected and we observe the presence of a longitudinal field component, E(z), within the different array apertures, which can be attributed to a diffractive effect. These measurements illustrate in detail the individual THz field components emerging from subwavelength apertures and provide a direct measure of two important mechanisms that contribute to the net transmission of light through arrays.

Protein dynamical transition does not require protein structure.

Terahertz time domain spectroscopy shows that the protein dynamical transition, the rapid increase in protein dynamics occurring at approximately 200 K, needs neither tertiary nor secondary structure. Further, short chain alanine studies find a dynamical transition down to penta-alanine, with no transition observed for di-alanine or tri-alanine. These results reveal the temperature dependence arises strictly from the side-chain interaction with the solvent. The lack of a transition for shorter chain peptides may indicate a qualitative change in this interaction occurs at a specific peptide chain length.

[Decisions in case of "problematic" cost-effectiveness ratios based on the example of a clinical trial in rehabilitation care]. / Entscheidungen bei "schwierigen" Kosteneffektivitätsrelationen am Beispiel eines klinischen Versuchs aus der Rehabilitation.

PURPOSE: Economic assessment of an additional psychological intervention in the rehabilitation of patients with chronic low-back pain and evaluation of results by decision makers. METHODS: Piggy-back cost-utility analysis of a randomised clinical trial, including a bootstrap analysis. Costs were measured by using the cost accounting systems of the rehabilitation clinics and by surveying patients. Health-related quality of life was measured using the EQ-5D. Implications of different representations of the decision problem and corresponding decision rules concerning the cost-effectiveness plane are discussed. RESULTS: As compared with the 126 patients of the control arm, the 98 patients in the intervention arm gained 3.5 days in perfect health on average as well as 1219 euro cost saving. However, because of the uncertainty involved, the results of a bootstrap analysis cover all quadrants of the cost-effectiveness plane. Using maximum willingness-to-pay per effect unit gained, decision rules can be defined for parts of the cost-effectiveness plane. These have to be aggregated in a further valuation step. CONCLUSIONS: Study results show that decisions on stochastic economic evaluation results may require an additional valuation step aggregating the various parts of the cost-effectiveness plane.

Large oxidation dependence observed in terahertz dielectric response for cytochrome c.

Far infrared dielectric response is used to characterize the collective mode density of states for cytochrome c as a function of oxidation state and hydration using terahertz time domain spectroscopy. A strong absorbance and refractive index increase was observed with the oxidation. A simple phenomenological fitting using a continuous distribution of oscillators reproduces the frequency dependence of the complex dielectric response as well as demonstrates quantitative agreement with a uniform increase in either mode density or polarizability with oxidation in the 5-80 cm(-1) frequency range. Hydration dependence measurements find that a difference in the equilibrium water content for ferri and ferro cytochrome c is not sufficient to account for the large change in terahertz response. The large dielectric increase at terahertz frequencies with oxidation suggests either a significant global softening of the potential and/or a significant increase in polarizability with oxidation.

Increased activity of factor VIII coagulant associated with venous ulcer in a patient with Klinefelter's syndrome.

Klinefelter's syndrome is the most frequent major abnormality of sexual differentiation in men with two or more X chromosomes. Recurrent venous ulcers as a result of a post-thrombotic syndrome are a well known symptom in patients with Klinefelter's syndrome. Until now the underlying pathomechanisms are not completely understood. Platelet hyperaggregability, factor V Leiden mutation and abnormalities in fibrinolysis were implicated as possible contributing factors. Here we describe the detection of an increased activity of factor VIII coagulant (factor VIII:C). This is the first case report on increased factor VIII:C activity associated with venous ulcers in a patient with Klinefelter's syndrome. Elevated factor VIII plasma levels are gradually accepted to be associated with an increased risk for venous thromboembolism. Therefore, we discuss that the examination of factor VIII:C may help in clarifying individual thromboembolic risks, especially in patients with Klinefelter's syndrome.

Successful treatment of a leg ulcer occurring in a rheumatoid arthritis patient under leflunomide therapy.

