Transitional changes in the structure of C-reactive protein create highly pro-inflammatory molecules: Therapeutic implications for cardiovascular diseases.

C-reactive protein (CRP) is the prototypic acute-phase reactant that has long been recognized almost exclusively as a marker of inflammation and predictor of cardiovascular risk. However, accumulating evidence indicates that CRP is also a direct pathogenic pro-inflammatory mediator in atherosclerosis and cardiovascular diseases. The 'CRP system' consists of at least two protein conformations with distinct pathophysiological functions. The binding of the native, pentameric CRP (pCRP) to activated cell membranes leads to a conformational change resulting in two highly pro-inflammatory isoforms, pCRP* and monomeric CRP (mCRP). The deposition of these pro-inflammatory isoforms has been shown to aggravate the localized tissue injury in a broad range of pathological conditions including atherosclerosis and thrombosis, myocardial infarction, and stroke. Here, we review recent findings on how these structural changes contribute to the inflammatory response and discuss the transitional changes in the structure of CRP as a novel therapeutic target in cardiovascular diseases and overshooting inflammation.

Lymphatic markers in the human optic nerve.

Tissues of the central nervous system (CNS), including the optic nerve (ON), are considered a-lymphatic. However, lymphatic structures have been described in the dura mater of human ON sheaths. Since it is known that lymphatic markers are also expressed by single non-lymphatic cells, these results need confirmation according to the consensus statement for the use of lymphatic markers in ophthalmologic research. The aim of this study was to screen for the presence of lymphatic structures in the adult human ON using a combination of four lymphatic markers. Cross and longitudinal cryo-sections of human optic nerve tissue (n = 12, male and female, postmortem time = 15.8 ± 5.5 h, age = 66.5 ± 13.8 years), were obtained from cornea donors of the Salzburg eye bank, and analyzed using immunofluorescence with the following markers: FOXC2, CCL21, LYVE-1 and podoplanin (PDPN; lymphatic markers), Iba1 (microglia), CD68 (macrophages), CD31 (endothelial cell, EC), NF200 (neurofilament), as well as GFAP (astrocytes). Human skin sections served as positive controls and confocal microscopy in single optical section mode was used for documentation. In human skin, lymphatic structures were detected, showing a co-localization of LYVE-1/PDPN/FOXC2 and CCL21/LYVE-1. In the human ON however, single LYVE-1+ cells were detected, but were not co-localized with any other lymphatic marker tested. Instead, LYVE-1+ cells displayed immunopositivity for Iba1 and CD68, being more pronounced in the periphery of the ON than in the central region. However, Iba1+/LYVE-1- cells outnumbered Iba1+/LYVE-1+ cells. PDPN, revealed faint labeling in human ON tissue despite strong immunoreactivity in rat ON controls, showing co-localization with GFAP in the periphery. In addition, pronounced autofluorescent dots were detected in the ON, showing inter-individual differences in numbers. In the adult human ON no lymphatic structures were detected, although distinct lymphatic structures were identified in human skin tissue by co-localization of four lymphatic markers. However, single LYVE-1+ cells, also positive for Iba1 and CD68 were present, indicating LYVE-1+ macrophages. Inter-individual differences in the number of LYVE-1+ as well as Iba1+ cells were obvious within the ONs, most likely resulting from diverse medical histories of the donors.

Lymphatic and vascular markers in an optic nerve crush model in rat.

Only few tissues lack lymphatic supply, such as the CNS or the inner eye. However, if the scleral border is compromised due to trauma or tumor, lymphatics are detected in the eye. Since the situation in the optic nerve (ON), part of the CNS, is not clear, the aim of this study is to screen for the presence of lymphatic markers in the healthy and lesioned ON. Brown Norway rats received an unilateral optic nerve crush (ONC) with defined force, leaving the dura intact. Lesioned ONs and unlesioned contralateral controls were analyzed 7 days (n = 5) and 14 days (n = 5) after ONC, with the following markers: PDGFRb (pericyte), Iba1 (microglia), CD68 (macrophages), RECA (endothelial cell), GFAP (astrocyte) as well as LYVE-1 and podoplanin (PDPN; lymphatic markers). Rat skin sections served as positive controls and confocal microscopy in single optical section mode was used for documentation. In healthy ONs, PDGFRb is detected in vessel-like structures, which are associated to RECA positive structures. Some of these PDGFRb+/RECA+ structures are closely associated with LYVE-1+ cells. Homogenous PDPN-immunoreactivity (IR) was detected in healthy ON without vascular appearance, showing no co-localization with LYVE-1 or PDGFRb but co-localization with GFAP. However, in rat skin controls PDPN-IR was co-localized with LYVE-1 and further with RECA in vessel-like structures. In lesioned ONs, numerous PDGFRb+ cells were detected with network-like appearance in the lesion core. The majority of these PDGFRb+ cells were not associated with RECA-IR, but were immunopositive for Iba1 and CD68. Further, single LYVE-1+ cells were detected here. These LYVE-1+ cells were Iba1-positive but PDPN-negative. PDPN-IR was also clearly absent within the lesion site, while LYVE-1+ and PDPN+ structures were both unaltered outside the lesion. In the lesioned area, PDGFRb+/Iba1+/CD68+ network-like cells without vascular association might represent a subtype of microglia/macrophages, potentially involved in repair and phagocytosis. PDPN was detected in non-lymphatic structures in the healthy ON, co-localizing with GFAP but lacking LYVE-1, therefore most likely representing astrocytes. Both, PDPN and GFAP positive structures are absent in the lesion core. At both time points investigated, no lymphatic structures can be identified in the lesioned ON. However, single markers used to identify lymphatics, detected non-lymphatic structures, highlighting the importance of using a panel of markers to properly identify lymphatic structures.

