Digitized mini optofluidic element and its application to ophthalmic lenses for presbyopia correction.

Using the internally placed elastic membrane and multi-chamber configuration, we designed a digitized mini optofluidic element for fast switching between refractive and diffractive states of preset optical powers. Relief surface was used in the diffractive state. We applied finite element analysis to establish membrane mechanical characteristics for switching at the force level produced by the ocular elements such as ciliary muscle or lower eyelid at eye downgaze. The prototypes were made to demonstrate proof-of-concept. Membrane conformance to the diffractive grooves and imaging quality were demonstrated. The analysis supported switching under the force level exerted by the ocular elements supporting the digitized optofluidic element potential for presbyopia correction by ophthalmic lenses.

Cell type-specific Nogo-A gene ablation promotes axonal regeneration in the injured adult optic nerve.

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre(+/-)xRtn4/Nogo-A(flox/flox)) and neuron-specific (Thy1-Cre(tg+)xRtn4(flox/flox)) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4(flox/flox) animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS.

Impairing MLL-fusion gene-mediated transformation by dissecting critical interactions with the lens epithelium-derived growth factor (LEDGF/p75).

The lens epithelium-derived growth factor (LEDGF/p75) tethers the mixed-lineage leukemia (MLL1) protein complex to chromatin. Likewise, LEDGF/p75 tethers the HIV-1 pre-integration complex to chromatin. We previously demonstrated that expression of the C-terminal fragment fused to enhanced green fluorescent protein (eGFP) (eGFP-LEDGF(325-530)) impaired HIV-1 replication. Here, we explored this strategy to selectively interfere with the leukemogenic activity of MLL-fusion proteins. We found that expression of LEDGF(325-530) impaired the clonogenic growth of MLL-fusion gene transformed human and mouse hematopoietic cells, without affecting the growth of control cells immortalized by the FLT3-ITD mutant or normal lineage-marker-depleted murine bone marrow cells. Expression of LEDGF(325-530) was associated with downregulation of the MLL target Hoxa9 and impaired cell cycle progression. Structure-function analysis revealed two small eGFP-fused LEDGF/p75 peptides, LEDGF(424-435) and LEDGF(375-386) phenocopying these effects. Both LEDGF(325-530) and the smaller active peptides were able to disrupt the LEDGF/p75-MLL interaction. Expression of LEDGF(325-530) or LEDGF(375-386) fragments increased the latency period to disease development in vivo in a mouse bone marrow transplant model of MLL-AF9-induced AML. We conclude that small peptides disrupting the LEDGF/p75-MLL interface have selective anti-leukemic activity providing a direct rationale for the design of small molecule inhibitors targeting this interaction.

Neuronal Nogo-A upregulation does not contribute to ER stress-associated apoptosis but participates in the regenerative response in the axotomized adult retina.

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.

A haemodynamic study during OPCAB surgery using a new multi-suction cardiac positioner.