OBJECTIVE: We report the case of a leg ulcer in a rheumatoid arthritis (RA) patient under treatment with leflunomide, discuss the influence of the drug on the aetiopathogenesis of the ulcer and describe its successful treatment. CASE SUMMARY: A 68-year-old woman with a 12-year history of RA developed a leg ulcer after 4 months of leflunomide treatment. Other ulcerogenic factors were ruled out. There were some clinical hints for rheumatoid vasculitis. The ulcer was resistant to ambulant conservative phase adapted wound bed preparation and a split skin transplantation failed. After omission of leflunomide and washout procedure with cholestyramine a second split skin transplantation resulted in complete healing. DISCUSSION: Leflunomide inhibits the division of activated T cells and thus inhibits among others the production of proinflammatory cytokines and the adhesion of cells to the endothelium. These mechanisms may partly explain the possible influence of leflunomide on the perpetuation of the ulcer. Until now, occurrence of vasculitis and leg ulcers has been described in one case each for the novel immunomodulator leflunomide. No successful treatment of a leg ulcer under leflunomide has been described yet. Omission of leflunomide and a washout treatment in our case led to a complete healing. This may indicate a critical role of leflunomide in the maintenance of this slow healing ulcer. CONCLUSIONS: An association between leflunomide intake, occurrence of leg ulcers in RA patients and delayed wound healing should be considered.

Successful treatment of stasis dermatitis with topical tacrolimus.

Stasis dermatitis is a common dermatologic disorder as a consequence of impaired venous drainage and often accompanied by chronic leg ulcers. Until today the standard in acute therapy represents the topical administration of highly potent corticosteroids and if possible a consequent long-term compression therapy. The macrolide tacrolimus represents a new selective inflammatory cytokine release inhibitor by binding to macrophilin-12 and inhibiting calcineurin. Beside the resulting anti-inflammation and immunosuppression an antipruritic effect have been discussed as further clinical benefits of tacrolimus. Here we report for the first time about a 81-year old patient suffering from an ulcus cruris mixtum and stasis dermatitis treated with topical 0.1% tacrolimus ointment twice daily for 5 days. Until now tacrolimus is available for topical treatment as a fatty ointment only. Although we would have preferred a more hydrophilic base for treatment of acute stasis dermatitis we achieved complete healing. As this is only a case report about one single patient further clinical investigations are needed to confirm this observation in more individuals with stasis dermatitis.

[Bureau-Barrière syndrome of the hand. A case report on an unusual localization]. / Bureau-Barrière-Syndrom der Hand. Ein Fallbericht über eine ungewönliche Lokalisation.

The Bureau-Barrière syndrome is described as an acral ulcer with mutilating osteolysis of the limbs that is nonfamiliar, non-sex-specific, induced by many factors, and elicited by sensory-trophic polyneuropathy. Often a bilateral location at the lower limb of male alcoholics has been described. We report about a 76-year-old diabetic women with unilateral mutilating acroosteolysis and ulceration of one finger and discuss the relevant clinical aspects of the Bureau-Barrière syndrome.

The use of a computer-based decision support system facilitates primary care physicians' management of chronic pain.

We tested whether computer-based decision support (CBDS) could enhance the ability of primary care physicians (PCPs) to manage chronic pain. Structured summaries were generated for 50 chronic pain patients referred by PCPs to a pain clinic. A pain specialist used a decision support system to determine appropriate pain therapy and sent letters to the referring physicians outlining these recommendations. Separately, five board-certified PCPs used a CBDS system to "treat" the 50 cases. A successful outcome was defined as one in which new or adjusted therapies recommended by the software were acceptable to the PCPs (i.e., they would have prescribed it to the patient in actual practice). Two pain specialists reviewed the PCPs' outcomes and assigned medical appropriateness scores (0 = totally inappropriate to 10 = totally appropriate). One year later, the hospital database provided information on how the actual patients' pain was managed and the number of patients re-referred by their PCP to the pain clinic. On the basis of CBDS recommendations, the PCP subjects "prescribed" additional pain therapy in 213 of 250 evaluations (85%), with a medical appropriateness score of 5.5 +/- 0.1. Only 25% of these chronic pain patients were subsequently re-referred to the pain clinic within 1 yr. The use of a CBDS system may improve the ability of PCPs to manage chronic pain and may also facilitate screening of consults to optimize specialist utilization.