Time-dependent retinal ganglion cell loss, microglial activation and blood-retina-barrier tightness in an acute model of ocular hypertension.

Glaucoma is a group of neurodegenerative diseases characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, and is the second leading cause of blindness worldwide. Elevated intraocular pressure is a well known risk factor for the development of glaucomatous optic neuropathy and pharmacological or surgical lowering of intraocular pressure represents a standard procedure in glaucoma treatment. However, the treatment options are limited and although lowering of intraocular pressure impedes disease progression, glaucoma cannot be cured by the currently available therapy concepts. In an acute short-term ocular hypertension model in rat, we characterize RGC loss, but also microglial cell activation and vascular alterations of the retina at certain time points. The combination of these three parameters might facilitate a better evaluation of the disease progression, and could further serve as a new model to test novel treatment strategies at certain time points. Acute ocular hypertension (OHT) was induced by the injection of magnetic microbeads into the rat anterior chamber angle (n = 22) with magnetic position control, leading to constant elevation of IOP. At certain time points post injection (4d, 7d, 10d, 14d and 21d), RGC loss, microglial activation, and microvascular pericyte (PC) coverage was analyzed using immunohistochemistry with corresponding specific markers (Brn3a, Iba1, NG2). Additionally, the tightness of the retinal vasculature was determined via injections of Texas Red labeled dextran (10 kDa) and subsequently analyzed for vascular leakage. For documentation, confocal laser-scanning microscopy was used, followed by cell counts, capillary length measurements and morphological and statistical analysis. The injection of magnetic microbeads led to a progressive loss of RGCs at the five time points investigated (20.07%, 29.52%, 41.80%, 61.40% and 76.57%). Microglial cells increased in number and displayed an activated morphology, as revealed by Iba1-positive cell number (150.23%, 175%, 429.25%,486.72% and 544.78%) and particle size analysis (205.49%, 203.37%, 412.84%, 333.37% and 299.77%) compared to contralateral control eyes. Pericyte coverage (NG2-positive PC/mm) displayed a significant reduction after 7d of OHT in central, and after 7d and 10d in peripheral retina. Despite these alterations, the tightness of the retinal vasculature remained unaltered at 14 and 21 days after OHT induction. While vascular tightness was unchanged in the course of OHT, a progressive loss of RGCs and activation of microglial cells was detected. Since a significant loss in RGCs was observed already at day 4 of experimental glaucoma, and since activated microglia peaked at day 10, we determined a time frame of 7-14 days after MB injection as potential optimum to study glaucoma mechanisms in this model.

Characterization of dsRed2-positive cells in the doublecortin-dsRed2 transgenic adult rat retina.

Doublecortin (DCX) is predominantly expressed in neuronal precursor cells and young immature neurons of the developing and adult brain, where it is involved in neuronal differentiation, migration and plasticity. Moreover, its expression pattern reflects neurogenesis, and transgenic DCX promoter-driven reporter models have been previously used to investigate adult neurogenesis. In this study, we characterize dsRed2 reporter protein-expressing cells in the adult retina of the transgenic DCX promoter-dsRed2 rat model, with the aim to identify cells with putative neurogenic activity. Additionally, we confirmed the expression of the dsRed2 protein in DCX-expressing cells in the adult hippocampal dentate gyrus. Adult DCX-dsRed2 rat retinas were analyzed by immunohistochemistry for expression of DCX, NF200, Brn3a, Sox2, NeuN, calbindin, calretinin, PKC-a, Otx2, ChAT, PSA-NCAM and the glial markers GFAP and CRALBP, followed by confocal laser-scanning microscopy. In addition, brain sections of transgenic rats were analyzed for dsRed2 expression and co-localization with DCX, NeuN, GFAP and Sox2 in the cortex and dentate gyrus. Endogenous DCX expression in the adult retina was confined to horizontal cells, and these cells co-expressed the DCX promoter-driven dsRed2 reporter protein. In addition, we encountered dsRed2 expression in various other cell types in the retina: retinal ganglion cells (RGCs), a subpopulation of amacrine cells, a minority of bipolar cells and in perivascular cells. Since also RGCs expressed dsRed2, the DCX-dsRed2 rat model might offer a useful tool to study RGCs in vivo under various conditions. Müller glial cells, which have previously been identified as cells with stem cell features and with neurogenic potential, did express neither endogenous DCX nor the dsRed2 reporter. However, and surprisingly, we identified a perivascular glial cell type expressing the dsRed2 reporter, enmeshed with the glia/stem cell marker GFAP and colocalizing with the neural stem cell marker Sox2. These findings suggest the so far undiscovered existence of perivascular associated cell with neural stem cell-like properties in the adult retina.