BACKGROUND: Displacement of the heart is necessary to expose the target vessel for distal anastomosis to achieve successful multivessel off-pump coronary artery bypass (OPCAB) grafting. In addition to complete revascularisation of the heart, a main challenge during the operation is to maintain haemodynamic stability during this procedure. A new heart positioner (Tentacles) was tested. METHODS: In a prospective clinical study we used the Tentacles device in 50 patients scheduled for multivessel OPCAB procedures and investigated the haemodynamic effects during displacement of the heart and while performing the anastomoses to the anterior, lateral and posterior wall. The following haemodynamic parameters were investigated: mean arterial blood pressure (MAP), cardiac index (CI) and stroke volume index (SVI). The incidence of myocardial ischaemia was monitored by transoesophageal echocardiography (TEE) and by ST-segment analysis in the electrocardiogram (ECG). RESULT: The Tentacles device permitted rapid, secure and excellent exposure of the lateral and posterior wall of the heart. During exposure of the anterior wall there was a small decrease in MAP (77 ± 10 vs.71 ± 9 mmHg, P = 0.02) in combination with an increase in the CI (3.0 ± 0.7 l vs. 3.1 ± 0.8 l/min/m2, P = 0.03). When the lateral and posterior walls of the heart were exposed, the SVI decreased significantly (36 ± 11 and 38 ± 8 mL/m2, P < 0.01 and P = 0.04, respectively) compared to baseline (44 ± 11 mL/m2) while CI and MAP remained stable. The amount of norepinephrine administered during displacement of the heart was significantly higher in all three positions (0.05 ± 0.05, 0.06 ± 0.05 and 0.04 ± 0.03 µg/kg/min, P < 0.01) compared to the physiological position (0.02 ± 0.02 µg/kg/min). Sinus rhythm was maintained throughout the operation. Neither significant changes of the ST-segment in the ECG nor incidences of wall motion abnormality in TEE were observed. Six hours postoperatively the troponin I concentration was 11.7 ± 4.3 ng/mL. CONCLUSION: The Tentacles device provided excellent access in multivessel OPCAB surgery. Haemodynamic stability was maintained in all patients; however additional catecholamine support was used when the heart was displaced. This was the case when carrying out an anastomosis on the anterior, lateral, or posterior wall.

On the cell biology of HIV integration from basic research to development of novel antiviral drugs.

Viruses in general, HIV in particular, use cellular proteins or cofactors to achieve their replication cycle. Our recent research efforts resulted in the identification and validation of two new cellular cofactors of HIV replication: LEDGF/p75 and transportin-SR2. LEDGF/p75 is a cellular transcriptional coactivator used by the virus to tether the preintegration complex onto the chromosome; transportin-SR2 is a cellular import factor used by the virus for nuclear import into the nucleus. After validation of LEDGF/p75 as an antiviral target, we initiated a drug discovery program to develop small molecule inhibitors of integrase-LEDGF/p75 interaction. Using molecular modeling, medicinal chemistry, crystallography and virology, our team developed LEDGINs, the first-in-class small molecule inhibitors of HIV targeting integrase/LEDGF/p75 interaction. As for transportin-SR2, this protein was identified by yeast-two-hybrid screening and validated as a nuclear import factor for HIV. A drug discovery program for inhibitors of nuclear import is ongoing. Together our research results provide a paradigm shift in antiviral research. Yes, small molecules can be developed inhibiting the protein-protein interaction between a viral protein and a cellular cofactor. In a broader perspective our research strongly supports the development of protein-protein interaction inhibitors as drugs.

Microvascular perfusion measured by orthogonal polarization spectral imaging is well maintained during exposure to high altitude in trained mountaineers.

The objective of this study was to examine microvascular perfusion during hypobaric hypoxia and physical exercise. We used orthogonal polarization spectral imaging for the non-invasive visualization and assessment of the sublingual mucosal microcirculation in twelve healthy altitude acclimatized mountaineers. Red blood cell velocity (RCV), microvascular diameter (Dia), functional capillary density (FCD) and the number of rolling leukocytes were studied at baseline and after (I) a climb to an altitude of 3196 m, (II) a passive ascent to the same altitude by helicopter and (III) an exercise program at an altitude below 2100 m in the European Alps. Exposure to high altitude and exercise resulted in an increased heart rate (Trial I: 64 (54-66) vs. 95 (84-100); median (interquartile range); P<0.05) and decreased oxygen saturation (Trial I: 98 (98-99) vs. 90 (88-92); P<0.05). However, RCV, Dia and FCD did not change significantly. Furthermore, no enhanced rolling of leukocytes in postcapillary venules could be observed (Trial I: 6.2 (4.4-6.8) vs. 7.8 (4.3-6.7)). In the pooled data of all three trials of this study we could show a significant positive correlation between oxygen saturation and red blood cell velocity (r = 0.25; P = 0.02). These results indicate that orthogonal polarization spectral imaging can be a useful tool for the microcirculatory assessment of man under hypoxic conditions. We could show that in trained, acclimatized subjects microvascular perfusion is well maintained during hypobaric hypoxia at an altitude of 3196 m and no evidence for an increased postcapillary leukocyte adhesion was seen.