Immunoelectron microscopic evidence for release of eosinophil granule matrix protein onto microfilariae of Onchocerca volvulus in the skin after exposure to amocarzine.

The involvement of eosinophils in the host reaction to microfilariae (mf) of Onchocerca volvulus was studied by immunohistochemistry and immunoelectron microscopy. Skin biopsies were obtained from patients after transepidermal administration of the microfilaricide amocarzine. At 20-28 h after the application of amocarzine, mf were degenerated or dead and a marked eosinophil-parasite adherence (EPA) reaction was seen, with intense staining for intra- and extracellular eosinophil granule proteins such as eosinophil cationic protein (ECP) surrounding the mf. Immunoelectron microscopically the eosinophil granule matrix in intact and necrotic eosinophils was specifically labeled, whereas granules whose matrix had dissolved showed no specific gold particle binding. As specific labeling was seen on lowly electron-dense material adjacent to matrix-depleted granules, the material was regarded as released eosinophil granule matrix material. Intact and necrotic eosinophils, matrix-containing as well as matrix-depleted eosinophil granules, and released eosinophil granule matrix material were observed on the surface of damaged mf and between collagen fibers. The coincidence of mf degeneration, EPA reaction, and release of eosinophil granule matrix material on damaged mf and collagen fibers indicated a role of eosinophils and eosinophil granule matrix protein in the host reaction to mf after amocarzine application.

The effect of epinephrine on small-dose hyperbaric bupivacaine spinal anesthesia: clinical implications for ambulatory surgery.

UNLABELLED: The effect of adding epinephrine to small doses of spinal bupivacaine on the duration of sensory motor block has not been carefully investigated. Twelve volunteers underwent hyperbaric bupivacaine spinal anesthesia (7.5 mg) with and without epinephrine (0.2 mg) in a randomized, double-blind, cross-over fashion. Sensory block was assessed with pinprick, transcutaneous electrical stimulation (TES) equivalent to surgical stimulation (at umbilicus, pubis, knee, and ankle), and tolerance of a pneumatic thigh tourniquet. Motor block was assessed with isometric force dynamometry. Discharge criteria were defined as return of pinprick sensation to dermatome S2, ability to ambulate, and ability to urinate. Extent of sensory block to pinprick over time was unaffected by the addition of epinephrine. However, epinephrine prolonged tolerance of TES at the pubis, knee, and ankle (33-48 min, P < 0.05) and of thigh tourniquet (30 min, P < 0.01). Motor block was prolonged by epinephrine at the quadriceps and gastrocnemius muscles (by 23 and 51 min, respectively, P < 0.002). Achievement of discharge criteria was prolonged by 48 min by the addition of epinephrine (P < 0.01). Thus, epinephrine may prolong surgical anesthesia for lower abdominal and lower extremity surgery and delay time until patients achieve discharge criteria. IMPLICATIONS: Using a cross-over study design, 12 volunteers underwent bupivacaine spinal anesthesia with and without epinephrine. This study suggests that adding epinephrine to bupivacaine may prolong surgical anesthesia and also delay patients' discharge.

Cellular and subcellular localization of cationic leukocyte antigen (CLA) in human eosinophil granulocytes.

