Vascular endothelial growth factor in gingival crevicular fluid around dental implants.

INTRODUCTION: Angiogenesis occurs under physiological and pathological conditions and is regulated by cytokines and growth factors. Vascular endothelial growth factor (VEGF) is a multifunctional angiogenic cytokine that plays a significant role in inflammation and immune responses implicated in the pathogenesis of inflammatory processes in the implant- surrounding tissues. OBJECTIVE: The study investigated the concentration of VEGF in gingival crevicular fluid (GCF) in healthy and diseased soft tissues surrounding implants. MATERIAL AND METHODS: Clinical examinations were focused on assessing the periodontal status of soft tissues around dental implants with the use of Florida Probe. Bone loss was examined radiologically. VEGF concentrations were assessed by enzyme-linked immunoabsorbent assay (ELISA). RESULTS: VEGF concentrations were found higher in crevicular fluid around implants than in clinically healthy sites. They were also strongly correlated with the pocket depth. CONCLUSIONS: The presence of VEGF in gingival crevicular fluid in patients with peri-implants can be implicated in the progression of peri-implantitis, possibly by promoting the formation of new blood vessels during angiogenic processes.

Trichosporon asahii as a prospective pathogen in solid organ transplant recipients.

Trichosporon spp. is not an important factor of mycotic infections in immunocompetent patients. It may be a cause of invasive mycoses with a high mortality rate in patients undergoing solid organ transplantation. We have analysed the antifungal agents' susceptibility of Trichosporon asahii and its frequency of occurrence as a prospective etiological agent of infections in liver, kidney and simultaneous pancreas-kidney transplant recipients. Clinical specimens (urine, blood, peritoneal fluid and swabs) were obtained from patients hospitalised in the Institute of Transplantation Medicine, Department of General and Transplantation Surgery, Medical University of Warsaw in 2005 and 2006. Microbiological tests were performed in Mycological Laboratory, Department of Microbiology, Medical University of Warsaw. A total of 475 strains of yeast-like fungi were isolated from clinical specimens taken from 263 liver, kidney and simultaneous pancreas-kidney transplant recipients and from 26 organ donors. Trichosporon asahii was found in 26 clinical samples taken from 18 patients and one organ donor. Positive cultures were obtained from 22 urine samples, one stoma fluid, one wound swab, one tracheal aspirate and one ejaculate. Isolates of Trichosporon asahii were found in 6% of total positive mycological cultures in the solid organ transplant recipients. Among cultured strains, 11 isolates were resistant to fluconazole, four to itraconazole and three of them demonstrated resistance to amphotericin B.

NG2 positive cells of rat spinal cord activated during experimental autoimmune encephalomyelitis are spatially associated with radially oriented astroglia and express p75 receptor: a role for nerve growth factor in oligodendrocyte progenitor migration?

Data have been provided from several studies that support the proposal that the adult oligodendrocyte progenitors migrate into the lesioned areas under conditions of experimental autoimmune encephalomyelitis (EAE). However, the routes of migration of these cells and the governing mechanisms are not clear. In the present studies, we have examined the effect of EAE upon activation of endogenous oligodendroglia progenitors and their spatial distribution in the spinal cord of Lewis rats using immunocytochemical procedures. Antibodies against the marker chondroitin sulfate proteoglycan NG2, are used for identification of oligodendroglia progenitors. We find that the activated elongated subpopulation of NG2 positive oligodendroglia progenitors of white matter is spatially associated with the radially-oriented astroglia during the acute phase of EAE. The latter re-expressed the phenotypic embryonic marker nestin while still expressing the mature astroglial marker GFAP. The elongated oligodendroglia progenitors express p75 receptor. In addition, colocalization of NG2 and p75 is observed also in ependymal neural cells of the central canal and the subventricular zone. This raises the possibility that the activated NG2+/p75+ parenchymal cell pool may also be recruited from multipotent neural cells of the germination areas. Our data suggest that, under EAE conditions, the radially oriented astroglia of juvenile phenotype may serve as scaffolding for migrating activated endogenous oligodendroglia progenitors just like radial glia provide a path for neuronal and oligodendroglia progenitor cells in embryonic stage. The expression of p75 receptor in oligodendroglia progenitors associated with radially oriented astroglia during EAE may implicate a role for NGF in the regulation of migration of oligodendroglia progenitors.