Selection of human immunodeficiency virus type 1 resistance against the pyranodipyrimidine V-165 points to a multimodal mechanism of action.

OBJECTIVES: We have previously identified the pyranodipyrimidines (PDPs) as a new class of integrase (IN) inhibitors. The most potent congener V-165 inhibits HIV-1 integration at low micromolar concentrations by inhibiting the binding of IN to the DNA. As part of pre-clinical studies with PDP, we wanted to investigate HIV resistance development against V-165 and to further characterize the physicochemical properties of the compound. METHODS: We selected PDP-resistant HIV-1 strains by growing the virus in the presence of increasing concentrations of V-165. The selected strains were analysed genotypically and phenotypically. Mutant IN enzymes were generated and evaluated in an enzymatic oligonucleotide-based assay for their activity and sensitivity to the different IN inhibitors. In addition, the antiviral effect of the compound on viral entry and integration was measured using quantitative PCR. RESULTS: Numerous mutations were detected in the RT, IN and env genes of the virus selected in the presence of V-165. Although V-165 inhibited integration in vivo as indicated by a decrease in the number of integrated proviruses, the compound also inhibited viral entry at a concentration of 19 microM. V-165 was poorly recovered from human hepatic microsomal matrix and 1% BSA. CONCLUSIONS: These data point to a multimodal mechanism of action. A quest for derivatives of V-165 that specifically target IN should be pursued.

[Microcirculatory monitoring of sepsis]. / Mikrozirkulatorisches Monitoring der Sepsis.

Microcirculatory dysfunctions play a central role in the pathophysiology of sepsis and shock. Modern methods enable microvascular monitoring in man and offer the possibility to test the effect of novel therapeutical strategies for sepsis. Furthermore, these techniques may be future tools for the monitoring of critically ill patients. In this review, we will describe four microvascular monitoring devices and give an overview of the microcirculatory changes observed during the course of sepsis. Laser Doppler fluxmetry is an easy to use noninvasive technique to measure tissue perfusion enabling monitoring of the effect of different catecholamines on the gastric perfusion during sepsis. Increased microvascular permeability and altered blood flow in septic patients can be quantified by venous congestion plethysmography. Alterations in sublingual microvascular blood flow are detected by intravital microscopy in septic patients and were identified as an outcome predictor. Furthermore, the role of gastrointestinal pCO2-tonometry for microcirculatory monitoring of the perfusion of splanchnic organs during sepsis is discussed. The true clinical value of these techniques has yet to be established and will depend on larger clinical trials showing an impact on diagnostics and patient management.

[Pre-clinical management of shock patients]. / Präklinische Versorgung des Patienten mit Schock.

The preclinical diagnosis of shock is still based on the patient's history, the physical examination, the injury pattern and a few hemodynamic parameters available in the emergency set-up. The clinical picture is characterised by hypotension and tachycardia, tachypnoe and dyspnoea as well as cerebral impairment. Results from recent clinical trials indicate, that a adapted and specific therapeutic approach for the various shock forms is necessary. In case of traumatic hypovolemic-hemorrhagic shock it is of particular relevance if penetrating trauma and/or uncontrolled bleeding exists. Under these conditions an immediate definite surgical treatment is required ("scoop and run") and a moderate hypotension should be tolerated. ("treat and run"). Fluid substitution and therapy with catecholamines should be used conservatively. In all other forms of shock the treatment approach can and should be more aggressive in order to improve microvascular perfusion as early as possible. Besides adequate fluid resuscitation in a combination of crystalloid and colloid solutions catecholamines and-under specific circumstances-also vasopressin should be used. Of utmost importance in the pre-clinical management of patients in shock is the optimal selection of the centre that the patient is referred to in order to establish the fastest and best possible definite treatment for the patient.