The cellular and subcellular localization of cationic leukocyte antigen (CLA) in human peripheral blood and tissue granulocytes was investigated by immunoenzymatic labeling and by immunoelectron microscopy. Human peripheral blood granulocytes from healthy individuals and from subjects with eosinophilia of varying etiology, as well as intravascular and/or perivascular granulocytes in skin biopsies taken from patients with generalized onchocerciasis, a skin disease caused by microfilariae of Onchocerca volvulus, were studied. Controlled indirect immunoenzymatic staining for CLA in cytospin preparations of buffy coat cells and in histological sections of skin biopsies revealed that this protein was exclusively found in the cytoplasm of eosinophil granulocytes. Furthermore, immunogold labeling coupled with electron microscopy showed that CLA was specifically localized within the matrix of both the small non-crystalloid-containing pale granules and the large crystalloid-containing secondary granules of peripheral blood and tissue eosinophils. No specific gold labeling was observed in other organelles of eosinophils, in neutrophils, basophils, lymphocytes or monocytes.

Immunohistological studies on macrophages in lymph nodes of onchocerciasis patients after treatment with ivermectin.

The role of macrophages in the killing and elimination of microfilariae (mf) was studied immunohistologically in 14 lymph nodes from 10 patients with generalized onchocerciasis 20-68 h after treatment with a single oral dose of 150 microg/kg ivermectin. Mf with signs of damage at light microscopical level were surrounded by a cellular infiltrate comprising macrophages, eosinophils and neutrophils, whereas light microscopically intact mf mostly showed no cellular reaction. Resident mature macrophages expressing the CD 68 epitope usually neither migrated nor attached to damaged mf, especially on the first and second day after ivermectin treatment. However, many young invading macrophages labelled for the L1 protein (antibodies 27 E 10, MAC 387, S 36.48 and 8.5C2) were found within the cellular infiltrate around damaged mf and in adherence to the mf in all lymph nodes after ivermectin treatment. Free L1 protein was observed on the cuticle of the mf. The attacking macrophages contained increased amounts of the enzymes lysozyme, alpha-1-antichymotrypsin and alpha-1-antitrypsin compared to resident macrophages. Free enzymes were found on the cuticle of the mf and around them, indicating a role of these enzymes in the inflammatory reaction to the parasites. The attacking macrophages were strongly labelled for human HLA-DR and they showed further an increased expression of the complement receptors CR1 (CD 35) for C3b and CR3 (CD 11b) for C3 bi in comparison to resident macrophages and thus were considered as activated macrophages. Rarely fragments of mf were seen within multinuclear macrophages. We conclude that young activated macrophages play a major role in the elimination of mf transported to the regional lymph nodes after ivermectin treatment. The immunohistological findings are in accordance with the assumption that these activated macrophages together with granulocytes contribute to the killing of the damaged mf. They also help to limit the damage of the host tissue by release of alpha-1-antichymotrypsin and alpha-1-antitrypsin.

Neutrophil granule proteins: evidence for the participation in the host reaction to skin microfilariae of Onchocerca volvulus after diethylcarbamazine administration.

The participation of neutrophil granulocytes in the cellular reaction to skin microfilariae of Onchocerca volvulus was studied by immunohistochemistry. Skin biopsies were obtained from adult Liberian and Ugandan patients with generalized onchocerciasis after exposure to topically applied diethylcarbamazine (DEC) and from untreated patients. After DEC many damaged microfilariae were observed either in dermal infiltrates or in epidermal microabscesses consisting both of neutrophils and eosinophils. Infiltrates and microabscesses contained some intact granulocytes and many neutrophils releasing myeloperoxidase, elastase, lactoferrin, defensin, lysozyme, alpha 1-antitrypsin and alpha 1-antichymotrypsin. Eosinophils discharged peroxidase and cationic proteins. Released granule proteins and remnants of disrupted granulocytes were found on the surface and in close proximity of damaged microfilariae in dermal infiltrates and epidermal microabscesses. In larger microabscesses neutrophils were predominant. These observations show that neutrophils and not only eosinophils recruit, accumulate, localize around and release their helminthotoxic granule proteins such as myeloperoxidase onto or closely around skin microfilariae of O. volvulus after topical DEC administration. The association between these processes and the damage of the microfilariae indicated that neutrophils together with eosinophils attack and damage microfilariae of O. volvulus after DEC treatment in the skin.