Conversion of the tetrameric restriction endonuclease Bse634I into a dimer: oligomeric structure-stability-function correlations.

The Bse634I restriction endonuclease is a tetramer and belongs to the type IIF subtype of restriction enzymes. It requires two recognition sites for its optimal activity and cleaves plasmid DNA with two sites much faster than a single-site DNA. We show that disruption of the tetramerisation interface of Bse634I by site-directed mutagenesis converts the tetrameric enzyme into a dimer. Dimeric W228A mutant cleaves plasmid DNA containing one or two sites with the same efficiency as the tetramer cleaves the two-site plasmid. Hence, the catalytic activity of the Bse634I tetramer on a single-site DNA is down-regulated due to the cross-talking interactions between the individual dimers. The autoinhibition within the Bse634I tetramer is relieved by bridging two DNA copies into the synaptic complex that promotes fast and concerted cleavage at both sites. Cleavage analysis of the oligonucleotide attached to the solid support revealed that Bse634I is able to form catalytically competent synaptic complexes by bridging two molecules of the cognate DNA, cognate DNA-miscognate DNA and cognate DNA-product DNA. Taken together, our data demonstrate that a single W228A mutation converts a tetrameric type IIF restriction enzyme Bse634I into the orthodox dimeric type IIP restriction endonuclease. However, the stability of the dimer towards chemical denaturants, thermal inactivation and proteolytic degradation are compromised.

Hypoxic ventilatory response after dopamine D2 receptor blockade in unilateral rat model of Parkinson's disease.

Modified non-motor brainstem ventilatory control might be involved in Parkinson's disease. Our study was designed to investigate the impact of degeneration of the nigrostriatal dopaminergic pathway on resting breathing and hypoxic ventilatory response in conscious rats. The role of central and peripheral dopamine D2 receptors in the modulation of the hypoxic ventilatory response in conditions of dopamine shortage was examined. Adult Wistar rats received a unilateral double 6-hydroxydopamine lesion of the right medial forebrain bundle. After surgery, animals were placed in whole-body plethysmographic chamber and exposed to hypoxia (8% O2). One group of animals received inraperitoneal injections of either haloperidol or domperidone before hypoxia. Levels of dopamine and its metabolite in the brainstem and striatum were assessed. Neurotoxin treatment evoked limb use asymmetry. No effect on the resting normoxic respiration was observed. An increase in tidal volume and a decrease in respiratory rate during respiratory response to hypoxia with short magnification of minute ventilation were predominant effects. Domperidone treatment in intact animals evoked a significant increase in normoxic tidal volume, while haloperidol potentiated tidal volume increase in response to hypoxia. After the lesion, the effects of both antagonists were absent. In rats with Parkinson's, the content of dopamine and its metabolite decreased substantially in the injured striatum. Augmentation of a tidal volume response to hypoxia, and the absence of stimulatory effect of intraperitoneal domperidone on normoxic and haloperidol on hypoxic tidal volume, in lesioned rats indicated altered control of breathing. This could be the result of a dopamine deficiency in the striatum and an increased turnover of DOPAC/DA in the brainstem.

Quantitative morphological study of microglial cells in the ischemic rat brain using principal component analysis.

Pathogenic stimuli induce alterations in the morphology of microglial cells. We analysed changes in lectin-stained cells on the 1st, 3rd, 7th or 14th day after transient global ischemia. Three areas differing in the degree of microglial reaction were selected for analysis: the upper cerebral cortex, the hippocampal CA1 area, and the hilus of the dentate gyrus. Nine morphological parameters, including fractal dimension, lacunarity, self-similarity range, solidity, convexity and form factor were determined. Then the resultant data were processed using principal component analysis (PCA). We found that the two first principal components together explained more than 73% of the observed variability, and may be sufficient both to describe the morphological diversity of the cells, and to determine the dynamics and direction of the changes. In both hippocampal areas, the transformation to hypertrophied and phagocytic cells was observed, but changes in the hilus were faster than in the CA1. In contrast, in the cortex, a microglial reaction was characterised by an increase in the complexity of processes. The results presented show that the quantitative morphological analysis can be an effective tool in research on the reactive behaviour of microglia and, particularly, in the detection of small and early changes in the cells.