[Orthogonal polarization spectral imaging--a new clinical method for monitoring of microcirculation]. / "Orthogonal polarization spectral imaging." Eine neue klinische Methode für das Monitoring der Mikrozirkulation.

The orthogonal polarization spectral (OPS) imaging technology is a new non-invasive method to directly visualize multiple conditions of the microcirculation which has several clinical applications in humans. Quantitative measurement of the diameter of vessels, the velocity of red blood cells and functional capillary density (FCD) can be made. Activation of leukocytes can be monitored and also quantified. A transdermal approach can be used in premature babies and neonates to view the microcirculation and has also been used experimentally to determine haemoglobin levels. The application to various surfaces and solid organs allows a variety of pathophysiologies and phases to be examined.

Influence of different cuff inflation protocols on capillary filtration capacity in human calves -- a congestion plethysmography study.

It has been suggested that venous congestion plethysmography (VCP) substantially underestimates microvascular permeability by activation of a veni-arteriolar constrictor mechanism, even when using small (< 25 mmHg) congestion pressure steps. We studied human lower limbs of 18 young healthy volunteers to test whether the congestion pressure step size of the VCP protocol has an influence on the values of the capillary filtration capacity (CFC) and isovolumetric venous pressure (P(vi)). Two different dual stage VCP pressure step protocols, with 3 and 10 mmHg steps, were used in randomised order and separated by a transient reduction in congestion pressure. Since lymph flow is known to increase after venous congestion, we also looked to see if changes in the estimated lymph flow (J(v)L) occur as a result of these VCP protocols. The measured CFC (median [25th; 75th percentile]) was 2.6 [2.5; 3.2] x 10(-3) ml (100 ml)(-1) min(-1) mmHg(-1) with the 3 mmHg pressure step protocol, which was not different from the value of 2.9 [2.7; 3.4] x 10(-3) ml (100 ml)(-1) min(-1) mmHg(-1) obtained with 10 mmHg pressure steps. However, when either of these step sizes was applied after a transient venous decongestion, significantly higher values of CFC, 4.0 [3.4; 4.1] x 10(-3) and 3.5 [3.1; 4.5] x 10(-3) ml (100 ml)(-1) min(-1) mmHg(-1), respectively, were obtained (P < 0.05). The assessment of P(vi) was also independent of the pressure protocol (10 mmHg: 8.0 [5.7; 13.2] mmHg and 3 mmHg: 15.7 [12.5; 18.5] mmHg), but when P(vi) was measured after the transient deflation, significantly higher values were found with both 10 and 3 mmHg steps (24.1 [20.9; 27.3] and 30.4 [28.9; 30.9] mmHg, respectively; P < 0.01). The transient pressure reduction was associated with a rise in estimated J(v)L from 0.04 [0.03; 0.05] to 0.12 [0.08; 0.18] and 0.04 [0.04; 0.05] to 0.09 [0.07; 0.10] ml (100 ml)(-1) min(-1), respectively (P < 0.01). The first stage data from these protocols shows that the value of CFC is not influenced by the size of the cumulative venous pressure steps, providing they are of 10 mmHg or less. The data also show that J(v)L can be estimated with small step VCP protocols. We hypothesise that the sudden reduction in cuff pressure after venous congestion is associated with a temporary upregulation of lymph flow. As the congestion pressure is raised again, there is a modulation of the enhanced lymph flow, such that the resulting CFC slope appears greater than that obtained in the first stage of the protocol.

Cryopreservation of reduced cytochrome C for determination of N-formyl-methionyl-leucyl-phenylalanine-stimulated superoxide anion production in human whole blood.