A novel, microscope based, non-invasive laser Doppler flowmeter for choroidal blood flow assessment.

Impaired ocular blood flow is involved in the pathogenesis of numerous ocular diseases like glaucoma or AMD. The purpose of the present study was to introduce and validate a novel, microscope based, non-invasive Laser Doppler Flowmeter (NI-LDF) for measurement of blood flow in the choroid. The custom made NI-LDF was compared with a commercial fiber optic based laser Doppler flowmeter (Perimed PF4000). Linearity and stability of the NI-LDF were assessed in a silastic tubing model (i.d. 0.3 mm) at different flow rates (range 0.4-3 ml/h). In a rabbit model continuous choroidal blood flow measurements were performed with both instruments simultaneously. During blood flow measurements ocular perfusion pressure was changed by manipulations of intraocular pressure via intravitreal saline infusions. The NI-LDF measurement correlated linearly to intraluminal flow rates in the perfused tubing model (r = 0.99, p < 0.05) and remained stable during a 1 h measurement at a constant flow rate. Rabbit choroidal blood flow measured by the PF4000 and the NI-LDF linearly correlated with each other over the entire measurement range (r = 0.99, y = x∗1.01-12.35 P.U., p < 0.001). In conclusion, the NI-LDF provides valid, semi quantitative measurements of capillary blood flow in comparison to an established LDF instrument and is suitable for measurements at the posterior pole of the eye.

[Biological and physical aspects of intraocular pressure]. / Biologische und physikalische Aspekte des Augendrucks.

A thorough understanding of intraocular pressure homeostasis is the biological foundation for the development of new strategies to treat patients with elevated intraocular pressure or glaucoma. However, investigations on the physiology of intraocular pressure homeostasis are also important to gain more comprehensive insights into the pathogenesis of glaucoma and other diseases with associated alterations of intraocular pressure. The present review intends to give alternative insights into the biological and physical aspects of intraocular pressure regulation. The pressure-volume as well as the hydraulic model of intraocular pressure and also the relationship between ciliary blood flow and aqueous humor production, which has moved into the centre of interest because of its possible clinical relevance for glaucoma patients, will be explained. The authors Have attempted to interrelate the different aspects of intraocular pressure genesis and regulation in a comprehensive but understandable way.

[Processing vestibular impulses in the central nervous system. Study based on positron emission tomography]. / Verarbeitung vestibulärer Impulse im Zentralnervensystem. Untersuchung anhand der Positronen-Emissions-Tomographie.

BACKGROUND AND OBJECTIVE: Functional imaging methods have opened new perspectives for vestibular research. Many authors have investigated the central connections of the system, but the differences between the reports leave further questions open. We investigated the cerebral projection of the vestibular system, using positron emission tomography in right-handed subjects. PATIENTS AND METHODS: Bilateral caloric stimulation was used in every volunteer (n = 6). This can be considered a standard method, which will make it possible to compare the results from different laboratories in the future. A detailed map of activated and deactivated brain regions is included. RESULTS: Changes caused by vestibular stimulation are portrayed. The activated regions partially correspond with previous results in the literature. We would like to point out the Brodmann 6 region as the cortical manifestation of involuntary isometric tightening of muscles. We have found many, previously unidentified regions showing decreased regional cerebral blood flow. CONCLUSIONS: We are the first to point out the functional connection between the hippocampus and the vestibular system in this report.

Colorectal cancer risk in relation to genetic polymorphism of cytochrome P450 1A1, 2E1, and glutathione-S-transferase M1 enzymes.