Self-reported data: reliability and role in determining program effectiveness.

This study was conducted to assess the reliability of self-reported hospitalization data, as well as the appropriateness of using self-reported data in evaluating the effectiveness of the Maine Ambulatory Diabetes Education and Follow-Up (ADEF) program. A Maine Blue Cross/Blue Shield (BC/BS) inpatient claims file was used as the reference source to verify self-reported hospitalization data. For a sample of 99 BC/BS subscribers who attended the ADEF program, 77% of the study participants accurately self-reported hospitalization patterns over a 12-mo time period before attending the education program, and 81% of the participants accurately self-reported hospitalization patterns during a posteducation follow-up time period. The reference BC/BS claims data documented a reduction in hospitalizations for the study participants similar to that reported using the ADEF self-reported hospitalization data. The Maine Diabetes Control Project used the self-reported hospitalization data in combination with selected reference claims data to secure third-party reimbursement for the Maine ADEF Program.

The impact of preconception counseling on pregnancy outcomes. The experience of the Maine Diabetes in Pregnancy Program.

OBJECTIVE: To determine if a noncentralized, statewide program could be established to educate health-care providers and women with pregestational diabetes on available strategies to prevent adverse outcomes in pregnancies complicated by diabetes. Characteristics of women who participated in the program and the outcomes of their pregnancies are evaluated. RESEARCH DESIGN AND METHODS: A network of regional providers caring for pregnant women with diabetes was developed. Continuing education sessions were delivered to both providers and women with existing diabetes on the importance of preconception counseling. RESULTS: Maine health-care providers collaborated on the development and adoption of three patient-care guidelines that address preconception counseling, prenatal care, and contraception for women with established diabetes. A total of 185 pregnancies among 160 women with pregestational diabetes reporting estimated delivery dates between 1 January 1987 and 31 December 1990 were identified. Of the total pregnancies, 62 (34%) occurred in women who received preconception counseling: among these 62 pregnancies were one major congenital defect (1.6%) and four fetal or neonatal deaths (6.4%). Among the 123 (66%) pregnancies occurring in women that had not received preconception counseling, 8 (6.5%) infants were born with congenital abnormalities, and 26 (21.1%) fetal or neonatal deaths were documented. CONCLUSIONS: A program promoting preconception counseling can be implemented on a statewide basis by using various health-care providers to deliver the program. Participation in such a program appears to be related to improved pregnancy outcomes among women with pregestational diabetes.

Nerve growth factor affects uninjured, adult rat septohippocampal cholinergic neurons.

The effect of nerve growth factor on the intact versus injured septohippocampal cholinergic system of adult rats was studied. Nerve growth factor was continuously infused into the lateral ventricle of adult uninjured rats or rats that had received unilateral partial transection of the fimbria. Controls (operated and unoperated) received intraventricular infusion of cytochrome c. After 2 weeks of nerve growth factor or cytochrome c treatments, choline acetyltransferase and acetylcholinesterase activities were measured in the septal area and in the hippocampus (divided into dorsal, medial and ventral parts). The continuous infusion of nerve growth factor resulted in a marked dose-dependent increase of choline acetyltransferase activity in both septum and hippocampus of adult unlesioned rats. In lesioned rats the nerve growth factor treatment was capable of inducing choline acetyltransferase activity in the hippocampus of not only the lesioned but also the unlesioned side, as well as in the septal area. In addition, nerve growth factor affected choline acetyltransferase activity differently in the hippocampus of the operated side with respect to the contralateral side or in unoperated animals. The chronic infusion of nerve growth factor did not affect acetylcholinesterase activity in the septum or in the hippocampus of either lesioned or unlesioned rats. The present findings indicate that nerve growth factor is capable of modulating the function of not only damaged but also normal adult forebrain cholinergic neurons. This suggests that nerve growth factor may modulate the function of these neurons in adulthood.