Various methods are available for measuring the production of reactive oxygen species by phagocytes, but they are limited in their use by the need for their immediate application, cell isolation and of cell-activation by unphysiological stimuli. In addition, after measurement of reactive oxygen metabolites using oxidizing agents, the reduced compounds formed have to be determined during or immediately after their formation. In the present study, an improved cytochrome C assay was investigated which allowed measurements of superoxide anions in whole blood samples following activation of phagocytes by physiological stimuli such as the bacterial tripeptide N-formyl-methionyl-leucyl-phenylalanine (fMLP). The fMLP-stimulated production of superoxide anion (O(2)(-)) showed a sigmoidal-shaped fMLP dose-response curve, and constant O(2)(-) production rates (nmol.1(-1)x10(6) granulocytes) could be determined reliably up to a blood granulocyte concentration of 1 x 10(4) x microl(-1). To allow the determination of reduced cytochrome C later after its formation, the effect of long-term storage at -20 degrees C on the stability of reduced cytochrome C was tested up to 16 weeks. The results obtained show that the determination of reduced cytochrome C in whole blood represents a simple and reliable method. Most importantly, O(2)(-)-reduced cytochrome C can be frozen and stored without any alterations, at least up to 2 weeks. Thus the method seems to be superior to other methods of detection, especially when the experimental conditions do not allow immediate spectrophotometry (e.g. mountain medicine, space medicine). Under such conditions the present assay would allow reliable measurement of reduced cytochrome C, even after weeks of cryopreservation.

Microvascular changes during anesthesia: sevoflurane compared with propofol.

BACKGROUND: We have developed a non-invasive computer-assisted venous congestion plethysmograph to measure the microvascular parameters in the lower limbs. This enables the assessment of microvascular changes following the induction of standardized anesthesia with either sevoflurane or propofol. METHODS: In a prospective randomized trial we measured the capillary filtration coefficient (CFC), isovolumetric venous pressure (Pvi), an index of the balance of Starling forces, and limb blood flow 24 h preoperatively, immediately after induction of anesthesia and on the 1st and 2nd postoperative day. Anesthesia was maintained with either 1.0% sevoflurane and 5 microg/kg/h remifentanil or propofol (3 mg/kg/h), and 5 microg/kg/h remifentanil in 20 female patients undergoing breast surgery. RESULTS: Preoperatively we found no significant differences between the mean CFC values of the sevoflurane group (3.7+/-0.3 ml/min 100 ml tissue/mmHg x 10-3=CFCU) and the propofol group (3.5+/-0.3 CFCU). In the sevoflurane group CFC decreased significantly to 2.9+/-0.2 CFCU, whereas it was unchanged in the propofol group. Both groups revealed a significant reduction in Pvi during steady-state anesthesia. Limb blood flow remained unchanged. There was an overall significant positive correlation between the perioperative fluid substitution and the difference between the preoperative and intraoperative CFC values (r = 0.64, P<0.01). CONCLUSION: The decreased CFC in response to sevoflurane may result in less extravasation of fluids into the interstitial space, thereby reducing intraoperative fluid requirements. These data suggest that sevoflurane may be the preferred anesthetic agent in subjects susceptible to large intraoperative fluid shifts.

Effects of confinement (110 and 240 days) on neuroendocrine stress response and changes of immune cells in men.

The aim of the study was to evaluate the effects of long-term confinement on stress-permissive neuroendocrine and immune responses in humans. Two groups of four male subjects were confined 240 days (group 240) or 110 days (group 110) in two space modules of 100 or 200 m3, respectively. During confinement, none of the volunteers developed psychic stress as could be examined and verified by a current stress test. However, in group 240 but not in group 110, the diurnal rhythm of cortisol secretion was slightly depressed and the urine excretion of norepinephrine significantly increased. The innate part of the immune system became activated as seen by a rise in the number of circulating granulocytes and the enhanced expression of beta2-integrins. In contrast, the ratio of T-helper to T-suppressor cells decreased. All these effects, observed during confinement, were even more pronounced in both groups when values of endocrinological and immunological parameters were compared between before and 1 wk after the end of the confinement period. Hence, return to normal life exerts pronounced effects to a much higher degree, irrespective of how long or under which conditions individuals were confined. Because the delayed-type hypersensitivity skin reaction against recall antigens remained unaffected, it is to be presumed that confinement appears to induce distinct sympathoadrenergic activation and immunological changes but no clinically relevant immunosuppression.