Chemical carcinogens generally require metabolic activation in order to be able to bind to DNA and contribute to cancer causation. Most of the human metabolizing enzymes are genetically polymorphic, and these polymorphisms may affect the enzyme activity or inducibility. In our present study we investigated the connection between genetic polymorphism of cytochrome P450 1A1, 2E1 (phase I enzymes) and glutathione-S-transferase M1 (a phase II enzyme) and colorectal cancer occurrence in a Hungarian population. The CYP 2E1 c2 allele proved to be in significant association with colorectal cancer (OR: 1.91, 95% CI: 1.05-3.52), the CYP 1A1 Val allele was also overrepresented among colon cancer patients (OR: 1.57, 95% CI: 0.90-2.74), and the frequency of GSTM1 homozygous 0 genotype showed only minor difference (OR: 1.19, 95% CI: 0.75-1.35). Combined analysis of the polymorphisms showed that individuals carrying all the three "high-risk" alleles have a strikingly increased risk for sporadic colorectal cancer (OR: 4.62, 95% CI: 1.23-25.68).

Usefulness of PCR for diagnosis of Pneumocystis carinii pneumonia in different patient groups.

Pneumocystis carinii pneumonia (PCP) is one of the most predominant opportunistic infectious diseases in patients with AIDS. Nested PCR has been described as a sensitive and specific tool for detecting P. carinii DNA in clinical specimens. Little is known about the correlation of positive PCR results and clinical evidence of PCP in patients with different forms of immunosuppression. One hundred and thirty-six sputum samples, 26 tracheal-bronchial aspirate samples, 35 bronchoalveolar lavage samples, and 11 lung biopsy samples from (i) human immunodeficiency virus (HIV)-infected patients with AIDS, (ii) immunocompromised patients with leukemia or lymphoma, and (iii) immunocompetent control patients were investigated by a nested PCR amplifying DNA from the mitochondrial large subunit of P. carinii. All patients suffered from acute episodes of respiratory disease. The resulting data were correlated with clinical evidence of PCP. A high degree of association of positive P. carinii PCR results and clinical evidence of PCP in HIV-infected patients with AIDS was found. When calculated for bronchoalveolar lavage and lung biopsy samples, the positive and the negative predictive values of P. carinii PCR for PCP diagnosis in HIV-infected patients with AIDS were 1 and the specificity and the sensitivity were 100%. In contrast, in the group of patients with leukemia or lymphoma, the positive predictive value of the nested PCR for these materials was found to be as low as 0.09, the negative predictive value was 0.73, the specificity was 44.4%, and the sensitivity was 25.0%. No P. carinii DNA could be detected in specimens from immunocompetent patients. In summary, in contrast to patients with leukemia and lymphoma, nested PCR seems to be a sensitive and specific tool for PCP diagnosis in HIV-infected patients with AIDS.

[Management of postoperative ischemic jejunal stenosis using balloon dilatation and/or Wallstent implant]. / Postoperativ ischaemiás jejunalis stenosis kezelése ballonkatéteres tágítással vagy/és Wallstent implantatióval.

Ischaemic stenosis of the jejunum is rare. For technical, anatomical, and pathological reasons ischemic stenosis of the jejunal segment used for the replacement of the stomach and oesophagus requires a special approach. The present study reports two cases of dilation of ischaemic strictures of the jejunal loop by balloon catheter, used for replacement after oesophagogastrectomy and gastrectomy. In the later case, in which the occlusion of the blood vessels supplying the affected segment was observed right at the level of the aorta, Wallstent was implanted. The advantages and disadvantages of metal stents are discussed and oesophaogoaortic fistula, a rare complication, which appeared a year after Wallstent placement, is described. The two cases presented in this study give evidence that using balloon catheters and implanting Wallstent-in selected cases-may give good results in the management of postoperative ischaemic strictures of the jejunum. The minimally invasive technique with the special indications described here is not known to have been used so far. The rare complication mentioned, however, requires special attention.

The effects of gender and birth control pill use on spontaneous blink rates.

BACKGROUND: Two major reference works suggest that men and women blink spontaneously at different rates, but they disagree with regard to which gender blinks faster. METHODS: Spontaneous blink rates of 59 males and 86 females, 44 of whom were taking birth control (BC) pills, were measured for 5 consecutive minutes. Schirmer test results and tear break-up times (TBUTs) were also obtained. RESULTS: Females taking BC pills blinked at a mean rate of 19.6 times per minute, females not taking birth control pills blinked 14.9 times per minute, and males blinked 14.5 times per minute. There were very large differences between blink rates for individuals in each of the groups. No strong associations were found between spontaneous blink rates and a history of contact lens use, tear break-up time, Schirmer test results, temperature or humidity in the examination room, subject age, or menstrual cycle phase. CONCLUSIONS: The 32 percent increase in mean blink rate for females taking BC pills suggests that the pills affect at least one of the mechanisms that control spontaneous blinking, but it is unclear how they accomplish this.