Concomitant up-regulation of astroglial high and low affinity nerve growth factor receptors in the CA1 hippocampal area following global transient cerebral ischemia in rat.

We have examined the effect of global transient cerebral ischemia, evoked in rat by 10 min of cardiac arrest, upon the changes in the cellular expression of two nerve growth factor (NGF) receptors (TrkA and p75) in the hippocampus. We have used immunocytochemical procedures, including a quantitative analysis of staining, along with some quantitative morphological analyses. We have found, under ischemic conditions, a decrease of TrkA immunoreactivity in degenerating CA1 pyramidal neurons and in neuropil. On the other hand, a strong, ischemia-induced up-regulation of TrkA and p75 immunoreactivity was observed in the majority of reactive astroglia population in the adjacent CA1 hippocampal region. The colocalization of the two receptors in the same reactive astroglial cells was evidenced by double immunostaining and further supported by quantitative morphological analysis of TrkA and p75 immunoreactive glial cells. Our data implicate the involvement of NGF receptors in the postischemic regulation of astrocytic function; however, the lack of NGF receptor expression on some astrocytes suggests heterogeneity of astroglia population. Our results also indicate that the lack of neuroprotective action of astroglial NGF induced in the ischemic hippocampus [J Neurosci Res 41 (1995) 684; Acta Neurobiol Exp 57 (1997) 31; Neuroscience 91 (1999) 1027] is not caused by a paucity of NGF receptors but may rather be due to the counteraction of some proinflammatory substances, released simultaneously by glia cells. On the other hand, the up-regulated astroglial TrkA receptor may be an important target for exogenous NGF, which, as previously described [J Neurosci 11 (1991) 2914; Neurosci Lett 141 (1992) 161], exerts a neuroprotective effect in ischemia.

Morphological transformations of cells immunopositive for GFAP, TrkA or p75 in the CA1 hippocampal area following transient global ischemia in the rat. A quantitative study.

Transient global ischemia induces intensive neuronal degeneration in the hippocampal CA1 pyramidal layer, accompanied by reactive transformation of glial cells. Previously, we have shown using the double immunostaining method that the NGF receptors (NGFR) p75 and TrkA are expressed mainly on subpopulations of GFAP+ astrocytes, and this expression increases progressively after ischemia. In the presented study, we analyzed quantitatively the morphological transformations of cells immunopositive for GFAP or NGF receptors in the stratum radiatum of the CA1 hippocampal area in different survival periods after ischemia, evoked by 10-min cardiac arrest in adult rats. In control brains, NGF receptors were expressed only on small cells with poorly ramified processes. After ischemia, the NGFR+ cells increased in size and morphological complexity (measured using fractal analysis). However, even 2 weeks after ischemia these cells did not reach the size and value of the fractal dimension typical of the largest GFAP+ astrocytes. Moreover, the reaction of NGFR+ cells was significantly delayed in comparison with the total astrocyte population. The obtained results suggest that NGF receptors are expressed mainly by immature astrocytes and ischemia induces the maturation of these cells.

Dentate granule neuron apoptosis and glia activation in murine hippocampus induced by trimethyltin exposure.

We investigated the effect of trimethyltin (TMT), a well-known neurotoxicant, on murine hippocampal neurons and glial cells. Three days following intraperitoneal (i.p.) injection of TMT into 1-month-old Balb/c mice at a dose of 2.5 mg/kg body weight we detected damage of the dentate gyrus granular neurons. The dying cells displayed chromatin condensation and internucleosomal DNA fragmentation, which are the most characteristic features of apoptosis. To study, if prolyl oligopeptidase is engaged in neuronal apoptosis following TMT administration, we pretreated mice with the specific inhibitor--Fmoc-Pro-ProCN in doses of 5 and 10 mg/kg body weight (i.p. injection). Three days following injection we did not observe any attenuation of neurotoxic damage, regardless of inhibitor dose, indicating the lack of prolyl oligopeptidase contribution to neuronal injury caused by TMT. The neurodegeneration was associated with reactive astrogliosis in whole hippocampus, but particularly in injured dentate gyrus. The reactive astrocytes showed an increased nerve growth factor (NGF) expression in ventral as well as dorsal hippocampal parts. NGF immunoreactivity was also augmented in neurons of CA3/CA4 areas, which were almost totally spared after TMT intoxication. It suggested a role for this neurotrophin in protection of pyramidal cells from loss of connection between CA3/CA4 and dentate gyrus fields. The granule neurons' death was accompanied by increased histochemical staining with isolectin B4, a marker of microglia, in the region of neurodegeneration. The microglial cells displayed ramified and ameboid morphology, characteristic of their reactive forms. Activated microglia were the main source of interleukin 1beta (IL-1beta). It is possible that this cytokine may participate in neurodegeneration of granule cells. Alternatively, IL-1beta elaborated by microglia could play a role in increasing NGF expression, both in astroglia and in CA3/CA4 neurons.

Prolonged and concomitant induction of astroglial immunoreactivity of interleukin-1beta and interleukin-6 in the rat hippocampus after transient global ischemia.

We have investigated the pattern of expression of IL-1beta and IL-6 immunoreactivities in rat hippocampus after transient complete brain ischemia evoked by a 10 min cardiac arrest, at survival times ranging from 1 day to 28 days. To identify the cell types expressing the two immunoreactivities we used specific cell markers and combined staining procedures. In the intact brain IL-1beta and IL-6 were mainly localized in neurons particularly in pyramidal and granular cell layers. Ischemic insult resulted in a concomitant induction of IL-1beta and IL-6 immunoreactivities in multiple astroglia especially in the CA1 area which is the most vulnerable to ischemic insult as manifested by a massive delayed neuronal death accompanied by an intense gliosis. The number of astroglia expressing both immunoreactivities and the intensity of staining was maximal at the 14th day and remained at the same level at the 28th day. Our data suggest that the astroglial IL-1beta and IL-6 may affect the neurodegeneration of CA1 neurons in the ischemic hippocampus and that the prolonged proinflammatory effects of IL-1beta prevail over the presumed protective action of IL-6.

The upregulation of nerve growth factor receptors in reactive astrocytes of rat spinal cord during experimental autoimmune encephalomyelitis.

Using immunocytochemistry, we have examined the effect of experimental autoimmune encephalomyelitis (EAE) upon the expression of nerve growth factor (NGF) and its TrkA and p75 receptors in astroglia cells of the spinal cord of Lewis rats. We have found that, in normal spinal cord, astroglia of white matter expressed both NGF receptors while those in gray matter expressed only TrkA and no astroglia expressed NGF. During EAE, strong upregulation of TrkA in the astroglia of gray and white matter was found, particularly in a population of radially oriented astrocytes. An upregulation of p75 was noted in radial astroglia and, to some extent, also in the stellate astrocytes of white matter. In general, the upregulation of NGF receptor immunoreactivities in astroglia correlated with the strong intensification of glial fibrillary acidic protein immunocytochemistry, a prominent feature of EAE. No NGF immunoreactivity appeared in any astroglia cells during EAE. Our results suggest that, during EAE, astroglia of the spinal cord become particularly receptive to NGF, possibly as part of a mechanism enabling astroglial cells to respond to localized release of neurotrophins. Moreover, our data suggest that spinal cord astroglia cells may be a potential target for pharmacological manipulations in EAE.

Pathogenicity of various Yersinia enterocolitica serotypes towards mice of Swiss and Balb/c strains.

Series of experiments aimed at determining LD50 of various Y. enterocolitica serotypes toward mice of Swiss and Balb/c strains infected by different route have been made. It appeared impossible to establish the LD50 in mice of Swiss strains after a single and repeated intravenous infection. Attention has been paid to a clear relationship between the mortality of Balb/c mice and culture conditions of Y. enterocolitica strains used for infection. In regard to particular serotypes LC50 could be determined on different days of observation and with various routes of infection of Balb/c mice. However, it concerned only those mice groups which were given suspensions of bacteria or broth culture incubated for 72 hrs at 22 degrees C. Irradiated Balb/c mice were more susceptible to infection with all serotypes of Y. enterocolitica. In the course of oral infection of Balb/c mice with serotypes IA and V of Y. enterocolitica it was possible to check the development of pathological lesions in gut and other organs, this did not refer, however, to Y. enterocolitica serotypes IB, II, III, IV, and VI.

The usefulness of various Yersinia enterocolitica antigens for passive hemagglutination test.

Using various antigen preparations of seven Yersinia enterocolitica serotypes (according to Knapp and Thal) and homologous rabbit immune sera the author succeeded in standardization of passive hemagglutination test. The usefulness of preparations termed "supernatant" derived from heated cells at 100 degrees C and lyophilized "crude" preparations of all serotypes were assessed using blood donors' sera and sera of subjects suspected of infection with Y. enterocolitica. It has been shown that though the diagnostic value of supernatant is higher than that of crude fraction, with the former one it is not possible to differentiate hemagglutinins against particular serotypes. More detailed assessment has been limited to serotype IA, only. An equal usefulness for passive hemagglutination of preparations termed "CA free crude" and "ethanol insoluble fraction" derived from TCA extracted cell sediment has been found.

Standardization of the agglutination test in the diagnosis of Yersinia enterocolitica infection.

For preparation of antigens standard strains of Yersinia enterocolitica representing serological groups I to VI (according to KNAPP and THAL) were used. The following antigens preparations were used: antigens autoclaved at 121 degrees C, antigens treated with formaline and living cells. The antigens were titrated against homologous immune sera, and their optical density was estimated spectrophotometrically. In the rabbit immune sera, and additionally, in the sera of blood donors and subjects suspected of Y. enterocolitica infection the levels of agglutinins were estimated against the antigens of all serotypes. The results from both groups of sera were analysed numerically with the use of the "Odra 1204" computer. Basing on the antigen titers frequency distribution critical and diagnostically doubtless values were established. The usefulness of living cells and antigens treated with formaline of the serotype IA and serotype V in the serological diagnosis of the Y. enterocolitica infections was demonstrated.

Sensitivity to chemotherapeutics of Yersinia enterocolitica strains.

290 Y. enterocolitica strains were tested as regards their sensitivity to 20 chemotherapeutics. All the strains were isolated from fecal specimens taken from human beings in Czechoslovakia. It should be noted that the strains presented homologous group as they belonged to 3 serological group according to Winblad or IA according to Knapp and Thal, they also belonged to biotype 4 according to Nilehn. It has been found that apart from their homogenicity, they showed quite different patterns of sensitivity to chemotherapeutics. Among them quite a great number of the strains were of high level of resistance to antibiotics. It was not possible to transfer their resistance to the sensitive E. coli K12 strain. All the strains proved to produce betalactamase.

Humoral and cellular immune reaction in Balb/c mice infected with Yersinia enterocolitica.

Experiments were carried out on Balb/c mice infected with Y. enterocolitica serotypes IA, IB, II, III, IV, V and VI (according to Knopp and Thal) to check humoral and cellular immune response both primary and secondary. It was shown that the occurrence of anti-OH agglutinins and their dynamics depends on the serological type of Y. enterocolitica used for infection, and that it is related to the primary and secondary challenge. The presence of anti-OH agglutinins and hemagglutinins was observed only in cases of secondary infection of the mice. Delayed hypersensitivity evaluated as a cellular reaction assayed with a capillary test of spleen macrophages migration was found only in the cases of infection with serotypes IB, II, IV and VI. The inhibition of macrophage migration was not observed in the cases when infection of Balb/c mice had been evoked with Y. enterocolitica serotypes IA, III and V.